U.S. patent application number 10/305800 was filed with the patent office on 2003-07-24 for tumor tissue microarrays for rapid molecular profiling.
This patent application is currently assigned to The Government of the U.S.A. as represented by the Secretary of the Dept. of Health & Human Services. Invention is credited to Kallioniemi, Olli-P., Kononen, Juha, Leighton, Stephen B..
Application Number | 20030138827 10/305800 |
Document ID | / |
Family ID | 26757495 |
Filed Date | 2003-07-24 |
United States Patent
Application |
20030138827 |
Kind Code |
A1 |
Kononen, Juha ; et
al. |
July 24, 2003 |
Tumor tissue microarrays for rapid molecular profiling
Abstract
An array-based technology facilitates rapid correlated gene copy
number and expression profiling of a very large numbers of human
tumors. Hundreds of cylindrical tissue biopsies (diameter 0.6 mm)
from morphologically representative regions of individual tumors
can be arrayed in a single paraffin block. Consecutive sections
from such arrays provide targets for parrallel in situ
visualization and quantitation of DNA, RNA or protein targets. For
example, amplifications of six loci (mybL2, erbB2, Cyclin-D1, myc,
17q23 and 20q13) were rapidly determined by fluorescence in situ
hybridization from 372 ethanol-fixed breast cancers. Stratification
of tumors by estrogen receptor and p53 expression data revealed
dictinct patterns of gene amplification in the various subgroups of
breast cancer that may have prognostic utility. The tissue array
technology is useful in the rapid molecular profiling of hundreds
of normal and pathological tissue specimens or cultured cells
Inventors: |
Kononen, Juha; (Rockville,
MD) ; Leighton, Stephen B.; (Silver Spring, MD)
; Kallioniemi, Olli-P.; (Rockville, MD) |
Correspondence
Address: |
KLARQUIST SPARKMAN, LLP
One World Trade Center, Suite 1600
121 S.W. Salmon Street
Portland
OR
97204
US
|
Assignee: |
The Government of the U.S.A. as
represented by the Secretary of the Dept. of Health & Human
Services
|
Family ID: |
26757495 |
Appl. No.: |
10/305800 |
Filed: |
November 26, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10305800 |
Nov 26, 2002 |
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09622686 |
Oct 12, 2000 |
|
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09622686 |
Oct 12, 2000 |
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PCT/US99/04001 |
Feb 24, 1999 |
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60075979 |
Feb 25, 1998 |
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Current U.S.
Class: |
435/6.14 ;
435/287.2; 435/7.2 |
Current CPC
Class: |
G01N 2001/368 20130101;
G02B 21/34 20130101; B01J 2219/00691 20130101; B01J 2219/00542
20130101; B01J 2219/00533 20130101; G01N 1/30 20130101; G01N 33/574
20130101; C12Q 1/68 20130101; G01N 33/48 20130101; B01J 2219/00702
20130101; B01L 3/5085 20130101; G01N 1/08 20130101; B01J 19/0046
20130101; B01J 2219/00315 20130101; B01J 2219/00659 20130101; B01J
2219/00725 20130101; B01J 2219/00673 20130101; B01J 2219/00689
20130101; G01N 2001/282 20130101; G01N 1/36 20130101; B01J
2219/00722 20130101; G01N 33/567 20130101 |
Class at
Publication: |
435/6 ; 435/7.2;
435/287.2 |
International
Class: |
C12Q 001/68; G01N
033/53; G01N 033/567; C12M 001/34 |
Claims
1. A method of parallel analysis of tissue specimens, comprising:
obtaining a plurality of donor specimens; placing each donor
specimen in an assigned location in a 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 a different histological
analysis of each section; and comparing the results of the
different histological analyses in corresponding assigned locations
of different sections to determine if there are correlations
between the results of the different histological analyses at each
assigned location.
2. The method of claim 1, wherein the donor specimen is obtained by
boring an elongated sample from the donor specimen, which is placed
in the assigned location in the recipient array.
3. The method of claim 2, wherein placing the donor specimen in an
assigned location in the recipient array comprises forming an
elongated receptacle in a donor block, and placing the elongated
sample in the elongated receptacle of the recipient block.
4. The method of claim 3, wherein the elongated sample is placed in
a receptacle having a cross-sectional size and shape complementary
to a cross-sectional size and shape of the elongated sample.
5. The method of claim 4, wherein forming the elongated receptacle
comprises forming a cylindrical bore in the recipient block, and
the sample is obtained by boring a cylindrical tissue specimen from
the donor block, wherein a diameter of the elongated receptacle is
substantially the same as a diameter of the sample.
6. The method of claim 1, further comprising associating a clinical
parameter with each assigned location in the recipient array.
7. The method of claim 1 wherein performing the different
histological analysis on each slide comprises performing different
tests selected from the group of an immunological analysis and a
nucleic acid hybridization.
