U.S. patent application number 12/782484 was filed with the patent office on 2010-12-23 for reducing irf4, dusp22, or flj43663 polypeptide expression.
This patent application is currently assigned to MAYO FOUNDATION FOR MEDICAL EDUCATION AND RESEARCH. Invention is credited to Ahmet Dogan, Andrew L. Feldman, Mark Law, David I. Smith, George Vasmatzis.
Application Number | 20100324119 12/782484 |
Document ID | / |
Family ID | 43354882 |
Filed Date | 2010-12-23 |
United States Patent
Application |
20100324119 |
Kind Code |
A1 |
Feldman; Andrew L. ; et
al. |
December 23, 2010 |
REDUCING IRF4, DUSP22, OR FLJ43663 POLYPEPTIDE EXPRESSION
Abstract
This document relates to the activity of interferon regulatory
factor 4 (IRF4) in T-cell lymphomas. For example, methods and
materials involved in reducing the expression of an IRF4
polypeptide in T-cell lymphoma cells and identifying agents having
the ability to reduce expression of an IRF4 polypeptide in T-cell
lymphoma cells are provided. This document also relates to reducing
DUSP22 or FLJ43663 polypeptide activity in T-cell lymphomas. For
example, methods and materials involved in reducing the expression
of DUSP22 polypeptides and/or FLJ43663 polypeptides in T-cell
lymphoma cells and identifying agents having the ability to reduce
expression of DUSP22 polypeptides and/or FLJ43663 polypeptides in
T-cell lymphoma cells are provided.
Inventors: |
Feldman; Andrew L.;
(Rochester, MN) ; Dogan; Ahmet; (Rochester,
MN) ; Vasmatzis; George; (Oronoco, MN) ; Law;
Mark; (Rochester, MN) ; Smith; David I.;
(Rochester, MN) |
Correspondence
Address: |
FISH & RICHARDSON P.C. (TC)
PO BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
MAYO FOUNDATION FOR MEDICAL
EDUCATION AND RESEARCH
Rochester
MN
|
Family ID: |
43354882 |
Appl. No.: |
12/782484 |
Filed: |
May 18, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61179201 |
May 18, 2009 |
|
|
|
Current U.S.
Class: |
514/44A ;
435/6.14; 435/7.1 |
Current CPC
Class: |
G01N 33/57407 20130101;
A61P 35/00 20180101; G01N 2333/4703 20130101; A61K 31/713
20130101 |
Class at
Publication: |
514/44.A ; 435/6;
435/7.1 |
International
Class: |
A61K 31/713 20060101
A61K031/713; C12Q 1/68 20060101 C12Q001/68; G01N 33/68 20060101
G01N033/68; A61P 35/00 20060101 A61P035/00 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] Funding for the work described herein was provided by the
federal government under grant number CA097274, awarded by the
National Cancer Institute. The federal government has certain
rights in the invention.
Claims
1. A method for reducing IRF4 polypeptide expression within a
T-cell lymphoma cell, said method comprising: a) identifying a
mammal having a T-cell lymphoma cell, and b) administering an IRF4
polypeptide inhibitor to said mammal under conditions wherein said
IRF4 polypeptide expression is reduced.
2. The method of claim 1, wherein said mammal is a human.
3. The method of claim 1, wherein said T-cell lymphoma cell is a
peripheral T-cell lymphoma cell.
4. The method of claim 3, wherein said peripheral T-cell lymphoma
cell is a primary cutaneous type anaplastic large cell lymphoma
cell.
5. The method of claim 1, wherein said administering step comprises
intravenous administration.
6. The method of claim 1, wherein said IRF4 polypeptide inhibitor
is an siRNA molecule capable of inducing RNA interference against
mRNA encoding an IRF4 polypeptide.
7. A method for reducing DUSP22 polypeptide expression within a
T-cell lymphoma cell, said method comprising: a) identifying a
mammal having a T-cell lymphoma cell, and b) administering an
DUSP22 polypeptide inhibitor to said mammal under conditions
wherein said DUSP22 polypeptide expression is reduced.
8. The method of claim 7, wherein said mammal is a human.
9. The method of claim 7, wherein said T-cell lymphoma cell is a
peripheral T-cell lymphoma cell.
10. The method of claim 9, wherein said peripheral T-cell lymphoma
cell is a primary cutaneous type anaplastic large cell lymphoma
cell.
11. The method of claim 7, wherein said administering step
comprises intravenous administration.
12. The method of claim 7, wherein said DUSP22 polypeptide
inhibitor is an siRNA molecule capable of inducing RNA interference
against mRNA encoding an DUSP22 polypeptide.
13. A method for reducing FLJ43663 polypeptide expression within a
T-cell lymphoma cell, said method comprising: a) identifying a
mammal having a T-cell lymphoma cell, and b) administering an
FLJ43663 polypeptide inhibitor to said mammal under conditions
wherein said FLJ43663 polypeptide expression is reduced.
14. The method of claim 13, wherein said mammal is a human.
15. The method of claim 13, wherein said T-cell lymphoma cell is a
peripheral T-cell lymphoma cell.
16. The method of claim 15, wherein said peripheral T-cell lymphoma
cell is a primary cutaneous type anaplastic large cell lymphoma
cell.
17. The method of claim 13, wherein said administering step
comprises intravenous administration.
18. The method of claim 13, wherein said FLJ43663 polypeptide
inhibitor is an siRNA molecule capable of inducing RNA interference
against mRNA encoding an FLJ43663 polypeptide.
19. A method for assessing a lymphoma of a mammal, wherein said
method comprises: (a) detecting an IRF4 translocation within said
lymphoma, and (b) classifying said mammal as having cutaneous
anaplastic large-cell lymphoma.
20. The method of claim 19, wherein said mammal is a human.
21. A method for assessing a lymphoma of a mammal, wherein said
method comprises: (a) determining whether or not said lymphoma of
said mammal has a translocation selected from the group consisting
of IRF4 translocations, DUSP22 translocations, and FLJ43663
translocations, and (b) classifying said mammal as having an
anaplastic large-cell lymphoma if said lymphoma has said
translocation.
22. The method of claim 21, wherein said mammal is a human.
23. A method for assessing a lymphoma of a mammal, wherein said
method comprises: (a) determining whether or not said a cell of
said lymphoma expresses an elevated level of an FLJ43663 mRNA or
polypepide, and (b) classifying said mammal as having an anaplastic
large-cell lymphoma if said cell expresses said elevated level.
24. The method of claim 21, wherein said mammal is a human.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is claims the benefit of U.S. Provisional
Application Ser. No. 61/179,201, filed May 18, 2009. The disclosure
of the prior application is considered part of (and is incorporated
by reference in) the disclosure of this application.
BACKGROUND
[0003] 1. Technical Field
[0004] This document relates to reducing interferon regulatory
factor 4 (IRF4) activity in T-cell lymphomas. For example, methods
and materials involved in reducing the expression of an IRF4
polypeptide in T-cell lymphoma cells and identifying agents having
the ability to reduce expression of an IRF4 polypeptide in T-cell
lymphoma cells are provided. This document also relates to reducing
DUSP22 or FLJ43663 polypeptide activity in T-cell lymphomas. For
example, methods and materials involved in reducing the expression
of DUSP22 polypeptides and/or FLJ43663 polypeptides in T-cell
lymphoma cells and identifying agents having the ability to reduce
expression of DUSP22 polypeptides and/or FLJ43663 polypeptides in
T-cell lymphoma cells are provided.
[0005] 2. Background Information
[0006] T-cell lymphomas are aggressive cancers that cause death in
the majority of affected patients despite treatment with
traditional chemotherapy. Peripheral T-cell lymphomas (PTCLs), in
which abnormal T-lymphocytes are found in the lymph nodes, body
organs, and sometimes in the peripheral circulating blood and/or
bone marrow, represent 10% of non-Hodgkin lymphomas. PTCLs are
fatal in the majority of patients.
SUMMARY
[0007] This document relates to reducing interferon regulatory
factor 4 (IRF4) activity in T-cell lymphomas. For example, methods
and materials involved in reducing the expression of an IRF4
polypeptide in T-cell lymphoma cells and identifying agents having
the ability to reduce expression of an IRF4 polypeptide in T-cell
lymphoma cells are provided. This document also provides methods
and materials for reducing DUSP22 and/or FLJ43663 polypeptide
activity in T-cell lymphomas. For example, methods and materials
for reducing the expression of DUSP22 polypeptides and/or FLJ43663
polypeptides in T-cell lymphoma cells and identifying agents having
the ability to reduce expression of DUSP22 polypeptides and/or
FLJ43663 polypeptides in T-cell lymphoma cells are provided.
[0008] In some cases, an agent that inhibits the expression of an
IRF4 polypeptide can be used to reduce the proliferation of
abnormal T-lymphocytes (e.g., T-cell lymphomas). In addition, this
document provides methods (e.g., in vivo and in vitro assays) for
identifying agents (e.g., antibodies, siRNAs, or other compounds)
that can reduce the expression of an IRF4 polypeptide in a
mammal.
[0009] In some cases, an agent that inhibits the expression of an
DUSP22 polypeptide and/or an FLJ43663 polypeptide can be used to
reduce the proliferation of abnormal T-lymphocytes (e.g., T-cell
lymphomas). In addition, this document provides methods (e.g., in
vivo and in vitro assays) for identifying agents (e.g., antibodies,
siRNAs, or other compounds) that can reduce the expression of an
DUSP22 polypeptide and/or an FLJ43663 polypeptide in a mammal.
[0010] In general, one aspect of this document features a method
for reducing IRF4 polypeptide expression within a T-cell lymphoma
cell. The method comprises, or consists essentially of, identifying
a mammal having a T-cell lymphoma cell and administering an IRF4
polypeptide inhibitor to the mammal under conditions wherein the
IRF4 polypeptide expression is reduced. The mammal can be a human.
The cell can be a peripheral T-cell lymphoma cell. The peripheral
T-cell lymphoma cell can be a primary cutaneous type anaplastic
large cell lymphoma cell. The administering step can include
intravenous administration. The IRF4 polypeptide inhibitor can be
an siRNA molecule capable of inducing RNA interference against mRNA
encoding an IRF4 polypeptide.
[0011] In another aspect, this document features a method for
reducing DUSP22 polypeptide expression within a T-cell lymphoma
cell. The method comprises, or consists essentially of, (a)
identifying a mammal having a T-cell lymphoma cell, and (b)
administering an DUSP22 polypeptide inhibitor to the mammal under
conditions wherein the DUSP22 polypeptide expression is reduced.
The mammal can be a human. The T-cell lymphoma cell can be a
peripheral T-cell lymphoma cell. The peripheral T-cell lymphoma
cell can be a primary cutaneous type anaplastic large cell lymphoma
cell. The administering step can comprise intravenous
administration. The DUSP22 polypeptide inhibitor can be an siRNA
molecule capable of inducing RNA interference against mRNA encoding
an DUSP22 polypeptide.
[0012] In another aspect, this document features a method for
reducing FLJ43663 polypeptide expression within a T-cell lymphoma
cell. The method comprises, or consists essentially of, (a)
identifying a mammal having a T-cell lymphoma cell, and (b)
administering an FLJ43663 polypeptide inhibitor to the mammal under
conditions wherein the FLJ43663 polypeptide expression is reduced.
The mammal can be a human. The T-cell lymphoma cell can be a
peripheral T-cell lymphoma cell. The peripheral T-cell lymphoma
cell can be a primary cutaneous type anaplastic large cell lymphoma
cell. The administering step can comprise intravenous
administration. The FLJ43663 polypeptide inhibitor can be an siRNA
molecule capable of inducing RNA interference against mRNA encoding
an FLJ43663 polypeptide.
[0013] In another aspect, this document features a method for
assessing a lymphoma of a mammal. The method comprises, or consists
essentially of, (a) detecting an IRF4 translocation within the
lymphoma, and (b) classifying the mammal as having cutaneous
anaplastic large-cell lymphoma. The mammal can be a human.
[0014] In another aspect, this document features a method for
assessing a lymphoma of a mammal. The method comprises, or consists
essentially of, (a) determining whether or not the lymphoma of the
mammal has a translocation selected from the group consisting of
IRF4 translocations, DUSP22 translocations, and FLJ43663
translocations, (b) classifying the mammal as having an anaplastic
large-cell lymphoma if the lymphoma has the translocation. The
mammal can be a human.
[0015] In another aspect, this document features a method for
assessing a lymphoma of a mammal. The method comprises, or consists
essentially of, (a) determining whether or not the a cell of the
lymphoma expresses an elevated level of an FLJ43663 mRNA or
polypepide, and (b) classifying the mammal as having an anaplastic
large-cell lymphoma if the cell expresses the elevated level. The
mammal can be a human.
[0016] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used to practice the invention, suitable
methods and materials are described below. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict,
the present specification, including definitions, will control. In
addition, the materials, methods, and examples are illustrative
only and not intended to be limiting.
