U.S. patent application number 11/151679 was filed with the patent office on 2005-11-03 for cellular arrays and methods of detecting and using genetic disorder markers.
Invention is credited to Barlund, Maarit, Kallioniemi, Olli-P, Kononen, Juha, Muller, Uwe Richard, Sauter, Guido.
Application Number | 20050244880 11/151679 |
Document ID | / |
Family ID | 26803238 |
Filed Date | 2005-11-03 |
United States Patent
Application |
20050244880 |
Kind Code |
A1 |
Kallioniemi, Olli-P ; et
al. |
November 3, 2005 |
Cellular arrays and methods of detecting and using genetic disorder
markers
Abstract
A method is disclosed for rapid molecular profiling of tissue or
other cellular specimens by placing a donor specimen in an assigned
location in a recipient array, providing copies of the array, and
performing a different biological analysis of each copy. The
results of the different biological analyses are compared to
determine if there are correlations between the results of the
different biological analyses at each assigned location. In some
embodiments, the specimens may be tissue specimens from different
tumors, which are subjected to multiple parallel molecular
(including genetic and immunological) analyses. The results of the
parallel analyses are then used to detect common molecular
characteristics of the genetic disorder type, which can
subsequently be used in the diagnosis or treatment of the disease.
The biological characteristics of the tissue can be correlated with
clinical or other information, to detect characteristics associated
with the tissue, such as susceptibility or resistance to particular
types of drug treatment. Other examples of suitable tissues which
can be placed in the matrix include tissue from transgenic or model
organisms, or cellular suspensions (such as cytological
preparations or specimens of liquid malignancies or cell
lines).
Inventors: |
Kallioniemi, Olli-P;
(Rockville, MD) ; Muller, Uwe Richard; (Painted
Post, NY) ; Sauter, Guido; (Basel, CH) ;
Kononen, Juha; (Rockville, MD) ; Barlund, Maarit;
(Tampere, FI) |
Correspondence
Address: |
Kevin M. Farrell
Pierce Atwood
Suite 350
One New Hampshire Avenue
Portsmouth
NH
03801
US
|
Family ID: |
26803238 |
Appl. No.: |
11/151679 |
Filed: |
June 13, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11151679 |
Jun 13, 2005 |
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09971742 |
Oct 4, 2001 |
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6905823 |
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09971742 |
Oct 4, 2001 |
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09429448 |
Oct 28, 1999 |
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60106038 |
Oct 28, 1998 |
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60150493 |
Aug 24, 1999 |
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Current U.S.
Class: |
435/6.14 |
Current CPC
Class: |
C12Q 1/6886 20130101;
G01N 2001/282 20130101; G01N 2001/368 20130101; B01J 2219/00743
20130101; B01J 2219/00702 20130101; G02B 21/34 20130101; G01N
2001/288 20130101; C12Q 1/6837 20130101; C12Q 1/6809 20130101; G01N
33/54306 20130101; G01N 33/5005 20130101; C12Q 1/6809 20130101;
B01L 3/5085 20130101; G01N 1/08 20130101; C12Q 2600/106 20130101;
C12Q 1/6837 20130101; B01J 2219/00659 20130101; C12Q 2600/118
20130101; C12Q 2565/501 20130101; C12Q 2539/103 20130101; C12Q
2539/103 20130101; C12Q 2537/157 20130101; C12Q 2537/157 20130101;
C12Q 2537/157 20130101; C12Q 2543/10 20130101; C12Q 2543/10
20130101; C12Q 2539/103 20130101; G01N 1/312 20130101; C12Q 1/6841
20130101; C12Q 2600/112 20130101; C12Q 1/6841 20130101; G01N 1/2813
20130101; C12Q 2600/156 20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 001/68 |
Claims
What is claimed is:
1. A method of parallel analysis of tissue specimens, the method
comprising: obtaining a plurality of donor specimens; placing each
donor specimen in an assigned location in a recipient array; using
a genosensor comparative genomic hybridization (gCGH) array to
identify a biomarker to test on the recipient array; obtaining a
plurality of sections from the recipient array in a manner that
each section contains a plurality of donor specimens that maintain
their assigned locations; performing on each section a different
biological analysis using the biomarker; and comparing the results
of the different biological analyses in corresponding assigned
locations of different sections to determine if there are
correlations between the results of the different biological
analyses at each assigned location.
2. The method of claim 1, wherein the biomarker is selected by
high-throughput genetic analysis.
3. The method of claim 1, wherein the biomarker comprises a
numerical alteration of a chromosome, chromosomal region, gene,
gene fragment, or locus.
4. The method of claim 1, wherein comparing the results comprises
determining if there is an alteration of a gene by examining a
marker for gene alteration.
5. The method of claim 4, wherein the alteration is an
amplification of PDGFB in breast, lung, colon, testicular,
endometrial, or bladder cancer.
6. A method of analyzing gene amplification in a tissue specimen,
the method comprising: screening multiple genes in a tissue
specimen with a genosensor comparative genomic hybridization (gCGH)
array that detects which genes are amplified in the tissue
specimen; and screening multiple tissue specimens in a tissue array
with a nucleic acid probe to detect which genes are amplified in
the tissue specimens; wherein the result of screening multiple
genes is used to select the nucleic acid probe to screen the
multiple tissue specimens, or wherein the result of screening
multiple tissue specimens is used to select the array that detects
which genes are amplified.
7. The method of claim 6, wherein the gCGH array is assayed for a
gene amplification, or a genetic or molecular marker that reflects
this amplification.
8. The method of claim 7, wherein the gCGH array is a microarray
that contains target loci that undergo amplification in cancer.
9. A method of analyzing a biological sample for a genetic
disorder, the method comprising: exposing a genosensor comparative
genomic hybridization (gCGH) array of genomic regions to a nucleic
acid sample from a cell with a known specific genetic disorder, and
identifying as a biomarker a genomic region to which the nucleic
acid hybridizes; obtaining a candidate probe that hybridizes to the
biomarker; exposing the candidate probe to a tissue specimen array
to determine a statistical measure of hybridization of the
candidate probe; selecting a candidate probe having a statistically
significant measure of hybridization; and using a selected
candidate probe to analyze a biological sample for the genetic
disorder.
10. The method of claim 9, wherein analysis of the biological
sample provides diagnostic information.
11. The method of claim 9, wherein analysis of the biological
sample provides prognostic information.
14. A method for detecting a genomic target sequence that is
associated with a specific genetic disorder, the method comprising
contacting a plurality of genomic regions in a genosensor
comparative genomic hybridization (gCGH) array with a nucleic acid
test sample comprising nucleic acid fragments that collectively
represent DNA from a cell with a known specific genetic disorder
under conditions that allow the nucleic acid fragments to hybridize
to one or more candidate genomic regions; measuring the amount of
nucleic acid test sample hybridized to the candidate genomic
regions, if any, and selecting a candidate genomic region
corresponding to an altered amount of hybridized test sample
nucleic acid compared to a control sample of normal DNA; preparing
a nucleic acid probe that hybridizes to the selected candidate
genomic region; contacting a plurality of tissue samples with the
probe under conditions that allow the probe to hybridize to
nucleotide sequences in the tissue samples; and selecting a
candidate genomic region corresponding to a probe that hybridizes
to a significant number of tissue samples as a genomic target
sequence that is associated with the specific genetic disorder.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional
Application Ser. No. 60/106,038, filed on Oct. 28, 1998, and U.S.
Provisional Application Ser. No. 60/150,493, filed on Aug. 24,
1999, both of which are incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The invention relates to the screening of tissue samples and
genomic regions to discover markers for genetic disorders such as
cancer.
BACKGROUND OF THE INVENTION
[0003] Biological mechanisms of many diseases have been clarified
by microscopic examination of tissue specimens. Histopathological
examination has also permitted the development of effective medical
treatments for a variety of illnesses. In standard anatomical
pathology, a diagnosis is made on the basis of cell morphology and
staining characteristics. Tumor specimens, for example, can be
examined to characterize the tumor type and predict the
aggressiveness of the tumor. Although this microscopic examination
and classification of tumors has improved medical treatment, the
microscopic appearance of a tissue specimen stained by standard
methods (such as hematoxylin and eosin) can often reveal only a
limited amount of diagnostic or molecular information.
[0004] Recent advances in molecular medicine have provided an even
greater opportunity to understand the cellular mechanisms of
disease, and select appropriate treatments with the greatest
likelihood of success. Some hormone dependent breast tumor cells,
for example, have an increased expression of estrogen receptors on
their cell surfaces, which indicates that the patient from whom the
tumor was taken will likely respond to certain anti-estrogenic drug
treatments. Other diagnostic and prognostic cellular changes
include the presence of tumor specific cell surface antigens (as in
melanoma), the production of embryonic proteins (such as
.alpha.-fetoprotein in liver cancer and carcinoembryonic
glycoprotein antigen produced by gastrointestinal tumors), and
genetic abnormalities (such as activated oncogenes in tumors). A
variety of techniques have evolved to detect the presence of these
cellular abnormalities, including immunophenotyping with monoclonal
antibodies, in situ hybridization with probes, and DNA
amplification using the polymerase chain reaction (PCR).
[0005] The development of new molecular markers of clinical
importance has been impeded by the slow and tedious process of
evaluating biomarkers in large numbers of clinical specimens. For
example, hundreds of tissue specimens representing different stages
of tumor progression have to be evaluated before the importance of
a given marker can be assessed. Since the number of antibodies, as
well as probes for mRNA or DNA targets is increasing rapidly, only
a small fraction of these can ever be tested in large numbers of
clinical specimens.
[0006] Various methods have been explored to prepare samples of
multiple tissues or nucleic acids on one slide or plate.
SUMMARY OF THE INVENTION
[0007] The invention is based on the discovery that two very
different types of arrays can be used in combination in new methods
to rapidly and accurately detect, with high resolution, genomic
copy number alterations, such as gene amplifications or deletions,
that can serve as markers for various genetic disorders such as
cancers and trisomies.
[0008] Thus, the invention features methods of detecting particular
genomic "target" regions (nucleic acid sequences) that correspond
to specific genetic disorders, e.g., one or more different types of
tumors or hereditary genetic diseases, by combining tissue
microarray technology with other technologies, such as
high-throughput genomics. These methods are used to identify
molecular characteristics, such as structural changes in genes or
proteins, and copy number or expression alterations of genes, and
to correlate these results with disease prognosis or therapy
outcome to identify novel targets for gene prevention, early
diagnosis, disease classification, or prognosis, and to identify
therapeutic agents. High-throughput technologies include cDNA
arrays and Comparative Genomic Hybridization ("CGH") arrays.
[0009] The invention also includes methods of preparing new arrays
of nucleic acids (genes), e.g., representative of specific types of
tumors (tumor-specific diagnostic gene arrays); probes that
hybridize selectively to these genomic target regions; methods of
preparing the probes; methods of using the probes to screen for
and/or diagnose specific genetic disorders; compositions that
interact with the genomic target regions to treat the genetic
disorders; and methods of treating the genetic disorders using
these compositions.
[0010] In general, in one aspect the invention features a method of
parallel analysis of tissue specimens, by obtaining a plurality of
donor specimens; placing each donor specimen in an assigned
location in a recipient array; using a genosensor comparative
genomic hybridization (gCGH) array to identify a biomarker to test
on the recipient array; obtaining a plurality of sections from the
recipient array in a manner that each section contains a plurality
of donor specimens that maintain their assigned locations;
performing on each section a different biological analysis using
the biomarker; and comparing the results of the different
biological analyses in corresponding assigned locations of
different sections to determine if there are correlations between
the results of the different biological analyses at each assigned
location. For example, the biomarker can be selected by
high-throughput genetic analysis, and the biomarker can include a
numerical alteration of a chromosome, chromosomal region, gene,
gene fragment, or locus.
[0011] The results can be compared by determining if there is an
alteration of a gene by examining a marker for gene alteration. For
example, the alteration can be an amplification of PDGFB in breast,
lung, colon, testicular, endometrial, or bladder cancer.
[0012] In another embodiment, the invention features a method of
analyzing gene amplification in a tissue specimen by screening
multiple genes in a tissue specimen with a genosensor comparative
genomic hybridization (gCGH) array that detects which genes are
amplified in the tissue specimen; and screening multiple tissue
specimens in a tissue array with a nucleic acid probe to detect
which genes are amplified in the tissue specimens; wherein the
result of screening multiple genes is used to select the nucleic
acid probe to screen the multiple tissue specimens, or wherein the
result of screening multiple tissue specimens is used to select the
array that detects which genes are amplified.
[0013] In this method, the gCGH array can be assayed for a gene
amplification, or a genetic or molecular marker that reflects this
amplification. The CGH array can be a microarray that contains
target loci that undergo amplification in cancer.
[0014] The invention also features a method of analyzing a
biological sample for a genetic disorder by exposing a genosensor
comparative genomic hybridization (gCGH) array of genomic regions
to a nucleic acid sample from a cell with a known specific genetic
disorder, and identifying as a biomarker a genomic region to which
the nucleic acid hybridizes; obtaining a candidate probe that
hybridizes to the biomarker; exposing the candidate probe to a
tissue specimen array to determine a statistical measure of
hybridization of the candidate probe; selecting a candidate probe
having a statistically significant measure of hybridization; and
using a selected candidate probe to analyze a biological sample for
the genetic disorder. This analysis of the biological sample can
provide diagnostic or prognostic information.
[0015] In addition, the invention features a method of detecting
the presence of cancerous cells in a specimen, by determining
whether platelet derived growth factor beta (PDGFB) is amplified in
the specimen, amplification indicating the presence of cancerous
cells in the specimen, e.g., a lung, bladder, or endometrial tissue
specimen.
[0016] In another aspect, the invention features a method for
detecting a genomic target sequence that is associated with a
specific genetic disorder by contacting a plurality of genomic
regions in a genosensor comparative genomic hybridization (gCGH)
array with a nucleic acid test sample including nucleic acid
fragments that collectively represent DNA from a cell with a known
specific genetic disorder under conditions that allow the nucleic
acid fragments to hybridize to one or more candidate genomic
regions; measuring the amount of nucleic acid test sample
hybridized to the candidate genomic regions, If any, and selecting
a candidate genomic region corresponding to an altered amount of
hybridized test sample nucleic acid compared to a control sample of
normal DNA; preparing a nucleic acid probe that hybridizes to the
selected candidate genomic region; contacting a plurality of tissue
samples with the probe under conditions that allow the probe to
hybridize to nucleotide sequences in the tissue samples; and
selecting a candidate genomic region corresponding to a probe that
hybridizes to a significant number of tissue samples as a genomic
target sequence that is associated with the specific genetic
disorder.