8. The method of claim 6, further comprising determining whether
there are correlations between clinical parameters, immunologic
analysis and nucleid acid hybridization.
9. The method of claim 1, wherein the biological sample is a tissue
specimen or cellular preparation.
10. A method of parallel analysis of identical arrays of tissue
specimens, comprising: forming a donor block comprising a
biological specimen embedded in embedding medium; obtaining a
plurality of cylindrical donor sample cores from the biological
specimen; boring receptacle cores from a recipient embedding medium
to form an array of cylindrical receptacles; placing the donor
sample cores in the cylindrical receptacles at assigned locations
in the array; sectioning the recipient embedding medium to obtain a
cross-section of the donor sample cores in the array, while
maintaining the assigned locations in the array in consecutive
cross-sections; performing a different histological analysis of
each cross-section; and comparing a result of each histological
analysis in corresponding assigned locations of different sections
to determine if there are correlations between the results of the
different histological analyses at each assigned location.
11. The method of claim 10, further comprising comparing the
results of the different histological analyses at each assigned
location to clinical information about the biological specimen at
the assigned location.
12. The method of claim 11, wherein the biological specimen is a
tissue specimen from a tumor.
13. The method of claim 12, wherein the histological analyses
comprise immunologic analysis and nucleic acid hybridization
analysis.
14. The method of claim 10, further comprising aligning a thin
tissue section above the donor block to identify an area of
interest from which the donor sample core is taken.
15. The method of claim 10, wherein the cylindrical donor sample
core has a diameter that is less than about 1 mm.
16. A cross-section of the donor sample cores obtained by the
method of claim 10.
17. An apparatus for preparing specimens for parallel analysis of
sections of biological material arrays, comprising: a holder that
can be positioned to maintain a tissue donor block in a donor
position; and a reciprocal punch positioned in relation to the
holder to punch a tissue specimen from the donor block in the donor
position, wherein the holder is also capable of holding a recipient
block in a recipient position, and the recipient block comprises an
array of receptacles, each of which can be positioned in a
preselected position in relation to the reciprocal punch to deliver
a tissue specimen from the reciprocal punch into a receptacle in
the preselected position
18. The apparatus of claim 17, wherein the holder comprises an x-y
positioning device that can be incrementally positioned to align
sequential receptacles with the reciprocal punch.
19. The apparatus of claim 17, further comprising a stylet
positioned for introduction into the reciprocal punch to expel the
tissue specimen from the punch into one of the receptacles aligned
with the punch.
20. The apparatus of claim 17, further comprising a positioner that
positions a thin section slide over the donor block, to align
structures of interest in the thin section slide with corresponding
tissue specimen regions in the donor block.
21. The apparatus of claim 17, further comprising a separate
reciprocal punch capable of being positioned in a fixed position
relative to the recipient block for forming the array of
receptacles in the recipient block.
22. The apparatus of claim 21, further comprising a recorder for
recording the positions of the receptacles in the recipient
block
23. The apparatus of claim 22, wherein the recorder is a computer
implemented system for recording the positions of the receptacles,
and an identification of the tissue specimen that is placed in each
receptacle.
24. A computer implemented system for parallel analysis of
consecutive sections of tissue arrays, comprising: an x-y
positioning platform for moving a tray to a plurality of
coordinates that correspond to positions in a recipient block
array; a receptacle punch positioned in punching relationship with
respect to the positioning platform, such that the receptacle punch
can punch a receptacle core from a recipient block on the
positioning platform, a donor punch positioned in a punching
relationship with respect to the positioning platform, such that
the donor punch can punch a donor specimen from a donor block on
the positioning platform, wherein the receptacle core has a
diameter that is substantially the same as the diameter of the
donor specimen; a stylet that is selectively alternatively aligned
with the donor punch and the recipient punch, for displacing
contents of the receptacle punch after a receptacle core is punched
from the recipient block, and for displacing contents of the donor
punch into receptacles of the recipient block array after a donor
specimen is punched from the donor block; and wherein the system
records an identification of tissue in the receptacles of the
recipient array.
25. The computer implemented system of claim 24, further comprising
a microscope for viewing the donor block, and locating a structure
of interest in a reference slide aligned with the donor block.
26. The computer implemented system of claim 24, wherein the system
punches a receptacle core from the recipient block and displaces
the receptacle core from the receptacle punch with the stylet, then
punches a donor specimen from the donor block, aligns the donor
punch with a selected receptacle in the recipient block, and
displaces the donor specimen into the selected receptacle.
27. A method of analyzing ex vivo tissue specimens, comprising
punching an elongated tissue sample from the ex vivo tissue
specimen, and subjecting the sample to a biological analysis.
28. The method of claim 27, wherein punching the elongated tissue
sample from the tissue specimen comprises placing the tissue
specimen in a holder below a reciprocal punch, and advancing the
reciprocal punch into a predetermined location of the tissue
specimen.