[0017] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 shows photographs of immunohistochemistry, karyotype,
and fluorescence in situ hybridization (FISH) identifying
peripheral T-cell lymphomas, unspecified (PTCL-Us) with IRF4/TCRA
translocations involving bone marrow and skin: (a) Diffuse
infiltration of bone marrow (75% involvement) in a 67 year-old male
(Case 1; H&E, .times.4) (reticulin fibrosis is present (inset,
.times.40)); (b) Medium to large tumor cells with plasma cells in
the background (H&E, .times.40; inset, .times.100); Tumor cells
are positive for (c) CD3, (d) TIA1, (e) and IRF4 (.times.40; inset,
.times.100); (f) Skin biopsy from the same patient 4 months later
(H&E, .times.40; inset, .times.100)); (g) Diffuse infiltration
of bone marrow (40% involvement) in a 71 year-old male (Case 2;
H&E, .times.4; reticulin, inset, .times.40); (h) Mostly
medium-sized tumor cells with plasma cells in the background
(H&E, .times.40; inset, .times.100); (i) Tumor cells are
positive for IRF4 (.times.40; inset, .times.100); (j) Karyotype
shows t(6;14)(p25;q11.2); (k) Dual-fusion fluorescence in situ
hybridization (D-FISH) shows IRF4/TCRA fusion signals (arrows).
[0019] FIG. 2 shows immunohistochemistry and breakapart
fluorescence in situ hybridization (BAP-FISH) identifying
anaplastic large-cell lymphomas (ALCLs) with IRF4 translocations:
(a) Primary cutaneous ALCL (C-ALCL), 48 year-old female (case 3)
(Medium to large tumor cells with admixed histiocytes (H&E,
.times.40; inset, .times.100)); (b) Lymph node involvement, same
patient, 10 years later (Confluent sheets of large "hallmark" cells
(H&E, .times.40; inset, .times.100)); (c) C-ALCL, 67 year-old
male (case 4; H&E, .times.20; inset, .times.100); (d) Lymph
node involvement, same patient, 7 months later (H&E, .times.40;
inset, .times.100) (Both biopsies show sheets of "hallmark" cells);
(e) C-ALCL, 89 year-old female (case 5), showing positivity for (f)
CD30 and (g) IRF4 (.times.10; insets, .times.100); (h) BAP-FISH
shows separation of signals flanking the IRF4 gene locus (arrows);
(i) Systemic ALK-negative ALCL, cervical lymph node, 79 year-old
male (case 12) (Large "hallmark" cells surround a residual reactive
follicle (lower left; H&E, .times.40; inset, .times.100)).
[0020] FIG. 3 shows IRF4 gene amplification and translocations in
T-cell lymphomas: (A) shows BAP-FISH of a single tumor cell with 7
copies of IRF4 on 6p25 (arrow) and 3 copies of the chromosome 6
centromere (arrowhead); (B) is a bar graph showing the percentage
of cases with IRF4 translocations observed in 68 skin biopsies
based on cancer type (1, systemic ALK-negative ALCL; 2, systemic
ALK-positive ALCL, 3, cutaneous ALCL; 4, lymphomatoid papulosis; 5,
mycosis fungoides; 6, Sezary syndrome; 7, CD4-positive TCL; 8,
extranodal NK/TCL; 9, subcutaneous panniculitis-like TCL; 10,
peripheral TCL, unspecified).
[0021] FIG. 4(A) is a photograph of a 5 .mu.m section of a tissue
microarray stained for IRF4 by immunohistochemistry (each circle
represents a section of a 0.6 mm core from a paraffin block of
human T-cell lymphoma tissue, three cores are taken from each
tissue block to ensure representative sampling (black box, upper
right)); (B) At higher magnification, brown nuclear staining
(diaminobenzidine) demonstrates IRF4 protein in the nuclei of
T-cell lymphoma cells both with (left) and without (right) IRF4
gene translocations; and (C) is a western blot of IRF4 from T-cell
lymphoma human tissue samples.
[0022] FIG. 5(A) is a photograph of immunohistochemistry staining
for IRF4 showing strong nuclear staining in SR786 T-cell anaplastic
large cell lymphoma cells; T-cell lymphoblastic leukemia cell lines
such as CCRF-CEM were negative for IRF4 staining; (B) is a western
blot for IRF4 prepared using T-cell lymphoma cell lines.
[0023] FIG. 6(A) are western blots for IRF4, MYC, and actin
prepared from SUDHL-1 T-cell lymphoma cells that were treated with
either IRF4 siRNA, a control (scrambled) siRNA, or no siRNA at 48
hours; (B) is a bar graph displaying IRF4 siRNA-mediated inhibition
of SUDHL-1 cell proliferation in two independent experiments by 47%
and 39%, respectively (.sup.3H-thymidine assay; each bar shows mean
of 6 wells, error bars show S.D.).
[0024] FIG. 7 is a schematic showing a proposed altered IRF4
regulatory network in T-cell lymphoma cells.
[0025] FIG. 8. Fluorescence in situ hybridization (FISH) in a fatal
case of ALK-negative anaplastic large cell lymphoma (ALCL).
Breakapart FISH probes to the 6p25.3 region included Probe #1 and
Probe #2 as published.16 Probe #1 is specific for the IRF4 locus
(A), while Probe #2 is less specific and also encompasses the
DUSP22 locus (B). Panel (C) shows morphology typical of ALCL;
immunohistochemical stains showed positivity for CD30 and
negativity for ALK (not shown). No IRF4 translocation was detected
by FISH using Probe #1 (not shown). Panel (D) shows the result of
FISH using Probe #2. The separated red and green signals (arrows)
indicate a translocation. Since this pattern was not seen with
Probe #1, a DUSP22 translocation is suggested.
[0026] FIG. 9 is a histogram of tag distances within perfect match
pairs.
[0027] FIG. 10. Summary of results of bioinformatic analysis of
mate-pair library sequencing data from the case shown in FIG. 8.
The region of the known 6p25.3 rearrangement is shown in the bottom
panel. The X-axis shows nucleotides according to the February 2009
genome assembly (GRCh37/hg19). Horizontal black bars represent
.about.5000 by DNA fragments from which the two ends (the "mate
pairs") were sequenced. The numerals in the bottom panel represent
mate pairs in which one end maps to 6p25.3 and the other end maps
to a different chromosome. Ten distinct (i.e. non-identical) mate
pairs are seen which map to a narrow region of chromosome 7 (eight
on the positive strand and two on the negative strand). In the
upper panel a similar map shows the corresponding locus on 7q32.3,
with the numerals showing the sites of the same 10 aberrant mate
pairs. Since these aberrant mate pairs have ends that map to
distinct regions of the genome, they suggest a possible
translocation between loci adjacent to these paired ends.
Occasional single numerals (e.g. the numeral "14" in the lower
right of the bottom panel) represent sporadic, non-repeated,
aberrant mate pairs that may be introduced during the ligation step
of the mate-pair library preparation. Thus, the number of
non-identical mate-pairs with ends mapping to distinct genomic
regions (e.g. 6p25.3 and 7q32.3 in the above diagram) is a
criterion in identifying putative translocations. The putative
breakpoints will lie between the positive-strand mate pairs and the
negative-strand mate pairs. In this case, subsequent PCR and
sequencing (see below) confirmed the breakpoints at the regions
indicated by the vertical lines.
[0028] FIG. 11. Sequence mapping of the der(6)t(6;7)(p25.3;q32.3).
Five representative mate pairs are shown in the top panel. For each
mate pair, one end maps to 6p25.3 (black boxes) and the other end
maps to 7q32.3 (shaded boxes). Primers designed to amplify the
intervening DNA (which includes the breakpoint) were used to
produce the PCR bands shown in the gel image. These bands were
excised and DNA was extracted and sequenced. Sequences were mapped
to the genome using the BLAT tool available at "http," colon,
slash, slash, "genome.ucsc.edu," slash,
"cgi-bin/hgBlat?command=start&org=Human&db=hg19&hgsid=159409307."
This analysis indicated the translocation joins the FLJ43663
hypothetical gene region on 7q32.3 with the DUSP22 gene on
6p25.3.
[0029] FIG. 12. Breakapart FISH probes designed to differentiate
IRF4 translocations (Probe #3)(A) from DUSP22 translocations (Probe
#4)(B) in cases with known rearrangements of the 6p25.3 region (as
determined by abnormal signal separation using Probe #2). The
red-labeled probe (CTD-2314K17) cross-hybridizes to 16p11 (not
shown), and these resultant extra red signals must be taken into
account when interpreting the results of FISH using Probes #3 and
#4 (see FIG. 6, below).
[0030] FIG. 13. Use of breakapart FISH Probes #3 and #4 to
differentiate IRF4 translocations from DUSP22 translocations in
cases with known 6p25.3 rearrangements. Above, a case of ALCL shows
abnormal separation of green and red signals using Probe #3, with
two additional red signals attributable to cross-hybridization (A).
The same case shows a normal signal pattern with Probe #4 (two
fusion signals and two red cross-hybridization signals)(B); thus,
this case has an IRF4 translocation. Below, a case of ALCL shows a
normal signal pattern with Probe #3 (two fusion signals and two red
cross-hybridization signals)(C), but shows abnormal separation of
green and red signals using Probe #4, with two additional red
signals attributable to cross-hybridization (D); thus, this case
has a DUSP22 translocation.
[0031] FIG. 14. Results of a FISH study of 82 Mayo Clinic patients
with ALCL, performed by screening cases for 6p25.3 rearrangements
with Probe #2 (FIG. 1B), and testing cases with abnormal signal
separation using Probes #3 and #4 to distinguish between IRF4 and
DUSP22 translocations (FIGS. 5 and 6). ALK immunohistochemistry was
used as a surrogate for the presence of ALK translocations.
Notably, IRF4 and DUSP22 translocations occurred only in
ALK-negative cases.
[0032] FIG. 15. DUSP22 expression and activity in TCLs. (A) Western
blotting shows DUSP22 expression in all TCL cell lines tested,
including T-lymphoblastic leukemias/lymphomas (Jurkat and
CCRF-CEM), mycosis fungoides/Sezary syndrome (MyLa), ALK-positive
ALCLs (SUDHL-1 and Karpas 299), and ALK-negative ALCL (FE-PD). (B)
DUSP22 protein is down-regulated in SUDHL-1 ALCL cells after
treatment with a DUSP22-specific small interfering RNA (siRNA). (C)
SUDHL-1 cells treated with DUSP22-specific siRNA show a 43%
reduction in proliferation compared to those treated with control
siRNA.
[0033] FIG. 16. Breakapart FISH probe to FLJ43663 hypothetical gene
region on 7q32.3. DNA from the centromeric and telomeric bacterial
artificial chromosomes (BACs) is labeled green or red,
respectively, as shown. The breakpoint identified in the sequenced
case (FIG. 8) is shown by the vertical dotted line.
[0034] FIG. 17. FISH using the 7q32.3 BAP probe on a section from
the sequenced tumor. Nuclei are stained blue (appears gray). Dotted
arrows indicate cells showing abnormal separation of the red and
green signals, indicating a translocation. Solid arrows indicate
cells with a normal signal pattern (2 fusion signals). Below, the
location of the probe on chromosome 7 is shown.
[0035] FIG. 18. Results of 7q32.3 BAP FISH in 30 TCLs with known
6p25.3 rearrangements. Concurrent rearrangements of 7q32.3 were
seen in 41% of the cases (representative FISH image shown in lower
right). An additional 23% had extra copies of the FLJ43663 locus
(aneuploid), while one-third of cases showed a normal signal
pattern.
[0036] FIG. 19. Validation of the t(6;7)(p25.3;q32.3) dual-fusion
FISH (D-FISH) probe. The probe was constructed by labeling DNA from
both BACs in 6p25.3 Probe #2 (FIG. 1B) red, and DNA from both BACs
in the 7q32 probe. The red and green signals are seen to hybridize
to their respective loci in a metaphase from peripheral blood
(left), and show a normal signal pattern in a paraffin section of a
lymph node (right: 2 red signals and 2 green signals in each
cell).
[0037] FIG. 20. Expression of the 3' terminus of FLJ43663 on 7p32.3
is increased in T-cell lymphomas with 6p25.3 rearrangements. Gene
microarray data from 25 TCL patients shows expression of FLJ43663
in 21 untranslocated cases and 4 translocated cases. The panel
above shows the relative locations of the normal FLJ43663 locus and
the 3' portion remaining on the derivative chromosome 7 after
translocation in the sequenced case. The relative locations of 4
EST-based array probes are shown. The bottom panels show mean
relative expression values normalized to the mean expression in
untranslocated cases. Translocated cases showed a mean expression
value of the 3' EST (AL569506) 5.5-fold higher than that seen in
untranslocated cases, whereas there was no difference in expression
of 5' probes or a 3' probe outside the FLJ43663 locus.
[0038] FIG. 21. An FLJ43663/DUSP22 fusion transcript is present in
the sequenced case with t(6;7)(p25.3;q32.3). PCR was performed on
cDNA using primers to FLJ43663 and DUSP22 (100 by ladder). The
arrows indicate bands demonstrating fusion transcripts of the
predicted sizes. The band in lane 5 was generated using primers
CCCTGGGGCATTTTATTAA and AGCCACTGCCGATACTGATG (566 bp), and that
indicated in lane 8 was generated using primers GCAGCCTGGCGTGACAAG
and AGCCACTGCCGATACTGATG (842 bp). These primers generate no
matches by in silico PCR of the normal human genome, 22 and are
.about.100 kb apart in the hybrid genomic DNA from the sequenced
patient. The bright bands in lanes 3 and 9 represent amplicons of
the expected sizes using primers for the intact regions (not
incorporating the breakpoint) of DUSP22 (lane 3) and FLJ43663 (lane
9), respectively. Sequencing of the band shown in lane 5 confirms
the presence of the fusion transcript, shown below, joining the 3'
portion of exon 3 of FLJ43663 to the 5' portion of exon 2 of
DUSP22.
