The Principles, Uses and Construction of Tissue
Microarrays in Pathology Research
Angelo M. De Marzo
and
Departments of
Pathology, The James Buchanan Brady Urological
Institute, and the
Supported by National Cancer Institute
Specialized Program of Research Excellence in Prostate Cancer Grant SPORE
in Prostate Cancer #P50CA58236 (
Presented
at the Short Course #47. “Gene Arrays and Tissue Arrays for
Pathologists” at The 92st Annual Meeting
of the United States and Canadian Academy of Pathology, March 27, 2003, Washington
D.C.
Introduction
Genomic approaches such as RNA profiling are providing a new powerful means to discover disease-related genes. One of the most challenging aspects presented by high throughput gene expression approaches is that they usually generate a large battery of potential targets. Determination of which genes are truly important for classification in terms of diagnosis, prognosis, and therapy represents a bottleneck. How does one begin to validate and prioritize potential targets? Often the first step in attempts to discover the disease relevance of a given gene is the elucidation of the precise cells that express it in normal and diseased human tissues. This is even more powerful if it can be done simultaneously with an assessment of clinical significance. A major limitation, however, is that in situ based molecular analysis is cumbersome and often limited by the availability of suitable reagents such as high quality antibodies or a robust system for in situ hybridization. In addition, adequate validation of biomarker expression often requires large patient cohorts with long-term clinical follow-up. Finally, interpretation of expression results requires a pathologist. While many of these limitations have yet to be solved, Tissue Microarrays (TMAs) are emerging as a breakthrough in our ability to rapidly analyze the expression of existing and new biomarkers using archival pathology specimens.
Multi-tissue blocks were first introduced by Battifora et al. in the so-called “sausage” or Multi-Tissue Tumor Block (MTTB) where up to 100 separate tissues were processed together into a single paraffin block1. Recently, Kononen et al. introduced a new method of combining multiple tissues into a single paraffin block that uses a novel sampling approach, with regular size and shaped tissues. This allows for many more specimens that are precisely arrayed to be inserted into the blocks2; for reviews see3-6.
What is a Tissue Microarray
and What are its Advantages Over Standard Tissue
Blocks?
The TMA consists of cylindrical paraffin embedded tissue cores that are acquired from primary “donor” blocks. The donor block is a standard tissue block that may be from surgical pathology, autopsy or research material. A morphologically representative area of interest within the donor block is identified under the microscope using a stained section (usually Hematoxylin and Eosin stained) on a glass slide as a guide. The tissue cores are removed from the donor and inserted into a “recipient” paraffin block using a custom patented instrument from Beecher Instruments. Using a precise spacing pattern, tissues are inserted at high density, with up to 1000 tissue cores in a single paraffin block. Sections from this block that are cut with a microtome are placed onto standard slides that can then be used for in situ analysis. Depending on the overall depth of tissue remaining in the donor blocks, tissue arrays can generate between 100 and 500 sections. Once constructed tissue microarrays can be used with a wide range of techniques including histochemical staining, immunohistochemical/immunofluorescent staining, or in situ hybridization for either DNA or mRNA.
Since relatively small areas of tissue (down to 0.6 mm in diameter) are obtained from the donor blocks, this method can help to expand the usefulness of existing archival paraffin blocks by facilitating the construction of multiple “duplicate” blocks. This significantly expands the capacity of the tissue samples, indicating that more studies can be performed using limited samples. Other advantages of TMAs are that they are designed for high throughput screening of expression, while providing uniform reaction conditions and multiple built-in potential positive and negative controls. Since only one or a few slides are subjected to the staining procedure, TMAs also allows one to economize use of reagents, which can at times be quite limiting. Since several hundred cases are now present on one or few slides, TMAs also cut down on microtome sectioning of numerous paraffin blocks. It should be pointed out that even after removal of cores from donor blocks, these donor blocks usually still retain sufficient residual tissue for adequate pathological interpretation.
Are Tissue Microarrays Valid for Clinico-Pathological Studies?
