Tissue Microarrays in Clinical Oncology

doi:10.1016/j.semradonc.2007.10.006

Seminars in Radiation Oncology

Volume 18, Issue 2, April 2008, Pages 89-97

https://doi.org/10.1016/j.semradonc.2007.10.006 Get rights and content

Tissue microarray (TMA) is a recently implemented, high-throughput technology for the analysis of molecular markers in oncology. This research tool permits the rapid assessment of a biomarker in thousands of tumor samples, using commonly available laboratory assays such as immunohistochemistry and in situ hybridization. Although introduced less than a decade ago, TMA has proven to be invaluable in the study of tumor biology, the development of diagnostic tests, and the investigation of oncologic biomarkers. This review describes the impact of TMA-based research in clinical oncology and its potential future applications. Technical aspects of TMA construction and the advantages and disadvantages inherent to this technology are also discussed.

Section snippets

TMA Construction and Analysis

The initial identification and collection of tumor samples represents the greatest portion of the work associated with TMA construction. Samples need to be identified based on their availability in sufficient numbers to address the proposed scientific or clinical question. For example, prognostic studies will require a large number of cases with long-term outcome data to provide adequate statistical power. Similarly, a study investigating a novel diagnostic biomarker may require the

Advantages of TMAs

The staining of a few TMA sections in comparison to many more whole sections offers a clear benefit with respect to the use of laboratory reagents and technician time. Because several research groups are now using TMAs representing in excess of 1,000 tumors, this approach represents a major savings in scientific resources. In addition, there is also the benefit of decreased technical variability during the staining and interpretation process. The close proximity of cores also permits more rapid

TMAs in the Study of Tumor Biology

TMAs permit the rapid assessment of individual molecular markers on large patient cohorts. This approach complements molecular screening and discovery studies by confirming results on large numbers of primary tumor cases. TMAs have been so used in the initial articles characterizing new breast cancer oncogenes such as EMSY³ and alpha-basic crystallin⁴ and the recently identified prostate cancer fusion oncogene TMPRSS2:ERG.⁵

TMAs are similarly useful in characterizing the immunohistochemical

TMAs for the Assessment of New Diagnostic Tools

In modern oncology, treatment decisions are critically dependent on accurate diagnostic pathology. Increasingly, molecular biomarkers are used in conjunction with conventional histology to improve diagnostic accuracy. Examples include the use of cytokeratin stain to localize micrometastases in sentinel lymph node biopsies for breast cancer and the use of antibody panels to ascertain a tissue diagnosis for a metastasis of unknown primary origin. Primary unknown malignancies are particularly

TMAs for the Assessment of Prognostic and Predictive Values

In the clinical practice of oncology, therapeutic decisions regarding adjuvant treatment are often based on a clinician’s estimate of recurrence risk and the expected therapeutic gain from a specific treatment. More recently, clinicians have at their disposal prognostic models that are based on the retrospective analysis of large outcome databases. Examples include the Kattan nomograms for prostate cancer,²¹ and the Web-based prediction tool “Adjuvant! Online” for breast and colorectal

TMAs in Quality Control and Clinical Practice

Although TMA studies are an important tool for the validation of novel biomarkers, most molecular epidemiology studies are derived from a single institution, and even if the tumor samples are collected from multiple sites, sample processing and data collection and analysis are performed centrally. There are multiple reports in the medical literature of conflicting results on the prognostic or predictive value of a novel biomarker. One example is cyclin E in breast cancer, for which 1 research

Validation of TMA Analysis

The most frequent criticism of TMA technology relates to the small size of each tissue core; there is concern that because of tumor heterogeneity, biomarker scores obtained from small TMA cores will not accurately reflect scores obtained from whole sections.43, 51, 52 However, it should be noted that although whole-section analysis is a “gold standard” for in situ tests, whole sections themselves represent just a small portion of a tumor, and, indeed core needle, punch, and bite biopsies used

Conclusion and Future Directions

The most important step in initiating a TMA study is the identification of appropriate patient cohorts with accessible archival tissue materials. Prognostic studies require patient data that are thorough and complete and include long-term follow-up. Diagnostic studies may require locating source blocks from multiple tumor types and tissues in various stages of malignant progression.