DETAILED DESCRIPTION
[0039] This document relates to reducing IRF4 polypeptide activity,
DUSP22 polypeptide activity, and/or FLJ43663 polypeptide activity
in T-cell lymphomas. For example, methods and materials are
provided for reducing the expression of an IRF4 polypeptide in
T-cell lymphoma cells and identifying agents having the ability to
reduce expression of an IRF4 polypeptide in T-cell lymphoma cells.
IRF4 polypeptides can be expressed in the cells of any mammal
(e.g., a mouse, (GI:7305518), rat (GI:157816962), dog
(GI:73992105), cow (GI:115497401), horse (GI:194222960), chimpanzee
(GI:114605169), monkey (GI:109069403), and human (GI:167555103)).
DUSP22 polypeptides can be expressed in the cells of any mammal
(e.g., a mouse (GI: 146198766 for isoform A and 133892215 for
isoform B), rat (GI: 157822426), chimpanzee (GI: 229892211), and
human (GI: 34147625)). FLJ43663 polypeptides can be expressed in
the cells of any mammal (e.g., a human (GI: 205277371, 205277373,
or 51094836)). An agent to reduce the expression of an IRF4
polypeptide, a DUSP22 polypeptide, and/or FLJ43663 polypeptide can
be administered to a mammal. A mammal can be any type of mammal
including, without limitation, a mouse, rat, dog, cat, horse,
sheep, goat, cow, pig, monkey, or human.
[0040] In some cases, an agent to reduce the expression of an IRF4
polypeptide, a DUSP22 polypeptide, and/or FLJ43663 polypeptide can
be administered to a mammal that has been identified as having a
peripheral T-cell lymphoma. Peripheral T-cell lymphomas can
include: anaplastic large cell lymphoma, primary systemic type;
anaplastic large cell lymphoma, primary cutaneous type;
angioimmunoblastic T-cell lymphoma; T-cell prolymphocytic leukemia;
T-cell large granular lymphocytic leukemia; adult T-cell
leukemia/lymphoma; enteropathy-associated T-cell lymphoma;
hepatosplenic T-cell lymphoma; Mycosis fungoides/Sezary syndrome;
subcutaneous panniculitis-like T-cell lymphoma; and unspecified
type peripheral T-cell lymphomas. An inhibitor of an IRF4
polypeptide can be any agent that reduces the expression of an IRF4
polypeptide (e.g., an siRNA molecule, antisense oligonucleotide, or
peptide nucleic acid) or that reduces the activity of an IRF4
polypeptide (e.g., an inhibitory anti-IRF4 antibody, anti-IRF4
aptamer, or an IRF4 polypeptide antagonist). In some cases, the
methods and materials provided herein can be used to treat a mammal
that has a T-cell lymphoma by reducing IRF4 polypeptide expression,
thereby reducing the level of T-cell lymphoma cell proliferation,
or increasing the level of T-cell lymphoma cell apoptosis. An
inhibitor of an DUSP22 polypeptide can be any agent that reduces
the expression of an DUSP22 polypeptide (e.g., an siRNA molecule,
antisense oligonucleotide, or peptide nucleic acid) or that reduces
the activity of an DUSP22 polypeptide (e.g., an inhibitory
anti-DUSP22 antibody, anti-DUSP22 aptamer, or an DUSP22 polypeptide
antagonist). In some cases, the methods and materials provided
herein can be used to treat a mammal that has a T-cell lymphoma by
reducing DUSP22 polypeptide expression, thereby reducing the level
of T-cell lymphoma cell proliferation, or increasing the level of
T-cell lymphoma cell apoptosis. An inhibitor of an FLJ43663
polypeptide can be any agent that reduces the expression of an
FLJ43663 polypeptide (e.g., an siRNA molecule, antisense
oligonucleotide, or peptide nucleic acid) or that reduces the
activity of an FLJ43663 polypeptide (e.g., an inhibitory
anti-FLJ43663 antibody, anti-FLJ43663 aptamer, or an FLJ43663
polypeptide antagonist). In some cases, the methods and materials
provided herein can be used to treat a mammal that has a T-cell
lymphoma by reducing FLJ43663 polypeptide expression, thereby
reducing the level of T-cell lymphoma cell proliferation, or
increasing the level of T-cell lymphoma cell apoptosis.
[0041] This document provides nucleic acid molecules that can
reduce the expression of an IRF4 polypeptide, a DUSP22 polypeptide,
and/or FLJ43663 polypeptide. For example, antisense
oligonucleotides, siRNA molecules, and other nucleic acid
constructs encoding transcription or translation products can be
used to reduce the expression of an IRF4 polypeptide, a DUSP22
polypeptide, and/or FLJ43663 polypeptide. The term "nucleic acid"
as used herein encompasses both RNA and DNA, including cDNA,
genomic DNA, and synthetic (e.g., chemically synthesized) DNA. A
nucleic acid can be double-stranded or single-stranded. A
single-stranded nucleic acid can be the sense strand or the
antisense strand. In addition, a nucleic acid can be circular or
linear.
[0042] An "isolated nucleic acid" refers to a nucleic acid that is
separated from other nucleic acid molecules that are present in a
naturally occurring genome, including nucleic acids that normally
flank one or both sides of the nucleic acid in a naturally
occurring genome. The term "isolated" as used herein with respect
to nucleic acids also includes any non-naturally-occurring nucleic
acid sequence, since such non-naturally-occurring sequences are not
found in nature and do not have immediately contiguous sequences in
a naturally-occurring genome.
[0043] An isolated nucleic acid can be, for example, a DNA
molecule, provided one of the nucleic acid sequences normally found
immediately flanking that DNA molecule in a naturally occurring
genome is removed or absent. Thus, an isolated nucleic acid
includes, without limitation, a DNA molecule that exists as a
separate molecule (e.g., a chemically synthesized nucleic acid, or
a cDNA or genomic DNA fragment produced by PCR or restriction
endonuclease treatment) independent of other sequences as well as
DNA that is incorporated into a vector, an autonomously replicating
plasmid, a virus (e.g., any paramyxovirus, retrovirus, lentivirus,
adenovirus, or herpes virus), or into the genomic DNA of a
prokaryote or eukaryote. In addition, an isolated nucleic acid can
include an engineered nucleic acid such as a DNA molecule that is
part of a hybrid or fusion nucleic acid. A nucleic acid existing
among hundreds to millions of other nucleic acids within, for
example, cDNA libraries or genomic libraries, or gel slices
containing a genomic DNA restriction digest, is not considered an
isolated nucleic acid.
[0044] A nucleic acid construct can comprise a vector containing a
nucleotide sequence encoding a transcription or translation product
targeting the expression of an IRF4 polypeptide, a DUSP22
polypeptide, and/or FLJ43663 polypeptide with any desired
transcriptional and/or translational regulatory sequences, such as
promoters, UTRs, and 3' end termination sequences. For example, a
polyadenylation region at the 3'-end of the coding region can be
included for expression of a polypeptide. In some cases, the
polyadenylation region can be derived from a natural gene. Vectors
can also include origins of replication, scaffold attachment
regions (SARs), markers, homologous sequences, and introns, for
example. The vector may also comprise a marker gene that confers a
selectable phenotype on cells. The marker may encode antibiotic
resistance, such as resistance to kanamycin, G418, bleomycin,
hygromycin.
[0045] In some cases, an siRNA molecule, an antisense nucleic acid,
or an interfering RNA for reducing the expression of an IRF4
polypeptide can be similar or identical to part of an IRF4 allele
in a mammal. In some cases, an siRNA molecule, an antisense nucleic
acid, or an interfering RNA for reducing the expression of an
DUSP22 polypeptide can be similar or identical to part of a DUSP22
allele in a mammal. In some cases, an siRNA molecule, an antisense
nucleic acid, or an interfering RNA for reducing the expression of
an FLJ43663 polypeptide can be similar or identical to part of an
FLJ43663 allele in a mammal. Antisense nucleic acids or interfering
RNAs can be about 10 nucleotides to about 2,500 nucleotides in
length. For example, nucleic acids described herein can be used as
an antisense nucleic acid to an IRF4 allele. In some cases, the
transcription product of a nucleic acid described herein can be
similar or identical to the sense coding sequence of an IRF4
allele, but is an RNA that is unpolyadenylated, lacks a 5' cap
structure, or contains an unsplicable intron.
[0046] In some cases, a nucleic acid can have catalytic activity
such as a DNA enzyme. For example, a 10-23 DNAzyme can have a
cation-dependent catalytic core of 15 deoxyribonucleotides that
bind to and cleave target RNA (e.g., an IRF4 RNA, a DUSP22 RNA,
and/or an FLJ43663 RNA) between an unpaired purine and paired
pyrimidine through a de-esterification reaction. The catalytic core
can be flanked by complementary binding arms of 6 to 12 nucleotides
in length that confer specificity to an IRF4 mRNA molecule, a
DUSP22 mRNA molecule, or an FLJ43663 mRNA molecule.
[0047] In some cases, a nucleic acid can be transcribed into a
ribozyme that affects expression of an IRF4 mRNA, a DUSP22 mRNA, or
FLJ43663 mRNA. Heterologous nucleic acids can encode ribozymes
designed to cleave IRF4 mRNA transcripts, thereby preventing
expression of an IRF4 polypeptide. Heterologous nucleic acids can
encode ribozymes designed to cleave DUSP22 mRNA transcripts,
thereby preventing expression of an DUSP22 polypeptide.
Heterologous nucleic acids can encode ribozymes designed to cleave
FLJ43663 mRNA transcripts, thereby preventing expression of an
FLJ43663 polypeptide. Various ribozymes can cleave mRNA at
site-specific recognition sequences. For example, hammerhead
ribozymes with flanking regions that form complementary base pairs
with an IRF4 mRNA can be used to reduce expression of an IRF4
polypeptide by cleaving IRF4 mRNAs at locations containing a
5'-UG-3' nucleotide sequence.
[0048] A nucleic acid described herein can be transcribed into an
RNA that is capable of inducing an RNA interference response. In
some cases, an interfering RNA can anneal to itself to form, for
example, a double stranded RNA having a stem-loop structure. One
strand of the stem portion of a double stranded RNA can comprise a
sequence that is similar or identical to the sense coding sequence
of an IRF4 polypeptide (or a DUSP22 polypeptide or FLJ43663
polypeptide) and that is about 10 nucleotides to about 2,500
nucleotides in length. In some cases, the length of the nucleic
acid sequence that is similar or identical to the sense coding
sequence can be from 10 nucleotides to 500 nucleotides, from 15
nucleotides to 300 nucleotides, from 20 nucleotides to 100
nucleotides, or from 25 nucleotides to 100 nucleotides. The other
strand of the stem portion of a double stranded RNA can comprise an
antisense sequence of an IRF4 polypeptide (or a DUSP22 polypeptide
or FLJ43663 polypeptide) and can have a length that is shorter, the
same as, or longer than the length of the corresponding sense
sequence. The loop portion of a double stranded RNA can be from 10
nucleotides to 500 nucleotides in length, for example from 15
nucleotides to 100 nucleotides, from 20 nucleotides to 300
nucleotides or from 25 nucleotides to 400 nucleotides in length. In
some cases, the loop portion of the RNA can include an intron.
[0049] A nucleic acid can be adapted to facilitate efficient entry
into cells. For example, a nucleic acid can be conjugated to and/or
complexed with a delivery reagent (e.g., cationic liposomes). In
some cases, a conjugate or complex can include a ligand of a T-cell
surface receptor. In some cases, a nucleic acid can be complexed or
conjugated to a protein to confer increased cellular uptake and
increased nuclease resistance of oligonucleotides (e.g.,
Atelocollagen).
[0050] An inhibitor (e.g., a treatment agent) of an IRF4
polypeptide, of a DUSP22 polypeptide, or of an FLJ43663 polypeptide
can be administered to a mammal alone or in combination with other
agents (e.g., another inhibitor of an IRF4 polypeptide or other
chemotherapy agents). For example, a composition containing an IRF4
siRNA can be administered alone or in combination with chemotherapy
agents (e.g., cytotoxic agents, proteosome inhibitors, histone
deacetylase inhibitors, and/or mTOR inhibitors) to a mammal
identified as having a T-cell lymphoma. In some cases, a
composition containing an IRF4 siRNA can be administered in
combination with a composition containing an DUSP22 siRNA and/or an
FLJ43663 siRNA. Such a composition can contain additional
ingredients including, without limitation, pharmaceutically
acceptable vehicles. A pharmaceutically acceptable vehicle can be,
for example, saline, water, lactic acid, or mannitol.
