U.S. patent number 8,283,122 [Application Number 11/572,667] was granted by the patent office on 2012-10-09 for prediction of clinical outcome using gene expression profiling and artificial neural networks for patients with neuroblastoma.
This patent grant is currently assigned to N/A, The United States of America, as Represented by the Secretary of the Department of Health and Human Services. Invention is credited to Braden T. Greer, Javed Khan, Jun S. Wei.
United States Patent |
8,283,122 |
Khan , et al. |
October 9, 2012 |
Prediction of clinical outcome using gene expression profiling and
artificial neural networks for patients with neuroblastoma
Abstract
A method of predicting the outcome of a patient with
neuroblastoma that includes obtaining experimental data on gene
selections. The gene selection functions to predict the outcome of
a patient with neuroblastoma when the expression of that gene
selection is compared to the identical selection from a
non-neuroblastoma cell or a different type of neuroblastoma cell.
The invention also includes a method of targeting at least one
product of a gene that includes administration of a therapeutic
agent. The invention also includes the use of a gene selection for
predicting the outcome of patient with neuroblastoma.
Inventors: |
Khan; Javed (Derwood, MD),
Wei; Jun S. (Gaithersburg, MD), Greer; Braden T.
(Gaithersburg, MD) |
Assignee: |
The United States of America, as
Represented by the Secretary of the Department of Health and Human
Services (Washington, DC)
N/A (N/A)
|
Family
ID: |
36000505 |
Appl.
No.: |
11/572,667 |
Filed: |
August 3, 2005 |
PCT
Filed: |
August 03, 2005 |
PCT No.: |
PCT/US2005/027660 |
371(c)(1),(2),(4) Date: |
April 28, 2009 |
PCT
Pub. No.: |
WO2006/026051 |
PCT
Pub. Date: |
March 09, 2007 |
Prior Publication Data
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|
|
Document
Identifier |
Publication Date |
|
US 20090215033 A1 |
Aug 27, 2009 |
|
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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60598728 |
Aug 3, 2004 |
|
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Current U.S.
Class: |
435/6.14 |
Current CPC
Class: |
C12Q
1/6886 (20130101); C12Q 2600/118 (20130101); C12Q
2600/136 (20130101); C12Q 2600/106 (20130101) |
Current International
Class: |
C12Q
1/68 (20060101) |
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|
Primary Examiner: Martinell; James
Attorney, Agent or Firm: Edwards Wildman Palmer LLP Corless;
Peter F. Shyjan, Esq.; Andrew W.
Government Interests
STATEMENT REGARDING FEDERALLY SUPPORTED RESEARCH
This invention was developed with the support of the Department of
Health and Human Services. The Government of the United States of
America has certain rights in the invention disclosed and claimed
herein below.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation of PCT/US2005/027660, filed Aug.
3, 2005, which claims priority to U.S. Provisional Patent
Application Ser. No. 60/598,728, filed Aug. 3, 2004 and claims the
benefit of that application under 35 U.S.C. .sctn.119(e), which
applications are hereby incorporated by reference.
Claims
We claim:
1. A method of predicting the outcome of a patient having
neuroblastoma comprising detecting an increase in expression of at
least one gene or group of genes selected from the group consisting
of PRSS3; PRSS3 and DLK1; PRSS3 and SLIT3; and PRSS3, DLK1, and
SLIT3, in a neuroblastoma cell from the patient, wherein an
increase in expression of at least one of the genes is indicative
of poor outcome of the subject.
2. The method of claim 1, wherein the DLK1 gene comprises Image ID
NO: 296815 or Image ID NO: 436121.
3. The method of claim 2, wherein the DLK1 gene comprises SEQ ID
NO:1.
4. The method of claim 1, wherein the SLIT3 gene comprises Image ID
NO: 450382, Image ID NO: 192225, or Image ID NO: 2030301.
5. The method of claim 1, wherein the SLIT3 gene comprises SEQ ID
NO:6.
6. The method of claim 1, wherein the PRSS3 gene comprises Image ID
NO: 1913366.
7. The method of claim 1, wherein the PRSS3 gene comprises SEQ ID
NO:3.
8. The method of claim 1, wherein the neuroblastoma cell does not
have an amplification of MYCN.
9. The method of claim 8, wherein an increase in expression of at
least one of the genes comprises detecting expression using micro
array analysis.
10. The method of claim 1, wherein the expression of at least one
of the genes is detected by detecting an increase in mRNA.
11. The method of claim 1, wherein detecting an increase in
expression comprises detecting an increase in serum levels of a
polypeptide encoded by at least one of the genes.
12. The method of claim 1, further comprising detecting expression
of MYCN.
13. The method of claim 1, further comprising detecting expression
of CD44.
14. The method of claim 1, further comprising detecting expression
of one of the genes selected from the group consisting of MYCN,
ARC, JPH1, Hs. 434957, Hs. 346735, Hs. 120591, CD44, ARH1, CNR1,
ROBO2, BTBD3, KLRC3, Hs. 196008, Hs. 124776, Hs. 119947, Hs.
349094.
15. The method of claim 14, wherein the gene MYCN comprises Image
ID NO: 41565.
16. The method of claim 14, wherein the gene MYCN comprises SEQ ID
NO:16.
17. The method of claim 14, wherein the gene for CD44 comprises
Image ID NO: 1967589.
18. The method of claim 14, wherein the gene for CD44 comprises SEQ
ID NO:12.
19. The method of claim 14, wherein a) a gene DLK1 comprises Image
ID NO: 296815 or 436121; b) a gene PRSS3 comprises Image ID NO:
1913366; c) a gene ARC, comprises Image ID NO: 222457; d) a gene
SLIT3 comprises Image ID NO: 450382, or Image ID NO: 192225, or
Image ID NO: 2030301; e) a gene JPH1 comprises Image ID NO: 811874;
f) a gene ARH1 comprises Image ID NO: 2336916; g) a gene CNR1
comprises Image ID NO: 26295; h) a gene ROBO2 comprises Image ID
NO: 377573; i) a gene BTBD3 comprises Image ID NO: 811918; j) a
gene KLRC3 comprises Image ID NO: 2361911; k) Hs. 434957 comprises
Image ID NO: 681891; l) Hs. 346735 comprises Image ID NO: 143169;
m) Hs. 120591 comprises Image ID NO: 1540478; n) Hs. 196008
comprises Image ID NO: 111264; o) Hs. 124776 comprises Image ID NO:
1574206; p) Hs. 119947 comprises Image ID NO: 379779; and q) Hs.
349094 comprises Image ID NO: 687667.
20. The method of claim 14, wherein a) a gene DLK1 comprises SEQ ID
NO:1; b) a gene PRSS3 comprises SEQ ID NO:3; c) a gene ARC
comprises SEQ ID NO:5; d) a gene SLIT3 comprises SEQ ID NO:6; e) a
gene JPH1 comprises SEQ ID NO:18; f) a gene ARH1 comprises SEQ ID
NO:4 g) a gene CNR1 comprises SEQ ID NO:7; h) a gene ROB02
comprises SEQ ID NO:14; i) a gene BTBD3 comprises SEQ ID NO:15; j)
a gene KLRC3 comprises SEQ ID NO:19; k) Hs. 434957 comprises SEQ ID
NO:11; l) Hs. 346735 comprises SEQ ID NO:8; m) Hs. 120591 comprises
SEQ ID NO:2; n) Hs. 196008 comprises SEQ ID N09; o) Hs. 124776
comprises SEQ ID NO:17; p) Hs. 119947 comprises SEQ ID NO:10; and
q) Hs. 349094 comprises SEQ ID NO:13.
21. The method of claim 14, wherein the expression of DLK1 (SEQ ID
NO: 1), EST (SEQ ID NO: 2), PRSS3 (SEQ ID NO: 3), ARHI (SEQ ID NO:
4), ARC (SEQ ID NO: 5), SLIT3 (SEQ ID NO: 6), CNR1 (SEQ ID NO: 7),
EST (SEQ ID NO: 8), EST (SEQ ID NO: 9), FLJ25461 (SEQ ID NO: 10),
EST (SEQ ID NO: 11), CD44 (SEQ ID NO: 12), EST (SEQ ID NO: 13),
ROBO2 (SEQ ID NO: 14), BTBD3 (SEQ ID NO: 15), MYCN (SEQ ID NO: 16),
EST (SEQ ID NO: 17), JPH1 (SEQ ID NO: 18), and KLRC3 (SEQ ID NO:
19) are detected.
22. The method of claim 21, further comprising detecting expression
of at least one or all genes selected from the group consisting of
EST (SEQ ID NO: 20), RET (SEQ ID NO: 21), CRABP1 (SEQ ID NO: 22),
ECEL1 (SEQ ID NO: 23), LOC283120 (SEQ ID NO: 24), HMGA2 (SEQ ID NO:
25), SNYPO2 (SEQ ID NO: 26), LOC163782 (SEQ ID NO: 27), VSNL1 (SEQ
ID NO: 28), HS3ST4 (SEQ ID NO: 29), AKR1C1 (SEQ ID NO: 30), EST
(SEQ ID NO: 31), GPR22 (SEQ ID NO: 32), EST (SEQ ID NO: 33), EST
(SEQ ID NO: 34), CCNA1 (SEQ ID NO: 35), PK1B (SEQ ID NO: 36), EST
(SEQ ID NO: 37), GAL (SEQ ID NO: 38), EST (SEQ ID NO: 39),
LOC221303 (SEQ ID NO: 40), EST (SEQ ID NO: 41), EST (SEQ ID NO:
42), BMP7 (SEQ ID NO: 43), SLC30A3 (SEQ ID NO: 44), FLJ10539 (SEQ
ID NO: 45), AMIGO2 (SEQ ID NO: 46), AKR1C2 (SEQ ID NO: 47), MGP
(SEQ ID NO: 48), PCSK1 (SEQ ID NO: 49), HK2 (SEQ ID NO: 50), EST
(SEQ ID NO: 51), EST (SEQ ID NO: 52), IL7 (SEQ ID NO: 53), PRSS12
(SEQ ID NO: 54), GABARAPL (SEQ ID NO: 55), DEFB129 (SEQ ID NO: 56),
NAV3 (SEQ ID NO: 57), RAB3B (SEQ ID NO: 58), KRT6B (SEQ ID NO: 59),
BEX1 (SEQ ID NO: 60), EST (SEQ ID NO: 61), EST (SEQ ID NO: 62),
SCYL1 (SEQ ID NO: 63), EST (SEQ ID NO: 64), RYR2 (SEQ ID NO: 65),
LRBA (SEQ ID NO: 66), CSPG3 (SEQ ID NO: 67), EST (SEQ ID NO: 68),
MMP12 (SEQ ID NO: 69), CHRNA1 (SEQ ID NO: 70), EST (SEQ ID NO: 71),
EST (SEQ ID NO: 72), HNRPH1 (SEQ ID NO: 73), LOC113251 (SEQ ID NO:
74), EST (SEQ ID NO: 75), PAG (SEQ ID NO: 76), PROK2 (SEQ ID NO:
77), HS6ST1 (SEQ ID NO: 78), EST (SEQ ID NO: 79), PCDH9 (SEQ ID NO:
80), EST (SEQ ID NO: 81), EST (SEQ ID NO: 82), GLDC (SEQ ID NO:
83), ADRB2 (SEQ ID NO: 84), ICSBP1 (SEQ ID NO: 85), CD48 (SEQ ID
NO: 86), EST (SEQ ID NO: 87), DYRK1B (SEQ ID NO: 88), KLRC1 (SEQ ID
NO: 89), EST (SEQ ID NO: 90), EST (SEQ ID NO: 91), EST (SEQ ID NO:
92), MOXD1 (SEQ ID NO: 93), EST (SEQ ID NO: 94), EST (SEQ ID NO:
95), GAS1 (SEQ ID NO: 96), COL9A2 (SEQ ID NO: 97), EST (SEQ ID NO:
98), DRPLA (SEQ ID NO: 99), EST (SEQ ID NO: 100), REPRIMO (SEQ ID
NO: 101), CACNA2D2 (SEQ ID NO: 102), NEBL (SEQ ID NO: 103), EST
(SEQ ID NO: 104), HLA-DQA1 (SEQ ID NO: 105), EDG3 (SEQ ID NO: 106),
CPVL (SEQ ID NO: 107), FLJ32884 (SEQ ID NO: 108), LCP1 (SEQ ID NO:
109), EST (SEQ ID NO: 110), EST (SEQ ID NO: 111), EST (SEQ ID NO:
112), EST (SEQ ID NO: 113), DKFZP564C152 (SEQ ID NO: 114), DMN (SEQ
ID NO: 115), GABRA5 (SEQ ID NO: 116), AKR1C3 (SEQ ID NO: 117),
LOC168850 (SEQ ID NO: 118), EST (SEQ ID NO: 119), KCNQ2 (SEQ ID NO:
120), NME5 (SEQ ID NO: 121), EST (SEQ ID NO: 122), PBX1 (SEQ ID NO:
123), CNTNAP2 (SEQ ID NO: 124), EST (SEQ ID NO: 125), SPON1 (SEQ ID
NO: 126), CDH8 (SEQ ID NO: 127), PRKCB1 (SEQ ID NO: 128), SLC21A11
(SEQ ID NO: 129), MAP4 (SEQ ID NO: 130), EST (SEQ ID NO: 131),
SCN7A (SEQ ID NO: 132), EST (SEQ ID NO: 133), EST (SEQ ID NO: 134),
EST (SEQ ID NO: 135), EST (SEQ ID NO: 136), CDW52 (SEQ ID NO: 137),
ARCB1 (SEQ ID NO: 138), EST (SEQ ID NO: 139), OST-2 (SEQ ID NO:
140), NRXN1 (SEQ ID NO: 141), ADAM22 (SEQ ID NO: 142), EST (SEQ ID
NO: 143), TRGV9 (SEQ ID NO: 144), EST (SEQ ID NO: 145), PTPRD (SEQ
ID NO: 146), EST (SEQ ID NO: 147), HS3ST2 (SEQ ID NO: 148), FGF13
(SEQ ID NO: 149), MKI67 (SEQ ID NO: 150), KIF12 (SEQ ID NO: 151),
EST (SEQ ID NO: 152), EST (SEQ ID NO: 153), EST (SEQ ID NO: 154),
EST (SEQ ID NO: 155), EST (SEQ ID NO: 156), KLIP1 (SEQ ID NO: 157),
EST (SEQ ID NO: 158), LOC157570 (SEQ ID NO: 159), MAD2L1 (SEQ ID
NO: 160), EST (SEQ ID NO: 161), EST (SEQ ID NO: 162), RGS5 (SEQ ID
NO: 163), ATP2B4 (SEQ ID NO: 164), HMGCL (SEQ ID NO: 165), ODZ3
(SEQ ID NO: 166), CHGA (SEQ ID NO: 167), MGC33510 (SEQ ID NO: 168),
GAGES (SEQ ID NO: 169), SARDH (SEQ ID NO: 170), EST (SEQ ID NO:
171), DAT1 (SEQ ID NO: 172), FUCA1 (SEQ ID NO: 173), TM6SF2 (SEQ ID
NO: 174), KCNK9 (SEQ ID NO: 175), ADCYAP1 (SEQ ID NO: 176), PLXNA4
(SEQ ID NO: 177), HLA-DMB (SEQ ID NO: 178), EST (SEQ ID NO: 179),
EST (SEQ ID NO: 180), GRIN3A (SEQ ID NO: 181), OSBPL3 (SEQ ID NO:
182), ODZ4 (SEQ ID NO: 183), EST (SEQ ID NO: 184), E2F1 (SEQ ID NO:
185), MGC16664 (SEQ ID NO: 186), HMP19 (SEQ ID NO: 187), IL2RB (SEQ
ID NO: 188), TOPK (SEQ ID NO: 189), ALDH1A1 (SEQ ID NO: 190), CED-6
(SEQ ID NO: 191), EST (SEQ ID NO: 192), A2BP1 (SEQ ID NO: 193),
LY6E (SEQ ID NO: 194), EST (SEQ ID NO: 195), EST (SEQ ID NO: 196),
PLXNC1 (SEQ ID NO: 197), EFS (SEQ ID NO: 198), ACTN2 (SEQ ID NO:
199), MYC (SEQ ID NO: 200), KIAA0527 (SEQ ID NO: 201), C6orf31 (SEQ
ID NO: 202), DLL3 (SEQ ID NO: 203), EST (SEQ ID NO: 204), STK33
(SEQ ID NO: 205), SEMA3A (SEQ ID NO: 206), EST (SEQ ID NO: 207),
IGSF4 (SEQ ID NO: 208), CKS2 (SEQ ID NO: 209), EST (SEQ ID NO:
210), EST (SEQ ID NO: 211), SIX3 (SEQ ID NO: 212), F1122002 (SEQ ID
NO: 213), HSD17B12 (SEQ ID NO: 214), HBA2 (SEQ ID NO: 215), CDH11
(SEQ ID NO: 216), RGS9 (SEQ ID NO: 217), EST (SEQ ID NO: 218),
NCAM2 (SEQ ID NO: 219), BIRC5 (SEQ ID NO: 220), EST (SEQ ID NO:
221), GNG12 (SEQ ID NO: 222), GPIG4 (SEQ ID NO: 223), EST (SEQ ID
NO: 224), ENPP4 (SEQ ID NO: 225), FMNL (SEQ ID NO: 226), EST (SEQ
ID NO: 227), PIWIL2 (SEQ ID NO: 228), CLSTN1 (SEQ ID NO: 229),
UHRF1 (SEQ ID NO: 230), EST (SEQ ID NO: 231), SLC40A1 (SEQ ID NO:
232), CLECSF6 (SEQ ID NO: 233), EST (SEQ ID NO: 234), BKLHD2 (SEQ
ID NO: 235), EST (SEQ ID NO: 236), EST (SEQ ID NO: 237), EST (SEQ
ID NO: 238), SORCS1 (SEQ ID NO: 239), NRP2 (SEQ ID NO: 240), E2-EPF
(SEQ ID NO: 241), CAST (SEQ ID NO: 242), KIAA1384 (SEQ ID NO: 243),
KIAA0644 (SEQ ID NO: 244), HLA-DRB3 (SEQ ID NO: 245), PMP22 (SEQ ID
NO: 246), DJ9P11.1 (SEQ ID NO: 247), SOX5 (SEQ ID NO: 248), CD3E
(SEQ ID NO: 249), and EST (SEQ ID NO: 250).
23. The method of claim 14, wherein the expression of the gene is
detecting mRNA.
24. The method of claim 23, wherein the mRNA is detected using
microarray analysis.
Description
FIELD OF THE INVENTION
The invention relates generally to selections of genes expressed in
a patient with neuroblastoma that function to characterize the
neuroblastoma, and methods of using the same for predicting the
outcome of and for targeting the therapy of neuroblastoma. The
invention also relates generally to the use of supervised pattern
recognition methods to predict the outcome of patients with
neuroblastoma. More specifically, the invention relates to the use
of supervised pattern recognition methods, such as artificial
neural networks for the prognosis of patients with neuroblastoma
using high dimensional data, such as gene expression profiling
data.
BACKGROUND OF THE INVENTION
Diagnosis and/or prognosis of disease is based on a myriad of
factors, both objective and subjective, including but not limited
to symptoms, laboratory test values, demographic factors and
environmental factors. Diagnosis and/or prognosis relies on a
clinician such as a physician or a veterinarian being able to
identify and evaluate the relevant factors. Often this task can be
difficult, and becomes exceedingly more so as the number of factors
to be considered increases.
An example of a disease whose diagnosis or prognosis is difficult
is cancer. Cancer may be diagnosed or prognosis developed on the
basis of clinical presentation, routine histology,
immunohistochemistry and electron microscopy. However, the
histological appearance may not reveal the genetic aberrations or
underlying biologic processes that contribute to the malignancy.
Monitoring global gene expression levels using DNA microarrays
could provide an additional tool for elucidating tumor biology as
well as the potential for molecular diagnostic classification of
cancers. Several studies have demonstrated that gene expression
profiling using DNA microarrays is able to classify tumors with a
high accuracy, and discover new cancer classes.
In clinical practice, several techniques are used for diagnosis or
prognosis, including immunohistochemistry, cytogenetics, interphase
fluorescence in situ hybridization and reverse transcription
(RT)-PCR. Immunohistochemistry allows the detection of protein
expression, but it can only examine one protein at a time.
Molecular techniques such as RT-PCR are used increasingly for
diagnostic confirmation following the discovery of tumor-specific
translocations such as EWS-FLI1; t(11;22)(q24;q12) in EWS, and the
PAX3-FKHR; t(2;13)(q35;q14) in alveolar rhabdomyosarcoma (ARMS).
However, molecular markers do not always provide a definitive
diagnosis or prognosis, as on occasion there is failure to detect
the classical translocations, due to either technical difficulties
or the presence of variant translocations.
DNA microarray technology is a recently developed high throughput
technology for monitoring gene expression at the transcription
level. Its use is akin to performing tens of thousands of northern
blots simultaneously, and has the potential for parallel
integration of the expression levels of an entire genome. A DNA
microarray includes DNA probes immobilized on a solid support such
as a glass microscope slide. The DNA probes can be double stranded
cDNA or short (25 mers) or long (50-70 mers) oligonucleotides of
known sequences. An ideal DNA microarray should be able to
interrogate all of the genes expressed in an organism.
In DNA microarrays using cDNA, the probes are PCR amplified from
plasmid cDNA clones that have been purified and can be robotically
printed onto coated glass slides. DNA microarrays using
oligonucleotides have an advantage over cDNA microarrays because
physical clones are not necessary. The oligonucleotides can either
be previously synthesized and printed on glass slides, or can be
synthesized directly on the surface of silicon or glass slides.
Several print-ready oligonucleotide (60-70 mers) sets are
commercially available for human, mouse and other organisms
(http://www.cgen.com, http://www.operon.com).
Another technique for fabricating oligonucleotides microarrays
chemically synthesizes the oligonucleotides (25 mers) on a silicon
surface using photolithography techniques. (Affymetrix Inc., Santa
Clara, Calif.). Originally such arrays were designed to detect
single-nucleotide mutations, but now have applications for gene
expression profiling studies. Yet another technique delivers single
nucleic acids, which ultimately form longer oligonucleotides (60
mers), by ink-jet onto glass surfaces.
One method of utilizing gene expression data from microarrays is
given by Tusher et al., PNAS 98(9) p. 5116-21, April, 2001. The
method of Tusher et al. is a statistical method titled Significance
Analysis of Microarrays ("SAM"). The general approach in SAM is
based on commonly used statistical tests, t-tests specifically, to
find genes that discriminate between two classes in a gene-by-gene
fashion. SAM uses replication of experiments to assign a
significance to the discriminating genes in terms of a false
discover rate. SAM therefore offers a method of choosing particular
genes from a set of gene expression data, but does not offer a
diagnosis based on those genes.
Gene-expression profiling using DNA microarrays may permit a
simultaneous analysis of multiple markers, and can be used for
example to categorize cancers into subgroups or provide other
information concerning the relationship of the gene expression
profile and the disease state. The only limitation associated with
the use of DNA microarrays is the vast amount of data generated
thereby. A method that would allow for the easy and automated use
of DNA microarray data in disease diagnosis or prognosis is
therefore desirable. Therefore, there remains a need for a method
of using gene expression data to diagnose, predict, or
prognosticate about a disease condition.
SUMMARY OF THE INVENTION
In accordance with one embodiment of the invention, there is
provided a selection of genes, expressed in a patient with
neuroblastoma, that functions to predict the outcome of the patient
when the expression of a gene selection from the cancer cell is
compared to the expression of an identical selection of genes from
a noncancerous cell or an identical selection of genes from a
cancer cell from a patient with a good outcome and/or porr outcome.
Devices for carrying out the above methods of the invention are
also included within the scope of the invention.
Another embodiment of the invention includes a method of targeting
a product of at least one of the genes in Table 2 that includes
identifying a therapeutic agent. Another embodiment of the
invention includes a method of targeting a product of at least one
of the genes in Table 3 that includes identifying a therapeutic
agent having an effect on said gene product.
Another embodiment of the invention provides a method of
predicting, and/or prognosticating about a disease including
obtaining experimental data, wherein the experimental data includes
high dimensional data, filtering noise from the data, reducing the
dimensionality of the data by using one or more methods of
analysis, training a supervised pattern recognition and/or
classification method, ranking individual data from the overall
data based on the relevance of the individual data to the
diagnosis, prediction, prognosis or classification, choosing
multiple individual data members, wherein the choice is based on
the relative ranking of the individual data, and using the chosen
data to determine if an unknown set of experimental data indicates
a particular disease prognosis, or prediction. Methods of the
invention may utilize linear methods, and preferably, methods of
the invention use nonlinear (with hidden layers) networks.
Methods of the invention can be utilized in a number of different
applications. For example, diagnostic chips can be fabricated based
on the identification of the diagnostic or prognostic genes. Such
chips would be very useful in clinical settings, as it would allow
clinicians to diagnose cancers or provide a prognosis from a
relatively small set of genes instead of purchasing entire gene
sets.
Methods of the invention can also be used to define which patients
with neuroblastoma are likely to respond to treatment. This would
allow a physician to intensify treatment for those with a more
negative prognosis based on their gene expression profiles as
detected utilizing a method of the invention. One aspect of the
invention includes a method of predicting the outcome of a patient
having neuroblastoma comprising detecting an increase in expression
of at least one gene selected from the group consisting of DLK1,
SLIT3, PRSS3, and mixtures thereof in a neuroblastoma cell from the
patient; wherein an increase in expression of at least one of the
genes is indicative of poor outcome of the subject.
Another method of predicting the outcome of patient having
neuroblastoma comprises detecting a change in expression at least
one gene or polynucleotide selected from the group consisting of
DLK1, PRSS3, ARC, SLIT3, JPH1, ARH1, CNR1, ROBO2, BTBD3, KLRC3, Hs.
434957, Hs. 346735, Hs. 120591, Hs. 196008, Hs. 124776, Hs. 119947,
Hs. 349094, and mixtures thereof, in a neuroblastoma cell from the
patient, wherein the expression profile of the gene or
polynucleotide is indicative of the outcome of the patient.
In some embodiments of the methods, the expression of at least one
of the genes or polynucleotides selected from the group consisting
of MYCN, DLK1, PRSS3, ARC, SLIT3, JPH1, Hs. 434957, Hs. 346735, Hs.
120591, and mixtures thereof, is upregulated, indicating the
outcome of the patient is poor. In other embodiments, the
expression of at least one gene or polynucleotide selected from the
group consisting of CD44, ARH1, CNR1, ROBO2, BTBD3, KLRC3, Hs.
196008, Hs. 124776, Hs. 119947, Hs. 349094, and mixtures thereof,
is downregulated, indicating the outcome of the patient is
poor.
In some embodiments all of the genes or polynucleotides of Table 2
are analyzed. In other embodiments at least one or all of the genes
of Table 3 are analyzed.
Another aspect of the invention includes a set or selection of
genes or polynucleotides comprising at least two genes or
polynucleotides selected from the consisting of DLK1, PRSS3, ARC,
SLIT3, JPH1, ARH1, CNR1, ROBO2, BTBD3, KLRC3, Hs. 434957, Hs.
346735, Hs. 120591, Hs. 196008, Hs. 124776, Hs. 119947, Hs. 349094,
and mixtures thereof, or the complements thereof. The set of genes
may further comprise MYCN and/or CD44.
Methods of the invention can also be used for identifying
pharmaceutical targets. Methods of the invention can be used to
determine which genes to target in efforts to target specific
diseases. Such methods include a method of identifying an agent
that can modulate the expression or activity of at least one gene
or polynucleotide comprising measuring expression or activity of at
least one polynucleotide or gene selected from the group consisting
of DLK1, PRSS3, ARC, SLIT3, JPH1, ARH1, CNR1, ROBO2, BTBD3, KLRC3,
Hs. 434957, Hs. 346735, Hs. 120591, Hs. 196008, Hs. 124776, Hs.
119947, Hs. 349094, and mixtures thereof, in the presence or
absence of a candidate agent; and identifying the candidate agent
that inhibits or increases expression or activity of the
polynucleotide or gene. Another method comprises measuring
expression or activity of at least one gene or polynucleotide
selected from the group consisting of DLK1, PRSS3, ARC, SLIT3,
JPH1, Hs. 434957, Hs. 346735, Hs. 120591, and mixtures thereof, in
the presence or absence of the candidate antagonist; determining
whether the candidate antagonist inhibits expression or activity of
at least one of the polynucleotides or genes. In another
embodiment, a method of identifying an agonist comprises measuring
expression or activity of at least one polynucleotide or gene
selected from the group consisting of ARH1, CNR1, ROBO2, BTBD3,
KLRC3, Hs. 196008, Hs. 124776, Hs. 119947, Hs. 349094, and mixtures
thereof, in the presence and absence of the candidate agonist; and
determining whether the candidate agonist increases expression
and/or activity of the polynucleotide or gene.
Another aspect provides for kits, devices for implementing the
methods of the invention.
Methods of the invention can also be utilized as a research tool
for analyzing all types of gene expression data including cDNA and
oligonucleotide microarray data. Methods of the invention can also
be utilized to identify and rank, by importance, the genes that
contribute to a prognosis. A minimal set of genes that can
correctly predict clinical outcomes can also be determined using
methods of the invention. Methods of the invention identify the
most significant genes, by calculating the sensitivity of the
classification to a change in the expression level of each gene. A
list of genes, ranked by their significance to the classification,
is produced thereby. This allows for cost effective fabrication of
subarrays for use in predicting clinical outcomes.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 illustrates a process flow for a method to identify a
prognostic expression profile using artificial neural networks
according to one embodiment of the invention.
FIG. 2 illustrates a general purpose computing system utilized as
part of an artificial neural network according to another
embodiment of the invention.
FIG. 3 illustrate a set of processing modules making up an
embodiment of an artificial neural network according to the
invention.
FIGS. 4A and B illustrate (A) workflow diagram for complete
leave-one-out artificial neural network (ANN) analysis using all
37920 clones; and (B) workflow diagram for identifying prognostic
gene expression signature and outcome prediction.
FIGS. 5A, B, C, and D depict (A) a plot of the top 3 principal
components (PCs) of the 56 NB samples using all quality-filtered
37920 clones--when the figure is viewed from a point of view facing
the figure, spheres located in the upper and lower right quadrants
represent for the most part poor-outcome patients, while spheres
located in the upper and lower left quadrants represent for the
most part good-outcome patients; (B) ANN voting results for outcome
prediction of the 49 unique NB patients using 37920 clones without
any further clone selection in a leave-one-out prediction scheme
(Samples labels; St=stage, NA=MYCN non-amplified, A=MYCN amplified,
followed by sample name) Symbols represent ANN average committee
votes for each sample, while the length of the horizontal lines
represents the standard error--triangles represent poor-outcome,
and circles represent good-outcome NBs. Vertical line at 0.5 is the
decision boundary for outcome prediction (i.e., good
signature<0.5, poor signature>0.5); (C) Kaplan-Meier curves
of survival probability for the 49 NB patients derived from the
results in FIG. 5B; and (D) Kaplan-Meier curves of survival
probability for the 49 NB patients using the current COG risk
stratification.
FIGS. 6A, B, C, and D depict (A) clone minimization plot for ANN
prediction; (B) plot of the top 3 principal components (PCs) of the
56 NB samples using the top 19 genes (duplicated clones of the same
gene were removed, and the top-ranked clone for each gene was used
in the ANN prediction)--when the figure is viewed from the point of
view facing the figure, spheres located in the upper and lower
right quadrants for the most part represent poor-outcome patients,
while spheres located in the upper and lower left quadrants for the
most part represent good-outcome patients; (C) ANN committee vote
results of the 56 samples using the top 19 ANN-ranked genes--the
horizontal dotted line divides the test (above the line) from the
training samples, triangles are poor outcome, circles are good
outcome; and (D) The Kaplan-Meier curves for survival probability
of the 49 patients were derived from the ANN prediction using the
19 genes in FIG. 6C.
FIGS. 7A, and B depict (A) the expression level of each gene was
logged (base 2) and mean-centered, and represented by pseudo-colors
according to the scale shown on the bottom right. A red color
corresponds to up regulation, and a green color corresponds to down
regulation as compared to the mean. The data presented in this
figure is also shown in Table 9A, B, C and upregulation and down
regulation of the gene in poor outcome patients is shown in Table
3. On the right are the ANN-ranked order, chromosomal location,
IMAGE Ids, gene symbols and the hierarchical clustering dendrogram.
The second and fourth bars below the sample labels mark
poor-outcome patients, and the first and third bars below the
sample labels mark good-outcome patients. Asterisks indicate genes
that have been previously reported to be associated with NB
prognosis; and (B) Differentially expressed genes in good- and
poor-prognostic groups. Box and whisker plots of the mean centered
expression levels of the 12 known genes identified in this study.
The boxes represent the upper and lower quartiles of the data. The
black horizontal line within the box denotes the median. The
whiskers extending above and below the box are fixed at 1.5 times
the inter-quartile range (IQR). Outliers that fall outside the
whiskers of the box are plotted as circles with a dot inside.
FIGS. 8A, B, C, D, E, and F depict (A) Kaplan-Meier curves of
survival probability for all 37920 genes; (B) Kaplan-Meier curves
of the top 19 ANN-ranked genes; (C) Multivariate Cox Proportional
Hazards Models excluding the ANN prediction--H.R.=hazard ratio.
C.I.=confidence interval; (D) Multivariate Cox Proportional Hazards
Models based on MYCN status, all 37920 clones ANN prediction; (E)
Kaplan-Meier curves for survival probability of the high-risk
patients (n=24) based on both MYCN status (top solid line
represents MYCNamplified and good outcome; the solid line ending at
36 months represents MYCNamplified and poor outcome; top dotted
line represents MYCN nonamplified and good outcome; bottom dotted
line ending at 72 months represents MYCN nonamplified and poor
outcome) and the 37920 clones ANN prediction; and (F) Kaplan-Meier
curves for survival probability of the MYCN non-amplified high-risk
patients (n=24) using the predictions based on the top 19
genes.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention is a method of classifying, diagnosing,
prognosticating about, and predicting disease conditions or other
biological states using supervised pattern recognition methods to
analyze high dimensional data.
One aspect of the invention is illustrated in FIG. 1. This
embodiment exemplifies a method of using supervised pattern
recognition methods to analyze high dimensional data. This process
flow describes an embodiment of the method that includes obtaining
experimental data 101, filtering the data 102, reducing the
dimensionality of the data 103, setting up a validation method 115,
training a supervised pattern recognition method 111, validating
the outcome of the supervised pattern recognition method 112, and
once the supervised pattern recognition method is validated,
ranking the data based on the outcome of the supervised pattern
recognition method 113. Further detail and more specific
embodiments of methods of the invention are described below.
Supervised pattern recognition methods are useful, interalia, to
analyze gene expression profiles useful to diagnose and/or provide
prognosis of disease. One aspect of the invention, provides for a
method for predicting the outcome of a patient or subject having
neuroblastoma comprising detecting an increase in expression of at
least one gene in a neuroblastoma cell from the patient selected
from the group consisting of DLK1, PRSS3, SL1T3 and mixtures
thereof, wherein the increase in expression is indicative of poor
outcome. In some embodiments, an increase in expression is
determined by detecting mRNA expression as compared to a
nonneuroblastoma cell, for example, from a mixture of other types
of cancer cells. In other embodiments, expression is compared to
expression in a cell from a neuroblastoma tumor from a subject with
a good outcome and/or a poor outcome. In some embodiments, a
control may also be employed to detect the expression of 5-10
housekeeping genes. The invention also includes another method for
predicting the outcome of a patient or subject having neuroblastoma
comprising detecting a change in expression of at least one gene or
polynucleotide or all genes or polynucleotides selected from the
group consisting of DLK1, PRSS3, ARC, SLIT3, JPH1, ARH1, CNR1,
ROBO2, BTBD3, KLRC3, Hs. 434957, Hs. 346735, Hs. 120591, Hs.
196008, Hs. 124776, Hs. 119947, Hs. 349094, and mixtures thereof,
in a neuroblastoma cell from the patient, wherein the expression
profile or change in expression of the gene or polynucleotide is
indicative of the outcome of the patient. In some embodiments, a
change in expression is determined as compared to a
nonneuroblastoma cell or as compared to expression in a cell from a
neuroblastoma tumor from a subject with a good outcome and/or a
poor outcome.
In some embodiments, the methods further comprise detecting the
expression of MYCN and/or CD44. In some embodiments, at least one
of the genes or polynucleotides selected from the group consisting
of DLK1, PRSS3, ARC, SLIT3, JPH1, Hs. 434957, Hs. 346735, Hs.
120591 and mixtures thereof, is upregulated indicating that the
outcome of the patient is poor. In other embodiments, at least one
gene or polynucleotide selected from the group consisting of ARH1,
CNR1, ROBO2, BTBD3, KLRC3, Hs. 196008, Hs. 119947, Hs. 124776, Hs.
349094, and mixtures thereof, is downregulated indicating that the
outcome of the patient is poor.
Another aspect of the invention involves a method of targeting a
gene or polynucleotide for treatment for neuroblastoma. A method
comprises identifying an antagonist or agonist of at least one gene
or polynucleotide for which a change in expression is correlated
with poor outcome in a patient or subject having neuroblastoma. In
some embodiments, the gene or polynucleotide is upregulated in a
tumor cell and is associated with poor outcome. If a gene is
upregulated, a method comprises identifying an antagonist of the
gene or polynucleotide. A gene or polynucleotide that is
upregulated comprises or is selected from the group consisting of
DLK1, PRSS3, SLIT3, and mixtures thereof. In other embodiments, a
gene or polynucleotide selected from the group consisting of DLK1,
PRSS3, ARC, SLIT3, JPH1, and mixtures thereof, is upregulated
indicating a poor outcome. A method comprises identifying an
antagonist of at least one gene or polynucleotide upregulated in
neuroblastoma cell comprising measuring expression or activity of
at least one gene or polynucleotide selected from the group
consisting of DLK1, PRSS3, ARC, SLIT3, JPH1, Hs. 434957, Hs.
346735, Hs. 120591, and mixtures thereof in the presence or absence
of a candidate agent; and identifying the candidate agent that
inhibits expression or activity of at least one of the genes.
In some embodiments, at least one gene or polynucleotide is
downregulated and correlated with poor outcome of a patient having
neuroblastoma. When a gene or polynucleotide is downregulated, a
method comprises identifying an agonist of a gene or polynucleotide
downregulated in a neuroblastoma cell comprising measuring
expression or activity of at least one gene or polynucleotide
selected from the group consisting of ARH1, CNR1, ROBO2, BTBD3,
KLRC3, Hs. 196008, Hs. 124776, Hs. 119947, Hs. 349094, and mixtures
thereof, in the presence and absence of a candidate agent,
identifying as an agonist the candidate agonist that increases the
expression or activity of the gene or polynucleotide.
Another aspect of the invention provides a set or selection of
genes, kits, and devices for carrying out the methods of the
invention.
A. Methods of Using Supervised Pattern Recognition to Analyze High
Dimensional Data for Prognosis and Identifying Therapeutic
Targets.
An embodiment of the invention provides a method of predicting,
and/or prognosticating about a disease comprising obtaining
experimental data, wherein the experimental data includes high
dimensional data, filtering noise from the data, reducing the
dimensionality of the data by using one or more methods of
analysis, training a supervised pattern recognition and/or
classification method, ranking individual data from the overall
data based on the relevance of the individual data to the
diagnosis, prediction, prognosis or classification, choosing
multiple individual data members, wherein the choice is based on
the relative ranking of the individual data, and using the chosen
data to determine if an unknown set of experimental data indicates
a particular disease prognosis, or prediction.
Obtaining Experimental Data
The first step in a method of the invention is to obtain
experimental data. Experimental data utilized in methods of the
invention is high dimensional data. High dimensional data is data
that has at least hundreds of individual pieces of information
associated with one sample. An example of high dimensional data
useful in methods of the invention is gene expression data. Gene
expression data is high dimensional data because each sample has a
large number of gene expression levels. Generally speaking, gene
expression data generally has thousands of gene expression levels
for each sample. Other examples of high dimensional data useful in
the invention include but are not limited to protein arrays and
protein chips, cell array based expression analysis, analysis of
patterns of single nucleotide polymorphisms in disease conditions,
and comparative genomic hybridization on metaphase, BAC genomic,
cDNA and oligonucleotide arrays.
Preferably, the gene expression data is obtained through use of DNA
microarray technology. DNA microarrays are preferred as a source of
data because they generally offer a more complete picture of the
interactions of a large number of genes with a limited number, or
even one experiment. An example of a general description of how
gene expression data can be obtained by using cDNA microarray
technology is given below.
DNA microarrays, although a relatively new technology, have already
been saddled with a number of different names, biochip, DNA chip,
gene chip, genome chip, cDNA microarray, and gene array. The use of
any of these terms herein refers generally to DNA microarrays. The
underlying principle of DNA microarrays is base pairing or
hybridization i.e., A-T and G-C for DNA, and A-U and G-C for
RNA.
DNA microarrays provide a medium for matching known and unknown DNA
samples based on the base pairings given above. DNA microarrays can
either be fabricated by high-speed robotics or can be fabricated in
a laboratory setting. They are generally patterned on glass, but
can also be fabricated on nylon substrates. Microarrays generally
have sample spot sizes of less than 200 .mu.m diameter, and
generally contain thousands of DNA spots on one microarray.
One method of fabricating cDNA microarrays begins by first
producing gene-specific DNA by polymerase chain reaction (PCR)
amplification of purified template plasmid DNAs from cloned
expressed sequence tags (ESTs). The PCR product is then purified,
resuspended and printed onto a substrate. DNA microarrays are also
commercially available from a number of sources, including but not
limited to Affymetrix, Inc. (Santa Clara, Calif.), Agilent
Technologies (Palo Alto, Calif.), and Research Genetics
(Huntsville, Ala.).
One general procedure for a cDNA microarray experiment begins by
preparing DNA samples and arraying them (either with an arraying
robot, or by hand), to form a DNA microarray. Next, the RNA samples
are extracted from the cells of interest, purified, reverse
transcribed into cDNA and differentially fluorescently labeled to
create probes. Then, the fluorescently labeled cDNA probes are
hybridized to the cDNA microarray. If a probe contains a cDNA whose
sequence is complementary to the DNA on a given spot, the cDNA
probe will hybridize to that spot. After the cDNA probes are
hybridized to the array, and any loose probe has been washed away,
the microarray is imaged to determine how much of each probe is
hybridized to each spot. This indicates how much of each gene from
the microarray is expressed in the two samples. If the amount of
starting material is small, for example from needle biopsies, the
RNA can first be subject to amplification by modified Eberwine
methods as described by Gelder et al. (Amplified RNA synthesized
from limited quantities of heterogeneous cDNA. (Proc. Natl. Acad.
Sci. USA 1990 March; 87(5):1663-7).) The experimental high
dimensional data, preferably obtained from gene expression
experiments, preferably performed using cDNA microarrays, is then
further analyzed by a method of the invention.
Filtering the Data
The next step in a method of the invention is filtering the data
102 to remove individual pieces of data that are deemed
undesirable. This filtering step functions to eliminate weak and/or
problematic data from further use in the method. Accomplishment of
the step of filtering depends greatly on the type of high
dimensional data utilized. Any method known to those of ordinary
skill in the art can be used to eliminate data determined to be
undesirable.
One basis for carrying out this filtering, if a DNA microarray is
being utilized for obtaining the high dimensional data, is the
intensity of the fluorescence from the individual microarray spots.
This basis of omitting data is based on failure or error in the
imaging of the specific spots. A preferred method of performing
initial data filtering on cDNA microarray data to remove those
spots where imaging was a problem is to utilize the intensity of
the various spots and utilize only those spots that have an
intensity over a certain threshold value. Other methods of
filtering DNA microarray data include but are not limited to
eliminating spots in which the number of pixels represented is less
than a threshold defined by the user, eliminating spots in which
the standard deviation of the signal on the spots is too large, as
defined by the user, eliminating spots in which the background
intensity of a single spot is too high, or any combination thereof.
In addition quality values based on intensity, can be assigned to
each spot, standard deviation of intensity, background and/or size
of each spot, then a spot could be eliminated if its quality value
falls below a threshold as defined by the user.
Reducing the Dimensionality of the Data
The next step in methods of the invention is reducing the
dimensionality of the data 103. The number of samples needed to
calibrate a classifier with good predictive ability, depends
critically on the number of features used in the design of the
classifier. In the case of high-dimensional data, such as
microarray data, where the number of samples is much smaller than
the number of individual pieces of data there exists a large risk
of over-fitting. There are two different solutions to this problem.
First, the calibration process can be carefully monitored using a
cross-validation scheme to avoid over-fitting (see below). Second,
the dimension of the data can be reduced, either by using a
dimensional reduction algorithm or by selecting a smaller set of
data for input to the supervised pattern recognition method.
Dimensionality reduction allows the number of parameters
representing each sample to be reduced. This allows for the design
of a classifier that has less risk of over-fitting, thereby
increasing its predictive ability. Examples of methods of reducing
the dimensionality of the data include but are not limited to
principal component analysis (PCA), weighted gene analysis, t-test,
rank based Wilcoxon or Mann-Whitney tests, signal-to-noise
statistic, Fisher's discriminant analysis, or ANOVA tests. In a
preferred embodiment of the invention, PCA is used to reduce the
dimensionality of the data.
In the case of PCA on gene expression data, reduction of the
dimensionality is achieved by rotating gene expression space, such
that the variance of the expression is dominated by as few linear
combinations of genes as possible. Even though the formal dimension
of the problem is given by the number of individual data points,
the effective dimension is just one less than the number of
samples. Hence the eigenvalue problem underlying PCA can be solved
without diagonalizing 2308.times.2308 matrices by using singular
value decomposition. Thus each sample is represented by 88 numbers,
which are the results of projections of the data using the PCA
eigenvectors.
A potential risk when using PCA on relatively few samples is that
components might be singled out due to strong noise in the data. It
could be argued that the outputs (labels) should be included in the
dimensional reduction, using e.g. the Partial Least Squares (PLS)
algorithm, in order to promote components with strong relevance for
the output. However, based on explorations with similar data sets,
this is not optimal; bias is introduced and implicitly
"over-trains" from the outset by including the outputs in the
procedure.
Setting Up a Validation Method for the Supervised Pattern
Recognition Method
Once the data has been filtered 102 and its dimensionality reduced
103, a validation method is set up for monitoring and validating
the training of the supervised pattern recognition method 115. Any
method commonly used by those of skill in the art for validating
the training of a supervised pattern recognition method can be
used.
In one embodiment, the first step in setting up a validation method
is to randomly divide the data into eight groups of data. (See FIG.
4A.) Then, one of those groups is chosen as a validation group 108.
The remaining 7 groups are combined into a training group 109,
which is used to train the supervised pattern recognition method
111 and the eighth group 108 is used to validate the performance of
the supervised pattern recognition method 111, once trained, and is
called a validation group 110.