29. The method of claim 28, further comprising placing the tissue
specimen in an embedding medium prior to punching.
30. The method of claim 29, wherein the predetermined location of
the tissue specimen is determined by examining a thin section cut
from the embedding medium.
Description
FIELD OF THE INVENTION
[0001] The present invention concerns devices for the microscopic,
histologic and/or molecular analysis of tissue specimens.
BACKGROUND OF THE INVENTION
[0002] 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 whether the
patient will respond to a particular form of chemotherapy. 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 only reveal a limited amount of diagnostic or
molecular information.
[0003] 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 rumors). 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).
[0004] The development of new molecular markers, however, has been
impeded by the inability to group a large number of tissues within
a small surface area. Only a limited amount of hybridoma
supernatant may be available, particularly during the early phase
of monoclonal antibody generation, which limits the number of
specimens that can be analyzed. Even if large quantities of the
immunohistologic agent are available, however, the reagents are
expensive and may vary in reactivity. These problems led Battifora
et al. to propose in Lab. Invest. 55:244-248 (1986), and in U.S.
Pat. No. 4,820,504, that multiple tissue specimens may be grouped
together on a single slide to enable the specimens to be
simultaneously screened by application of a single drop of
hybridoma supernatant. The specimens were prepared by using a
hand-held razor blade to cut deparaffinized and dehydrated tissue
specimens into slices, which were then bundled together randomly,
wrapped in a sausage casing, and re-embedded in paraffin. This
technique required a high degree of manual dexterity, and
incorporated samples into a composite block in a manner that made
it difficult to find and identify particular specimens of
interest.
[0005] A modification of this process was disclosed by Wan et al.,
J. Immunol. Meth. 103:121-129 (1987), and Furmanski et al. in U.S.
Pat. No. 4,914,022, in which cores of paraffin embedded tissue were
obtained from standard tissue blocks. The cores were softened and
straightened by manually rolling them on a warm surface, and then
bundled inside a conventional drinking straw. This method was said
to be suitable for simultaneous histologic testing of multiple
tissue specimens, for example in the characterization of monoclonal
antibodies. The technique of Miller and Groothuis, A.J.C.P
96:228-232 (1991) similarly rolled tissue strips into "logs" from
which transverse sections were taken to be embedded in paraffin.
The straw and log techniques, however, were labor intensive,
required a high degree of manual dexterity, and also randomly
arranged the samples in a manner that complicated the
identification of specimens of interest.
[0006] Battifora and Mehta, Lab. Invest. 63:722-724 (1990), and
U.S. Pat. No. 5,002,377, attempted to overcome some of the problems
of random placement by cutting specimens into a plurality of narrow
strips, which were individually positioned in parallel rectangular
grooves in a mold. The tissue strips were embedded in agar gel that
was poured into the grooves to produce a plate-like member with a
series of ridges. Several of the ridged plates were stacked
together and embedded in paraffin to form a tissue block. A similar
approach was proposed by Sundblad, A.J.C.P. 102:192-193 (1993), in
which the tissue strips were placed in triangular wedges instead of
rectangular grooves. Slicing the tissue, assembling it into rows,
and embedding it in several steps to form the block was a
time-consuming method that reduced the efficiency of examining a
large number of tissue specimens.
[0007] All of these techniques have been inadequate for the
efficient preparation of an array of tissue specimens that can be
used for rapid parallel analysis of a variety of independent
molecular markers. This inefficiency has been a significant problem
in fields such as cancer research, because cancer development and
progression is a multi-step process that involves sequential
losses, rearrangements and amplifications of several chromosomal
regions and multiple genes. These events lead to a dysregulation of
critical signal transduction pathways for cell growth, death, and
differentiation. The details of this complex process remain
incompletely understood, partly because high-throughput strategies
and techniques for analyzing such genetic changes in large numbers
of uncultured human tumors have not been available.
[0008] For example, simultaneous analysis of several genes within
the same or related signal transduction pathways may be necessary
to pinpoint critical, rate-limiting steps in the dysregulation of
cancer cell growth. Furthermore, analysis of structural and
numerical changes affecting several chromosomes, loci and genes at
the same time may be needed to understand the patterns of
accumulation of genetic changes in different stages of the cancer
progression. Finally after novel genes and genetic changes of
potential importance in cancer have been identified, substantial
additional research is usually required to determine the
diagnostic, prognostic and therapeutic significance of these
molecular markers in clinical oncology.
[0009] Since the amount of tissue often becomes rate limiting for
such studies, the ability to efficiently procure, fix, store and
distribute tissue for molecular analysis in a manner that minimizes
consumption of often unique, precious tumor specimens is important.