[0051] A composition containing an inhibitor of an IRF4
polypeptide, a DUSP22 polypeptide, or an FLJ43663 polypeptide can
be administered to a mammal by any appropriate route, such as
enterally (e.g., orally), parenterally (e.g., subcutaneously,
intravenously, intradermally, intramuscularly, or
intraperitoneally), intracerebrally (e.g., intraventricularly,
intrathecally, or intracisternally) or intranasally (e.g., by
intranasal inhalation).
[0052] Suitable formulations for oral administration can include
tablets or capsules prepared by conventional means with
pharmaceutically acceptable excipients such as binding agents
(e.g., pregelatinized maize starch, polyvinylpyrrolidone or
hydroxypropyl methylcellulose), fillers (e.g., lactose,
microcrystalline cellulose or calcium hydrogen phosphate),
lubricants (e.g., magnesium stearate, talc or silica),
disintegrants (e.g., potato starch or sodium starch glycolate), or
wetting agents (e.g., sodium lauryl sulfate). Tablets can be coated
by methods known in the art. Preparations for oral administration
can also be formulated to give controlled release of the agent.
[0053] Intranasal preparations can be presented in a liquid form
(e.g., nasal drops or aerosols) or as a dry product (e.g., a
powder). Both liquid and dry nasal preparations can be administered
using a suitable inhalation device. Nebulized aqueous suspensions
or solutions can also be prepared with or without a suitable pH
and/or tonicity adjustment.
[0054] A composition containing an inhibitor of an IRF4
polypeptide, a DUSP22 polypeptide, or an FLJ43663 polypeptide can
be administered to a mammal in any amount, at any frequency, and
for any duration effective to achieve a desired outcome (e.g.,
reduce expression of an IRF4 polypeptide, a DUSP22 polypeptide, or
an FLJ43663 polypeptide). In some cases, a composition containing
an agent that reduces the expression of an IRF4 polypeptide, a
DUSP22 polypeptide, or an FLJ43663 polypeptide can be administered
to a mammal to reduce an IRF4 polypeptide, a DUSP22 polypeptide, or
an FLJ43663 polypeptide expression in a mammal by 1, 5, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70 percent or more). An effective amount of an agent that reduces
the expression of an IRF4 polypeptide, a DUSP22 polypeptide, or an
FLJ43663 polypeptide can be any amount that reduces IRF4
polypeptide, DUSP22 polypeptide, or FLJ43663 polypeptide expression
without producing significant toxicity to a mammal. In some cases,
an effective amount of an agent that reduces the expression of an
IRF4 polypeptide, a DUSP22 polypeptide, or an FLJ43663 polypeptide
can be between 1 .mu.g and 500 mg (e.g., between 1 .mu.g and 250
mg, between 1 .mu.g and 200 mg, between 1 .mu.g and 150 mg, between
1 .mu.g and 100 mg, between 1 .mu.g and 50 mg, between 1 .mu.g and
10 mg, between 1 .mu.g and 1 mg, between 1 .mu.g and 100 .mu.g,
between 1 .mu.g and 50 .mu.g, between 5 .mu.g and 100 mg, between
10 .mu.g and 100 mg, between 100 .mu.g and 100 mg, or between 10
.mu.g and 10 mg). Various factors can influence the actual
effective amount used for a particular application. For example,
the frequency of administration, duration of treatment, use of
multiple treatment agents, route of administration, and level of
expression of an IRF4 polypeptide, a DUSP22 polypeptide, or an
FLJ43663 polypeptide may require an increase or decrease in the
actual effective amount administered.
[0055] The frequency of administration of an agent that reduces the
expression of an IRF4 polypeptide, a DUSP22 polypeptide, or an
FLJ43663 polypeptide can be any frequency that reduces the
expression of an IRF4 polypeptide, a DUSP22 polypeptide, or an
FLJ43663 polypeptide without producing significant toxicity to the
mammal. For example, the frequency of administration can be from
about three times a day to about twice a month, or from about once
a week to about once a month, or from about once every other day to
about once a week, or from about once a month to twice a year, or
from about four times a year to once every five years, or from
about once a year to once in a lifetime. The frequency of
administration can remain constant or can be variable during the
duration of treatment. For example, an agent that reduces the
expression of an IRF4 polypeptide, a DUSP22 polypeptide, or an
FLJ43663 polypeptide can be administered daily, twice a day, five
days a week, or three days a week. An agent that reduces the
expression of an IRF4 polypeptide, a DUSP22 polypeptide, or an
FLJ43663 polypeptide can be administered for five days, 10 days,
three weeks, four weeks, eight weeks, 48 weeks, one year, 18
months, two years, three years, or five years. A course of
treatment can include rest periods. For example, an agent that
reduces the expression of an IRF4 polypeptide, a DUSP22
polypeptide, or an FLJ43663 polypeptide can be administered for
five days followed by a rest period (e.g., 1, 2, 3, 4, 5, 7, 14,
30, 60 or 90 days) and such a regimen can be repeated multiple
times. As with the effective amount, various factors can influence
the actual frequency of administration used for a particular
application. For example, the effective amount, duration of
treatment, use of multiple treatment agents, route of
administration, and level of expression of an IRF4 polypeptide, a
DUSP22 polypeptide, or an FLJ43663 polypeptide may require an
increase or decrease in administration frequency.
[0056] An effective duration for administering an agent that
reduces the expression of an IRF4 polypeptide, a DUSP22
polypeptide, or an FLJ43663 polypeptide can be any duration that
reduces the number of abnormal T-lymphocytes in a mammal without
producing significant toxicity to the mammal. Thus, the effective
duration can vary from several days to several weeks, months, or
years. In general, the effective duration for reducing the
expression of an IRF4 polypeptide, a DUSP22 polypeptide, or an
FLJ43663 polypeptide can range in duration from several days to
several months. In some cases, an effective duration can be for as
long as an individual mammal is alive. Multiple factors can
influence the actual effective duration used for a particular
treatment. For example, an effective duration can vary with the
frequency of administration, effective amount, use of multiple
treatment agents, route of administration, and level of expression
of an IRF4 polypeptide, a DUSP22 polypeptide, or an FLJ43663
polypeptide.
[0057] The term "decreased level" as used herein with respect to
the level of an IRF4 polypeptide, a DUSP22 polypeptide, or an
FLJ43663 polypeptide, or activity of an IRF4 polypeptide (e.g., to
bind DNA and activate transcription of target genes), a DUSP22
polypeptide, or an FLJ43663 polypeptide is any level that is below
a median IRF4 polypeptide, DUSP22 polypeptide, or FLJ43663
polypeptide level in a tissue sample from a random population of
mammals (e.g., a random population of 10, 20, 30, 40, 50, 100, or
500 mammals) that have a peripheral T-cell lymphoma. In some cases,
a sample of T-cell lymphoma cells can have a 20%, 30%, 40%, or 50%
decrease in the level of an IRF4 polypeptide, a DUSP22 polypeptide,
or an FLJ43663 polypeptide following treatment with an agent that
reduces the expression of an IRF4 polypeptide, a DUSP22
polypeptide, or an FLJ43663 polypeptide.
[0058] In some cases, a decreased level of IRF4 polypeptide, DUSP22
polypeptide, or FLJ43663 polypeptide expression can be determined
by assaying T-cell proliferation, apoptosis, or cell cycle phase.
For example, a decreased level of an IRF4 polypeptide can result in
a 10%, 20%, or 30% reduction in T-cell proliferation following
treatment with an agent that reduces the expression of an IRF4
polypeptide. In some cases, a decreased level of an IRF4
polypeptide can result in a 10%, 20%, or 30% increase in the number
of T-cell lymphoma cells undergoing apoptosis after treatment with
an IRF4 polypeptide inhibitor. In some cases, a decreased level of
an IRF4 polypeptide can result in a 10%, 20%, or 30% increase in
the level of T-cell lymphoma cells in the G0/G1 cell cycle phase
and a 10%, 20%, or 30% decrease in the level of S-phase T-cell
lymphoma cells after treatment with an IRF4 polypeptide
inhibitor.
[0059] In addition, this document provides methods for identifying
treatment agents that can reduce the expression of an IRF4
polypeptide, a DUSP22 polypeptide, or an FLJ43663 polypeptide from
among various test and candidate agents. Treatment agents that can
inhibit expression of an IRF4 polypeptide, a DUSP22 polypeptide, or
an FLJ43663 polypeptide in cells can be identified by screening
test agents and candidate agents (e.g., from synthetic compound
libraries and/or natural product libraries). Test agents and
candidate agents can be obtained from any commercial source and can
be chemically synthesized. Test agents and candidate agents can be
screened and can be characterized using in vitro cell-based assays,
cell free assays, and/or in vivo animal models. In some cases, a
candidate agent can be identified from among test agents any
appropriate assay to determine whether or not a test agent reduces
the expression of an IRF4 polypeptide, a DUSP22 polypeptide, or an
FLJ43663 polypeptide.
[0060] For example, primary tissue, cell lines, or animal models
can be used to identify test agents that reduce the expression of
an IRF4 polypeptide, a DUSP22 polypeptide, or an FLJ43663
polypeptide. In some cases, blood samples can be obtained from
anonymous healthy donors and T-cells isolated and purified. In
vitro assays can be performed using IRF4-positive T-cell lymphoma
cell lines such as Karpas 299, SR786, SeAx, MyLa, and Hut78. In
some cases, IRF4 expression can be induced in normal T-cells by
culturing in the presence of phytohemagglutinin (e.g., 2.5
.mu.g/ml). Animal models (e.g., IRF4 knockout mice) or cell lines
(e.g., IRF4-negative Jurkat cells) can be used as controls for
confirming the ability of a test agent or candidate agent to reduce
the expression of an IRF4 polypeptide in vivo.
[0061] Any appropriate assay can be used to identify test agent
that are capable of reducing the expression of an IRF4 polypeptide,
a DUSP22 polypeptide, or an FLJ43663 polypeptide. For example,
reduction of IRF4, DUSP22, or FLJ43663 expression can be determined
by western blot or quantitative real-time PCR. In some cases,
reduction of an IRF4 polypeptide, a DUSP22 polypeptide, or an
FLJ43663 polypeptide can be determined by an IRF4, DUSP22, or
FLJ43663 activity assay. For example, the level of IRF4 expression
can be determined by assessing T-cell proliferation (e.g.,
measuring tritiated-thymidine (H.sup.3-TdR) or carboxyfluorescein
succinimidyl ester (CFSE)), determining cell cycle phase (e.g.,
using flow cytometric analysis after treatment with
five-bromo-2-deoxyuridine(BrU) to distinguish between G0/G1, S,
G2/M), or assaying for apoptosis (e.g., flow cytometric analysis
after Annexin-V-fluoroscein isothiocyanate (FITC) and propidium
iodide (PI) staining) In some cases, reduction of IRF4 expression
can be determined by assays directed towards transcriptional
targets of an IRF4 polypeptide (e.g., MYC (myc proto-oncogene
protein), PRDM1 (PR domain-containing protein 1), CDK6
(cyclin-dependent kinase 6), VEGFA (vascular endothelial growth
factor A), IL-2 (interleukin-2), IL-4 (interleukin-4), BCL2 (B-cell
lymphoma protein 2), GATA3 (GATA-binding factor 3), and/or CCNB
(Cyclin B1) polypeptides). For example, a reduction in the
expression of an IRF4 polypeptide can be identified by a western
blot or quantitative real-time PCR results demonstrating a
decreased level of MYC polypeptide. Assays can be performed at any
time following introduction of a test, candidate, or treatment
agent (e.g., about one, two, three, four, and/or six days after
administration of an IRF4 polypeptide inhibitor).
[0062] This document also provides a massively parallel sequencing
of mate-pair DNA libraries that can be used to detect chromosomal
translocations in, for example, lymphomas. Recent technologic
advances in massively parallel ("Next Generation") DNA sequencing
offer a new tool for detecting genetic abnormalities in diseased
human tissues. However, high cost, bioinformatic challenges, and
analysis time can preclude whole genome sequencing for clinical and
most investigational applications. The advantages of the approach
described herein are the ability to detect all translocations
regardless of the formation of fusion transcripts, and a dramatic
reduction in time and resources by reducing the amount of sequenced
DNA by a factor of about 200 compared to whole genome
sequencing.
[0063] In some cases, a clinical test can include obtaining nucleic
acid (e.g., genomic DNA) from a mammal, creating a mate-pair DNA
library, performing nucleic acid sequencing (e.g., massively
parallel "Next Generation" DNA sequencing), and mapping the
sequence data to a reference genome using an algorithm (e.g., an
algorithm described herein and elsewhere (Vasmatzis et al.,
Bioinformatics, 23:1348-1355 (2007))). Such a clinical test can be
used to identify patients with any particular chromosomal
translocation or set of chromosomal translocations located across a
mammal's genome.
[0064] One identified biomarker that can be used as described
herein includes a translocation of DUSP22 and FLJ43663. About half
of ALCLs lack ALK translocations, and this ALK-negative subset has
a poorer prognosis than ALK-positive ALCLs. The advantage to
utilizing biomarkers discovered as a result of the approach
described herein is their applicability to TCLs (and especially
ALCLs) that lack ALK expression. These biomarkers include DUSP22
and FLJ43663. These biomarkers can be used for
diagnostic/prognostic purposes.