In an embodiment, the 8-fold cross validation procedure (steps 104
through 110) is performed on all of the samples. A data group
having a known number of samples is given as an example. The known
(labeled) number samples are randomly shuffled 104 and split into
equally 8 sized groups. The supervised pattern recognition method
111 is then calibrated as discussed below using the training group
109. The eighth group, a validation group 110, is reserved for
testing predictions. Comparisons with the known answers refer to
the results from the validation group 110 (i.e. when using a model,
the samples used for training the model are never used in
predictions). This procedure is repeated 8 times, each time with a
different group used for validation. The random shuffling 104 is
done about 100 to 10000 times, preferably 100 times. For each
shuffling, one supervised pattern recognition method 111 model is
generated. Thus, in this embodiment, in total, each sample belongs
to validation group 110, 100 times and 800 supervised pattern
recognition methods 111 have been calibrated. Other cross
validation schemes can be designed and readily utilized.
Training the Supervised Pattern Recognition Method
The supervised pattern recognition method 111 is then trained. The
specific method of training the supervised pattern recognition
method 111 is dependent on the specific form that the supervised
pattern recognition method 111 takes. The choice of the supervised
pattern recognition method 111 and the training thereof is well
within one of skill in the art, having read this specification.
One example of a supervised pattern recognition method is an
artificial neural network (ANN). ANNs are computer-based algorithms
that are modeled on the structure and behavior of neurons in the
human brain and can be trained to recognize and categorize complex
patterns. Pattern recognition is achieved by adjusting parameters
of the ANN by a process of error minimization through learning from
experience. They can be calibrated using any type of input data,
such as gene-expression levels generated by cDNA microarrays, and
the output can be grouped into any given number of categories. ANNs
have been recently applied to clinical problems such as diagnosing
myocardial infarcts and arrhythmias from electrocardiograms and
interpreting radiographs and magnetic resonance images.
In some embodiments where an artificial neural network (ANN) is
employed as the supervised pattern recognition method 111,
calibration is preferably performed using MATLAB (The Mathworks,
Natick, Mass.), preferably, the resilient backpropagation learning
algorithm is used with initial delta=0.07, max delta=50, delta
increase=1.2, and the delta decrease=0.5. The calibration is
performed using a training set and it is monitored both for the
training set and a validation set, which is not subject to
calibration (see below). The weight values are updated and the
calibration is terminated after 100 passes (epochs) through the
entire training set. In one embodiment of a method of the
invention, the resulting parameters for the completed training of a
supervised pattern recognition method 111 defines a "model".
The possibility of using all the PCA components as inputs followed
by a subsequent pruning of weights to avoid "over-fitting" is also
one alternative.
Verifying the Outcome of the Supervised Pattern Recognition
Method
Once the supervised pattern recognition method 111 is trained, the
next step is to determine whether the validation of the supervised
pattern recognition method 111 is successful 112. This step
determines whether the supervised pattern recognition method 111
adequately predicted the results for the validation data set 110
using any number of performance measurements and error
measurements.
Any method known to those of ordinary skill in the art can be
utilized to evaluate the performance of the training of the
supervised pattern recognition method 111. Generally speaking, the
performance is evaluated by comparison with some predetermined
level of correct predictions that the user has determined is
acceptable.
If the performance of the supervised pattern recognition method 111
is sufficiently poor, and a measure of error is greater than an
allowable threshold, the processing may return to module 103 where
the dimensionality of the data is reduced in a different manner and
the entire training and validation process is repeated.
Ranking the Data
Once module 112 determines that the network 111 has been adequately
trained, the processing proceeds to rank the output of the
supervised pattern recognition method 113.
The outcome of the supervised pattern recognition method 111 can be
looked at either independently or in a compiled form. Each
supervised pattern recognition method 111 gives a number between 0
(good outcome) and 1 (poor outcome) as an output for each sample.
If the predictions are viewed independently, the maximal output is
forced to 1 while the other outputs are forced to 0. Then it is
determined how many of the predictions are correct. If the
predictions are viewed in a compiled form, all of the predicted
outputs are considered in their numerical form, after which all of
the numbers are averaged and the resulting average is forced to 0
or 1. In one embodiment of the method, the predictions, as
compiled, are used to classify samples.
In one embodiment, each sample is classified as belonging to the
good or poor outcome corresponding to the largest average in the
compilation. Optionally, in addition, it may be desirable to be
able to reject the second largest vote, as well as test samples
that do not fall within a distance d.sub.c from a sample to the
ideal vote for each outcome type is defined as
.times..times..delta. ##EQU00001## where c is a outcome type,
o.sub.i is the average from the compilation for outcome type i, and
.delta..sub.i,c is unity if i corresponds to outcome type c and
zero otherwise. The distance is normalized such that the distance
between two ideal samples belonging to different outcome types is
unity. Optionally, based on the validation group, an empirical
probability distribution of its distances is generated for each
outcome type.
Optionally, empirical probability distributions may be built using
each supervised pattern recognition method 111 independently (not
the average from the compilation). Thus, the number of entries in
each distribution is given by 100 multiplied by the number of
samples belonging to the outcome type. For a given test sample, the
possible classifications based on these probability distributions
can be rejected. This means that for each outcome type a cutoff
distance from an ideal sample is defined, within which, based on
the validation samples, a sample of this category is expected to
be. The distance given by the 95th percentile of the probability
distribution is preferably chosen as a cutoff, which means that if
a sample is outside of this cutoff distance it cannot confidently
provide a prognosis. It should be noted that the classification as
well as the extraction of important genes (see below) converges
using less than 100 supervised pattern recognition method 111
models. 800 supervised pattern recognition method 111 models are
preferred is because sufficient statistics exist for these
empirical probability distributions.
For each output category the sensitivity and specificity of the
prognosis may be calculated (see Table 1 below). Table 1 gives
sensitivity, specificity for both validation and test samples. Both
the sensitivity and the specificity are very high for all
categories. It should be noted, that they generally depend on the
kind of samples that are used as test samples.
Neuroblastoma Prognosis Using Gene Expression Profiling
TABLE-US-00001 TABLE 1 PERFORMANCE OF ANN PREDICTION Positive
Positive predictive predictive value (%) value (%) ANN Sensitivity
(%) Specificity (%) poor- good- prediction poor-outcome
poor-outcome outcome outcome Leave-one-out 84 90 84 90 with all
clones (n = 49) 19 genes (test 100 94 83 100 samples; n = 21) 19
genes (n = 49) 100 97 95 100
The Receiver Operator Characteristic (ROC) curve area is identical
to another more intuitive and easily computed measure of
discrimination: the probability that in a randomly chosen pair of
samples, one belonging to and one not belonging to the outcome
category, the one belonging to the category is the one with the
closest distance to the ideal for that particular category. Since
the ROC curve areas are unity for all output categories, it is
possible to define cutoff distances such that both the sensitivity
and the specificity are 100% for all outcomes. However, based on
the training and validation groups it is difficult to motivate such
cutoff distances.
The next step in a method in accordance with the invention is to
actually rank the data. This step can in principle be done in two
ways; (1) model-independent and (2) model-dependent analysis
respectively. Due to the relative small number of samples, the
model-dependent analysis is preferred when using ANN models.
The sensitivity (S) of the outputs (o) with respect to any of the
input variables (x.sub.k) is defined as:
.times..times..times..times..delta..times..times..delta..times..times.
##EQU00002##
where N.sub.s is the number of samples and N.sub.o is the number of
outputs (4). The procedure for computing S.sub.k involves a
committee of models. In addition we have defined a sensitivity for
each output i (S.sub.i), which is analogous to Eq. (2) but without
the sum over outputs. Furthermore, a sensitivity can be defined for
each sample (or subsets of samples) individually, by only using
that sample(s) in the sum over samples in Eq. (2). For all these
sensitivities the sign of the sensitivity has also been defined.
The sign signals whether the largest contribution to the
sensitivity stems from positive or negative terms. A positive sign
implies that increased expression of the gene increases the
possibility that the sample belongs to the poor outcome type, (P+
means higher expression in the death or poor outcome group) while a
negative sign means decreased expression of the gene is associated
with poor outcome (P- means decreased expression in poor outcome
group).
In one embodiment, once ranked, a relevant set of data can be
selected module 114 by minimizing the amount of data to be used to
classify and identify a particular disease. In one embodiment, a
predetermined amount of data having the highest ranking are
selected. Of course, other selection methods may be employed
without deviating from the spirit and scope of the present
invention as recited in the attached claims.
Implementation of Methods of the Invention
In embodiments of the method in which the supervised pattern
recognition method 111 is an artificial neural network, a general
purpose computing system as depicted in FIG. 2 can be utilized. An
exemplary ANN processing system 200 provides an artificial neural
network that also receives experimental data to train the
artificial neural network, to verify the output of an artificial
neural network, and to identify relevant genes using the neural
network.
Those of ordinary skill in the art will appreciate that the ANN
processing system 200 may include many more components than those
shown in FIG. 2. However, the components shown are sufficient to
disclose an illustrative embodiment for practicing the present
invention. As shown in FIG. 2, the ANN processing system 200 is
connected to a WAN/LAN, or other communications network, via
network interface unit 210. Those of ordinary skill in the art will
appreciate that network interface unit 210 includes the necessary
circuitry for connecting the ANN processing system 200 to a
WAN/LAN, and is constructed for use with various communication
protocols including the TCP/IP protocol. Typically, network
interface unit 210 is a card contained within the ANN processing
system 200.
The ANN processing system 200 also includes processing unit 212,
video display adapter 214, and a mass memory, all connected via bus
222. The mass memory generally includes RAM 216, ROM 232, and one
or more permanent mass storage devices, such as hard disk drive
228, a tape drive, CD-ROM/DVD-ROM drive 226, and/or a floppy disk
drive. The mass memory stores operating system 220 for controlling
the operation of ANN processing system 200. It will be appreciated
that this component may comprise a general purpose server operating
system as is known to those of ordinary skill in the art, such as
UNIX, LINUX, MAC OS.RTM., or Microsoft WINDOWS NT.RTM.. Basic
input/output system ("BIOS") 218 is also provided for controlling
the low-level operation of ANN processing system 200.
The mass memory as described above illustrates another type of
computer-readable media, namely computer storage media. Computer
storage media may include volatile and nonvolatile, removable and
non-removable media implemented in any method or technology for
storage of information, such as computer readable instructions,
data structures, program modules or other data. Examples of
computer storage media include RAM, ROM, EEPROM, flash memory or
other memory technology, CD-ROM, digital versatile disks (DVD) or
other optical storage, magnetic cassettes, magnetic tape, magnetic
disk storage or other magnetic storage devices, or any other medium
which can be used to store the desired information and which can be
accessed by a computing device.
The mass memory also stores program code and data for providing an
ANN processing and network development. More specifically, the mass
memory stores applications including ANN processing module 230,
programs 234, and other applications 236. ANN processing module 230
includes computer executable instructions which, when executed by
ANN processing system 200, performs the logic described above.
The ANN processing system 200 also comprises input/output interface
224 for communicating with external devices, such as a mouse,
keyboard, scanner, or other input devices not shown in FIG. 2.
Likewise, ANN processing system 200 may further comprise additional
mass storage facilities such as CD-ROM/DVD-ROM drive 226 and hard
disk drive 228. Hard disk drive 228 is utilized by ANN processing
system 200 to store, among other things, application programs,
databases, and program data used by ANN processing module 230. For
example, customer databases, product databases, image databases,
and relational databases may be stored. The operation and
implementation of these databases is well known to those skilled in
the art.
A set of processing modules making up an embodiment of an
artificial neural network according to the invention is illustrated
in FIG. 3. The artificial neural network disclosed herein
corresponds to a generic neural network of no particular topology
for the network of nodes contained therein. The neural network
typically utilizes a form of competitive learning for the operation
of the nodes within the network. Within competitive learning
networks, a large number of data vectors are distributed in a
highly dimensional space. These data vectors represent known values
for experimental data that typically reflect a probability
distribution of the input experimental data. From this probability
distribution representation, predictions for unknown values for
similar input data may be determined.
In all of these competitive learning networks, the networks are
typically presented a set of input data that possesses a
corresponding set of results data. From these data values, the
network of nodes "learns" a relationship between the input data and
its corresponding results data. In this process, the probability
distribution relationship is estimated using the multi-dimensional
network of nodes. This relationship is represented within a set of
artificial neural network coefficients for a particular topology of
nodes.
One skilled in the art will recognize that competitive learning
networks include a nearly infinite number of network topologies
that may be used to represent a particular probability distribution
relationship without deviating from the spirit and scope of the
present invention as recited within the attached claims. In
addition, artificial neural networks may utilize various well-known
algorithm architectures, including hard-competitive learning (i.e.
"winner-take-all" learning), soft competitive learning without a
fixed network dimensionality, and soft competitive learning with a
fixed network dimensionality, to specify an artificial neural
network according to the invention as recited within the attached
claims. Each of these algorithm architectures represents the same
probability distribution relationship; however each of the various
algorithm architectures better optimize corresponding processing
parameters, which are often mutually exclusive with each other.
These parameters include error minimization or the minimization of
an expected quantization error, entropy maximization for the
reference vectors used within a network, and topology-preserving or
feature mapping architectures that attempt to map high-dimensional
inputs signals onto lower-dimensional structures in a manner that
attempts to preserve similar relationships found within the
original data within the post-mapping data. As such, any of these
types of algorithm architectures may be used to construct an
artificial neural network without deviating from the spirit and
scope of the present invention as recited within the attached
claims.
Now referring to FIG. 3, an artificial neural network processing
system 301 comprises a learning module 311, a prediction module
321, and a database of network node coefficients 313. The learning
module 311 is used with a set of experimental data 315 that
possesses a corresponding set of experimental results 316 to
generate a set of network node coefficients that represent a
probability distribution relationship for the experimental data
315--experimental result 316 data set for a particular neural
network topology and algorithm architecture. The learning module
311 includes a data learning input module 312 that receives the
experimental data 315--experimental result 316 data set generated
using the process described above. The learning module 311 also
includes an ANN training module 313 that processes the experimental
data 315--experimental result 316 data set to generate the
coefficients used to specify the probability distribution
relationship and an ANN coefficient storage module 314 for storing
the coefficients that have been previous generated within the
database 313 for later use.
The data processing within the learning module 311 may proceed in a
batch processing fashion in which all of the vectors within the
experimental data 315--experimental result 316 data set are
processed at a single time. In such a process, the experimental
data 315--experimental result 316 data set is received by the input
module 312, processed by the training module 313, and the generated
coefficients are placed within the database 313 by the storage
module 314. Alternatively, the experimental data 315--experimental
result 316 data set may be processed as a sequence of smaller data
sets in which the experimental data 315--experimental result 316
data set data values are generated at different times. In such a
process, the training module 313 uses the previously stored
coefficients retrieved by the storage module along with a new small
data set provided by the input module 312 to generate an updated
set of coefficients. These updated coefficients may be once again
stored within the database 313 for use at a later time.
Once an artificial neural network 301 has been trained, the
prediction module 321 may be used to predict, or classify, a
particular test data value 325. The prediction module 321 includes
a data prediction input module 322, an ANN prediction module 323,
and an ANN curve slope module 324. The data prediction input module
322 receives the input test data generated as described above for
use in the prediction module. The ANN prediction module 323
receives and utilizes the network coefficient values for the neural
network from the ANN coefficient database 313 to predict the
possible result for the probability distribution relationship
specified within the neural network. This output value is used by
the ANN curve slope module 324 to determine all possible values for
a given gene, in the manner discussed above, to determine a curve
slope value. This slope value is then output for later use in
ranking and classifying the individual genes used to determine the
presence, absence or prognosis of a disease.
The embodiments described herein are implemented as logical
operations performed by a computer. The logical operations of these
various embodiments of the present invention are implemented (1) as
a sequence of computer implemented steps or program modules running
on a computing system and/or (2) as interconnected machine modules
or hardware logic within the computing system. The implementation
is a matter of choice dependent on the performance requirements of
the computing system implementing the invention. Accordingly, the
logical operations making up the embodiments of the invention
described herein can be variously referred to as operations, steps,
or modules.
While the above embodiments of the invention describe the use of an
artificial neural network to identify relevant genes associated
with diseases and use the identified genes to classify and identify
diseases, one skilled in the are will recognize that the use of the
processing system discussed above are merely example embodiments of
the invention. As long as experimental data is used to self-train a
processing system using competitive learning processing, the
present invention to would be useable in other data
processing-systems. It is to be understood that other embodiments
may be utilized and operational changes may be made without
departing from the scope of the present invention as recited in the
attached claims.
Prediction of Clinical Outcome Using Gene Expression Profiling and
ANN
In an embodiment, a prognostic profile and prediction of clinical
outcome of patients having neuroblastoma can be made using gene
expression data and a method of analyzing the data using ANNs as
described herein.
The high dimensional data is obtained from neuroblastoma tumor
cells. In some embodiments, total mRNA from neuroblastoma cells is
obtained and expression levels are determined using commercially
available sequence verified cDNA libraries comprising about 42,578
cDNA clones representing 25,933 unique genes (Unigene clusters:
13,606 known genes and 12,327 unknown expressed sequence tags).
In an embodiment, gene expression ratio information obtained from
neuroblastoma cells and reference RNA on each microarray can be
normalized using a pin based normalization method. Quality of each
individual cDNA can be evaluated by Chen et al. (Bioinformatics,
18:207 (2002)). Spots with an average quality across all of the
samples can be excluded.
In an embodiment, principal component analysis can be used to
reduce the dimensionality of the expression data to the principal
components as inputs for artificial neural networks. A feed forward
resilient back propagation multilayer perceptron artificial neural
network (coded in Matlab, The Mathworks, Natick, Mass.) can be used
having at least 3 layers: an input layer of the top 10 principal
components of the data or the gene expression ratios of each cDNA
spot (for the minimized gene set); a hidden layer with 3 nodes; and
an output layer generating a committee vote that discriminates
between two classes (i.e. good and poor outcome groups). Average
artificial neural network committee votes can be used to classify
samples and 0.5 can be selected as decision boundary. An ideal vote
was 0 for good outcome group (alive) and 1 for poor outcome group
(dead).
The artificial neural networks can be trained using an 8 fold cross
validation scheme. (See FIG. 4A) In an embodiment, the top 10
principal components are used for input to the ANN. One sample is
left out as an independent test sample, and the ANNs are trained
using the remaining 48 NB samples as shown in FIG. 4A. All
remaining neuroblastoma samples are randomly partitioned into eight
groups. One of the eight groups (containing 6 samples each) is
selected as a validation set, whereas the remaining 7 groups (42
samples) are used to train the network. The training weights are
iteratively adjusted for 100 cycles (epochs). The ANN output (0-1,
where 0=ideal good-outcome and 1=ideal poor-outcome) is calculated
for each sample in the validation set. A different validation set
is selected from the same partitioning of the initial set, and the
remaining seven groups are used for training. The training scheme
is repeated until each of the eight groups from the initial set are
used as a validation set exactly one time. The samples are randomly
repartitioned into eight new groups, and training steps are
repeated. Sample partitioning was performed 100 times in total.
Thus, training steps are repeated 100 times. Eight hundred ANN
models are trained and are used to predict the left out test
sample. This scheme can be repeated for each left out test
sample.
In an embodiment, to identify the prognostic genes and outcome
prediction, a separate ANN analysis is conducted using a gene
minimization procedure. The gene minimization procedure involves
ranking each of the input clones according to its importance to
prediction of ANNs. Increasing numbers of the top ranked clones are
used to to train ANNs and the classification error monitored. The
minimal number of clones that yielded the minimal classification
error is identified and the top ranked clones are used to retrain
ANNs and predict the test samples without preforming a principal
component analysis.
An analysis of 56 neuroblastoma samples from patients (34 patients
alive and 22 deceased as shown in FIG. 4B) is conducted. The
samples are divided into two groups: 35 NB samples selected for
training (18 samples from patients that were alive and 17 from
patients that were deceased) and 21 NB samples reserved for testing
(16 samples from patients that were alive and 5 samples from
patients that were deceased). The first set of samples are used to
train ANNs and are subjected to gene minimization to identify 19
unique genes or polynucleotides. The 19 genes were then used to
train ANNs and the set of 21 samples are analyzed using the trained
ANNs and the outcome of these patients as predicted with a score of
0 for good outcome and a score of 1 for poor outcome. In an
embodiment, the set of genes useful for prognosis of neuroblastoma
is summarized as shown in FIG. 7A and Tables 2 and 3.
B. Compositions, Methods, and Devices for Predicting the Clinical
Outcome of Patients with Neuroblastoma.
Methods
Neuroblastoma is the most common solid extracranial tumor of
childhood and is derived from the sympathetic nervous system.
Patients in North America are currently stratified by the
Children's Oncology Group into high, intermediate, and low risk
based on age, tumor staging, Shimada Histology, MYCN amplification,
and DNA ploidy (Brodeur, et al., Neuroblastoma. In: PA Pizzo and DG
Poplack, editors, Principles and practice of pediatric oncology,
4th ed. Philadelphia: Lippincott-Raven, pp. 895-937 (2002)).
Patients<1 year of age or with lower stage diseases
(International Neuroblastoma Staging System stages 1 and 2) usually
have better outcome than older patients or those with advanced
stage diseases (International Neuroblastoma Staging System stages 3
and 4). Certain consistent cytogenetic changes, including gain of
2p24 and 17q and loss of heterozygosity at 1p36 have been
associated with a more aggressive phenotype (Schwab et al., Lancet
Oncol., 4:472-480 (2003); Westermann et al., Cancer Lett.,
184:127-147 (2002)). The MYCN gene is amplified in .about.22% of
all neuroblastoma patients (Brodeur, Nat. Rev. Cancer, 3:203-216
(2003)) and is an independent predictor for poor prognosis,
especially for patients>1 year of age. Although other genes,
such as TRKA, TRKB, hTERT, BCL-2, caspases, and FYN (Brodeur, Nat.
Rev. Cancer, 3:203-216 (2003); Berwanger et al., Cancer Cell,
2:377-386 (2002)) have been associated with neuroblastoma
prognosis, they all lack the predictive power of MYCN and are not
used currently in clinical practice.
High-risk patients compose .about.50% of all neuroblastoma cases;
however, despite significant improvement in the therapy of
neuroblastoma using neoadjuvant chemotherapy, surgery, and
radiation, the death rate for these patients remains at 70%
(Pearson et al., In: G M Brodeur, T. Sawada, Y. Tsuchida, P A
Voute, editors. Neuroblastoma, 1st ed. Amsterdam, The Netherlands:
Elsevier Science, p. 555 (2000)). Although the Children's Oncology
Group risk stratification has been carefully developed to take into
account the above risk factors, it is primarily used to guide
therapy and does not predict which individual patients will be
cured from the disease.
Gene expression profiles from cDNA microarrays are described herein
and are useful to predict the outcome and identify a prognostic
gene set in patients with neuroblastoma using artificial neural
networks. A prediction of the outcome of the patient having
neuroblastoma will assist the physicians in selecting an
appropriate treatment regimen. For those patients whose gene
expression profile indicates a poor outcome, more aggressive
treatment may be warranted. For those patients whose gene
expression profile indicates a good outcome, less aggressive
treatment may be warranted. The identification of the set of genes
useful for prognosis will provide for microarray assays or other
clinical assays useful in predicting outcome of patients having
neuroblastoma. Once the prognostic profile is identified as
described herein, the prediction of outcome may be accomplished
without the use of artificial neural network analysis.
One aspect of the invention, provides for a method for predicting
the outcome of a patient or subject having neuroblastoma comprising
detecting an increase in expression of at least one gene selected
from the group consisting of DLK1, PRSS3, SL1T3 and mixtures
thereof in a neuroblastoma cell from the patient, wherein the
increase in expression is indicative of poor outcome. Optionally,
the expression levels of at least one gene or polynucleotide are
compared to that of a patient with a good outcome and/or poor
outcome. For example, if the expression level is upregulated in
comparison to expression levels in a patient with good outcome,
then it is likely the patient will have a poor outcome. Poor
outcome refers to patient that is likely to die, die in a much
shorter time, and/or has died. Good outcome refers to a patient
that is still alive and/or is in remission (no progression or
relapse) for at least 3 years. In some embodiments, an increase in
expression is determined by detecting mRNA expression as compared
to a nonneuroblastoma cell or as compared to expression in a cell
from a neuroblastoma tumor from a subject with a good and/or poor
outcome. Examples of the expression levels of prognostic genes
identified herein is shown in FIGS. 7A, 7B, Table 3 and/or Tables
9A, B and C. Upregulation (P+) or down regulation (P-) of genes in
poor outcome patients is shown in Table 3. Typically genes
upregulated in poor outcome patients are not up or down, or are
downregulated in good outcome patients. Typically genes
downregulated in poor outcome patients are not up or down, or are
upregulated in good outcome patients. Predictions using expression
profile data can be made utilizing standard statistical techniques
as described herein, such as Kaplan Meier methods.
In some embodiments, reference RNA is included in the microassay
analysis, such reference RNA can be obtained from a
nonneuroblastoma cell such as from a mixture of other type of cell
lines. In addition, a control may be included for detecting
expression of at least one housekeeping gene, preferably 5-10
housekeeping genes. In some embodiments, an increase in mRNA
expression is detected using a microarray, hybridization assay or
PCR assays including real time PCR. In other embodiments,
expression of at least one of the genes is detected by measuring
the concentration of the protein in a biological sample using
standard methodologies such as ELISA, immuno PCR, and other like
assays.
Multiple clones may provide for detection of any one of the genes
identified herein as prognostic for neuroblastoma. See, for
example, FIG. 7A or Table 3, showing several clones detecting SLIT3
and other genes. The polynucleotide (or its complement) and amino
acid sequence associated with an Image ID No. and/or Accession No.
can be readily identified in publicly available databases such as
source.stanford.edu/cgi-bin/source/sourceSearch or the NCBI
database for Unigene IDs. The polynucleotides and/or genes and
polypeptides are preferably human. In addition, cDNA libraries,
including human cDNA libraries or DNA libraries; are commercially
available and provide a source of the sequences for the genes
and/or polynucleotides identified herein. (See, for example,
Invitrogen's website.) Representative polynucleotide sequences for
each gene are provided in the sequence listing which forms a part
of this disclosure,
In some embodiments, the gene for DLK1 comprises a polynucleotide
sequence of Image ID NO: 296815 or Image ID NO: 436121. The DLK1
gene may also comprises a polynucleotide sequence of SEQ ID NO:1.
In some embodiments, the gene for SLIT3 comprise a nucleotide
sequence of Image ID NO: 450382, or Image ID NO: 192225, or Image
ID NO: 2030301. The SLIT3 gene may also comprise a polynucleotide
sequence of SEQ ID NO:6. In some embodiments, the PRSS3 gene
comprises a polynucleotide sequence of Image ID NO: 1913366. The
PRSS3 gene may also comprise a polynucleotide sequence of SEQ ID
NO:3.
In some embodiments, DLK1 comprises an amino acid sequence as
provided in Accession No. NP.sub.--003827 (gI: 21361080) and having
a sequence of SEQ ID NO:254. In an embodiment, PRSS3 comprises an
amino acid sequence of Accession No. NP.sub.--002762 (gi|21536452)
having a sequence of SEQ ID NO:255. In an embodiment, SLIT3
comprises an amino acid sequence of Accession No. NP.sub.--003053
(gi|11321571) and having a sequence of SEQ ID NO:256. Other
secreted polypeptides include PRSS12, GAL, and IL-7. The
polypeptides may be useful to develop antibodies or other reagents
that may be useful to detect an increase of the polypeptide in a
biological sample.
In some embodiments of the methods, the expression of at least two
of the genes, preferably at least three of the genes is detected.
In a further embodiment, the method may further comprise detecting
an up-regulation of expression of MYCN and/or a downregulation of
expression of CD44. In some embodiments, the gene for MYCN
comprises the sequence of Image ID NO: 41565. The gene for MYCN may
also comprise a polynucleotide sequence of SEQ ID NO:16. In some
embodiments, the gene for CD44 comprises the sequence of Image ID
NO: 1967589. The gene for CD44 may also comprise a polynucleotide
sequence of SEQ ID NO:12.
The invention also includes another method for predicting the
outcome of a patient or subject having neuroblastoma comprising
detecting a change in expression of at least one gene or
polynucleotide or all genes or polynucleotides selected from the
group consisting of DLK1, PRSS3, ARC, SLIT3, JPH1, ARH1, CNR1,
ROBO2, BTBD3, KLRC3, Hs. 434957, Hs. 346735, Hs. 120591, Hs.
196008, Hs. 124776, Hs. 119947, Hs. 349094, and mixtures thereof in
a neuroblastoma cell from the patient, wherein the expression
profile or change in expression of the gene or polynucleotide is
indicative of the outcome of the patient. In some embodiments, the
method further comprises detecting the expression of MYCN and/or
CD44. In some embodiments, at least one of the genes or
polynucleotides selected from the group consisting of DLK1, PRSS3,
ARC, SLIT3, JPH1, Hs. 434957, Hs. 346735, Hs. 120591, and mixtures
thereof, is upregulated indicating that the outcome of the patient
is poor. In other embodiments, at least one gene or polynucleotide
selected from the group consisting of ARH1, CNR1, ROBO2, BTBD3,
KLRC3, Hs. 196008, Hs. 124776, Hs. 119947, Hs. 349094, and mixtures
thereof, is downregulated indicating that the outcome of the
patient is poor. Optionally, the expression levels of at least one
gene or polynucleotide are compared to that of a patient with a
good outcome and/or poor outcome. For example, if the expression
level of the gene is upregulated or down regulated in comparison to
expression levels in a patient with good outcome, then it is likely
the patient will have a poor outcome.
In some embodiments, the genes or polynucleotides comprises a
sequence of the Image Id Nos as follows: a gene DLK1 comprises a
polynucleotide sequence of Image ID NO: 296815 or 436121; a gene
PRSS3 comprises a polynucleotide sequence of Image ID NO: 1913366;
a gene ARC comprises a polynucleotide sequence of Image ID NO:
222457; a gene SLIT3 comprises a polynucleotide sequence of Image
ID NO: 450382, or Image ID NO: 192225, or Image ID NO: 2030301; a
gene JPH1 of Image ID NO: 811874; a gene ARH1 comprises a
polynucleotide sequence of Image ID NO: 2336916; a gene CNR1
comprises a polynucleotide sequence of Image ID NO: 26295; a gene
ROBO2 comprises a polynucleotide sequence of Image ID NO: 377573; a
gene BTBD3 comprises a polynucleotide sequence of Image ID NO:
811918; a gene KLRC3 comprises a polynucleotide sequence of Image
ID NO: 2361911; Hs. 434957 comprises a polynucleotide sequence of
Image ID NO: 681891; Hs. 346735 comprises a polynucleotide sequence
of Image ID NO: 143169; Hs. 120591 comprises a polynucleotide
sequence of Image ID NO: 1540478; Hs. 196008 comprises a
polynucleotide sequence of Image ID NO: 111264; Hs. 124776
comprises a polynucleotide sequence of Image ID NO: 1574206; Hs.
119947 comprises a polynucleotide sequence of Image ID NO: 379779;
and Hs. 349094 comprises a polynucleotide sequence of Image ID NO:
687667. SEQ ID NOs corresponding to a representative polynucleotide
sequence for each gene or polynucleotide are provided in Tables 2
and 3. Sequences corresponding to the SEQ ID NOs are provided in
the sequence listing provided herein. The sequence listing forms a
part of this disclosure and the contents of the sequence listing
are hereby incorporated herein.
In some embodiments of the methods, the expression of at least two
of the genes or polynucleotides, preferably at least three, at
least four, at least five, at least six, at least seven, at least
eight, at least nine, at least ten, at least eleven, at least
twelve, at least thirteen, at least fourteen, at least fifteen, at
least sixteen, at least seventeen, at least eighteen or all of the
genes of Table 2 are detected. In a further embodiment, the method
may further comprise detecting an up-regulation of expression of
MYCN and/or a downregulation of expression of CD44. In some
embodiments, the gene for MYCN comprises the sequence Image ID NO:
41565. The gene for MYCN may also comprise a polynucleotide
sequence of SEQ ID NO:16. In some embodiments, the gene for CD44
comprises the sequence of Image ID NO: 1967589. The gene for CD44
may also comprise a polynucleotide sequence of SEQ ID NO:12.
In some embodiments of the methods, the patient having
neuroblastoma is classified as high risk according to the criteria
of the Children's Oncology group. This criteria has been described
in Brodeur et al. cited supra. In other embodiments, the tumor from
the patient having neuroblastoma does not have an amplification of
MYCN. The methods of the invention are useful to predict the
outcome of high risk patients including those patients that do not
have an amplification of MYCN.
In some embodiments, the methods may further comprise detecting at
least one other gene or polynucleotide identified in Table 3. The
methods may involve successively detecting each of the next 10 top
ranked genes or polynucleotides as provided in Table 3 up to and
including detecting all 250 genes or polynucleotides identified in
Table 3. For example, the methods for predicting outcome of a
patient having neuroblastoma may further comprise detecting the
expression levels of at least the top twenty to thirty ranked
genes, the top thirty to forty top ranked genes etc. or combination
thereof. In Tables 3 and 9A, B, C, the expression profile of the
genes as upregulated or downregulated in neuroblastoma cells is
shown.
Another aspect of the invention provides methods for selecting a
treatment for patients having neuroblastoma comprising a)
determining a gene expression profile of the neuroblastoma tumor
cell of at least one gene or polynucleotide selected from group
consisting of DLK1, PRSS3, ARC, SLIT3, JPH1, ARH1, CNR1, ROBO2,
BTBD3, KLRC3, Hs. 434957, Hs. 346735, Hs. 120591, Hs. 196008, Hs.
124776, Hs. 119947, Hs. 349094, and mixtures thereof; and b)
predicting whether the outcome of the patient is poor or good based
on the expression profile; c) optionally, designing a more
aggressive treatment for the patient if the predicted outcome for
the patient is poor or designing a less aggressive treatment if the
predicted outcome is good. Predicting whether the outcome is good
or bad can be determined by comparing the expression profile to the
expression profile of a patient with a good outcome and/or the
expression of profile of a patient with poor outcome. For example,
if the expression level is upregulated or down regulated in
comparison to expression levels in a patient with good outcome,
then it is likely the patient will have a poor outcome. Standard
statistical methods may be employed to conduct the comparison,
including Kaplan Meier methods. An example of the expression
profile is provided herein in FIG. 7B, Table 3, and 9A, B, C. In
some embodiments, designing a more aggressive treatment for
patients predicted to have a poor outcome comprises using at least
one treatment that is considered experimental, especially for those
treatments for which clinical trials have indicated a positive
response. In some embodiments, designing a treatment for a patient
with predicted good outcome comprises selecting at least one
treatment that has less risk of toxicity or death associated with
treatment, such as decreases in the dosage or amounts of
chemotherapeutic agent.
The genes given in table 2 and 3 below can also be used to make up
a selection or set of genes for predicting the outcome of a patient
with neuroblastoma (NB). Gene selections such as these can be used
to predict the clinical outcome of a patient with neuroblastoma as
discussed above.
TABLE-US-00002 TABLE 2 Top 19 Ranked Genes for Prediction of
Neuroblastoma Clinical Outcome Rank Gene SEQ ID NO. Title of Gene 1
DLK1 SEQ ID NO. 1 delta-like 1 homolog 2 EST SEQ ID NO. 2 Homo
sapiens cDNA FLJ35632 fis, clone SPLEN2011678 3 PRSS3 SEQ ID NO. 3
protease, serine, 3 (mesotrypsin) 4 ARHI SEQ ID NO. 4 ras homolog
gene family, member 1 5 ARC SEQ ID NO. 5 activity-regulated
cytoskeleton- associated protein 6 SLIT3 SEQ ID NO. 6 slit homolog
3 (Drospholia) 7 CNR1 SEQ ID NO. 7 cannabinoid receptor 1 (brain) 8
EST SEQ ID NO. 8 Homo sapiens, clone IMAGE: 3881549, mRNA 9 EST SEQ
ID NO. 9 Homo sapiens, cDNA FLJ11723 fis, clone HEMBA 1005314 10
FLJ25461 SEQ ID NO. 10 hypothetical protein FLJ25461 11 EST SEQ ID
NO. 11 Homo sapiens, clone IMAGE: 3618365, mRNA 12 CD44 SEQ ID NO.
12 CD44 antigen (homing function and Indian blood group system) 13
EST SEQ ID NO. 13 Homo sapiens cDNA clone IMage: 4811759, partial
cds 14 ROBO2 SEQ ID NO. 14 roundabout, axon guidance receptor,
homolog 2 (Drosophila) 15 BTBD3 SEQ ID NO. 15 BTB (POZ) domain
containing 3 16 MYCN SEQ ID NO. 16 v-myc myelocytomatosis viral
related oncogene, neuroblastoma derived (avian) 17 EST SEQ ID NO.
17 Homo sapiens mRNA; cDNA DKFZp564N1116 (from clone DKFZp564N1116)
18 JPH1 SEQ ID NO. 18 junctophilin 1 19 KLRC3 SEQ ID NO. 19 killer
cell lectin-like receptor subfamily C, member 3
TABLE-US-00003 TABLE 3 Top 250 Ranked Genes for Prediction of
Neuroblastoma Clinical Outcome Plate Rank Gene SEQ ID NO. Position
Direction Clone ID UG_ID 1. DLK1 SEQ ID NO. 1 HsKG60E8 P+ 296815
Hs.169228 2. EST SEQ ID NO. 2 R43273g9 P+ 1540478 Hs.120591 3.
PRSS3 SEQ ID NO. 3 R43297f11 P+ 1913366 Hs.435699 4. ARHI SEQ ID
NO. 4 HsKG99e8 P- 2336916 Hs.194695 5. ARC SEQ ID NO. 5 HsKG85h1 P+
222457 Hs.40888 6. SLIT3 SEQ ID NO. 6 HsKG54B2 P+ 450382 Hs.484063
7. CNR1 SEQ ID NO. 7 HsKG14D12 P- 26295 Hs.75110 8. EST SEQ ID NO.
8 R4353e2 P+ 143169 Hs.346735 9. EST SEQ ID NO. 9 R4353a9 P- 111264
Hs.196008 10. FLJ25461 SEQ ID NO. 10 R43251f2 P- 379779 Hs.119947
11. EST SEQ ID NO. 11 R43175b4 P+ 681891 Hs.434957 12. CD44 SEQ ID
NO. 12 CD1C7 P- 1967589 Hs.306278 13. EST SEQ ID NO. 13 R43163f10
P- 687667 Hs.349094 14. ROBO2 SEQ ID NO. 14 R43234e10 P- 377573
Hs.31141 15. BTBD3 SEQ ID NO. 15 FHskG5F10 P- 811918 Hs.7935 16.
MYCN SEQ ID NO. 16 HsKG20G3 P+ 41565 Hs.25960 17. EST SEQ ID NO. 17
R43277g5 P- 1574206 Hs.124776 18. JPH1 SEQ ID NO. 18 R43167a6 P+
811874 Hs.293836 19. KLRC3 SEQ ID NO. 19 HsKG99g7 P- 2361911
Hs.74082 20. EST SEQ ID NO. 20 R43175a2 P+ 666469 Hs.444181 21. RET
SEQ ID NO. 21 HsKG97c4 P+ 1516955 Hs.350321 22. CRABP1 SEQ ID NO.
22 HsKG32F5 P+ 809694 Hs.346950 23. ECEL 1 SEQ ID NO. 23 HsKG88h8
P- 37986 Hs.26880 24. LOC283120 SEQ ID NO. 24 R4383a3 P+ 428721
Hs.415722 25. HMGA2 SEQ ID NO. 25 R43158f12 P- 42803 Hs.6421 26.
SNYPO2 SEQ ID NO. 26 R43199g2 P+ 284383 Hs.24192 27. LOC163782 SEQ
ID NO. 27 R4327c6 P- 246035 Hs.78026 28. VSNL1 SEQ ID NO. 28
HsKG2H2 P- 210575 Hs.2288 29. HS3ST4 SEQ ID NO. 29 HsKG92e9 P-
1569187 Hs.8040 30. AKR1C1 SEQ ID NO. 30 HsKG5H3 P- 196992
Hs.295131 31. EST SEQ ID NO. 31 R43234b6 P+ 345656 Hs.83623 32.
GPR22 SEQ ID NO. 32 HsKG77B6 P+ 42685 Hs.432557 33. EST SEQ ID NO.
33 HsKG91b12 P+ 486278 Hs.502418 34. EST SEQ ID NO. 34 R43241e9 P-
375741 Hs.144627 35. CCNA1 SEQ ID NO. 35 HsKG64C12 P+ 377799
Hs.417050 36. PKIB SEQ ID NO. 36 R43335a1 P- 26883 Hs.363171 37.
EST SEQ ID NO. 37 R43248b12 P- 174685 Hs.31564 38. GAL SEQ ID NO.
38 R43332c7 P+ 2237353 Hs.278959 39. EST SEQ ID NO. 39 R43386f8 P+
1836760 Hs.459132 40. LOC221303 SEQ ID NO. 40 R43276g5 P+ 1563968
Hs.126712 41. EST SEQ ID NO. 41 HsKG93b5 P+ 725709 Hs.367767 42.
EST SEQ ID NO. 42 HsKG68H9 P+ 145310 Hs.22404 43. BMP7 SEQ ID NO.
43 R4366e1 P+ 366887 Hs.170195 44. SLC30A3 SEQ ID NO. 44 R43145b2
P+ 744391 Hs.111967 45. FLJ10539 SEQ ID NO. 45 R43136h12 P- 595162
Hs.301198 46. AMIGO2 SEQ ID NO. 46 R43244f2 P- 253884 Hs.121520 47.
AKR1C2 SEQ ID NO. 47 HsKG101e7 P- 2449395 Hs.201967 48. MGP SEQ ID
NO. 48 HsKG12G8 P- 590264 Hs.365706 49. PCSK1 SEQ ID NO. 49 HsKG3H7
P- 31072 Hs.78977 50. HK2 SEQ ID NO. 50 HsKG56B8 P+ 1637282
Hs.406266 51. EST SEQ ID NO. 51 R43187d12 P+ 136502 Hs.409873 52.
EST SEQ ID NO. 52 HsKG100f8 P+ 2410555 Hs.460974 53. IL7 SEQ ID NO.
53 R43331b3 P- 2090264 Hs.72927 54. PRSS12 SEQ ID NO. 54 HsKG70C9
P+ 1553054 Hs.512796 55. GABARAPL1 SEQ ID NO. 55 HsKG50B10 P- 81409
Hs.336429 56. DEFB129 SEQ ID NO. 56 R43145c2 P+ 743161 Hs.112087
57. NAV3 SEQ ID NO. 57 R43251d10 P- 379484 Hs.306322 58. RAB3B SEQ
ID NO. 58 R43163f3 P- 687297 Hs.123072 59. KRT6B SEQ ID NO. 59
R43266g2 P- 1486118 Hs.432677 60. BEX1 SEQ ID NO. 60 R4337a1 P+
341706 Hs.334370 61. EST SEQ ID NO. 61 R4343a1 P+ 140210 Hs.155795
62. EST SEQ ID NO. 62 R43345h11 P- 1558233 Hs.7413 63. SCYL1 SEQ ID
NO. 63 R4382f12 P- 770697 Hs.238839 64. EST SEQ ID NO. 64 R43100e4
P- 51993 Hs.7047 65. RYR2 SEQ ID NO. 65 HsKG43H4 P- 53099 Hs.90821
66. LRBA SEQ ID NO. 66 HsKG23C10 P+ 376516 Hs.209846 67. CSPG3 SEQ
ID NO. 67 HsKG56F5 P+ 1609966 Hs.169047 68. EST SEQ ID NO. 68
R43405e1 P- 1880885 Hs.129977 69. MMP12 SEQ ID NO. 69 HsKG4D7 P+
196612 Hs.1695 70. CHRNA1 SEQ ID NO. 70 HsKG77E8 P- 347370
Hs.434419 71. EST SEQ ID NO. 71 R43340e10 P- 1518228 Hs.130061 72.
EST SEQ ID NO. 72 R43105h8 P- 52329 Hs.470493 73. HNRPH1 SEQ ID NO.
73 R4344f7 P+ 195127 Hs.202166 74. LOC113251 SEQ ID NO. 74 R43399f5
P- 1856516 Hs.26613 75. EST SEQ ID NO. 75 R4337f2 P- 137793
Hs.17962 76. PAG SEQ ID NO. 76 R4381f12 P- 282779 Hs.266175 77.
PROK2 SEQ ID NO. 77 R43162g10 P- 53319 Hs.13305 78. HS6ST1 SEQ ID
NO. 78 HsKG69H10 P+ 969769 Hs.512841 79. EST SEQ ID NO. 79 R43405c7
P- 1880352 Hs.104419 80. PCDH9 SEQ ID NO. 80 R4376b12 P- 284714
Hs.492696 81. EST SEQ ID NO. 81 R43265d8 P+ 1469434 Hs.458730 82.
EST SEQ ID NO. 82 R43279d1 P- 1585344 Hs.121518 83. GLDC SEQ ID NO.
83 HsKG5C5 P+ 248261 Hs.149156 84. ADRB2 SEQ ID NO. 84 HsKG15A5 P-
241489 Hs.2551 85. ICSBP1 SEQ ID NO. 85 R43331c7 P+ 2107378
Hs.14453 86. CD48 SEQ ID NO. 86 CD1C6 P- 1671476 Hs.901 87. EST SEQ
ID NO. 87 R43184a7 P- 191787 Hs.13640 88. DYRK1B SEQ ID NO. 88
R43274g9 P+ 1553469 Hs.130988 89. KLRC1 SEQ ID NO. 89 HsKG64E8 P-
1525029 Hs.512576 90. EST SEQ ID NO. 90 HsKG87b11 P+ 625786
Hs.380933 91. EST SEQ ID NO. 91 R43199b1 P+ 281517 Hs.388565 92.
EST SEQ ID NO. 92 R4337c5 P+ 120162 Hs.406351 93. MOXD1 SEQ ID NO.
93 FHskG5F5 P- 767181 Hs.6909 94. EST SEQ ID NO. 94 R43126a6 P+
304927 Hs.44380 95. EST SEQ ID NO. 95 R43206h6 P- 451394 Hs.191950
96. GAS1 SEQ ID NO. 96 R4378h7 P- 365826 Hs.65029 97. COL9A2 SEQ ID
NO. 97 R43330g9 P- 2019798 Hs.418012 98. EST SEQ ID NO. 98 R43146g7
P- 244312 Hs.440908 99. DRPLA SEQ ID NO. 99 HsKG5H6 P- 45291
Hs.169488 100. EST SEQ ID NO. 100 R43396h4 P- 1850044 Hs.334594
101. REPRIMO SEQ ID NO. 101 HsKG91d4 P- 1034699 Hs.100890 102.
CACNA2D2 SEQ ID NO. 102 R43145a2 P+ 123539 Hs.389415 103. NEBL SEQ
ID NO. 103 R43171d12 P- 796643 Hs.5025 104. EST SEQ ID NO. 104
R43174a9 P- 43705 Hs.25211 105. HLA-DQA1 SEQ ID NO. 105 R43305e9 P-
320393 Hs.387679 106. EDG3 SEQ ID NO. 106 R43199e6 P+ 283748
Hs.4257 107. CPVL SEQ ID NO. 107 HsKG91d10 P- 39833 Hs.95594 108.