It is therefore an object of this invention to perform large-scale
molecular profiling of tissue specimens (such as tumors) with
minimal tissue requirements, in a manner that allows rapid parallel
analysis of molecular characteristics (such as gene dosage and
expression) from hundreds of morphologically controlled tumor
specimens.
SUMMARY OF THE INVENTION
[0010] The foregoing objects are achieved by a method of parallel
analysis of tissue specimens, in which a plurality of donor
specimens are placed in assigned locations in a recipient array,
and a plurality of sections are obtained from the recipient array
so that each section contains a plurality of donor specimens that
maintain their assigned locations. A different histological
analysis is performed on each section, to determine if there are
correlations between the results of the different analyses at
corresponding locations of the array. In particular embodiments,
the donor specimen is obtained by boring an elongated sample, such
as a cylindrical core, from donor tissue, and placing the donor
specimen in a receptacle of complementary shape, such as a
cylindrical core, in the recipient array. Analyses that may be
performed on the donor specimens include immunological analysis,
nucleic acid hybridization, and clinicopathological
characterization of the specimen.
[0011] In a more particular embodiment of the method, a recipient
block is formed from a rigid embedding medium such as paraffin that
can be cut with a punch or microtome, and a separate donor block is
also formed by embedding a biological specimen in the embedding
medium. Cylindrical receptacle cores are bored in the recipient
block to form an array of receptacles at fixed positions, and
cylindrical donor sample cores are obtained from the embedded
biological specimen in the donor block. The donor sample cores are
then placed in the cylindrical receptacles at assigned locations in
the array, and the recipient block is sliced to obtain a
cross-section of the donor sample cores in the array, without
disrupting the assigned array locations. A different histological
analysis may be performed on each section, for example by using
different monoclonal antibodies that recognize distinct antigens,
or a combination of antigenically distinct monoclonal antibodies
and nucleic acid (e.g. RNA and DNA) probes on sequential sections.
The result of each distinct histological analysis in each position
of the array is compared, for example to determine if a tissue that
expresses an estrogen receptor also has evidence that a particular
oncogene has been activated.
[0012] In a more particular embodiment of the method, a recipient
block is formed from a rigid embedding medium such as paraffin that
can be cut with a punch or microtome, and a separate donor block is
also formed by embedding a biological specimen in the embedding
medium. Cylindrical receptacle cores are bored in the recipient
block to form an array of receptacles at fixed positions, and
cylindrical donor sample cores are obtained from the embedded
biological specimen in the donor block. The donor sample cores are
then placed in the cylindrical receptacles at assigned locations in
the array, and the recipient block is sliced to obtain a
cross-section of the donor sample cores in the array, without
disrupting the assigned array locations. A different histological
analysis may be performed on each section, for example by using
different monoclonal antibodies that recognize distinct antigens,
or a combination of antigenically distinct monoclonal antibodies
and nucleic acid (e.g. RNA and DNA) probes on sequential sections.
The result of each distinct histological analysis in each position
of the array is compared, for example to determine if a tissue that
expresses an estrogen receptor also has evidence that a particular
oncogene has been activated. The presence or absence of the
estrogen receptor and oncogene can then be correlated with clinical
or pathological information about the tissue (such as the presence
of metastatic disease or the histological grade of a tumor). This
simultaneous parallel analysis of multiple specimens helps clarify
the inter-relationship of multiple molecular and clinical
characteristics of the tissue.
[0013] The invention also includes a method of obtaining small
elongated samples of tissue from a tissue specimen, such as a
tumor, and subjecting the specimen to laboratory analysis, such as
histological or molecular analysis. The elongated tissue sample can
be taken from a region of interest of the tissue specimen, and the
size of the sample is small enough that the characterstic being
analyzed is substantially homogenous throughout the small sample.
In a disclosed embodiment, the sample is a cylindrical sample
punched from the tissue specimen, wherein the cylindrical specimen
is about 1-4 mm long, and has a diameter of about 0.14 mm, for
example about 0.3-2.0 mm. In specific embodiments, the cylinder
diameter is less than about 1.0 mm, for example 0.6 mm. The sample
is preferably preserved in a manner (such as ethanol fixation) that
does not interfere with analysis of nucleic acids, and the sample
can therefore be subjected to any type of molecular analysis, such
as any type of molecular analysis based on isolated DNA or RNA.
[0014] The invention also includes an apparatus for preparing
specimens for parallel analysis of sections of biological material
arrays. The apparatus includes a platform, a tissue donor block on
the platform, and a punch that punches or bores a tissue specimen
from the donor block. The platform can also carry a recipient block
in which the punch forms an array of receptacles at selected
positions. Each receptacle can be positioned so that a tissue
specimen can be expelled from the reciprocal punch into the
receptacle. An x-y positioning device incrementally moves the punch
or recipient block with respect to one another as the punch
reciprocates, to form the receptacle array. The x-y positioning
device also aligns sequential receptacles of the recipient block
with the punch to deliver tissue specimens from the punch into the
receptacle A stylet may be introduced into the punch to expel the
contents of the punch, which may be either paraffin from the
recipient block or tissue from the donor block. Regions of interest
of the tissue specimen are located by positioning a thin section
slide over the donor block, to align structures of interest in the
thin section slide with corresponding tissue specimen regions in
the donor block.