The most frequently asked question regarding tissue microarrays is how do they account for heterogeneity of tissues? Camp et al., examined the number of “disks” or TMA spots required to adequately represent the expression of three common antigens, estrogen receptor (ER), progesterone receptor (PR) and the Her2/neu oncogene, in 38 cases of invasive breast carcinoma7. They made TMAs containing 10 tissue cores from the same tumor and compared the results of analysis of staining of the TMA cores to that obtained using a single standard whole tissue section from which the TMA cores were derived. They found that two spots produced similar results to the whole tissue in more than 95% of the cases.
The largest published study to date
to address the issue of tissue heterogeneity is that of Torhorst et al. who
examined ER, PR and p53 in breast carcinoma8. In this study, the
clinical relevance of staining was examined by comparing immunostaining results
using standard sections versus TMA slides in 553 breast cancer patients. Four
high-density TMA blocks were constructed, each containing 1 core from a
different region of the tumor from each patient. For ER, the range of positive
staining from the 4 different blocks was from 78.9 to 80.8%, which compares to
that observed in a large standard section (79.8%). When using a single 0.6 mm
sample, loss of ER correlated inversely with disease specific survival to a
similar extent as standard sections.
Thus, very little benefit was obtained using more than one spot for ER.
For PR, the addition of multiple spots analyzed increased the frequency of
positive staining towards that obtained with standard sections (41.1% with 1
spot, 53.1% with 4 spots, compared with 60.3% with conventional sections). Loss of PR as analyzed on TMA spots was also
predictive of poor outcome in a manner similar to that of the conventional
section, even when only 1 0.6 mm core was used. For p53, the frequency of
positive staining was less using TMAs than when using standard slides
(15.2-20.9% for single spots, up to 24.1% for all 4 combined, as compared to
42.8% for conventional sections). However, in terms of prognostic significance
the correlation between p53 staining and poor outcome was stronger using TMA
spots, even one spot, than was that of conventional sections.
In terms of prostate cancer, Rubin et al. used digital image analysis (CAS200, Bacus Labs, Lombard, IL) on TMAs containing 10 replicate tumor samples from 88 cases of prostate cancer9 to evaluate Ki-67 expression for each case. Four cores provided the optimal sampling for TMA cores using a Cox proportional hazards analysis to determine predictors of time until PSA recurrence following radical prostatectomy for clinically localized prostate cancer. Fewer TMA samples significantly increased Ki-67 variability and a larger number did not significantly improve accuracy.
Several
other studies have also examined the question of the representation of tissues
in TMAs using various markers in different tumor types10,11. In
general, although they vary somewhat in terms of the recommendations for
sampling, all studies indicate there is usually excellent agreement between the
use of TMAs and standard tissue sections for clinico-pathological studies.
From a
theoretical point of view the question of how many samples are required to
adequately perform a study is related to the variability of the parameter being
analyzed. Thus, for homogeneous markers, a single TMA spot per case will be
adequate. At our institution we routinely take 4 cores each from areas of
prostate tumors and matched normal tissue (Fig. 1) in order to maximize the
usefulness of the TMA since we do not know what biomarkers we will be applying
in the future. Thus for some studies, this will be “overkill” and for others it
may be barely adequate.
Another
potential difficulty in terms of how many cores to take involves the fact that
not all TMA cores will be present on all TMA slides. Having at least one additional TMA core will
help ensure the presence of the number of cores that one hopes to obtain on the
final TMA slide.
Digital Image Acquisition and Analysis
TMA slides can be viewed under conventional microscopes. In this case a key, usually in the form of a spreadsheet, corresponding to the x y coordinate system is used and histopathological diagnoses and interpretations are recorded. The data can be recorded on paper for later entry into a spreadsheet or database, or, it can be entered directly into the computer. One of the difficulties with this approach is that since the array spots do not have their coordinates printed on the slide it is likely that the user may loose track of the x and y coordinates of given spots and have to repeatedly become reoriented.
Prostate SPORE Approach to
Imaging
Several groups have been developing methods to acquire and archive digital images of the TMA spots for evaluation on a computer monitor such that the data is linked to underlying clinico-pathological information regarding the array spot. To acquire digital images of TMA spots a number of users have been using the Bacus Labs Incorporated Slide Scanner (BLISS, Bacus™ laboratories, Lombard, IL)12 as previously described13,14. The BLISS imaging system consists of a Zeiss microscope equipped with a software driven motorized stage, integrated digital video camera, and a customized personal computer running Microsoft Windows. The Bacus Labs Tracer software program is designed to scan entire glass microscope slides, or any part of the slide, using any available microscope objective. The slides can then be viewed using the free downloadable WebSlideÒ Viewer. Slide images are generally stored on a server
running WebSlide Server software
from Bacus Labs.