[0017] As used herein, a "polypeptide" is any chain of amino acids,
regardless of length or post-translational modification (e.g.,
glycosylation or phosphorylation).
[0018] A "gene amplification" is an increase in the copy number of
a gene, as compared to the copy number in normal tissue. An example
of a genomic amplification is an increase in the copy number of an
oncogene. A "gene deletion" is a deletion of one or more nucleic
acids normally present in a gene sequence, and in extreme examples
can include deletions of entire genes or even portions of
chromosomes.
[0019] A "genomic target sequence" is a sequence of nucleotides
located in a particular region in the human genome that corresponds
to one or more specific loci, including genetic abnormalities, such
as a nucleotide polymorphism, a deletion, or an amplification.
[0020] A "genetic disorder" is any illness, disease, or abnormal
physical or mental condition that is caused by an alteration in one
or more genes or regulatory sequences (such as an amplification,
mutation, deletion, or translocation).
[0021] "Comparative Genomic Hybridization" or CGH is a technique of
differential labeling of test DNA and normal reference DNA, which
are hybridized simultaneously to chromosome spreads, as described
in Kallioniemi et al., Science, 258:818-821, 1992.
[0022] A "nucleic acid array" refers to an arrangement of nucleic
acid (such as DNA or RNA) in assigned locations on a matrix, such
as that found in cDNA or CGH arrays.
[0023] A "microarray" is an array that is miniaturized so as to
require microscopic examination for visual evaluation.
[0024] A "DNA chip" is a DNA array in which multiple DNA molecules
(such as cDNAs) of known DNA sequences are arrayed on a substrate,
usually in a microarray, so that the DNA molecules can hybridize
with nucleic acids (such as cDNA or RNA) from a specimen of
interest. DNA chips are further described in Ramsay, Nature
Biotechnology, 16:40-44, 1998.
[0025] "Gene expression microarrays" refers to microscopic arrays
of cDNAs printed on a substrate, which serve as a high density
hybridization target for mRNA probes, as in Schena, BioEssays
18:427-431, 1996.
[0026] "Serial Analysis of Gene Expression" or "SAGE" refers to the
use of short sequence tags to allow the quantitative and
simultaneous analysis of a large number of transcripts in tissue,
as described in Velculescu et al., Science, 270:484-487, 1995.
[0027] "High throughput genomics" refers to application of genomic
or genetic data or analysis techniques that use microarrays or
other genomic technologies to rapidly identify large numbers of
genes or proteins, or distinguish their structure, expression or
function from normal or abnormal cells or tissues.
[0028] A "tumor" is a neoplasm that may be either malignant or
non-malignant. "Tumors of the same tissue type" refers to primary
tumors originating in a Particular organ (such as breast, prostate,
bladder or lung). Tumors of the same tissue type may be divided
into tumors of different sub-types (a classic example being
bronchogenic carcinomas (lung tumors) which can be an
adenocarcinoma, small cell, squamous cell, or large cell
tumor).
[0029] A "cellular" specimen is one which contains whole cells, and
includes tissues, which are aggregations of similarly specialized
cells united in the performance of a particular function. Examples
include cells from the skin, breast, prostate, blood, testis, ovary
and endometrium.
[0030] A "cellular suspension" is a liquid in which cells are
dispersed, and may include a uniform or non-uniform suspension.
Examples of cellular suspensions are those obtained by fine-needle
aspiration from tumor sites, cytology specimens (such as vaginal
fluids for preparing Pap smears, washes (such as bronchial
washings), urine that contains cells (for example in the detection
of bladder cancer), ascitic fluid (for example obtained by
abdominal paracentesis), or other body fluids.
[0031] A "cytological preparation" is a pathological specimen, such
as vaginal fluids, in which a cellular suspension can be converted
into a smear or other form for pathological examination or
analysis.
[0032] 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 belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
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.
[0033] The invention provides a rapid means of identifying not only
specific genomic abnormalities present in a tissue, but the
importance and statistical significance of these abnormalities in
hundreds or thousands of tissues, to provide relevant diagnostic
and prognostic information, as well as potential targets for
therapeutic agents.
[0034] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 is a schematic perspective view of a first embodiment
of the punch device of the present invention, showing alignment of
the punch above a region of interest of donor tissue in a donor
block.
[0036] FIG. 2 is a view similar to FIG. 1, but in which the punch
has been advanced to obtain a donor specimen sample.
[0037] FIG. 3 is a schematic, perspective view of a recipient block
into which the donor specimen has been placed.
[0038] FIGS. 4-8 are schematic diagrams illustrating steps in the
preparation of thin section arrays from the recipient block.
[0039] FIG. 9 is a perspective view of a locking device for holding
a slide mounted specimen above the tissue in the donor block to
locate a region of interest.
[0040] FIG. 10A is a view of an H&E stained, thin section
tissue array mounted on a slide for microscopic examination.
[0041] FIG. 10B is a magnified view of a portion of the slide in
FIG. 10A, showing results of erbB2 mRNA in situ hybridization on a
tissue array from the region in the small rectangle in FIG.
10A.
[0042] FIG. 10C is an electrophoresis gel showing that high
molecular weight DNA and RNA can be extracted from the breast
cancer specimens fixed in cold ethanol.
[0043] FIG. 10D is an enlarged view of one of the tissue samples of
the array in FIG. 10A, showing an immunoperoxidase staining for the
erbB2 antigen.
[0044] FIG. 10E is a view similar to FIG. 10D, showing high level
erbB2 gene amplification detected by fluorescent in situ
hybridization (FISH) of tissue in the array by an erbB2 DNA
probe.
[0045] FIGS. 11A, 11B, 11C and 11D are schematic views illustrating
an example of parallel analysis of arrays obtained by the method of
the present invention.
[0046] FIG. 12 is an enlarged view of a portion of FIG. 11.
[0047] FIG. 13 is a schematic representation of a genosensor CGH
microarray that contains 31 target loci that have been reported to
undergo amplification in cancer. Circles around target loci
indicate amplifications found in the breast cancer cell lines
tested in this study.
[0048] FIG. 14 is a digital representation of the results of a
chromosomal CGH analysis showing high level amplifications in
Sum-52 breast cancer cells at 10q25-q26 and at 7q21-q22, a
genosensor CGH analysis indicating high level amplifications of the
MET (7q21) and FGFR2 (10q25) oncogenes, and a FISH analysis showing
amplification of FGFR2 (at 10q25).
[0049] FIG. 15 is a schematic diagram of a breast cancer tissue
microarray, as well as a digital image of a hybridization, showing
that FGFR2 was amplified in 4.5% of the tumor samples in the breast
cancer tissue microarray.
[0050] FIG. 16 is a schematic representation of the combination of
the DNA array and the tissue array, showing that the DNA array can
probe a single tumor with hundreds of probes, while the tissue
array technology can conversely probe specimens from hundreds of
tumors with a single probe.
[0051] FIG. 17 is a schematic diagram representing the combination
of the tissue array technology with cDNA and/or CGH arrays.
DETAILED DESCRIPTION
[0052] The use of tissue arrays in combination with other array
techniques can provide information about the frequency of a
multitude of genetic alteration or gene expression patterns
(including normal gene expression patterns) in a variety of tissue
types (such as different types of tumors), and in tissue of a
particular histological type (such as a tumor of a specific type,
such as intraductal breast cancer), as well as the tissue
distribution of molecular markers tested.
[0053] In one specific embodiment of the combined DNA and tissue
arrays, the DNA array may be a cDNA or genomic microarray chip that
allows a plurality (hundreds, thousands, or even more) of different
nucleic acid sequences to be affixed to the surface of a support to
form an array. Such a chip may, for instance carry an array of cDNA
clones, oligonucleotides, or large-insert genomic P1, BAC, or PAC
clones. These arrays enable the analysis of hundreds of genes or
genomic fragments at once to determine their expression or copy
number in a test specimen.
[0054] A high-throughput DNA chip can be used together with
high-throughput tissue array technology. Such hybrid inventions
include using a DNA array to screen a limited number of tumor
samples for expression or copy number of one or more (for example
thousands of) specific genes or DNA sequences. Probes containing
the gene of interest may then be used to screen a tissue microarray
that contains many different tissue specimens (such as a variety of
breast tumors or prostate tumors) to determine if the identified
gene or genetic locus is similarly altered in these tumors. For
instance, a cDNA chip can be used to screen a human breast cancer
cell line, to identify one or more genes that are overexpressed or
amplified in that particular breast cancer. A probe, corresponding
to the identified gene, would then be used to probe a tissue array
containing a plurality of tissue samples from different breast
cancers, or even tumors of different types (such as lung or
prostate cancer). Such a probe could be made by labeling the
identical clone used in the DNA array (for example with a
fluorescent or radioactive marker). The presence of the gene in
related (or unrelated) tumors would be revealed by the pattern of
hybridization of the probe to the tissue array.
[0055] Another embodiment includes a method of preparing a
diagnostic tumor-specific gene array.
Embodiments of FIGS. 1 to 12
[0056] A first embodiment of a device for making the microarrays of
the present invention is shown in FIGS. 1 and 2, in which a donor
block 30 (of tissue) is shown in a rectangular container 31 mounted
on a stationary platform 32 having an L-shaped edge guide 34 that
maintains donor container 31 in a predetermined orientation on
platform 32. A punch apparatus 38 is mounted above platform 32, and
includes a vertical guide plate 40 and a horizontal positioning
plate 42. The positioning plate 42 is mounted on an x-y stage (not
shown) that can be precisely positioned with a pair of digital
micrometers.
[0057] Vertical guide plate 40 has a flat front face that provides
a precision guide surface against which a reciprocal punch base 44
can slide along a track 46 between a retracted position shown in
FIG. 1 and an extended position shown in FIG. 2. An elastic band 48
helps control the movement of base 44 along this path, and the
limits of advancement and retraction of base 44 are set by track
member 46, which forms a stop that limits the amplitude of
oscillation of base 44. A thin wall stainless steel tube punch 50
with sharpened leading edges is mounted on the flat bottom face of
base 44, so that punch 50 can be advanced and retracted with
respect to platform 32, and the container 31 on the platform. The
hollow interior of punch 50 is continuous with a cylindrical bore
through base 44, and the bore opens at opening 51 on a horizontal
lip 53 of base 44.
[0058] FIG. 1 shows that a thin section of tissue, stained with
hematoxylin-eosin or other stains, can be obtained from donor block
30 and mounted on a slide 52 (with appropriate preparation and
staining) so that anatomic and micro-anatomic structures of
interest can be located in the block 30. Slide 52 can be held above
donor block 30 by an articulated arm holder 54 (FIG. 9) with a
clamp 56 which securely holds an edge of a transparent support
slide 58. Arm holder 54 can articulate at joint 60, to swivel
between a first position in which support slide 58 is locked in
position above container 31, and a second position in which support
slide 58 moves horizontally out of the position shown in FIG. 9 to
permit free access to punch 50.
[0059] In operation, the rectangular container 31 is placed on
platform 32 (FIG. 1) with edges of container 31 abutting edge
guides 34 to hold container 31 in a selected position. A donor
block 30 is prepared by embedding a gross tissue specimen (such as
a three dimensional tumor specimen 62) in paraffin. A thin section
of donor block 30 is shaved off, stained, and mounted on slide 52
as thin section 64, and slide 52 is then placed on support slide 58
and positioned above donor block 30 as shown in FIG. 9. Slide 52
can be moved around on support slide 58 until the edges of thin
section 64 are aligned with the edges of the gross pathological
specimen 62, as shown by the dotted lines in FIG. 9. Arm 54 is then
locked in the first position, to which the arm can subsequently
return after displacement to a second position.
[0060] A micro-anatomic or histologic structure of interest 66 can
then be located by examining the thin section through a microscope
(not shown). If the tissue specimen is, for example, an
adenocarcinoma of the breast, then the location of the structure of
interest 66 may be an area of the specimen in which the cellular
architecture is suggestive of specific features of the cancer, such
as invasive and noninvasive components. Once the structure of
interest 66 is located, the corresponding region of tissue specimen
62 from which the thin section structure of interest 66 was
obtained is located immediately below the structure of interest 66.
As shown in FIG. 1, positioning plate 42 can be moved in the x and
y directions (under the control of the digital micrometers or a
joystick), or the donor block can be moved for larger distances, to
align punch 50 in position above the region of interest of the
donor block 30, and the support slide 58 is then horizontally
pivoted away from its position above donor block 30 around pivot
joint 60 (FIG. 9).
[0061] Punch 50 is then introduced into the structure of interest
in donor block 30 (FIG. 2) by advancing vertical guide plate 40
along track 46 until plate 44 reaches its stop position (which is
preset by apparatus 38). As punch 50 advances, its sharp leading
edge bores a cylindrical tissue specimen out of the donor block 30,
and the specimen is retained within the punch as the punch
reciprocates back to its retracted position shown in FIG. 1. The
cylindrical tissue specimen can subsequently be dislodged from
punch 50 by advancing a stylet (not shown) into opening 51. The
tissue specimen is, for example, dislodged from punch 50 and
introduced into a cylindrical receptacle of complementary shape and
size in an array of receptacles in a recipient block 70 shown in
FIG. 3.
[0062] One or more recipient blocks 70 can be prepared prior to
obtaining the tissue specimen from the donor block 30. Block 70 can
be prepared by placing a solid paraffin block in container 31 and
using punch 50 to make cylindrical punches in block 70 in a regular
pattern that produces an array of cylindrical receptacles of the
type shown in FIG. 3. The regular array can be generated by
positioning punch 50 at a starting point above block 70 (for
example a corner of the prospective array), advancing and then
retracting punch 50 to remove a cylindrical core from a specific
coordinate on block 70, then dislodging the core from the punch by
introducing a stylet into opening 51. The punch apparatus or the
recipient block is then moved in regular increments in the x and/or
y directions, to the next coordinate of the array, and the punching
step is repeated. In the specific disclosed embodiment of FIG. 3,
the cylindrical receptacles of the array have diameters of about
0.6 mm, with the centers of the cylinders being spaced by a
distance of about 0.7 mm (so that there is a distance of about 0.05
mm between the adjacent edges of the receptacles).