FLJ32884 SEQ ID NO. 108 R43320b4 P- 383153 Hs.375551 109. LCP1 SEQ
ID NO. 109 HsKG12H6 P- 344589 Hs.381099 110. EST SEQ ID NO. 110
R4327f11 P- 67033 Hs.386104 111. EST SEQ ID NO. 111 HsKG67A3 P+
1461048 Hs.443884 112. EST SEQ ID NO. 112 R43411b11 P- 1908847
Hs.150167 113. EST SEQ ID NO. 113 R4342e12 P- 138974 Hs.28367 114.
DKFZP564C152 SEQ ID NO. 114 R43330f1 P+ 1865232 Hs.184216 115. DMN
SEQ ID NO. 115 FHskG6A7 P- 1161564 Hs.381347 116. GABRA5 SEQ ID NO.
116 HsKG99g5 P+ 2358925 Hs.24969 117. AKR1C3 SEQ ID NO. 117
HsKG17A1 P- 1473304 Hs.78183 118. LOC168850 SEQ ID NO. 118 R4376d1
P- 265114 Hs.159006 119. EST SEQ ID NO. 119 R43352h6 P- 1584099
Hs.128216 120. KCNQ2 SEQ ID NO. 120 HsKG24F10 P+ 179534 Hs.4975
121. NME5 SEQ ID NO. 121 HsKG51B1 P+ 502173 Hs.72050 122. EST SEQ
ID NO. 122 R43162f7 P- 29841 Hs.165570 123. PBX1 SEQ ID NO. 123
R4364d4 P- 200656 Hs.408222 124. CNTNAP2 SEQ ID NO. 124 R43159d1 P-
27404 Hs.106552 125. EST SEQ ID NO. 125 R43338g9 P- 1503694
Hs.406982 126. SPON1 SEQ ID NO. 126 R43210e4 P+ 773495 Hs.5378 127.
CDH8 SEQ ID NO. 127 R4334f12 P- 40751 Hs.388928 128. PRKCB1 SEQ ID
NO. 128 HsKG3H1 P- 753923 Hs.349845 129. SLC21A11 SEQ ID NO. 129
R43239f7 P- 878698 Hs.113657 130. MAP4 SEQ ID NO. 130 R43240e6 P-
858672 Hs.31095 131. EST SEQ ID NO. 131 R43258f10 P- 855448
Hs.162966 132. SCN7A SEQ ID NO. 132 R4364h4 P- 795262 Hs.406684
133. EST SEQ ID NO. 133 R43102g1 P- 51420 Hs.446660 134. EST SEQ ID
NO. 134 R43279a11 P- 1584398 Hs.370168 135. EST SEQ ID NO. 135
R43164h7 P- 754346 Hs.34145 136. EST SEQ ID NO. 136 R43367f11 P+
1690886 Hs.134687 137. CDW52 SEQ ID NO. 137 HsKG100g3 P+ 2417330
Hs.276770 138. ARCB1 SEQ ID NO. 138 HsKG20G8 P+ 813256 Hs.21330
139. EST SEQ ID NO. 139 R43407f2 P- 1893735 Hs.146175 140. OST-2
SEQ ID NO. 140 HsKG30A7 P+ 897910 Hs.136348 141. NRXN1 SEQ ID NO.
141 R43344h11 P- 1552433 Hs.22998 142. ADAM22 SEQ ID NO. 142
R4369b7 P+ 284541 Hs.256398 143. EST SEQ ID NO. 143 R43243f12 P+
38152 Hs.301296 144. TRGV9 SEQ ID NO. 144 HsKG11B6 P- 281003
Hs.407442 145. EST SEQ ID NO. 145 HsKG88e11 P+ 840677 Hs.377975
146. PTPRD SEQ ID NO. 146 R43105e7 P- 47186 Hs.323079 147. EST SEQ
ID NO. 147 R43237g1 P+ 1292654 Hs.120364 148. HS3ST2 SEQ ID NO. 148
HsKG59G11 P+ 1557290 Hs.115830 149. FGF13 SEQ ID NO. 149 HsKG100a7
P- 2385663 Hs.6540 150. MKI67 SEQ ID NO. 150 HsKG6G9 P+ 769513
Hs.80976 151. KIF12 SEQ ID NO. 151 R4342d12 P- 214205 Hs.28149 152.
EST SEQ ID NO. 152 R43252h5 P+ 432477 Hs.113170 153. EST SEQ ID NO.
153 HsKG66G1 P- 306841 Hs.449439 154. EST SEQ ID NO. 154 HsKG3B11
P- 770014 Hs.74647 155. EST SEQ ID NO. 155 HsKG10E4 P+ 66560
Hs.356861 156. EST SEQ ID NO. 156 HsKG2B3 P- 267420 Hs.510917 157.
KLIP1 SEQ ID NO. 157 FHskG14E3 P+ 782259 Hs.38178 158. EST SEQ ID
NO. 158 R43272a11 P+ 1522487 Hs.130183 159. LOC157570 SEQ ID NO.
159 R43282h5 P+ 1623191 Hs.99480 160. MAD2L1 SEQ ID NO. 160
HsKG28B2 P+ 814701 Hs.79078 161. EST SEQ ID NO. 161 R43275a2 P-
1554430 Hs.388347 162. EST SEQ ID NO. 162 R43160f5 P- 726564
Hs.97579 163. RGS5 SEQ ID NO. 163 HsKG37F3 P+ 853809 Hs.24950 164.
ATP2B4 SEQ ID NO. 164 R4376f10 P- 502326 Hs.343522 165. HMGCL SEQ
ID NO. 165 HsKG2G7 P- 838366 Hs.444925 166. ODZ3 SEQ ID NO. 166
R43371a4 P- 1704063 Hs.41793 167. CHGA SEQ ID NO. 167 HsKG61C1 P+
1585535 Hs.124411 168. MGC33510 SEQ ID NO. 168 R43238d9 P- 884658
Hs.158798 169. GAGE5 SEQ ID NO. 169 HsKG82H6 P+ 2911881 Hs.278606
170. SARDH SEQ ID NO. 170 R43402f5 P- 1870053 Hs.198003 171. EST
SEQ ID NO. 171 R43164e6 P- 753982 Hs.86538 172. DAT1 SEQ ID NO. 172
R43229e6 P+ 897262 Hs.301914 173. FUCA1 SEQ ID NO. 173 HsKG5E7 P-
308437 Hs.576 174. TM6SF2 SEQ ID NO. 174 R43144a9 P+ 342187
Hs.367829 175. KCNK9 SEQ ID NO. 175 R43229d8 P+ 897105 Hs.117010
176. ADCYAP1 SEQ ID NO. 176 HsKG2A3 P- 969568 Hs.68137 177. PLXNA4
SEQ ID NO. 177 R43230e12 P- 41287 Hs.169129 178. HLA-DMB SEQ ID NO.
178 HsKG1B4 P- 148231 Hs.1162 179. EST SEQ ID NO. 179 R43205d4 P-
436059 Hs.186937 180. EST SEQ ID NO. 180 R43122e6 P- 742685
Hs.519270 181. GRIN3A SEQ ID NO. 181 R43173e6 P- 42747 Hs.283852
182. OSBPL3 SEQ ID NO. 182 R43217f10 P- 824212 Hs.197955 183. ODZ4
SEQ ID NO. 183 FHskG5G6 P+ 785913 Hs.5028 184. EST SEQ ID NO. 184
R43414c5 P- 1930391 Hs.182889 185. E2F1 SEQ ID NO. 185 HsKG41H12 P+
768260 Hs.96055 186. MGC16664 SEQ ID NO. 186 R43288h5 P+ 1641875
Hs.400696 187. HMP19 SEQ ID NO. 187 HsKG86h6 P+ 838701 Hs.70669
188. IL2RB SEQ ID NO. 188 CD1E6 P- 2132327 Hs.75596 189. TOPK SEQ
ID NO. 189 HsKG89c5 P+ 785368 Hs.104741 190. ALDH1A1 SEQ ID NO. 190
HsKG15A1 P- 855624 Hs.76392 191. CED-6 SEQ ID NO. 191 HsKG85g6 P+
782476 Hs.107056 192. EST SEQ ID NO. 192 R43159h12 P- 768146
Hs.376455 193. A2BP1 SEQ ID NO. 193 R43323c4 P- 759206 Hs.57937
194. LY6E SEQ ID NO. 194 HsKG16D12 P+ 1470048 Hs.77667 195. EST SEQ
ID NO. 195 R43104h4 P- 39885 Hs.497208 196. EST SEQ ID NO. 196
R43197e12 P- 259884 Hs.419170 197. PLXNC1 SEQ ID NO. 197 HsKG60F10
P- 261834 Hs.286229 198. EFS SEQ ID NO. 198 R4365c5 P+ 795730
Hs.24587 199. ACTN2 SEQ ID NO. 199 R43234f4 P- 377812 Hs.83672 200.
MYC SEQ ID NO. 200 HsKG2H7 P- 812965 Hs.202453 201. KIAA0527 SEQ ID
NO. 201 R43313f4 P+ 2016891 Hs.196647 202. C6orf31 SEQ ID NO. 202
R43235b6 P- 436765 Hs.301920 203. DLL3 SEQ ID NO. 203 HsKG92e2 P+
1469966 Hs.127792 204. EST SEQ ID NO. 204 R43363a10 P+ 1663168
Hs.435132 205. STK33 SEQ ID NO. 205 R43261a6 P+ 1416035 Hs.148135
206. SEMA3A SEQ ID NO. 206 HsKG78B10 P- 767055 Hs.252451 207. EST
SEQ ID NO. 207 R43338f10 P- 1502008 Hs.143707 208. IGSF4 SEQ ID NO.
208 R43141h9 P- 772960 Hs.156682 209. CKS2 SEQ ID NO. 209 HsKG10C4
P+ 725454 Hs.83758 210. EST SEQ ID NO. 210 R43259g11 P- 969593
Hs.116922 211. EST SEQ ID NO. 211 R43371c12 P+ 1705626 Hs.444405
212. SIX3 SEQ ID NO. 212 R4371d7 P- 277283 Hs.227277 213. FLJ22002
SEQ ID NO. 213 R4331c9 P- 153779 Hs.461485 214. HSD17B12 SEQ ID NO.
214 R4377h10 P+ 278938 Hs.132513 215. HBA2 SEQ ID NO. 215 HsKG81G2
P+ 2782586 Hs.398636 216. CDH11 SEQ ID NO. 216 R43407d7 P- 1893136
Hs.443435 217. RGS9 SEQ ID NO. 217 R43192c4 P- 383501 Hs.117149
218. EST SEQ ID NO. 218 R43279a2 P- 1583668 Hs.128282 219. NCAM2
SEQ ID NO. 219 HsKG97f12 P- 1898102 Hs.135892 220. BIRC5 SEQ ID NO.
220 R4398a5 P+ 796694 Hs.1578 221. EST SEQ ID NO. 221 R43237b1 P-
462850 Hs.444347 222. GNG12 SEQ ID NO. 222 R43119c10 P- 265045
Hs.8107 223. GPIG4 SEQ ID NO. 223 R43359c2 P- 1648516 Hs.352552
224. EST SEQ ID NO. 224 R43128g4 P+ 299629 Hs.49265 225. ENPP4 SEQ
ID NO. 225 HsKG90c5 P+ 281737 Hs.54037 226. FMNL SEQ ID NO. 226
R43199b3 P+ 281605 Hs.100217 227. EST SEQ ID NO. 227 HsKG40C4 P-
743230 Hs.240443 228. PIWIL2 SEQ ID NO. 228 R43138a4 P- 743309
Hs.274150 229. CLSTN1 SEQ ID NO. 229 R43192b10 P- 231718 Hs.29665
230. UHRF1 SEQ ID NO. 230 R43344e11 P+ 1550739 Hs.108106 231. EST
SEQ ID NO. 231 R43332e10 P- 2253160 Hs.89121 232. SLC40A1 SEQ ID
NO. 232 HsKG86b1 P- 71863 Hs.409875 233. CLECSF6 SEQ ID NO. 233
R43255b1 P- 454296 Hs.115515 234. EST SEQ ID NO. 234 R43271h10 P+
1520938 Hs.127505 235. BKLHD2 SEQ ID NO. 235 R43111h1 P+ 951083
Hs.348262 236. EST SEQ ID NO. 236 R43246f2 P- 121182 Hs.520888 237.
EST SEQ ID NO. 237 R4333e12 P- 67037 Hs.282970 238. EST SEQ ID NO.
238 R43405d9 P- 1880732 Hs.146138 239. SORCS1 SEQ ID NO. 239
R43370a4 P- 1701301 Hs.348923 240. NRP2 SEQ ID NO. 240 R43168g10 P-
823811 Hs.368746 241. E2-EPF SEQ ID NO. 241 HsKG21A11 P+ 810600
Hs.462306 242. CAST SEQ ID NO. 242 R43325f1 P- 591381 Hs.440961
243. KIAA1384 SEQ ID NO. 243 R43359c10 P- 1649134 Hs.88442 244.
KIAA0644 SEQ ID NO. 244 R43332g4 P- 2273304 Hs.21572 245. HLA-DRB3
SEQ ID NO. 245 HsKG12H2 P- 855547 Hs.308026 246. PMP22 SEQ ID NO.
246 R43247g12 P- 162310 Hs.372031 247. DJ79P11.1 SEQ ID NO. 247
R4365h1 P+ 810367 Hs.398989 248. SOX5 SEQ ID NO. 248 HsKG99e11 P-
2338834 Hs.87224 249. CD3E SEQ ID NO. 249 HsKG61E1 P+ 1536968
Hs.3003 250. EST SEQ ID NO. 250 R4327e11 P- 240945 Hs.445357 Rank
Accession Accession Title of Gene 1. N74203 NM_003836 delta-like 1
homolog gi|1231488 gi|34147651 2. AA928113 AK092951 Homo sapiens
cDNA gi|3077269 gi|21751664 FLJ35632 fis, clone SPLEN2011678 3.
AI308916 NM_002771 protease, serine, 3 gi|4003787 gi|21536451
(mesotrypsin) 4. AI692753 NM_004675 ras homolog gene family,
gi|4970093 gi|58530880 member 1 5. H86117 NM_015193
activity-regulated gi|1067696 gi|56676395 cytoskeleton-associated
protein 6. AA703652 NM_003062 slit homolog 3 (Drosophila)
gi|2713570 gi|11321570 7. R20626 NM_016083 cannabinoid receptor 1
gi|775407 gi|38683843 (brain) 8. R73759 BC012900 Homo sapiens,
clone gi|848129 gi|15277677 IMAGE: 3881549, mRNA 9. T84084 AK021785
Homo sapiens, cDNA FLJ11723 gi|712372 gi|10433040 fis, clone HEMBA
1005314 10. AA706038 NM_144966 hypothetical protein gi|2715956
gi|56549657 FLJ25461/FREM1 11. AA256176 BC004287 Homo sapiens,
clone gi|1891715 gi|13279127 IMAGE: 3618365, mRNA 12. AI368364
NM_000610 CD44 antigen (homing function gi|4147117 gi|48255934 and
Indian blood group system) 13. AA235370 NM_002351 Homo sapiens cDNA
clone gi|1859808 gi|4506922| IMage: 4811759, partial cds 14.
AA055534 BX648828 roundabout, axon guidance gi|1547891 gi|34367993
receptor, homolog 2 (Drosophila) 15. AA454990 NM_014962 BTB (POZ)
domain gi|2177766 gi|31317210 containing 3 16. R52824 NM_005378
v-myc myelocytomatosis viral gi|814726 gi|62750358 related
oncogene, neuroblastoma derived (avian) 17. AA938345 AL049227 Homo
sapiens mRNA; gi|3096456 gi|4499957 cDNA DKFZp564N1116 (from clone
DKFZp564N1116) 18. AA454632 NM_020647 junctophilin 1 gi|2177408
gi|61676191 19. AI810168 killer cell lectin-like gi|5396734
receptor subfamily C, member 3 20. AA232953 BM721099 Homo sapiens
LOC376510 gi|1855945 gi|19040795 (LOC376510), mRNA 21. AA903339
NM_020630 ret proto-oncogene (multiple gi|3038462 gi|50593520
endocrine neoplasia and medullary thyroid carcinoma 1, Hirschsprung
disease) 22. AA454702 NM_004378 cellular retinoic acid gi|2177478
gi|4758051 binding protein 1 23. R61395 NM_004826 endothelin
converting gi|832090 gi|4758231 enzyme-like 1 24. AA004638 BC040073
hypothetical protein gi|1448175 gi|25455647 LOC283120 25. R60014
AK123640 high mobility group AT- gi|830709 gi|34529239 hook 2 26.
N52151 AL833547 synaptopodin 2 gi|1193412 gi|21734192 27. N55540
hypothetical protein gi|1198419 LOC163782 28. H65066 NM_003385
visinin-like 1 gi|1023806 gi|63252921 29. AA973808 NM_006040
heparan sulfate(glucosamine) gi|3148988 gi|48427666
3-O-sulfotransferase 4 30. R93124 NM_001353 aldo-keto reductase
family 1, gi|967290 gi|56121816 member C1 (dihydrodiol
dehydorgenase 1; 20-alpha(3-alpha)- hydroxysteriod dehydrogenase)
31. W72068 BX648323 Homo sapiens cDNA: gi|1382338 gi|34367482
FLJ21545 fis, clone COL06195 32. R61341 NM_005295 G protein-coupled
receptor gi|832036 gi|4885308 22 33. AA044023 Homo sapiens
transcribed sequence gi|1521944 with weak similarilty to protein
ref: NP_060312.1 (H. sapiens) hypothetical protein FLJ20489 [Homo
sapiens] 34. AA034366 BU620794 Clone ID: 449512 gi|1506175
gi|23287009 35. AA777001 NM_003914 cyclin A1 gi|16306528 36. R37656
NM_181795 protein kinase (cAMP-dependent, gi|795112 gi|32483391
catalytic inhibitor beta) 37. H40665 BX093245 Homo sapiens full
length gi|916717 gi|27823200 insert cDNA YN61C04 38. AI623173
galanin gi|4648098 39. AI205664 BM701300 Homo sapiens transcribed
sequence gi|3764336 gi|19014558 with strong similarity to protein
ref: NP_055378.1 (H. sapiens) spondyloepiphyseal dysplasia, late;
sedlin [Homo sapiens] 40. AA918535 BQ012257 hypothetical protein
gi|3058425 gi|19737158 LOC221303 41. AA394198 BE970051 Homo sapiens
transcribed sequence gi|2047217 gi|10582984 with strong similarilty
to protein sp: P07478 (H. sapiens) TRY2_HUMAN Trypsin II precursor
(Anionic trypsinogen) 42. R77783 AL519577 Homo sapiens transcribed
sequence gi|852893 gi|45695127 with strong similarity to protein
ref: NP_003610.1 (H. sapiens) protease, serine, 12 (neurotrypsin,
motopsin) [Homo sapiens] 43. AA029597 NM_001719 bone morphogenetic
gi|1497001 gi|4502426 protein 7 (osteogenetic protein 1) 44.
AA621201 NM_003459 solute carrier family 30 gi|2525140 gi|52630414
(zinc transporter), member 3 45. AA173755 hypothetical protein
gi|1754078 FLJ10539 46. N22620 NM_181847 amphoterin induced gene 2
gi|1128754 gi|40556374 47. AI924357 NM_001354 aldo-keto reductase
family 1, gi|5660321 gi|45446741 member C2 (dihydrodiol
dehydrogenase 2; bile acid binding protein; 3-alpha hydroxysteroid
dehydrogenase, type III) 48. AA155913 NM_000900 matrix Gla protein
gi|1727531 gi|49574513 49. R42630 proprotein convertase gi|817391
subtilisin/kexin type 1 50. AI005515 NM_000189 hexokinase 2
gi|3215025 gi|40806188 51. R34343 BX107971 Homo sapiens transcribed
gi|791244 gi|27834959 sequences Clone ID: 136502 52. AI830281
BX365439 Homo sapiens melanoma antigen gi|5450952 gi|46286082
family A9 (MAGEA9) mRNA, partial cds 53. AI539460 NM_000880
interleukin 7 gi|4453595 gi8610152 54. AA928660 NM_003619 protease,
serine, 12 gi|3076951 gi|21327713 (neurotrypsin, motospin) 55.
T60160 NM_031412 GABA(A) receptor- gi|661997 gi|56676368 associated
protein like 1 56. AA401404 NM_080831 defensin, beta 129 gi|2053629
gi|30061487 57. AA705735 NM_014903 neuron navigator 3 gi|2715653
gi|66933019 58. AA235116 NM_002867 RAB3B, member RAS gi|1859553
gi|19923749 oncogene family 59. AA936779 NM_005555 keratin 6B
gi|3094813 gi|17505187 60. W60582 NM_018476 brain express, X-linked
1 gi|1367411 gi|685332 61. R66103 Homo sapiens transcribed
gi|838741 sequences Clone ID: 140210 62. AA975538 Homo sapiens
transcribed gi|3151330 sequences Clone ID: 1558233 63. AA476300
NM_020680 SCY1-like (S. cerevisiae) gi|2204511 gi|19923565 64.
H23444 AK092129 Homo sapiens TAFA1 gi|892139 gi|21750647 mRNA,
complete cds 65. R15791 NM_001035 ryanodine receptor 2 gi|768206
gi|4506756 (cardiac) 66. AA041400 NM_006726 LPS-responsive vesicle
trafficking, gi|1517689 gi|16904380 beach and anchor containing 67.
AI000557 NM_004386 chondroitin sulfate gi|3191111 gi|4758083
proteoglycan 3 (neurocan) 68. AI268450 Homo sapiens transcribed
gi|3887617 sequnces Clone ID: 1880885 69. R92994 NM_002426 matrix
metalloproteinase gi|965348 gi|4505206 12 (macrophage elastase) 70.
W81677 NM_000079 cholinergic receptor, nicotinic, gi|1392187
gi|4557456 alpha polypeptide 1 (muscle) 71. AA903531 AI961087 Homo
sapiens transcribed sequence gi|3038654 gi|5753868 with weak
similarily to protein pir: T47135 (H. sapiens) T47135 hypothetical
protein DKFZp761L0812.1 human (fragment) 72. H23463 Homo sapiens
transcribed gi|892158 sequences Clone ID: 52329 73. R91170
NM_005520 heterogeneous nuclear gi|958710 gi|5031752
ribonucleoprotein H1 (H) 74. AI240426 NM_199188 c-Mpl binding
protein gi|3835823 gi|40353739 75. R68243 AK055280 Homo sapiens
cDNA FLJ30718 gi|841760 gi|16549979 fis, clone FCBBF2001675 76.
N50114 NM_018440 phosphoprotein associated with gi|1191280
gi|63054863 glycosphingolipid-enriched microdomains 77. R15853
BX648828 prokineticin 2 gi|768268 gi|34367993 78. AA772904
NM_004807 heparan sulfate 6-O- gi|2825746 gi|4758565
sulfotransferase 1 79. AI290481 Homo sapiens transcribed gi|3933255
sequences Clone ID: 1880352 80. N63057 NM_020403 protocadherin 9
gi|1210886 gi|45243537 81. AA866153 Homo sapiens transcribed
gi|2958429 sequences Clone ID: 1469434 82. AA976650 BM716109 Homo
sapiens transcribed sequence gi|3154096 gi|19029367 with weak
similarily to protein ref: NP_009056.1 (H. sapiens) ubiquitously
transcribed tetratri- copeptide repeat gene, Y chromosome;
Ubiquitously transcribed TPR gene on Y chromosome [Homo sapiens]
83. N58494 glycine dehydrogenase gi|1202384 (decarboxylating;
glycine decarboxylase, glycine cleavage system protein P) 84.
H90431 NM_000024 adrenergic, beta-2-, gi|1080861 gi|15718673
recpetor surface 85. AI391632 NM_002163 interferon consensus
gi|4217636 gi|55953136 sequence binding protein 1 86. AI028034
NM_001778 CD48 antigen (B-cell gi|3245343 gi|21361570 membrane
protein) 87. H40323 BC043430 Homo sapiens cDNA clone gi|916375
gi|34193298 IMAGE: 5294683, partial cds 88. AA962159 NM_004714
dual-specificity tyrosine-(Y)- gi|3134323 gi|4758221
phosphorylation regualted kinase 1B 89. AA913480 killer cell
lectin-like receptor gi|3052872 subfamily C, member 1 90. AA188378
Homo sapiens, clone gi|1775412 IMAGE: 4865966, mRNA 91. N47979
BX538341 Homo sapiens mRNA; gi|1189145 gi|31874840 cDNA
DKFZp686C13222 (from clone DKFZp686C13222) 92. T95274 AF146695 Homo
sapiens clone gi|733898 gi|4887201 IMAGE: 120162 mRNA sequence 93.
AA424574 NM_015529 monooxygenase, DBH-like 1 gi|2103544 gi|24308084
94. N93122 Homo sapiens transcribed sequence gi|1265431 with weak
similarity to protein sp: P39191 (H. sapiens) ALU4_HUMAN Alu
subfamily SB2 sequence contamination warning entry 95. AA707167
AU253973 Homo sapiens transcribed gi|2717085 gi|34322686 sequences
Clone ID: 451394 96. AA025819 NM_002048 growth arrest-specific 1
gi|1491222 gi|4503918 97. AI493478 NM_001852 collagen, type IX,
alpha 2 gi|4394481 gi|31083125 98. N52812 BX105296 Homo sapiens
transcribed gi|1193978 gi|27833450 sequences Clone ID: 244312 99.
H08643 NM_001940 dentatorubral- gi|873465 gi|55750040
pallidoluysian atrophy (atrophin-1) 100. AI248323 AI248323 Homo
sapiens transcribed gi|3843720 gi|3843720 sequences Clone ID:
1850044
101. AA779892 NM_019845 candidate mediator of the gi|2839223
gi|54792141 p53-dependent G2 arrest 102. R00809 NM_006030 calcium
channel, voltage- gi|750545 gi|54112393 dependent, alpha 2/delta
subunit 2 103. AA461473 NM_006393 nebulette gi|2185337 gi|5453757
104. H05706 H05706 Homo sapiens transcribed gi|869258 gi|869258
sequences Clone ID: 43705 105. W16836 NM_002122 major
histocompatibility gi|1291224 gi|52426772 complex, class II, DQ
alpha 1 106. N50742 AL832194 endothelial differentiation,
gi|1191908 gi|21732739 sphingolipid G-protein-coupled receptor, 3
107. R53455 NM_019029 carboxypeptidase, gi|815357 gi|22027515
vitellogenic-like 108. AA071005 NM_144702 hypothetical protein
gi|1578558 gi|21389614 FLJ32884 109. W73144 NM_002298 lymphocyte
cytosolic gi|1383279 gi|7382490 protein 1 (L-plastin) 110. T70327
T70327 Homo sapiens transcribed sequence gi|681475 gi|681475 with
weak similarity to protein ref: NP_001432.1 (H. sapiens) fatty acid
amide hydrolase [Homo sapiens] 111. AA890136 Homo sapiens similar
to gi|3017015 expressed sequence AW121567 (LOC374514), mRNA 112.
AI300926 BC042456 Homo sapiens, clone gi|3960272 gi|27502868 IMAGE:
4818531, mRNA 113. R62835 BX101784 Homo sapiens transcribed
gi|834714 gi|27831388 sequences Clone ID: 138974 114. AI269361
DKFZP564C152 protein gi|3888528 115. AA877815 NM_145728 desmuslin
gi|2986780 gi|22027637 116. AI807646 gamma-aminobutyric acid
gi|5394212 (GABA) A receptor, alpha 5 117. AA916325 NM_003739
aldo-keto reductase family 1, gi|3055717 gi|24497582 member C3
(3-alpha hydroxysteroid dehydrogenase, type II) 118. N20820
NM_176814 hypothetical protein gi|1126001 gi|39753952 LOC168850
119. AA972401 BX100412 Homo sapiens transcribed gi|3147691
gi|27844465 sequences Clone ID: 1584099 120. H51419 NM_172107
potassium voltage-gated channel, gi|991260 gi|26051263 KQT-like
subfamily, member 2 121. AA133350 NM_003551 non-metastatic cells 5,
protein gi|1690318 gi|37622352 expressed in (nucleoside-
diphosphate kinase) 122. R41560 AF131795 Homo sapiens clone 25052
gi|816860 gi|4406623 mRNA sequence 123. R98407 NM_002585 pre-B-cell
leukemia gi|985119 gi|4505622 transcription factor 1 124. R13972
NM_014141 contactin associated gi|767048 gi|21071040 protein-like 2
125. AA907347 Homo sapiens cDNA gi|3042807 FLJ40156 fis, clone
TESTI2014385 126. AA427924 NM_006108 spondin 1, (f-spondin)
gi|2111686 gi|124307904 extracellular matrix protein 127. R56219
NM_001796 cadherin 8, type 2 gi|826325 gi|16306538 128. AA479102
NM_002738 protein kinase C, beta 1 gi|2207658 gi|47157320 129.
AA775372 NM_013272 solute carrier family 21 (organic gi|2834706
gi|7706713 anion transporter), member 11 130. AA778985 NM_002375
microtubule-associated gi|2838316 gi|47519638 protein 4 131.
AA664081 Homo sapiens transcribed gi|2618072 sequences Clone ID:
855448 132. AA453997 NM_002976 sodium channel, voltage- gi|2167666
gi|4506810 gated, type VII, alpha 133. H20717 AK125162 Homo sapiens
cDNA FLJ43172 gi|889412 gi|34531161 fis, clone FCBBF3007242 134.
AA971518 Homo sapiens transcribed gi|3146808 sequences Clone ID:
1584398 135. AA436138 BG576442 Homo sapiens transcribed gi|2141052
gi|13584095 sequences Clone ID: 754346 136. AI088327 Homo sapiens
transcribed gi|3427386 sequences Clone ID: 1690886 137. AI826477
NM_001803 CD52 (CAMPATH-1 gi|5447148 gi|68342029 antigen) 138.
AA455911 ATP-binding cassette, sub-family gi|2178687 B (MDR/TAP),
member 1 139. AI277247 AI277247 Homo sapiens transcribed gi|3899515
gi|3899515 sequences Clone ID: 1893735 140. AA598653 NM_006475
osteoblast specific factor 2 gi|2432236 gi|5453833 (faciclin
1-like) 141. AA927036 NM_004801 neurexin 1 gi|3075933 gi|21070965
142. N59441 NM_021723 a disintegrin and gi|1203331 gi|21536387
metalloproteinase domain 22 143. R49458 Homo sapiens cDNA:
gi|1820356 FLJ23131 fis, clone LNG08502 144. N50880 BC030554 T cell
receptor gamma gi|1192046 gi|20988582 variable 9 145. AA486362
AK128524 Homo sapiens immunoglobulin gi|2215168 gi|34535933 kappa
light chain mRNA, partial cds 146. H10403 NM_002839 protein
tyrosine gi|875225 gi|4506308 phosphatase, receptor type, D 147.
AA719150 BC035185 Homo sapiens hypothetical gi|2732249 gi|34191447
protein LOC285194, mRNA (cDNA clone IMGE: 5266409), partial cds
148. AA935694 NM_006043 heparan sulfate (glucosamine) gi|3092851
gi|5174462 3-O-sulfotransferase 2 149. AI762428 NM_004114
fibroblast growth factor 13 gi|5178095 gi|16306544 150. AA426264
NM_002417 antigen identified by gi|2107605 gi|19923216 monoclonal
antibody Ki-67 151. H77627 NM_138424 kinesin family member 12
gi|1055716 gi|19923948 152. AA699493 Homo sapiens transcribed
gi|2703649 sequences Clone ID: 432477 153. N91921 AA994097 Homo
sapiens TCR BV3 mRNA for gi|1264230 gi|3180642 T cell receptor beta
chain (CDR3 region), partial cds, isolate: HTLV-1 myopathy case3,
clone: Tax tetramer-5 154. AA427491 BC041074 Human T-cell receptor
active alpha- gi|2111387 gi|27370838 chain mRNA from Jurkat cell
line 155. T67053 Homo sapiens cDNA FLJ26905 gi|676493 fis, clone
RCT01427, highly similar to Ig lambda chain C regions 156. N24966
Homo sapiens transcribed gi|1139116 sequences Clone ID: 267420 157.
AA431741 NM_024629 KSHV latent nuclear gi|2115449 gi|38016934
antigen interacting protein 1 158. AA908678 Homo sapiens
transcribed gi|3048083 sequences Clone ID: 1522487 159. AA992658
AL832666 hypothetical protein gi|3178392 gi|21733242 LOC157570 160.
AA481076 NM_002358 MAD2 mitotic arrest gi|2210628 gi|6466452
deficient-like 1 (yeast) 161. AA931491 BX648964 Homo sapiens mRNA;
gi|3085877 gi|34368136 cDNA DKFZp686J0156 (from clone
DKFZp686J0156) 162. AA398118 Homo sapiens transcribed sequence
gi|2051227 with weak similarity to protein ref: NP_060265.1 (H.
sapiens) hypothetical protein FLJ20378 [Homo sapiens] 163. AA668470
NM_003617 regulator of G-proetin gi|2629969 gi|41387215 signalling
5 164. AA156674 NM_001684 ATPase, Ca++ gi|1728353 gi|48255956
transporting, plasma membrane 4 165. AA458779 NM_000191
3-hydroxymethyl-3- gi|2183686 gi|62198231 methylglutaryl-Coenzyme A
lyase (hydroxymethylglutaricaciduria) 166. AI159901 XM_371717 odd
Oz/Ten-m homolog 3 gi|3693281 gi|51464322 167. AA976699 NM_001275
chromagranin A gi|3154145 gi|10800418 (parathyroid secretory
protein 1) 168. AA629901 NM_152765 hypothetical protein gi|2552512
gi|34303955 MGC33510 169. AW510753 NM_001474 G antigen 5 gi|7148831
gi|4503882 170. AI245607 NM_007101 sarcosine dehydrogenase
gi|3841004 gi|21361377 171. AA479967 Homo sapiens cDNA FLJ44429
gi|2208118 fis, clone UTERU2015653 172. AA677643 NM_018640 neuronal
specific gi|2658165 gi|41350202 transcription factor DAT1 173.
W24873 NM_000147 fucosidase, alpha-L-1, gi|1302728 gi|24475878
tissue 174. W63783 NM_023002 transmembrane 6 gi|1371384 gi|30794471
superfamily member 2 175. AA676876 NM_016601 potassium channel
gi|2657398 gi|16445406 subfamily K, member 9 176. AA772803
NM_001117 adenylate cyclase gi|2825645 gi|10947062 activating
polypeptide 1 (pituitary) 177. R56614 XM_379927 plexin A4 gi|826720
gi|51466511 178. H13691 NM_002118 major histocompatibility
gi|878511 gi|18641376 complex, class II, DM beta 179. AA700815 Homo
sapiens transcribed gi|2703980 sequences Clone ID: 436059 180.
AA400292 AK092836 Homo sapiens cDNA FLJ35517 gi|2054172 gi|21751529
fis, clone SPLEN2000698. 181. R61128 NM_133445 glutamate receptor,
gi|831823 gi|20143963 ionotropic, N-methyl-D-aspartate 3A 182.
AA490967 NM_145323 oxysterol binding protein- gi|2220140
gi|21735585 like 3 183. AA449490 XM_166254 odd Oz/ten-m homolog 4
gi|2163240 gi|51468857 184. AI333640 AI333640 Homo sapiens
transcribed gi|4070199 gi|4070199 sequences Clone ID: 1930391 185.
AA424950 NM_005225 E2F transcription factor 1 gi|2107038
gi|12669910 186. AI018400 NM_173509 hypothetical protein gi|3232919
gi|34222229 MGC16664 187. AA457267 NM_015980 HMP19 protein
gi|2179987 gi|34222326 188. AI433655 NM_000878 interleukin 2
receptor, beta gi|4290700 gi|23238195 189. AA476576 NM_018492 T-LAK
cell-originated gi|2204787 gi|18490990 protein kinase 190. AA664101
NM_000689 aldehyde dehydrogenase 1 gi|2618092 gi|25777722 family,
member A1 191. AA431753 NM_016315 PTB domain adaptor gi|2115461
gi|56550114 protein CED-6 192. AA426561 NM_016300 Homo sapiens cDNA
FLJ36329 gi|2106816 gi|68161512 fis, clone THYMU2005855 193.
AA496047 NM_145893 ataxin 2-binding protein 1 gi|2229368
gi|22538408 194. AA865464 NM_002346 lymphocyte antigen 6 gi|2957740
gi|4505048 complex, locus E 195. R52543 NM_199051 Homo sapiens
similar to RIKEN gi|814445 gi|39979637 cDNA B830045N13 (LOC339479),
mRNA 196. N32904 NM_020455 Homo sapiens cDNA FLJ16029 gi|1153303
gi|37620168 fis, clone KIDNE2012945, weakly similar to PROCOLLAGEN
C-PROTEINASE ENHANCER PROTEIN PRECURSOR 197. H98855 NM_005761
plexin C1 gi|1123523 gi|5032222 198. AA460282 NM_005864 embryonal
Fyn-associated gi|2185098 gi|14589877 substrate 199. AA775521
NM_001103 actinin, alpha 2 gi|2834855 gi|4501892 200. AA464600
NM_002467 v-myc myelocytomatosis gi|2189484 gi|31543215 viral
oncogene homolog (avian) 201. AI356230 XM_171054 KIAA0527 protein
gi|4107851 gi|51463939 202. AA703077 NM_030651 chromosome 6 open
gi|2706190 gi|21361926 reading frame 31 203. AA865362 NM_016941
delta-like 3 (Drosophila) gi|2957638 gi|45243550 204. AI129115
AK001013 Homo sapiens cDNA FLJ10151 gi|3597629 gi|7022026 fis,
clone HEMBA1003402. 205. AA948041 NM_030906 serine/threonine kinase
33 gi|3109294 gi|44890053 206. AA451750 NM_006080 sema domain,
immunoglobulin gi|2165419 gi|5174672 domain (lg), short basic
domain, secreted, (semaphorin) 3A 207. AA887204 Homo sapiens
transcribed gi|3002312 sequences Clone ID: 1502008 208. AA476257
NM_014333 immunoglobulin gi|2204468 gi|22095346 superfamily, member
4 209. AA397813 NM_001827 CDC28 protein kinase gi|2051021
gi|4502858 regulatory subunit 2 210. AA663726 Homo sapiens
transcribed gi|2617717 sequences Clone ID: 969593 211. AI143189
Homo sapiens transcribed sequence gi|3664998 with weak similarity
to protein ref: NP_055405.1 (H. sapiens) endogenous retroviral
family W, env(C7), member 1 (syncytin); envelope protein [Homo
sapiens] 212. N41052 sine oculis homeobox gi|1164650 homolog 3
(Drosophila) 213. R48248 NM_024838 hypothetical protein
gi|810274 gi|34222383 FLJ22002 214. N66644 NM_016142 hydroxysteroid
(17-beta) gi|1218769 gi|7705854 dehydrogenase 12 215. AW157797
NM_000517 hemoglobin, alpha 2 gi|6229198 gi|14043068 216. AI278518
NM_033664 cadherin 11, type 2, OB- gi|3900786 gi|16306533 cadherin
(osteoblast) 217. AA678971 NM_003835 regulator of G-protein
gi|2659493 gi|4506520 signalling 9 218. AA972020 Homo sapiens
transcribed gi|3147310 sequences Clone ID: 1583668 219. AI306467
NM_004540 neural cell adhesion gi|3989538 gi|33519480 molecule 2
220. AA460685 NM_001168 baculoviral IAP repeat- gi|2185805
gi|59859877 containing 5 (survivin) 221. AA705316 Homo sapiens mRNA
similar gi|2715234 to joined to JAZF1 (cDNA clone MGC: 52103 IMAGE:
5736798), complete cds 222. N20796 NM_018841 guanine nucleotide
binding gi|1125977 gi|51036602 protein (G protein), gamma 12 223.
AI055991 NM_152545 GPI-gamma 4 gi|3329857 gi|22749128 224. N75004
AK124396 Homo sapiens cDNA FLJ42405 gi|1237582 gi|34530173 fis,
clone ASTRO3000474 225. N51740 NM_014936 ectonucleotide gi|1192906
GI: 54124344 pyrophosphatase/phosphodiesterase 4 (putative
function) 226. N51614 NM_005892 formin-like gi|1192780 gi|33356147
227. AA400234 AF001893 cDNA DKFZp686L01105 gi|2054248 gi|2529723
(from clone DKFZp686L01105) 228. AA400495 piwi-like 2 (Drosophila)
gi|2054366 229. H92875 NM_014944 calsyntenin 1 gi|1099203
gi|57242754 230. AA908902 NM_013282 ubiquitin-like, containing
gi|3048307 gi|16507203 PHD and RING finger domains, 1 231. AI685539
AB007954 Homo sapiens mRNA, chromosome gi|4896833 gi|3413928 1
specific transcript KIAA0485 232. T52564 NM_014585 solute carrier
family 40 (iron- gi|654424 gi|31543639 regulated transporter),
member 1 233. AA677149 NM_016184 C-type (calcium dependent,
gi|2657671 gi|37577113 carbohydrate-recognition domain) lectin,
superfamily member 6 234. AA910828 Homo sapiens transcribed
gi|3050118 sequences Clone ID: 1520938 235. AA620455 NM_033495 BTB
and kelch domain gi|2524394 gi|45643137 containing 2 236. T96951
Homo sapiens zinc finger protein gi|735575 (ZFD25), mRNA (cDNA cone
IMAGE: 6146402), partial cds 237. T70329 Homo sapiens transcribed
sequence gi|681477 with weak similarity to protein ref: NP_055474.1
(H. sapiens) KIAA0377 gene product [Homo sapiens] 238. AI268241
Homo sapiens transcribed gi|3887408 sequences Clone ID: 1880732
239. AI174481 NM_052918 VPS10 domain recpetor gi|3721334
gi|61743972 protein SORCS 1 240. AA490279 NM_201266 neuropilin 2
gi|2219452 gi|41872561 241. AA464729 NM_014501 ubiquitin carrier
protein gi|2189613 gi|7657045 242. AA158584 NM_173060 calpastatin
gi|1733395 gi|27765084 243. AI051108 AB037805 KIAA1384 protein
gi|3307913 gi|7243148 244. AI630806 AB014544 KIAA0644 gene product
gi|4682136 gi|3327101 245. AA664195 NM_002124 major
histocompatibility gi|2618186 gi|4504410 complex, class II, DR beta
3 246. H28091 NM_000304 peripheral myelin protein gi|898444
gi|24430161 22 247. AA464180 NM_032621 X-linked protein gi|2189064
gi|50658085 248. AI693344 SRY (sex determining gi|4970684 region
Y)-box 5 249. AA933862 CD3E antigen, epsilon gi|3090130 polypeptide
(TiT3 complex) 250. H90890 Homo sapiens transcribed sequence
gi|1081320 with weak similarity to protein ref: NP_060954.1 (H.
sapiens) hOAT4 [Homo sapiens]
Table 3 shows the top 250 ranked genes for predicting the outcome
of a patient having neuroblastoma. Table 3 provides exemplary
sequences for each of genes or polynucleotides by Unigene No.,
Accession No. and a corresponding SEQ ID NO:, other polynucleotide
sequences and/or amino acid sequences can be readily identified by
one of skill in the art. The sequence listing forms a part of this
disclosure and is hereby incorporated by reference. Table 3 also
shows expression level of the gene or polynucleotide in a poor
outcome patient with P+ meaning the gene is upregulated in poor
outcome patients and P- is downregulated in poor outcome patients.
An example of expression levels of each gene or polynucleotide is
shown in Tables 9A, B, and C.
One embodiment of the invention offers a selection or set of genes
that are expressed in a neuroblastoma cell. Such a selection or set
of genes function to predict the outcome of a patient with
neuroblastoma when the gene selection from the neuroblastoma cell
is compared to the expression of an identical selection of genes
from a non-neuroblastoma cell, or a neuroblastoma cell associated
with a good outcome and/or poor outcome. As used herein, the phrase
"function to predict the outcome of a patient" can mean to
identify, to be indicative of, to be highly and/or differentially
expressed in patients having different outcomes. As used herein,
the phrase "different outcomes" can refer to time remaining before
death, survival versus death, response to a particular course of
treatment, for example. In one embodiment, at least one of the
genes is chosen from table 2. In another embodiment, at least one
of the genes is chosen from table 2, or 3. In a further embodiment,
there are at least 9 genes chosen from table 2, preferentially
selected from the top ranked genes. In an even further embodiment,
there are at least 9 genes chosen from table 2 or 3, preferentially
selected from the top ranked genes.
The invention also includes a gene set or selection comprising at
least two genes or polynucleotides selected from the group
consisting of DLK1, PRSS3, ARC, SLIT3, JPH1, ARH1, CNR1, ROBO2,
BTBD3, KLRC3, Hs. 434957, Hs. 346735, Hs. 120591, Hs. 196008, Hs.
124776, Hs. 119947, Hs. 349094, and mixtures thereof, or the
complements thereof. In some embodiments, the gene set or selection
further comprises MYCN and/or CD44. Image ID NOs corresponding to
these genes or polynucleotides have been described in FIG. 7A and
representative sequences corresponding to SEQ ID NOs have been
provided in Tables 2 and 3 and the sequence listing that forms a
part of this disclosure.
In some embodiments, the gene selection comprises at least two of
the genes or polynucleotides, preferably at least three, at least
four, at least five, at least six, at least seven, at least eight,
at least nine, at least ten, at least eleven, at least twelve, at
least thirteen, at least fourteen, at least fifteen, at least
sixteen, at least seventeen, at least eighteen or at least 19 of
the genes of Table 2 or the complements of these polynucleotides or
genes.
In other embodiments, the gene set or selection comprises at least
two genes upregulated in a neuroblastoma in patients with poor
outcome. A gene set or selection comprises DLK1, PRSS3, SLIT3, or
mixtures thereof. A gene set or selection may further comprise ARC,
MYCN, JPH1, Hs. 434957, Hs. 346745, Hs. 120591, or mixtures thereof
or complements thereof. The gene set or selection may further
comprise one or more additional genes shown in Table 3 that are
upregulated in a neuroblastoma cell with poor outcome (identified
as P+) or the complements thereof.
In a further embodiment, the gene set or selection comprises at
least two genes downregulated in a neuroblastoma cell in a patient
with poor outcome. A gene set or selection comprises CD44, ARH1,
CNR1, ROBO2, BTBD3, KLRC3, Hs. 196008, Hs. 124776, Hs. 119947, Hs.
349094, or mixtures thereof or complements thereof. The gene set or
selection may further comprise at least one additional gene or
polynucleotide downregulated in a neuroblastoma cell from a patient
with poor outcome (identified as P-) as shown in Table 3 or
complements thereof.