The system has been adapted to scan TMA spots using customizable features that were developed in collaboration with the Prostate SPORE Tissue Microarray Working Group (University of Michigan, Baylor College of Medicine, and Johns Hopkins University School of Medicine)13,14. The operator then indicates through the software the number and location of the array spots. All array spots are then automatically scanned at full resolution (in most cases a 20x Zeiss Plan-Apochromat® objective is used, although other objectives can be used). Each array spot is imaged as 6 individual 640 x 480 pixel images that the software automatically “tiles” into a single composite image. The composite images are stored in a file containing the embedded x y coordinates from the tissue array spot, along with user provided information regarding the TMA slide that was scanned. The composite image files can be viewed individually using any number of image viewers, or can be imported into a relational database and related by their x y coordinates to the specimen from which they were derived (Fig. 2). More recently, Bacus Labs have developed an Active X plug-in that is designed to facilitate image handling for viewing TMA spots directly from inside a database application of your choosing. Several other systems that are either being adapted or are potentially adaptable to imaging TMA spots are shown in Table 1.
|
Company/University |
Product |
Website |
|
Bacus Labs |
BLISS |
|
|
Yale |
Spotfinder and Aqua
|
|
|
Chromavision |
ACIS
|
|
|
MicroBrightField Inc. |
Virtual Slice Module |
|
|
CompuCyte |
Laser Scanning Cytometer |
|
|
TissueInformatics |
Quant f(x) platform |
Table 1. Digital
microscopic imaging systems that already adapted for TMAs, are in the process
of adapting, or may be adaptable for TMA scanning.
TMA Clinico-Pathological and Image Data Handling
TMA-based technology prompted the need for a system that effectively managed data generated from this high-throughput approach. The use of a large spreadsheet has been the standard solution to handle voluminous amounts of data. This approach is useful for experiments on a one-time basis, but becomes very cumbersome when analyzing multiple markers on a given specimen or when having multiple observers render diagnoses and scores on a given specimen. As part of the same on-going collaborations between the Specialized Programs in Research Excellence for prostate cancer, the three groups from the University of Michigan, Johns Hopkins University, and Baylor College of Medicine (Houston, TX) have been developing systems to manage TMA clinical data and TMA image data14,15. The overall architecture of the system is that the TMA images are examined on a computer screen where the image is presented within a database form. The user then enters data regarding the image directly into the form. The type of data that is recorded is flexible and can include items such as image quality, diagnosis, and immunohistochemical scoring. An example of one such database form is shown in Fig. 2. A demonstration edition of this software, referred to as TMA-J can be found at (http://tmaj.pathology.jhmi.edu/).
Another system, also based on
scanning TMA spots using the BLISS system, has been presented by the Stanford
group16. The approach was to develop
two software programs to aid in analysis of staining results and rapid
retrieval of TMA spot digital images.
The authors designed a program that allows for rapid transformation of
immunostained data, recorded in Microsoft Excel, into a format that can be used
for cluster analysis. The program Cluster is used to group data with regards to
tumor staining pattern with antibodies, analogous to tumors grouped according
to RNA expression. The Clustered pattern can be viewed in another freely
available software program called Treeview. For an online demonstration, see (http://genome-www.stanford.edu/TMA/explore.shtml).
At Yale University David L. Rimm’s group has developed software that uses a custom imaging microscope system for scanning TMA slides that are stained with fluorescent markers17. Trotter et al. have presented at this meeting last year on mapping, navigation and data management of TMA data18.
Quantification of Immunohistochemical Staining Using TMA
At present modules for quantification of TMA results using the Bacus system are under development. A commercial system for imaging TMA slides has been developed by Chromavision as an extension of their Automated Cellular Imaging System (ACIS)19. The system is designed specifically for quantification of immunohistochemical staining using images obtained by light microscopy. In this system, the user examines a low power image of the scanned TMA slide and selects the region of interest to view at higher power. Next a region on the higher power view is selected for further automated analysis of the staining results such as area of positive staining as well as intensity of staining. The data is then exportable to spreadsheets or database management systems.