[0015] The invention also includes a computer implemented system
for parallel analysis of consecutive sections of tissue arrays, in
which an x-y positioning platform moves a tray to a plurality of
coordinates that correspond to positions in a recipient block
array. A receptacle punch then punches a receptacle core from a
recipient block on the positioning platform, and a stylet expels
the receptacle core from the receptacle punch. A donor punch (which
may be the same or separate from the recipient punch) punches a
donor specimen from a donor block on the positioning platform, and
a stylet expels the donor specimen from the donor punch into the
receptacle as the donor punch is introduced into the receptacle.
The donor specimen suitably has a diameter that is substantially
the same as the diameter of the receptacle, so that the donor
specimen fits securely in the receptacle. The computer system
identifies the tissue by its location in the recipient array, so
that when the donor block is sectioned a corresponding position in
each sectional array will contain tissue from the identical donor
specimen.
[0016] The foregoing and other objects, features, and advantages of
the invention will become more apparent from the following detailed
description of preferred embodiments which proceeds with reference
to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] 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.
[0018] FIG. 2 is a view similar to FIG. 1, but in which the punch
has been advanced to obtain a donor specimen sample.
[0019] FIG. 3 is a schematic, perspective view of a recipient black
into which the donor specimen has been placed.
[0020] FIGS. 4-8 illustrate steps in the preparation of thin
section arrays from the recipient block.
[0021] 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.
[0022] FIG. 10A is a view of an H&E stained, thin section
tissue array mounted on a slide for microscopic examination.
[0023] FIG. 10B is a magnified view of a portion of the slide in
FIG. 10A, showing results of erbB2 mRNA in situ hybridzation on a
tissue array from the region in the small rectangle in FIG.
10A.
[0024] FIG. 10C is an electrophoresis gel showing that high
molecular weight DNA and RNA can be extracted from the breast
cancer specimens.
[0025] FIG. 10D is an enlarged view of one of the tissue samples of
the array in FIG. 10A, showing an immunoperoxidase stain for the
erbB2 antigen.
[0026] 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.
[0027] FIG. 11 is a schematic view illustrating an example of
parallel analysis of arrays obtained by the method of the present
invention.
[0028] FIG. 12 is an enlarged view of a portion of FIG. 11.
[0029] FIG. 13 is a top view of a second embodiment of a device for
forming the arrays of the present invention.
[0030] FIG. 14 is a front view of the device shown in FIG. 13,
illustrating the formation of a receptacle in a recipient block
with a recipient punch.
[0031] FIG. 15 is a view similar to FIG. 14, but showing expulsion
of a plug from the recipient punch into a discard tray.
[0032] FIG. 16 is a view showing a donor punch obtaining a tissue
specimen from a donor block.
[0033] FIG. 17 is a view showing insertion of the donor tissue into
a receptacle of the recipient block.
[0034] FIG. 18 is an enlarged view of the donor punch aligned above
a structure of interest in the donor block, which is shown in
cross-section.
[0035] FIG. 19 is an enlarged cross-sectional view of the recipient
punch, while FIG. 20 is a similar view of the donor punch,
illustrating the relative cross-sectional diameters of the two
punches.
[0036] FIG. 21 is a cross-sectional view of the recipient block
with the donor specimens arranged in the recipient array, and with
lines of microtome sections of the recipient block being shown.
[0037] FIG. 22 is a schematic view of a computer system in which
the method of the present invention can be implemented.
[0038] FIG. 23 is an algorithm illustrating an example of the
computer implemented method of the present invention.
DETAILED DESCRIPTION
Embodiment of FIGS. 1-10
[0039] A first embodiment of a device for making the microarrays of
the present invention is shown in FIGS. 1-2, in which a donor block
30 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.
[0040] 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.
[0041] FIG. 1 shows that a thin section of tissue 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.
[0042] 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.
[0043] 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 interest 66 may
be an area of the specimen in which the cellular architecture is
suggestive of metaplasia (e.g. pyknotic nuclei, pleomorphism,
invasiveness). 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).
[0044] 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
[0045] 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 a 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).
[0046] In a specific example, core tissue biopsies having a
diameter of 0.6 mm and a height of 34 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. Up to 1000 such tissue cylinders 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.
[0047] 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. 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.
[0048] A breast cancer tissue specimen was 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 copy number or
expression of multiple genes. This analysis is performed by testing
for different gene amplifications 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.
[0049] 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.degree. C. overnight in a vacuum oven.
[0050] 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.