[0063] In a specific example, core tissue biopsies having a
diameter of 0.6 mm and a height of 3-4 mm, were taken from selected
representative regions of individual "donor" paraffin-embedded
tumor blocks and precisely arrayed into a new "recipient" paraffin
block (20 mm.times.45 mm). H&E-stained sections were positioned
above the donor blocks and used to guide sampling from
morphologically representative sites in the tumors. Although the
diameter of the biopsy punch can be varied, 0.6 mm cylinders have
been found to be suitable because they are large enough to evaluate
histological patterns in each element of the tumor array, yet are
sufficiently small to cause only minimal damage to the original
donor tissue blocks, and to isolate reasonably homogenous tissue
blocks.
[0064] Up to 1000 such tissue cylinders, or more, can be placed in
one 20.times.45 mm recipient paraffin block. Specific disclosed
diameters of the cylinders are 0.1-4.0 mm, for example 0.5-2.0 mm,
and most specifically less than 1 mm, for example 0.6 mm.
Automation of the procedure, with computer guided placement of the
specimens, allows very small specimens to be placed tightly
together in the recipient array.
[0065] FIG. 4 shows the array in the recipient block after the
receptacles of the array have been filled with tissue specimen
cylinders. The top surface of the recipient block is then covered
with an adhesive film 74 from an adhesive coated tape sectioning
system (Instrumedics) to help maintain the tissue cylinder sections
in place in the array once it is cut. The array block may be warmed
at 37.degree. C. for 15 minutes before sectioning, to promote
adherence of the tissue cores and allow smoothing of the block
surface when pressing a smooth, clean surface (such as a microscope
slide) against the block surface.
[0066] With the adhesive film in place, a 4-8 .mu.m section of the
recipient block is cut transverse to the longitudinal axis of the
tissue cylinders (FIG. 5) to produce a thin microarray section 76
(containing tissue specimen cylinder sections in the form of disks)
that is transferred to a conventional specimen slide 78. The
microarray section 76 is adhered to slide 78, for example by
adhesive on the slide. The film 74 is then peeled away from the
underlying microarray member 76 to expose it for processing. A
darkened edge 80 of slide 78 is suitable for labeling or handling
the slide.
[0067] Breast cancer tissue specimens were fixed in cold ethanol to
help preserve high-molecular weight DNA and RNA, and 372 of the
specimens were fixed in this manner. At least 200 consecutive 4-8
.mu.m tumor array sections can be cut from each block providing
targets for correlated in situ analyses of multiple molecular
markers at the DNA, RNA, or protein level, including copy number or
expression of multiple genes. This analysis is performed by testing
for different gene molecular targets (e.g., DNA or RNA sequences or
antigens defined by antibodies) in separate array sections, and
comparing the results of the tests at identical coordinates of the
array (which correspond to tissue specimens from the same tissue
cylinder obtained from donor block). This approach enables
measurement of virtually hundreds of molecular characteristics from
every tumor, thereby facilitating construction of a large series of
correlated genotypic or phenotypic characteristics of uncultured
human tumors.
[0068] An example of a single microarray 76 containing 645
specimens is shown in FIG. 10A. An enlarged section of the
microarray (highlighted by a rectangle in FIG. 10A) is shown in
FIG. 10B, in which an autoradiogram of erbB2 mRNA in situ
hybridization illustrates that two adjacent specimens in the array
demonstrate a strong hybridization signal. FIG. 10C illustrates
electrophoresis gels which demonstrate that high molecular weight
DNA and RNA can be extracted from breast cancer specimens fixed in
ethanol at 4(C overnight.
[0069] One of the tissue specimens that gave the fluorescent
"positive" signals was also analyzed by immunoperoxidase staining,
as shown in FIG. 10D, where it was confirmed (by the dark stain)
that the erbB2 gene product was present. A DNA probe for the erbB2
gene was used to perform fluorescent in situ hybridization (FISH).
FIG. 10D shows one of the tumor array elements, which demonstrated
high level erbB2 gene amplification. The insert in FIG. 10E shows
three nuclei with numerous tightly clustered erbB2 hybridization
signals and two copies of the centromeric reference probe.
Additional details about these assays are given in Examples 1-4
below.
[0070] The potential of the array technology of the present
invention to perform rapid parallel molecular analysis of multiple
tissue specimens is illustrated in FIGS. 11A-11D, where the y-axis
of the graphs in FIGS. 11A and 11C corresponds to percentages of
tumors in specific groups that have defined clinicopathological or
molecular characteristics. This diagram shows correlations between
clinical and histopathological characteristics of the tissue
specimens in the micro-array. Each small box in the aligned rows of
FIG. 11B represents a coordinate location in the array.
Corresponding coordinates of consecutive thin sections of the
recipient block are vertically aligned above one another in the
horizontally extending rows. These results show that the tissue
specimens could be classified into four classifications of tumors
(FIG. 11A) based on the presence or absence of cell membrane
estrogen receptor expression, and the presence or absence of the
p53 mutation in the cellular DNA. In FIG. 11B, the presence of the
p53 mutation is shown by a darkened box, while the presence of
estrogen receptors is also shown by a darkened box. Categorization
into each of four groups (ER-/p53+, ER-/p53-, ER+/p53+ and
ER+/p53-) is shown by the dotted lines between FIGS. 11A and 113,
which divide the categories into Groups I, II, III and IV
corresponding to the ER/p53 status.
[0071] FIG. 11B also shows clinical characteristics that were
associated with the tissue at each respective coordinate of the
array. A darkened box for Age indicates that the patient is
premenopausal, a darkened box N indicates the presence of
metastatic disease in the regional lymph nodes, a darkened box T
indicates a stage 3 or 4 tumor which is more clinically advanced,
and a darkened box for grade indicates a high grade (at least grade
III) tumor, which is associated with increased malignancy. The
correlation of ER/p53 status can be performed by comparing the top
four lines of clinical indicator boxes (Age, N, T, Grade) with the
middle two lines of boxes (ER/p53 status). The results of this
cross correlation are shown in the bar graph of FIG. 11A, where it
can be seen that ER-/p53+ (Group I) tumors tend to be of higher
grade than the other tumors, and had a particularly high frequency
of myc amplification, while ER+/p53+ (Group III) tumors were more
likely to have positive nodes at the time of surgical resection.
The ER-/p53- (Group II) showed that the most common gene amplified
in that group was erbB2. ER-/p53- (Group II) and ER+/p53- (Group
IV) tumors, in contrast, were shown to have fewer indicators of
severe disease, thus suggesting a correlation between the absence
of the p53 mutation and a better prognosis.
[0072] This method was also used to analyze the copy numbers of
several other major breast cancer oncogenes in the 372 arrayed
primary breast cancer specimens in consecutive FISH experiments,
and those results were used to ascertain correlations between the
ER/p53 classifications and the expression of these other oncogenes.
These results were obtained by using probes for each of the
separate oncogenes, in successive sections of the recipient block,
and comparing the results at corresponding coordinates of the
array. In FIG. 11B, a positive result for the amplification of the
specific oncogene or marker (mybL2, 20q13, 17q23, myc, cnd1 and
erbB2) is indicated by a darkened box. The erbB2 oncogene was
amplified in 18% of the 372 arrayed specimens, myc in 25% and
cyclin D1 (cnd1) in 24% of the tumors.
[0073] The two recently discovered novel regions of frequent DNA
amplification in breast cancer, 17q23 and 20q13, were found to be
amplified in 13% and 6% of the tumors, respectively. The oncogene
mybL2 (which was recently localized to 20q13.1 and found to be
overexpressed in breast cancer cell lines) was found to be
amplified in 7% of the same set of tumors. MybL2 was amplified in
tumors with normal copy number of the main 20q13 locus, indicating
that it may define an independently selected region of
amplification at 20q. Dotted lines between FIGS. 11B and 11C again
divide the complex co-amplification patterns of these genes into
Groups I-IV which correspond to ER-/p53+, ER-/p53-, ER+/p53+ and
ER+/p53-.
[0074] FIGS. 11C and 11D show that 70% of the ER-/p53+ specimens
were positive for one or more of these oncogenes, and that myc was
the predominant oncogene amplified in this group. In contrast, only
43% of the specimens in the ER+/p53- group showed co-amplification
of one of these oncogenes, and this information could in turn be
correlated with the clinical parameters shown in FIG. 11A. Hence
the microarray technology of the present invention permits a large
number of tumor specimens to be conveniently and rapidly screened
for these many characteristics, and analyzed for patterns of gene
expression that may be related to the clinical presentation of the
patient and the molecular evolution of the disease. In the absence
of the microarray technology of the present invention, these
correlations are more difficult to obtain.
[0075] A specific method of obtaining these correlations is
illustrated in FIG. 12, which is an enlargement of the right hand
portion of FIG. 11B. The microarray 76 (FIG. 10A) is arranged in
sections that contain seventeen rows and nine columns of circular
locations that correspond to cross-sections of cylindrical tissue
specimens from different tumors, wherein each location in the
microarray can be represented by the coordinates (row, column). For
example, the specimens in the first row of the first section have
coordinate positions (1,1), (1,2) . . . (1,9), and the specimens in
the second row have coordinate positions (2,1), (2,2), . . . ,
(2,9). Each of these array coordinates can be used to locate tissue
specimens from corresponding positions on sequential sections of
the recipient block, to identify tissue specimens of the array that
were cut from the same tissue cylinder.
[0076] FIG. 12 illustrates one conceptual approach to organizing
and analyzing the array, in which the rectangular array may be
converted into a linear representation in which each box of the
linear representation corresponds to a coordinate position of the
array. Each of the lines of boxes may be aligned so that each box
that corresponds to an identical array coordinate position is
located above other boxes from the same coordinate position. Hence
the boxes connected by dotted line 1 correspond to the results that
can be obtained by looking at the results at a coordinate position
[for example (1,1)] in successive thin sections of the donor block,
or clinical data that may not have been obtained from the
microarray, but which can be entered into the system to further
identify tissue from a tumor that corresponds to that coordinate
position. Similarly, the boxes connected by dotted line 10
correspond to the results that can be found at coordinate position
(2,1) of the array, and the boxes connected by dotted line 15
correspond to the results at coordinate position (2,6) of the
array. The letters a, b, c, d, e, f, g, and h correspond to
successive sections of the donor block that are cut to form the
array.
[0077] By comparing the aligned boxes along line 1 in FIG. 12, it
can be seen that a tumor was obtained from a postmenopausal woman
with no metastatic disease in her lymph nodes at the time of
surgical resection, in which the tumor was less than stage 3, but
in which the histology of the tumor was at least Grade III. A
tissue block was taken from this tumor and is associated with the
recipient array at coordinate position (1,1). This array position
was sectioned into eight parallel sections (a, b, c, d, e, f, g,
and h) each of which contained a representative section of the
cylindrical array. Each of these sections was analyzed with a
different probe specific for a particular molecular attribute. In
section a, the results indicated that this tissue specimen was
p53+; in section b that it was ER-; in section c that it did not
show amplification of the mybL2 oncogene; in separate sections d,
e, f, g and h that it was positive for the amplification of 20q13,
17q23, myc, cnd1 and erbB2.
[0078] Similar comparisons of molecular characteristics of the
tumor specimen cylinder that was placed at coordinate position
(2,1) can be made by following vertical line 10 in FIG. 12, which
connects the tenth box in each line, and corresponds to the second
row, first column (2,1) of the array 76 in FIG. 10(A). Similarly
the characteristics of the sections of the tumor specimen cylinder
at coordinate position (2,6) can be analyzed by following vertical
line 15 down through the 15.sup.th box of each row. In this manner,
parallel information about the separate sections of the array can
be performed for all 372 positions of the array. This information
can be presented visually for analysis as in FIG. 12, or entered
into a database for analysis and correlation of different molecular
characteristics (such as patterns of oncogene amplification, and
the correspondence of those patterns of amplification to clinical
presentation of the tumor).
[0079] Analysis of consecutive sections from the tumor arrays
enables co-localization of hundreds of different DNA, RNA, protein
or other targets in the same cell populations in morphologically
defined regions of every tumor, which facilitates construction of a
database of a large number of correlated genotypic or phenotypic
characteristics of uncultured human tumors. Scoring of mRNA in situ
hybridizations or protein immunohistochemical staining is also
facilitated with tumor tissue microarrays, because hundreds of
specimens can be analyzed in a single experiment. The tumor arrays
also substantially reduce tissue consumption, reagent use, and
workload when compared with processing individual conventional
specimens one at a time for sectioning, staining and scoring. The
combined analysis of several DNA, RNA and protein targets provides
a powerful means for stratification of tumor specimens by virtue of
their molecular characteristics. Such patterns will be helpful to
detect previously unappreciated but important molecular features of
the tumors that may turn out to have diagnostic or prognostic
utility.
[0080] Analysis techniques for observing and scoring the
experiments performed on tissue array sections include a
bright-field microscope, fluorescent microscope, confocal
microscope, a digital imaging system based on a CCD camera, or a
photomultiplier or a scanner, such as those uses in the DNA chip
based analyses.
[0081] These results show that the very small cylinders used to
prepare tissue arrays can in most cases provide accurate
information, especially when the site for tissue sampling from the
donor block is selected to contain histological structures that are
most representative of tumor regions. It is also possible to
collect samples from multiple histologically defined regions in a
single donor tissue block to obtain a more comprehensive
representation of the original tissue, and to directly analyze the
correlation between phenotype (tissue morphology) and genotype. For
example, an array could be constructed to include hundreds of
tissues representing different stages of breast cancer progression
(e.g. normal tissue, hyperplasia, atypical hyperplasia, intraductal
cancer, invasive and metastatic cancer). The tissue array
technology would then be used to analyze the molecular events that
correspond to tumor progression.