In some embodiments, the gene selection may further comprise at
least one other gene or polynucleotide identified in Table 3. The
gene selection may successively include each of the next 10 top
ranked genes or polynucleotides as provided in Table 3 up to and
including all 250 genes or polynucleotides identified in Table 3 or
their complements. For example, the gene selection may further
comprise at least the top twenty to thirty ranked genes, the top
thirty to forty top ranked genes etc., and combinations thereof or
their complements.
The gene selection or set of genes and probes or primers that can
detect these genes or polynucleotides can be used to prepare a
microarray, hybridization assay, PCR assay that can be used to
analyze a neuroblastoma tumor cell or sample in order to provide a
prediction regarding the outcome of the patient having a
neuroblastoma tumor. In some embodiments, gene products, such as
polypeptides, can be detected using standard methodologies such as
ELISA, immunoPCR and the like. An amino acid sequence of the
polypeptides encoded by the polynucleotide are available by
accessing the Image ID NOs. or Accession Nos. using a publicly
available database such as the source database at Stanford.
Another embodiment of the invention includes a selection of least
one product of a selection of genes. As used herein, the term
"product of a gene" or "gene product" can include entities that are
naturally produced by the cancer cell. Examples of gene products
include, but are not limited to, DNA, mRNA, and proteins. Gene
products can be utilized in methods of the invention for predicting
the outcome of a patient with neuroblastoma or as a target for
therapeutic treatment of a patient with neuroblastoma.
Another aspect of the invention provides a kit for predicting the
outcome of a patient having a neuroblastoma. A kit for predicting
the outcome of a patient having neuroblastoma comprises an agent
for detecting expression of at least two genes or polynucleotides,
or the complements thereof, selected from the group consisting of
DLK1, PRSS3, SLIT3, and mixtures thereof, or the complements
thereof, and optionally, instructions for detecting increased
expression as compared to a control, wherein enhanced expression is
indicative of poor outcome. The control can be prepared from one or
more nonneuroblastoma cells including at least one housekeeping
gene, or it can be a neuroblastoma cell from a patient or patients
with good and/or poor outcomes. Examples of such information
concerning expression levels of genes or polynucleotides in
neuroblastoma cells form patients with good outcome and/or poor
outcome is provided herein in FIG. 7B and Tables 9A, B, and C.
A number of different known assays can be utilized to determine
expression levels of a gene from a cell or patient sample. These
assays include, for example, microarray assays, hybridization
assays, PCR assays, ELISA assays, immunoPCR assays. One embodiment,
may involve detecting increased levels of one or more polypeptides
in a biological sample, such as serum, from patients having
neuroblastoma. In some embodiments, the agent is at least one probe
or primer that can detect at least one of DLK1, PRSS3 and SLIT3. In
other embodiments, the agent is at least one antibody that can
detect at least one of DLK1, PRSS3, or SLIT3. Preferably, the
antibody is detectably labeled with a radioactive or fluorescent
moiety.
In other embodiments, a kit comprises an agent that can detect
expression of at least two genes or polynucleotides selected from
the group consisting of DLK1, PRSS3, ARC, SLIT3, JPH1, ARH1, CNR1,
ROBO2, BTBD3, KLRC3, Hs. 434957, Hs. 346735, Hs. 120591, Hs.
196008, Hs. 124776, Hs. 119947, Hs. 349094, and mixtures thereof,
or the complements thereof, and optionally, instructions providing
the expression profile of at least one polynucleotide that is
indicative of a poor and/or good outcome of the patient.
Preferably, the expression profile of all of the genes is provided.
An agent can comprise at least one probe or primer that can detect
at least one of the genes or polynucleotides. In other embodiments,
the agent is at least one antibody that can detect at least one
polypeptide encoded by the gene or polynucleotide.
In some embodiments, the kit comprises a plurality of agents that
can detect expression of all the genes or polynucleotides of Table
2, or the complements thereof. In some embodiments, the kit
comprises a plurality of agents that can detect expression of at
least one additional gene or polynucleotide or all of the genes or
polynucleotides of Table 3, or complements thereof. The plurality
of agents may comprise a primer or probe that can detect expression
of each of the polynucleotides or genes, or their complements, of
Table 2. Another embodiment includes a plurality of polynucleotides
comprising two or more genes or polynucleotides of Table 3, or
their complements, optionally attached to a solid substrate, and
preferably excluding MYCN or CD44. The agents may be attached to a
solid substrate such as a polystyrene plate or glass slide.
In some embodiments, the kits provide instructions that provide
that a poor outcome is characterized by upregulation of at least
one gene or all genes selected from the group consisting of DLK1,
PRSS3, ARC, SLIT3, JPH1, Hs. 434957, Hs. 3467345, Hs. 120591, and
mixtures thereof. The instructions may also provide that a poor
outcome is characterized by downregulation of at least one gene or
all genes selected from the group consisting of ARH1, CNR1, ROBO2,
BTBD3, KLRC3, Hs. 196008, Hs. 124776, Hs. 119947, Hs. 349094, and
mixtures thereof.
C. Methods of Targeting a Gene Product to Produce a Therapeutic
Agent Useful to Treat Neuroblastoma.
One embodiment of the invention includes a method of targeting a
product of at least one of the genes in table 2 or 3 that includes
identifying a therapeutic agent having a therapeutic effect on said
gene product. Another embodiment includes a method of therapeutic
treatment of neuroblastoma by using a selection of genes or their
products that are expressed in a neuroblastoma cell, wherein the
genes and/or their products function to predict the outcome of the
neuroblastoma cell when the gene selection from the neuroblastoma
cell is compared to the expression of an identical selection of
genes from a non-neuroblastoma cell, or a cancer cell from a
patient with a good outcome and/or poor outcome. Another embodiment
includes a method of targeting a product of at least one of the
genes in Tables 2 or 3 for identification of an antagonist or
agonist that can be utilized to treat neuroblastoma.
Another aspect of the invention involves a method of screening for
an agent that modulates a gene or polynucleotide for treatment for
neuroblastoma. A screening method comprises a method for detecting
an agent that can modulate the expression of at least one gene or
polynucleotide for which a change in expression is correlated with
poor outcome in a patient or subject having neuroblastoma. In some
embodiments, the genes or polynucleotides are selected from the top
ranked genes of Table 2. In other embodiments, at least one of the
top ranked genes of table 2 is screened excluding MYCN and/or CD44.
In other embodiments, at least one gene or polynucleotide can be
selected from any of the genes of Table 3.
In some embodiments, the gene or polynucleotide is upregulated in a
neuroblastoma tumor cell and is associated with poor outcome. If a
gene is upregulated, a method comprises identifying an antagonist
of the gene or polynucleotide. A gene or polynucleotide that is
upregulated comprises or is selected from the group consisting of
DLK1, PRSS3, SLIT3, and mixtures thereof. In other embodiments, a
gene or polynucleotide is selected from the group consisting of
DLK1, PRSS3, ARC, SLIT3, JPH1, and mixtures thereof. A method
comprises identifying an antagonist of at least one gene or
polynucleotide upregulated in neuroblastoma cell comprising
measuring expression or activity of at least one gene or
polynucleotide selected from the group consisting of DLK1, PRSS3,
ARC, SLIT3, JPH1, Hs. 434957, Hs. 346735, Hs. 120591, and mixtures
thereof in the presence or absence of a candidate agent; and
identifying the candidate agent that inhibits expression or
activity of at least one of the genes or polynucleotides. The
method can further comprise at least one or more genes or
polynucleotides that are upregulated in a neuroblastoma cell and
associated with poor outcome, such as provided in Table 3 and
identified as P+.
In some embodiments, at least one gene or polynucleotide is
downregulated and correlated with poor outcome of a patient having
neuroblastoma. When a gene or polynucleotide is downregulated, a
method comprises identifying an agonist of a gene or polynucleotide
downregulated in a neuroblastoma cell comprising measuring
expression or activity of at least one gene selected from the group
consisting of ARH1, CNR1, ROBO2, BTBD3, KLRC3, Hs. 196008, Hs.
124776, Hs. 119947, Hs. 349094, and mixtures thereof, in the
presence and absence of a candidate agent, identifying as an
agonist the candidate agonist that increases the expression or
activity of the gene or polynucleotide. The method can further
comprise screening for an agonist of one or more of the genes or
polynucleotides that are downregulated in a neuroblastoma cell and
associated with poor outcome, such as provided in Table 3 and
identified as P-.
Exemplary sequences for the genes, polynucleotides, or polypeptides
of Tables 2 and 3 can be found in Image ID NOs and Accession Nos.
as provided in FIG. 7A and Table 3, Seq ID NOs in Tables 2 and 3
and the sequence listing that forms a part of this disclosure.
Complementary sequences for the genes and polynucleotides can
readily be determined by one of skill in the art.
An antagonist or agonist useful as a therapeutic agent can comprise
a biological or chemical entity that is based on some aspect of a
gene. Examples of therapeutic agents include, but are not limited
to, vaccines, antibodies, oligonucleotide DNA antisense, RNAi,
chemical molecules, proteins, inhibitors, antagonists, or
combinations thereof. Having a therapeutic effect on a gene product
can include, but is not limited to, inhibition of some activity or
process of a cell, cessation of some activity or process of a cell,
an increase in some activity or process of a cell, interference
with some process or activity of a cell, modification of the
expression of at least one gene, modification of the expression of
at least one gene product, modification of the function of at least
one gene, and modification of the function of at least one gene
product.
An antagonist of any of the genes, polynucleotides or gene products
is effective to inhibit expression or activity of the gene or gene
product and can include antisense nucleic acid, nucleic acid or
protein vaccines, siRNA, aptamers, and antagonist antibodies
(including humanized antibodies). An agonist of any of the genes,
polynucleotides or gene products is effective to enhance or
increase expression or activity of the gene or gene products and
can include agonist antibodies (including humanized antibodies).
Other agonists include polynucleotides providing for expression,
preferably overexpression, of the downregulated gene or
polynucleotide. Antibodies can be prepared by methods known to
those of skill in the art and in references such as U.S. Pat. No.
6,331,415; Kohler et al., Eur. J. Immun., 6:511 (1976); Winter et
al., Ann. Rev. of Immunol., 12:433 (1994); and U.S. Pat. No.
5,225,539.
The antagonists and/or agonists identified herein may be utilized
in methods to treat neuroblastoma. For example, a therapeutic agent
such as a humanized antibody or antisense nucleic acid may be
administered to a patient in order to downregulate expression of
genes or polynucleotides that are upregulated in patients that are
predicted to have a poor outcome. Such therapeutic agents may be
utilitized in combination with other therapies, including
conventional chemotherapeutic agents.
WORKING EXAMPLES
The following examples provide a nonlimiting illustration of
various embodiments of the invention.
Example 1
Preparation of Microarrays
Preparation of Glass cDNA Microarrays, Probe Labeling,
Hybridization and Image acquisition can be performed according to
the protocol given below, which is a standard NHGRI protocol
(http://www.nhgri.nih.gov/DIR/LCG/15K/HTML/protocol.html).
Gene-specific DNA is produced by PCR amplification of purified
template plasmid DNAs from cloned ESTs. The PCR product is purified
by ethanol precipitation, thoroughly resuspended in 3.times.SSC,
and printed onto a poly-L-lysine coated slide.
The materials, reagents, and solutions used include: 96 well
alkaline lysis miniprep kit (Edge BioSystems, Gaithersburg, Md.); L
B Broth (Biofluids, Rockville, Md.); Superbroth (Biofluids,
Rockville, Md.); dATP, dCTP, dGTP, dTTP, 100 mM each #27-2035-02,
store frozen, -20.degree. C. (Pharmacia, Peapack, N.J.); PCR primer
AEK M13F (5'-GTTGTAAAACGACGGCCAGTG-3') (SEQ ID NO. 251) and AEK
M13R (5'-CACACAGGAAACAGCTATG-3') (SEQ ID NO. 252) at 1 mM
concentration, store frozen, -20.degree. C.; 10.times.PCR Buffer, #
N808-0189, and Ampli-Taq DNA polymerase, # N808-4015 store frozen,
-20.degree. C. (Perkin Elmer, Norwalk, Conn.); Carbenicillin
(Gibco-BRL, Rockville, Md.); Ethanol (200 Proof USP Ethyl Alcohol);
1M Tris-HCl (pH 8); 0.5M NaEDTA (pH 8); T Low E; Buffer;
20.times.SSC; Glycerol (enzyme grade); Sodium Acetate
(tri-hydrate); Boric Acid; Sodium Hydroxide (1M); Glacial Acetic
Acid; Succinic anhydride, #23969-0 and 1-methyl-2-pyrrolidinone,
#32863-4 (Aldrich Chemical Co., St. Louis, Mo.); Diethyl
Pyrocarbonate (DEPC) treated H.sub.2O; Master set of
clone-purified, sequence verified human ESTs (e.g. gf211 release,
Research Genetics, Huntsville, Ala.); 96 pin inoculating block (#VP
4088, V&P Scientific, Inc, San Diego, Calif.); Airpore Tape
Sheets, (#19571, QIAGEN Inc., Valencia, Calif.); Sterile 96-well
plate seals, (e.g. # SEAL-THN-STR (Elkay Products, Inc.,
Shrewsbury, Mass.); 96-well U-Bottom Microtiter Plates, #3799 and
96-well V-Bottom Microtiter Plates, #3894 (Corning Inc., Corning,
N.Y.); Thin wall PCR plate and Cylcleseal PCR plate sealer (e.g.
#1038-50-0 and #1044-39-4, Robbins Scientific Corp. Sunnyvale,
Calif.); household one-gallon sealable storage bags (e.g. Glad
Lock); heat sealable storage bags and heat sealer; 0.2 mm Sterile
Filtration unit; Diamond scribe for writing on slides; Pyrex baking
dish (.about.24.times.34.times.5 cm); UV transparent plastic wrap
(e.g. Glad Cling Wrap); 30 slide rack (stainless steel) #113 and 30
slide glass tank, #122 (Shandon Lipshaw, Pittsburgh, Pa.); 1 L
glass tank; 1 L glass beaker; 1 L graduated; cylinder; Stir bar;
Slide Box (plastic with no paper or cork liners), (e.g.
#60-6306-02, PGC Scientific, Gaithersburg, Md.); PCR heat cycler
(e.g. DNA Engine Tetrad, MJ Research, Waltham, Mass.); Centrifuge
with a horizontal ("swinging bucket") rotor with a depth capacity
of 6.2 cm for spinning microtiter plates and filtration plates
(e.g. Sorvall Super T 21, Sorvall Inc., Newtown, Conn.); 37.degree.
C. Shaker incubator with holders for deep-well plates; 37.degree.
C. Waterbath; 65.degree. C. Incubator; Vortex mixer; Immunowash
microtiter plate washer, #1575 (BioRad, Hercules, Calif.); pH
Meter; Platform Shaker; UV Stratalinker 2400, (Stratagene La Jolla,
Calif.); Stirrer/Hotplate; Robotic slide printer; -80.degree. C.
Freezer; -20.degree. C. Freezer; 45% (w/v) Sterile Glycerol; 450
grams enzyme grade glycerol per liter 9 Autoclave and store at room
temperature); T low E Buffer; 1M Tris-HCl (pH 8.0) 10 mL; 0.5 M
EDTA (pH 8.0) 0.2 mL; DEPC treated H.sub.2O 990 mL (Autoclave and
store at room temperature); Carbenicillin stock solution (1 gram of
carbenicillin in 10 mls of sterile water, Sterile filter with a 0.2
micron filter, Store frozen at -20.degree. C.); LB with 100
.mu.g/ml carbenicillin (Add 1 ml of carbenicillin stock solution to
1 liter of LB, Make fresh); 3M Sodium Acetate pH=6.0 (408.24 grams
sodium acetate (tri-hydrate) per liter, 3M acetic acid (172.4 ml
per liter), Titrate the pH of the 3M sodium acetate solution to pH
6.0 with the 3M acetic acid solution, Filter sterilize using a 0.2
micron filter, Store at room temperature); Ethanol/acetate mix
(Ethanol (100%) 950 ml, Sodium acetate pH=6.0, 50 ml); 1000 ml
3.times.SSC; DEPC H.sub.2O 42.5 ml; 20.times.SSC 7.5 ml; 50 ml 70%
Ethanol; Ethanol (100%) 350 ml; DEPC H.sub.2O 150 ml; 500 ml.
The first step is to grow the EST clones. An exemplary method is
described below. In one embodiment, the cDNA clones were obtained
from Research Genetics (Huntsville, Ala.) and were their standard
microarray set, which consisted of 3789 sequence-verified known
genes and 2778 sequence-verified ESTs. In other embodiments,
sequence verified libraries with 42,578 cDNA clones were used and
obtained from Research Genetics (Huntsville, N.C.) as described in
Example 3.
The sealed master plates are incubated over night at 37.degree. C.
Most suppliers provide low density bacterial cultures. Replicating
directly from these dilute stocks frequently results in non-growth
in the secondary culture. If making the template from a plate that
had previously been cultured to high density before freezing, this
initial growth step should not be used, as it will reduce the
viability of the cultures.
A set of standard 96 well round (U) bottom plates is then prepared
by labeling all plates and placing 100 .mu.l of LB broth containing
100 .mu.g/ml carbenicillin in each well. These plates are used as
working copies. To preserve the master set of plates, it is useful
to make replicate copies of the master plate to serve as working
copies when the master plate is first replicated. The EST clones
are then checked to insure that they were in a vector conferring
ampicillin resistance, as is common with human IMAGE clones.
The master plates are spun briefly (about two minutes) at 1000 rpm
in a horizontal microtiter plate rotor to remove condensation and
droplets from the seals before opening. Bacterial culture fluid on
the sealers can easily be transferred from one well to others,
cross-contaminating the stocks.
Then a container is partially filled with 100% alcohol. The 96
pin-replicating tool is dipped in the alcohol, removed and then the
pins were flamed.
The inoculation block is allowed to cool briefly, then the
replicating tool is dipped in the master plate and then into the
daughter plate. This is repeated as necessary for each plate
inoculated. It is useful to color the plate corner near the A-1
well of all master and daughter plates with a marker pen before
beginning the replication process in order to reduce mistakes in
the relative orientation of the plates. The suggested plates have a
notch at this corner as well.
The inoculated LB plates, with the lids on, are placed into a one
gallon sealable bag containing a moistened paper towel and grow
overnight at 37.degree. C. Many 37.degree. C. incubators tend to
dry out microtiter plate cultures. Placing the plates in a highly
humidified bag avoids this problem.
Next, deep well plates are filled with 1 ml of Superbroth (100
.mu.g/ml carbenicillin) per well. These plates serve as the source
of culture for template preparation. Using the replicating tool,
the deep well plates are then inoculated directly from the freshly
grown LB plates. Next, the openings of the deep well plates are
covered with Qiagen Airpore Tape Sheets and the plastic lids are
placed over the sheet. The plates are then placed in a 37.degree.
C. shaker incubator at 200 RPM for twenty-four hours. 50 .mu.l of
45% (w/v) sterile glycerol is added to each well of any working
plates that are to be frozen (-80.degree. C.) and subsequently used
as culture sources.
After the EST clones are grown, the plasmid templates have to be
isolated. First, the lysis buffer (Edge Biosystems Kit) is warmed
to 37.degree. C. to dissolve the SDS. Then the RNAse solution is
added to the resuspension buffer (Edge Biosystems Kit), 1 ml/100
ml, and stored at 4.degree. C. The receiving plates are prepared
from the Edge Biosystems Kit by adding 350 .mu.l of ethyl alcohol
to each well of the receiving plates. The filter plate is then
placed on top and secured with tape. The bacterial cultures in the
deep well plates are centrifuged at 1500.times.g for seven minutes
in a centrifuge equipped with a horizontal rotor for 96-well
plates. They were then briefly inverted and excess media is tapped
out on a clean paper towel. The pellets will loosen and may be lost
when pouring off excess media if this step is delayed.
The pellet is then resuspended in 100 .mu.l of Resuspension Buffer,
and Vortexed until the entire pellet was re-suspended. This step is
critical. Poor resuspension of the cells results in clumps of cells
that do not lyse in subsequent steps. This reduces the yield and
decreases the purity of the product. 100 .mu.l of Lysis Buffer is
then added and the solution is mixed gently by rocking the plates
from side to side, to avoid shearing the bacterial chromosomal DNA.
100 .mu.l of Precipitation buffer is added to each well and briefly
mixed. Then, 100 .mu.l of Neutralization buffer is added to each
well and vortexed.
The contents of the deep wells are then transferred to the waiting
filter plates/receiving plate stacks using the wide bore pipette
tips provided in the kits. The stacked plates are then centrifuged
at 1500.times.g for twelve minutes in a centrifuge equipped with a
horizontal rotor for 96-well plates. The stacked plates are then
removed from the centrifuge. The filter plates are removed and
discarded. The alcohol and filtrate are decanted from the receiver
plate and the excess alcohol is touched off on clean paper towels.
500 .mu.l of 70% ethanol is added to each well and immediately
decanted and excess alcohol is touched off with a clean paper
towel. Then, the plates are placed in a clean drawer without their
lids, covered with a clean paper towel and allowed to dry
overnight.
The next day, the DNA is resuspended in 200 .mu.l of T Low E
Buffer. The top is sealed with plate sealer and rehydrated at
4.degree. C. for at least two days before using. They are stored at
-20.degree. C. in the interim.
After the plasmid templates have been isolated, the EST inserts are
amplified. For each 96 well plate to be amplified, a PCR reaction
mixture is prepared containing the following ingredients: 1000
.mu.l of 10.times.PCR Buffer, 20 .mu.L of dATP (100 mM), 20 .mu.L
of dGTP (100 mM), 20 .mu.L of dCTP (100 mM), 20 .mu.L of dTTP (100
mM), 5 .mu.L of AEK M13F primer (1 mM), 5 .mu.L of AEK M13R primer
(1 mM), 100 .mu.L of Ampli-Taq polymerase (5 U/.mu.l), and 8800 mL
of H.sub.2O. The 96-well PCR plates are then labeled and 100 .mu.l
of the PCR reaction mixture from above is aliquotted to each well.
The plates are then gently tapped to insure that no air bubbles are
trapped at the bottom of the wells. 1 .mu.l of purified EST plasmid
template from above is then added to each well. The donor and
recipient plates are then marked at the corner, near the A1 well to
facilitate correct orientation during transfer of the template. It
is important to make sure that the pipette tips are all submerged
in the PCR reaction mix when delivering the template. Missing the
liquid is easier when multi-channel pipettes are used.
The following thermal cycle series is then performed: 1 initial
cycle of heating to 96.degree. C. and holding for 30 sec, 25 cycles
of denaturing at 94.degree. C. for 30 sec, reannealing at
55.degree. C. for 30 sec, and extending at 72.degree. C. for 150
sec, one final cycle of holding at 72.degree. C. for 5 minutes,
then cooling to ambient temperature. After the above cycle, the
plates are held at 4.degree. C. while quality controls are
performed.
The quality control is done by agarose gel electrophoresis of the
ESTs. If this is the first time the template for these ESTs is
being amplified, 2 .mu.l of each PCR product is analyzed on a 2%
agarose gel. If amplified products from this template had been
previously tested, then one row of wells from each plate amplified
is analyzed. Gel imaging allowed a rough quantitation of product
while giving an excellent characterization of the product. Band
size, as well as the number of bands observed in the PCR products,
contributed to an understanding of the final results of the
hybridization. The use of gel well formats suitable for loading
from 96 well plates and programmable pipetters made this form of
analysis feasible on a large scale.
The materials, reagents and solutions for the quality control check
included: Electrophoresis apparatus with capacity for four 50 well
combs, (e.g. #D3, Owl Scientific, Woburn, Mass.); 50.times.
Tris-Acetate Electrophoresis BufferM; Agarose; Dye Solution (Xylene
Cyanol/Bromophenol Blue) (e.g. #351-081-030, Quality Biological
Inc., Gaithersburg Md.); Glycerol (enzyme grade); Ethidium Bromide
solution (10 mg/ml); 100 base-pair ladder size standard;
Programmable, 12-channel pipetter (e.g. #2019, Matrix Technologies,
Lowell, Mass.); Disposable microtiter mixing trays (e.g. Falcon
#353911, Becton Dickinson, Franklin Lake, N.J.); Electrophoresis
power supply; 1.times.TAE Buffer; 50.times.TAE Buffer 40 ml;
Ethidium Bromide (10 mg/ml) 0.1 ml and Water 960 ml; 1000 ml;
Loading Buffer; Glycerol (enzyme grade) 4.0 ml, DEPC Water 0.9 ml,
and Dye Solution* 0.1 ml for a total of 5.0 ml (*THis solution is
0.25% (w/v) Xylene Cyanol and 0.25% (w/v) Bromophenol Blue); 100 bp
Size Standards; DNA ladder (1 mg/ml) 50 .mu.L, 1 M Tris-HCl (pH
8.0) 5 .mu.l, 0.5 M EDTA (pH 8.0) 5 .mu.l, and Loading Buffer 440
.mu.l for a total of 500 .mu.l
The electrophoresis is carried out with a 2% agarose gel
(1.times.TAE) with four combs (50 tooth) that is submerged in an
electrophoresis apparatus with sufficient 1.times.TAE buffer to
just cover the surface of the gel. A reservoir of Loading Buffer is
prepared, using 12 wells of a microtiter plate. Then a pipetter is
programmed to sequentially carry out the following steps: fill with
2 .mu.l, fill with 1 .mu.L, fill with 2 .mu.l, mix a volume of 5
.mu.l five times, expel 5 .mu.l. Twelve (12) disposable tips are
then placed on the pipetter. 2 .mu.l of PCR product from wells
A1-A12 of the PCR plate were loaded, followed by 1 .mu.l of air,
then 2 .mu.l of Loading Buffer from the reservoir. The tips are
then placed in clean wells of a disposable mixing tray and the
pipette is allowed to mix the sample and loading dye. The pipette
tip is then placed in a 50 well row so that the tip containing the
PCR product from well A1 is in the second well of the row, and the
other tips are in every other succeeding well.
The process is repeated (changing tips each time), to load PCR
plate row B starting in the 3rd well, interleaved with the A row,
the C row starting at well 26, and the D row at well 27,
interleaved with the C row. Then 5 .mu.l of 100 bp Size Standards
are placed in wells 1 and 50. This process is repeated, to load
samples from rows E, F, G, and H in the second, 50 well row of gel
wells, to load samples from two 96 well PCR plates per gel, or
single row samples from 16 PCR plates. To reduce diffusion and
mixing, a voltage is applied to the gel for a minute between
loading each well strip. This caused the DNA to enter the gel, and
reduced band spreading and sample loss.
A voltage is then applied to the gel and it is run until the
bromophenol blue (faster band) has nearly migrated to the next set
of wells. For a gel that is 14 cm in the running dimension, and 3
cm between each row of wells, 200 volts were applied for 15
minutes. Digital photos of the gel are taken and the images stored
for future reference. The gels should show bands of fairly uniform
brightness distributed in size between 600 to 2000 base-pairs.
Further computer analysis of such images can be carried out with
image analysis packages to provide a list of the number and size of
bands. Ideally this information can be made available during
analysis of the data from hybridizations involving these PCR
products.
After the quality control checks are run on the plates, the next
step involves purifying the PCR products. 96 well V-bottom plates
were filled with 200 .mu.l per well of ethanol/acetate mix. The
ethanol acetate solution used for precipitation is less acidic (pH
6) than is typically used. In this instance, more acidic solutions
produce precipitates which are harder to resuspend without
improving yield.
100 .mu.l per well of PCR product is transferred into V-bottom
plates and mixed by pipetting a volume of 75 .mu.l per well four
times. The plates are then placed in a -80.degree. C. freezer for
one hour or stored overnight at -20.degree. C. The plates are
stored at -20.degree. C. if they were to be left for more than one
hour, because aggressive precipitation produces precipitates which
are hard to resuspend. The plates are then thawed to reduce
brittleness and melt any ice, which may have formed in the
wells.
The plates are loaded into a centrifuge with a horizontal
microtiter plate rotor and spun at 2600.times.g for 40 minutes at
4.degree. C. Next, the supernatant from each well is aspirated
using the Immunowash plate washer. Settings for the depth of
aspiration by the plate washer needed to be adjusted to suit the
microtiter plates used. It is advisable to leave approximately
10-20 ml in the bottom of the well to avoid disturbing the
pellet.
200 .mu.l of 70% ethanol is delivered to each well in the plate
using the Immunowash plate washer, and the plates are centrifuged
at 2600.times.g for 40 minutes. The supernatant is aspirated from
each well using the Immunowash plate washer, and the plates are
dried overnight in a closed drawer. They should not be dried in a
speed-vac because desiccated PCR products are hard to
resuspend.
After the PCR products are purified, they are then resuspended by
adding 40 .mu.l of 3.times.SSC per well. The plates are then sealed
with a foil sealer, taking care to achieve a tight seal over each
well. The plates are then placed in heat sealable bags with paper
towels moistened with 3.times.SSC and the bag is sealed with a heat
sealer. The high external humidity within the sealed bag helped to
keep the volumes in the individual wells from varying. The bags are
then placed in a 65.degree. C. incubator for 2 hours. The heat in
the incubator is then turned off, and the plates are allowed to
cool gradually in the incubator to avoid condensation on the
sealers. The plates are stored at -20.degree. C.
The yield of the PCR suspension is then checked by fluorometric
determination of DNA concentration. 1 .mu.l of resuspended PCR
product from one row of wells from each plate on a 2% agarose gel
was analyzed as previously described. Adequate precipitation and
resuspension produce very intense bands, with no material failing
to leave the loading well, and no smear of material from the band
towards the loading well.
While it would be ideal to be able to exactingly quantify each EST
PCR product and spot each DNA species at equivalent concentrations,
it is impractical for most labs to do so when thousands of ESTs
must be prepared. Fortunately, it is possible to use a strategy
where excess DNA is spotted, so that the exact quantities used do
not produce much variation in the observed results. When using this
strategy, it is necessary to track the average productivity of the
PCR reactions. Fluorometry provides a simple way to obtain an
approximate concentration of the double-stranded PCR product in the
PCR reaction mix.
Next, the double stranded DNA is quantified. The materials,
reagents, and solutions necessary include: reference
double-stranded DNA (0.5 mg/ml) (e.g. #15612-013 Gibco/BRL,
Bethesda, Md.), 96 well plates for fluorescent detection (e.g.
#7105, Dynex, Chantilly, Va.), Fluorometer (e.g. #LS50B, Perkin
Elmer, Norwalk, Conn.), FluoReporter Blue dsDNA Quantitation Kit
(#F-2962, Molecular Probes, Eugene, Oreg.), TE, 12 channel
multi-pipetters, Computer equipped with Microsoft Excel software,
Ds-DNA Standards: 50 .mu.g/ml, 100 .mu.g/ml, 250 .mu.g/ml, 500
.mu.g/ml, .mu.l TE 90, 80, 50, 0 .mu.l ds-DNA (0.5 mg/ml) 10, 20,
50, 100, (It is good practice to check both the integrity (agarose
gel) and the concentration (absorbance) of the standard before
use); Fluor Buffer (HoecHist 33258 solution (contains the dye at an
unspecified concentration in a 1:4 mixture of DMSO:H.sub.2O) (from
kit) 25 .mu.l, TNE Buffer (TNE Buffer is 10 mM Tris-HCl (pH 7.4), 2
M NaCl, 1 mM EDTA) (from kit) 10 ml.
The double stranded DNA is quantified as follows. 96 well plates
are labeled for fluorescence assay. 200 .mu.l of Fluor Buffer is
added to each well. 1 .mu.l of PCR product from each well in a row
of a PCR plate is added to a row of the fluorometry plate. Samples
are added to rows A through G of the fluorometry plate. In the
final row of the fluorometry plate 1 .mu.l of each of the series of
ds-DNA standards 0 .mu.g/ml (TE only), 50, 100, 250 and 500
.mu.g/ml ds-DNA are added. This series is repeated twice in the
final row.
The fluorometer is set for excitation at 346 nm and emission at 460
nm, and adjusted as necessary to read the plate. If the fluorometer
used does not support automated analysis, the data table is
exported to Excel. The response for the standards is tested to see
that it was linear and reproducible from the range of 0 to 500
.mu.g/ml of ds-DNA.
Next, the concentration of ds-DNA in the PCR reactions is
calculated using the following equation, after subtracting the
average 0 .mu.g/ml value from all other sample and control values:
[ds-DNA(.mu.g/ml)]=((PCR sample value)/(average 100 .mu.g/ml
value))*100 Constantly tracking the yields of the PCRs makes it
possible to rapidly detect many ways in which PCR can fail or
perform poorly. This assay can also be applied after precipitation
and resuspension of the PCR products to monitor overall recovery of
product. 1 .mu.l of amplified products from one row of wells from
each amplified plate by fluorometry is analyzed.
Slides are then coated with poly-L-lysine to have a surface that is
both hydrophobic and positively charged. The hydrophobic character
of the surface minimizes spreading of the printed spots, and the
charge appears to help position the DNA on the surface in a way
that makes cross-linking more efficient.
Materials, reagents, and solutions for coating the slides includes:
Gold Seal Microscope Slides (#3011, Becton Dickinson, Franklin
Lake, N.J.), Ethanol (100%), Poly-L-lysine (#P8920, Sigma, St.
Louis, Mo.), 50 Slide Stainless Steel Rack, #900401, and 50 Slide
Glass Tank, #900401, (Wheaton Science Products, Millville, N.J.),
Sodium Hydroxide, Stir Plate, Stir Bar, Platform Shaker, 30 Slide
Rack, #196, plastic, and 30 slide Box, #195, plastic, (Shandon
Lipshaw, Pittsburgh, Pa.), Sodium Chloride, Potassium Chloride,
Sodium Phosphate Dibasic Heptahydrate, Potassium Phosphate
Monobasic, Autoclave, 0.2 mm Filter: Nalgene, Centrifuge: Sorvall
Super 20, Slide Box (plastic with no paper or cork liners), (e.g.
#60-6306-02, PGC Scientific, Gaithersburg, Md.), 1 L Glass Beaker;
1 L Graduated Cylinder, 1M Sodium Borate (pH 8.0) (Dissolve 61.83 g
of Boric acid in 900 ml of DEPC H.sub.2O. Adjust the pH to 8.0 with
1N NaOH. Bring volume up to one liter. Sterilize with a 0.2 micron
filter and store at room temperature), Cleaning Solution (H.sub.2O
400 ml, Ethanol 600 ml, NaOH 100 g--Dissolve NaOH in H.sub.2O. Add
ethanol and stir until the solution clears. If the solution does
not clear, add H.sub.2O until it does), and Poly-L-lysine Solution
(poly-L-lysine (0.1% w/v) 35 ml PBS 35 ml H.sub.2O 280 ml 350
ml)
First, the slides are placed into 50 slide racks and the racks are
placed in glass tanks with 500 ml of cleaning solution. Gold Seal
Slides are highly recommended, as they have been found to have
consistently low levels of autofluorescence. It is important to
wear powder free gloves when handling the slides to avoid
contamination.
The tanks are placed on platform shakers for two hours at 60 rpm.
After being shook, the cleaning solution is poured out, and the
slides are then washed in H.sub.2O for three minutes. This wash is
repeated four times. The slides are then transferred to 30 slide
plastic racks and placed into small plastic boxes for coating. The
slides are then submerged in 200 ml poly-L-lysine solution per box.
The slide boxes are then placed on platform shaker for one hour at
60 rpm. The slides are rinsed three times with H.sub.2O, and
submerged in H.sub.2O for one minute, and then centrifuged for two
minutes at 400.times.g and the slide boxes used for coating are
dried.
The slides are then placed back into the slide box used for coating
and allowed to stand overnight before transferring to a new slide
box for storage. This allowed the coating to dry before it was
handled. The slides are allowed to age for two weeks on the bench,
in a new slide box, before they are printed on. The coating dried
slowly, becoming more hydrophobic with time.
Slide boxes used for long term storage should be plastic and free
of cork lining. The glue used to affix the cork will leach out over
time and give slides stored in these types of boxes a greasy film
that has a high degree of autofluorescence. All glassware and racks
used for slide cleaning and coating should be cleaned with highly
purified H.sub.2O only, and detergent should not be used.
Once the slides are coated, they were printed. The variety of
printers and pens for transferring PCR products from titer plates
to slides precludes highly detailed descriptions of the process.
The following steps provide a general description of the
processing.
The print pens are pre-cleaned according to the manufacturer's
specification. The printer slide deck is then loaded with
poly-L-lysine coated slides from above. The plates containing the
purified EST PCR products are thawed and centrifuged briefly,
(about two minutes) at 1000 rpm in a horizontal microtiter plate
rotor to remove condensation and droplets from the seals before
being opened. 5 to 10 .mu.l of the purified EST PCR products are
transferred to a plate that served as the source of solution for
the printer. Printing with quill-type pens usually requires that
the volume of fluid in the print source was sufficiently low, so
that when the pen was lowered to the bottom of the well, it was
submerged in the solution to a depth of less than a millimeter.
This keeps the pen from carrying a large amount of fluid on the
outside of the pen shaft and producing variable, large spots on the
first few slides printed.
A repetitive test print is run on the first slide. In this
operation, the pens are loaded with the DNA solution, and then the
pens serially deposited this solution on the first slide in the
spotting pattern specified for the print. A test is run to check
the size and shape of the specified spotting pattern, as well as
its placement on the slide. It also serves to verify that the pens
are loading and spotting, and that a single loading produced as
many spots as were required to deliver material to every slide in
the printer. If one or more of the pens is not performing at the
desired level, it is re-cleaned or substituted with another pen and
tested again. If all pens are performing, the full print is carried
out.
At the end of the print, the slides are removed from the printer,
labeled with the print identifier and the slide number by writing
on the edge of the slide with a diamond scribe and placed in a dust
free slide box to age for one week. It was useful to etch a line,
which outlined the printed area of the slide, onto the first slide.
This served as a guide to locate the area after the slides are
processed, and the salt spots are then washed off.
The slides are placed, printed side face up, in a casserole dish
and covered with cling wrap. The slides were then exposed to a 450
mJ dose of ultraviolet irradiation in the Stratalinker. Slides
should have been and are aged at ambient temperature in a closed
slide box for one week prior to blocking. The slides are then
transferred to a 30 slide stainless steel rack and the rack is
placed into a small glass tank. 6.0 g succinic anhydride is
dissolved in 325 ml 1-methyl-2-pyrrolidinone in a glass beaker by
stirring with a stir bar. Nitrile gloves are worn and the work is
carried out in a chemical fume hood while handling
1-methyl-2-pyrrolidinone (a teratogen).
25 ml 1M sodium borate buffer (pH 8.0) is added to the beaker. The
solution is allowed to mix for a few seconds, then rapidly poured
into a glass tank with slides. Succinic anhydride hydrolyzed quite
rapidly once the aqueous buffer solution is added. To obtain
quantitative passivation of the poly-L-lysine coating, the reactive
solution is brought in contact with the slides as quickly as
possible. The glass tank is placed on a platform shaker in a fume
hood for 20 minutes. Small particulates resulting from
precipitation of reaction products may be visible in the fluid.
While the slides are incubating on the shaker a boiling H.sub.2O
bath is prepared to denature the DNA on the slides. After the
slides are incubated for 20 minutes, they are transferred into the
boiling H.sub.2O bath. The heating element is immediately turned
off after the slides are submerged in the bath. The slides are
allowed to stand in the H.sub.2O bath for 2 minutes. The slides are
then transferred into a glass tank filled with 100% ethanol and
incubated for 4 minutes. The slides are removed and centrifuged at
400 rpm for 3 minutes in a horizontal microtiter plate rotor to dry
the slides. The slides are then transferred to a clean, dust free
slide box and allowed to stand overnight before being used for
collection of gene expression data.
Example 2
Exemplary Method of Culturing Cells and Tumor Samples
This protocol details exemplary methods used to extract RNA from
cells, purify the RNA by a combination of phase extraction and
chromatography, and prepare a labeled cDNA copy of the message
fraction of the purified RNA. The protocol also describes the
process of making fluorescent cDNA representations of the message
pools within the isolated total RNA pools. This is accomplished by
using the pure total RNA as a substrate for reverse transcription
in the presence of nucleotides derivatized with either a Cy3 or a
Cy5 fluorescent tag.
The materials, reagents, and solutions needed include: Trizol
Reagent (#15596-018, Life Technologies, Rockville, Md.); RNeasy
Maxi Kit (#75162, Qiagen, Valencia, Calif.); Chloroform; Ethanol
(200 Proof USP Ethyl Alcohol); DPBS (Dulbecco's phosphate buffered
saline); 3M sodium acetate (pH 5.2); DATP, dCTP, dGTP, dTTP, 100 mM
each, store frozen, -20.degree. C. (#27-2035-02, Pharmacia,
Peapack, N.J.); pd(T)12-18 resuspend at 1 mg/ml, and store frozen
-20.degree. C. (#27-7858, Amersham Pharmacia Biotech); Anchored
oligo primer (anchored; 5'-TTT TTT TTT TTT TTT TTT TTV N-3') (SEQ
ID NO.253); resuspend at 2 mg/ml, store frozen -20.degree. C. (e.g.
#3597-006, Genosys); CyTM3-dUTP, 1 mM, and CyTM5-dUTP, 1 mM, store
-20.degree. C., light sensitive; RNasina Rnase inhibitor, store
-20.degree. C. (#N211A, Promega); SUPERSCRIPT.TM. II Rnase H'
Reverse Transcriptase Kit, store -20.degree. C., (#18064-014, Life
Technologies, Rockville, Md.); C0t-1 DNA, 1 mg/ml, store frozen
-20.degree. C. (#15279-011, Life Technologies, Rockville, Md.);
0.5M EDTA (pH 8.0); 1 N NaOH; 1M TRIS-HCL; (pH7.5); TE pH 7.4; DEPC
water 50.times. Tris Acetate Buffer; 15 ml round bottom;
polypropylene centrifuge tubes; 50 ml conical polypropylene
centrifuge tubes; 1.5 ml; Eppendorf tubes; 0.2 ml thin wall PCR
tube; MicroCon 100 (Amicon Cat No. 42412); High speed centrifuge
for 15 ml tubes; Clinical centrifuge with horizontal rotor for 50
ml conical tubes; Tissue homogenizer (e.g. Polytron PT1200 with
Polytron-Aggregate-Dispergier-und-Mischtechnik 147a Ch6014
#027-30-520-0, Brinkmann Instruments Inc., Westbury, N.Y.); RPE
Buffer (Add 4 volumes of ethanol per volume of RPE concentrate
supplied in Quiagen Kit0; RW1 Buffer (Supplied in Qiagen Kit) 75%
EtOH (Ethanol (100%) 375 ml, and DEPC H2O 125 ml for a total of 500
ml); 10.times. low T dNTP Mix (25 .mu.L dGTP (100 mM), 25 .mu.L
dATP (100 mM), 25 .mu.L dCTP (100 mM), 10 .mu.L dTTP (100 mM), and
415 .mu.L DEPC H.sub.2O for a total of 500 .mu.L); 5.times. First
Strand Buffer (Provided with Superscript II); TAE Buffer (50.times.
Tris Acetate Electrophoresis Buffer 20 ml, and DEPC H2O 980 mL for
a total of 1000 ml)
If the cells that are used were harvested from tissue culture, the
cell pellet is washed twice in DPBS. If the cells that are used
were from tissue culture, 1 ml of Trizol was added per
2.times.10.sup.7 cells and mixed by shaking. If tissue is being
used, 100 mg of frozen tissue is added directly to 4 ml of Trizol,
and dissociated by homogenization with a rotating blade tissue
homogenizer.
Whatever the source, 2/10 volume of chloroform is added to the
cells and shook for 15 seconds, and then allowed to stand for 3
minutes, followed by centrifugation at 12,000.times.g for 15
minutes at 4.degree. C. The supernatant is taken off and added to a
polypropylene tube, while recording the volume of the
supernatant.
Then 0.53 volumes of ethanol is slowly added to the supernatant
while vortexing, this produces a final ethanol concentration of
35%. The ethanol is added drop by drop and allowed to mix
completely with the supernatant before more ethanol is added. If a
high local concentration of ethanol is produced, the RNA in that
vicinity will precipitate.
The supernatant from an extraction of 2.times.10.sup.7 to
1.times.10.sup.8 cells is added to an RNeasy maxi column, which is
seated in a 50 ml centrifuge tube. The tube is then centrifuged at
2880.times.g in a clinical centrifuge with a horizontal rotor at
room temperature for 5 minutes. The flow-through is then poured
back onto the top of the column and centrifuged again. This step is
necessary because a significant amount of RNA is not captured by
the column matrix in the first pass of the RNA containing solution
through the column.
The flow-through is discarded and 15 ml of RW1 buffer is added to
the column, followed by centrifugation at 2880.times.g for 5
minutes. The flow-through is discarded again and then 10 ml of RPE
buffer is added, followed again by centrifugation at 2880.times.g
for 5 minutes. Once again, the flow through is discarded and
another 10 ml of RPE buffer is added, and the column was
centrifuged at 2880.times.g for 10 minutes.
Next, the column is placed in a fresh 50 ml tube and add 1 ml of
DEPC treated water from the kit is added to the column, and the
column is allowed to stand for 1 minute. The column is then
centrifuged at 2880.times.g for 5 minutes, and another 1 ml of
water is added to the column. The column is allowed to stand for 1
minute, followed by centrifugation at 2880.times.g for 10
minutes.
Then, 400 .mu.l portions of the column eluate is aliquotted to 1.5
ml Eppendorf tubes, to which 1/10 volume of 3M sodium acetate (pH
5.2) is added, along with 1 ml of ethanol. The tubes are then
allowed to stand for 15 minutes, after which they are centrifuged
at 12000.times.g at 4 C for 15 minutes. The pellet is then washed
two times in 75% EtOH and stored at -80.degree. C.
The RNA is resuspended at approximately 1 mg/ml in DEPC H.sub.2O.
It is then concentrated to greater than 7 mg/ml by centrifugation
on a MicroCon 100 filter unit, centrifuged at 500.times.g, checking
as necessary to determine the rate of concentration. This step
removes many residual, small to medium sized, molecules that
inhibit the reverse transcription reaction in the presence of
fluorescently derivatized nucleotides. The concentration of RNA in
the concentrated sample is then determined by spectrophotometry,
and the sample was stored at -80.degree. C.
If an anchored oligo dT primer is used, the primer is annealed to
the RNA in the following 17 .mu.l reaction (a 0.2 ml thin wall PCR
tube is used so that incubations could be carried out in a PCR
cycler):
TABLE-US-00004 addition for addition for Component Cy5 labeling Cy3
labeling Total RNA (>7 mg/ml) 150-200 .mu.g 50-80 .mu.g Anchored
primer (2 .mu.g/.mu.l) 1 .mu.l 1 .mu.l DEPC H2O to 17 .mu.l to 17
.mu.l
If an oligo dT(12-18) primer was used, the primer was annealed to
the RNA in the following 17 .mu.l reaction:
TABLE-US-00005 addition for addition for Component Cy5 labeling Cy3
labeling Total RNA (>7 mg/ml) 150-200 .mu.g 50-80 .mu.g dT
(12-18) primer (1 .mu.g/.mu.l) 1 .mu.l 1 .mu.l DEPC H2O to 17 .mu.l
to 17 .mu.l
The incorporation rate for Cy5-dUTP is less than that of Cy3-dUTP,
so more RNA is labeled to achieve more equivalent signal from each
species.