[0051] The potential of the array technology of the present
invention to perform rapid parallel molecular analysis of multiple
tissue specimens is illustrated in FIG. 11, where the y-axis of the
graphs 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 11B, which divide the categories into Groups
I, II, III and IV corresponding to the ER/p53 status.
[0052] 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.
[0053] 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.
[0054] 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-.
[0055] 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 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.
[0056] 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.
[0057] As shown in FIG. 12, the rectangular array is 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 is 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 coordinate position (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 sucessive sections of the donor block that are cut to
form the array.
[0058] 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 introduced into the
recipient array at coordinate position (1,1), and once the array
was completed it 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.
[0059] 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).
[0060] Analysis of consecutive sections from the arrays enables
co-localization of hundreds of different DNA, RNA or protein
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 small amounts of
the identical reagents are used for each analysis. The tumor arrays
also substantially reduce tissue consumption, reagent use, and
workload when compared with processing individual conventional
specimens 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 wil be helpful to
detect previously unappreciated but important molecular features of
the tumors that may turn out to have diagnostic or prognostic
utility.
[0061] 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.
[0062] 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 could be placed in
replicate 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
microdissection 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 PCR-based techniques for RNA and DNA. When nucleic
acid analysis is planned, the tissue specimen is preferably fixed
(before embedding in paraffin) in 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.
[0063] 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.
Embodiment of FIGS. 13-23
[0064] An example of an automated system for high speed preparation
of the microarrays is shown in FIGS. 13-23. The system includes a
stage 100 having an x drive 102 and a y drive 104, each of which
respectively rotates a drive shaft 106. 108. The shaft 108 moves a
specimen bench 110 in a y direction, while the shaft 106 moves a
tray 112 on the bench 110 in an x direction. Mounted in a front row
of tray 112 are three recipient containers 116, 118 and 120, each
of which contains a recipient paraffin block 122, 124 or 126, and a
donor container 128 that contains a donor paraffin block 130, in
which is embedded a tissue specimen 132. In a back row on the tray
are two multi-well donor trays 132, 134 (which contain multiple
containers for maintaining specimens in liquid medium), and a
discard container 136.
[0065] Disposed above stage 100 is a punch apparatus 140 that can
move up and down in a z direction. Apparatus 140 includes a
central, vertically disposed, stylet drive 142 in which
reciprocates a stylet 144. Apparatus 140 also includes an inclined
recipient punch drive 146, and a inclined donor punch drive 148.
Punch drive 146 includes a reciprocal ram 150 that carries a
tubular recipient punch 154 at its distal end, and punch drive 148
includes a reciprocal ram 152 that carries a donor tubular punch
156 at its distal end When the ram 150 is extended (FIG. 14),
recipient punch 154 is positioned with the open top of its tubular
bore aligned with stylet 144, and when ram 152 is extended (FIG.
16), donor punch 156 is positioned with the open top of its tubular
bore aligned with stylet 144.
[0066] The sequential operation of the apparatus 140 is shown in
FIGS. 13-17. Once the device is assembled as in FIG. 13, a computer
system can be used to operate the apparatus to achieve high
efficiency. Hence the computer system can initialize itself by
determining the location of the containers on tray 112 shown in
FIG. 13. The x and y drives 102, 104 are then activated to move
bench 110 and tray 112 to the position shown in FIG. 14, so that
activation of ram 150 extends recipient punch 154 to a position
above position (1,1) in the recipient block 122. Once punch 154 is
in position, apparatus moves downward in the z direction to punch a
cylindrical bore in the paraffin of the recipient block. The
apparatus 140 then moves upwardly in the z direction to raise punch
154 out of the paraffin recipient block 122, but the punch 154
retains a core of paraffin that leaves a cylindrical receptacle in
the recipient block 122. The x-y drives are then activated to move
bench 110 and position discard container 136 below punch 154.
Stylet drive 142 is then activated to advance stylet 144 into the
open top of the aligned punch 154, to dislodge the paraffin core
from punch 154 and into discard container 136.
[0067] Stylet 144 is retracted from recipient punch 154, ram 150 is
retracted, and the x-y drive moves bench 110 and tray 112 to place
donor container 128 is a position (shown in FIG. 16) such that
advancement of ram 152 advances donor punch 156 to a desired
location over the donor block 130. Apparatus 140 is then moved down
in the z direction to punch a cylindrical core of tissue out of the
donor block 130, and apparatus 140 is then moved in the z direction
to withdraw donor punch 156, with the cylindrical tissue specimen
retained in the punch. The x-y drive then moves bench 110 and tray
112 to the position shown in FIG. 17, such that movement of
apparatus 140 downwardly in the z direction advances donor punch
156 into the receptacle at the coordinate position (1,1) in block
122 from which the recipient plug has been removed. Donor punch 156
is aligned below stylet 144, and the stylet is advanced to dislodge
the retained tissue cylinder from donor punch 156, so that the
donor tissue cylinder remains in the receptacle of the recipient
block 122 as the apparatus 140 moves up in the z direction to
retract donor punch 156 from the recipient array. Ram 152 is then
retracted.