[0082] A tighter packing of cylinders, and a larger recipient block
can also provide an even higher number of specimens per array.
Entire archives from pathology laboratories can be placed in
replicate 500-1000 specimen tissue microarrays for molecular
profiling. Using automation of the procedure for sampling and
arraying, it is possible to make dozens of replicate tumor arrays,
each providing hundreds of sections for molecular analyses. The
same strategy and instrumentation developed for tumor arrays also
enables the use of tissue cylinders for isolation of high-molecular
weight RNA and DNA from optimally fixed, morphologically defined
tumor tissue elements, thereby allowing correlated analysis of the
same tumors by molecular biological techniques (such as PCR-based
techniques) based on RNA and DNA. When nucleic acid analysis is
planned, the tissue specimen is preferably fixed (before embedding
in paraffin) in an alcohol based fixative, such as ethanol or
Molecular Biology Fixative (Streck Laboratories, Inc., Omaha,
Nebr.) instead of in formalin, because formalin can cross-link and
otherwise damage nucleic acid. The tissue cylinder of the present
invention provides an ample amount of DNA or RNA on which to
perform a variety of molecular analyses.
[0083] The potential of this array technology has been illustrated
in FISH analysis of gene amplifications in breast cancer. FISH is
an excellent method for visualization and accurate detection of
genetic rearrangements (amplifications, deletions or
translocations) in individual, morphologically defined cells. The
combined tumor array technology allows FISH to become a powerful,
high-throughput method that permits the analysis of hundreds of
specimens per day.
[0084] Automated high speed devices can also be used that
incorporate the basic principles of the device described herein.
Such devices can process multiple donor and recipient trays or
containers, and are described in Provisional Application Ser. No.
60/106,038 and PCT Application US99/04000, which are incorporated
herein by reference. The devices are controlled by standard
operating environments including a computer that comprises at least
one high speed processing unit (CPU), in conjunction with a memory
system, an input device, and one or more output devices. These
elements are interconnected by at least one bus structure. The CPU
is of familiar design and includes an ALU for performing
computations, a collection of registers for temporary storage of
data and instructions, and a control unit for controlling operation
of the system. The CPU may be a processor having any of a variety
of architectures including Alpha from Digital; MIPS from MIPS
Technology, NEC, IDT, Siemens and others; x86 from Intel and
others, including Cyrix, AMD, and Nexgen; 680x0 from Motorola; and
PowerPC from IBM and Motorola. For example, the invention could be
implemented with a Power Macintosh 8500 available from Apple
Computer, or an IBM compatible Personal Computer (PC).
[0085] Examples and Applications of Array Technologies
[0086] The automated tumor array technology easily allows testing
of dozens or hundreds of markers from the same set of tumors. These
studies can be carried out in a multi-center setting by sending
replicate tumor array blocks or sections to other laboratories. The
same approach would be particularly valuable for testing newly
discovered molecular markers for their diagnostic, prognostic, or
therapeutic utility. The tissue array technology also facilitates
basic cancer research by providing a platform for rapid profiling
of hundreds or thousands of tumors at the DNA, RNA, and protein
levels, leading to a construction of a correlated database of
biomarkers from a large collection of tumors. For example, search
for amplification target genes requires correlated analyses of
amplification and expression of dozens of candidate genes and loci
in the same cell populations. Such extensive molecular analyses of
a defined large series of tumors would be difficult to carry out
with conventional technologies.
[0087] Applications of the tissue array technology are not limited
to studies of cancer, although the following Examples 1-4 disclose
embodiments of its use in connection with analysis of neoplasms.
Array analysis could also be instrumental in understanding
expression and dosage of multiple genes in other diseases, as well
as in normal human or animal tissues, including tissues from
different transgenic animals or cultured cells.
[0088] Tissue arrays can also be used to perform further analysis
on genes and targets discovered from, for example, high-throughput
genomics, such as DNA sequencing, DNA microarrays, or SAGE (Serial
Analysis of Gene Expression) (Velculescu et al., Science,
270:484-487, 1995). Tissue arrays can also be used to evaluate
reagents for cancer diagnostics, for instance specific antibodies
or probes that react with certain tissues at different stages of
cancer development, and to follow progression of genetic changes
both in the same and in different cancer types, or in diseases
other than cancer. Tissue arrays can be used to identify and
analyze prognostic markers or markers that predict therapy outcome
for cancers. Tissue arrays compiled from hundreds of cancers
derived from patients with known outcomes permit one or more of
DNA, RNA, and protein assays to be performed on those arrays, to
determine important prognostic markers, or markers predicting
therapy outcome.
[0089] Tissue arrays can also be used to help assess optimal
therapy for particular patients showing particular tumor marker
profiles. For example, an array of tumors can be analyzed to
determine which ones amplify and/or overexpress HER-2, such that
the tumor type (or more specifically the subject from whom the
tumor was taken) would be a good candidate for anti-HER-2 Herceptin
immunotherapy. In another application, tissue arrays can be used to
find novel targets for gene therapy. For example, cDNA
hybridization patterns (such as on a DNA chip) may reveal
differential gene regulation in a tumor of a particular tissue type
(such as lung cancer), or a particular histological sub-type of the
particular tumor (such as adenocarcinoma of the lung). Analysis of
each of such gene candidates on a large tissue array containing
hundreds of tumors would help determine which is the most promising
target for developing diagnostic, prognostic, or therapeutic
approaches for cancer.
EXAMPLES
[0090] The invention is further described in the following
examples, which do not limit the scope of the invention described
in the claims.
Example 1
Tissue Specimens
[0091] A total of 645 breast cancer specimens were used for
construction of a breast cancer tumor tissue microarray. The
samples included 372 fresh-frozen ethanol-fixed tumors, as well as
273 formalin-fixed breast cancers, normal tissues and fixation
controls. The subset of frozen breast cancer samples was selected
at random from the tumor bank of the institute of Pathology,
University of Basel, which includes more than 1500 frozen breast
cancers obtained by surgical resections during 1986-1997. Only the
tumors from this tumor bank were used for molecular analyses. This
subset was reviewed by a pathologist, who determined that the
specimens included 259 ductal, 52 lobular, 9 medullary, 6 mucinous,
3 cribriform, 3 tubular, 2 papillary, 1 histiocytic, 1 clear cell,
and 1 lipid rich carcinoma. There were also 15 ductal carcinomas in
situ, 2 carcinosarcomas, 4 primary carcinomas that had received
chemotherapy before surgery, 8 recurrent tumors and 6
metastases.
[0092] Histological grading was performed only in invasive primary
tumors that had not undergone previous chemotherapy. Of these
tumors, 24% were grade 1, 40% grade 2, and 36% grade 3. The pT
stage was pT1 in 29%, pT2 in 54%, pT3 in 9%, and pT4 in 8%.
Axillary lymph nodes had been examined in 282 patients (45% pN0,
46% pN1, 9% pN2). All previously unfixed tumors were fixed in cold
ethanol at +4.degree. C. overnight and then embedded in
paraffin.
Example 2
Immunohistochemistry
[0093] After formation of the tissue array and sectioning of the
donor block, standard indirect immunoperoxidase procedures were
used for immunohistochemistry (ABC-Elite, Vector Laboratories).
Monoclonal antibodies from DAKO (Glostrup, Denmark) were used for
detection of p53 (DO-7, mouse, 1:200), erbB-2 (c-erbB-2, rabbit,
1:4000), and estrogen receptor (ER ID5, mouse, 1:400). A microwave
pretreatment was performed for p53 (30 minutes at 90.degree. C.)
and erbB-2 antigen (60 minutes at 90.degree. C.) retrieval.
Diaminobenzidine was used as a chromogen. Tumors with known
positivity were used as positive controls. The primary antibody was
omitted for negative controls. Tumors were considered positive for
ER or p53 if an unequivocal nuclear positivity was seen in at least
10% of tumor cells. The erbB-2 staining was subjectively graded
into 3 groups: negative (no staining), weakly positive (weak
membranous positivity), strongly positive (strong membranous
positivity)
Example 3
Fluorescent In Situ Hybridization (FISH)
[0094] Two-color FISH hybridizations were performed using
Spectrum-Orange labeled cyclin D1, myc, or erbB2 probes together
with corresponding FITC labeled centromeric reference probes
(Vysis). One-color FISH hybridizations were done with spectrum
orange-labeled 20q13 minimal common region (Vysis, and see Tanner
et al., Cancer Res. 54:4257-4260 (1994)), mybL2 and 17q23 probes
(Barlund et al., Genes Chrom. Cancer 20:372-376 (1997)). Before
hybridization, tumor array sections were deparaffinized, air dried
and dehydrated in 70, 85, and 100% ethanol followed by denaturation
for 5 minutes at 74.degree. C. in 70% formamide-2.times.SSC
solution. The hybridization mixture contained 30 ng of each of the
probes and 15 .mu.g of human Cot1-DNA. After overnight
hybridization at 37.degree. C. in a humidified chamber, slides were
washed and counterstained with 0.2 .mu.M DAPI in an antifade
solution. FISH signals were scored with a Zeiss fluorescence
microscope equipped with double-band pass filters for simultaneous
visualization of FITC and Spectrum Orange signals. Over 10 FISH
signals per cell or tight clusters of signals were considered as
indicative of gene amplification.
Example 4
mRNA In Situ Hybridization
[0095] For mRNA in situ hybridization, tumor array sections were
deparaffinized and air dried before hybridization. Synthetic
oligonucleotide probes directed against erbB2 mRNA (Genbank
accession number X03363, nucleotides 350-396) was labeled at the
3'-end with .sup.33P-DATP using terminal deoxynucleotidyl
transferase. Sections were hybridized in a humidified chamber at
42.degree. C. for 18 hours with 1.times.10.sup.7 CPM/ml of the
probe in 100 .mu.L of hybridization mixture (50% formamide, 10%
dextran sulfate, 1% sarkosyl, 0.02 M sodium phosphate, pH 7.0,
4.times.SSC, 1.times. Denhardt's solution and 10 mg/ml ssDNA).
After hybridization, sections were washed several times in
1.times.SSC at 55.degree. C. to remove unbound probe, and briefly
dehydrated. Sections were exposed for three days to phosphorimager
screens to visualize ERBB2 mRNA expression. Negative control
sections were treated with RNase prior to hybridization, which
abolished all hybridization signals.
[0096] The present method enables high throughput analysis of
hundreds of specimens per array. This technology therefore provides
an order of magnitude increase in the number of specimens that can
be analyzed, as compared to prior blocks where a few dozen
individual formalin-fixed specimens are in a less defined or
undefined configuration, and used for antibody testing. Further
advantages of the present invention include negligible destruction
of the original tissue blocks, and an optimized fixation protocol
which expands the utility of this technique to visualization of DNA
and RNA targets. The present method also permits improved
procurement and distribution of human tumor tissues for research
purposes. Entire archives of tens of thousands of existing
formalin-fixed tissues from pathology laboratories can be placed in
a few dozen high-density tissue microarrays to survey many kinds of
tumor types, as well as different stages of tumor progression. The
tumor array strategy also allows testing of dozens or even hundreds
of potential prognostic or diagnostic molecular markers from the
same set of tumors. Alternatively, the cylindrical tissue samples
provide specimens that can be used to isolate DNA and RNA for
molecular analysis.
Example 5
Tissue Microarrays for Gene Amplification Surveys in Many Different
Tumor Types
[0097] To facilitate rapid screening for molecular alterations in
many different malignancies, a tissue microarray consisting of
samples from 17 different tumor types, from 397 individual tumors,
were arrayed in a single paraffin-block. Amplification of three
oncogenes (CCND1, MYC, ERBB2) was analyzed in three Fluorescence in
situ Hybridization (FISH) experiments from consecutive sections cut
from the tissue microarray. Amplification of CCND1 was found in
breast, lung, head and neck, and bladder cancer as well as in
melanoma. ERBB2 was amplified in bladder, breast, colon, stomach,
testis, and lung cancers. MYC was amplified in breast, colon,
kidney, lung, ovary, bladder, head and neck, and endometrial
cancer.
[0098] The microarray was constructed from a total of 417 tissue
samples consisting of 397 primary tumors from 17 different tumor
types and 20 normal tissues which were snap-frozen and stored at
-70.degree. C. Specimens were fixed in cold ethanol (+4.degree. C.)
for 16 hours and then embedded in paraffin. An H&E-stained
section was made from each block to define representative tumor
regions. Tissue cylinders with a diameter of 0.6 mm were then
punched from tumor areas of each "donor" tissue block and brought
into a recipient paraffin block using a custom-made precision
instrument as described. Then 5 .mu.m sections of the resulting
multi-tumor tissue microarray block were transferred to glass
slides using the paraffin sectioning aid system (adhesive coated
slides, (PSA-CS4x), adhesive tape, UV-lamp; Instrumedics Inc., New
Jersey) supporting the cohesion of 0.6 mm array elements.
[0099] The primary tumors consisted of 96 breast tumors (41 ductal,
28 lobular, 6 medullar, 5 mucinous, and 4 tubular carcinomas, 7
ductal carcinomas in situ (DCIS) and 5 phylloides tumors), 80
carcinomas of the lung (31 squamous, 11 large cell, 2 small cell,
31 adeno, and 5 bronchioloalveolar carcinomas), 17 head and neck
tumors (12 squamous cell carcinomas of the oral cavity and 5 of the
larynx), 32 adenocarcinomas of the colon, 4 carcinoids (3 from the
lung and one from the small intestine), 12 adenocarcinomas from the
stomach, 28 clear cell renal cell carcinomas, 20 testicular tumors
(10 seminomas and 10 terato-carcinomas), 37 transitional cell
carcinomas of the urinary bladder (33 invasive (pT1-4) and 4
non-invasive tumors), 22 prostate cancers, 26 carcinomas of the
ovary (12 serous, 12 endometroid, and 2 mucinous tumors), 13
carcinomas from the endometrium, 3 carcinomas of the thyroid gland,
3 pheochromocytomas, and 4 melanomas. Normal tissue from breast,
prostate, pancreas, small bowel, stomach, salivary gland, colon,
and kidney were used as controls.