[0065] This document also provides methods and materials for
reducing the expression of DUSP22 or FLJ43663 to treat patients
with TCLs. Inhibiting or reducing expression of DUSP22 and/or
FLJ43663 can be used as a treatment for patients with TCLs. The
current method of treating TCL consists of a chemotherapeutic
regimen, usually "CHOP" or minor variations on this regimen. This
is a non-targeted regimen, i.e. it kills dividing cells, whether
cancerous or not, and thus has significant toxicity. Furthermore,
despite such treatment, the majority of patients die of their
disease. One example of the strategy described herein is that of
inhibiting DUSP22. Such a strategy is a targeted strategy, i.e. it
is directed at a polypeptide whose importance was demonstrated
specifically in TCL cells. Another advantage of a targeted strategy
over conventional chemotherapy is that measurement of the targeted
polypeptide can be used as a biomarker to predict response. Thus,
in the example of DUSP22 inhibition, this treatment can be used
only in patients whose tumors express DUSP22 polypeptides, allowing
therapy to be "individualized" to those most likely to benefit.
[0066] As described herein, the presence of translocations
involving IRF4, DUSP22, and/or FLJ43663 can indicate that a mammal
(e.g., human) has a TCL or a particular type of TCL (e.g.,
anaplastic large-cell lymphoma). For example, the presence of an
IRF4 translocation in a lymphoma sample from a human can indicate
that that human has cutaneous anaplastic large-cell lymphoma. In
some cases, the presence of a translocation involving IRF4, DUSP22,
and/or FLJ43663 can indicate that the mammal (e.g., human) has an
anaplastic large-cell lymphoma.
[0067] As also described herein, the expression of FLJ43663 (e.g.,
FLJ43663 mRNA or polypeptide) can indicate that a mammal (e.g.,
human) has an anaplastic large-cell lymphoma. Any appropriate
method can be used to assess cells for the expression of FLJ43663
mRNA or polypeptide. For example, RT-PCR can be used to assess
FLJ43663 mRNA levels, and immuno-based assays can be used to assess
FLJ43663 polypeptide levels.
[0068] This document also provides antibodies (e.g., monoclonal,
polyclonal, or fragments thereof) having the ability to bind (e.g.,
specifically bind) an FLJ43663 polypeptide. Such antibodies can be
generated against an amino acid sequence (e.g., 20, 25, 30, 40,
50-mer, or more) encoded by the last 1737 nucleotides of the
sequence set forth in GenBank GI No. 205277371 or against the amino
acid sequence (e.g., 20, 25, 30, 40, 50-mer, or more) set forth in
GenBank GI No. 51094836.
[0069] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
Examples
Example 1
Recurrent Translocations Involving the IRF4 Oncogene in Peripheral
T-Cell Lymphomas
[0070] Specimens from 169 patients with primary T-cell lymphomas
(PTCL) diagnosed by WHO criteria were studied. There were 104 males
and 65 females (M:F ratio, 1.6:1). The mean age was 58 years
(range, 5-92 years). Cases included 23 angioimmunoblastic T-cell
lymphomas (AITLs, 13%), 72 PTCL-unspecified (43%), 18 ALK-positive
anaplastic large-cell lymphomas (ALCLs) (11%), 24 ALK-negative
ALCLs (14%), 14 C-ALCLs (8%), and 18 other PTCLs (Table 1).
TABLE-US-00001 TABLE 1 IRF4 Translocations and IRF4 Protein
Expression in Peripheral T-cell Lymphomas FISH Immunohistochemistry
Diagnosis # pos.* % # pos.* % Angioimmunoblastic T-cell lymphoma
0/19 0 0/23 0 PTCL, unspecified 3/64 5 20/72 28 (CD30-positive)
(1/17) (6) (13/18) (72) (CD30-negative) (2/47) (4) (7/54) (13)
Anaplastic large cell lymphoma, ALK-positive 0/18 0 16/17 94
Anaplastic large cell lymphoma, ALK-negative 1/23 4 20/22 91
Cutaneous anaplastic large cell lymphoma 8/14 57 13/14 93 T-cell
large granular lymphocyte leukemia 0/4 0 0/4 0 Hepatosplenic T-cell
lymphoma 0/3 0 0/3 0 Subcutaneous panniculitis-like T-cell lymphoma
-- -- 0/1 0 Enteropathy-associated T-cell lymphoma 0/2 0 0/2 0
Extranodal NK/T-cell lymphoma, nasal type 0/8 0 0/8 0 Total 12/155
8 69/166 42 *Includes informative cases only, of 169 total cases
tested.
Fluorescence In Situ Hybridization (FISH)
[0071] Breakapart and dual fusion IRF4 and TCRA FISH probes were
developed. BAC clones (Table 2) were identified using the
University of California Santa Cruz Genome Browser (located on the
World Wide Web at www.genome.UCSC.edu) and ordered from ResGen.TM.
Invitrogen (Carlsbad, Calif.). Positive cases were confirmed using
a second breakapart IRF4 probe (probe #2, Table 1). BAC DNA was
isolated using the Qiagen (Valencia, Calif.) Plasmid Maxi Kit and
fluorescently labeled using SpectrumOrange-dUTP or
SpectrumGreen-dUTP and the Abbott Molecular (Des Plaines, Ill.)
Nick Translation Kit. Centromeric and telomeric BAC DNA was labeled
with different fluorophores for breakapart probes, and with the
same fluorophore for dual-fusion (D-FISH) probes. Specificity of
hybridization was confirmed on metaphases from a splenic marginal
zone lymphoma with IRF4/IGH fusion, a PTCL with a TCRA
translocation, and normal samples. TCRB and TCRG probes were
purchased from Dako (Carpinteria, Calif.). The upper limit of the
normal range for each probe was determined using a 95% confidence
interval. Upper limits of normal for IRF4, TCRA, TCRB, and TCRG
were 6%, 9%, 5%, and 6%, respectively.
TABLE-US-00002 TABLE 2 BAC Clones Used to Prepare Fluorescence In
Situ Hybridization Probes BAC Clones Locus Centromeric Telomeric
IRF4 (6p25) [probe #1] RP11-164H16 CTD-2314K17 RP11-119L15 IRF4
(6p25) [probe #2] RP11-164H16 CTD-2308G5 TCRA (14q11) RP11-524O1
CTD-2555K7 RP11-689J19 RP11-137H15 RP11-702N19 RP11-298I3
CTD-2574K8
[0072] Paraffin tissue microarrays (TMAs) were constructed. In
cases with insufficient tissue, whole-tissue sections were
analyzed. B5- and formalin-fixed cases were included. Sections were
sequentially immersed in Citrisolve, Lugol solution, and sodium
thiocyanate. Slides were microwaved for 5 minutes in citrate
buffer, then digested in 0.4% pepsin solution at 37.degree. C. Ten
microliters of FISH reagent (7 .mu.L LSI buffer and 3 .mu.L probe)
were placed on each slide and slides were cover-slipped, denatured,
and incubated in a humidified chamber at 37.degree. C. for 12
hours. Slides were washed, counterstained with
4',6-diamidino-2-phenylindole dihydrochloride, and analyzed by a
microscopist (ML) using a fluorescent microscope with appropriate
filter sets. A minimum of 50 cells and a maximum of 200 cells were
scored per case. A minimum of 20 abnormal cells were required for a
sample to be considered abnormal. Some cases were non-informative
due to hybridization failures. Positive cases detected on TMAs were
confirmed on whole tissue sections. Scoring for IRF4 in four
translocated cases with areas showing confluent sheets of tumor
cells areas revealed a mean of 71% positive cells (range,
55%-93%).
Immunohistochemistry (IHC)
[0073] Five-micron paraffin whole-tissue sections were
immunostained using antibodies to assist in disease classification.
For IRF4 immunostaining, whole-tissue or TMA sections were
pretreated in 1 mM EDTA buffer at pH 8.0 for 30 minutes at
98.degree. C. (PT Module, Lab Vision, Fremont, Calif.), then
stained for IRF4 using a monoclonal mouse anti-human antibody
(MUM1p, 1:50; Dako). Detection was carried out on a Dual Link
Envision+/DAB+ (Dako). Scoring was performed in correlation with
H&E and appropriate immunostains (e.g., CD20 and CD3).
Specimens were considered positive for IRF4 when >30% of tumor
cells demonstrated nuclear staining. Technical factors precluded
scoring in rare cases. Diagnosis of C-ALCL required CD30 positivity
in >75% of tumor cells, as per WHO criteria. This cutoff also
was used to define CD30 positivity in cases of PTCL-U.
Conventional Cytogenetics
[0074] Results of karyotype analysis prepared at the time of biopsy
were reviewed retrospectively when available.
Results
[0075] Twelve PTCLs with IRF4 translocations among 155 PTCLs with
informative FISH results (8%; Tables 1,3). These included 3/64
PTCL-Us (5%), 1/23 ALK-negative ALCLs (4%), and 8/14 C-ALCLs (57%)
were identified. IRF4 translocations were not seen in ALK-positive
ALCLs, AITLs, or other PTCL subtypes. An IRF4 polypeptide was
detected in the majority of ALCLs, regardless of type (Table 1).
Staining for IRF4 was positive in 72% of CD30-positive PTCL-Us and
13% of CD30-negative PTCL-Us, and was negative in other PTCL
subtypes. All cases with IRF4 translocations were positive for IRF4
by IHC.
TABLE-US-00003 TABLE 3 Peripheral T-cell lymphomas with IRF4
Translocations Time from IRF4/ Age/ diagnosis Cytotoxic IRF4 TCRA
Case Sex Diagnosis Site (mos) phenotype* FISH Fusion Karyotype
Treatment Follow-up 1 67/M PTCL-U BM -- YES -- -- 48-49, XY, +3[5],
+5, der(6)t(6; 14) CHOP, alive, progressive (p25; q11.2),
add(7)(p11.2), -14, ICE, cutaneous disease, 4 +16, +16[5], -20,
-22[4], +1, -2mar ITMTX mos [cp6]/95-98, idemx2, +3-8mar[2] skin 2
YES -- -- -- skin 4 -- POS POS -- 2 71/M PTCL-U BM -- YES POS POS
49, XY, +add(3)(q27), -- -- t(6; 14)(p25; q11.2), +8, -9, +19,
+21.sup..dagger. 3 48/F Cutaneous skin -- -- -- -- -- PUVA, alive,
in remission, ALCL skin 1 -- -- -- -- IFN.alpha.-2A, 154 mos skin
83 YES POS NEG -- CHOP LN 126 -- POS -- -- LN 134 -- POS NEG -- 4
67/M Cutaneous skin -- -- -- -- -- -- alive, LN ALCL LN 7 NO POS
NEG -- involvement, 7 mos 5 89/F Cutaneous skin -- NO POS NEG --
XRT, died, progressive ALCL CHOP cutaneous disease, no autopsy, 4
mos 6 65/M Cutaneous skin -- YES POS NEG -- CHOP alive, skin ALCL
recurrence, 27 mos 7 52/M Cutaneous skin -- -- POS NEG -- CHOP
died, unrelated ALCL LN 1 NO -- -- -- cause, no autopsy, 9 mos 8
74/M Cutaneous skin -- NO -- -- -- -- alive, LN ALCL LN 34 NO POS
NEG -- involvement, 34 mos 9 35/M Cutaneous skin -- NO POS NEG --
-- -- ALCL 10 50/M Cutaneous skin -- NO POS NEG -- -- -- ALCL 11
73/F PTCL-U pleura -- NO POS NEG -- -- -- 12 79/M ALK-neg LN -- NO
POS NEG -- -- -- ALCL FISH, fluorescence in situ hybridization; BM,
bone marrow; LN, lymph node; PTCL-U, peripheral T-cell lymphoma,
unspecified; ALCL, anaplastic large-cell lymphoma; PUVA, psoralen
UVA photochemotherapy; IFN.alpha.-2A, interferon alpha-2A; CHOP,
cyclophosphamide + hydroxydoxorubicin + oncovin + prednisone; ICE,
ifosfamide + carboplatin + etoposide; ITMTX, intrathecal
methotrexate; XRT, radiotherapy *Positive for TIA-1 by
immunohistochemistry. .sup..dagger. FISH for BCL6 at 3q27 was
normal.
[0076] Two PTCL-Us with IRF4 translocations by FISH had karyotypes
with t(6;14)(p25;q11.2) (Cases 1 and 2, Table 3). Karyotypes were
not performed in the other 10 PTCLs with IRF4 translocations.
Karyotypes of 35 PTCLs without IRF4 translocations by FISH and 2
PTCLs in which IRF4 FISH failed exhibited no anomalies of 6p25
(Table 4). The PTCL-Us with t(6;14)(p25;q11.2) had similar
clinicopathologic features: Both presented in older adult males
with mild cytopenias and without significant lymphadenopathy or
hepatosplenomegaly. In both patients, imaging showed diffuse
skeletal uptake and renal mass lesions. No tumor cells were seen in
the peripheral blood. Both patients had extensive bone marrow
infiltration by tumor with admixed plasma cells and reticulin
fibrosis (FIGS. 1(a) and (g)). The cells were larger and more
pleomorphic in Case 1 (FIGS. 1(b) and (h)). Both tumors were
positive for CD3, beta-F1, TIA1, and IRF4 (FIGS. 1(c), (d), (e),
and (i)); and were negative for CD5, CD30, CD25, FoxP3 and EBV.