[0068] This process can be repeated until a desired number of
recipient receptacles have been formed and filled with cylindrical
donor tissues at the desired coordinate locations of the array.
Although this illustrated method shows sequential alternating
formation of each receptacle, and introduction of the tissue
cylinder into the formed receptacle, it is also possible to form
all the receptacles in recipient blocks 122, 124 and 126 as an
initial step, and then move to the step of obtaining the tissue
specimens and introducing them into the preformed receptacles. The
same tissue specimen 132 can be repeatedly used, or the specimen
132 can be changed after each donor tissue specimen is obtained, by
introducing a new donor block 130 into container 128. If the donor
block 130 is changed after each tissue cylinder is obtained, each
coordinate of the array can include tissue from a different tissue
specimen.
[0069] A positioning device is shown in FIG. 18, which helps locate
structures of interest from which donor specimens can be taken. The
positioning device includes a support slide 160 that extends
between opposing walls of donor container 128, to support a
specimen slide 162 on which is mounted a thin stained section of
the specimen 132 in donor block 130. Using a microscope mounted on
apparatus 140 (the objective of the microscope is shown at 166),
microanatomic structures of interest can be found. The correct
vertical height of apparatus 140 above the top surface of donor
block 130 can be determined by the use of two positioning lights
168, 170 that are mounted to apparatus 140. Light beams 172, 174
are projected from lights 168, 170 at an angle such that the beams
coincide at a single spot 176 when vertical height of apparatus 140
above the top surface of the light is at a desired z level. This
desired z level will position the punches 152, 154 at an
appropriate height to penetrate the surface of block 130 at the
desired location, and to a desired depth.
[0070] It is advantageous if the tissue cylinders punched from
block 130 fit securely in the recipient receptacles that are formed
to receive them. If the donor punch 156 has the same inner and
outer diameters as the recipient punch 154, then the cylindrical
donor tissue specimen will be-formed by the inner diameter of the
punch, and the recipient receptacle will be formed by the outer
diameter of the punch. This discrepancy will provide a receptacle
that is slightly larger in diameter than the donor tissue cylinder.
Hence, as shown in FIGS. 19 and 20, the recipient punch 154
preferably has a smaller diameter than the donor punch 156.
Recipient punch will therefore form a cylindrical receptacle
(having a diameter corresponding to the outer diameter of punch
154) that is substantially the same diameter as the tissue specimen
cylinder 180, which is formed with a diameter that is determined by
the inner diameter of the donor punch 156.
[0071] FIG. 21 illustrates a cross-section through the recipient
array, once the receptacles 182 have been formed and filled with
tissue specimen cylinders 180. Small partitions of paraffin
material 122 separate tissue cylinders 180, and the receptacles 182
as illustrated are deeper than the specimen cylinders 180, such
that a small clearance is present between the specimen and the
bottom of the receptacles. Once the array has been formed, a
microtome can be used to cut a thin section S off the top of the
block 122, so that the section S can be mounted on a specimen slide
162 (FIG. 18) to help locate structures of interest in the tissue
specimen 132. The microtome then also cuts thin parallel sections
a, b, c, d, e, f, g, and h that can each be subjected to a
different molecular analysis, as already described
Exemplary Operating Environment
[0072] FIG. 22 and the following discussion are intended to provide
a brief, general description of a suitable computing environment in
which the invention may be implemented. The invention is
implemented in a variety of program modules. Generally, program
modules include routines, programs, components, data structures.
etc. that perform particular tasks or implement particular abstract
data types. The invention may be practiced with other computer
system configurations, including hand-held devices, multiprocessor
systems, microprocessor-based or programmable consumer electronics,
minicomputers, mainframe computers, and the like. The invention may
also be practiced in distributed computing environments where tasks
are performed by remote processing devices that are linked through
a communications network. In a distributed computing environment,
program modules may be located in both local and remote memory
storage devices.
[0073] Referring to FIG. 22, an operating environment for an
illustrated embodiment of the present invention is a computer
system 220 with a computer 222 that comprises at least one high
speed processing unit (CPU) 224, in conjunction with a memory
system 226, an input device 228, and an output device 230. These
elements are interconnected by at least one bus structure 232.
[0074] The illustrated CPU 224 is of familiar design and includes
an ALU 234 for performing computations, a collection of registers
236 for temporary storage of data and instructions, and a control
unit 238 for controlling operation of the system 220. The CPU 224
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.