[0100] The tissue microarray sections were treated according to the
Paraffin Pretreatment Reagent Kit protocol (Vysis, Illinois) before
hybridization. FISH was performed with Spectrum Orange-labeled
CCND1, ERBB2, and MYC probes. Spectrum Green-labeled centromeric
probes CEP11 and CEP17 were used as a reference (Vysis, Illinois).
Hybridization and post-hybridization washes were according to the
"LSI procedure" (Vysis, Illinois). Slides were then counterstained
with 125 ng/ml 4',6-diamino-2-phenylindole in antifade solution.
FISH signals were scored with a Zeiss fluorescence microscope
equipped with double-band pass filters for simultaneous
visualization of Spectrum Green and Spectrum Orange signals (Vysis,
Illinois). Amplification was defined as presence (in at least 5% of
tumor cells) of either (a) more than 10 gene signals or tight
clusters of at least 5 gene signals; or (b) more than 3 times as
many gene signals than centromere signals of the respective
chromosome.
[0101] Seventy-two amplifications were found in 968 successfully
hybridized tumor samples, whereas none of the normal tissues showed
amplification. Amplification usually involved almost all tumor
cells within an array element. CCND1 amplification was found in 6
of 16 head and neck carcinomas (38%), 14 of 62 breast carcinomas
(23%), 1 of 6 DCIS (17%), 3 of 27 bladder cancers (11%), 7 of 76
carcinomas of the lung (9%), and 1 of 4 melanomas.
[0102] MYC amplification was observed in 2 of 11 endometrial
cancers (18%), 9 of 74 breast carcinomas (12%), 1 of 5 DCIS (20%),
1 of 17 head and neck cancers (6%), 1 of 22 tumors of the kidney
(5%), 2 of 24 ovarian carcinomas (8%), 1 of 17 tumors of the testis
(6%), 1 of 30 colon carcinomas (3%), 7 of 78 lung tumors (9%), and
in 1 of 33 bladder-tumors (3%).
[0103] ERBB2 was amplified in 4 of 71 breast carcinomas (6%), 4 of
6 DCIS (67%), 2 of 11 stomach cancers (18%), 1 of 30 colon
carcinomas (3%), 1 of 17 tumors of the testis (6%), and in 1 of 75
carcinomas of the lung (1%). Co-amplifications of all three genes
were seen in two breast carcinomas. Co-amplifications of two genes
were found in two breast carcinomas (CCND1/MYC and CCND1/ERBB2) and
in one terato-carcinoma of the testis (MYC and ERBB2).
[0104] Consecutive sections cut from the block provide starting
material for the in situ detection of multiple DNA, RNA or protein
targets in many tissues at a time, in a massively parallel fashion.
The tissue array technology permits increased capacity, automation,
negligible damage to the original tissue blocks from which the
specimens are taken, the precise positioning of tissue specimens,
and the use of these tissues in different kinds of molecular
analyses, besides immunostaining. It is possible to retrieve 10-20
punched samples (or more) from each donor block without
significantly damaging it. This enables generation of multiple
replicate array-blocks, each with the identical coordinates, and
the same specimens. The application of a precision instrument to
deposit the samples in a predefined format also facilitates the
development of automated image analysis strategies for the arrayed
tumors. Depending on the thickness of the original tissue blocks,
between 150 and 300 sections can be cut from each array block. This
technology enables analyses of even small primary tumors, thereby
preserving often unique and precious tumor specimens for a large
number of analyses that may be of interest in future
investigations.
[0105] The array data reported in this example agreed with the
previous literature on the presence or absence of gene
amplification in 73% of evaluations, although the number of samples
per tumor type was too small for a comprehensive analysis of some
tumor types in this pilot study. Previously described
amplifications were not detected on the array in 9 of 25 tumor
types from which less than 25 samples were examined. In contrast,
when at least 25 cases were analyzed per tumor type, 92% of the
known amplifications (11/12) were detected.
[0106] In this study, frozen tumor tissues were fixed in cold
ethanol because this procedure allows the retention of good quality
nucleic acids from fixed tissue samples. Even formalin-fixed tumor
tissues, such as those obtained at autopsy, can be analyzed by FISH
for DNA copy number alterations. However, the cold ethanol fixation
is advantageous for FISH, because the samples require fewer
pretreatments than samples fixed in 4% buffered formalin. Cold
ethanol fixation may cause RNAs to degrade in paraffin blocks after
only a few months of storage, hence it may not be desired to fix a
large series of precious tissues in cold ethanol, unless RNA
inhibitors are added or blocks stored in a manner that prohibits
this degradation.
Example 6
PDGFB FISH Experiments Using A Multi-Tumor Tissue Array
[0107] The multi-tumor tissue array of Example 5 was used in this
experiment. A platelet derived growth factor beta (PDGFB) probe was
obtained from Vysis Inc. of Downers Grove, Ill. The probe was
obtained by PCR screening of a genomic large-insert library using
two sequence tagged sites (STS) in the gene sequence as a target
for developing PCR primers that were used in the PCR-based library
screening. The hits obtained from genomic library screening were
further verified by their content of the STSs, as well as by
hybridizing the probe to metaphase chromosomes using FISH. This
resulted in a signal at the expected chromosomal location of
PDGFB.
[0108] PCR/STS screening can be performed using a PCR primer set
specific to the gene of interest, as described by Green &
Olson, PNAS USA, 87:1213-1217, 1990. Probes for FISH may be
generated from large-insert libraries (e.g., cosmids, P1 clones,
BACs, and PACs) using a PCR-based screening of arrayed and pooled
large-insert libraries. Both Research Genetics (Huntsville, Ala.)
and Genome Systems (St. Louis) perform such filter screening, and
sell pools of DNA for performing library screening.
[0109] One method of isolating the P1 clone for PDGFB (pVYS309A)
would be to screen DNA pools of a human P1 library obtained from
Genome Systems, Inc. Individual clones are identified by producing
the expected DNA fragment size on gels after PCR. Bacterial
cultures containing candidate PDGFB clones are purified by
streaking on nutrient agar media for single colonies. Cultures from
individual colonies are then grown and DNA isolated by standard
techniques. The DNA is confirmed to contain the desired DNA
sequence by PCR and gel electrophoresis (STS confirmation). A
sample of the DNA is labeled by nick-translation or random priming
with SpectrumOrange dUTP (Vysis) and shown to hybridize to the
expected region of chromosome 22q normal metaphase chromosomes by
FISH.
[0110] PCR primers for PDGFB can be derived from the published
sequence of the cDNA of this gene (GenBank Accession X02811). The
preferred region of STS design is the 3' untranslated region of the
cDNA. Several PCR primer sets for PDGFB are in public databases,
e.g., amplimers (PCR primer sets) PDGFB PCR1, PDGFB PCR2, PDGFB
PCR3, stPDGFB.b, WI-8985, and can be found in the Genome Database
(http://gdbwww.gdb.org/gdb/gdbtop.html). WI-8985 primer sets can
also be found at the Whitehead Institute database
(http://www-genome.wi.mit.edu/), and at the NIH Gene Map 98
database (http://www.ncbi.nlm.nih.gov/genemap98/).
[0111] FISH was done using standard protocols, as in Example 5, and
hybridization of the probe to specimens of the tissue array was
detected as in Example 5. Hybridization was detected in the
following types of tumors:
1 Ratio Percent TUMOR Positive Positive breast CA 2/70 2.9%
phylloides 0/4 DCIS 0/7 lung 15/77 19% colon 1/30 3.3% carcinoid
0/3 stomach 0/9 renal cell 0/11 testis 1/16 6% TCC 10/32 31%
(bladder transitional cell carcinoma) head/neck 0/17 PCA 0/18 ovary
0/22 endometrium 2/8 25% Total 22/324
[0112] This Example provides the first evidence of previously
unsuspected, high-level amplifications of PDGFB in specific types
of malignancies, such as breast, lung, colon, testicular,
endometrial, and bladder cancer.
Example 7
Gene Amplifications During Prostate Cancer Progression
[0113] In this study, five different gene amplifications (AR, CMYC,
ERBB2, Cyclin D1, and NMYC) were assayed by FISH from consecutive
formalin fixed tissue microarray sections containing samples from
more than 300 different prostate tumors. The objective was to
obtain a comprehensive survey of gene amplifications in different
stages of prostate cancer progression, including specimens from
distant metastases. The tissue microarray contained minute samples
from 371 specimens.
[0114] Formalin-fixed and paraffin-embedded tumor and control
specimens were obtained from the archives of the Institutes for
Pathology, University of Basel (Switzerland) and the Tampere
University Hospital (Finland). The least differentiated tumor area
was selected to be sampled for the tissue microarray. The minute
specimens that were interpretable for at least one FISH probe
included: I) transurethral resections from 32 patients with benign
prostatic hyperplasia (BPH) which were used as controls; II) 223
primary tumors, including 64 cancers incidentally detected in
transurethral resections for BPH; stage T1a/b, 145 clinically
localized cancers from radical prostatectomies, and 14
transurethral resections from patients with primary, locally
extensive disease; III) 54 local recurrences after hormonal therapy
failure including 31 transurethral resections from living patients
and 23 specimens obtained from autopsies; IV) Sixty-two metastases
collected at the autopsies from 47 patients who had undergone
androgen deprivation by orchiectomy, and had subsequently died of
end-stage metastatic prostate cancer. Metastatic tissue was sampled
from pelvic lymph nodes (8), lung (21), liver (16), pleura (5),
adrenal gland (5), kidney (2), mediastinal lymph nodes (1),
peritoneum (1), stomach (1), and ureter (1). In 23 autopsies
material was available from both the primary and from the
metastatic site. More than one sample per tumor specimen was
arrayed in 44 of the 339 cases. A tumor was considered amplified if
at least one sample from the tumor exhibited gene
amplification.
[0115] The array also included 48 pathologically representative
samples which consistently failed in the analysis of sections with
all FISH probes, and were therefore excluded from the analyses.
Most of these were autopsy samples. The number of samples evaluated
with the different probes was variable, because the hybridization
efficiency of the probes was slightly different, some samples on
the array were occasionally lost during the sectioning or
FISH-procedure, and some tumors were only representative on the
surface of the block, and the morphology changed as more sections
were cut.
[0116] The prostate tissue microarray was constructed as previously
described in Example 1, except with prostate instead of breast
cancer specimens.
[0117] Two-color FISH to sections of the arrayed formalin-fixed
samples was performed using Spectrum Orange-labeled AR, CMYC,
ERBB2, and CyclinD1 (CCND1) probes with corresponding FITC-labeled
centromeric probes (Vysis, Downer's Grove, Ill.). In addition,
one-color FISH was done with a Spectrum Orange-labeled NMYC probe
(Vysis). The hybridization was performed according to the
manufacturer's instructions. To allow formalin-fixed tumors on the
array to be reliably analyzed by FISH, the slides of the prostate
microarray were first deparaffinized, acetylated in 0.2 N HCl,
incubated in 1 M sodium thiocyanate solution at 80.degree. C. for
30 minutes and immersed in a protease solution (0.5 mg/ml in 0.9%
NaCl) (Vysis) for 10 minutes at 37.degree. C. The slides were then
post-fixed in 10% buffered formalin for 10 minutes, air dried,
denatured for 5 minutes at 73.degree. C. in 70%
formamide/2.times.SSC(SSC is 0.3 M sodium chloride and 0.03 M
sodium citrate) solution and dehydrated in 70, 80, and 100%
ethanol, followed by proteinase K (4 .mu.g/ml phosphate buffered
saline)(GIBCOBRL, Life Technologies Inc., Rockville, Md.) treatment
for 7 minutes at 37.degree. C. The slides were then dehydrated and
hybridized.
[0118] The hybridization mixture contained 3 .mu.l of each of the
probes and Cot1-DNA (1 mg/ml; GIBCOBRL, LifeTechnologies Inc.,
Rockville, Md.) in a hybridization mixture. After overnight
hybridization at 37.degree. C. in a humid chamber, slides were
washed, and counterstained with 0.2 .mu.M DAPI. FISH signals were
scored with a Zeiss fluorescence microscope equipped with a
double-band pass filter using .times.40-.times.100 objectives. The
relative number of gene signals in relation to the centromeric
signals was evaluated. Criteria for gene amplification were: at
least 3 times more test probe signals than centromeric signals per
cell in at least 10% of the tumor cells. Test/control signal ratios
in the range between 1 and 3 were regarded as low level gaits, and
were not scored as evidence of specific gene amplification.
Amplification of NMYC without a reference probe was defined as at
least 5 gene signals in at least 10% of the tumor cells.
[0119] High-quality hybridization signals with both centromeric and
gene specific probes were obtained in 96% of the BPH samples for
chromosome. X/AR gene, 84% for chromosome 8/CMYC, 81% for
chromosome 17/ERBB2, and 83% for chromosome 11/Cyclin D1. In the
BPH samples that could be evaluated, the average percentage of
epithelial cells with two signals for autosomal probes was
.about.75%, with .about.20% showing one signal, and .about.5% no
signals. The percentage of cells with one or zero signals is
believed to be attributable to the truncation of nuclei with
sectioning. In the punched (single array element) samples of biopsy
cancer specimens, AR, CMYC, ERBB2, and CCND1 FISH data could be
obtained from 92%, 78%, 82%, and 86% of the cases, respectively.
The success rate of FISH was lower in punches from autopsy tumors
(44-58%). Amplifications were only scored when the copy number of
the test probe exceeded that of the chromosome-specific centromere
reference probe by .gtoreq.3-fold in 10% or more of the tumor
cells. This criterion was chosen, as low-level amplification is
likely to be less relevant, and since locus-specific probes often
display slightly higher copy numbers than centromeric probes, due
to signal splitting or the presence of G2/M-phase cells.
[0120] FISH with the AR probe revealed amplification in 23.4% of
the 47 evaluable hormone-refractory local recurrences.
Amplification was seen equally often (22.0%) in 59 metastases of
hormone-refractory tumors. The strong association between AR
amplification and hormone-refractory prostate cancer is evident
from the fact that only two of the 205 evaluable primary tumors
(1%) and none of the 32 BPH controls showed any AR amplification.