Case 1 was positive for granzyme B and CD4, and partially positive
for CD8. In both cases, the t(6;14)(p25;q11.2) corresponded to
IRF4/TCRA fusion by D-FISH (FIGS. 1(j) and (k)). The patient in
Case 1 received chemotherapy (Table 3) but developed progressive
skin lesions and probable cerebrospinal fluid involvement. Despite
additional therapy, his skin lesions progressed (FIG. 1(f)). The
patient in Case 2 developed skin and soft tissue lesions during the
course of evaluation. These cases were classified as PTCL-Us.
TABLE-US-00004 TABLE 4 Cytogenetic Findings in 39 Peripheral T-cell
lymphomas with Informative Karyotypes Age/Sex Diagnosis IRF4 FISH
Karyotype 67/M PTCL-U with POSITIVE 48-49, XY, +3[5], +5,
der(6)t(6; 14)(p25; q11.2), add(7)(p11.2), -14, +16, IRF4/TCRA
+16[5], -20, -22[4], +1-2mar[cp6]/95-98, idem .times. 2, +3-8mar[2]
(Case 1) 71/M PTCL-U with POSITIVE 49, XY, +add(3)(q27), t(6;
14)(p25; q11.2), +8, -9, +19, +21 IRF4/TCRA (Case 2) 77/F PTCL-U
NEGATIVE 46, XX, add(11)(q23) 67/M PTCL-U NEGATIVE 46, XY,
del(20)(q11q13.1) 67/M PTCL-U NEGATIVE 46, XY, add(4)(q21) 57/F
PTCL-U NEGATIVE 46, XX, t(10; 14)(?; q11.2) 62/M PTCL-U NEGATIVE
47-51, XY, +X, +Y, +9, add(9)(p13) .times. 2, +10, add(10)(p11.2),
add(14)(q32), add(16)(p11.2), add(17)(p11.1), +19, +21, +0-3mar
40/F PTCL-U NEGATIVE 38-44 X, -X, -4, -5, -6, -7, -8, -10, -11,
-12, -18, -20, dup(1)(q25q44), der(2)t(2; ?)(p13; ?), +der(7)t(5;
7)(q13; q36), der(10)(t(10; ?)(p15; ?), der(12)t(12; ?)(p13; ?),
der(19)t(19; ?)(?q13; ?), +5-7mar 84/F PTCL-U NEGATIVE 50-56, XX,
del(2)(p23), +3, +5, +i(7q), +9, +9, +11, +14, -17, +18, +19, +mar
69/M PTCL-U NEGATIVE 47, XY, t(12; 22)(q13; q13), t(14; 17)(q32;
q21), +mar 43/M PTCL-U NEGATIVE 46, XY, t(5; 14)(q33; q32) 78/M
PTCL-U NEGATIVE 50, XY, +8, +der(3), +der(4), der(6), +der(7),
der(10), der(21) 28/M PTCL-U NEGATIVE 46, XY 58/M PTCL-U NEGATIVE
44-45, X, -Y, add(2)(q1?3), t(3; 12)(p13; q13), -4, i(7q), i(8q),
del(11)(q21q23) 44/M PTCL-U NEGATIVE 46, XY, add(8)(q24.1) 88/F
PTCL-U NEGATIVE 46, XX, -20, +mar 65/M PTCL-U NEGATIVE 55-57, XY,
+2, +4, +5, +7, +8, +10, +11, +12, -13, +14, +15, +15, +16, +mar
90/F PTCL-U NEGATIVE 47, XX, +19[4]/48, XX, +7, +19[1] 71/M PTCL-U
NEGATIVE 46, XY, +3, +5, +18, t(19; 22)(q13.3; q11.2) 76/M PTCL-U
NEGATIVE 46, XY, del(3)(p13p21) 76/M PTCL-U NEGATIVE 46, XY, t(3;
20)(q21; q11.2) 39/M PTCL-U NEGATIVE 47, XY, add(13)(p13.2),
add(14)(q24), add(17)(q23), +19, add(22)(q11.2) 70/M PTCL-U
NEGATIVE 46, XY 33/M NKTL NEGATIVE 45, X, -Y, dup(2)(q21->q33),
t(3; 6)(p13; q13), der(8)t(8; ?)(p21; ?) 57/M NKTL NEGATIVE 38-43,
X, -Y, -4, -5, add(8)(q24.3), add(12)(q24.3), der(13; 22)(q10;
q10), add(15)(p12), i(17)(q10), dic(18; ?)(q23; ?), add(19)(p13.3),
+mar 37/M NKTL NEGATIVE 45-48, XY, +X, i(7)(q10), +21[cp17] 56/M
LGL NEGATIVE 47-48, XY, inv(1)(q13q42), +3, del(5)(q13q33),
dup(5)(q11.2q13), +8, add(13)(q32), add(18)(q23), +r 18/F ALCL,
ALK- NEGATIVE 46, XX, t(2; 5)(p23; q35), t(9; 19)(q22; q13.1)
positive 18/M ALCL, ALK- NEGATIVE 46, XY* positive 56/M ALCL, ALK-
NEGATIVE 80-94, XXY-Y, add(2)(p23) .times. 2, +5, add(5)(q33)
.times. 2, +6, +8, -9, positive t(11; 11)(p15; q11) .times. 2, -15,
-16, -19, -20, -21, -22 31/F ALCL, ALK- NEGATIVE 46, XX,
add(1)(p13q23), add(2)(p11.2), del(3)(q12), der(4)t(1; 4)(q23;
p16), negative add(5)(q31), der(7)t(2; 7)(p11.2; p11.2),
add(9)(q34), add(17)(p11.2), +mar 50/F ALCL, ALK- NEGATIVE 46-48,
X, -X, add(2)(q37), t(5; 14)(q 11.2; q11.2), negative
add(6)(p21.3), t(9; 18)(p22; q12.2), add(11)(q13), +22, +1-2mar
65/F AITL NEGATIVE 46, XX 71/F AITL FAILED 48, XX, +5, +14,
der(2),der(17) 75/M AITL NEGATIVE 47, XY, add(1)(p13), +der(3)t(1;
3)(p13; p13), -16, der(19)t(1; 19)(q21; q13.3), +mar 76/F AITL
FAILED 48, XX, +2, +11 71/M AITL NEGATIVE 46, XY 63/M AITL NEGATIVE
46, XY 46/F AITL NEGATIVE 46, XX
[0077] IRF4 translocations were detected in 8/14 C-ALCLs tested
(57%). All initial diagnostic biopsies were reviewed. Clinical or
pathologic features of lymphomatoid papulosis were not observed.
None of the patients had a history of mycosis fungoides (MF) or
dermatitis suggestive of clinical MF. Four patients developed nodal
disease 1 to 126 months after diagnosis (Table 3). The patient with
a 1-month interval between cutaneous and nodal disease (Case 7) had
multiple skin nodules and local adenopathy. Staging was otherwise
negative, suggesting C-ALCL with locoregional spread, however, it
is possible the disease originated in the lymph node. Cases 3 and 8
showed different histology in cutaneous and nodal specimens. The
skin showed mostly medium-sized tumor cells with admixed
histiocytes in the background (FIG. 2(a)), and occasional
perivascular "hallmark" cells. The subsequent lymph node biopsies
showed sheets of large "hallmark" cells (FIG. 2(b)). The remaining
C-ALCLs with IRF4 translocations had typical histologic features
(FIG. 2(c)-(e)). CD30 and IRF4 were positive (FIGS. 2(f) and (g)).
FISH showed IRF4 translocations (FIG. 2(h)). C-ALCLs with and
without IRF4 translocations showed similar clinicopathologic
features (Table 5). TIA1 positivity was somewhat less common in
IRF4-translocated cases than in untranslocated cases. Only one
untranslocated case developed nodal disease.
[0078] Two additional PTCLs with translocations of IRF4 but not
TCRA were identified. Case 11 was a PTCL-U in the pleura of a 73
year-old female who also had multiple lung nodules. The tumor cells
were medium to large and pleomorphic. "Hallmark" cells were not
seen. Staging was negative. Case 12 was an ALK-negative ALCL in a
79 year-old male with generalized lymphadenopathy but without
cutaneous disease. Lymph node biopsy showed sheets of "hallmark"
cells (FIG. 2(i)). Both cases were positive for CD30 and IRF4 and
were non-cytotoxic. FISH for TCRB and TCRG was negative in Case
12.
TABLE-US-00005 TABLE 5 Clinicopathologic Features of Primary
Cutaneous Anaplastic Large Cell Lymphomas With and Without IRF4
Translocations. IRF4 Translocation Present Absent n (%) 8 (57) 6
(43) M:F 6:2 3:3 Age, mean (range), y 60 (35-89) 64 (12-92)
Immunophenotype (%) CD30 8/8 (100) 6/6 (100) CD3 6/8 (75) 2/6 (33)
CD4 5/8 (63) 4/6 (67) CD8 0/8 (0) 1/6 (17) ALK 0/8 (0) 0/6 (0) TIA1
2/8 (25) 3/6 (50) IRF4 8/8 (100) 5/6 (83) Subsequent extracutaneous
disease 4 1 Follow-up, mean (range), mos 29 (0-154) 30 (0-76)
[0079] In addition, T-cell lymphomas with amplification of the IRF4
gene locus were identified (FIG. 3A). These results demonstrate
that IRF4 translocations and amplifications are associated with
peripheral T-cell lymphomas.
Example 2
IRF4 Translocations are Specific for Cutaneous Anaplastic Large
Cell Lymphoma
[0080] Skin biopsies involved by T-cell lymphoproliferative
disorders from 68 patients were classified by WHO/EORTC criteria.
Clinicopathologic data for classification included
progression/regression of lesions, history of mycosis fungoides
(MF) or other cutaneous T-cell lymphoproliferative disorders,
anatomic site and timing of extracutaneous disease, morphology,
immunophenotype, and T-cell clonality if needed. Cases that could
not be classified definitively were excluded. FISH for IRF4 was
performed using a breakapart probe. Positive cases also were
screened for T-cell receptor (TCRA, TCRB, and TCRG) rearrangements.
FISH was scored by a cytogeneticist using previously established
normal ranges based on 95% confidence intervals.
[0081] Among cALCLs, 9/22 (41%) demonstrated abnormal separation of
the IRF4 breakapart probe, indicating an IRF4 translocation. None
of the 46 remaining T-cell lymphoproliferative disorders showed
IRF4 translocations, including: 12 LyPs; six systemic ALK
translocation negative ALCL; 3 systemic ALK translocation positive
ALCL; 12 cases of MF (2 transformed); 2 cases of Sezary syndrome; 1
CD4+ small/medium-sized pleomorphic T-cell lymphoma; 2 extranodal
NK/T-cell lymphomas, nasal type; 1 subcutaneous panniculitis-like
T-cell lymphoma; and 7 peripheral T-cell lymphomas, unspecified. No
rearrangements of TCRA, TCRB, or TCRG. were identified in cases
with IRF4 translocations.
[0082] These results indicate that IRF4 translocations are specific
for cutaneous ALCL (FIG. 3B). Furthermore, cutaneous ALCL patients
with IRF4 translocations had an aggressive clinical course. For
example, spread to extracutaneous sites was seen in 50% of patients
with, and 17% of patients without, IRF4 translocations after
similar mean follow-up intervals (FIG. 4). These findings indicate
that the presence of IRF4 translocations can be a clinical
biomarker for both cancer diagnosis and prognosis.
Example 3
Human T-Cell Lymphoma Lines Express IRF4
[0083] To develop an in vitro model for studying IRF4 in T-cell
lymphomas, human T-cell lymphoma cell lines were screened by
western blot and immunohistochemistry. All T-cell lymphomas and,
T-cell lymphoma cell lines tested (SUDHL-1, SR786, Karpas 299,
SeAx, MyLa, and HuT78) were positive for IRF4. T-cell lymphoblastic
leukemia cell lines were negative for IRF4 (FIG. 5A). Western
blotting for MYC was also performed. Cell lines with weaker IRF4
expression also showed weaker expression of MYC (FIG. 5B).
Example 4
Silencing IRF4 in T-Cell Lymphoma Cells
[0084] SUDHL-1 cells were transfected with either a scrambled
control siRNA or IRF4 siRNA (Santa Cruz Biotechnology, Inc.). Cell
lysates were assayed for protein expression by western blot. IRF4
siRNA almost completely abolished IRF4 protein expression (FIG.
6A). IRF4 siRNA, but not the control siRNA, also decreased MYC
protein expression in SUDHL-1 cells. IRF4 siRNA inhibited cell
proliferation by 47% and 39% in two independent experiments (FIG.
6B). These data indicate that IRF4 drives MYC expression in SUDHL-1
cells. IRF4, MYC, and PRDM1 may act together in T-cell lymphoma
cells to amplify the IRF4-dependent transcriptional program, which
may include genes such as CDK6, BCL2, and VEGFA (FIG. 7). Taken
together, these results demonstrate that molecules designed to
reduce the expression of an IRF4 polypeptide can be used to treat
T-cell lymphomas.