[0075] The memory system 226 generally includes high-speed main
memory 240 in the form of a medium such as random access memory
(RAM) and read only memory (ROM) semiconductor devices, and
secondary storage 242 in the form of long term storage mediums such
as floppy disks, hard disks, tape, CD-ROM, flash memory, etc. and
other devices that store data using electrical, magnetic, optical
or other recording media. The main memory 240 also can include
video display memory for displaying images through a display
device. Those skilled in the art will recognize that the memory 226
can comprise a variety of alternative components having a variety
of storage capacities.
[0076] The input and output devices 228, 230 also are familiar. The
input device 228 can comprise a keyboard, a mouse, a scanner, a
camera, a capture card, a limit switch (such as home, safety or
state switches), a physical transducer (e.g., a microphone), etc.
The output device 230 can comprise a display, a printer, a motor
driver, a solenois, a transducer (e.g., a speaker), etc. Some
devices, such as a network interface or a modem, can be used as
input and/or output devices.
[0077] As is familiar to those skilled in the art, the computer
system 220 further includes an operating system and at least one
application program. The operating system is the set of software
which controls the computer system's operation and the allocation
of resources. The application program is the set of software that
performs a task desired by the user, using computer resources made
available through the operating system. Both are resident in the
illustrated memory system 226.
[0078] For example, the invention could be implemented with a Power
Macintosh 8500 available from Apple Computer, or an IBM compatible
Personal Computer (PC). The Power Macintosh uses a PowerPC 604 CPU
from Motorola and runs a MacOS operating system from Apple Computer
such as System 8. Input and output devices can be interfaced with
the CPU using the well-known SCSI interface or with expansion cards
using the Peripheral Component Interconnect (PCI) bus. A typical
configuration of a Power Macintosh 8500 has 72 megabytes of RAM for
high-speed main memory and a 2 gigabyte hard disk for secondary
storage. An IBM compatible PC could have a configuration with 32
megabytes of RAM for high-speed main memory and a 2-4 gigabyte hard
disk for secondary storage.
[0079] In accordance with the practices of persons skilled in the
art of computer programming, the present invention is described
with reference to acts and symbolic representations of operations
that are performed by the computer system 220, unless indicated
otherwise. Such acts and operations are sometimes referred to as
being computer-executed. It will be appreciated that the acts and
symbolically represented operations include the manipulation by the
CPU 224 of electrical signals representing data bits which causes a
resulting transformation or reduction of the electrical signal
representation, and the maintenance of data bits at memory
locations in the memory system 226 to thereby reconfigure or
otherwise alter the computer system's operation, as well as other
processing of signals. The memory locations where data bits are
maintained are physical locations that have particular electrical,
magnetic, or optical properties corresponding to the data bits.
Description of Computer-Array System
[0080] A block diagram showing a system for carrying out the
invention is shown at FIG. 23. The hardware is initialized at step
250, for example by determining the position of the punches 154,
156, bench 110, and tray 112. The system may then be configured by
the operator at step 252, for example by entering data or prompting
the system to find the location (x, y, z coordinates) of the upper
right corner of each recipient block 122-126, as well as the
locations of trays 130-136. The number of donor blocks,
receptacles, operating speed, etc. may also be entered at this
time.
[0081] At step 254, the system prompts for entry of identifying
information about the first donor block 130 that will be placed in
tray 128. This identifying information can include accession number
information, clinical information about the specimen, and any/or
other information that would be useful in analyzing the tumor
arrays. At step 256, the operator pushes a select function button,
which raises the punches 154, 156 and enables a joystick to move
the specimens using the x-y drives. The entered data is displayed
at step 258, and approved at 260
[0082] The system then obtains one or more donor specimens from the
identified donor block at step 262, and prompts the user for entry
of information about the next donor block. If information about
another block is entered, the system returns to step 256 and
obtains the desired number of specimens from the new block. After a
new donor block has been placed in donor container 128, the system
also checks the position of the punches at step 268. If information
about another block is not entered at step 264, the system moves
the donor tray to the reloading position so that a block 130 in the
donor tray can be removed. This system is also adaptable to
sampling cylindrical biopsies from histologically controlled sites
of specimens (such as tumors) for DNA/RNA isolation.
[0083] 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.
Examples of Array Technology
[0084] 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 repositories of
tissues from different transgenic animals or cultured cells. The
following specific examples illustrate some particular embodiments
of the invention.
EXAMPLE 1
Tissue Specimens
[0085] 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.
Histological grading was only performed 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
[0086] After formation of the 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.) and erbB-2 antigen
(60 minutes at 90.degree.) 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)
[0087] 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
criteria for gene amplification.
EXAMPLE 4
mRNA In Situ Hybridization
[0088] 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.
[0089] 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. Automation of the procedure permits efficient specimen
sampling and array formation into multiple tissue arrays, each
providing as many as 50, 100 or even up to 200 sections for
molecular analysis. 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.
[0090] In view of the many possible embodiments to which the
principles of our invention may be applied, it should be recognized
that the illustrated embodiments are preferred 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 We claim:
* * * * *