The two exceptions included a patient with locally advanced and
metastatic prostate cancer, and another patient with clinically
localized disease. Paired tumors from the primary site of the
cancer and from a distant metastasis of 17 patients were
successfully analyzed for AR amplification. In 11 of these
patients, no AR amplification could be seen at either site. Of the
six remaining patients, three patients showed amplification both in
the local tumor mass, as well as in the distant metastases. In two
cases amplification was only found in the sample from the primary
site, whereas in another case only the distant metastasis showed
amplification.
[0121] High-level CMYC amplifications were found in 5 of 47
evaluable metastatic deposits (10.6%), in 2 of the 47 local
recurrences (4.3%, both metastatic cancers), but in none of the 168
evaluable primary cancers or 31 BPH controls. The comparison
between different gene amplifications within the tumor cells
defined by single punch-samples (array elements) showed that there
was a significant association between AR and CMYC amplifications.
CMYC was amplified In 11.1% of 27 evaluable punch-samples with AR
amplifications but only in 1.7% of 235 samples without AR
amplifications (p=0.0041, contingency table analysis). AR was
independently amplified in 24 samples, whereas only four samples
had CMYC amplification, but no AR amplification.
[0122] On a tumor by tumor basis, there was a significant
association between AR and CMYC amplifications. CMYC was amplified
in 12.5% of 24 evaluable tumors with AR amplifications, but only in
1.8% of 219 tumors without AR amplifications (p=0.003, contingency
table analysis). AR was independently amplified in 21 tumors,
whereas only 4 tumors had CMYC amplification, but no AR
amplification.
[0123] CCND1 amplifications were found in 2 (1.2%) of the 172
evaluable primary tumors, in 3 (7.9%) of 38 local recurrences, and
in 2 (4.7%) of the 43 metastases. CCND1 amplification appeared
independent from AR or CMYC amplification with 4/7 CCND1 amplified
punched tumor samples not showing amplifications for any other
genes tested. There were no ERBB2 amplifications among any of the
262 evaluable tumors or 31 BPH controls. Finally, a subset of the
tumors was analyzed with the NMYC probe in a single color FISH
analysis. Out of the 164 tumors available, none showed
amplification, as defined by the lack of 5 or more signals per cell
in >10% of the tumor cells.
[0124] For this study a tumor array was constructed that allowed
investigation of the pattern of amplifications of multiple genes in
samples representing the entire spectrum of prostate cancer
progression, including distant metastases. The tumor array strategy
facilitates standardized analysis of multiple genes in the same
tumors, even in the same specific tumor sites using the same
technology, with the same kind of probes, and similar
interpretation criteria. In just five FISH experiments, 371
specimens were screened for five genes resulting in a total of over
1400 evaluable FISH results. The ability to achieve reliable
detection of gene amplifications from formalin-fixed tissues
substantially extends the range of possible applications for the
tumor array technology.
[0125] Many symptomatic prostate cancers become both
hormone-refractory and metastatic, and it is difficult to
distinguish between these two clinical features, or the molecular
mechanisms that contribute to either of these processes. The
results of the present example indicate that AR amplification is
more closely associated with the development of hormone-refractory
cell growth, whereas CMYC amplification is associated with
metastatic progression. The most common gene amplification in
prostate cancers is that of the AR gene, which is usually amplified
independently of both CMYC and Cyclin D1. In this study, CMYC
amplifications were more common in the distant metastases (11%)
than in the locally recurrent tissues (4%; both from patients with
end-stage metastatic cancers), whereas AR amplifications were
equally common at both anatomical sites (22% and 23%,
respectively). This suggests that AR is conferring an advantage for
hormone-refractory growth, and not metastatic dissemination,
whereas the reverse may be true for CMYC.
[0126] This Example indicates that the AR gene is the most frequent
target, and often the first target, selected for amplification
during prostate cancer progression. Second, in contrast to AR,
amplifications of the CMYC oncogene appear to be primarily
associated with metastatic dissemination. Finally, prostate cancers
occasionally also amplify the Cyclin D1 gene, whereas ERBB2 and
NMYC amplifications are unlikely to play a significant role at any
stage of the progression of prostate cancer.
Example 8
Rapid Screening for Prognostic Markers in Renal Cell Carcinomas
(RCC) by Combining cDNA-Array and Tumor-Array Technologies
[0127] This example first uses cDNA arrays to identify genes that
play a role in renal cell carcinoma (RCC), and subsequently
analyzes emerging candidate genes on a tumor array for their
potential clinical significance. The results show that the
combination of nucleic acid arrays and tumor arrays is a powerful
approach to rapidly identify and further evaluate genes that play a
role in RCC biology.
[0128] cDNA was synthesized and radioactively labeled using 50
.mu.g of total RNA from normal kidney (Invitrogen) and a renal
cancer cell line (CRL-1933) (ATCC, VA, USA) according to
standardized protocols (Research Genetics; Huntsville, Ala.).
Release I of the human GeneFilters from Research Genetics was used
for differential expression screening. A single membrane contained
5184 spots each representing 5 ng of cDNA of known genes or
expressed sequence tags (EST's). After separate hybridization the
two cDNA array filters (Research Genetics) were exposed to a high
resolution screen (Packard) for three days. The gene expression
pattern of 5184 genes in normal tissue and the tumor cell line was
analyzed and compared on a phosphor imager (Cyclone, Packard). To
define genes/EST's as under- or overexpressed, both an at least
tenfold expression difference between normal tissue and the cell
line using the Pathfinder software (Research Genetics; Huntsville,
Ala.) and visual confirmation of an unequivocal difference in the
staining intensity on filters was requested.
[0129] For the construction of the renal tumor microarray block, a
collection of 615 renal tumors after nephrectomy was screened for
availability of representative paraffin-embedded tissue specimens.
Tumor specimens from 532 renal tumors and tissue from 6 normal
kidneys were selected for the tumor array. The tumors were staged
according to TNM classification, graded according to Thoenes
(Pathol. Res. Pract., 181:125-143, 1986) and histologically
subtyped according to the recommendations of the UICC (Bostwick et
al., Cancer, 80:973-1001, 1997) by one pathologist.
Core-tissue-biopsies (diameter 0.6 mm) were taken from selected
morphologically representative regions of individual
paraffin-embedded renal tumors (donor blocks) and precisely arrayed
into a new recipient paraffin block (45 mm.times.20 mm) using a
custom-built instrument. Then 5 .mu.m sections of the resulting
tumor tissue micro array block were transferred to glass slides
using the paraffin sectioning aid system (adhesive coated slides,
(PSA-CS4x), adhesive tape, UV-lamp; Instrumedics Inc., New Jersey)
supporting the cohesion of 0.6 mm array elements.
[0130] Standard indirect immunoperoxidase procedures were used for
immunohistochemistry (ABC-Elite, Vectra Laboratories) as described,
for example in Moch et al., Hum. Pathol., 28:1255-1259, 1997. A
monoclonal antibody was employed for vimentin detection
(anti-vimentin; Boehringer Mannheim, Germany, 1:160). Tumors were
considered positive for vimentin, if an unequivocal cytoplasmic
positivity was seen in tumor cells. Vimentin positivity in
endothelial cells served as an internal control. The vimentin
positivity in epithelial cells was defined as negative (no
staining) or positive (any cytoplasmic staining).
[0131] Contingency table analysis was used to analyze the
relationship between vimentin expression, grade, stage, and tumor
type. Overall survival was defined as the time between nephrectomy
and patient death. Survival rates were plotted using the
Kaplan-Meier method. Survival differences between the groups were
determined with the log-rank test. A Cox proportional hazard
analysis was used to test for independent prognostic
information.
[0132] Two cDNA array membranes were hybridized with
radioactive-labeled cDNA from normal kidney and tumor cell line
CRL-1933. The experiment resulted in 89 differentially expressed
genes/EST's. An overexpression in CRL-1933 was found for 38
sequences, including 26 named genes and 12 EST's while 51 sequences
(25 named genes, 26 EST's) were underexpressed in the cell line.
The sequence of one of the upregulated genes in the cell line was
identical to vimentin.
[0133] The presence of epithelial tumor cells was tested for every
tissue cylinder using an H&E-stained slide. Vimentin expression
could be evaluated on the tissue cylinders in 483 tumors and all 6
normal kidney tissues. Vimentin expression was frequent in
clear-cell (51%) and papillary RCC (61%), but rare in 23
chromophobe RCC (4%). Only 2 of 17 oncocytomas showed a weak
vimentin expression (12%). Normal renal tubules did not express
vimentin. The association between vimentin expression and
histological grade and tumor stage was only evaluated for clear
cell RCC. Vimentin expression was more frequent in grade II (44%)
and grade III (42%) than in grade I (13%) RCC (p<0.0001).
Vimentin expression was more common in higher tumor stages (60% in
stage pT1/2 versus 40% in stage pT3/4), but this difference was not
significant (p=0.09).
[0134] There was a mean follow-up of 52.9.+-.51.4 months (median,
37, minimum 0.1, maximum 241 months). Poor overall survival was
strongly related to high histologic grade (p<0.0001) and high
tumor stage (p<0.0001). The association between patient
prognosis and vimentin expression was evaluated for patients with
clear cell RCC. Vimentin expression was strongly associated with
short overall survival (p=0.007). Proportional Hazards analysis
with the variables tumor stage, histological grade, and vimentin
expression indicates that vimentin expression was an independent
predictor of prognosis, the relative risk being 1.6 (p=0.01) in
clear cell RCC.
[0135] The results of this example show that the combination of
cDNA and tumor arrays is a powerful approach for identification and
further evaluation of genes playing a role in human malignancies.
This example illustrates that cDNA arrays may be used to search for
genes that are differentially expressed in tumor cells (such as
kidney cancer) as compared to normal tissue (kidney tissue in this
example). Evaluation of all candidate genes emerging from a cDNA
experiment on a representative set of uncultured primary tumors
would take years if traditional methods of molecular pathology were
used. However the tumor microarray technology markedly facilitates
such studies. Tissue arrays allow the simultaneous in situ analysis
of hundreds of tumors on the DNA, RNA and protein level, and even
permits correlation with clinical follow up data.
[0136] This high throughput analysis allowed marked differences in
the vimentin expression between renal tumor subtypes to be
illustrated. Vimentin was frequently detected in papillary and
clear cell RCC, but rarely in oncocytoma and chromophobe RCC. Given
the high rate of vimentin positivity in clear cell RCC detected in
this example, the presence of vimentin expression may be used as a
diagnostic feature to distinguish a diagnosis of clear cell RCC
from chromophobe RCC.
[0137] This example further illustrates that tumor tissue arrays
can facilitate the translation of findings from basic research into
clinical applications. The speed of analysis permits a multi-step
strategy. First, molecular markers or genes of interest are
assessed on a master multi-tumor-array containing samples of many
(or all) possible human tumor type. In a second step, all tumor
types that have shown alterations in the initial experiment are
then further examined on tumor type-specific arrays (for example
bladder cancer) containing much higher numbers of tumors of the
same tissue type, with clinical follow up information on survival
or response to specific therapies. In a third step the analysis of
conventional (large) diagnostic histologic and cytologic specimens
is then restricted to those markers for which promising data
emerged during the initial array based analyses. For example,
vimentin expression can now be studied on larger tissue specimens
to confirm its prognostic significance in clear cell RCC. If the
array data are confirmed, vimentin immunohistochemistry may then be
included in prospective studies investigating prognostic markers in
RCC.
Example 9
DNA Array Technology
[0138] Instead of using a single probe to test for a specific
sequence on the sample DNA, a gene or DNA chip incorporates many
different "probes." Although a "probe" usually refers to what is
being labeled and hybridized to a target, in this situation the
probes are attached to a substrate. Many copies of a single type of
probe are bound to the chip surface in a small spot which may be,
for example, approximately 0.1 mm or less in diameter. The probe
may be of many types including DNA, RNA, cDNA, or oligonucleotide.
In variations of the technology, specific proteins, polypeptides or
immunoglobulins or other natural or synthetic molecules may be used
as a target for analyzing DNA, RNA, protein or other constituents
of cells, tissues, or other biological specimens. Many spots, each
containing a different molecular target, are then arrayed in the
shape of a grid. The surface for arraying may be a glass, or other
solid material, or a filter paper or other related substance useful
for attaching biomolecules. When interrogated with labeled sample,
the chip indicates the presence or absence of many different
sequences or molecules in that specimen. For example, a labeled
cDNA isolated from a tissue can be applied on a DNA chip to assay
for expression of many different genes at a time.
[0139] The power of these chips resides not only in the number of
different sequences or other biomolecules that can be probed
simultaneously, as explained below for nucleic acid chips. In the
analysis of nucleic acids, a relatively small amount of sample
nucleic acid is required for such an analysis (typically less than
a millionth of a gram of nucleic acid). The binding of nucleic acid
to the chip can be visualized by first labeling the sample nucleic
acid with fluorescent molecules or a radioactive label. The emitted
fluorescent light or radioactivity can be detected by very
sensitive cameras, confocal scanners, image analysis devices,
radioactive film or a PHOSPHOIMAGER.TM., which capture the signals
(such as the color image) from the chip. A computer with image
analysis software detects this image, and analyzes the intensity of
the signal for each probe location in the array. Detection of
differential gene expression with a radioactive cDNA array was
already described in Example 8. Usually, signals from a test array
are compared with a reference (such as a normal sample).
[0140] DNA chips may vary significantly in their structure,
composition, and intended functionality, but a common feature is
usually the small size of the probe array, typically on the order
of a square centimeter or less. Such an area is large enough to
contain over 2,500 individual probe spots, if each spot has a
diameter of 0.1 mm and spots are separated by 0.1 mm from each
other. A two-fold reduction in spot diameter and separation can
allow for 10,000 such spots in the same array, and an additional
halving of these dimensions would allow for 40,000 spots. Using
microfabrication technologies, such as photolithography, pioneered
by the computer industry, spot sizes of less than 0.01 mm are
feasible, potentially providing for over a quarter of a million
different probe sites.
[0141] Targets on the array may be made of oligomers or longer
fragments of DNA. Oligomers, containing between 8 and 20
nucleotides, can be synthesized readily by chemical methods.