Example 5
Use of a Massively Parallel Sequencing of Mate-Pair DNA Libraries
to Detect Chromosomal Translocations in Lymphomas and Other
Diseases
[0085] Mate-pair library generation, massively parallel sequencing
technology, and a bioinformatic algorithm were combined to detect
and discover chromosomal translocations in diseased human tissue.
The presence of a translocation, t(6;7)(p25.3;q32.3), in tissue
from a fatal case of systemic ALK-negative ALCL was detected. The
ability to use polymerase chain reaction (PCR) and conventional
sequencing to identify the precise breakpoints of translocations
discovered/detected using this approach, including the
t(6;7)(p25.3;q32.3), which involve the region between exons 1 and 2
of the DUSP22 gene on chromosome 6 and the hypothetical gene region
FLJ43663 on chromosome 7 was demonstrated. In addition, using a
breakapart (BAP) FISH probe to the region of the breakpoint on
7q32.3, it was demonstrated that 43% of TCL cases with
rearrangements at 6p25.3 have concurrent rearrangements at 7q32.3.
Using a dual-fusion FISH (D-FISH) probe, it was demonstrated that
100% of such cases have FISH-based evidence of t(6;7)(p25.3;q32.3)
translocations. The presence of this translocation was specific to
ALCLs lacking expression of the protein ALK. A FISH assay for the
6p25.3 gene region, which successfully distinguishes between
translocations involving the IRF4 gene and those involving the
DUSP22 gene, was developed. Using this approach, it was shown that
t(6;7)(p25.3;q32.3) translocations are limited to TCLs with a
6p25.3 breakpoint in the DUSP22 rather than the IRF4 gene. In
addition, FLJ43663 translocations exhibited high specificity for
TCLs with t(6;7)(p25.3;q32.3) translocations, being present in
<1% of TCLs without rearrangements of 6p25.3. These
translocations caused up-regulation of the FLJ43663 transcript in
TCLs, compared to its reported absence in normal T cells. This
up-regulation existed specifically in the region of FLJ43663 that
is retained on der(7)t(6;7)(p25.3;q32.3). A fusion FLJ43663/DUSP22
transcript exists derived from the t(6;7)(p25.3;q32.3).
Widely-utilized T-cell lymphoma cell lines expressed DUSP22
polypeptide, and the ability to inhibit expression of DUSP22
polypeptide in a TCL cell line using small interfering RNAs
(siRNAs) was demonstrated. This inhibition of DUSP22 expression
induced significant inhibition of the proliferation of the TCL
cells.
[0086] A mate-pair library was prepared from genomic DNA extracted
from frozen tissue (.about.90% tumor) from the tumor shown in FIG.
8, following the manufacturer's protocol ("Mate pair sequencing
assay" from Illumina). Ten micrograms of genomic DNA in 50 .mu.L TE
buffer was added to 700 .mu.L nebulization buffer and fragmented
using nebulization for 30 seconds at 7.5 psi. This generated
double-stranded DNA fragments with blunt or sticky ends with
fragment sizes in the 2-5 kb range. The ends were repaired and
phosphorylated using Klenow, T4 polymerase, and T4 polynucleotide
kinase. Biotinylated dNTPs then were substituted for natural dNTPs
at the 3' ends of the double-stranded DNA again using Klenow, T4
polymerase, and T4 polynucleotide kinase to allow for the
purification of the original size selected fragments after
circularization and secondary fragmentation. The resulting
biotinylated constructs were separated on a 1% agarose gel. DNA
fragments of approximately 5-5.5 kb were excised from the gel and
purified using the Qiagen Gel Extraction Kit. Circularization of
the size-selected fragments was performed by blunt end ligation for
16 hours at 16.degree. C. using circularization ligase (Illumina).
Non-circularized fragments were eliminated by DNA exonuclease
treatment. The remaining circularized DNA was again fragmented,
this time using the Covaris E210 (duty cycle, 5%; intensity, 3;
cycles, 200; time, 180 seconds), generating double-stranded DNA
fragments with fragment sizes in the 300-600 by range. The
biotinylated fragments were purified using M-280 streptavidin beads
from Dynal as outlined in the Illumina mate-pair protocol. The ends
of the biotinylated fragments immobilized on the beads were
repaired and phosphorylated using Klenow, T4 DNA polymerase, and T4
polynucleotide kinase. An "A" base then was added to the 3' ends of
double-stranded DNA using Klenow exo- (3' to 5' exo minus).
Paired-end DNA adaptors (Illumina) with a single "T" base overhang
at the 3' end were ligated, and the immobilized adapter-modified
DNA fragments were enriched by 18 cycles of PCR using primers PE
1.0 and PE 2.0 (Illumina). The PCR supernatant was recovered from
the beads using a magnetic rack. The PCR-enriched constructs were
separated on a 2% agarose gel, and DNA fragments of approximately
400-600 by were excised from the gel and purified using Qiagen
MinElute Gel Extraction Kits. The concentration and size
distribution of the libraries was determined on an Agilent
Bioanalyzer DNA 1000 chip.
[0087] The library was loaded onto a paired-end flow cell at a
concentration of 9 pM generating an average of 215,000
clusters/tile following Illumina's standard protocol using the
Illumina Cluster Station and Paired-End Cluster Generation Kit,
version 4. The flow cell was sequenced as a 76.times.2 paired-end
read on an Illumina GAIIx using SBS Sequencing Kit, version 4, and
SCS 2.5 Data Collection Software. Base-calling was performed using
Illumina Pipeline, version 1.5.
[0088] A rough representation of a patient's entire genome was
reconstructed by aligning (mapping) next generation (NG) sequencing
fragments to a reference genome. Algorithms were developed to
store, manipulate, and map the NG fragments and interpret the
results. Mapping the 200 million NG fragments generated from an 8
lane Illumina GAIIx run can be impractical using search algorithms
like BLAST; but the mapping algorithm mapped these NG fragments to
a 3 billion nucleotide genome within one day.
[0089] The algorithm converted the reference genome and the NG
fragments to binary numbers. This feature maximized computer memory
and computational speed, and allowed the reference genome and its
reverse compliment to be stored in RAM. Up to two mismatches per NG
fragment were allowed to account for single point mutations and NG
sequencing errors. The "good" NG fragments mapped to the reference
genome exactly once and were output for analysis. Fragments that
mapped multiple times were ignored. NG fragment lengths ranged from
.about.500 bps for a paired-end protocol, up to 5-10 kb for
mate-pair protocols. For each NG fragment, only 36-100 by of each
end, called a NG tag, were sequenced by the GAIIx. The intervening
sequence between the two tags was referred to as "fandom" sequence
below. Accounting for the length and number of "good" fragments,
the bridged coverage of the pair-end and mate-pair protocols from
one lane (on the Illumina) were 0.35.times. and 7.times.,
respectively. Bridged coverage referred to the entire length of
each "good" NG fragment. This length included the 36-100 known
sequences of each tag at both ends of the fragment and the known by
distance between the two tags.
[0090] The analysis algorithms searched the mapped good NG
fragments to identify genomic alterations. Putative translocations
were identified when (1) each tag of a fragment mapped to two
different chromosomes and (2) this pattern was observed for
multiple NG fragments. The 7.times. bridged coverage using the
mate-pair protocol was sufficient to produce the multiple
observations required in step two. Deletions and amplifications as
small as 10 kb were detected by: (1) counting the number of NG
fragments that mapped within a specified region and (2) observing
if this number exceeds or falls below an expected amount. The
expected number of tags that will map within a given region was
determined from a statistical method based on Extreme Value Theory.
This theory was valid even for the sparse coverage achieved in
pair-end protocols. Since only sparse coverage is required to
detect genomic alterations, this procedure was financially
preferable compared to procedures that require dense coverage.
[0091] This methodology included the following steps: (1) mapping
200 million NG fragments on a reference genome, and (2)
interpretation of the mapped data to identify alterations. Multiple
observations of wrong/aberrant rearrangements between chromosomes
identified amplification and deletions with sparse coverage.
Mapping NG Sequencing Data to the Human Genome
[0092] The NG sequencing data were mapped to a reference genome
using an algorithm. Details regarding the algorithm are set forth
elsewhere (Vasmatzis et al., Bioinformatics, 23:1348-1355 (2007)).
Briefly, the algorithm's design incorporated four main steps. The
first step was to convert the reference genome and NG data to a
binary representation. Each nucleotide was uniquely identified by
two binary digits, for example:
TABLE-US-00006 nucleotide G A C T 1.sup.st binary bit 1 1 0 0
2.sup.nd binary bit 1 0 1 0
[0093] Memory storage was maximized by converting 32 consecutive
nucleotides in the genome into two 32-bit binary numbers. The two
numbers were referred to as the base and check arrays.
TABLE-US-00007 32 nucleotide string GAGCCCCAAA TGCCTTCTTT
GGTTTTCTTA GA 1.sup.st 32-bit binary number: 1110000111 0100000000
base array 1100000001 01 2.sup.nd 32-bit binary number: 1011111000
0111001000 check array 1100001000 10
[0094] With the binary conversion, a 3.2 billion nucleotide genome
was converted to .about.100 million, non-overlapping 32-bit
numbers. At 8 bytes per 32 nucleotides, the memory requirement for
the entire genome was 800 megabytes (MB); including the reverse
compliment, 1.6 gigabytes (GB).
[0095] The second step applied a forward chaining system to store
large segments of the genome in RAM. The following steps 2-4 were
repeated for each additional genome segment until the entire genome
was analyzed. Two tables were created: (1) an Index table and (2) a
Look-up table. The Index table contained two columns, the left
column was the sequence of 27-bit binary numbers from 0-2.sup.27
(111111111111111111111111111). The right column recorded the first
position in the genome where the number in the corresponding left
column is found. A partial Index table is illustrated below. A
sequential Look-up table records all successive positions in the
genome, for each 27-bit binary number, as well as all possible
32-bit base and check overlapping numbers. The length of the
Look-up table was the length of the genome segment (number of
positions) being stored into RAM.
[0096] Segment of the Genome, Represented as 2 Consecutive 32-bit
Binary Numbers
##STR00001##
TABLE-US-00008 Index table All possible 27-bit numbers Position
Genome 000000000000000000000000000 0 000000000000000000000000001 0
000000000000000000000000010 0 000000000000000000000000011 0 . . .
111000011101000110000000101 1104701 111000011101000110000000110
1104733 . . . 111111111111111111111111111 0
[0097] Mapping the NGS to the genome occured in the third step by
searching the Index and Look-up tables. More specifically, the left
27 bits of the NGS base array created in step 1 was found in the
left column of the Index table, and the corresponding right column
revealed the first genome position for that NGS. Mapping the
(27-bit) NGS continued with the Look-up table to locate additional
matching positions in the genome segment.
[0098] The final step used binary operation functions to compare
both the base and check arrays of the entire NG tag to the genome
base and check arrays. The comparison was performed using an
exclusive OR operation. The output revealed the number of
mismatches between the NGS and the genome. If the number of
mismatches was less than a designated threshold, that position on
the genome was considered a match for the NG tag, also referred to
as a mapped read for the NG tag. The threshold was designated to
identify a perfect match between the genome and NG tag or to allow
them to differ by one or more nucleotides, thus accounting for
possible mutations or sequencing errors. While some NG tags had 0
mapped reads, or over 100, only a NG tag with exactly one mapped
read was written to an output file for further analysis.
[0099] This algorithm was fully automated and adaptable to rapidly
map any number of NG tags to the genome. For example, over 200
million NG tags were map to a 3 billion nucleotide genome within
one day using the Mayo Clinic Computer Cluster. The algorithm can
be optimized to use one or both tags of a paired end or mate pair
fragment.
Mapping of the Mate-Pair Lane
[0100] One lane on the Illumina GAIIx was run by mate-pair protocol
as described above. Of 28.90 million paired sequences, 21.78
million had at least one perfect (single tag) hit across the
genome. Of those, 4.19 million were considered duplicates (29%) and
were eliminated. Of the 8.61 million fragments where both tags hit
perfectly within chromosomes, 8.46 million were within 0 and 10 Kb
with the peak at 5 kb (FIG. 9).
[0101] Coverage across the genome was as follows:
TABLE-US-00009 max effective Bridged Chromosome position length
coverage 1 249.24 225.09 15.72 2 243.19 238.23 15.98 3 197.95
194.81 16.31 4 191.04 187.72 15.9 5 180.9 177.31 15.98 6 171.05
167.46 16.22 7 159.13 155.31 15.11 8 146.3 142.82 16.13 9 141.15
119.44 14.46 10 135.52 131.24 15.61 11 134.95 131.19 15.65 12
133.84 130.54 15.83 13 115.11 95.62 16.24 14 107.29 88.3 15.57 15
102.52 81.6 15.15 16 90.29 78.86 14.29 17 81.2 77.83 14 18 78.02
74.71 16.37 19 59.12 55.83 12.05 20 62.97 59.57 15.76 21 48.12 35.2
15.45 22 51.24 34.91 13.2 X 155.16 149.61 7.45 Y 59.36 24.78
4.05
Detection of DNA Alterations, Specifically Translocations by
NGS
[0102] Fragments spanning putative translocation points could be
identified when each tag of a fragment maps perfectly to two
different chromosomes. The actual sequence might not include these
points but it is assumed that the translocation point will be
within the fandom sequence of the fragment. After the ends of a
fragment were identified, long range sequencing was used
specifically on this fragment by amplifying it from the library.