It is then heated to 65.degree. C. for 10 minutes and cooled on ice
for 2 minutes. Then, 23 .mu.l (8 .mu.l of 5.times. first strand
buffer, 4 .mu.l of 10.times. low T dNTPs mix, 4 .mu.l of Cy5 or Cy3
dUTP (1 mM), 4 .mu.l of 0.1 M DTT, 1 .mu.l of Rnasin (30 u/.mu.l),
and 2 .mu.l of Superscript II (200 u/.mu.l)) of reaction mixture
containing either Cy5-dUTP or Cy3-dUTP nucleotides is added, mixed
well by pipetting and a brief centrifuge spin is used to
concentrate it in the bottom of the tube. Superscript polymerase is
very sensitive to denaturation at air/liquid interfaces, so be
careful to suppress foaming in all handling of this reaction.
It is then incubated at 42.degree. C. for 30 min., after which 2
.mu.l Superscript II is added, making sure the enzyme is well mixed
in the reaction volume and incubated at 42.degree. C. for 30-60
min. Then, 5 .mu.l of 0.5M EDTA is added, making sure the reaction
is stopped with EDTA before adding NaOH (the next step), since
nucleic acids precipitate in alkaline magnesium solutions.
Then, 10 .mu.l 1N NaOH is added and it is incubated at 65.degree.
C. for 60 minutes to hydrolyze residual RNA, after which it was
cooled to room temperature. The purity of the sodium hydroxide
solution used in this step is important. Slight contamination or
long storage in a glass vessel can produce a solution that will
degrade the Cy5 dye molecule, turning the solution yellow. Some
researchers achieve better results by reducing the time of
hydrolysis to 30 minutes.
It is then neutralized by adding 25 .mu.l of 1M Tris-HCl (pH 7.5).
Then, the labeled cDNA is desalted by adding the neutralized
reaction, 400 .mu.l of TE pH 7.5 and 20 .mu.g of human C0t-1 DNA to
a MicroCon 100 cartridge. It is then pipetted to mix, and spun for
10 minutes at 500.times.g. 200 .mu.l TE pH 7.5 is added, and the
solution is then concentrated to about 20-30 .mu.l (approximately
8-10 min at 500.times.g). Alternatively, a smaller pore MicroCon 30
is used to speed the concentration step. In this case, the first
wash is centrifuged for approximately 4.5 minutes at 16,000.times.g
and the second (200 .mu.l wash) for about 2.5 minutes at
16,000.times.g.
It is then recovered by inverting the concentrator over a clean
collection tube and spinning for 3 min at 500.times.g. In some
cases, the cy5 labeled cDNA forms a gelatinous blue precipitate
that is recovered in the concentrated volume. The presence of this
material signals the presence of contaminants. The more extreme the
contamination, the greater the fraction of cDNA which will be
captured in this gel. Even if heat solubilized, this material tends
to produce uniform, non-specific binding to the DNA targets. When
concentrating by centrifugal filtration, the times required to
achieve the desired final volume are variable. Overly long spins
can remove nearly all the water from the solution being filtered.
When fluor-tagged nucleic acids are concentrated onto the filter in
this fashion, they are very hard to remove, so it is necessary to
approach the desired volume by conservative approximations of the
required spin times. If control of volumes proves difficult, the
final concentration can be achieved by evaporating liquid in the
speed-vac. Vacuum evaporation, if not to dryness, does not degrade
the performance of the labeled cDNA.
Next, a 2-3 .mu.l aliquot of the Cy5 labeled cDNA is taken for
analysis, leaving 18-28 .mu.l for hybridization. This probe is run
on a 2% agarose gel (6 cm wide.times.8.5 cm long, 2 mm wide teeth)
in Tris Acetate Electrophoresis Buffer (TAE). For maximal
sensitivity when running samples on a gel for fluor analysis, a
loading buffer with minimal dye was used and no ethidium bromide is
added to the gel or running buffer.
The gel is then scanned on a Molecular Dynamics Storm fluorescence
scanner (setting: red fluorescence, 200 micron resolution, 1000
volts on PMT). Successful labeling produces a dense smear of probe
from 400 bp to >1000 bp, with little pile-up of low molecular
weight transcripts. Weak labeling and significant levels of low
molecular weight material indicates a poor labeling. A fraction of
the observed low molecular weight material is unincorporated fluor
nucleotide.
Next, the fluorescent cDNA had to be hybridized to the microarray.
The volume of hybridization solution required is first determined.
The rule of thumb is to use 0.033 .mu.l for each mm 2 of slide
surface area covered by the cover slip used to cover the array. An
array covered by a 24 mm by 50 mm cover slip required 40 .mu.l of
hybridization solution. The volume of the hybridization solution is
critical. When too little solution is used, it is difficult to seat
the cover slip without introducing air bubbles over some portion of
the arrayed ESTs, and the cover slip will not sit at a uniform
distance from the slide. If the cover slip is bowed toward the
slide in the center, there will be less labeled cDNA in that area
and hybridization will be non-uniform. When too much volume is
applied, the cover slip will move easily during handling, leading
to misplacement relative to the arrayed ESTs, and non-hybridization
in some areas of the array.
For a 40 .mu.l hybridization, the Cy3 and Cy5 labeled cDNAs are
pooled into a single 0.2 ml thin wall PCR tube and the volume is
adjusted to 30 .mu.l by either adding DEPC H.sub.2O, or removing
water in a SpeedVac. If a vacuum device is used to remove water,
high heat or heat lamps are not used to accelerate evaporation
because the fluorescent dyes could be degraded.
For a 40 .mu.l hybridization the following components are
combined:
TABLE-US-00006 High Sample Blocking High Array Blocking Cy5 + Cy3
probe 30 .mu.l 28 .mu.l Poly d(A) (8 mg/ml) 1 .mu.l 2 .mu.l Yeast
tRNA (4 mg/ml) 1 .mu.l 2 .mu.l Human C0t-1 DNA 1 .mu.l 0 .mu.l (10
mg/ml) 20x SSC 6 .mu.l 6 .mu.l 50x Denhardt's blocking 1 .mu.l
(optional) 2 .mu.l solution Total volume 40 ul 40 ul
Arrays and samples can vary somewhat, making it necessary to vary
the composition of the hybridization cocktail. In cases where there
is residual hybridization to control repeat DNA samples on the
array, more C0t-1 DNA was used, as in the High Sample Blocking
formulation. When there is diffuse background or a general haze on
all of the array elements, more of the non-specific blocker
components is used, as in the High Array Blocking formulation.
The components are mixed well by pipetting, heated at 98.degree. C.
for 2 minutes in a PCR cycler, cooled quickly to 25.degree. C. and
0.6 ul of 10% SDS is added. It was then centrifuged for 5 min at
14,000.times.g. The fluor labeled cDNAs have a tendency to form
small, very fluorescent, aggregates which result in bright,
punctate background on the array slide. Hard centrifugation will
pellet these aggregates, preventing introduction to the array.
The labeled cDNA is applied to a 24 mm.times.50 mm glass cover slip
and then touched with the inverted microarray. Applying the
hybridization mix to the array and cover slipping it is an
operation which requires some dexterity to get the positioning of
the cover slip and the exclusion of air bubbles just right. It was
helpful to practice this operation with buffer and plain slides
before attempting actual samples. The hybridization solution is
added to the cover slip first, since some aggregates of fluor
remain in the solution and will bind to the first surface they
touch.
The slide is then placed in a microarray hybridization chamber, 5
.mu.l of 3.times.SSC is added to the reservoir, if the chamber
provided one, or at the scribed end of the slide and the chamber is
sealed. The chamber is submerged in a 65.degree. C. water bath and
the slide is allowed to hybridize for 16-20 hours. There are a wide
variety of commercial hybridization chambers. It was worthwhile to
prepare a mock hybridization with a blank slide, load it in the
chamber and incubate it to test for leaks, or drying of the
hybridization fluid, either of which cause severe fluorescent noise
on the array.
Next, the unbound fluorescent cDNA is washed off. The hybridization
chamber is removed from the water bath, cooled and carefully dried
off. The chamber is unsealed and the slide is removed. As there may
be negative pressure in the chamber after cooling, it is necessary
to remove water from around the seals so that it is not pulled into
the chamber and onto the slide when the seals are loosened.
The slide is placed, with the cover slip still affixed, into a
Coplin jar filled with 0.5.times.SSC/0.01% SDS wash buffer. The
cover slip is allowed to fall from the slide and then removed from
the jar with a forceps. The slide is allowed to wash for 2-5
minutes. The slide is transferred to a fresh Coplin jar filled with
0.06.times.SSC, and allowed to wash for 2-5 minutes. The sequence
of washes may need to be adjusted to allow for more aggressive
noise removal, depending on the source of the sample RNA. Useful
variations are to add a first wash which is 0.5.times.SSC/0.1% SDS
or to repeat the normal first wash twice.
The slide is then transferred to a slide rack and centrifuged at
low rpm (700-1000) for 3 minutes in a clinical centrifuge equipped
with a horizontal rotor for microtiter plates. If the slide is
simply air dried, it frequently acquires a fluorescent haze.
Centrifuging off the liquids results in a lower fluorescent
background. As the rate of drying can be quite rapid, it is
suggested that the slide be placed in the centrifuge immediately
upon removal from the Coplin jar.
Image analysis can be performed using DeArray software (Chen, Y.,
Dougherty, E. R. and Bittner, M. L. Ratio-based decisions and the
quantitative analysis of cDNA microarray images, Biomedical Optics
2, 364-374 (1997).
Example 3
Predicting Clinical Outcome for Patients with Neuroblastoma
Fifty-six pre-treatment primary neuroblastoma (NB) tumor samples
from 49 NB patients with outcome information were obtained
retrospectively from 3 sources presenting between 1992-2000 (Table
4). All patients were treated according to local or national
guidelines that followed similar protocols, which included
"wait-and-see" after surgery or combinations of vincristine,
doxorubicin, carboplatin, cisplatin, cyclophosphamide, melphalan
and etoposide, depending on the risk factors. All samples were
anonymized, and our protocol was deemed exempt from the NIH
Multiple Project Assurance.
TABLE-US-00007 TABLE 4 Neuroblastoma samples used in the study and
ANN prognostic prediction ##STR00001## ##STR00002## ##STR00003##
##STR00004## ##STR00005## ##STR00006## ##STR00007##
##STR00008##
All samples (except NB1, NB2, NB3, NB4, NB207, NB209 and NB210)
were used in the leave-one-out ANN analysis. Samples highlighted in
gray are the 21 test samples, and the rest were used for training
during the clone optimization procedure. There were 7 replicated
samples, marked by the numbers in superscript.
Sample Source: 1=Cooperative Human Tissue Network (CHTN, Ohio,
USA); 2=German Cancer Research Center (GCRC); 3=The Children's
Hospital at Westmead (CHW, Australia).
INSS=International Neuroblastoma Staging System.
MYCN amplification status: AMP=amplification; NA=not amplified.
Shimada Histology: F=favorable, "-"=not known, UF=unfavorable.
COG risk stratification: H=high-risk; I=intermediate-risk;
L=low-risk.
Ave. ANN Vote=average ANN committee votes.
ANN prediction: average ANN vote <0.5=A (alive); >0.5=D
(dead).
Clinical Outcome: A=alive without event; D=deceased due to NB
disease.
Pre-treatment tumor samples were snap-frozen in liquid nitrogen
following removal from the patients. Tumors were diagnosed as NB by
local centers experienced in the management of these cancers.
Additionally, the 56 samples were confirmed to be NBs by ANNs using
the previously-identified NB-specific gene expression profiles,
shown in the examples above. Patients were divided into two outcome
groups: "good-outcome" group had event-free survival (i.e. neither
relapse nor NB progression) for at least 3 years (n=30), and
"poor-outcome" died due to NB disease (n=19). The median age for
the good-outcome group was 0.9 years (range from 0.1 to 4.6 years)
and for the poor-outcome group was 2.8 years (range from 0.8 to
10.5 years) (Table 4).
Total RNA was extracted from the frozen pre-treatment tumor samples
using a previously published method (Wei, J. S, and Khan, J.
Purification of Total RNA from Mammalian Cells and Tissues. In: D.
Bowtell and J. Sambrook (eds.), DNA Microarrays: A Molecular
Cloning Manual, pp. 110-119. Cold Spring Harbor, N.Y.: Cold Spring
Harbor Laboratory Press, 2002). An Agilent BioAnalyzer 2100
(Agilent, Palo Alto, Calif.) was used to assess the integrity of
total RNAs from the tumor samples. Total RNA from seven human
cancer cell lines (CHP212, RD, Hela, A204, K562, RDES and CA46) was
pooled in equal portions to constitute a reference RNA, which was
used in all cDNA microarrray experiments.
Messenger RNA was amplified one round using a modified Eberwine RNA
amplification procedure (Sotiriou, C., Khanna, C., Jazaeri, A. A.,
Petersen, D., and Liu, E. T. Core biopsies can be used to
distinguish differences in expression profiling by cDNA
microarrays. J Mol Diagn, 4: 30-36, 2002). Next, an indirect
fluorescent labeling method was used to label cDNA (Hegde, P., Qi,
R., Abernathy, K., Gay, C., Dharap, S., Gaspard, R., Hughes, J. E.,
Snesrud, E., Lee, N., and Quackenbush, J. A concise guide to cDNA
microarray analysis. Biotechniques, 29: 548-550, 552-544, 556
passim., 2000) wherein, aminoallyl-dUTP (Sigma-Aldrich, St. Louis,
Mo.) was first incorporated into cDNA in a reverse transcription
reaction in which amplified anti-sense RNA was converted into cDNA
by Superscript II reverse transcriptase enzyme (Invitrogen, Grand
Island, N.Y.) according to the manufacturer's instructions. Second,
unincorporated aminoallyl-dUTP was removed with Qiagen PCR
purification kits (Qiagen, Valencia, Calif.) according to the
manufacturer's instructions. Third, monoreactive-Cye5 or Cye3 dyes
(AmershamPharmacia, Piscataway, N.J.) were conjugated with the
aminoallyl-dUTP on the cDNA. Fluorescent-labeled cDNA was purified
with Qiagen PCR purification kits.
The DNA microrarrays were fabricated from sequence-verified cDNA
libraries purchased from Research Genetics (Huntsville, Ala.). The
library consisted of a total of 42578 cDNA clones, representing
25933 Unigene clusters (13606 known genes and 12327 unknown ESTs).
The cDNA were printed on microarrays using a BioRobotics MicroGrid
II spotter (Harvard Bioscience, Holliston, Mass.). Fabrication,
hybridization and washing of microarrays were performed as
described above in Example 1. Images were acquired by an Agilent
DNA microarray scanner (Agilent, Palo Alto, Calif.), and analyzed
using the Microarray Suite program, coded in IPLab (Scanalytics,
Fairfax, Va.).
Gene expression ratios between the tumor sample RNA and the
reference RNA on each microarray were normalized using a pin-based
normalization method modified from Chen et al (Chen, Y., Dougherty,
E. R., and Bittner, M. L. Ratio-based decisions and the
quantitative analysis of cDNA microarray images. Biomedical
Optics., 2: 364-374, 1997). In order to include only high quality
data in the analysis, the quality of each individual cDNA spot was
calculated according to Chen et al (Chen, Y., Kamat, V., Dougherty,
E. R., Bittner, M. L., Meltzer, P. S., and Trent, J. M.
Bioinformatics, 18:1207-1215, 2002). Next, spots with an average
quality, across all samples, of less than 0.95 were excluded from
the analysis. There were 37920 (90.3%) clones that passed this
quality filter.
Feed-forward resilient back-propagation multi-layer perceptron ANNs
(coded in Matlab, The Mathworks, Natick, Mass.) with 3 layers were
used. The three layers were: an input layer of the top 10 principal
components (PCs) of the data (FIGS. 4A and B) or the gene
expression ratios of each cDNA spot (for the minimized gene set,
see FIG. 4B); a hidden layer with 3 nodes; and an output layer
generating a committee vote that discriminates two classes (i.e.,
good- and poor-outcome groups).
Average ANN committee votes were used to classify samples, and 0.5
was used as the decision boundary for ANN prediction throughout the
study. The ideal vote was 0 for the good-outcome group (alive), and
1 for the poor-outcome group (dead). The ANNs were trained and used
to predict NB outcomes using an 8-fold cross validation scheme in
all analyses similar as described above.
A leave-one-out prediction strategy was performed first (FIG. 4A),
where each sample (out of the 49 unique samples) was left out one
time during the training of the ANNs, and the left out sample was
then tested as an independent sample to predict the outcomes with
all quality-filtered clones (n=37920) without further clone
selection.
Visualization of all 56 NB samples using principal component
analysis (PCA) of all quality-filtered 37920 clones revealed NB
samples generally grouped according to their clinical outcomes
(FIG. 5A), clearly indicating a pre-existent prognostic signature.
The ability of ANNs to predict prognosis of the 49 unique
individuals was then tested (excluding 7 replicated samples) with
all 37920 clones using a conservative unbiased leave-one-out
prediction strategy (FIG. 4A). The ANNs correctly predicted 16/19
poor-outcome and 27/30 good-outcome cases (FIG. 5B). This
corresponds to a sensitivity of 84% and specificity of 90% for the
poor-outcome patients, with a positive predictive value of 84% for
the poor- and 90% for the good-outcome patients (Table 5).
TABLE-US-00008 TABLE 5 Performance of ANN prediction Positive
Positive predictive predictive Specificity value value Sensitivity
(%) (%) (poor- (%) (poor- (%)(good- ANN Prediction (poor-outcome)
outcome) outcome) outcome) Leave-one-out 84 90 84 90 with all
clones (n = 49)
The average ANN vote, the ANN predicted outcome of the patient, and
the clinical outcome of the NB patients is also summarized in Table
6 below.
TABLE-US-00009 TABLE 6 ANN predicted results and Clinical outcome
of NB Patients ##STR00009## ##STR00010## ##STR00011## ##STR00012##
##STR00013## ##STR00014## ##STR00015## ##STR00016## ##STR00017##
##STR00018##
Survival length was calculated for the 49 unique NB patients from
date of diagnosis until date of death or last follow-up as
appropriate. The probability of survival and significance were
calculated using the Kaplan-Meier and Mantel-Haenszel methods,
respectively (Kaplan, E. and Meier, P. Non-Parametric Estimation
from Imcomplete Observations. J. Am. Stat. Assoc., 53: 457-481,
1958; and Mantel, M. Evaluation of Survival Data and Two New Rank
Order Statistics Arising in its Consideration. Cancer Chem. Rep.,
50: 163-170, 1966).
The Kaplan-Meier curves demonstrated that patients with poor and
good gene expression signatures as identified by the ANNs had
significantly different survival probabilities (P<0.0001 see
FIG. 5C).
The Cox proportional hazards model (Cox, D. Regression Models and
Life Tables. J. Royal Stat. Soc. (B), 34: 187-202, 1972) was used
to determine the hazard ratios and confidence intervals (Matthews,
D. E. and Farewell, V. T. Using and Understanding Medical
Statistics. In, 3rd edition edition, pp. 150-160. Basel: Karger,
1996) for survival between the dichotomized groups of patients, and
was used to assess which factors were jointly significant in the
association with survival for the 24 high-risk patients (Cox et
al.).
The Cox model parameters (b.sub.i) were converted to hazard ratios
by computing exp(b.sub.i), where exp(a)=2.7183.sup.a. The 95%
confidence interval for the hazard ratio was computed as
[exp(b.sub.iL), exp(b.sub.iH)] where b.sub.iL=b.sub.i-1.96
[estimated standard error (b.sub.i)] and b.sub.iH=b.sub.i=1.96
[estimated standard error (b.sub.i)] (Matthews et al.). In this
study, the hazard ratio indicates the risk associated with
NB-caused death while being in a greater-risk category compared to
that of being in the lower-risk category. Using the procedure
described by Simon and Altman, a likelihood ratio test was used to
assess for importance of the microarray prediction after adjusting
for standard prognostic factors such as MYCN amplification, age, or
stage (Simon, R. and Altman, D. G. Statistical aspects of
prognostic factor studies in oncology. Br J Cancer, 69: 979-985,
1994).
The Cox proportional hazard ratio for the risk of death associated
with the poor signature was 16.1 (95% confidence interval:
4.6-56.9, P<0.0001), which was higher than those of all the
other risk factors we examined (stage, MYCN amplification, age)
except Shimada Histology, and was comparable to the COG risk
stratification (Table 7 and FIG. 5D).
TABLE-US-00010 TABLE 7 Univariate Proportional Hazard Analysis for
the Risk of NB-related Death Log-Rank P Variable H.R. 95% C.I.
Value All NB Samples (n = 49) All 37920 Clones (Poor signature vs.
16.1 (4.6-56.9) <0.0001 Good signature) Top 19 ANN-Ranked Genes
(Poor .infin.* -- <0.0001 signature vs. Good signature) COG risk
stratification (High Risk vs. 29.7 (4.0-222.9) <0.0001 Low &
Intermediate Risk) COG risk stratification (High & 13.6
(1.8-101.7) 0.0009 Intermediate Risk vs. Low Risk) COG risk
stratification 23.2 (3.1-175.9) <0.0001 (High Risk vs. Low Risk)
INSS Stage (Stage 4 vs. Stages 1-3) 7.1 (2.1-24.2) 0.0003 INSS
Stage (Stage 3 & 4 vs. 13.6 (1.8-101.7) 0.0009 Stage 1 & 2)
MYCN status 9.8 (3.6-26.7) <0.0001 (amplified vs. not amplified)
Age (>1 yr vs. <1 yr) 12.3 (1.6-92.5) 0.0017 Shimada
Histology 19.9 (2.4-166.1) 0.0001 (unfavorable vs. favorable) (n =
27) High Risk Samples (n = 24) MYCN status 3.5 (1.2-10.0) 0.01
(amplified vs. not amplified) Top 19 ANN-Ranked Genes (Poor
.infin.* -- 0.0005 signature vs. Good signature) All 37920 Clones
(Poor signature vs. 5.3 (1.4-19.4) 0.0067 Good signature)
The Cox proportional hazards model was used to calculate all hazard
ratios (H.R.) and confidence intervals (C.I.). P-values were
calculated using the Mantel-Haenszel method. These hazard ratios
are infinite because none of the patients predicted to have
good-outcome experienced an event (i.e., death).
Example 4
Optimization of Genes Used for NB Clinical Outcome Prediction
To identify the optimal set of genes that results in the minimum
classification errors a gene minimization procedure was performed
as exemplified above. All 56 samples were randomly partitioned into
training (n=35) and testing sets (n=21) and the training set was
used for the gene selection algorithm.
From this, it was observed that the top 24 ANN-ranked clones
resulted in the minimal classification error (FIG. 6A). The
top-ranked clone for each gene was taken and this set of genes was
used as a minimal gene set. These 24 clones represented 19 unique
genes as shown in Table 2.
The top-ranked clone for each gene was taken and this set of genes
was used as a minimal gene set. When the overall variance of these
genes was visualized using PCA on all 56 samples a clearer
separation of the poor- from the good-outcome samples (in
comparison to that observed with the PCA for all 37920 clones)
(FIG. 6B compared to FIG. 5A).
The ANN was then recalibrated with the 35 training samples using
the expression ratios for the 19 genes and correctly predicted the
outcomes for 5/5 poor-outcome and 15/16 good-outcome patients in
the independent test set, corresponding to a sensitivity of 100%
and a specificity of 94% for predicting poor-outcome (FIG. 6C and
Table 8). The positive predictive values were 83% and 100% for the
poor- and good-outcome groups, respectively for the test samples,
and 95% and 100% for all patients (Table 8).
TABLE-US-00011 TABLE 8 Performance of ANN prediction Sensitivity
Positive Positive (%) Specificity predictive value predictive value
ANN (poor- (%) (poor- (%) (%) Prediction outcome) outcome)
(poor-outcome) (good-outcome) 19 Genes 100 94 83 100 (Test samples:
n = 21) 19 Genes 100 97 95 100 (n = 49)
The Kaplan-Meier curves demonstrated that patients with good and
poor signatures based on the expression ratios of the 19 genes had
significantly different survival probabilities (P<0.0001 see
FIG. 6D). Furthermore, no patients died in the good signature
group, thus the hazard ratio for death risk was infinite (Table
8).
The top 24 ANN-ranked clones represent 19 UniGene clusters of 12
known genes and 7 ESTs, as DLK1, ARHI, PRSS3, and SLIT3 were
represented by multiple cDNA clones (FIG. 7A). Nine of the genes
were up regulated and 10 down regulated in the poor- compared to
the good-outcome group (FIG. 7, A and B). To our knowledge, all of
the genes, except MYCN and CD44, have not been previously
associated with NB prognosis.
The expression data presented in FIG. 7A, as well as additional
expression data for the top ranked 250 genes is described in Tables
9A, B, C. Whether the gene expression is upregulated or
downregulated in poor outcome patients in shown in Table 3.
Expression level of each gene was logged (base.sup.2) and mean
centered. Table 9A provides expression data from the top 250 genes
in good outcome patients used for training for ANNs. Table 9B
provides expression data for top 250 genes in poor outcome patients
used for training ANNs. Table 9C shows the expression data in the
test samples from good and poor outcome patients.
TABLE-US-00012 TABLE 9A Training Samples: Good outcome patients.
(1.sup.st bar FIG. 7A) Rank Gene St1_NA_NB1 St1_NA_NB208
St1_NA_NB237 St1_NA_NB29 St1_NA_NB7 St1_- NA_NB77 1 DLK1 0.11 0.66
0.3 0.03 0.12 0.16 2 est 0.18 0.39 0.57 0.35 0.68 0.25 3 PRSS3 5.12
1.16 3.4 2.61 0.78 3.9 4 ARHI 1.32 17.3 5.13 1.19 17.3 6.56 5 ARC
2.21 5.4 4.73 1.56 3.97 2.18 6 SLIT3 24.97 15.98 16.59 11.54 10.29
27.14 7 CNR1 16.23 9.34 4.62 17.56 11.15 19.41 8 est 0.24 0.42 0.69
0.2 0.12 0.23 9 est 2.18 1.21 2.13 1.61 2.56 1.88 10 FLJ25461 0.8
1.91 1.7 1.11 4.52 1.01 11 est 0.24 0.68 0.4 0.39 0.36 0.34 12 CD44
4.39 2.25 2.94 3.8 1.69 3.68 13 est 0.25 2.48 0.37 0.42 2.01 0.87
14 ROBO2 3.18 16.42 0.75 3.12 5.5 3.18 15 BTBD3 0.87 1.62 0.61 0.99
2.87 1.58 16 MYCN 9.53 9.94 0.87 7.34 6.44 3.01 17 est 5.59 8.52
11.48 7.3 9.8 30.93 18 JPH1 0.04 0.05 0.12 0.07 0.15 0.05 19 KLRC3
0.06 0.15 0.06 0.11 0.08 0.05 20 est 3.92 22.55 39.56 1.91 6.6 3.17
21 RET 0.88 1.33 18.4 0.93 8.86 4.15 22 CRABP1 0.06 0.12 0.11 0.13
0.53 0.05 23 ECEL1 2.12 2.17 0.27 2.08 1.88 2.24 24 LOC283120 1 1
1.03 0.92 0.8 15.84 25 HMGA2 8.72 24.37 5.71 10.85 10.48 20.86 26
SYNPO2 9.47 12.41 33.16 5.35 4.54 14.85 27 LOC163782 0.19 0.23 1.45
0.2 0.41 0.22 28 VSNL1 1.9 35.23 7.52 2.47 7.76 14.01 29 HS3ST4
0.11 0.49 0.11 0.15 0.14 0.09 30 AKR1C1 0.57 0.22 0.52 0.41 0.33
0.42 31 est 0.7 9.95 0.77 0.04 10.97 0.26 32 GPR22 7.71 4.56 7.88
22.63 23.98 5.08 33 est 1.27 1.75 3.82 1.07 2.96 1.58 34 est 0.15
0.21 0.29 0.11 0.05 0.21 35 CCNA1 1.43 6.2 4.96 1.18 3.66 1.22 36
PKIB 3.76 8.15 17.22 3.01 0.57 13.21 37 est 0.84 1.9 1.97 0.95 2.35
1.01 38 GAL 0.3 0.41 0.64 0.17 0.08 0.17 39 est 0.88 0.43 11.1 1.11
3.23 0.39 40 LOC221303 2.59 1.87 22.22 1.75 2.17 16.05 41 est 5.5
2.36 2.66 2.08 1.31 3.71 42 est 1.07 6.12 3.3 1.17 0.82 2.34 43
BMP7 8.84 0.29 4.47 4.94 1.81 0.36 44 SLC30A3 0.47 1.39 0.46 0.62
1.04 0.38 45 FLJ10539 1.77 1.31 0.32 2.46 0.58 1.24 46 AMIGO2 6.36
0.42 2.6 6.26 8.21 4.79 47 AKR1C2 0.53 0.24 0.61 0.55 0.38 0.5 48
MGP 0.37 0.07 0.06 0.04 0.16 0.72 49 PCSK1 0.19 0.2 0.4 0.26 0.36
0.46 50 HK2 0.34 0.26 0.27 0.18 0.19 0.18 51 est 0.33 0.57 0.38
0.53 0.7 0.44 52 est 0.43 0.34 0.32 0.49 0.39 0.28 53 IL7 5.72 8.26
0.74 12.92 6.4 8.2 54 PRSS12 0.7 1.58 1.64 0.81 0.56 1.09 55
GABARAPL1 2.2 0.8 3.35 1.33 1.41 1.4 56 DEFB129 0.64 1.72 0.63 0.74
1.1 0.57 57 NAV3 0.43 4.97 4.85 0.51 3.67 6.35 58 RAB3B 17.91 25.84
21.5 9.84 21.21 16.71 59 KRT6B 0.63 1.47 2.13 3.23 2.22 2.58 60
BEX1 24.41 21.74 17.54 15.56 40.05 16.52 61 est 28.76 23.52 16.38
12.76 38.4 15.28 62 est 0.47 1.25 0.38 0.52 1.94 2.93 63 SCYL1 4.83
6.49 3.29 5.77 5.1 3.44 64 est 1.24 8.63 1.92 1.1 4.44 3.78 65 RYR2
7.65 37.67 6.75 8.2 14.93 8.05 66 LRBA 0.79 0.37 0.63 0.79 0.45
0.31 67 CSPG3 0.49 4.53 1.2 0.41 1.44 0.79 68 est 3.1 2.39 2.66
4.04 4.32 1.49 69 MMP12 1.04 15.51 3.22 1.04 2.09 4.03 70 CHRNA1
0.03 0.02 0.02 0.04 0.03 0.04 71 est 1.54 14.04 5.88 1.75 2.07 1.92
72 est 24.74 74.3 7.5 37.92 44.69 8.5 73 HNRPH1 50.66 6.09 4.21
11.84 19.57 33.39 74 LOC113251 1.16 2.01 0.32 1.99 1.9 0.79 75 est
4.46 5.41 0.99 2.44 3.9 1.25 76 PAG 4.89 6.31 2.91 5.67 3.87 3.41
77 PROK2 6.55 24.79 2.83 6.59 11 5.89 78 HS6ST1 1.68 6.89 5.81 1.78
4.15 1.63 79 est 3.05 10.82 2.94 10.94 8.33 3.02 80 PCDH9 1.54
14.65 14.09 2.55 5.9 5.16 81 est 29.13 11.12 5.34 10.36 5.43 15.41
82 est 0.17 0.42 0.44 0.28 0.55 0.43 83 GLDC 0.38 0.58 0.74 0.45
0.44 0.32 84 ADRB2 2.93 2.21 0.98 1.54 2.51 0.86 85 ICSBP1 0.3 0.42
0.71 0.26 0.16 0.29 86 CD48 0.66 0.28 0.27 0.47 0.3 0.97 87 est
2.07 1.93 0.41 3.15 0.96 0.67 88 DYRK1B 0.52 0.53 0.58 0.63 0.75
0.61 89 KLRC1 0.08 0.27 0.11 0.16 0.13 0.08 90 est 0.21 0.11 0.16
0.13 0.14 0.21 91 est 1.47 1.03 0.78 1.24 2.07 0.72 92 est 0.07
0.16 0.1 0.05 0.12 0.38 93 MOXD1 0.64 0.13 1.35 0.25 0.35 0.31 94
est 0.38 0.78 0.21 0.45 0.81 0.25 95 est 4.4 8.55 3.2 5.03 5.03
3.24 96 GAS1 0.1 0.04 0.05 0.07 0.07 0.17 97 COL9A2 2.45 6.53 0.29
0.95 0.29 1.67 98 est 1.31 3.59 1.51 1.39 1.22 1.32 99 DRPLA 0.42
0.2 0.38 0.34 0.3 0.3 100 est 21.13 44.17 8.42 17.06 9.84 17.03 101
REPRIMO 41.46 9.06 1.88 16.9 1.68 19.81 102 CACNA2D2 0.79 1.48 0.7
0.81 1 0.67 103 NEBL 0.6 1.51 2.17 0.92 0.98 0.83 104 est 1.37 3.44
0.43 1.64 0.98 0.67 105 HLA-DQA1 1.93 0.94 1.8 1.37 1.57 7.01 106
EDG3 4.38 2.91 4.19 2.92 0.55 2.95 107 CPVL 1.09 0.26 0.25 0.77