Photolithographic techniques allow the synthesis of hundreds of
thousands of different types of oligomers to be separated into
individual spots on a single chip, in a process referred to as in
situ synthesis. Long pieces of DNA, on the other hand, contain up
to several thousand nucleotides, and can not be synthesized through
chemical methods. Instead, they are excised from the human genome
and inserted into bacterial cells through genetic engineering
techniques. These cells, or clones, serve as a convenient source
for these DNAs, which can be produced in large quantities by
fermentation. After extraction and appropriate chemical preparation
the DNA from each clone is deposited onto the chip by a robot,
which is equipped either with very fine syringes or with an ink-jet
system.
[0142] The targets on the DNA chip interact with the DNA that is
being analyzed (the target DNA) by hybridizing. The specificity of
this process (the accuracy with which the sample nucleic acid
sequences will bind to their complementary arrayed target
sequences) is mainly a function of the length of the probe. For
short oligonucleotide probes, the conditions can be chosen such
that a single point mutation (the change of a single nucleotide in
a gene) can be detected. That may require as many as 65,536 or even
more different oligonucleotide probes on a single chip to
unambiguously deduce the sequence of even a relatively small DNA
sequence. This process, called sequencing by hybridization (SbH),
generates very complex hybridization patterns that are interpreted
by image analysis computer software. In addition, the sequence to
be analyzed is preferably short, and it must be isolated and
amplified from the rest of the genome through a technique called
Polymerase Chain Reaction (PCR), before it is applied to the chip
for sequence analysis
[0143] In Comparative Genomic Hybridization (CGH), DNA from a
sample tissue, such as a tumor, is compared to normal human DNA. In
a particular example of CGH performed by Vysis, Inc., this is
accomplished by labeling the sample DNA with a fluorescent dye, and
the reference ("normal") DNA with a fluorescent dye of a different
color. Both DNAs are then mixed in equal amounts and hybridized to
a DNA chip. The Vysis chip or genosensor, contains an array of
large insert DNA clones, each comprising approximately 100,000
nucleotides of human DNA sequence. After hybridization, a
multi-color imaging system determines the ratio of colors (for
example green to red fluorescence) for each of the probe spots in
the array. If there is no difference between the sample DNA and the
normal DNA, then all spots should have an equal mixture of red and
green fluorescence, resulting in a yellow color. A shift toward
green or red for a given spot would indicate that either more green
or more red labeled DNA was bound to the chip by that probe
sequence. This color shift indicates a difference between the
sample and the reference DNA for that particular region on the
human genome, pointing either toward amplification or deletion of a
specific sequence or gene contained in the clones positioned in the
array. Examples of genetic changes that can be detected include
amplifications of genes in cancer, or characteristic deletions in
genetic syndromes, such as Cri du chat.
[0144] Since each genetic region to be analyzed needs to be
represented on the chip in only 1 or few replicate spots, the
genosensor can be designed to scan the total human genome for large
deletions or duplications in a single assay. For example, an array
of just 3000 different clones evenly spaced along the human genome
would provide a level of resolution that is at least 10 times
better than what can be achieved with metaphase hybridization, at a
much lower cost and in much less time. Specialty chips can be
tailored to the analysis of certain cancers or disease syndromes,
and can also provide physicians with much more information on
routine clinical analysis than currently can be obtained even by
the most sophisticated research laboratories.
[0145] The color ratio analysis of the genosensor CGH (gCGH) assay
has the advantage that absolute quantitation of the amount of a
specific sequence in the sample DNA is not required. Instead, only
the relative amount compared to the reference (normal) DNA is
measured with relatively high accuracy. This approach is equally
useful for a third kind of chip technology, referred to as
"Expression Chips." These chips contain arrays of probe spots which
are specific for different genes in the human genome. They do not
measure the presence or absence of a mutation in the DNA directly,
but rather determine the amount of message that is produced from a
given gene. The message, or mRNA, is an intermediary molecule in
the process by which the genetic information encoded in the DNA is
translated into protein. The process by which mRNA amounts are
measured involves an enzymatic step which converts the unstable
mRNA into cDNA, and simultaneously incorporates a fluorescent
label. cDNA from a sample tissue is labeled in one color and cDNA
from a normal tissue is labeled with a different color. After
comparative hybridization to the chip, a color ratio analysis of
each probe spot reveals the relative amounts of that specific mRNA
in the sample tissue compared to normal tissue. Expression chips
measure the relative expression of each gene for which there is a
probe spot on the chip.
[0146] There are approximately 100,000 different genes in the human
genome, and it is expected that all of them will be known within a
few years. Since chips with thousands of different probe spots can
be made, the relative expression of each gene can be determined in
a single assay. This has significant implications for disease
diagnosis and therapy. Expression chips may be used to test the
effect of drugs on the expression of a limited number of genes in
tissue culture cells, by comparing mRNA from drug treated cells to
that of untreated cells. The ability to measure the effect on the
regulation of all genes will allow a much more rapid and precise
drug design, since the potency and potential side effects of drugs
can be tested early in development. Moreover, the rapid increase in
understanding of the regulatory switches that determine tissue
differentiation will allow for the design of drugs that can
initiate or modify these processes. Findings about differential
expression in CGH can be further analyzed in tissue arrays, in
which expression of mRNA can also be determined.
[0147] In one particular embodiment of CGH, a DNA chip or
genosensor (hence, genosensor CGH or gCGH), such as an AmpliOnc.TM.
chip from Vysis, contains an array of P1, BAC, or PAC clones, each
with an insert of human genomic DNA. The size of these inserts
ranges from 80 to 150 kilobases, and they are spaced along the
human genome to improve the resolution of this technique. Since the
hybridization probe mixture contains only on the order of 200 ng of
total human DNA from each of the test and reference tissue, the
total number of available probes for each arrayed target clone is
relatively low, placing higher demands on the sensitivity of this
system than what is needed for regular fluorescent in situ
hybridization techniques. These demands have been met with the
development of improved chip surfaces, attachment chemistry, and
imaging systems. The combination of such features can provide a
sensitivity of <10.sup.8 fluorophors/cm.sup.2, which is achieved
through highly efficient background reduction.
[0148] Autofluorescence emanating from the chip surface may be
reduced by coating the glass chip with chromium, as disclosed in
U.S. patent application Ser. No. 09/085,625. This highly reflective
surface provides enhanced signal collection efficiency, and its
hydrophobic nature reduces non-specific binding of probes.
Efficient reading of CGH chips is achieved with a sensitive, high
speed, compact, and easy to use multicolor fluorescence imaging
system, such as that described in U.S. patent application Ser. No.
09/049,748. The non-epifluorescent excitation geometry eliminates
autofluorescence from the collection optics, and collects only
fluorescent light from the chip surface. A xenon arc lamp serves as
a safe and long-lasting light source, providing even illumination
over a wide range of wavelengths. This allows for the use of many
different fluorophores, limited only by the choice of excitation
and emission filters. Fluorescent images are acquired from a 14
mm.times.9 mm sample area by a cooled CCD camera without scanning
or magnification, and even the need for routine focusing has been
eliminated. The images are analyzed by software, which interrogates
each individual pixel to calculate the ratio of sample to reference
probe that are hybridized to each target spot. An appropriate
statistical analysis reveals the relative concentration of each
target specific sequence in the probe mixture.
[0149] This system may be used for expression analysis or genomic
applications, such as an analysis of genetic changes in cancer. For
this purpose a microarray was developed for the specific analysis
of all genetic regions that have been reported so far to be
associated with tumor formation through amplification at the genome
level. The AmpliOnc.TM. chip contains 33 targets (mostly known
oncogenes), each replicated 5 times. A schematic representation of
such a chip (and 31 of the targets) is shown in FIG. 13. New chips
containing 50 targets or more can also be used.
Example 10
Combination of Microarrays to Detect Amplification of FGFR2 Gene in
Sum-52 Breast Cancer Cell Line
[0150] This Example demonstrates how target genes for chromosomal
gains seen by comparative genomic hybridization (CGH) can be
rapidly identified and studied for their clinical relevance using a
combination of novel, high-throughput microarray strategies. CGH to
metaphase spreads (FIG. 14, chromosomal CGH) showed high-level DNA
amplifications at chromosomal regions 7q31, 8p11-p12 and 10q25 in
the Sum-52 breast cancer cell line. Genomic DNA from the Sum-52
cell line was then hybridized to a novel CGH microarray (FIG. 14,
genosensor CGH, Vysis, Downers Grove, Ill.), which enabled
simultaneous screening of copy number at 31 loci containing known
or suspected oncogenes (the loci are shown in FIG. 13) This gCGH
analysis implicated specific, high-level amplifications of the MET
(at 7q31) and FGFR2 (at 10q25) genes, as well as low level
amplification of the FGFR1 gene (at 8p11-p12), indicating the
involvement of these three genes in the amplicons seen by
conventional CGH analysis.
[0151] A large-scale expression survey of the same cell line using
a cDNA microarray (Clonetech Inc.) provided additional information.
The FGFR2 gene was the most abundantly overexpressed transcript in
the SUM-52 cells implicating this gene as the likely amplification
target gene at 10q25. Overexpression of FGFR2 was confirmed by
Northern analysis, and amplification by fluorescence in situ
hybridization (FISH). Finally, FISH to a tissue microarray
consisting of 145 primary breast cancers (FIG. 15) showed the in
vivo amplification of the FGFR2 gene in 4.5% of the cases.
[0152] These three microarray experiments can be accomplished in a
few days, and illustrate how the combination of microarray-based
screening techniques is very powerful for the rapid identification
of target genes for chromosomal rearrangements, as well as for the
evaluation of the prevalence of such alterations in large numbers
of primary tumors. This power is conferred by the ability to screen
many genes against one tumor, using DNA array technologies (such as
cDNA chips or CGH), to find a gene of interest, in combination with
the ability to screen many tumors against the gene of interest
using the tissue microarray technology. FIG. 16 illustrates that
the DNA chip can use multiple clones (for example more than 100
clones) to screen a single tumor or other cell, while the
complementary tissue microarray technology can use a single probe
to screen multiple (for example more than 100) tumor or other
tissue specimens (of either the same or different tissue
types).
Example 11
Tissue Arrays to Determine Frequency and Distribution of Gene
Expression and Copy Number Changes During Cancer Progression
[0153] Tissue arrays may be used to follow up genes and targets
discovered from, for example, high-throughput genomics, such as DNA
sequencing, DNA microarrays, or SAGE (Serial Analysis of Gene
Expression) (Velculescu et al., Science, 270:484-487, 1995).
Comparative analysis of gene expression patterns with cDNA array
technology (Schena 1995 and 1996) provides a high-throughput tool
for screening expressional changes for better understanding
molecular mechanisms responsible for tumor progression as well as
aiming for discovery of new prognostic markers and potential
therapeutic targets. Tissue arrays provide accurate frequency and
distribution information concerning such genes in both pathological
and normal physiological conditions.
[0154] An example is the use of a prostate tumor array to determine
that IGFBP2 (Insulin Growth factor binding protein 2) is a marker
associated with progression of human prostate cancer. To elucidate
mechanisms underlying the development and progression of hormone
refractory prostate cancer, gene expression profiles were compared
for four independent CWR22R hormone refractory xenografts to
androgen dependent CWR22 primary xenograft. The CWR22 xenograft
model of human prostate cancer was established by transplantation
of human prostate tumor cells into the nude mouse (Pretlow, J.
Natl. Cancer Inst., 3:394-398, 1993). This parental tumor xenograft
is characterized by secretion of prostate specific antigen (PSA)
and with rapid reduction of tumor size in response to the
hormone-withdrawal therapy. Approximately half of the treated
animals will develop recurrent tumors from a few weeks to several
months. These recurrent tumors are resistant to further hormonal
treatments when transferred to the new host. They also are
characterized by a more aggressive phenotype than parental CWR22
tumors, and eventually lead to death of the animal. This
experimental model mimics the course of prostate cancer progression
in human patients.
[0155] Comparison of the expression levels of 588 known genes
during che progression of the CWR22 prostate cancer in mice was
performed with the cDNA microarray technology. RNA was prepared
from CWR22 xenografts as described earlier with minor modifications
(Chirgwin, 1979). The mRNA was purified using oligo(dT) selection
with DynaBeads (Dynal) according to manufacturers instructions. The
cDNA array hybridizations were performed on AtlasII cDNA arrays
(Clontech) according to manufacturers instructions. The cDNA probes
were synthesized using 2 .mu.g of polyA.sup.+ RNA and labeled with
32P a dCTP.
[0156] The gene expression pattern in a hormone-sensitive CWR22
xenograft was compared with that of a hormone-refractory CWR22R
xenograft. Expressional changes of several genes, which have
previously been shown to be involved in prostate cancer
pathogenesis were detected in addition multiple genes were
identified with no previous connection to prostate cancer, nor had
they been known to be regulated by androgens. One of the most
consistently upregulated genes, Insulin-like Growth Factor Binding
Protein 2 (IGFBP-2), was chosen for further study. The tissue
microarray technology was used to validate that the IGFBP2
expression changes also take place in vivo, during the progression
of prostate cancer in patients undergoing hormonal therapy.
[0157] Formalin-fixed and paraffin-embedded samples from a total of
142 prostate cancers were used for construction of the prostate
cancer tissue microarray. The tumors included 188 non-hormone
refractory primary prostate cancers, 54 transurethral resection
specimens of locally recurrent hormone-refractory cancers operated
during 1976-1997, and 27 transurethral resections for BPH as benign
controls. The subset of the primary non-hormone refractory tumors
and benign controls was selected from the archives of the Institute
for Pathology, University of Basel, (Switzerland), and the subset
of hormone-refractory tumors from the University of Tampere
(Finland). The group of primary non-hormone refractory prostate
cancers consisted of 50 incidentally detected tumors in
transurethral resections for presumed BPH (pT1a/b), and 138 radical
prostatectomy specimens of patients with clinically localized
disease. The specimens were fixed in 4 percent phosphate-buffered
formalin. The sections were processed into paraffin and slides were
cut at 5 .mu.m and stained with haematoxylin and eosin (H & E).
All sections were reviewed by one pathologist, and the most
representative (usually the least differentiated) tumor area was
delineated on the slide. The tissue microarray technology was used
as previously described to construct the tissue array.