Alternatively, PCR primers were designed to PCR amplify a fragment
from the original DNA pool to validate the translocation.
[0103] However, in the library preparation steps, two or more
fragments from different parts of the genome were often ligated
resulting in hybrid fragments that, when sequenced from both ends,
look like a translocation. Since such ligations were random, it is
very unlikely to find them repeatedly joining fragments from the
same regions of the genome. However, real translocation will show
up about as much as the bridged coverage. The 7.times. bridged
coverage using the mate-pair protocol was sufficient to produce the
multiple observations. With almost 7.times. bridged coverage, it
was expected that real translocations would be observed multiple
times. An algorithm was written to analyze the mapped data and find
repeated evidence of chromosomal rearrangements.
[0104] Bioinformatic analysis of the mate-pair library sequencing
data indicated a putative t(6;7) translocation, with 10 aberrant
mate pairs, each showing one end mapping to 6p25.3 and the other to
7q32.3 (FIG. 10). To confirm this translocation, PCR primers were
designed spanning the putative breakpoints on the derivative
chromosomes 6 and 7. These confirmed the presence of two hybrid
segments of genomic DNA, each composed partly of material from the
DUSP22 locus on 6p25.3 (with a breakpoint with the intron between
exons 1 and 2), and partly of material from the FLJ43663
hypothetical gene locus on 7q32.3. The results from analysis of the
der(6) are shown in FIG. 11.
[0105] The finding of a translocation involving DUSP22 led to the
development of BAP FISH probes that could examine cases with 6p25.3
rearrangements and differentiate translocations involving DUSP22
from those involving IRF4 (FIG. 12). Representative FISH images
showing the use of these probes are shown in FIG. 13. The screening
probe, Probe #2 (FIG. 8B), was used to confirm the presence of a
6p25.3 rearrangement, since without this step the extra signals
attributable to cross-hybridization cannot be accurately
interpreted. In an analysis of 82 patients with ALCL, DUSP22
translocations were seen in 5%, IRF4 translocations were seen in
7%, and ALK expression (a surrogate for the presence of ALK
translocations (Jaffe, Mod. Pathol., 14:219-228 (2001)) was seen in
36% (FIG. 14). All cases with DUSP22 or IRF4 translocations were
ALK negative.
[0106] DUSP22 is a dual-specificity phosphatase whose major role is
in modulating mitogen-activated protein kinase (MAPK) signaling
(Patterson et al., Biochem. J., 418:475-489 (2009)). DUSP22
expression was analyzed in 6 TCL cell lines. DUSP22 was expressed
in all lines tested (FIG. 8A). SUDHL-1, an ALCL cell line, was
selected for further studies. SUDHL-1 cells were treated with small
interfering RNAs (siRNAs) to determine the effect of DUSP22
silencing on cell proliferation. Cells were treated either with a
pool of three siRNAs specific for DUSP22, or an irrelevant control
siRNA (Santa Cruz Biotechnologies). Western blotting at 48 hours
confirmed that the cells treated with DUSP22 siRNAs down-regulated
expression of DUSP22 protein (FIG. 15B). Radioactivity was measured
in a scintillation counter 48 hours after siRNA treatment and 16
hours after the addition of tritiated (.sup.3H--) thymidine. Cells
treated with DUSP22 siRNA exhibited a 43% decrease in proliferation
compared to cells treated with control siRNA (FIG. 15C). This
decrease was statistically significant (p<0.000001, Student's t
test). These results suggest that the activating functions of
DUSP22 TCL cells outweigh its inhibitory functions, and that DUSP22
may have oncogenic function in TCLs.
[0107] To determine whether the t(6;7)(p25.3;q32.3) translocation
occurs in other TCLs, a BAP FISH probe to the 7q32.3 locus
containing the FLJ43663 hypothetical gene region was designed (FIG.
16). The probe exhibited separation of the red and green signals in
the sequenced case described above, which then served as a positive
control (FIG. 17). Then, 29 additional TCL cases (30 cases
overall), which were previously revealed to have 6p25.3
rearrangements (Feldman et al., Leukemia, 23:574-580 (2009) and
Wada et al., Mod. Pathol., 22 suppl 1:289A (abst 1308) (2009)),
were tested using the 7q32.3 BAP FISH probe. Rearrangements of
7q32.3 were seen in 41% of these cases (FIG. 18). Interestingly,
another 23% exhibited extra copies of the unrearranged FLJ43663
locus.
[0108] To determine whether the cases with both 6p25.3 and 7q32.3
rearrangements had t(6;7)(p25.3;q32.3) translocations (rather than
two unrelated translocations), a dual-fusion FISH (D-FISH) probe
was developed to detect the t(6;7)(p25.3;q32.3)(FIG. 19). A summary
of the FISH results is given in Table 6. Cases with 6p25.3
rearrangements were subdivided according the their 6p25.3
breakpoint based on FISH using Probes #3 and #4 as shown in FIGS.
12 and 14. Among cases with successful hybridizations, this method
successfully localized the breakpoint to either IRF4 or DUSP22 in
all but one case (Case 13) in which results were indeterminate. Of
the 12 cases with a FLJ43663 break detected by the 7q32.3 BAP
probe, D-FISH confirmed the presence of a t(6;7)(p25.3;q32.3) in
10, and the hybridization failed in 2. Interestingly, in all cases
with a confirmed t(6;7)(p25.3;q32.3), the breakpoint on 6p25.3 lay
within the DUSP22 gene region, rather than within IRF4 (though Case
10 had concurrent IRF4 and FLJ43663 breaks but D-FISH could not be
interpreted). Of the 10 cases with t(6;7)(p25.3;q32.3), 5 exhibited
only 1 fusion signal, suggesting probable unbalanced
translocations. In the remaining 5 cases, 2 fusion signals were
seen in at least some cells, suggesting balanced
translocations.
TABLE-US-00010 TABLE 6 Genetic characteristics of 30 cases of
T-cell lymphomas with 6p25.3 rearrangements. 7p32.3 Number of
Breakpoint Breakapart t(6; 7)(p25.3; q32.3) Fusion Case Age Sex
Diagnosis on 6p25.3* FISH D-FISH Signals 1 79 M ALCL, ALK- DUSP22
Break Fusion 1-2 2 89 F cALCL DUSP22 Break Fusion 1-2 3 50 M ALCL,
ALK- DUSP22 Break Fusion 1-2 4 68 M cALCL DUSP22 Break Fusion 1 5
65 M cALCL Hyb. fail. Break Fusion 1 6 65 M ALCL, ALK- DUSP22 Break
Fusion 1 7 50 M cALCL DUSP22 Break Fusion 1 8 65 M ALCL, ALK-
DUSP22 Break Fusion 2 9.sup..dagger. 46 M ALCL, ALK- DUSP22 Break
Fusion 2 10 77 M cALCL IRF4 Break Hyb. fail. 11 65 M ALCL, ALK-
Hyb. fail. Break Hyb. fail. 12 66 F cALCL DUSP22 Break** Fusion 1
13 57 M cALCL Indeterminate Aneuploid 14*** 76 M cALCL DUSP22
Aneuploid 15*** 76 M cALCL Hyb. fail. Aneuploid 16 59 F cALCL IRF4
Aneuploid 17 66 M cALCL DUSP22 Aneuploid 18 65 F cALCL Not done
Aneuploid 19 52 M cALCL IRF4 Aneuploid 20 71 F cALCL DUSP22 Normal
21 50 M ALCL, ALK- DUSP22 Normal 22 54 M ALCL, ALK- DUSP22 Normal
23 33 F ALCL, ALK- DUSP22 Normal 24 86 M cALCL Hyb. fail. Normal 25
35 M cALCL IRF4 Normal 26 54 M ALCL, ALK- IRF4 Normal 27 56 F cALCL
IRF4 Normal 28 45 M cALCL Hyb. fail. Normal 29 62 M ALCL, ALK- IRF4
Normal 30 73 F PTCL, NOS Hyb. fail. Hyb. fail. Abbreviations: FISH,
fluorescence in situ hybridization; D-FISH, dual-fusion FISH; ALCL,
anaplastic large cell lymphoma; ALK-, anaplastic lymphoma
kinase-negative; cALCL, cutaneous ALCL; PTCL, NOS, peripheral
T-cell lymphoma, not otherwise specified; Hyb. fail., hybridization
failure .sup..dagger.Index case (sequenced) *As determined using
Probes #3 and #4 **1R2F signal pattern with BAP probe; D-FISH
results confirmed translocation ***Cases 14 and 15 were different
tumors from the same patient
[0109] To determine the incidence of FLJ43663 rearrangements in the
absence of 6p25.3 translocations, FISH was performed using the
7q32.3 BAP probe on tissue microarrays (TMAs), constructed as
described elsewhere (Feldman et al., Am. J. Clin. Pathol.,
130:178-185 (2008) and Feldman et al., Leukemia, 22:1139-1143
(2008)). TCLs from 143 patients were tested. Hybridization was
successful in 110 and failed in 33. Of the 110 patients with
successful hybridizations, only 1 (<1%) exhibited rearrangement
of FLJ43663. This case was an ALK-negative ALCL (1 of 11
ALK-negative ALCLs with successful hybridizations). An additional 7
TCLs had extra copies of the FLJ43663 locus. These data are
summarized in Table 7. Taken together, these results suggest that
translocations of FLJ43663 are rare in the absence of 6p25.3
rearrangements, and, to date, are only seen in ALK-negative ALCLs
(systemic or cutaneous).
TABLE-US-00011 TABLE 7 Abnormalities of the FLJ43663 locus on
7q32.3 in patients lacking 6p25.3 rearrangements ALCL, ALCL, PTCL,
ALK- ALK+ cALCL NOS AITL NKTL Other** Total Rearranged 1 0 0 0 0 0
0 1 Additional 0 1 0 4 0 2 0 7 Copies* Normal 10 10 2 43 20 5 12
102 Failed 6 2 1 15 4 2 3 33 Hybridization Total 17 13 3 62 24 9 15
143 Abbreviations: ALCL, anaplastic large cell lymphoma; ALK,
anaplastic lymphoma kinase; cALCL, primary cutaneous ALCL; PTCL,
NOS, peripheral T-cell lymphoma, not otherwise specified; AITL,
angioimmunoblastic T-cell lymphoma; NKTL, extranodal NK/T-cell
lymphoma, nasal type. *Additional copies defined as 4 or more
fusion signals **Enteropathy associated TCL (2), hepatosplenic TCL
(2), subcutaneous panniculitis-like TCL (2), mycosis fungoides (4),
cutaneous CD4+ small/medium TCL (1), large granular lymphocyte
leukemia (4)
[0110] To determine the effect of 6p25.3 rearrangements on
expression of genes near the sequenced breakpoint on 7q32.3, a gene
expression microarray analysis (Affymetrix U133 plus 2.0) was
performed on 25 cases of TCLs, including 4 with and 21 without
6p25.3 rearrangements. The expression probe on 7q32.3 most
up-regulated among the translocated cases was derived from the EST
AL569506, which resides within the 3' terminus of FLJ43663 itself
(FIG. 20). This region exhibited 5.5-fold overexpression in
translocated cases compared to non-translocated cases. This 3'
region of FLJ43663 was the region remaining on the der(7) after the
t(6;7)(p25.3;q32.3) translocation. Probes at the 5' end of
FLJ43663, as well as a probe farther 3' (centromeric) to FLJ43663,
exhibited no evidence of overexpression.
[0111] FLJ43663 is a hypothetical gene region, with known
transcript isoforms and a hypothetical but unproved protein
product. To evaluate the sequenced case for the presence of a
fusion transcript, primers from DUSP22 and FLJ43663 were utilized
in a PCR reaction of cDNA prepared from DNase-treated RNA. This
produced bands of the sizes predicted, indicating the presence of a
fusion transcript (FIG. 21). Based on the overexpression of the 3'
terminus of FLJ43663 mRNA, and the presence of a fusion transcript,
FLJ43663 appears to be aberrantly up-regulated by the
t(6;7)(p25.3;q32.3) in TCLs, and appears to contribute to the
pathogenesis and/or clinical behavior of these tumors. Thus, in
addition to the clinical use of detecting FLJ43663 gene
rearrangements as a diagnostic/prognostic biomarker, FLJ43663, its
mRNA, or its polypeptide product can be a therapeutic target in
TCLs, for example, by down-regulating it. Thus, human malignancies
may be targeted by reducing FLJ43663 expression.
Other Embodiments
[0112] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
6119DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1ccctggggca ttttattaa 19220DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2agccactgcc gatactgatg 20318DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 3gcagcctggc gtgacaag
18420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 4agccactgcc gatactgatg 20532DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 5gagccccaaa tgccttcttt ggttttctta ga
32670DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 6agggcctggg tggtcttgat tttgtatttt
aggaaccaga caagtacctt tttacgggtc 60tttgaatggt 70
* * * * *
References