0.63 0.99 108 FLJ32884 34.54 12.02 11.43 22.78 4.38 8.69 109 LCP1
0.85 0.31 1.04 0.55 0.55 0.99 110 est 1.01 3.38 0.39 1.06 1.4 4.05
111 est 60.29 100 21.64 30 49.33 51.27 112 est 15.13 7.18 0.42
10.16 1.91 13.57 113 est 5.06 6.26 2.06 4.85 3.83 1.23 114
DKFZP564C152 1.12 1.2 3.65 1.11 1.47 1.36 115 DMN 1.58 1.79 8.01
1.1 1.24 1.88 116 GABRA5 0.1 0.17 0.3 0.24 0.29 0.11 117 AKR1C3
0.32 0.14 0.37 0.3 0.17 0.36 118 LOC168850 2.19 4.27 2.16 5.59 5.9
3.3 119 est 3.17 5.68 9.56 2.94 8.11 6.23 120 KCNQ2 1.31 0.96 0.8
1.26 0.68 1.14 121 NME5 11.96 6.8 9.7 4.39 4.64 2.61 122 est 6.4
3.28 1.88 9.26 3.27 2.75 123 PBX1 2.79 4.55 0.88 2.13 4.89 1.85 124
CNTNAP2 2.36 1.71 3.3 2.57 2.17 3.77 125 est 67.22 73.47 27.7 61.6
10.56 33.87 126 SPON1 4.15 0.91 3.34 1.9 2.22 13.38 127 CDH8 0.63
2.8 0.31 1.02 1.21 4.7 128 PRKCB1 0.92 1.17 0.31 1.24 1.6 0.87 129
SLC21A11 7.03 1.79 1.1 4.85 2.56 2.78 130 MAP4 28.51 13.84 30.27
18.09 9.53 35.79 131 est 3.53 10.47 1.89 4.57 2.17 1.63 132 SCN7A
5.04 3.82 35.6 7.58 8.3 3.22 133 est 0.85 8.02 3.33 0.99 9.4 3.65
134 est 5.95 3.36 1.04 3.02 1.87 2.31 135 est 1.48 0.82 1.42 1.43
1.53 1.75 136 est 0.31 0.47 0.47 0.44 0.61 0.57 137 CDW52 0.21 0.09
0.06 0.22 0.12 0.14 138 ABCB1 2.17 1.94 8.86 1.93 3.3 2.79 139 est
0.36 0.58 0.34 0.83 2.05 0.22 140 OSF-2 42.12 2.74 2.09 9.76 26.42
70.66 141 NRXN1 0.54 1.34 0.5 0.56 1.83 3.06 142 ADAM22 1.39 1.58
1.95 2.21 1.83 2.01 143 est 3.75 7.17 9.79 4.01 2.66 5.63 144 TRGV9
0.93 0.54 0.75 1.03 0.65 0.43 145 est 0.06 0.04 0.08 0.08 0.12 0.08
146 PTPRD 6.81 22.96 1.63 7.82 8.04 4.9 147 est 0.81 0.83 0.69 0.55
0.79 0.95 148 HS3ST2 10.12 2.66 13.19 1.03 3.81 1.75 149 FGF13 3.12
3.29 0.76 3.63 8.41 2.52 150 MKI67 0.57 0.84 0.14 0.5 0.43 0.43 151
KIF12 4.04 8.31 1.58 3.22 1.3 1.03 152 est 0.85 3.12 1.21 0.91 1.21
1.02 153 est 1.27 0.25 0.48 1.1 0.47 1.08 154 est 8.14 3.31 4.97
6.95 3.44 9.32 155 est 0.36 0.21 0.17 0.31 0.35 0.4 156 est 0.58
7.72 2.19 0.54 1.49 2.53 157 KLIP1 0.21 0.58 0.1 0.29 0.46 0.22 158
est 0.53 0.56 0.42 0.54 0.81 0.4 159 LOC157570 0.18 0.3 0.05 0.27
0.32 0.26 160 MAD2L1 0.22 0.22 0.05 0.18 0.34 0.11 161 est 0.51
2.12 2.92 0.46 0.32 2.78 162 est 7.12 6.13 3.17 6.77 3.41 3.85 163
RGS5 27.94 44.29 35.88 78.35 79.41 27.9 164 ATP2B4 2.35 4.53 5.12
2.81 6.41 4.75 165 HMGCL 0.07 0.03 0.08 0.06 0.05 0.24 166 ODZ3
5.51 6.84 11.04 5.5 3.11 3.25 167 CHGA 100 100 43.1 54.1 81.08
95.38 168 MGC33510 0.46 5.52 0.46 0.18 6.78 0.24 169 GAGE5 0.01
0.02 0.01 0.01 0.01 0.01 170 SARDH 22.75 16.83 2.21 19.42 13.61
15.84 171 est 10.51 0.79 1.6 1.43 1.82 19.99 172 DAT1 0.25 0.28
0.69 0.29 1 0.19 173 FUCA1 4.11 2.54 1.78 3.02 1.64 7.02 174 TM6SF2
1.27 2.15 0.7 0.89 0.84 0.87 175 KCNK9 1.33 1.89 1.47 1.07 1.65
1.12 176 ADCYAP1 0.51 3.75 14.76 0.53 12.24 1.61 177 PLXNA4 2.69
1.26 0.96 1.32 0.9 2.73 178 HLA-DMB 2.28 0.95 2.5 1.28 1.36 3.06
179 est 0.36 0.8 2.54 0.5 0.65 0.4 180 est 0.27 0.08 0.39 0.17 0.26
0.82 181 GRIN3A 0.57 0.64 1.03 0.4 0.37 0.83 182 OSBPL3 2.84 2.89
1.93 3.03 4.05 2.35 183 ODZ4 1.97 8.23 3.96 1.34 1.64 2.79 184 est
5.8 3.08 25.12 8.26 7.78 2.9 185 E2F1 0.57 0.67 0.09 0.48 0.39 0.29
186 MGC16664 25.5 12.8 7.88 9.05 4.33 20.18 187 HMP19 80.34 100
53.99 44.72 97.74 78.76 188 IL2RB 1.73 0.93 3.07 0.78 1.02 2.67 189
TOPK 0.12 0.26 0.03 0.14 0.19 0.15 190 ALDH1A1 5.9 1.67 32.18 4.35
3.92 4.14 191 CED-6 0.14 0.11 3.25 0.27 0.55 0.31 192 est 0.4 1.53
0.49 0.55 1.09 0.78 193 A2BP1 9.59 5.4 2.47 8.24 3.39 7.09 194 LY6E
0.27 0.41 0.43 0.19 0.21 0.36 195 est 2.36 3.71 1.91 1.65 2.2 2.31
196 est 0.48 0.32 8.79 0.45 0.64 1.55 197 PLXNC1 15.57 11.42 4.33
18.46 9.23 12.21 198 EFS 0.77 0.17 3.38 0.51 0.37 0.7 199 ACTN2
5.15 4.47 0.56 3.12 4.57 5.24 200 MYC 0.08 0.03 0.08 0.06 0.07 0.25
201 KIAA0527 0.11 0.16 0.47 0.21 0.41 0.26 202 C6orf31 0.53 5.79
0.63 0.31 8.54 0.3 203 DLL3 1.29 2.98 0.44 0.82 1.23 1.08 204 est
1.21 0.99 0.48 1.12 0.77 1.05 205 STK33 0.32 0.82 1.14 0.37 0.78
0.32 206 SEMA3A 0.27 3.02 0.22 0.55 0.4 1.06 207 est 2.05 33.41
2.64 1.37 1.71 2.53 208 IGSF4 18.11 42.66 9.33 8.66 5.65 9.73 209
CKS2 0.11 0.12 0.05 0.07 0.09 0.11 210 est 2.74 2.84 1.1 1.93 0.76
2.23 211 est 0.45 0.47 0.23 0.75 0.86 0.26 212 SIX3 1.56 95.71 3.74
1.18 17.6 12.69 213 FLJ22002 0.17 0.08 0.42 0.19 0.22 0.14 214
HSD17B12 0.42 0.41 1.93 0.49 1.27 1.18 215 HBA2 0.81 0.15 0.09 0.33
0.33 0.8 216 CDH11 1.45 0.79 1.37 1.78 1.77 2.22 217 RGS9 3.81 4.48
3.04 1.9 1.83 4.06 218 est 1.29 3.03 1.99 0.84 1.68 0.89 219 NCAM2
0.98 4.61 3.27 1.4 0.67 1.05 220 BIRC5 0.12 0.3 0.02 0.14 0.14 0.12
221 est 3.27 3.29 0.55 3.07 1.02 1.26 222 GNG12 0.47 0.56 1.82 0.43
0.6 0.83 223 GPIG4 0.98 0.59 1.54 1.44 0.8 1.09 224 est 1.02 3.21
14.46 1.53 2.12 1.86 225 ENPP4 0.93 0.4 6.89 1.39 3.95 3.35 226
FMNL 1.02 0.96 0.86 0.98 1.6 0.75 227 est 0.27 0.07 0.52 0.2 0.17
0.27 228 PIWIL2 0.58 4.44 1.03 0.45 0.56 8.55 229 CLSTN1 1.06 0.61
1.04 1.01 1.1 1.04 230 UHRF1 0.08 0.19 0.06 0.14 0.18 0.09 231 est
0.14 0.24 1.12 0.21 0.3 0.25 232 SLC40A1 2.84 2.87 1.77 4.71 5.66
5.46 233 CLECSF6 4.2 2.7 3.42 2.31 3.28 8.85 234 est 3.58 2.97 1.67
2.42 1.76 1.8 235 BKLHD2 2.31 1.92 3.09 2.91 2.78 2.15 236 est 2.08
0.32 1.05 2.65 2.93 1.57 237 est 0.8 10.19 0.21 0.33 19.86 0.38 238
est 1.15 1.67 0.86 1.58 1.63 0.55 239 SORCS1 30.21 23.81 57.82
18.64 8.3 16.75 240 NRP2 17.83 29.44 15.63 9.06 7.63 26.01 241
E2-EPF 0.4 0.81 0.11 0.2 0.35 0.41 242 CAST 2.71 0.8 4.46 1.57 1.16
4.64
243 KIAA1384 0.66 6.3 1.31 0.76 1.14 3.56 244 KIAA0644 1.82 0.8
0.33 1.74 0.72 1.42 245 HLA-DRB3 3.55 1.34 9.56 2.33 2.4 6.46 246
PMP22 8.35 4.38 21.1 8.1 5.46 7.65 247 DJ79P11.1 9.82 7.65 7.54
5.99 12.53 8.9 248 SOX5 1.32 7.15 2.7 1.67 1.46 1.61 249 CD3E 10.59
11.33 3.77 4.54 4.08 6.59 250 est 0.81 3.54 0.46 1.49 1.17 4.9 Rank
St2_NA_NB15 St2_NA_NB231 St2_NA_NB255 St3_NA_NB216 St3_NA_NB61
St4_A_- NB14 1 0.01 0.05 2.8 0.16 0.17 0.77 2 0.4 0.32 0.49 0.26
0.89 0.7 3 1.48 0.85 4.01 4.14 8.16 7.72 4 3.62 1.69 0.54 0.73 0.63
4.32 5 1.24 1.87 5.79 6.99 1.96 1.57 6 19.17 21.25 35.38 14.94
57.24 15.41 7 7.16 11.72 3.76 24.05 2.72 8.43 8 0.39 0.14 0.58 0.19
0.23 1.34 9 1.35 0.5 0.3 0.63 1.06 0.39 10 0.6 0.44 1.3 1.26 0.41
0.95 11 0.24 0.28 2.47 0.37 0.44 0.99 12 4.89 2.83 2.9 2.23 2.87
2.06 13 0.99 3.79 1.6 2.44 0.16 0.84 14 4.12 8.28 9.32 6.99 0.76
2.98 15 0.87 0.59 0.38 1.36 0.83 0.7 16 2.53 4.38 3.54 5.27 6.12
15.84 17 20.86 8.17 3.19 6.67 4.41 2.21 18 0.08 0.08 0.07 0.07 0.06
0.12 19 0.07 0.16 0.06 0.1 0.09 0.28 20 10.33 23.73 19.55 28.44 3.4
5.16 21 2.02 0.82 1.76 10.83 1.1 26.9 22 0.08 0.06 0.07 0.1 0.09
0.34 23 1.59 0.31 0.7 1.11 0.99 0.4 24 2.09 1.56 3.78 0.94 5.05 0.8
25 16.89 14.31 8.41 12.76 2.6 11.62 26 20.3 23.02 17.51 10.04 15.86
21.3 27 0.33 0.15 0.06 0.12 0.15 0.26 28 13.3 18.96 4.12 13.37 3.45
7.87 29 0.18 0.07 0.06 0.15 0.07 0.24 30 0.69 0.3 0.27 0.44 0.49
0.42 31 0.11 8.78 16.68 0.08 0.2 0.04 32 14.88 22.56 29.09 18.99
16.8 5.25 33 1.31 1.14 2.23 0.53 1.15 2.62 34 0.55 0.89 0.29 0.36
0.19 0.13 35 2.8 2.07 3.28 1.84 2.51 1.32 36 7.67 8.31 2.46 8.87
2.62 10.72 37 0.95 0.43 1.8 1.73 0.37 0.84 38 0.62 1.44 1.49 0.84
0.05 1.46 39 1.17 0.47 0.44 0.28 0.59 6.14 40 3.45 4.63 6.81 2.08
6.46 4.04 41 1.71 1.44 5.35 2.94 6.1 2.95 42 2.2 18.62 11.65 40.36
1.1 52.13 43 0.28 0.34 0.38 0.22 3.19 3.74 44 0.73 0.65 0.66 2.69
1.97 1.27 45 0.85 2.44 0.57 1.42 0.54 0.82 46 3.67 6.06 1.19 3.22
1.46 1.42 47 0.58 0.35 0.3 0.5 0.41 0.45 48 0.54 0.22 0.53 0.09
1.09 0.63 49 0.26 0.82 0.1 1.14 0.07 1.36 50 0.09 0.56 0.39 0.24
0.23 0.5 51 0.44 0.3 0.49 0.41 0.42 0.65 52 0.36 0.36 0.27 0.33
0.59 0.39 53 12.81 12.41 6.76 13.01 6.81 1.29 54 0.96 3.49 3.91
8.35 0.71 9.8 55 1.14 0.91 0.99 1.21 0.85 2.53 56 0.73 0.66 0.62 3
2.4 0.98 57 5.28 5.48 3.86 0.26 4.35 3.55 58 12.19 25 12.15 12.54
10.4 1.81 59 4.39 3.38 1.54 3.19 1.47 1.27 60 12.4 9.38 20.91 15.14
18.19 32.84 61 11.29 10 19.75 16.99 21.54 37.68 62 0.46 0.56 1.76
2.06 0.52 4.12 63 6.35 11.21 5.61 10.69 3.01 2.05 64 2.48 13.11
1.69 11.24 2.95 7.27 65 11.91 13.14 19.48 11.43 2.01 3.23 66 0.45
1.03 0.78 0.31 0.66 0.44 67 0.77 0.49 0.5 0.52 0.56 1.01 68 1.3
3.07 0.74 2.23 1.38 1.64 69 2.17 4.06 85.14 7.03 1.56 18.58 70 0.02
0.05 0.03 0.05 0.03 0.1 71 4.08 2.17 1.68 5.08 1.17 2.62 72 25.89
49.14 43.89 45.02 5.94 8.6 73 4.4 16.39 45.39 27.56 48.63 9.75 74
1.18 1.7 1.08 1.21 0.72 0.58 75 2.17 2.07 3.28 3.42 0.87 1.57 76
5.38 11.26 4.72 10.08 3.06 2.18 77 7.52 9.26 11.3 11.64 3.29 2.09
78 2.73 3.33 3.99 2.24 2.53 1.55 79 10.02 15.81 6.93 7.52 0.81 2.27
80 12.39 4.67 1.88 9.85 0.97 8.46 81 14.12 15.74 17.31 21.13 25.08
10.33 82 0.21 0.32 0.36 0.29 0.25 0.44 83 0.37 0.57 0.56 0.63 0.33
1.51 84 2.26 1.65 1.18 1.14 1.84 0.92 85 0.66 1.22 1.32 0.87 0.22
1.06 86 0.99 0.84 0.56 0.37 1.66 1.53 87 1.24 3.9 1.05 1.86 0.34
0.71 88 0.46 0.47 0.5 0.47 0.88 0.64 89 0.13 0.22 0.1 0.15 0.12 0.3
90 0.17 0.16 0.25 0.12 0.26 0.33 91 1.33 1.27 2.9 1.43 2.41 2.93 92
0.05 0.53 0.24 0.47 0.05 1.38 93 1.06 0.2 0.2 0.13 0.33 0.84 94
0.26 0.32 1.2 0.37 0.71 0.25 95 11.31 11.15 7.77 10.7 1.88 2.03 96
0.1 0.2 0.05 0.05 0.31 0.08 97 2.4 1.12 0.33 0.48 0.54 0.37 98 1.83
2.95 0.54 3.74 0.35 1.42 99 0.49 0.25 0.23 0.41 0.31 0.28 100 15.43
41.94 22.27 94.04 8.57 11.28 101 18.53 36.26 8.22 12.15 2.78 1.19
102 0.76 0.7 0.67 2.28 2.1 0.9 103 1.2 1.21 0.45 2.65 0.22 1.37 104
2.22 1.86 1.13 1.91 0.43 0.98 105 3.71 2.07 1.34 1.07 7.8 9.09 106
3.11 3.25 2.61 3.64 3.28 3.87 107 0.58 0.4 0.96 0.3 1.95 2.9 108
7.03 17.66 5.79 47.13 2.65 9.76 109 0.69 0.99 0.96 0.47 1.52 1.71
110 1.17 0.75 1.25 3.96 0.92 1.43 111 52.21 58.31 74.77 51.74 33.22
52.1 112 9.32 10.16 5.54 15.21 9.42 1.64 113 1.85 4.64 9.18 11.07
1.89 8.2 114 1.32 1.41 2.01 0.68 0.91 3.07 115 1.87 1.48 2.3 2.48 1
2.22 116 0.14 0.21 0.15 0.35 0.14 0.34 117 0.39 0.22 0.18 0.3 0.31
0.3 118 7.68 6.92 4.01 5.33 2.47 1.06 119 4.31 4.92 4.16 4.77 3.45
7.02 120 1.16 0.61 0.69 0.75 1.05 1.12 121 6.2 1.62 2.69 2.41 2.94
2.2 122 2.18 4.59 1.28 2.33 0.84 4.54 123 1.96 1.97 1.73 2.05 1.34
1.36 124 2.49 1.37 2.1 0.92 2.32 1.27 125 26.55 2.09 9.07 5.17
11.05 16.58 126 6.21 3.75 3.99 1.02 9.76 4.35 127 1.15 0.64 0.73
3.32 0.56 1.79 128 0.68 0.92 0.59 1.54 1.29 0.75 129 2.45 4.83 1.77
4.73 3.03 1.2 130 14.34 20.16 19.78 10.76 16.6 25.48 131 3.71 5.26
7.23 11.09 1.65 2.62 132 14.67 10.54 9.64 12.24 3.5 6.74 133 2.8
2.6 1.28 2.79 0.93 1.09 134 2.78 2.16 2.37 4.08 1.08 1.43 135 1.21
0.56 0.54 0.56 0.63 0.5 136 0.32 0.36 0.5 0.68 0.39 0.73 137 0.53
0.41 0.41 0.21 0.51 1.02 138 4.02 3.01 11.14 5.72 12.09 3.23 139
1.69 1.58 0.35 0.4 0.28 0.32 140 9.22 2.97 31.96 6.03 34.87 19.54
141 0.62 0.63 1.9 2.13 0.61 2.83 142 1.75 2.63 2.52 4.34 1.66 3.52
143 6.28 8.33 9.68 14.54 2.66 23.94 144 1.52 2.13 1.34 0.98 1.79
0.8 145 0.19 0.07 0.85 0.06 0.54 0.34 146 4.97 7.46 5.08 17.08 1.02
17.63 147 0.76 0.65 0.66 0.7 0.77 0.72 148 2.12 3.13 4.39 2.59 2.18
17.47 149 2.19 3.82 3.4 4.3 1.02 5.47 150 0.37 0.36 1.24 0.55 0.72
0.2 151 0.84 1.6 14.3 7.83 1.24 8.39 152 0.98 1.1 1.22 0.99 1.02
0.91 153 2.58 3.04 0.67 0.89 3.64 0.76 154 17.28 31.39 4.64 6.05
32.3 2.37 155 0.59 0.23 2.58 0.24 1.1 1.02 156 5.79 2.88 6.47 6.73
1.34 2.65 157 0.22 0.32 1.05 0.33 0.41 0.14 158 0.5 0.65 0.65 0.6
0.8 0.61 159 0.2 0.2 0.61 0.2 0.46 0.16 160 0.14 0.34 0.46 0.09
0.22 0.09 161 1.29 2.07 0.39 0.72 1.16 2.4 162 9.64 9.12 5.63 9.63
2.88 4.33 163 52.39 50.96 49.94 44.69 35.38 22.35 164 3.61 3.53
1.99 4.39 2.6 3.04 165 0.26 0.12 0.51 0.1 0.09 0.09 166 3.8 2.91
2.65 5.28 1.26 3.14 167 100 64.75 100 73.13 64.13 88.95 168 0.16
6.79 19.63 0.15 0.22 0.12 169 0.01 0.01 0.01 0.01 0.02 0.02 170
15.11 21.51 16.4 12.31 8.36 4.44 171 6.89 22.1 4.22 1.8 21.4 8.58
172 0.75 0.22 0.34 0.33 0.38 1.93 173 2.46 2.53 5.36 1.86 8.43 9.91
174 2.36 1.96 1.23 1.19 1.05 1.28 175 0.79 0.9 2.15 1.37 1.47 4.47
176 2.31 2.11 0.83 1.12 3.09 4.17 177 2.22 3.12 1.77 1.39 2.38 0.99
178 2.79 1.63 2.89 1.09 3.69 9.77 179 1.84 1.61 0.79 1.59 0.41 1.19
180 0.42 0.55 0.34 0.12 0.59 0.82 181 1.06 0.82 0.38 1.14 0.51 2.14
182 3.43 3.26 1.86 2.6 1.88 1.14 183 4.62 3.41 5.18 3.43 2.21 4.56
184 10.55 11.75 12.34 13.53 3.2 3.21 185 0.43 0.27 1.38 0.41 0.82
0.25 186 13.6 8.37 13.86 18.03 12.22 12.54 187 100 57.47 87.77
68.09 84.44 63.09 188 3.42 1.31 1.12 0.78 3.45 3.61 189 0.17 0.22
0.86 0.18 0.33 0.07 190 18.52 8.42 3.32 1.56 4.42 9.84 191 1.65
0.59 0.36 0.31 0.35 0.34 192 0.7 0.56 1.47 1.22 0.43 1.1 193 1.9
2.7 1.11 6.08 1.35 6.53 194 0.29 0.22 1.13 0.21 0.44 0.55 195 5.19
1.81 1.61 1.57 1.25 1.3 196 3.26 1.19 1.16 0.6 1.14 0.58 197 6.75
21.34 4.94 22.2 10.89 4.76 198 1.29 0.99 0.43 0.64 0.55 0.51 199
0.57 0.49 0.64 4.22 0.41 1.26 200 0.25 0.12 0.46 0.12 0.08 0.09 201
0.19 0.22 0.17 0.08 0.15 0.92 202 0.32 4.72 13.45 0.22 0.34 0.23
203 0.9 0.85 4.79 0.91 0.33 1.58 204 0.67 1 0.95 1.02 0.8 1.6 205
0.77 0.27 0.56 0.54 0.5 1.24 206 0.84 1.34 0.62 0.54 0.27 0.38 207
1.63 0.74 43.66 10.77 0.7 10.62 208 19.87 39.92 12.24 37.08 8.95
6.05 209 0.08 0.09 0.25 0.09 0.14 0.11 210 2.65 2.63 0.89 2.03 1.54
1.15 211 0.56 0.68 1.41 0.62 0.54 0.39 212 3.33 7.01 37.74 39.78
6.1 16.72 213 0.35 0.19 0.08 0.16 0.16 0.12 214 0.92 0.59 1.08 0.35
0.6 2.36 215 0.16 0.28 0.86 0.57 1.24 0.49 216 1.62 1.17 0.85 0.66
1.28 1.22 217 3.58 1.69 1.94 4.27 1.64 1.31 218 2.33 2.94 2.89 2.8
0.81 1.47 219 2.16 2.5 1.04 4.06 0.28 3.58 220 0.13 0.1 0.41 0.11
0.13 0.07 221 1.6 3.45 1.26 1.14 0.65 1.53 222 0.99 0.73 0.38 0.37
0.58 0.57 223 1.51 1.4 0.84 1.38 1.07 2.6 224 1.54 1.92 24.61 3.89
2.42 2.24 225 1.45 2.61 1.35 0.36 1.42 2.27 226 1.28 0.97 2.42 0.96
1.93 2.71 227 0.43 0.28 0.18 0.33 0.25 0.66 228 0.9 0.68 0.63 1.91
0.87 0.48 229 0.89 0.37 0.3 0.54 0.94 0.41 230 0.07 0.09 0.1 0.11
0.15 0.1 231 0.69 0.57 0.27 0.19 0.14 0.4 232 6.02 4.13 3.63 2.31
9.64 15.63 233 5.15 3.75 3.93 2.43 8.6 6.56 234 1.34 2.83 2.16 2.28
2.64 3.56 235 1.6 3.76 3.14 4.73 2.2 3.53 236 1.97 2.64 0.66 1.66
0.71 0.96 237 0.2 18.16 23.66 0.39 0.43 0.37 238 1.32 1.57 1.47 1.3
0.39 0.51 239 16.05 13.17 8.14 28.46 2.22 12.18
240 35.56 27.44 20.28 41.75 10.35 6.04 241 0.23 0.11 0.62 0.21 0.31
0.24 242 3.01 2.2 1.69 1.02 5.58 4.26 243 1.27 1.35 0.8 8.02 1.08
3.07 244 0.56 0.73 0.26 0.48 1.19 0.46 245 5.84 3.28 2.47 1.78 7.92
6.79 246 9.97 5.88 7.24 9.41 9.24 7.49 247 3.5 4.2 6.51 6.51 7.98
13.04 248 1.77 2.29 0.88 1.89 1.22 1.16 249 7.15 6.49 8.39 4.64
7.22 9.99 250 1.12 0.76 0.74 3.92 0.81 1.31 Rank St4_NA_NB2
St4_NA_NB3 St4_NA_NB30 St4_NA_NB31 St4_NA_NB32 St4_NA_NB4- 1 0.04
0.05 0.03 0.02 0.13 0.02 2 0.19 0.26 0.4 0.44 0.21 0.22 3 1.43 1.33
1.1 1.65 2.51 1.38 4 3.46 4.24 4.99 12.14 4.28 12.94 5 4.33 5.83
3.53 1.18 2.15 2.63 6 15.11 17.85 11.25 6.89 19.19 11.35 7 5.69
9.49 8.67 10.28 14.78 10.12 8 0.32 0.25 0.32 0.33 0.1 0.24 9 1.41
1.81 1.31 1.73 0.41 2.08 10 3.15 4.19 3.14 4.45 3.82 2.88 11 0.11
0.11 0.15 0.15 0.27 0.14 12 3.1 3.22 3.25 2.42 3.23 3.54 13 1 1.1
1.36 1.75 2.06 1.01 14 5.16 6.39 5.17 9.86 10.31 9.87 15 1.05 1.54
1.6 2.38 2.1 2.7 16 1.84 2.46 0.86 3.66 8.55 5.01 17 9.83 13.43
15.64 51.6 18.05 37.79 18 0.04 0.03 0.04 0.04 0.09 0.02 19 0.21
0.24 0.26 0.29 0.1 0.13 20 2.64 2.13 2.09 5.38 11.13 5.28 21 3.59
3.83 4.5 2.72 9.28 1.67 22 0.06 0.06 0.07 0.09 0.06 0.09 23 1.42
1.12 1.11 2.52 2.19 2.6 24 1.56 1.3 1.21 1.42 1.42 1.29 25 11.29
12.52 6.77 28.77 15.46 18.8 26 15.81 19.38 20.99 10.73 9.65 9.9 27
0.25 0.25 0.17 0.42 0.44 0.54 28 19.42 31.2 15.83 48.08 26.76 71.64
29 0.45 0.54 0.42 0.41 0.25 0.57 30 0.17 0.26 0.19 0.38 0.61 0.37
31 13.45 7.6 11.48 11.64 0.07 9.18 32 3.74 5.34 7.06 2.93 5.05 1.23
33 1.21 1.36 1.38 0.53 1.52 0.52 34 0.31 0.3 0.31 0.31 0.2 0.24 35
1.78 2.45 1.16 2.21 2.46 6.92 36 6.68 6.12 6.09 4.62 5.7 6.6 37
1.84 2.25 1.79 3.76 3.57 2.64 38 0.17 0.19 0.13 0.19 0.23 0.17 39
0.91 1.03 1.13 1.15 0.3 1.08 40 3.2 2.86 2.51 2.22 1.83 2.11 41 2
2.31 1.66 1.52 2.4 2.25 42 2.07 2.5 2.98 1.05 1.49 0.89 43 4.86 4.5
3.87 0.77 2.64 0.87 44 0.8 0.74 0.75 0.58 0.69 0.53 45 0.67 0.73
0.84 1.29 0.95 0.89 46 0.97 0.99 0.99 6.27 1.11 6.76 47 0.2 0.21
0.23 0.39 0.63 0.42 48 1.06 1.25 0.73 0.2 0.32 0.57 49 0.19 0.4
0.26 0.59 0.35 0.47 50 0.35 0.16 0.22 0.17 0.37 0.16 51 0.51 0.38
0.56 0.46 0.48 0.31 52 0.43 0.34 0.43 0.37 0.72 0.34 53 7.59 6.51
5.39 12.41 8.29 8.97 54 1.34 1.39 1.12 0.69 0.7 0.67 55 1.43 1.38
1.06 1.94 1.64 1.7 56 1 0.77 0.77 0.75 0.86 0.63 57 4.01 5.24 5.61
6.75 6.38 5.65 58 24.88 20.71 14.56 37.79 16.64 34.32 59 1.4 0.98
1.45 1.79 3.19 1.94 60 30.43 27.28 23.7 7.24 16.17 19.67 61 31.65
33.38 24.05 9.15 14.52 22.83 62 2.33 3.25 3.06 5.36 6.51 3.79 63
4.9 6.74 6.56 12.35 8.33 8.88 64 5.84 2.3 5.04 11.08 8.57 7.5 65
14.59 17.84 16.23 11.37 11.41 10.83 66 0.62 0.73 0.64 0.3 0.26 0.3
67 1.44 1.91 0.96 1.11 0.8 1.42 68 1.59 2.06 2.35 2 1.61 1.9 69
11.42 5.83 8.59 1.14 13.47 2.38 70 0.04 0.03 0.2 0.06 0.02 0.02 71
5.04 5.79 3.3 5.11 7.68 4.67 72 30.3 38.86 26.93 41.3 53.93 23.56
73 27.19 25.47 28.24 8.11 7.12 13.47 74 0.79 0.83 1.89 1.41 1.65
1.48 75 4.05 3.63 2.41 3.27 2.36 4.35 76 4.37 6.75 6.97 9.17 7.08
7.34 77 10.38 10.78 8.5 18.25 14.07 19.44 78 2.16 2.83 2.05 5.97
6.25 6.59 79 2.38 2.32 7.64 5.48 8.37 5.05 80 5.89 7.42 9.77 20.77
5.69 14.47 81 14.9 13.32 10.53 12.61 16.61 14.42 82 0.7 0.89 0.81
1.14 0.52 0.77 83 0.43 0.46 0.71 0.43 0.29 0.32 84 0.74 0.71 0.7
1.16 0.79 1.96 85 0.23 0.22 0.24 0.22 0.28 0.23 86 0.58 0.47 0.75
0.71 0.68 0.61 87 1.14 1.3 1.11 1.44 1.12 0.98 88 0.52 0.56 0.61
0.57 0.53 0.5 89 0.27 0.37 0.36 0.19 0.12 0.15 90 0.41 0.4 0.25
0.12 0.13 0.15 91 0.73 0.92 0.81 0.79 0.7 0.92 92 2.51 2.42 1.97
0.05 1.33 0.18 93 1.09 0.98 0.97 1.04 0.37 1.2 94 0.4 0.53 0.51
0.27 0.37 0.21 95 4.12 4.45 3.5 6.71 4.94 5.96 96 0.14 0.12 0.1
0.09 0.05 0.1 97 3.37 2.1 1.4 0.95 1.25 1.56 98 1.99 2.47 2.04 2.92
1.89 2.94 99 0.15 0.15 0.15 0.36 0.54 0.26 100 14.23 13.32 7.24
26.65 34.2 18.47 101 4.95 3.97 3.21 11.73 14.1 14 102 1.05 0.79
0.87 0.9 0.76 0.74 103 1.14 1.33 1.53 2.02 1.67 1.19 104 1.67 2.39
2.03 2.75 1.51 2.67 105 3.16 2.31 3.09 2.96 3 2.27 106 2.15 3 2.27
5.14 3.34 5.86 107 2.03 1.92 1.85 1 0.73 1.09 108 39.56 33.2 16.66
20.09 29.88 31.81 109 0.75 0.74 0.65 0.9 0.69 0.64 110 2.78 3.42
3.54 3.25 5.27 2.58 111 41.25 65.42 26.05 39.22 20.41 50.47 112
9.42 11.84 3.05 7.8 8.42 11.32 113 7.26 10.09 8.3 2.77 7.46 3.23
114 1.15 1.1 1.1 0.5 1.24 0.43 115 1.64 1.75 1.68 2.27 1.34 2.91
116 0.14 0.13 0.33 0.26 0.13 0.24 117 0.14 0.14 0.16 0.3 0.45 0.25
118 1.68 1.88 2.77 2.26 7.15 5.33 119 4.97 5.78 5.24 8.42 5.48 10.3
120 0.98 0.83 0.96 0.98 1.16 0.85 121 1.77 1.78 2.53 1.66 2.78 4.38
122 4.53 7.04 7.17 4.4 5.04 5.16 123 2.87 3.17 2.35 3.96 2.48 5.3
124 1.47 2.14 1.71 1.16 1.21 1.73 125 66.32 45.73 41.85 32.34 40.51
45.33 126 12.04 9.77 5.61 3.37 1.11 6.02 127 1.93 3.12 2.09 1.88
3.12 2.64 128 0.57 0.67 0.55 1.06 0.93 0.45 129 1.25 1.67 1.48 2.16
2.22 2.65 130 17.04 32.54 12.13 21.43 9.44 41.6 131 5.35 4.95 2.85
3.78 10.81 2.7 132 3.31 4.11 4.78 4.82 7.81 4.27 133 2.35 2.91 3.61
10.69 5.57 14.87 134 1.41 1.57 1.2 2.28 1.55 3.87 135 0.9 1.15 1.05
1.24 0.51 1.35 136 0.76 0.61 1.41 0.32 0.37 0.44 137 0.12 0.09 0.23
0.19 0.25 0.11 138 1.97 2.65 2.43 2.08 2.9 1.6 139 0.17 0.19 0.22
2.35 0.71 1.31 140 59.2 53.79 62.79 4.92 6.68 12.16 141 2.36 3.02
3.09 5.43 6.09 3.76 142 1.48 2.13 2.06 1.98 3.34 1.46 143 3.15 4.07
3.46 4.56 5.52 3.65 144 2.5 2.93 2.91 1.11 0.98 1.38 145 0.49 0.51
0.66 0.36 0.07 0.46 146 6.51 9.03 7.04 5.92 10.07 8.91 147 0.88
0.68 0.81 0.87 0.85 0.55 148 6 7.78 3.13 3.74 3.66 2.94 149 4.22
4.67 5.03 3.73 4.92 2.8 150 0.4 0.37 0.41 0.21 0.64 0.2 151 8.04
19.13 6.18 1.67 5.44 2.77 152 1.23 0.95 1.1 1.31 1.17 1.33 153 1.07
0.93 1.02 1.42 1.26 1.32 154 9.39 11.4 8.19 11.61 10.34 10.46 155 3
2.6 2.8 1.35 0.47 1.42 156 6.16 9.98 6.41 2.56 2.49 1.62 157 0.26
0.28 0.3 0.13 0.21 0.12 158 0.52 0.51 0.56 0.55 1.95 0.42 159 0.23
0.25 0.33 0.19 0.22 0.15 160 0.17 0.2 0.22 0.09 0.17 0.08 161 0.79
0.73 0.73 1.23 1.93 1.16 162 2.98 3.95 3.18 6.52 5.8 6.91 163 25.03
25.32 26.74 33.55 18.76 31 164 2.64 3.81 3.24 9.52 5.27 7.93 165
0.13 0.12 0.08 0.11 0.05 0.13 166 5.38 5.76 5.15 10.6 8.95 8.33 167
85.69 91.81 74.89 28.33 54.43 100 168 7.76 10.95 8.1 9.4 0.15 7.38
169 0.01 0.01 0.01 0.03 0.01 0.01 170 10.14 11.84 9.66 20.01 11.83
21.22 171 17.16 16.87 12.45 6.09 3.05 10.16 172 0.28 0.15 0.27 0.3
0.29 0.19 173 5.58 5.5 7.9 2.54 3.16 3.17 174 2.52 2.7 1.85 0.79
0.59 0.95 175 1.7 2.1 1.05 1.09 1.33 1.19 176 5.12 4.69 5.42 15.16
8.55 8.94 177 2.43 3.47 1.96 1.23 1.98 2.67 178 3.28 2.71 2.85 2.47
2.95 2.15 179 0.55 0.5 0.93 0.72 0.4 0.44 180 0.45 0.44 0.43 0.29
0.17 0.31 181 0.61 0.69 0.74 1.18 0.89 1.23 182 1.72 2.03 1.46 2.1
2.92 2.33 183 3.19 3.58 2.08 2.05 1.88 6.59 184 5.34 6.31 4.96 6.14
6.47 4.27 185 0.54 0.42 0.43 0.31 0.47 0.2 186 11.94 11.85 6.55
14.91 10.39 13.12 187 73.69 100 27.06 48.28 32.93 100 188 3.11 2.09
1.93 1.57 1.68 2.11 189 0.18 0.23 0.22 0.12 0.15 0.11 190 4.25 4.48
4.69 10.17 2.97 25.02 191 0.32 0.51 0.6 0.55 0.13 0.35 192 1.18
1.41 1.38 1.67 1.67 1.08 193 7.23 7.13 6.78 7.1 9.45 8.62 194 0.41
0.34 0.37 0.31 0.24 0.23 195 2.28 1.78 1.67 15.35 2.04 9.14 196 0.8
0.83 0.78 1.61 0.44 1.32 197 5.56 6.9 8.15 14.68 10.27 13.24 198
0.6 0.5 0.53 0.31 0.16 0.45 199 3.97 5.6 3.3 3.99 5.19 4.04 200
0.13 0.12 0.09 0.13 0.05 0.13 201 0.13 0.17 0.23 0.15 0.09 0.13 202
5.89 5.54 5.57 6.59 0.22 3.55 203 0.56 0.54 0.53 1.3 1.02 1.24 204
0.81 1.05 0.91 0.52 1.24 0.54 205 0.69 0.66 0.55 0.71 0.75 0.53 206
0.92 1.21 1.16 1.5 0.48 0.69 207 10.78 7.94 5.9 4.17 6.58 4.33 208
6.78 6.01 3.85 31.48 20.26 19.38 209 0.1 0.1 0.11 0.1 0.12 0.1 210
1.54 1.63 1.5 3.24 2.4 3.97 211 0.31 0.32 0.44 0.3 0.66 0.26 212
47.36 68.52 39.55 11.34 44.85 28.79 213 0.25 0.24 0.22 0.23 0.07
0.25 214 0.64 0.81 0.8 0.28 0.54 0.31 215 0.68 0.41 0.57 0.28 0.15
0.24 216 1.92 2.09 3.05 1.36 0.98 1.29 217 4.91 2.9 2.41 5.36 5.9
6.73 218 2.81 2.68 2.31 2.02 2.56 2.17 219 1.13 1.37 1.08 2.35 3.16
1.32 220 0.16 0.14 0.24 0.1 0.22 0.08 221 0.58 0.63 0.97 0.99 1.17
1.21 222 0.59 0.68 0.69 1.05 0.32 0.95 223 1.74 1.61 1.89 1.4 0.94
1.09 224 1.14 1.22 1.48 1.57 0.97 1.11 225 2.29 3.28 2.91 0.98 1.82
0.66 226 0.89 0.66 0.64 0.85 0.58 0.75 227 0.95 1.09 0.63 0.39 0.44
0.34 228 0.76 0.69 0.79 4.89 3.08 8.24 229 0.73 0.77 0.65 1.17 0.48
1.03 230 0.08 0.07 0.14 0.08 0.13 0.04 231 0.22 0.25 0.45 0.31 0.16
0.24 232 5.56 8.75 9.99 3.76 6.28 5.47 233 5.47 5.94 5.63 2.18 2.23
3.87 234 3.13 3.08 1.73 1.63 2.03 1.41 235 1.05 1.3 1.46 1.5 1.78
1.28 236 0.72 0.69 0.68 2.33 0.49 2.79
237 7 9.99 18.41 12.19 0.38 9.85 238 0.62 1.01 1.13 1.78 1.53 1.46
239 28 26.79 15.99 37.03 27.12 40.72 240 17.14 16.33 14.59 18.83
21.32 23.44 241 0.53 0.43 0.33 0.25 0.22 0.32 242 2.88 2.39 2.53
2.57 3.73 2.14 243 2.35 2.94 2.46 2.93 3.93 2.21 244 0.34 0.31 0.4
0.94 0.65 0.78 245 4.71 3.67 3.84 5.17 6.19 5.23 246 5.64 4.49 5.22
4.38 6.15 5.05 247 11.64 11.39 8.72 7.25 7.61 8.52 248 1.78 2.33
1.85 2.91 4.53 1.72 249 6.62 6.39 4.71 3.06 6.62 9.45 250 2.17 3.09
2.51 3.17 4.64 2.87
TABLE-US-00013 TABLE 9B Training Samples Poor-Outcome patients (2nd
Bar of FIG. 7A) Rank Gene St2_NA_NB18 St3_A_NB75 St4_A_NB21
St4_A_NB254 St4_A_NB266 St4_A_- NB27 1 DLK1 5.34 0.39 6.96 3.59
0.97 2.14 2 est 1.16 10.52 3.73 5.12 1.78 15.56 3 PRSS3 4.61 32.17
7.93 13.15 4.78 1.39 4 ARHI 0.48 0.33 0.4 0.57 0.67 0.55 5 ARC 4.06
7.21 7.56 12.84 2.58 3.98 6 SLIT3 61.76 85.49 23.26 33.29 38.92
7.43 7 CNR1 0.96 1.85 1.75 0.84 2.57 4.6 8 est 1.48 14.9 4.62 7.53
0.71 0.86 9 est 0.43 0.13 0.07 0.11 0.47 0.27 10 FLJ25461 1.02 0.32
0.37 0.49 0.75 0.32 11 est 6.99 0.59 12.8 3.24 1.05 2.24 12 CD44
0.56 0.16 0.2 1.17 0.68 0.12 13 est 2.26 0.2 0.31 0.84 1.44 4.53 14
ROBO2 15.52 1.02 3.08 2.13 2.8 6.52 15 BTBD3 0.29 0.28 0.24 0.34
0.3 0.36 16 MYCN 3.6 54.67 72.43 37.8 21.24 42.7 17 est 4.87 2.52
2.94 4.43 5.89 2.69 18 JPH1 0.18 0.33 0.88 0.27 0.54 0.65 19 KLRC3
0.04 0.03 0.05 0.17 0.09 0.04 20 est 25.13 50.6 40.74 11.44 33.86
34.25 21 RET 6.74 22.79 1.34 28.33 2.43 1.33 22 CRABP1 0.09 2.46
1.37 0.41 0.11 1.21 23 ECEL1 0.25 0.44 0.48 0.26 0.12 0.26 24
LOC283120 17.28 3.04 5.07 7.09 1.97 1.04 25 HMGA2 16.26 2.86 2.18
4.76 5.55 8.7 26 SYNPO2 14.55 23.09 37.13 60.99 31.01 7.45 27
LOC163782 0.05 0.06 0.12 0.07 0.08 0.06 28 VSNL1 7.75 12.02 1.08
4.22 2.14 12.7 29 HS3ST4 0.04 0.08 0.07 0.06 0.09 0.06 30 AKR1C1
0.17 0.24 0.01 0.07 0.04 0.36 31 est 11.61 0.23 0.03 13.81 7.95
0.03 32 GPR22 27.65 11.6 31.12 11.05 28.75 34.35 33 est 4.81 2.72
8.39 3.02 2.88 3.12 34 est 0.05 0.1 0.08 0.13 0.35 0.08 35 CCNA1
6.65 4.62 0.81 8.89 1.94 2.86 36 PKIB 0.39 1.77 0.38 3.09 1.41 0.41
37 est 0.85 0.31 0.37 0.39 0.84 0.29 38 GAL 8.4 2.65 12.39 6.84
2.32 12.17 39 est 1 1.06 1.97 1.04 0.97 3.76 40 LOC221303 9.52 5.7
41.43 22.71 49.92 29.62 41 est 7.57 20.32 6.69 9.36 5.72 1.41 42
est 3.32 52.47 4.94 60.04 16.22 0.97 43 BMP7 0.12 6.4 3 9.34 1.62
1.13 44 SLC30A3 2.18 6.99 14.04 3.07 3.11 10.88 45 FLJ10539 0.32
0.32 0.29 0.92 0.34 3.31 46 AMIGO2 0.4 0.25 0.31 0.98 2.32 0.35 47
AKR1C2 0.17 0.25 0.02 0.07 0.06 0.28 48 MGP 0.14 0.09 0.2 0.27 0.65
0.23 49 PCSK1 0.2 0.17 0.27 0.23 0.17 0.23 50 HK2 1.54 2.09 4.1
1.08 0.36 0.46 51 est 0.5 1.08 1.09 2.88 0.45 3.05 52 est 1.54 0.51
1.44 0.56 0.22 1.39 53 IL7 2.26 0.84 0.89 1.04 1.27 0.72 54 PRSS12
1.35 16.31 2.6 20.99 3.36 0.72 55 GABARAPL1 0.17 0.12 0.51 0.36
0.57 0.15 56 DEFB129 2.94 5.53 10.05 3.43 3.3 11.9 57 NAV3 0.22
0.46 0.41 0.84 2.46 0.57 58 RAB3B 6.2 1.96 1.23 1.11 3.49 0.9 59
KRT6B 3.45 2.12 0.34 0.68 1.58 1.02 60 BEX1 24.26 31.07 59.91 34.92
13.92 47.34 61 est 27.18 36.94 59.77 39.76 15.3 38.99 62 est 2.09
0.73 0.59 0.63 0.45 0.39 63 SCYL1 2.53 2.18 1.21 2.59 1.85 1.36 64
est 1.12 1.03 0.56 1.35 1.13 0.79 65 RYR2 9.39 2.81 8.55 4.13 6.07
16.41 66 LRBA 0.99 1.03 2.2 1.87 1.41 0.72 67 CSPG3 1.76 7.66 0.85
2.91 1.75 10.17 68 est 0.62 0.92 0.65 0.6 0.4 0.9 69 MMP12 23.54
31.92 6.4 13.35 10.9 1.4 70 CHRNA1 0.02 0.02 0.03 0.04 0.05 0.03 71
est 3.53 1.08 0.73 1.54 1.33 1.15 72 est 14.9 4.6 4.01 6.09 6.71
10.92 73 HNRPH1 62.09 46.23 6.31 87.81 96.22 12.8 74 LOC113251 0.86
0.44 0.28 0.26 0.5 0.72 75 est 1.06 0.86 0.49 0.95 2.17 2.82 76 PAG
2.14 2.22 1.29 2.16 1.52 1.18 77 PROK2 17.38 1.6 7.17 3.15 4.98 9.2
78 HS6ST1 6.39 6.04 0.86 9.28 2.32 3.19 79 est 9.82 2.13 1.27 2.11
8.75 6.75 80 PCDH9 3.43 1.63 0.45 2.69 1.31 6.81 81 est 25.52 28.46
14.05 20.05 27.98 5.14 82 est 0.58 0.16 0.12 0.15 0.24 0.17 83 GLDC
0.31 6.41 0.39 2.25 0.98 1.06 84 ADRB2 0.24 0.26 0.35 0.45 0.67
0.54 85 ICSBP1 6.96 2 6.77 7.93 1.2 3.79 86 CD48 0.25 0.11 0.14
0.84 0.53 0.09 87 est 0.56 0.44 0.43 1.19 0.62 2.61 88 DYRK1B 0.9
4.79 2.22 2.33 1.05 5.56 89 KLRC1 0.06 0.07 0.05 0.21 0.1 0.06 90
est 0.42 1.01 1.96 1.39 0.9 1.06 91 est 3.09 4.04 6.11 4.15 4.18
2.39 92 est 0.95 0.99 4.22 1.94 0.73 2.86 93 MOXD1 0.09 0.09 0.12
0.58 0.67 0.12 94 est 1.06 1.64 2.98 1.89 1.32 1.88 95 est 2.14 0.4
0.51 2.44 2.4 8.86 96 GAS1 0.06 0.02 0.05 0.05 1.24 0.06 97 COL9A2
4.02 0.42 0.33 0.28 0.27 0.35 98 est 1.04 0.72 0.47 0.71 1.05 2.41
99 DRPLA 0.16 0.19 0.03 0.07 0.05 0.24 100 est 8.66 5.79 1.1 4.15
12.41 5.53 101 REPRIMO 1.28 1.82 1.45 2.39 2.76 11.99 102 CACNA2D2
2.52 4.16 7.74 3.14 2.78 9.26 103 NEBL 0.7 0.31 0.48 0.42 0.92 2
104 est 0.3 0.19 0.28 0.75 2.05 0.51 105 HLA-DQA1 1.13 0.48 0.47
5.01 2.34 0.52 106 EDG3 8.04 15.81 23.13 23.18 5.64 4.23 107 CPVL
0.3 0.19 0.31 1.19 0.86 0.24 108 FLJ32884 7.57 2.16 4.64 5.17 16.88
3.71 109 LCP1 0.41 0.11 0.2 1.01 0.76 0.17 110 est 0.86 0.92 0.59
1.12 0.6 0.62 111 est 46.81 67.42 40.27 93.36 13.93 44.96 112 est
3.92 4.25 1.82 1.19 2.77 3.44 113 est 2.31 1.64 0.97 1.06 1.37 1.27
114 DKFZP564C152 4.58 1.7 11.74 2.47 1.39 2.62 115 DMN 0.78 0.36
0.83 0.7 0.58 0.67 116 GABRA5 0.19 0.85 2.02 0.44 0.98 0.54 117
AKR1C3 0.11 0.19 0.04 0.06 0.07 0.15 118 LOC168850 4.1 2.14 0.54
1.29 2.43 1.03 119 est 1.58 0.94 1.07 2.47 3.46 1.43 120 KCNQ2 4.05
2.8 6.7 2.12 1.19 1.91 121 NME5 2.36 3.87 2.26 1.64 3.34 2.75 122
est 1.42 0.95 1.13 0.95 1.23 1.17 123 PBX1 1.1 1.11 0.55 0.8 1.29
2.93 124 CNTNAP2 0.66 0.52 0.4 0.79 1.02 0.8 125 est 9.83 21.2 0.71
12.86 2.67 0.82 126 SPON1 2.25 4.27 20.56 5.81 32.07 2.77 127 CDH8
0.93 1.12 0.37 0.65 0.61 0.47 128 PRKCB1 0.32 0.12 0.34 0.4 0.32
0.54 129 SLC21A11 1.56 0.35 1.43 1.12 1.59 0.85 130 MAP4 8.43 16.27
2.16 4.78 3.49 22.39 131 est 4.26 0.98 1.2 2.18 4.16 2.07 132 SCN7A
1.53 1.71 0.95 0.56 1.24 2.4 133 est 1.02 1.1 0.68 1.15 1.35 5.99
134 est 1.03 0.7 0.78 0.71 1.82 1.24 135 est 0.61 0.33 0.23 0.34
0.4 0.64 136 est 0.66 3.04 1.11 1.93 0.51 2.42 137 CDW52 0.1 0.06
0.07 0.52 0.26 0.06 138 ABCB1 6.37 4.83 8.56 6.38 3.1 1.22 139 est
0.17 0.2 0.3 0.35 0.19 0.59 140 OSF-2 21.65 31.52 7.76 100 48.2
3.42 141 NRXN1 1.59 0.94 0.49 0.69 0.51 0.38 142 ADAM22 3.35 10.38
10.35 5.79 3.55 3.58 143 est 32.13 13.97 18.2 13.34 12.09 8.19 144
TRGV9 0.3 0.35 0.26 1.16 1.16 0.35 145 est 0.21 0.07 0.04 7.47 2.75
0.03 146 PTPRD 4.98 4.68 1.01 1.13 2.77 2.74 147 est 0.89 1.22 0.64
1.32 0.98 0.58 148 HS3ST2 10.47 4.65 24.13 12.69 3.13 1.9 149 FGF13
3.61 4.16 1.08 1.5 1.13 3.69 150 MKI67 1.03 1.26 0.82 1.42 1.19
1.31 151 KIF12 0.99 1.75 0.92 1.98 1.43 0.99 152 est 2.51 3.39 2.33
3.93 2.41 12.54 153 est 0.3 0.16 0.33 1.16 1.19 0.23 154 est 2.85
1.73 0.73 7.93 7.96 0.81 155 est 0.81 0.81 0.21 20.91 5.82 0.15 156
est 1.83 0.77 0.51 1.07 0.78 0.94 157 KLIP1 1.03 1 0.93 0.92 0.86
1.41 158 est 1.83 0.8 2 0.76 2.26 0.89 159 LOC157570 0.64 0.77 1.07
0.75 0.73 1.07 160 MAD2L1 0.5 0.68 0.99 0.68 0.37 0.97 161 est 1.21
0.26 0.76 0.61 0.4 0.29 162 est 5.82 1.57 1.56 1.5 2.68 5.42 163
RGS5 60.33 9.32 44.35 67.04 13.44 21.18 164 ATP2B4 0.61 1.38 0.61
0.94 0.84 1.02 165 HMGCL 0.07 0.04 0.05 0.07 0.14 0.03 166 ODZ3
0.94 2.55 0.91 3.97 0.96 4.27 167 CHGA 100 100 97.08 100 32.96
25.79 168 MGC33510 6.2 0.26 0.18 7.51 5.72 0.12 169 GAGE5 0.03 0.01
0.02 0.02 0.01 0.05 170 SARDH 7.96 2.74 1.32 3.57 3.73 8.46 171 est
0.91 1.33 0.94 5.73 56.13 0.96 172 DAT1 0.49 3.12 0.51 4.91 0.9