[0158] Standard indirect immunoperoxidase procedures were used for
immunohistochemistry (ABC-Elite, Vector Laboratories). The goat
polyclonal antibody IGFBP-2, C-18 (1:x, Santa Cruz Biotechnology,
Inc., California) was used to detect IGFBP-2 after a microwave
pretreatment. The reaction was visualized by diaminobenzidine as a
chromogen. Positive controls for IGFBP-2 consisted of normal renal
cortex. The primary antibody was omitted for negative controls. The
intensity of the cytoplasmic IGFBP-2 staining was estimated and
stratified into 4 groups (negative, weak, intermediate, and strong
staining).
[0159] There was a strong relationship between IGFBP-2 staining and
progression of cancer to a hormone refractory disease with an
increasing frequency of high-level staining. Strong IGFBP-2
staining was present in none of the normal glands, in 30% of the
non-hormone-refractory primary tumors but in 96% of the recurrent,
hormone-refractory prostate cancers (p=0.0001). Hence, this example
provides another case in which a high-throughput expression survey
by cDNA array hybridization indicated a specific gene, which may be
involved in disease progression. This hypothesis could be directly
validated using the tissue array technology. The results have
identified IGFBP2 to be used as a target for developing diagnostic,
prognostic or therapeutic approaches to the management of patients
with advanced prostate cancer.
Example 12
PDGFB in Breast Cancer
[0160] The breast cancer SKBR3 cell line was screened with the
AmpliOnc.TM. DNA array, and Platelet Derived Growth Factor B
(PDGFB) was identified as being amplified. Using this information,
a PDGFB probe was made using a clone identical to the PDGFB clone
used in the AmpliOnc.TM. array. This probe was used to screen a
breast cancer tumor array. It was found that only 2% of all the
breast cancers screened were amplified for PDGFB. A multi-tumor
array (described in Example 6) was then probed using this probe.
This revealed that, unexpectedly, the PDGFB gene was amplified in a
large percentage of lung and bladder cancers. Thus, using the
invention, a novel marker of diagnostic importance in these other
types of tumors was identified.
Example 13
Herceptin Treatment
[0161] Tissue arrays can be used to screen large numbers of tumor
tissue samples to determine which tumors would be susceptible to a
particular treatment. For example, a breast cancer array can be
screened for expression of the HER-2 gene (also called ERBB2 in
Example 1), as explained in Example 1. Tumors that over-express
and/or amplify the HER-2 gene may be good candidates for treatment
with herceptin, which is an antibody that inhibits the expression
of HER-2. Screening of the multi-tumor tissue array with the HER-2
antibodies or a DNA probe would provide information about cancers
other than breast cancer that could be successfully treated with
the Herceptin therapy.
Example 14
Correlating Prognosis and Survival with Markers
[0162] Tumor tissue arrays constructed from tumors taken from
patients for whom history and outcome is known may be used to
assess markers with prognostic relevance. This example illustrates
that prognostic markers in urinary bladder cancer can be evaluated
using tumor tissue arrays, in spite of any intratumor
heterogeneity.
[0163] An array of 315 bladder tumors was analyzed for nuclear p53
accumulation by immunohistochemistry. The p53 analysis was done
twice; once on conventional large histological sections taken from
entire tumor blocks and once on a section from a tumor array
containing one sample from each tumor. The tumor series consisted
of 127 pTa, 81 pT1, and 128 pT2-4 bladder carcinomas with clinical
follow up information (tumor specific survival).
[0164] One block per tumor was analyzed. One section was taken from
each block for immunohistochemical analysis. Then a tissue array
was constructed by taking one "punch biopsy" from each block and
bringing it in an empty recipient block. Sections 4 .mu.m thick
were taken from primary tumor blocks and from the array block. The
monoclonal antibody DO-7 (DAKO, 1:1000) was applied for
immunostaining using standard procedures.
[0165] On large sections, a tumor was considered positive if
moderate or strong nuclear p53 staining was seen in at least 20% of
tumor cells, at least in an area of the tumor. On array sections, a
tumor was considered positive if moderate or strong nuclear p53
staining was seen in at least 20% of arrayed tumor cells. Weak
nuclear and any cytoplasmic p53 staining was disregarded.
[0166] A Chi-square test was used to compare the p53 results
between array and large sections. Survival curves were plotted
according to Kaplan-Meier. A log rank test was applied to examine
the relationship between p53 positivity and tumor specific
survival. Surviving patients were censored at the time of their
last clinical control. Patients dying from causes other than their
bladder tumor were censored at the time of death.
[0167] Results showed that p53 could be analyzed on 315 arrayed
tumor samples (21 samples were absent on the p53 stained array
section). On conventional sections, p53 immunostaining was positive
in 105 of these 315 tumors which were also present on the array.
p53 positivity as detected on conventional "large" sections was
significantly linked to poor prognosis (FIG. 1A, p<0.0001). Only
69 of these 105 tumors (66%) that were p53 positive on large
sections were also positive on arrayed tumor samples, while 36
(34%) remained negative probably because of tumor heterogeneity.
Nevertheless, there was a strong association between p53
immunostaining results on arrays and on large sections
(p<0.0001) and p53 positivity on arrays was still significantly
linked to poor prognosis (FIG. 1B, p=0.0064).
[0168] The specific number of biopsies from each tumor that are
preferably obtained to reproduce 90%, 95%, or 100% of the
information obtained from the whole-section analysis will make it
possible to determine how many "punches" with the tissue arrays are
required to extract clinically significant information from the
tissue array experiments. This optimal number may vary depending on
the tumor type and the specific biological target that will be
analyzed.
Example 15
Novel Gene Targets
[0169] Tissue arrays may be used to find novel targets for cancer
and other therapies. Hundreds of different genes may be
differentially regulated in a given cancer (based on cDNA, e.g.,
microarray, hybridizations, or other high-throughput expression
screening methods such as sequencing or SAGE). Analysis of each
gene candidate on a large tissue array can help determine which is
the most promising target for development of novel drugs,
inhibitors, etc. For instance, a tumor array containing thousands
of diverse tumor samples may be screened with a probe for an
oncogene, or a gene coding for a novel signal transduction
molecule. Such a probe can bind to one or a number of different
tumor types. If a probe reveals that a particular gene is
overexpressed and/or amplified in many tumors, then that gene may
be an important target, playing a key role in many tumors of one
histological type or in different tumor types. Therapies directed
to interfere with the expression of that gene, or with the function
of the gene product of that gene, may be promising novel cancer
drugs. In particular, the tissue arrays can be used to help
prioritize the selection of targets for drug development.
Example 16
Tissue Array Followed by DNA Array
[0170] Although many of the foregoing examples have described the
DNA array being used prior to the tissue array, the present
invention includes use of these arrays in either order, or in
combination with other analytic techniques. Hence, genes of
interest noted when probing multiple tumor samples with a single
probe during tissue array analysis can subsequently be selected to
be placed on a DNA array, using a unique sequence from the gene of
interest as one of the probes attached to the array substrate. For
example, one could tailor a DNA chip that has most diagnostic,
prognostic, or therapeutic relevance based on information from the
microarray experiment.
[0171] Some possible interrelationship of cDNA arrays, CGH arrays,
and tissue arrays is shown in FIG. 17. As illustrated in that
figure, the various assays can be performed in any order, or in any
combination.
Example 17
Cell Line Arrays
[0172] Cultured cells or cells isolated from non-solid tissues or
tumors (such as blood samples, bone marrow biopsies, or cytological
specimens obtained by needle aspiration biopsies) can also be
analyzed with the tissue array techniques. This is an important
extension of the tissue array technology to the analysis of
individual cells, or populations of cells, obtained either directly
from people or animals or after various incubations of cell culture
experiments have been performed in vitro (such as a specific
hormonal or chemotherapeutic test performed on a microtiter tray
format for pharmaceutical drug screening) In the analysis of
malignancies, this would enable analysis of leukemias and lymphoma
tissues or other liquid tumor types following the same strategies
described above for solid tumors.
[0173] Using this approach, cancer cell lines obtained from the
American Type Culture Collection (Rockville, Md.) were used. Cells
were trypsinized and the cell suspensions were spun down with a
centrifuge at 1200.times.g. The cell pellet was fixed with
alcohol-based and formaldehyde fixatives, and the fixed cell pellet
was embedded in paraffin following routine protocols used in
pathology laboratories. The fixed and embedded cell suspensions can
then be used as starting material for the development of cell
arrays, using the same procedure as described previously for the
fixed and embedded tissue specimens. It is anticipated that up to
or at least 1000 different cell populations can be arrayed in a
single standard-size paraffin block using this method.
[0174] Very small punch sizes (for example less than 0.5 mm) can be
used for creating arrays from homogenous cultured cells. This
allows high density arrays to be constructed. For example,
approximately 2000 different cell populations can be placed in a
single 40 mm.times.25 mm paraffin block.
[0175] The methods of analyzing tissue in accordance with the
present invention can take many different forms, other than those
specifically disclosed in the above examples. The tissue specimens
need not be abnormal, but can be normal tissue analyzed for
function and tissue distribution of a specific gene, protein, or
other biomarker (where a biomarker is a biological characteristic
that is informative about a biological property of the specimen).
The normal tissue could include embryonal tissues, or tissues from
a genetically modified organism, such as a transgenic mouse.
[0176] The array technologies can also be used to analyze diseases
that do not have a genetic basis. For example, the gene or protein
expression patterns that are likely to have importance for the
pathogenesis or diagnosis of a disease can be profiled. The tissue
specimens need not be limited to solid tumors, but can also be
taken from cell lines, hematological or other liquid tumors,
cytological specimens, or isolated cells.
[0177] Cells of humans or other animals can be used in a
suspension, as may cells of yeast or bacteria. Alternatively, cells
in suspension can be spun down in a centrifuge to provide a solid
or semi-solid pellet, fixed, and then placed in the array, much
like a tissue specimen. Liquid cellular suspensions can be placed
with a pipette into a matrix (for example depressions in a slide
surface) and then can be analyzed in the same manner as the tissue
array already described. The tissue arrays can also be used in cell
line experiments, such as high throughput chemotherapeutic
screening of cells grown on microtiter plates. The cells from each
well are treated with a different drug or a different concentration
of the drug, and are then recovered and inserted into a cell line
microarray to analyze their functional characteristics, morphology,
viability and expression of specific genes brought about by the
drug treatment.
[0178] Histological or immunological analyses that can be used with
the array include, without limitation, a nucleic acid
hybridization, PCR (such as in situ PCR), PRINS, ligase chain
reaction, pad lock probe detection, histochemical in situ enzymatic
detection, and the use of molecular beacons. The tissue array
technology can be used to directly collect specimens (tissues or
cells) from humans, animals, cell lines, or other experimental
systems. For example, when biopsy specimens are treated in a
conventional manner in pathology laboratories, after fixation, the
specimens are routinely inserted horizontally in a paraffin block.
Therefore, it is very difficult, if not impossible to acquire
specimens from such tissues into a tissue array. However, if
multiple biopsy specimens obtained from surgery are directly fixed
(and, if required, embedded in a suitable medium, such as paraffin)
and then inserted directly vertically into a matrix, this would
enable construction of a tissue array of biopsy specimens. Such an
array would be useful for research purposes or in a clinical
setting to e.g. monitor progression of premalignant lesions or
monitor treatment responses (with molecular markers) from
metastatic tumors that cannot be surgically removed.
[0179] Cytological specimens (such as fine needle aspirations,
cervical cytology, blood specimens, isolated blood cells, or urine
cells) can be pelleted by centrifugation and then fixed and
embedded for arraying as explained previously. Alternatively, cells
can be fixed in a suspension, and directly inserted (e.g.,
pipetted) into holes in a matrix or embedded first, and then
arrayed. This will provide an array of cells for research or for,
diagnostic purposes. This would enable rapid cytological
diagnostics where multiple specimens from different patients can be
screened simultaneously from a single slide, not only for their
morphology, but for their molecular characteristics. This would
also enable automation of the analysis, since a number of specimens
can be screened with a microscope, automated image analysis system,
scanner or associated expert systems at once. The use of such
cellular preparations is particularly important for the diagnosis
of hematological disorders, such as leukemias and lymphomas. This
would also allow automation of lymphocyte typing from many patients
at once, whose specimens are inserted in an array format for
immunophenotyping or for analysis by in situ hybridization.
Screening of donated blood specimens for viral antigens, viral DNA,
or other pathogens in a blood bank could similarly be performed in
an array format.
[0180] Arrays of tumor progression can also be constructed by
collecting specimens from a subject at different stages of
progression of the subject's tumor (such as progression to hormone
refractory prostate cancer). Alternatively, tumors of different
stages from different subjects can be collected and incorporated
into the array. The array can also be used to follow the
progression of pre-neoplastic lesions (such as the evolution of
cervical neoplasia), and the effects of chemoprevention agents
(such as the effects of anti-estrogens on breast epithelium and
breast cancer development).
[0181] In another embodiment, specimens from a transgenic or model
organism can be obtained at different stages of development of the
organism, such as different embryonic stages, or different ages
after birth. This enables the study of things such as normal and
abnormal embryonic development.
[0182] The biological analyses that are performed on the microarray
sections can be any analysis performed on regular tissue sections.
Arrays can also be assembled from one or more tumors at different
stages of progression, such as normal tissue, hyperplasia, in situ
cancer, invasive cancer, recurrent tumor, local lymph node
metastases, or distant metastases.
[0183] An "EST" or "Expressed Sequence Tag" refers to a partial DNA
or cDNA sequence, typically of between 50 and 500 sequential
nucleotides, obtained from a genomic of cDNA library, prepared from
a selected cell, cell type, tissue or tissue type, organ or
organism, which corresponds to an mRNA of a gene found in that
library. An EST is generally a DNA molecule.
[0184] "Specific hybridization" refers to the binding, duplexing,
or hybridizing of a molecule only to a particular nucleotide
sequence under stringent conditions when that sequence is present
in a complex mixture (e.g., total cellular) DNA or RNA.
Other Embodiments
[0185] In view of the many possible embodiments to which the
principles of the invention can be applied, it should be recognized
that the illustrated embodiments are examples of the invention, and
should not be taken as a limitation on the scope of the invention.
Rather, the scope of the invention is defined by the following
claims. We therefore claim as our invention all that comes within
the scope and spirit of these claims.
* * * * *
References