In terms of academic institutions, Camp
et al., at
Special Array Types
Frozen TMAs
At least two groups have so far produced TMAs using frozen tissues20,21. The advantage to having frozen tissue arrays is that post fixation can be tightly controlled, some antibodies do not bind to formalin fixed epitopes, and the quality of nucleic acids (DNA/RNA) is generally much higher. Hoos and Cordon-Cardo have developed a simple devise, independent of the Beecher Instruments devise for frozen TMA construction20 and Fejzon have adapted the Beecher Instrument machine using dry ice to keep the donor and recipient blocks frozen 21.
Cell lines
One of the most powerful types of controls for immunohistochemical staining and for in situ hybridization is the use of well-defined cell lines. For example, when one is working up a new antibody against the retinoblastoma protein (pRB), an excellent negative control would be a retinoblastoma cell line that was shown to be genetically null for RB alleles. Similar approaches are useful for p53, etc. In fact, when one knows the status of either the genomic DNA corresponding to a given gene in a given cell line, or information about the expression at the mRNA and/or protein level, then suitable positive and negative controls can be obtained for immunohistochemistry or in situ hybridization for essentially all non-house keeping genes. Along these lines, a very useful type of control is to use an isogenic system to induce expression of a given gene in a cell line that does not normally express it. In this case the untreated cell serves as the negative control and the treated cell serves as the positive control. We recently used this approach to develop controls for examining COX-2 expression in prostate cancer cells where we induced expression of COX-2 using phorbol ester in PC3 cells22. We have been isolating cells grown in culture, fixing them in formalin, embedding them in agarose, and then submitting them for routine processing into paraffin23 (see below for detailed method) The advantage of a solid-like gel suspension such as agarose is that the cells are not lost in processing, which often happens when preparing paraffin blocks from cell pellets. A manuscript comparing methods of embedding cells in culture for TMA production has recently been published24.
Obtaining TMA Slides
For those seeking to obtain slides from existing tissue arrays, there is a NIH program called Tissue Array Research Program (TARP) where individual slides are available for purchase at very reasonable rates25.
There are
also several commercial sources including Research Genetics
(VastArray™
Tissue Arrays)26, Zymed Laboratories (MaxArray™)27; and SuperBioChips28.
Tissue Fixation Issues
One of the most important issues in constructing tissue microarrays is to be sure of the quality of the tissues used. While there is no best fixative for all types of applications, the vast majority of archival specimens have been fixed in 10% neutral buffered formalin, which is actually contains 4% formaldehyde among other chemicals. While many antibodies produce excellent staining using formalin fixed tissues, not all tissues are properly fixed after “routine fixation”. We have found for p27Kip1 that longer fixation times yield more reliable results29. For construction of our prostate TMAs we typically only use tissues that we are certain of the quality of fixation; we use either tissues that were freshly harvested and sectioned into thin portions (less than or equal to 3 mm thick) that are fixed in large volumes, or those where the prostates have been injected with formalin to provide uniform fixative coverage30. In addition, all blocks are subjected to immunohistochemical staining prior to selection for a TMA.
New Target Validation
Our approach at John Hopkins to new
target identification and validation has been to discover genes that are highly
over expressed in prostate cancer and attempt to validate expression using TMAs31. In collaboration with
Other Potential Type of TMAs
The types of tissues one can use for construction of TMAs is unlimited. For example those consisting of human xenograft tumors may greatly extend the ability of many different investigators to have access to these tissues. In addition, animal tissues as well as xenografts can be subjected to drug treatments in vivo and then the pattern of gene expression alterations can be documented using cDNA arrays and/or TMAs. Similar types of studies with human tissue can be obtained and tissues can be arrayed before and after treatment. The number of cell lines is also growing rapidly and a resource that provides TMAs containing many cell lines would be very valuable.
Practical Methods of TMA
Construction
The following is largely derived from a book chapter that will appear in the Series: Practical Methods In Molecular Biology by Humana Press.
Several sources of information are available for tissue microarray protocols, tips techniques, and trouble-shooting. These include recent reviews