1.49 173 FUCA1 1.99 0.46 0.37 2.65 2.82 0.5 174 TM6SF2 5.69 3.17
1.87 1.78 5.35 1.53 175 KCNK9 5.27 4.04 5.45 4.63 1.97 2.58 176
ADCYAP1 0.57 0.98 0.87 5.26 0.79 0.53 177 PLXNA4 3.14 0.79 1.41
0.93 1.22 1.22 178 HLA-DMB 1.28 0.47 0.32 4.65 2.2 0.46 179 est
1.15 0.27 0.26 0.43 0.9 0.98 180 est 0.17 0.07 0.08 0.44 1.14 0.08
181 GRIN3A 0.35 0.42 0.63 0.61 0.53 0.75 182 OSBPL3 0.65 0.63 0.69
1.38 1.33 1.52 183 ODZ4 5.41 25.84 1.78 6.23 4.31 1.98 184 est 2.07
1.67 3.14 1.14 2.05 2.63 185 E2F1 1.24 1.89 1.32 1.48 1.65 1.51 186
MGC16664 15.6 40.99 14.22 11.1 20.57 23.76 187 HMP19 100 100 100
100 22.17 86.03 188 IL2RB 1.38 0.42 0.54 3.17 1.94 0.57 189 TOPK
0.49 0.49 0.74 0.69 0.45 0.73 190 ALDH1A1 1.18 0.98 1.26 5.89 4.82
1.65 191 CED-6 3.52 1.12 5.77 2.69 1.93 0.75 192 est 1.02 0.52 0.63
0.3 0.41 1.12 193 A2BP1 1.66 2.33 1.16 0.74 1.4 1.64 194 LY6E 3.2
0.44 4.08 1.83 0.78 1.14 195 est 1.07 0.89 0.77 1.37 1.96 0.96 196
est 0.36 0.18 0.32 0.45 1.42 0.91 197 PLXNC1 1.51 3.05 1.07 3.61
3.03 2.47 198 EFS 0.31 0.9 1.77 1.86 3.12 0.48 199 ACTN2 3.95 0.36
0.36 2.09 0.79 1.63 200 MYC 0.08 0.04 0.04 0.07 0.16 0.03 201
KIAA0527 0.3 0.41 0.94 0.99 1.07 1.66 202 C6orf31 5.65 0.31 0.2
6.29 5.12 0.15 203 DLL3 1.58 5.19 4.11 6.96 1.58 6.09 204 est 2.14
4.67 4.57 1.48 1.03 1.79 205 STK33 0.62 1.22 2.7 1.65 0.67 1.14 206
SEMA3A 0.13 0.64 0.14 0.44 0.81 0.34 207 est 3.3 4.99 0.84 2.07 4
0.52 208 IGSF4 7.22 9.19 1.28 3.46 5.55 9.74 209 CKS2 0.27 0.58
0.81 0.54 0.32 0.46 210 est 0.98 1.28 0.84 0.44 0.57 1.2 211 est
1.36 1.65 1.08 1.26 2.07 1.43 212 SIX3 2.87 71.85 1.08 5.05 6.25
31.73 213 FLJ22002 0.07 0.06 0.07 0.09 0.09 0.06 214 HSD17B12 1.21
1.01 3.1 1.74 0.93 1.14 215 HBA2 1.15 1.46 0.44 1.66 2.81 0.53 216
CDH11 0.68 0.51 0.45 1.35 4.09 0.52 217 RGS9 1.49 0.91 0.61 0.9
0.81 1.13 218 est 1.59 0.54 1.36 1.27 1.72 1.36 219 NCAM2 5.96 1.27
0.49 1.23 2.27 5.36 220 BIRC5 0.52 0.82 0.7 0.31 0.31 0.61 221 est
4.02 0.66 0.51 0.81 1.28 1.35 222 GNG12 0.31 0.16 0.42 0.5 0.96
0.19 223 GPIG4 0.42 0.3 0.2 1.01 0.85 1.15 224 est 1.7 11.89 3.4
12.57 3.47 1.87 225 ENPP4 3.51 4.1 0.48 2.86 1.65 0.28 226 FMNL
2.91 2.38 4.18 2.81 2.73 1.68 227 est 0.06 0.13 0.17 0.19 0.16 0.37
228 PIWIL2 1.09 0.56 0.73 0.62 0.53 7.85 229 CLSTN1 0.39 0.31 0.15
0.16 0.3 0.29 230 UHRF1 0.23 0.57 0.3 0.08 0.11 0.26 231 est 0.45
0.05 0.15 0.29 0.6 0.19 232 SLC40A1 2.25 0.84 1.18 3.05 2.23 2.81
233 CLECSF6 3.15 0.96 3.16 5.22 6.26 1.4 234 est 3.13 9.35 3.42
3.37 3.22 5.68 235 BKLHD2 4.31 8.55 3.29 4.57 2.58 4.96 236 est
0.52 0.33 0.4 0.7 0.98 0.32 237 est 8.45 0.54 0.54 9.93 12.67 0.4
238 est 0.91 0.31 0.51 0.39 0.63 1.36 239 SORCS1 1.1 13.82 1.28
24.72 1.91 17.57 240 NRP2 8.02 1.97 4.82 8.9 16.36 6.09 241 E2-EPF
0.73 1.25 1.76 1.34 0.36 1.22 242 CAST 1.76 0.45 0.29 3.13 2.73
0.64
243 KIAA1384 0.91 0.77 0.56 0.81 0.96 0.56 244 KIAA0644 0.85 0.28
0.14 0.21 0.65 0.28 245 HLA-DRB3 2.13 0.45 0.66 4.23 4.74 0.86 246
PMP22 5.9 1.07 3.04 1.92 2.59 4.15 247 DJ79P11.1 6.93 11.41 24.48
12.38 7.27 14.69 248 SOX5 0.85 0.93 0.44 0.5 1.02 0.85 249 CD3E
19.65 7.23 7.63 12.37 4.03 2.26 250 est 0.79 1.04 0.3 0.59 0.61
0.42 Rank St4_A_NB278 St4_A_NB79 St4_NA_NB205 St4_NA_NB207
St4_NA_NB209 St4_NA_- NB210 1 0.04 1.72 0.11 0.52 5.08 5.03 2 0.55
4 1.14 1.09 2.7 3.6 3 11.25 11.65 6.93 9.83 6.46 4.73 4 1.27 0.53
0.56 4.25 0.67 0.83 5 5.03 8.48 3.21 27.46 57.24 1.7 6 45.06 38.69
26.47 61.8 56.04 45.74 7 3.77 0.65 1.57 1.4 1.84 4.68 8 3.82 5.16
0.14 0.25 0.45 5.74 9 0.15 0.08 2.29 0.35 1.07 0.5 10 0.45 0.44
0.26 0.56 0.68 0.38 11 0.13 1.74 0.28 0.55 4.05 4.55 12 0.3 0.25
1.61 2.33 0.61 0.52 13 4.61 2.01 0.09 0.13 0.15 1.12 14 0.76 0.71
0.39 0.92 4.37 5.76 15 0.62 0.25 0.4 0.33 0.37 0.37 16 45.64 87.87
5.61 1.83 3.02 4.98 17 1.74 1.64 6.5 2.39 1.66 4.57 18 0.22 0.59
0.23 0.13 0.28 0.2 19 0.05 0.06 0.06 0.05 0.09 0.03 20 34.11 34.43
5.94 4.73 9.44 58.72 21 17.47 7.41 0.92 23.8 25.05 15.16 22 7.18
1.21 0.1 0.17 0.15 0.19 23 0.85 0.25 1.22 0.21 0.29 0.28 24 0.98
7.6 0.88 1.19 22.26 16.32 25 2.54 1.01 6.58 4.38 2 6.57 26 81.74
81.35 10.74 15.96 26.48 27.77 27 0.16 0.13 0.09 0.1 0.08 0.08 28
4.49 9.26 2.48 4.38 2.9 5.4 29 0.08 0.06 0.08 0.16 0.11 0.1 30 0.26
0.13 0.37 0.4 0.03 0.11 31 9.71 0.13 9.66 0.06 4.16 13.18 32 1.78
8.44 28.04 11.47 51.2 63.52 33 3.23 2.45 2.54 6.65 3.54 3.7 34 0.17
0.09 0.48 0.14 0.07 0.03 35 2.5 2.48 13.18 15.32 8.31 6.82 36 1.39
1.65 2.73 1.35 0.99 2.51 37 0.78 0.32 0.32 0.66 0.8 0.6 38 1.61
2.76 0.11 0.26 1.77 3.83 39 1.76 1.8 1.46 12.63 4.74 0.46 40 11.03
3.7 4.13 4.24 5.49 14.45 41 8.85 4.09 2.56 5.32 5.14 3.58 42 11.9
92.78 0.71 1.56 7.68 10.73 43 6.87 3.8 5 3.26 3.25 0.29 44 2.25
6.42 0.36 0.57 3.21 0.96 45 0.5 0.68 0.46 0.15 0.36 0.33 46 0.3
0.56 0.78 0.71 0.8 1.86 47 0.31 0.13 0.39 0.47 0.04 0.12 48 0.05
0.41 0.25 0.38 0.03 0.07 49 0.22 0.06 0.11 0.46 0.09 0.15 50 1.38
0.78 0.17 0.2 2.14 2.32 51 0.95 0.58 0.66 1.93 4.35 0.86 52 0.62
0.75 0.42 9.85 2.24 1.29 53 0.7 1.11 8.25 1.12 4.78 2.9 54 5.19
30.7 0.66 0.91 3.73 5.25 55 0.28 0.25 0.83 1.74 0.56 0.57 56 3.51
5.87 0.66 0.67 3.34 0.79 57 1.47 0.45 3.37 1.87 0.77 0.59 58 1 2.3
3.51 6.99 8.3 12.63 59 0.5 1.17 1 1.38 1.3 1.96 60 36.17 36.6 25.84
41.96 19.43 17.7 61 41.25 31.99 26.17 43.09 22.7 18.16 62 1.2 5.35
0.31 1 0.22 0.92 63 1.38 0.86 1.16 2.25 1.57 3.83 64 3.21 1.17 4.29
2.28 1.82 1.49 65 2.42 0.89 3.16 2.12 9.89 6.41 66 0.65 5.24 0.62
0.66 1.43 2.26 67 7.68 4.56 3 0.8 4.13 1.21 68 0.54 0.29 3.88 0.41
0.6 0.57 69 3.36 4.43 5.51 3.85 56.24 6.37 70 0.02 0.02 0.04 0.02
0.02 0.03 71 5.27 0.86 0.88 1.72 1.17 1.82 72 39.4 5.98 13.52 3.61
12.07 9.43 73 19.17 15.48 8.1 17.87 100 85.58 74 0.42 0.39 0.49
0.53 0.46 0.74 75 3.15 1.3 1.36 1.05 0.87 1.92 76 1.36 0.69 0.96
1.89 1.46 3.56 77 1.44 1.95 2 1.84 5.33 8.84 78 3.13 3.42 15.77
15.9 9.86 8.33 79 1.8 1.05 4.7 3.32 4.93 4.91 80 16.91 1.11 4.2
2.67 5.95 8.74 81 26.96 34.02 25.67 15.06 18.46 20.83 82 0.08 0.84
0.12 0.13 0.2 0.38 83 4.17 4.43 0.98 0.76 0.95 0.63 84 0.22 0.43
2.96 0.51 0.38 0.35 85 1.29 2.05 0.16 0.38 1.55 2.66 86 0.06 0.2
0.47 0.22 0.5 0.18 87 0.74 0.34 0.72 0.33 0.94 0.76 88 0.42 2.11
0.79 0.99 1.32 1.67 89 0.09 0.07 0.12 0.11 0.14 0.08 90 0.86 2.25
0.19 0.17 0.8 0.15 91 1.69 5.1 1.59 0.92 4.96 5.03 92 3.83 1.95
1.44 0.18 2.41 0.86 93 0.08 0.38 0.37 0.84 0.18 0.16 94 1.24 1.54
0.82 0.25 0.98 1.97 95 4.12 1.38 6.85 2.29 1.83 3.3 96 0.03 0.07
0.05 0.09 0.03 0.02 97 0.26 0.56 0.28 0.55 0.86 1.31 98 2.17 0.54
0.37 0.95 1.25 1.74 99 0.2 0.13 0.3 0.36 0.05 0.12 100 31.65 3.39
13.22 2.94 4.46 9.48 101 1.91 2.01 13.52 2.02 1.58 3.16 102 3.28
4.77 0.81 0.83 2.8 0.81 103 0.79 0.55 0.25 1.07 0.43 0.8 104 1.47
0.12 2.63 1.12 1.88 1.35 105 0.18 1.51 1.57 1.82 0.47 0.38 106 3.92
21.39 4.37 2.8 2.69 8.68 107 0.09 0.71 0.57 0.53 0.28 0.17 108 1.6
1.61 8.75 4.32 13.52 17.6 109 0.06 0.35 0.65 0.23 0.37 0.38 110
4.85 0.55 0.62 0.44 1.32 0.54 111 88.63 52.92 30.49 72.9 62.1 66.86
112 6.55 3.14 7.69 1.15 1.01 1.74 113 1.48 1.37 1.67 1.32 2.17 2.28
114 2.95 1.76 1.63 4.83 2.06 2.48 115 0.44 0.27 0.62 2.31 0.6 1.19
116 0.75 5.39 0.14 0.25 0.25 0.15 117 0.21 0.11 0.25 0.3 0.04 0.08
118 0.5 0.97 2.6 1.52 2.28 3.61 119 0.7 1.47 3.67 4.87 1.57 2.76
120 3.15 5.98 1.19 0.67 2.9 3.3 121 3.94 2.31 6 20.86 4.82 9.6 122
0.63 0.65 1.86 1.55 3.75 3.92 123 2.28 2.53 1.28 0.67 0.46 1.22 124
0.43 0.47 1.31 0.46 0.48 0.56 125 50.83 0.78 14.27 22.65 1.93 5.15
126 2.95 5.81 4.54 4.23 20.13 6.77 127 4.82 0.57 0.44 0.44 1.24
0.49 128 0.29 0.12 0.69 0.36 0.49 0.39 129 0.29 0.38 2.16 0.5 1.25
1.88 130 3.84 1.71 21.34 9.36 3.14 11.62 131 3.4 0.9 1.85 1.19 1.77
2.83 132 2.39 1.11 1.55 7 1.82 2.19 133 1.5 4.77 3.26 0.89 1.16
2.04 134 1.16 0.92 1.32 0.63 0.82 1.16 135 0.24 0.24 1.19 0.29 0.58
0.54 136 3.33 6.99 1.01 0.67 0.89 0.42 137 0.04 0.05 0.14 0.07 0.26
0.05 138 1.07 1.43 2.42 15.23 12.72 7.6 139 0.33 0.2 0.37 0.37 0.36
0.32 140 3.97 37.46 35.39 32.11 13.42 28.47 141 0.96 5.52 0.41 1.24
0.37 1.27 142 7.04 3.17 1.76 2.23 5.9 2.78 143 3.59 7.53 3 13.75
18.02 37.72 144 0.31 0.34 1.41 0.44 1.34 0.43 145 0.03 0.2 0.44
0.03 0.1 0.1 146 9.44 1.53 3.46 1.49 3.44 4.95 147 2.84 1.31 13.4
1.87 1.45 1.58 148 5.21 7.25 12.77 8.2 9.06 8.3 149 1.05 0.16 4.63
1.06 1.9 3.65 150 0.59 0.98 1.03 0.18 2.39 1.29 151 1.47 1.29 0.9
1.26 3.03 1.57 152 7.48 6.15 1.41 1.26 2.52 1.77 153 0.19 0.18 0.81
0.26 1.08 0.24 154 1.38 1.49 4.95 1.8 5.64 1.41 155 0.14 0.19 1.74
0.13 0.35 0.32 156 2.56 0.86 3.69 2.32 2.92 11.65 157 0.64 1.26
0.44 0.15 0.69 1.01 158 1.22 1.06 0.51 1.46 2.39 1.66 159 0.5 0.95
0.63 0.17 0.71 0.95 160 0.45 0.63 0.45 0.09 0.52 0.63 161 0.2 0.5
0.32 0.35 0.88 0.67 162 4.77 0.88 2.68 1.96 2.18 4.67 163 7.05
38.93 32.41 100 100 100 164 0.78 1.07 5.59 3.76 1.08 3.02 165 0.02
0.04 0.16 0.11 0.09 0.05 166 3.25 1.66 1.74 1.45 1.26 3.16 167 100
100 66.02 34.82 100 94.45 168 6.45 0.23 10.04 0.31 3.73 8.07 169
0.01 0.01 0.01 0.01 0.01 0.01 170 6.18 6.62 5.35 2.88 2.95 8.22 171
1.1 4 4.04 2.21 1.2 1.06 172 1.07 2.13 0.17 0.56 0.69 0.42 173 0.44
1.12 3.32 2.67 1.58 1.57 174 5.24 4.92 1.35 1.39 6 3.85 175 2.59
1.76 1.08 2.88 7.75 11.54 176 10.58 2.7 0.67 1.47 1.02 1.04 177
0.43 1.46 1.11 0.42 0.75 0.58 178 0.17 1.48 1.96 1.33 0.77 0.6 179
0.36 0.29 0.31 0.44 0.57 0.7 180 0.05 0.19 0.3 0.32 0.15 0.08 181
0.28 0.51 0.52 0.29 0.28 0.21 182 0.51 0.42 2.4 1.02 0.79 1.43 183
18.11 18.2 2.99 3.2 6.14 3.41 184 2.29 0.69 2.58 4.4 2.67 2.94 185
1.07 1.98 0.59 0.25 1.22 0.91 186 25.58 22.13 20.11 8.38 17.18
13.66 187 100 56.83 49.87 100 65.56 98.25 188 0.11 1.39 1.18 0.96
0.81 0.33 189 0.3 0.96 0.61 0.08 0.57 0.8 190 1.27 1.47 3.9 7.46
1.74 1.45 191 2.9 0.23 0.25 0.73 2.3 5.01 192 0.33 0.62 0.32 0.31
0.61 0.8 193 1.08 0.83 3.21 1.97 3.32 3.76 194 0.19 0.97 0.27 0.46
2.26 2.03 195 1.4 0.99 1.14 1.27 1.13 1.05 196 0.16 0.42 1.07 0.68
0.53 0.29 197 6.85 1.58 3.75 3.82 1.67 2.2 198 4.22 2.65 0.46 0.48
1.28 0.25 199 0.49 0.51 1.38 3.4 1.37 2.52 200 0.02 0.06 0.17 0.13
0.08 0.06 201 1.21 3.04 0.35 0.29 0.32 0.38 202 4.16 0.31 4.65 0.31
2.3 8.38 203 3.21 6.17 1.54 0.33 2.3 1.46 204 1.88 1.91 1.13 0.88
2.46 2.7 205 1.53 1.63 0.84 1.54 1.36 1.1 206 1.68 0.62 0.2 0.15
0.28 0.27 207 11.21 2.8 2.5 1.08 3.17 1.12 208 13.32 3.42 6.28 2.58
5 9.49 209 0.32 0.47 0.17 0.08 0.31 0.24 210 0.75 0.63 0.99 0.84
0.72 1.32 211 2.25 1.33 0.66 0.58 1 1.68 212 38.27 23.79 1.26 7.49
2.4 4.2 213 0.07 0.09 0.3 0.26 0.11 0.09 214 1.02 1.19 0.74 4.86
1.33 1.13 215 0.67 1.06 0.16 0.34 6.46 1.39 216 0.53 0.68 0.86 0.81
0.39 0.46 217 1.17 1.12 1.58 2 1.3 1.42 218 1.63 0.34 0.95 1.05
1.47 0.77 219 1.21 1.42 0.48 0.54 1.3 2.12 220 0.41 1.02 0.39 0.04
0.59 0.25 221 0.69 0.48 1.43 0.38 0.82 0.76 222 0.06 0.38 0.39 0.77
0.24 0.41 223 0.33 0.39 0.91 1.98 0.77 0.34 224 11.13 3.17 1.3 2.86
2.52 3.39 225 1.83 1.32 5.63 9.09 3.92 2.81 226 1.62 3.51 1.27 0.9
3.79 3.54 227 0.08 0.06 0.55 0.97 0.35 0.15 228 0.88 0.65 0.56 0.51
1.1 5.56 229 0.16 0.2 1.04 0.33 0.48 0.35 230 0.27 0.29 0.28 0.2
0.38 0.21 231 0.34 0.08 0.15 0.27 0.31 0.33 232 0.75 1.37 5.19 8.58
1.81 1.65 233 0.7 1.28 1.11 1.18 2.73 0.95 234 4.43 4.26 5.73 1.95
4.29 4.72 235 10.13 8.62 2.56 2.25 2.22 7.06 236 0.32 0.37 0.46
0.56 0.74 1.02 237 12.61 0.55 17.56 0.25 7.26 14.49 238 0.76 0.2
0.83 0.87 0.66 1.7 239 18.47 8.96 10.33 2.94 2.6 17.99
240 13.13 2.8 12.36 4.05 7.87 4.87 241 0.86 1.59 0.34 0.32 0.89
0.57 242 0.11 1.63 1.77 1.25 1.44 0.35 243 1.22 0.55 0.61 1.01 1.01
0.87 244 0.25 0.25 0.59 0.2 0.53 0.66 245 0.31 3.26 2.04 3.28 1.96
0.56 246 1.46 3.85 4.92 2.57 1.52 3.04 247 12.34 13.15 11.05 17.96
7.65 7.93 248 2.04 0.77 1.09 0.6 0.84 0.97 249 11.72 18.84 11.2
2.91 16.63 4.3 250 5.08 0.57 0.59 1.1 1.2 0.86 Rank St4_NA_NB273
St4_NA_NB275 St4_NA_NB276 St4_NA_NB283 St4_NA_NB69 1 0.62 6.22 7.67
0.15 4.19 2 1.23 2.98 2.85 1.87 5.36 3 11.77 10.94 6.29 2.84 11.33
4 5.91 0.52 1.1 0.52 3.14 5 23.44 48.96 2.73 4.57 12.01 6 54.98
46.22 45.47 13.56 13.44 7 1.61 1.63 10.81 1.81 13.21 8 0.32 0.53
3.99 0.16 2.47 9 0.33 0.71 0.49 0.12 5.87 10 0.55 0.67 0.5 0.45
2.11 11 0.61 4.08 6.41 0.28 6.34 12 2.91 0.64 0.32 2.45 3.9 13 0.17
0.1 0.69 0.43 3.66 14 0.97 5.31 5.91 1.62 2.99 15 0.33 0.36 0.46
0.3 1.69 16 1.91 2.42 3.71 3.45 4.09 17 3.27 1.73 3.84 0.93 8.18 18
0.13 0.32 0.18 0.17 0.21 19 0.06 0.07 0.06 0.1 0.04 20 11.8 7.58
100 9.01 13.68 21 29.14 20.22 10.44 3.4 1.54 22 0.1 0.1 0.77 0.11
0.13 23 0.21 0.25 0.25 0.53 2.29 24 1.29 19.16 31.41 1.65 1.22 25
3.98 2.03 6.49 10.91 18.15 26 31.59 30.74 35.22 10.19 8.66 27 0.08
0.13 0.04 0.12 0.1 28 3.59 1.76 8.65 3 43.65 29 0.22 0.16 0.14 0.24
0.32 30 0.57 0.05 0.09 0.27 0.4 31 0.07 4.87 10.64 1.1 8.58 32
12.19 61.16 77.52 4.9 0.89 33 8.07 4.54 7.47 1.5 4 34 0.09 0.09
0.01 0.1 0.11 35 20.94 10.81 11.63 39.03 17.64 36 1.96 0.92 1.87
1.56 8.37 37 0.83 1.02 0.59 0.75 1.93 38 0.44 3.42 2.97 0.05 1 39
15.92 6.57 1.09 4.05 2.02 40 4.63 11.09 34.95 2.26 3.76 41 5.94
6.47 3.18 1.94 3.22 42 1.5 8.28 8.71 0.95 1.78 43 3.18 3.85 0.36
0.49 0.86 44 0.73 3.08 0.73 0.69 1.48 45 0.14 0.3 0.2 0.24 0.92 46
1.06 0.96 1.27 1.28 4.01 47 0.49 0.05 0.13 0.28 0.41 48 0.71 0.03
0.06 0.16 0.67 49 0.49 0.13 0.23 0.14 0.54 50 0.24 2.28 3.54 0.22
0.29 51 2.97 7.91 0.91 1.34 0.4 52 10.83 2.91 1.67 0.73 0.34 53 1.1
4.76 2.52 7.1 3.75 54 1.14 3.57 3.38 0.87 0.66 55 1.88 0.59 0.61
0.29 1.73 56 0.78 2.94 0.65 0.92 1.78 57 2.56 0.86 0.5 4.11 7.13 58
10.59 12.67 8.17 7.07 20.06 59 0.12 0.11 0.48 0.24 1.94 60 49.72
32.42 18.78 26.55 28.37 61 46.84 33.99 13.79 28.99 42.97 62 1.19
0.24 0.89 0.42 1.56 63 3.33 1.89 3.38 3.89 4.39 64 3.21 1.42 1.17
1.61 0.9 65 2.81 11.47 8.11 1.55 4.68 66 0.85 1.78 2.69 1.53 0.44
67 0.94 3.76 1.04 3.75 5.82 68 0.46 0.6 0.5 0.89 0.86 69 1.64 100
34.92 34.13 15.91 70 0.02 0.02 0.03 0.03 0.02 71 1.89 0.88 1.35
1.94 2.5 72 3.42 11.37 7.93 8.79 7.5 73 44.87 100 100 6.1 5.85 74
0.36 0.37 0.56 0.92 1.51 75 0.76 0.48 0.98 1.09 1.34 76 3.3 2.1
8.88 3.36 4.01 77 2.35 5.75 9.61 2.88 5.42 78 15.57 10.45 11.32
38.41 20.2 79 0.84 0.72 2.66 1.27 2.42 80 2.52 6.28 10.74 0.53 18.5
81 19.34 33.27 15.01 28.01 8.3 82 0.19 0.28 0.49 0.22 3.34 83 0.51
0.83 0.01 0.72 0.88 84 0.61 0.57 0.04 0.41 0.55 85 0.46 2.14 2.32
0.2 0.77 86 0.33 0.29 0.09 0.76 0.55 87 0.38 0.45 0.61 0.54 0.54 88
0.94 1.41 1.33 1.12 2.4 89 0.1 0.16 0.01 0.18 0.07 90 0.22 1.11
0.19 0.21 0.14 91 1.44 6.08 4.02 0.69 1.13 92 0.16 2.32 0.82 0.84
0.07 93 1.22 0.16 0.08 0.15 1.25 94 0.29 1.28 2.35 1.06 0.89 95
2.22 2.1 2.31 4.08 2.53 96 0.17 0.04 0.02 0.06 0.05 97 0.48 0.76
0.76 1.13 1.63 98 0.99 0.67 1.5 1 0.94 99 0.39 0.05 0.1 0.18 0.29
100 4.36 5.64 12.73 6.34 7.1 101 2.19 1.94 0.03 9.8 11.99 102 1.03
2.54 0.74 0.96 1.52 103 1.43 0.46 0.52 0.43 1.04 104 1.24 1.22 0.98
1.67 1.15 105 1.41 0.34 0.34 1.64 2.2 106 3.12 3.53 15.78 2.63 3.68
107 0.56 0.29 0.04 0.57 0.72 108 4.54 14.61 10.81 10.15 8.77 109
0.36 0.49 1.64 0.5 0.43 110 0.39 0.77 0.5 1.03 2.37 111 100 84.82
96.39 100 98.44 112 1.1 1.05 1.54 1.3 2.89 113 2.12 3.26 1.52 1.98
4.29 114 4.74 2.57 2.14 1.38 4.22 115 2.45 0.81 1.23 1.13 1.29 116
0.17 0.39 0.01 0.58 0.5 117 0.33 0.04 0.08 0.17 0.21 118 0.66 0.4
1.38 1.4 8.04 119 5.41 1.3 1.92 2.07 6.13 120 0.68 2.74 5.51 1.11
0.83 121 22.94 8.93 32.76 17.59 9.03 122 1.7 2.31 3.59 1.83 3.63
123 0.83 0.45 1.35 0.82 3.41 124 0.65 0.4 0.6 1.76 1.82 125 23.4
1.16 3.57 66.38 70.86 126 8.61 33.23 4.93 2.79 1.8 127 0.36 0.73
0.01 0.98 2.41 128 0.38 0.53 0.36 0.68 0.86 129 0.66 1.21 1.54 1.54
1.49 130 7.8 2.58 9.23 3.86 19.17 131 1.3 1.44 1.93 3.99 1.66 132
9.82 2.46 3.31 1.3 3.75 133 1.06 0.81 2.31 0.8 1.3 134 0.53 0.66
0.83 0.97 1.28 135 0.36 0.54 0.5 0.29 2.97 136 0.72 0.88 0.43 0.55
0.33 137 0.08 0.35 0.04 0.47 0.06 138 14.03 14.25 10.27 3.86 6.3
139 0.29 0.21 0.39 0.24 0.75 140 19.73 15.37 42.38 12.16 24.89 141
1.11 0.33 1.05 0.48 1.53 142 2.35 8.08 4.12 3.1 1.74 143 19.81
25.42 34.01 3.5 8.18 144 0.47 1 0.01 1.66 0.26 145 0.02 0.08 0.07
1.59 0.05 146 1.2 2.94 4.06 7.52 12.11 147 2.36 1.94 2.84 2.02 1.3
148 10 9.07 6.28 2.93 11.84 149 1.2 2.26 2.76 1.44 7.54 150 0.25
3.24 1.34 1.68 1.15 151 1.1 2.78 5.86 1.34 4.8 152 1.31 3.11 1.29
1.59 5.55 153 0.35 0.94 0.15 1.88 1.02 154 2.41 3.11 1.03 11.81
1.76 155 0.07 0.2 0.19 3.68 0.23 156 1.84 1.48 11.82 0.58 0.76 157
0.15 1.14 0.92 1.23 0.55 158 1.32 3.32 1.34 6.14 0.58 159 0.2 0.92
0.98 0.7 0.53 160 0.13 0.93 1.17 0.41 0.36 161 0.5 0.73 0.34 0.47
1.06 162 1.7 1.11 7.33 4.66 2.67 163 100 94.42 100 89.59 26.53 164
2.72 0.77 3.79 1.97 2.7 165 0.14 0.1 0.05 0.05 0.12 166 1.48 1.7
2.58 1.96 6.54 167 97.72 100 100 100 100 168 0.34 4.11 5.26 0.8
4.17 169 0.01 0.01 0.01 0.01 0.01 170 2.13 3.29 5.22 9.01 26.44 171
4.79 0.86 0.85 5.66 1.79 172 1.16 0.44 0.32 0.56 1.16 173 2.2 1.76
1 2.7 3.39 174 1.57 10.58 3.53 1.55 3.04 175 2.34 8.84 8.24 1.15
1.59 176 0.75 0.72 2.28 0.9 23.28 177 0.39 0.9 0.51 0.57 0.4 178
1.48 0.88 0.33 1.78 2.73 179 0.5 0.53 0.57 0.39 0.17 180 0.43 0.15
0.07 0.23 0.37 181 0.39 0.2 0.13 0.47 1.35 182 0.99 0.5 0.87 2.08
4.47 183 3.11 8.62 3.76 1.74 6.73 184 5.6 2.33 2.91 1.78 1.88 185
0.23 1.24 1 1.12 0.72 186 7.28 19.43 22.87 19.5 24.09 187 100 63.06
100 89.25 100 188 1.11 0.46 0.14 0.98 2.11 189 0.08 0.77 1.25 0.83
0.61 190 7.22 1.32 0.75 1.96 4.15 191 0.91 2.58 6.19 0.16 0.42 192
0.43 0.82 0.51 0.5 0.93 193 2.01 2.7 3.09 2.35 11.66 194 0.55 2.94
18.09 0.2 2.04 195 1.17 1.25 1.2 1.4 2.78 196 1.21 0.33 0.21 0.34
0.54 197 4.13 1.78 2.33 9.27 20.98 198 0.38 1.23 0.1 1.04 1.03 199
4.16 1.88 2.57 4.37 1.27 200 0.14 0.09 0.04 0.07 0.13 201 0.29 0.29
0.28 0.15 0.27 202 0.39 2.13 4.21 0.72 3.65 203 0.37 3.22 1.66 1.59
5.83 204 1 3.13 1.99 1.14 1.17 205 1.97 1.79 1.34 1.63 0.73 206
0.13 0.29 0.26 1.25 1.74 207 1.31 3.78 0.9 0.61 0.94 208 2.92 4.01
10.85 4.55 5.59 209 0.11 0.55 0.31 0.27 0.22 210 0.83 0.65 1.19
1.04 1.25 211 0.47 1.02 2.28 1.82 0.62 212 4.88 0.94 1.8 15.7 13.17
213 0.21 0.09 0.09 0.07 0.15 214 3.57 1.4 1.31 0.87 1.59 215 1.08
3.03 2.1 0.16 0.13 216 0.8 0.35 0.33 0.57 0.8 217 2.84 1.13 1.32
2.03 4.41 218 1.61 1.99 0.58 0.97 0.6 219 0.54 1.65 2.26 0.22 2.33
220 0.03 0.62 0.4 0.83 0.27 221 0.4 0.43 0.66 0.62 0.66 222 0.92
0.24 0.28 0.26 0.47 223 1.92 0.67 0.27 0.62 0.82 224 3.23 2.43 2.95
1.71 1.23 225 11.77 4.63 2.47 3.36 0.76 226 1.12 5.11 2.28 0.59
0.87 227 1.04 0.25 0.27 0.22 0.17 228 0.59 0.4 8.44 0.54 0.5 229
0.46 0.56 0.33 0.23 2.35 230 0.18 0.33 0.28 0.33 0.15 231 0.39 0.31
0.34 0.19 0.07 232 4.96 1.01 1.17 3.35 2.78 233 1.15 0.87 1.01 2.03
2.22 234 2.89 6.12 6.32 2.83 2.61 235 2.32 2.26 7.48 1.29 2.55 236
0.67 0.59 0.65 0.99 1.32 237 0.29 3.39 17.05 1.4 4.68
238 1.14 0.7 1.28 1.1 0.29 239 4.19 3.27 11.33 7.34 44.07 240 5.97
9.55 3.58 6.83 20.32 241 0.24 0.69 0.73 0.78 1.09 242 1.75 1.35
0.26 1.21 1.84 243 1.32 0.96 1.11 3.49 6.64 244 0.25 0.46 0.56 0.12
1.16 245 3.97 2.5 0.46 1 1.33 246 3.17 1.36 3.29 1.13 2.81 247
19.12 8.17 8.6 9.17 13.71 248 0.6 0.82 0.67 1.8 1.98 249 5.04 34.59
8.45 13.74 6.92 250 0.82 0.83 0.24 1.27 2.16
TABLE-US-00014 TABLE 9C Testing: Good (G) and Poor (P) (3rd and 4th
Bars of FIG. 7A) St1_NA_NB221 St1_NA_NB238 St1_NA_NB33 St1_NA_NB34
St1_NA_NB9 Rank Gene (G) (G) (G) (G) (G) 1 DLK1 0.09 0.29 0.08 0.56
0.03 2 est 0.27 0.24 0.38 0.73 0.45 3 PRSS3 1.21 1.73 2.19 4.74
1.91 4 ARHI 14.49 18.97 5.25 3.98 5.5 5 ARC 5.53 1.88 1.38 1.71
2.19 6 SLIT3 16.52 9.18 10.57 18.44 10.89 7 CNR1 14.38 15.81 4.38
2.29 16.34 8 est 0.23 0.18 0.24 0.66 0.11 9 est 3.03 1.93 1.67 1.43
1.42 10 FLJ25461 1.27 0.67 1.77 1.84 1.03 11 est 0.24 0.34 0.28
0.63 0.35 12 CD44 2.82 3.47 4.66 3.47 3.27 13 est 0.69 1.26 1.17
0.68 1.04 14 ROBO2 8.69 10.63 7.12 2.54 5.93 15 BTBD3 1.94 2.73
1.51 0.82 2.75 16 MYCN 5 4.98 2.66 6.99 4.22 17 est 20.9 21.61 3.33
4.26 22.75 18 JPH1 0.02 0.03 0.06 0.04 0.04 19 KLRC3 0.07 0.14 0.24
0.17 0.2 20 est 4.31 7.39 1.06 2.41 1.99 21 RET 5.88 2.4 2.65 2.93
1.63 22 CRABP1 0.29 0.11 0.17 0.32 0.09 23 ECEL1 4.95 3.32 2.29
2.39 2.04 24 LOC283120 1.7 0.7 1.09 1.04 1.86 25 HMGA2 40.25 8.37
11.35 17.6 11.27 26 SYNPO2 4.04 11.3 3.67 5.8 9.65 27 LOC163782
0.23 0.13 0.29 0.23 0.12 28 VSNL1 22.01 19.81 2.47 5.04 10.94 29
HS3ST4 0.29 0.15 0.67 0.76 0.14 30 AKR1C1 0.45 0.69 0.33 0.26 0.29
31 est 0.14 0.05 7.55 0.05 0.1 32 GPR22 2.59 6.85 7.09 33.62 5.57
33 est 2.01 1.05 1.35 2.29 2.42 34 est 0.14 0.5 0.96 0.39 0.36 35
CCNA1 7.84 4.01 2.1 1.64 5.94 36 PKIB 1.38 6.18 9.16 17.61 9.03 37
est 1.17 0.8 1.2 1.57 0.85 38 GAL 0.02 0.09 0.11 0.29 0.32 39 est
0.19 0.22 0.34 1.04 0.41 40 LOC221303 2.27 3.55 1.84 5.93 2.01 41
est 1.79 2.11 1.16 3.09 1.86 42 est 0.99 2.93 1.14 3.41 1.89 43
BMP7 0.21 0.16 5.05 4.62 1.07 44 SLC30A3 0.79 0.5 0.72 0.96 1.2 45
FLJ10539 0.32 0.9 1.35 0.84 1.03 46 AMIGO2 6.06 14.48 1.81 2.2 6.4
47 AKR1C2 0.51 0.65 0.46 0.35 0.42 48 MGP 0.04 0.09 0.05 0.08 0.13
49 PCSK1 0.95 0.74 0.55 0.62 0.48 50 HK2 0.48 0.25 0.19 0.32 0.22
51 est 0.53 0.43 0.68 0.66 0.47 52 est 0.23 0.33 0.7 0.62 0.37 53
IL7 3.57 10.87 10.08 7.91 14.22 54 PRSS12 0.88 1.47 0.99 1.37 0.85
55 GABARAPL1 1.15 1.83 0.75 1.12 1.02 56 DEFB129 0.64 0.64 0.78
0.84 1.05 57 NAV3 7.82 8.78 5.48 5.16 5.22 58 RAB3B 9.61 16.05 7.26
7.7 9.24 59 KRT6B 2.65 2.88 4.96 3.37 5.23 60 BEX1 26.39 23.35 9.78
16.38 11.86 61 est 33.68 24.94 9.4 15.39 11.82 62 est 3.55 2.55
5.21 1.65 1.89 63 SCYL1 5.21 6.88 5.26 4.82 3.27 64 est 38.31 21.83
7.24 5.15 2.86 65 RYR2 12.7 15.14 26.56 16.27 9.44 66 LRBA 0.8 0.85
0.54 0.67 0.52 67 CSPG3 3.27 0.73 0.45 0.69 0.7 68 est 2.61 5.37
1.15 1.65 1.98 69 MMP12 3.49 10.92 3.19 5.15 2.5 70 CHRNA1 0.13
0.03 0.06 0.03 0.03 71 est 4.91 2.56 2.13 1.92 1.75 72 est 76.94
58.08 20.79 10.75 30.61 73 HNRPH1 2.52 3.96 2.11 28.27 100 74
LOC113251 2.84 3.35 3.11 1.55 1.85 75 est 5.2 2.38 2.05 1.55 1.66
76 PAG 3.93 5.58 4.61 3.91 3.7 77 PROK2 14.79 10.65 8.93 3.7 9.07
78 HS6ST1 9.13 3.76 2.2 2.36 6.37 79 est 8.87 9.69 12.05 9.59 8.8
80 PCDH9 15.3 24.39 9.19 15.45 7.67 81 est 9.85 21.46 13.88 15.19
12.21 82 est 2.49 1.69 0.25 0.27 0.24 83 GLDC 0.51 0.46 0.4 0.45
0.31 84 ADRB2 1.33 3.79 1.16 2.02 1.25 85 ICSBP1 0.07 0.14 0.26
0.34 0.42 86 CD48 0.1 0.97 0.68 0.43 1.04 87 est 0.57 1.05 1.58
1.19 1.62 88 DYRK1B 0.52 0.53 0.59 0.63 0.61 89 KLRC1 0.09 0.2 0.27
0.22 0.37 90 est 0.15 0.21 0.15 0.13 0.17 91 est 1.97 0.58 1.3 1.28
0.54 92 est 0.04 0.31 0.12 0.42 0.06 93 MOXD1 0.1 0.15 0.18 0.24
0.26 94 est 0.49 0.26 0.48 0.36 0.43 95 est 7.25 9.93 5.4 6.78 5.35
96 GAS1 0.07 0.06 0.06 0.06 0.2 97 COL9A2 0.16 1.9 0.47 0.41 0.49
98 est 1.61 2.27 1.31 1.55 1.7 99 DRPLA 0.43 0.58 0.34 0.27 0.25
100 est 13.38 23.5 21.1 15.4 16.16 101 REPRIMO 5.31 27.34 3.89 7.35
12.92 102 CACNA2D2 0.71 0.73 0.86 0.98 0.86 103 NEBL 1.07 1.24 1.23
1.01 1.02 104 est 1.66 3.44 1.47 3.36 1.46 105 HLA-DQA1 0.41 4.98
3.89 1.3 4.22 106 EDG3 4.94 2.93 2.92 3.08 0.87 107 CPVL 0.21 0.5
0.63 0.36 0.87 108 FLJ32884 18.74 7.74 13.71 8.68 17.39 109 LCP1
0.22 1.02 0.51 0.37 0.85 110 est 4.14 3.21 3.81 2.84 5.83 111 est
49.69 91.15 9.62 43.63 38.78 112 est 3.21 5.85 9.64 8.22 6.68 113
est 2.42 2.9 2.06 2.28 2.95 114 DKFZP564C152 1.78 0.85 1.05 1.33
1.57 115 DMN 1.68 2.27 1.2 1.75 0.64 116 GABRA5 0.25 0.17 0.48 0.27
0.18 117 AKR1C3 0.33 0.3 0.3 0.22 0.21 118 LOC168850 4.59 6.27 9.21
5.86 8.02 119 est 8.39 8.79 2.57 3.2 4.3 120 KCNQ2 0.8 0.5 1.1 0.93
0.59 121 NME5 10.83 10.82 1.95 3.54 4.35 122 est 2.04 2.12 4.28
2.96 6.92 123 PBX1 4.71 2.81 2.46 1.65 1.3 124 CNTNAP2 1.87 1.02
3.52 1.41 2.25 125 est 12.91 15.7 80.61 95.55 35.71 126 SPON1 2.96
0.89 1.27 1.69 4.84 127 CDH8 4.02 2.91 3.49 3 5.84 128 PRKCB1 0.33
0.48 1.14 1.81 0.51 129 SLC21A11 2.3 3.37 2.56 2.31 2.99 130 MAP4
17.95 25.02 9.37 17.93 16.31 131 est 4.17 4.98 6.8 3.86 3.36 132
SCN7A 6.73 10.24 1.26 1.81 5.3 133 est 8.75 7.91 1.41 1.24 7.58 134
est 1.62 1.38 1.38 1.61 1.93 135 est 2.49 1.27 1.32 1.12 1.05 136
est 0.46 0.52 1.11 1.45 0.55 137 CDW52 0.05 0.22 0.3 0.2 0.28 138
ABCB1 2.14 2.15 1.9 6.81 3.48 139 est 1.79 2.3 0.77 0.52 0.39 140
OSF-2 4.6 10.19 3.87 10.22 6.13 141 NRXN1 3.36 2.43 3.9 1.25 2.08
142 ADAM22 2.16 1.84 3.44 3.15 2.47 143 est 7.71 6.98 3.9 8.83 4.38
144 TRGV9 0.31 1.4 0.94 0.74 2.03 145 est 0.03 0.06 0.13 0.16 0.37
146 PTPRD 10.93 10.23 9.14 4.72 5.1 147 est 0.84 0.63 0.61 0.64
0.64 148 HS3ST2 3.69 3.74 1.1 1.83 1.46 149 FGF13 2.78 3.82 2.94
3.25 3.33 150 MKI67 0.43 0.33 0.59 0.23 0.49 151 KIF12 1.85 1.58
1.5 1.41 1.89 152 est 1.18 1.39 1.15 1.01 1.05 153 est 0.29 0.9
0.96 0.68 1.44 154 est 1.92 7.25 5.36 4.44 12.54 155 est 0.19 0.13
0.35 0.55 0.82 156 est 3.78 10.54 1.37 7.58 1.92 157 KLIP1 0.47
0.14 0.49 0.17 0.47 158 est 0.59 0.82 0.66 0.93 0.66 159 LOC157570
0.28 0.14 0.49 0.24 0.43 160 MAD2L1 0.21 0.16 0.18 0.09 0.22 161
est 0.81 0.44 0.69 0.58 0.58 162 est 6.38 7.28 5.92 6.63 5.2 163
RGS5 53.87 43.46 33.73 65.93 56.46 164 ATP2B4 7.69 5.48 2.13 2.71
2.98 165 HMGCL 0.05 0.1 0.07 0.08 0.12 166 ODZ3 2.31 2.77 3.48 3.79
3.13 167 CHGA 100 100 34.9 79.57 97.29 168 MGC33510 0.31 0.22 5.3
0.25 0.2 169 GAGE5 0.01 0.01 0.01 0.01 0.01 170 SARDH 26.07 27.53
21.62 13.27 17.8 171 est 0.51 2.8 1.88 2.61 7.72 172 DAT1 0.55 0.12
0.67 0.26 0.31 173 FUCA1 1.3 5.17 2.97 1.64 5.7 174 TM6SF2 0.9 0.64
0.68 0.87 0.69 175 KCNK9 2.24 1.45 1.17 1.82 1.95 176 ADCYAP1 16.37
4.15 1.09 21.82 3.19 177 PLXNA4 1.57 2.41 2.02 1.25 1.52 178
HLA-DMB 0.73 2.76 1.53 0.99 2.48 179 est 0.53 0.97 0.4 0.45 0.75
180 est 0.08 0.14 0.19 0.14 0.39 181 GRIN3A 1.15 1.36 0.62 0.65
0.59 182 OSBPL3 3.81 2.35 8.94 4.45 3.72 183 ODZ4 2.53 4.69 2.21
1.64 4.29 184 est 9.36 18.04 1.22 2.01 5.05 185 E2F1 0.6 0.2 0.72
0.25 0.33 186 MGC16664 22.76 17.82 15.1 11.58 7.22 187 HMP19 100
86.88 39.73 70.97 75.48 188 IL2RB 0.53 2.01 1.38 0.98 1.74 189 TOPK
0.22 0.13 0.23 0.09 0.25 190 ALDH1A1 1.55 2.78 1.7 2.44 5.12 191
CED-6 0.28 0.11 0.11 0.39 0.34 192 est 2.02 1.56 0.97 0.67 0.71 193
A2BP1 5.75 2.36 6.74 5.22 6.75 194 LY6E 0.21 0.24 0.18 0.3 0.17 195
est 1.77 1.72 1.06 1.52 4.29 196 est 0.32 0.42 0.59 0.65 0.88 197
PLXNC1 10.01 21.01 9.98 11.87 13.77 198 EFS 0.2 0.23 0.88 0.29 0.38
199 ACTN2 0.79 1.53 2.53 2.57 2.41 200 MYC 0.04 0.11 0.07 0.09 0.16
201 KIAA0527 0.44 0.26 0.3 0.33 0.27 202 C6orf31 0.31 0.2 6.3 0.37
0.24 203 DLL3 0.71 1.12 1.58 2.03 0.94 204 est 0.96 0.76 0.99 1.55
0.69 205 STK33 0.46 0.88 0.49 0.8 0.62 206 SEMA3A 0.75 1.41 0.76
0.69 1.09 207 est 7.3 3.29 2.56 2.6 1.9 208 IGSF4 13.14 18.07 9.94
7.1 6.88 209 CKS2 0.11 0.07 0.16 0.07 0.16 210 est 2.42 3.45 2.4
1.42 3.29 211 est 0.87 0.25 0.53 0.41 0.58 212 SIX3 25.3 20.41 2.95
6.26 4.66 213 FLJ22002 0.2 0.08 0.24 0.22 0.18 214 HSD17B12 0.68
0.48 0.55 0.99 0.85 215 HBA2 0.12 0.07 0.19 0.53 1.85 216 CDH11
1.78 1.36 1.52 0.9 1.75 217 RGS9 2.95 3.45 1.46 1.6 1.79 218 est
3.12 3.09 2.11 1.97 1.75 219 NCAM2 1.81 4.02 1.22 1.38 0.93 220
BIRC5 0.27 0.09 0.26 0.06 0.16 221 est 1.05 1.17 1.34 1.07 1.25 222
GNG12 0.28 0.88 0.32 0.35 0.52 223 GPIG4 0.7 1.68 0.79 1.81 1.13
224 est 1.54 1.65 1.64 1.94 1.54 225 ENPP4 0.53 0.69 0.51 1.24 3.58
226 FMNL 1.17 0.46 1.2 1.13 0.56 227 est 0.3 0.36 0.39 0.43 0.73
228 PIWIL2 0.43 0.56 0.82 2.78 0.53 229 CLSTN1 1.14 1.03 1.03 0.75
0.84 230 UHRF1 0.12 0.07 0.16 0.13 0.19 231 est 0.24 0.42 0.27 0.38
0.46 232 SLC40A1 1.2 1.99 3.21 1.84 5.76 233 CLECSF6 2.37 6.85 3.62
2.77 5.71 234 est 1.35 1.99 1.18 2.65 2.09 235 BKLHD2 1.93 2.18
2.18 2.6 1.93 236 est 3.11 5.36 1.03 1.28 2.65 237 est 0.33 0.34
8.76 0.33 0.3 238 est 1.28 2.13 1.59 1.46 1.39 239 SORCS1 5.88
12.43 9.52 13.24 10.5 240 NRP2 14.44 30.86 9.01 6.94 12.83 241
E2-EPF 0.52 0.15 0.39 0.21 0.22 242 CAST 0.48 2.65 1.59 1.3
1.89
243 KIAA1384 7.94 2.82 2.75 1.52 2.32 244 KIAA0644 0.89 1.03 1.65
1.25 1.22 245 HLA-DRB3 0.97 6.85 3.39 1.8 3.51 246 PMP22 3.53 9.71
5.59 6.62 4.94 247 DJ79P11.1 12.96 7.63 5.67 7.32 5.33 248 SOX5
1.33 2.24 2.25 2.57 3.81 249 CD3E 9.81 12.43 4.25 5.82 7.73 250 est
4.56 3.38 3.88 3.05 4.49 St2_NA_NB220 St2_NA_NB232 St2_NA_NB235
St3_NA_NB201 St3_NA_NB215 Rank (G) (G) (G) (G) (G) 1 0.02 0.14 0.28
0.04 0.02 2 0.48 0.24 0.22 1.25 0.29 3 3.79 1.91 1.12 3.15 5.64 4
2.5 3.27 0.69 1.06 49.91 5 8.38 7.74 3.66 3 5 6 14.74 35.88 9.16
11.63 61.29 7 31.82 6.31 11.88 18.54 4.68 8 0.07 1.25 0.08 0.23 1.1
9 0.99 0.61 0.29 0.35 0.89 10 0.74 0.52 0.4 0.37 0.57 11 0.48 0.17
0.34 0.33 0.09 12 2.75 1.88 1.25 2.34 3.24 13 2.86 0.61 3.07 2.52
0.57 14 10.89 3.81 21.67 22.91 2.29 15 0.61 0.73 1.08 0.95 0.68 16
4.88 0.97 9.84 5.66 0.64 17 26.8 4.97 26.5 11.15 14.05 18 0.05 0.08
0.03 0.12 0.04 19 0.07 0.13 0.04 0.08 0.14 20 55.15 31.72 34.96
38.78 8.62 21 2.14 12.52 1.16 4.17 3.09 22 0.04 0.09 0.14 0.19 1.13
23 1.48 0.18 1.24 2.28 6.15 24 0.92 1.37 0.71 0.97 2.18 25 14.26
6.65 15.09 7.98 27.49 26 10.01 15.73 13.2 13.17 26.71 27 0.09 0.18
0.06 0.22 0.1 28 21.84 7.73 27.88 39.81 40.43 29 0.03 0.29 0.07
0.22 0.05 30 0.62 0.28 0.4 0.77 0.17 31 9.98 16.11 13.09 12.73 0.18
32 100 7.02 9.95 20.92 2.36 33 0.89 1.61 2.67 0.84 1.18 34 0.08
0.16 0.07 0.32 0.36 35 4.88 2.01 10.86 2.53 10.98 36 18.98 6.23 0.3
3.55 8.54 37 0.97 0.7 0.61 0.43 0.46 38 2.51 0.03 0.15 0.63 3.84 39
3.33 0.92 0.62 4.91 4.02 40 11.42 3.32 1.73 3.25 5.84 41 6.87 1.51
1.39 2.96 7.61 42 4.2 5.02 1.59 10.46 1.45 43 0.2 0.26 0.12 0.34
0.28 44 0.87 1.19 0.68 0.76 0.84 45 1.07 0.5 3.08 1.07 0.49 46 5.08
2.02 3.13 2.35 7.46 47 0.67 0.31 0.41 0.79 0.17 48 0.06 0.67 0.1
0.3 1.05 49 0.75 0.42 0.5 0.29 5.7 50 0.1 1.2 0.39 0.16 0.27 51
0.46 0.56 0.35 0.39 0.44 52 0.45 0.28 0.28 0.28 0.3 53 16.85 1.23
15.37 12.93 1.21 54 1.16 2.26 1.04 3.59 0.96 55 2.72 2.39 1.03 1.41
3.28 56 1.01 1.3 0.96 0.78 0.74 57 5.75 1.83 1.53 6.91 3.65 58
39.88 15.38 11.41 17.11 11.2 59 3.99 4 4.11 1.02 2.03 60 17.42
17.79 22.31 23.07 28.58 61 18.12 15.95 19.53 22.59 27.38 62 0.69
1.18 1.7 1.01 2.01 63 17.25 3.53 10.23 11.04 2.63 64 2.68 2.27
10.43 25.16 4.93 65 3.12 5.87 16.51 8.67 4.02 66 0.47 0.46 2.38
0.51 0.53 67 0.62 0.62 0.51 0.65 0.81 68 5.28 1.58 6.87 5.84 1.63
69 9.33 10.74 11.13 7.48 2.57 70 0.03 0.05 0.03 0.15 0.03 71 2.89
1.41 6.91 5.59 2.24 72 63.16 7.92 69.99 56.35 6.77 73 6.57 64.03
10.28 31.91 19.91 74 1.78 0.56 2.88 1.51 1.42 75 4.08 1.66 2.37
3.49 1.44 76 13.67 2.84 7.78 10.15 2.56 77 14.77 4.98 22.57 27.6
4.55 78 6.23 2.64 11.53 3.73 10.59 79 11.93 8.13 14.92 8.21 3.22 80
3.18 5.69 3.13 8.02 6.27 81 15.44 12.62 21.31 14.7 6.36 82 0.61
0.35 0.64 0.77 0.51 83 0.8 0.88 0.62 0.78 0.55 84 0.8 0.62 0.87
1.12 0.57 85 2.06 0.16 0.21 0.53 4.36 86 0.62 0.33 0.16 0.48 1.15
87 1.94 0.83 6.19 1.49 0.57 88 0.64 0.39 0.44 1.05 0.53 89 0.12
0.26 0.1 0.12 0.24 90 0.18 0.31 0.21 0.16 0.21 91 1.36 0.42 1.51
2.43 0.35 92 0.17 0.13 0.03 0.18 0.04 93 0.07 0.53 0.14 0.37 1.68
94 0.21 0.26 1.74 0.92 0.12 95 6.05 3.66 13.62 7.4 2.62 96 0.03
0.06 0.08 0.04 0.5 97 1.16 0.28 1.55 2.3 0.48 98 5.56 3.99 1.65
1.17 1.1 99 0.54 0.27 0.36 0.6 0.13 100 50.02 19.01 76.19 27.79 6.8
101 16.3 8.5 16.43 6.41 3.91 102 0.92 1.27 0.9 0.89 1.06 103 3.26
1.24 0.78 1.02 1.24 104 1.88 0.63 2.25 2.43 0.64 105 0.7 2.16 0.54
2.89 7.84 106 5.63 1.77 2.87 2.54 1.59 107 0.36 0.5 0.16 0.39 1.35
108 9.86 27.95 53.03 27.53 4.27 109 0.54 0.54 0.23 0.55 0.94 110
1.29 1.5 3.03 1.31 1.53 111 100 72.5 84.87 57.85 83.41 112 17.18
2.56 3.21 7.82 1.6 113 4.93 1.54 3.51 2.94 1.03 114 0.98 1.19 3.41
0.65 1.09 115 2.22 1.91 1.06 1.46 1.8 116 0.14 0.32 0.15 0.52 0.25
117 0.38 0.19 0.24 0.45 0.16 118 11.55 6.2 10.71 2.49 2.55 119 9.83
10.26 3.4 4.33 8.74 120 0.64 0.85 1.37 1.52 0.76 121 14.48 5.03
7.43 5.15 8.47 122 2.5 2.85 6.77 2.75 1.34 123 3.24 1.79 2.51 3.23
1.21 124 1.18 0.86 2.45 1.44 1.57 125 0.66 2.02 1.46 8.44 75.87 126
0.98 15.71 1.02 2.83 23.25 127 1.19 1.26 2.59 0.94 1.7 128 1.71
0.94 0.69 0.85 0.33 129 0.81 1.09 3.59 2.29 1.05 130 13.73 15 14.56
24.42 27.17 131 4.6 2.77 7.94 6.25 1.77 132 19.65 7.49 4.63 19.43
5.55 133 7.68 1.3 5.49 2.35 1.13 134 1.43 0.7 2.21 1.63 0.79 135
0.75 0.45 0.62 0.56 0.58 136 0.62 0.49 0.36 0.38 0.61 137 0.14 0.11
0.07 0.21 0.41 138 12.46 7.95 1.22 3.52 5.09 139 1.85 0.87 1.53 1.1
0.52 140 7.37 76.94 20.71 10.11 37.72 141 0.75 1.04 1.74 1.21 2.39
142 2.44 1.55 4.98 3.1 1.46 143 13.99 16.96 6.35 9.56 9.31 144 1.04
1.66 1.05 1.14 2.31 145 0.01 0.06 0.02 0.33 2.02 146 25.41 5.98
12.08 12.05 4.84 147 0.7 0.83 0.44 0.75 0.86 148 5.32 12.47 7.47
3.31 5.75 149 3.75 2.99 5.08 5.32 2.93 150 0.13 0.3 2.96 0.8 0.11
151 2.01 2.19 1.84 1.75 1.5 152 1.36 2.31 1.3 4.87 1.42 153 1.02
0.52 0.71 0.92 2.19 154 8.54 3.44 4.82 6.87 20.26 155 0.09 0.15
0.08 0.96 9 156 1.14 15.94 3.1 3.06 5.8 157 0.16 0.14 1.67 0.56
0.06 158 0.55 0.44 0.46 0.48 0.43 159 0.1 0.06 0.75 0.69 0.07 160
0.43 0.06 0.76 0.48 0.03 161 0.45 0.5 0.52 0.75 1.64 162 9.58 5.16
12.66 4.92 2.74 163 31.2 35.52 41.94 11.54 28.89 164 5.9 3.4 3.3
4.25 5.82 165 0.1 0.36 0.04 0.08 0.32 166 4.12 3.06 6.29 5.57 4.65
167 100 25.25 100 48.71 37.99 168 5.57 6.88 14.69 11.72 0.26 169
0.01 0.02 0.01 0.01 0.01 170 20.5 3.17 26.29 15.85 11.99 171 1.2
17.8 1.05 3.42 31.06 172 0.53 0.15 0.13 2.02 0.3 173 3.11 1.55 1.05
1.89 2.47 174 1.4 0.86 1.8 3.58 0.93 175 0.77 1.4 1.36 1.01 1.34
176 1.49 4.88 3.39 1.8 56.37 177 1.91 0.87 1.85 1.77 0.67 178 1.56
2.23 0.4 1.4 3.82 179 1.49 1.8 1.71 2.24 1.3 180 0.16 0.37 0.07
0.18 0.54 181 0.46 1.04 0.84 1.03 0.69 182 2.62 1.03 3.07 1.86 1.23
183 2.18 2.63 4.21 1.69 3.82 184 18.05 14.79 6.63 25.49 5.22 185
0.09 0.24 1.05 0.67 0.09 186 20.9 8.29 15.63 18.53 13 187 100 37.95
65.01 54.06 100 188 1.09 1.9 0.38 1.1 5.13 189 0.05 0.06 0.94 0.75
0.04 190 1.83 3.92 1.28 10 12.6 191 0.12 0.47 0.14 0.48 0.79 192
1.22 1.16 0.92 2.5 1.25 193 3.4 2.86 4.03 5.09 2.44 194 0.15 0.26
0.21 0.23 0.37 195 2.11 2.29 2.42 1.63 2.06 196 0.52 0.88 0.53 1.04
2.01 197 16.84 6.94 15.81 13.07 4.49 198 0.41 0.67 6.12 1.9 0.81
199 1.59 1.71 0.15 0.75 0.47 200 0.11 0.39 0.04 0.12 0.35 201 0.14
0.31 0.08 0.28 0.26 202 6.26 6.55 11.2 6.95 0.32 203 2.08 0.38 0.26
0.63 0.75 204 0.51 0.61 1.66 0.87 0.6 205 0.43 0.48 0.3 0.43 0.61
206 0.67 0.43 1.38 0.59 0.6 207 4.85 1.35 9.85 30.3 0.92 208 17.4
25.81 100 25.87 4.76 209 0.05 0.1 0.31 0.16 0.06 210 1.75 0.8 1.74
2.02 1.02 211 0.38 0.27 3.55 1.04 0.22 212 64.92 22.59 43.3 77.09
6.16 213 0.07 0.18 0.8 0.32 0.7 214 0.4 0.92 0.92 0.43 0.87 215
0.12 1.08 0.15 0.55 0.43 216 0.53 1.63 0.71 0.88 1.33 217 3.44 3.92
1.88 3.38 3.95 218 2.46 2.02 2.7 1.93 1.3 219 3.83 0.86 1.34 5.07
0.84 220 0.02 0.04 0.6 0.31 0.02 221 1.2 0.91 2 1 1.02 222 1.34
1.02 0.22 0.59 1.07 223 1.15 4.26 0.94 1.7 1.3 224 5.22 5.06 1.45
14.42 1.63 225 0.4 0.85 1.25 4.26 3.6 226 0.84 0.42 0.79 1.63 0.68
227 0.12 0.64 0.49 0.36 1.19 228 0.53 1.29 0.61 0.45 0.75 229 0.57
0.39 0.27 0.43 0.62 230 0.06 0.06 0.22 0.11 0.09 231 0.32 0.44 0.09
0.13 0.67 232 3.78 2.01 1.09 2.85 5.41 233 4.23 4.19 2.67 3.62 6.19
234 2.28 2.12 2.43 2.29 1.74 235 4.37 2.19 5.18 3.67 1.48 236 2.58
1.13 1.07 1.24 3.64 237 12.06 9.86 18.3 14.28 0.37 238 2.08 1.26
2.48 1.48 0.48 239 11.22 17.68 39.79 27.82 18.56
240 34.92 12.64 43.35 17.69 16.29 241 0.15 0.12 0.42 0.4 0.22 242
1.55 2.12 0.63 1.52 6.27 243 7.09 4.42 1.28 3.55 1.28 244 0.46 0.15
0.34 0.27 0.35 245 1.83 3.75 0.93 2.85 9.61 246 6.58 6.99 4.21 7.34
8.99 247 7.21 6.7 10.57 7.33 10.26 248 1.7 1.61 3.56 1.7 1.3 249
6.99 2.59 11.29 3.96 6.69 250 1.41 1.79 3.83 1.25 1.26 St4_NA_NB24
St4_NA_NB269 St4_NA_NB282 St4_NA_NB35 St4_NA_NB64 Rank (G) (G) (G)
(G) (G) 1 0.03 0.03 0.05 0.01 0.06 2 0.28 0.8 1.18 0.31 0.34 3 0.8
3.11 1.32 2.45 1.7 4 1.1 3.41 4.98 1.49 2.28 5 1.39 4.74 3.46 5.74
2.33 6 6.47 38.97 5.75 61.03 12.61 7 15.11 4.98 1 1.4 7.14 8 0.14
0.99 1 0.55 0.2 9 0.26 1.26 0.06 0.37 1.46 10 0.85 0.81 2.37 1.01
1.99 11 0.42 0.19 0.1 0.1 0.23 12 1.02 2.51 0.65 2.08 3.68 13 5.59
0.39 0.55 0.22 1.75 14 18.84 2.49 1.6 1.99 3.71 15 0.86 0.42 0.38
0.38 1.11 16 4.87 0.72 1.28 1.56 5.72 17 4.46 6.39 1.31 6.8 34.8 18
0.11 0.1 0.09 0.14 0.15 19 0.07 0.14 0.13 0.21 0.06 20 73.63 27.24
4 4.03 2.38 21 0.51 9.92 2.9 1.67 1.49 22 0.12 0.15 0.39 0.19 0.06
23 0.44 0.32 1.66 0.5 2.69 24 1.22 2.06 0.81 2.43 2.68 25 28.54
2.72 6.32 18.14 30.6 26 15.39 23.58 5.43 16.55 8.08 27 0.08 0.64
0.1 0.05 0.48 28 16.99 11.36 2.53 9.09 37.79 29 0.04 0.09 0.33 0.17
0.73 30 0.26 0.35 0.05 0.09 0.33 31 0.04 0.18 0.06 9.95 0.39 32
5.23 5.06 3.5 1.63 13.54 33 0.9 3.9 0.96 1 2.08 34 0.17 0.65 0.12
0.66 0.11 35 11.84 2.91 31.49 2.93 5.56 36 2.87 12.53 0.31 1.56
4.07 37 0.81 0.92 2.34 0.9 1.86 38 1.39 0.49 0.07 0.05 0.1 39 0.37
4.76 0.89 0.37 3.7 40 0.88 5.21 1.63 6.2 5.01 41 1.04 5.28 1.18 1.1
1.68 42 2.03 2.72 1.23 0.86 0.91 43 0.23 2.28 0.45 0.73 7.23 44
5.45 0.72 5.15 1.44 3.04 45 1.39 0.49 0.24 0.69 0.31 46 0.3 2.53
0.45 1.13 4.84 47 0.24 0.35 0.06 0.07 0.32 48 0.09 0.7 0.09 1.07
0.17 49 0.38 0.38 0.15 0.07 0.36 50 0.13 0.24 3.05 0.32 0.18 51 0.5
0.56 0.72 0.45 0.37 52 0.28 0.24 4.06 0.63 0.29 53 9 0.84 5.42 3.67
12.38 54 1.28 1.17 0.89 0.57 0.79 55 1.06 2.8 1.13 1.2 1.78 56 4.87
0.74 4.6 1.49 2.99 57 0.58 4.93 2.01 1.33 5.29 58 10.14 17.31 2.65
3.9 23.09 59 1.46 3.04 0.55 3.18 1.54 60 33.43 17.2 29.36 10.48
22.89 61 25.5 17.32 31.1 10.44 27.49 62 2.22 1.16 1.46 0.69 2.24 63
8.75 2.96 9.3 2.48 5.38 64 2.66 2.28 1.53 1.49 2.02 65 21.54 4.86
6.07 2.42 2.27 66 0.64 0.53 0.17 0.76 1.12 67 1.24 0.55 3.4 1.45
4.96 68 1.96 1.02 0.51 0.42 4.86 69 8.1 2.65 5.47 1.64 16.08 70
0.02 0.03 0.03 0.02 0.04 71 5.15 3.47 1.57 4 4.39 72 42.8 5.34 4.9
9.81 18.18 73 52.95 13.7 40.87 98.22 21.27 74 2.73 0.63 0.51 0.38
1.47 75 2.09 1.75 1.16 1.53 1.16 76 8.73 2.67 9.65 2.27 5.01 77
24.22 4.13 2.65 3.79 7.84 78 9.51 3.7 15.84 2.84 8.45 79 4.93 6.36
2.57 2.87 3.84 80 20.25 10.81 2.58 0.74 1.45 81 10.1 5.56 6.61
14.06 14.38 82 1.01 0.62 0.25 0.14 0.2 83 0.36 0.39 0.46 0.65 0.51
84 0.94 1.35 0.4 0.72 1.37 85 1.04 0.48 0.29 0.13 0.16 86 0.46 0.84
0.37 2.14 0.6 87 1.7 0.67 0.53 0.71 0.32 88 0.49 0.65 0.84 0.49
0.78 89 0.13 0.18 0.12 0.27 0.07 90 0.13 0.24 0.49 0.31 0.27 91
2.48 0.75 0.18 0.89 0.73 92 0.1 0.07 1.61 0.27 0.9 93 0.12 1.27
0.45 0.71 0.36 94 1.19 0.13 0.79 0.34 1.11 95 14.45 3.1 1.62 4.88
2.45 96 0.33 0.1 0.06 1.84 0.06 97 0.96 0.33 1.56 0.58 1.98 98 1.26
1.13 1.03 0.67 0.97 99 0.17 0.22 0.07 0.08 0.24 100 17.51 4.87 7.89
11.71 12.32 101 8.14 5.51 1.32 1.93 5.79 102 3.55 0.71 3.28 1.38
2.21 103 1.03 1.04 0.23 0.46 1.27 104 1.94 1.77 2.96 0.45 1.23 105
0.84 4.37 1.24 10.94 2.54 106 3.85 1.26 1.22 1.46 4.02 107 0.55
1.48 0.23 2.02 0.55 108 13.09 5.44 12.86 10.03 9.83 109 0.46 0.99
0.23 1.4 0.6 110 1.69 0.53 1.65 0.63 2.02 111 100 55.59 21.9 20.06
31.75 112 7.58 0.88 3.14 2.71 5.91 113 10.18 1.45 1.2 2.27 2.28 114
0.83 2.12 0.84 0.91 1.88 115 0.68 3.24 0.73 0.45 1.72 116 0.13 0.16
0.31 0.21 0.19 117 0.16 0.25 0.04 0.14 0.19 118 1.54 5.34 1.44 2.49
8.18 119 2.29 9.38 1.64 3.99 5.71 120 1.44 0.71 1.79 1.72 1.02 121
0.93 10.46 3.24 3.7 3.8 122 3.02 2 3.53 1.81 1.96 123 1.41 1.73
0.88 1.26 2.14 124 1.26 3.15 1.03 2.47 2.45 125 5.8 3.68 97.35 13.8
38.7 126 1.85 4.62 1.31 11.14 1.84 127 1.6 0.41 1.6 0.61 1.27 128
0.54 0.27 0.54 0.48 0.87 129 3.73 1.2 0.59 1.32 3.13 130 9.09 5.52
7.44 3.59 33.96 131 16.21 1.74 3.66 2.67 2.72 132 3.39 30.92 0.45
2.29 3.56 133 3.29 1.91 1.1 0.56 0.85 134 3.23 0.86 0.52 0.64 2.86
135 0.5 0.75 0.27 0.36 1.07 136 0.39 0.63 2.32 0.38 0.43 137 0.21
0.15 0.15 0.58 0.13 138 2.91 8.66 1.14 1.96 1.3 139 0.31 0.32 0.2
0.17 1.01 140 9.91 10.64 24.58 96.28 31.16 141 1.91 1.2 1.57 0.77
1.81 142 2.69 1.4 2.21 2.71 2.01 143 11.62 7.59 31.52 6.82 3.61 144
0.94 1.58 0.31 2.97 1.03 145 0.03 0.51 0.06 1.99 0.18 146 24.92 1.4
1.12 3.92 8.22 147 0.35 0.81 1.77 1.22 0.63 148 1.07 15.95 1.72
9.18 2.12 149 7.48 0.96 8.99 3.41 3.44 150 2.47 0.11 0.51 0.66 1.19
151 9.74 1.61 1.07 4.04 1.41 152 1.02 1.08 1.19 1.06 1.04 153 1.24
0.98 0.7 2.77 0.7 154 8.2 12.15 2.99 29.23 8.11 155 0.21 1.67 0.17
4.86 0.49 156 5.85 4.41 1.68 1.58 0.85 157 1.35 0.08 0.53 0.37 0.59
158 0.55 0.67 1.42 1.01 0.62 159 0.84 0.12 0.53 0.43 0.62 160 0.49
0.02 0.42 0.17 0.51 161 0.47 4.11 1.15 1.17 0.69 162 13.13 5.07
4.58 4.16 4.92 163 48.95 38.44 12.47 21.95 32.02 164 1.48 3.77 1.6
3.32 2.73 165 0.05 0.36 0.11 0.25 0.14 166 2.81 7 3.28 2.16 10.99
167 100 33.6 41.57 95.82 98.54 168 0.12 0.25 0.21 6.7 0.16 169 0.01
0.01 0.02 0.01 0.02 170 35.03 2.4 2.95 2.76 15.91 171 3.04 12.32
3.57 16.48 3.39 172 0.47 0.54 0.58 0.23 0.23 173 2.32 2.18 0.96
3.94 4.67 174 0.74 0.96 1.28 1.35 0.88 175 1.47 2.41 2.3 1.29 1.24
176 0.89 8.87 8.49 1.14 0.55 177 2.54 0.7 0.8 0.72 1.08 178 1.21
3.81 0.67 5.52 1.6 179 1.75 2.25 0.44 1.13 0.19 180 0.12 0.81 0.15
1.34 0.23 181 1.28 1.26 0.53 1.04 1.49 182 2.83 1.6 0.91 1.27 5.41
183 3.41 6.72 2.14 6.59 2.65 184 3.43 7.67 1.35 2.24 3.64 185 1.07
0.07 1.21 0.56 1.06 186 8.61 4.69 14.19 6.51 19.85 187 100 66 86.39
36.93 85.31 188 0.73 3.9 0.5 5.01 1.19 189 0.85 0.03 0.68 0.31 0.59
190 1.07 26.78 1.21 4.64 1.3 191 0.12 2.33 1.47 0.89 0.7 192 1.37
0.63 0.75 0.44 0.55 193 4.85 3.1 6.81 2.19 5.27 194 0.2 0.33 0.28
0.41 0.29 195 1.07 2.36 1.22 1.97 5.09 196 0.53 4.67 0.41 1.92 0.57
197 21.96 5.14 1.44 5.56 7.86 198 1.99 2.09 0.4 1.11 0.6 199 0.73
1.06 0.71 1.31 2.88 200 0.04 0.35 0.13 0.26 0.14 201 0.11 0.36 0.18
0.32 0.13 202 0.18 0.38 0.27 4.58 0.31 203 0.89 0.3 3 0.16 1.87 204
1.51 0.69 1.1 1.16 1.55 205 0.36 0.97 1.06 0.57 0.55 206 0.7 0.33
0.77 0.31 0.95 207 24.9 1.39 14.41 1.43 4.8 208 25.57 4.81 4.44
3.84 3.91 209 0.43 0.05 0.29 0.16 0.21 210 2.6 0.97 0.72 0.85 2 211
2.78 0.48 0.76 0.43 0.7 212 49.9 2.99 8.37 15.05 17.32 213 0.04
0.44 0.18 0.22 0.18 214 0.36 1.78 0.5 0.89 0.84 215 0.85 0.18 0.69
2.1 0.71 216 0.8 1.52 0.78 7.13 1.25 217 2.02 1.61 1.53 1.44 2.14
218 1.71 1.61 0.96 1.22 1.12 219 2.99 1.13 0.52 0.65 1.27 220 0.68
0.02 0.91 0.32 0.56 221 2.19 1.08 0.64 0.55 1.02 222 0.17 1.36 0.58
1 0.37 223 0.99 2.68 0.6 6.16 1.49 224 1.11 3.67 0.96 1.38 1.13 225
1.42 6.11 2.67 2.86 4.17 226 2.08 0.87 0.22 1.3 0.62 227 0.46 0.42
0.87 0.85 0.64 228 0.61 1.01 0.48 1.56 0.37 229 0.45 0.86 0.18 0.48
0.87 230 0.37 0.11 0.32 0.13 0.16 231 0.14 1.03 0.16 0.57 0.14 232
2.61 2.39 0.88 8.27 4.68 233 3.55 6.69 1.07 11.5 3.3 234 1.51 1.6
2.4 0.81 2.33 235 5.31 1.85 1.04 0.73 1.67 236 0.38 1.3 0.84 0.7
2.26
237 0.27 0.33 0.3 9.71 0.62 238 2.62 0.73 0.5 0.55 0.54 239 6.77
21.43 16.53 8.37 58.27 240 24.05 14.02 2.2 13.72 12.74 241 1.22
0.13 1.54 0.36 0.77 242 0.88 6.25 0.7 6.12 1.21 243 2.6 1.32 38.02
1.5 3.16 244 1.22 0.24 0.23 0.73 0.52 245 0.85 6.95 1.32 3.16 2.33
246 10 13.06 1.12 3.63 4.75 247 8.21 6.78 13.88 4 12.01 248 3.83
2.53 1.93 1.74 1.96 249 9.66 3.61 2.67 7.9 11.2 250 1.34 0.56 2.02
0.77 1.83 St3_A_NB72 St4_A_NB251 St4_A_NB265 St4_NA_NB206
St4_NA_NB8 Rank (P) (P) (P) (P) (P) 1 6.12 1.85 0.01 0.53 3.23 2
4.48 2.19 1.92 1.37 2.81 3 24.44 7.74 1.71 14.04 8.07 4 0.6 0.67
0.54 4.37 0.88 5 13.97 1.53 7.01 18.41 3.45 6 53.61 18.75 31.16
56.99 20.18 7 1.54 3.77 0.78 1.43 1.98 8 7.93 0.93 2.04 0.32 1.2 9
0.07 0.15 0.08 0.34 0.29 10 0.5 0.34 0.33 0.43 0.38 11 6.63 1.41
0.1 0.66 4.49 12 0.2 2.17 1.24 2.7 1.8 13 0.65 0.41 0.33 0.13 0.07
14 1.74 2.49 0.68 0.91 6.57 15 0.27 0.32 0.3 0.35 0.28 16 69.76
28.82 56.04 1.94 1.15 17 2.16 1.84 4 2.35 1.57 18 0.6 0.35 0.4 0.1
0.2 19 0.06 0.26 0.11 0.06 0.04 20 20.79 15.87 10.85 5.09 1.93 21
4.69 4.58 7 23.79 25.26 22 0.88 0.14 0.15 0.12 0.32 23 0.18 0.15
0.11 0.25 0.9 24 2.72 2.23 1.72 1.5 6.93 25 0.76 5.23 3.37 6.01 2.9
26 57.13 42.82 12.99 18.93 3.08 27 0.11 0.17 0.22 0.12 0.06 28 3.86
1.4 1.8 3.57 1.04 29 0.05 0.3 0.06 0.21 0.11 30 0.05 0.11 0.05 0.35
0.12 31 8.15 8.53 0.17 0.07 0.03 32 17.9 37.18 1.41 10.63 26.36 33
5.01 4.29 1.55 7.2 3.47 34 0.15 0.24 0.46 0.13 0.16 35 2.94 1.25
2.09 11.48 8.57 36 1.2 0.69 0.65 1.38 2.59 37 0.39 0.37 0.45 0.59
0.44 38 0.04 2.35 3.45 0.32 4.05 39 1.51 0.48 0.48 16.94 2.8 40
27.79 42.02 4.65 4.14 15.75 41 12.91 5.78 1.28 5.26 4.15 42 62.07
3.71 3.04 1.36 1.48 43 5.36 0.35 2.78 3.81 4.8 44 5.75 1.18 3.64
0.48 0.61 45 0.6 0.45 0.28 0.17 0.53 46 0.39 0.95 0.69 0.85 0.63 47
0.06 0.11 0.07 0.48 0.1 48 0.08 0.15 0.16 0.42 0.24 49 0.07 0.25
0.08 0.48 0.23 50 1.14 0.4 0.61 0.25 0.55 51 0.53 0.45 0.47 1.89
0.55 52 0.44 0.3 0.35 10.52 4.35 53 0.78 10.03 1.01 1.02 1.01 54
25.53 1.48 1.38 0.99 0.81 55 0.15 0.41 0.25 1.79 1.03 56 4.5 1.38
3.85 0.7 0.54 57 1.47 0.88 0.54 2.49 0.92 58 1.09 1.66 0.67 9.37
2.99 59 1.92 1.14 1.84 0.73 1.12 60 28.97 9.81 21.36 40.22 19.11 61
29.51 9.88 19.02 43.8 21.99 62 0.42 0.39 0.8 1.06 0.42 63 0.68 4.89
1.44 2.49 0.66 64 0.94 2.14 1.34 2.19 0.72 65 2.07 1.94 1.42 2.54
2.2 66 2.01 1.81 1.03 0.79 2.24 67 2.02 3.65 4.51 0.78 0.82 68 0.61
1.84 0.42 0.41 0.34 69 4.44 12.72 22.49 2.72 1 70 0.03 0.04 0.02
0.04 0.05 71 0.9 1.99 0.67 1.85 1.73 72 3.34 6.63 22.25 3.41 0.78
73 20.49 55.43 100 20.04 100 74 0.44 0.57 0.33 0.4 0.49 75 0.72
1.18 1.99 1.1 0.93 76 0.57 4.27 1.17 1.98 0.72 77 3.28 4.93 1.82
2.15 6 78 3.32 2.03 3.01 14.62 8.46 79 3.25 5.65 7.18 2.2 1.38 80
0.74 0.92 0.57 2.57 10.37 81 68.76 14.62 13.89 13.34 18.47 82 0.15
0.2 0.1 0.13 0.08 83 7.63 2.81 6.07 0.62 0.4 84 0.49 0.82 0.5 0.59
0.48 85 0.18 1.96 2.47 0.42 3.13 86 0.11 3.84 1.78 0.25 0.33 87
0.54 0.85 0.83 0.33 0.63 88 2.53 1.21 0.93 0.97 1.75 89 0.06 0.29
0.14 0.11 0.1 90 2.46 0.97 1.04 0.16 1.17 91 5.83 3.26 1.84 1.22
5.14 92 0.61 0.33 3.3 0.16 1.21 93 0.12 0.16 0.37 0.84 0.64 94 1.99
1.37 2.01 0.32 0.58 95 0.66 3.05 1.93 2.14 1.82 96 0.06 0.11 0.09
0.1 0.22 97 0.55 0.37 0.36 0.57 0.44 98 0.72 0.85 0.8 1.04 0.32 99
0.08 0.1 0.06 0.33 0.08 100 6.9 8.02 7.73 2.73 2.82 101 1.8 2.28
1.56 2.01 1.52 102 3.44 1.17 3.25 0.82 0.59 103 0.34 0.53 0.27 1.27
0.72 104 0.29 0.72 0.12 0.91 0.61 105 0.67 5.55 4.38 2.26 2.71 106
12.85 7.64 3.89 3.22 10.44 107 0.32 1.41 1.65 0.58 1.27 108 1.71
7.3 2.1 5.85 4.57 109 0.15 1.77 1.19 0.3 1.35 110 0.75 0.87 1.75
0.35 0.54 111 53.27 14.05 20.21 39.19 74.58 112 1.22 7.62 3.38 1.44
0.84 113 1.14 2.47 1 1.58 0.92 114 2.23 4.52 1.21 4.57 2.74 115
0.41 0.5 0.47 2.65 0.57 116 1.08 0.43 1.66 0.35 0.23 117 0.06 0.09
0.06 0.29 0.09 118 2.6 2.76 3.95 1.38 1.07 119 1.02 1.71 1.51 6.18
2.81 120 8.41 1.53 2.28 0.65 4.13 121 2.43 0.46 2.07 16.71 7.86 122
0.74 0.97 0.77 1.22 1.51 123 0.92 0.79 0.98 0.91 0.8 124 0.69 1.42
0.82 0.52 0.64 125 12.62 2.87 1.48 19.56 2 126 16.29 5.28 13.4 4.66
18.42 127 0.98 0.65 1.59 0.42 0.48 128 0.22 0.61 0.34 0.39 0.15 129
0.5 1.64 0.62 0.57 1.33 130 1.56 10.96 2.82 14.97 22.42 131 1.01
3.14 1.85 1.26 1.4 132 1.8 1.31 0.37 8.81 2.43 133 0.91 0.86 4.48
0.86 0.67 134 0.65 1.36 1.61 0.55 0.55 135 0.3 0.37 0.26 0.3 0.41
136 0.56 0.48 1.55 0.63 5.95 137 0.04 1.78 0.75 0.1 0.15 138 8.45
2.49 1.37 11.99 10.61 139 0.29 0.21 0.21 0.33 0.34 140 6.41 15.05
33.73 27.39 67.51 141 0.51 0.47 0.89 1.19 0.38 142 10.87 2.88 1.55
2.28 2.46 143 5.95 11.88 3.16 19.58 20.7 144 0.29 2.04 1.57 0.41
0.37 145 0.06 3.82 1.27 0.03 0.08 146 2.9 2.53 1.49 1.49 1.77 147
1.71 0.63 1.59 2.12 0.88 148 7.58 1.3 3.98 7.98 9.19 149 2.45 2.11
1.47 0.87 1.35 150 1.8 1.25 2.18 0.19 0.61 151 1.78 2.59 1.27 1.27
0.77 152 7.3 6.7 4.8 1.64 0.8 153 0.24 8.66 3.7 0.34 0.33 154 1.3
58.96 20.73 1.91 3.82 155 0.18 10.07 2.48 0.2 0.26 156 0.89 1.27
0.84 2.04 0.78 157 1.27 0.78 1.67 0.15 0.61 158 1.75 0.59 0.79 1.58
1.71 159 0.8 0.81 1.08 0.13 0.58 160 1.01 0.63 0.62 0.09 0.52 161
0.37 0.99 0.68 0.35 0.46 162 1.61 2.13 2.5 1.37 2.43 163 21.61
26.34 4.8 100 49.1 164 1.18 1.04 0.94 4.55 1.33 165 0.06 0.12 0.11
0.12 0.07 166 1.38 1.25 1.93 1.43 2.03 167 100 35.46 100 22.55 100
168 3.83 9.58 0.19 0.22 0.13 169 0.01 0.03 0.01 0.02 0.01 170 5.02
5.48 1.73 3.13 2.37 171 1.45 5.38 9.05 2.61 9.69 172 2.38 0.18 4.76
0.46 0.29 173 0.69 3.4 3.07 2.7 2.53 174 3.1 2.71 3.02 1.36 0.87
175 5.32 3.56 1.19 2.73 2.55 176 0.78 1.09 1.77 0.74 0.6 177 0.93
1.64 1.14 0.54 0.84 178 0.54 3.51 3.3 1.26 2.53 179 0.22 0.78 0.95
0.38 0.73 180 0.13 0.48 0.44 0.49 0.63 181 0.35 0.47 0.46 0.33 0.42
182 1.26 1.53 0.65 1.33 1.16 183 6.17 2.25 9.77 2.49 2.87 184 1.58
2.83 0.92 5.5 2.04 185 1.8 0.98 2.56 0.31 1.14 186 23.26 12.34
20.21 13.28 11.55 187 86.73 19.69 20.09 100 82.21 188 0.36 3.59
2.93 1.23 1.74 189 0.93 0.68 0.61 0.07 0.42 190 1.48 2.07 2.53 7.18
2.74 191 2.45 2.08 0.31 0.65 2.32 192 0.3 0.33 0.36 0.3 0.45 193
0.89 1.16 0.81 2.02 1.87 194 3.21 0.9 0.35 0.51 2.15 195 0.82 1.1
1.97 1.18 1 196 0.25 0.79 0.41 0.91 0.39 197 3.15 4.59 3.46 3.85
3.07 198 2.29 1.63 1.74 0.59 0.78 199 0.55 0.3 0.43 3.58 2.83 200
0.07 0.12 0.11 0.13 0.07 201 1.38 0.31 1.3 0.26 0.2 202 3.62 6.16
0.28 0.3 0.24 203 6.01 2.5 6.24 0.39 2.17 204 3.96 1.7 1.6 1.22
1.65 205 1.63 0.73 1.19 1.44 2.28 206 0.5 0.71 0.81 0.17 1.79 207
4.26 10 5.13 1.16 0.44 208 4.78 10.51 3.17 2.11 2.69 209 1.02 0.44
0.47 0.1 0.4 210 0.6 0.81 0.46 0.83 1.22 211 0.9 2.42 4.94 0.53
1.42 212 22.47 2.61 44.62 7.92 1.79 213 0.08 0.12 0.08 0.3 0.09 214
2.28 1.28 0.65 3.38 2.92 215 0.75 1.24 2.56 0.47 1.93 216 0.6 1.01
0.88 0.85 2.6 217 0.92 0.9 0.91 2.42 0.87 218 0.71 0.8 1.52 1.37
1.3 219 1.04 0.38 1.22 0.57 0.2 220 1.61 0.5 0.94 0.04 0.42 221
0.59 0.64 2.26 0.32 0.55 222 0.51 0.53 0.43 0.84 0.7 223 0.17 0.8
0.58 1.82 1.15 224 12.33 2.46 9.33 2.29 1.04 225 2.4 2.72 2.19 9.43
2.79 226 3.35 2.81 1.5 1.08 3.83 227 0.43 0.25 0.2 1.21 0.37 228
0.44 0.56 0.6 0.51 0.56 229 0.17 0.18 0.14 0.42 0.37 230 0.37 0.21
0.29 0.17 0.26 231 0.08 0.38 0.28 0.28 0.3 232 1.53 2.66 3.8 7 7.15
233 1.23 5.16 1.9 1.11 3.02
234 4.91 3.02 1.69 2.94 3.62 235 7.27 3.11 3.89 2.43 3.07 236 0.4
0.64 0.74 0.64 0.55 237 6.43 14.77 0.6 0.36 0.28 238 0.24 0.78 0.74
0.67 1.12 239 4.92 3.66 9.47 3.99 6.04 240 12.26 15.44 10.9 4.82
9.11 241 2.02 0.67 1.08 0.32 0.58 242 0.56 2.97 2.74 1.55 2.02 243
0.75 1.66 1.32 1.07 0.75 244 0.28 0.33 0.12 0.24 0.44 245 0.65 3.29
3.96 3.48 3.54 246 2.16 1.28 0.97 2.53 4.02 247 11.2 6.51 7.83
20.66 10.96 248 1.02 0.97 0.78 0.95 0.68 249 6.68 6.84 15.39 2.28
12.63 250 0.79 0.7 1.54 1.05 0.49
For the gene minimization procedure the 7 replicate samples were
placed in the training set and the remaining samples were then
randomly partitioned into training (n=35) and testing (n=21) sets
(FIG. 4B). The minimal number of clones for outcome prediction was
identified using the training set as described above.
Quality-filtered clones were first ranked by determining the
sensitivity of prediction of the 35 training samples with respect
to a change in the gene expression level of each clone. Then, using
increasing numbers of the top ANN-ranked clones, the minimum number
of clones that generated minimum prediction errors were identified
(FIG. 4B). Where multiple clones represented one gene, the
top-ranked clone was selected to obtain a minimal predictor gene
set. The ANNs were then recalibrated using the expression ratios of
these genes for the training samples (without performing principal
component analysis (PCA)). Finally the survival status of the test
samples was predicted using the trained ANNs (FIG. 4B). It was also
determined that the top 250 ranked genes would provide a
classification error less than about 2/35. These top 250 ranked
genes are given in Table 3.
Example 5
Outcome Prediction for High-Risk Patients
This example shows that the gene expression signatures for both the
full set of genes and the minimized gene set can separate those
patients currently stratified as high-risk according to their
survival status.
For the 24 high-risk patients in examples 3 and 4, the Kaplan-Meier
curves demonstrated that ANNs were able to further partition these
patients according to their clinical outcomes using all 37920
quality-filtered clones (P=0.0067), as well as the top 19
ANN-ranked genes (P=0.0005) (FIGS. 8 A and B). As shown in FIG. 8B,
the top 19 ANN-ranked genes were able to correctly predict all 5
with good signature as surviving, and 18/19 with poor signature as
dying, suggesting a potential benefit for predicting outcome in
these high-risk patients. The hazard ratio was again infinite as
all of the patients that we predicted to have a good-outcome
survived (Table 8).
To determine if the gene expression signatures could provide
additional predictive power over the conventional risk factors, a
Cox model was created using age, stage and MYCN amplification
excluding the ANN prediction results. The model showed that MYCN
amplification (P=0.0064) was the only significant factor (i.e.,
P<0.05, see FIG. 8C). Therefore another multivariate model using
MYCN amplification was built and the prediction results based on
all 37920 clones (FIG. 8D) (the ANN results based on the 37920
clones were used, because there were no deaths in the good
signature group using the 19 genes, and in these circumstances it
is not possible to create models where the hazard ratios are
infinite). Applying the likelihood ratio test, it was determined
that prediction by all clones added predictive ability to the model
(P=0.012). Additionally, the Kaplan-Meier curves (FIGS. 8E and 7F)
illustrate that ANN prediction can further separate the MYCN
non-amplified patients according to their survival status based on
either all clones (P=0.047) or in particular the 19 genes (P=0.0076
see FIG. 8F).
An ANN-based method for predicting the outcome of patients with NB
has been developed that can use the expression profiles of only 19
genes that provides a significant improvement in prediction over
the current known risk factors. Moreover, it has been found that
the most important advantage of the approach was the ability to
further partition COG (Children's Oncology Group) stratified
high-risk patients, in particular those without MYCN amplification,
into two subgroups according to their survival status. The ability
to predict the outcome of individual patients with high-risk NB at
initial diagnosis using gene expression signatures has major
clinical implications, since approximately 70% of the patients in
this group (about 50% of all NB patients) succumb to the disease.
Firstly, patients that are identified to have a poor signature,
i.e. predicted to die if given conventional therapy, may directly
benefit from the newer therapeutic strategy trials that are
currently under investigation by the cooperative study groups such
as COG. Secondly, since treatment-related death rates have been
reported to be as high as 23%, it may be possible to design future
dose intensity reduction trials to minimize therapy-related
morbidity and mortality for the high-risk patients who have a good
signature. An example of such a patient in the latter category is
NB14 (stage 4, MYCN-amplified) who despite his high-risk status
experienced event free survival for >3 yrs as was predicted by
our ANNs. Although the survival rate for patients with COG
stratified low-risk disease is 95%, our approach may identify the
few patients predicted to have a poor outcome by the ANNs who may
benefit from more aggressive therapy. For instance, although case
NB18 was classified as low-risk (based on stage 2 and MYCN not
amplified), our ANNs predicted this sample as poor-outcome, and
this patient died within 1.5 years after diagnosis. These results
indicate the potential utility of using the approach for
individualized management of patients with cancer
Since there was some overlap in the expression levels of the top 19
ANN-ranked genes between the prognostic groups, the prospect of
identifying a single gene that can accurately predict outcome is
unlikely. Thus, a combinatorial approach using several genes and
artificial machine learning algorithms provided for accurate
outcome prediction. MYCN amplification is an established marker for
high stage and poor outcome, and plays a role in the aggressive
phenotype of NB tumors. Our analysis confirmed MYCN as a prognostic
marker (ranked 16 out of 19), however, the median expression level
of this gene was similar in the two groups, in agreement with
previous reports that MYCN expression levels are not consistently
correlated with survival in patients with non-amplified tumors.
MYCN amplification is currently the only molecular marker utilized
for risk stratification, however, it cannot be used as the sole
risk predictor, as only 22% of NB patients have this molecular
trait.
Of the 19-predictor genes, 8 out of the 12 known genes have been
previously reported to be expressed in neural tissue. Of these, 5
were up regulated in the poor-outcome group (DLK1, PRSS3, ARC,
SLIT3, and MYCN) and 3 were down regulated (CNR1, ROBO2, and
BTBD3). DLK1 (ranked number 1) is the human homologue of the
Drosophilia delta gene and is expressed by neuroblasts in the
developing nervous system as well as in neuroblastoma. It is a
transmembrane protein that activates the Notch signaling pathway,
which has been shown to inhibit neuronal differentiation.
Additionally, ARC, MYCN, and SLIT3 are also expressed during neural
development. The higher expression levels of these genes in the
poor-outcome tumors suggest a more aggressive phenotype
characterized by a less differentiated state, reminiscent of
proliferating and migrating neural crest progenitors. Up regulation
of the neuron axon repellant gene, SLIT3, was observed with the
down regulation of one of its receptors, ROBO2, in the poor-outcome
group suggesting the possibility that these neuroblastoma cells
secrete a substrate to repel connecting axons and potentially
prevent differentiation.
Of additional interest, the ARHI gene, which maps to 1p31, is a
maternally imprinted tumor suppressor gene implicated in ovarian
and breast cancer, possibly through methylation silencing, and is
among the down regulated genes for the poor-outcome group. A
further study of its role in tumorigenesis as a potential tumor
suppressor gene in NB is warranted particularly because of its
proximity to the 1p36 region, which is frequently deleted in
poor-outcome NB patients.
We noted the absence of three previously reported prognostic
related genes, TRKA, TRKB and FYN, amongst our 19 genes.
Unfortunately, TRKA was not on the microarrays, and TRKB and FYN
were not ranked within the top 500 clones by ANNs. At this point,
the predictive role of TRKA, TRKB or FYN is not conclusive, and
none are currently utilized to guide therapy.
In this study a small subset of 19 predictor genes was identified
from a pool of 25933 unique genes with the majority of genes
showing a greater than two fold average differential expression
between good- and poor-outcome tumors. This small number of genes
can be provide cost-effective clinical assays for outcome
prediction. In addition, the products of 3 genes (DLK1, SLIT3, and
PRSS3) are secreted proteins, indicating the utility of these
proteins as serum markers for prognosis.
In this data set, our ANN-based method provided a significant
improvement in prediction over the current risk factors in patients
with NB. Moreover, an advantage of the approach is the ability to
further partition COG stratified high-risk patients, in particular
those without MYCN amplification, into two subgroups according to
their survival status. This approach would allow physicians to
tailor therapy for each individual patient according to their
molecular profile, with the prospect of improving clinical outcome
and survival rates in patients with neuroblastoma.
The above specification, examples and data provide a complete
description of the manufacture and use of the composition of the
invention. Since many embodiments of the invention can be made
without departing from the spirit and scope of the invention, the
invention resides in the claims hereinafter appended. All
references identified herein are hereby incorporated by
reference.
SEQUENCE LISTINGS
0 SQTB SEQUENCE LISTING The patent contains a lengthy "Sequence
Listing" section. A copy of the "Sequence Listing" is available in
electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US08283122B2)-
. An electronic copy of the "Sequence Listing" will also be
available from the USPTO upon request and payment of the fee set
forth in 37 CFR 1.19(b)(3).
* * * * *
References