In laboratory medicine, TMAs already play an important role in quality assurance and the characterization of new

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Depression is associated with a higher ovarian cancer risk. Prior work suggests that depression can lead to systemic immune suppression, which could potentially alter the anti-tumor immune response.
We evaluated the association of pre-diagnosis depression with features of the anti-tumor immune response, including T and B cells and immunoglobulins, among women with ovarian tumor tissue collected in three studies, the Nurses’ Health Study (NHS; n = 237), NHSII (n = 137) and New England Case-Control Study (NECC; n = 215). Women reporting depressive symptoms above a clinically relevant cut-point, antidepressant use, or physician diagnosis of depression at any time prior to diagnosis of ovarian cancer were considered to have pre-diagnosis depression. Multiplex immunofluorescence was performed on tumor tissue microarrays to measure immune cell infiltration. In pooled analyses, we estimated odds ratios (OR) and 95% confidence intervals (CI) for the positivity of tumor immune cells using a beta-binomial model comparing those with and without depression. We used Bonferroni corrections to adjust for multiple comparisons.
We observed no statistically significant association between depression status and any immune markers at the Bonferroni corrected p-value of 0.0045; however, several immune markers were significant at a nominal p-value of 0.05. Specifically, there were increased odds of having recently activated cytotoxic (CD3⁺CD8⁺CD69⁺) and exhausted-like T cells (CD3⁺Lag3⁺) in tumors of women with vs. without depression (OR = 1.36, 95 %CI = 1.09–1.69 and OR = 1.24, 95 %CI = 1.01–1.53, respectively). Associations were comparable when considering high grade serous tumors only (comparable ORs = 1.33, 95 %CI = 1.05–1.69 and OR = 1.25, 95 %CI = 0.99–1.58, respectively). There were decreased odds of having tumor infiltrating plasma cells (CD138⁺) in women with vs. without depression (OR = 0.54, 95 %CI = 0.33–0.90), which was similar among high grade serous carcinomas, although not statistically significant. Depression was also related to decreased odds of having naïve and memory B cells (CD20⁺: OR = 0.54, 95 %CI = 0.30–0.98) and increased odds of IgG (OR = 1.22, 95 %CI = 0.97–1.53) in high grade serous carcinomas.
Our results provide suggestive evidence that depression may influence ovarian cancer outcomes through changes in the tumor immune microenvironment, including increasing T cell activation and exhaustion and reducing antibody-producing B cells. Further studies with clinical measures of depression and larger samples are needed to confirm these results.
Reliable Gene Expression Profiling from Small and Hematoxylin and Eosin–Stained Clinical Formalin-Fixed, Paraffin-Embedded Specimens Using the HTG EdgeSeq Platform
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It has been shown herein that the EdgeSeq platform (HTG Molecular Diagnostics, Inc.) can generate reliable expression data from as little as 1 mm2 FFPE tissue and crude FFPE tissue lysates equivalent to surface areas as low as 0.31 mm2 of a section (5 μm thick). This is approximately half the size of a small tissue microarray core,62 and hundreds or even thousands of times smaller than that used for analysis by alternative methods.49,59–61 Thus, the requirement for tissue with an equivalent surface area of at least 0.31 mm2 does not represent a significant limitation of this method.
Clinical biomarker studies are often hindered by the scarcity or suboptimal quality of biological specimens. EdgeSeq, a transcriptomics analysis platform, combines quantitative nuclease protection assay technology with next-generation sequencing, using small amounts of starting material and delivering reproducible gene expression profiles from challenging material, such as formalin-fixed, paraffin-embedded (FFPE) tissue. To evaluate EdgeSeq for analysis of archives of stained FFPE tissue, EdgeSeq was performed on unstained, hematoxylin and eosin (H&E)–stained, and immunohistochemistry-stained slides from patients with small-cell and non–small-cell lung cancer. Pairwise comparisons of gene expression profiles from stained and unstained slides showed higher Pearson correlation coefficients with H&E staining (0.86 to 0.97) than with immunohistochemistry staining (0.21 to 0.56). A 25-gene interferon-γ signature score from unstained slides showed a Pearson correlation coefficient of 0.92 with H&E-stained slides and a significant Spearman correlation (P = 0.0025) with immune scores. To test gene expression profiling in small samples, FFPE sample equivalents were examined from 5.0 to 0.08 mm² of a section (5 μm thick); sample equivalents ≥0.31 mm² showed alignment rates >69% and pairwise Pearson correlation coefficients ≥0.87. EdgeSeq can, thus, be used to profile small and H&E-stained FFPE tumor specimens to obtain biomarker data from limited tissue in oncology clinical trials and enable research into tumor microenvironment and immune cell engagement with tumors at the locoregional level.
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Organ-confined muscle-invasive bladder cancer is treated with cystectomy or bladder preservation techniques, including radiation therapy. There are currently no biomarkers to inform management decisions and aid patient choice. Previously we showed high levels of MRE11 protein, assessed by immunohistochemistry (IHC), predicted outcome after radiation therapy, but not cystectomy. Therefore, we sought to develop the MRE11 IHC assay for clinical use and define its relationship to clinical outcome in samples from 2 major clinical trials.
Samples from the BCON and BC2001 randomized controlled trials and a cystectomy cohort were stained using automated IHC methods and scored for MRE11 in 3 centers in the United Kingdom.
Despite step-wise creation of scoring cards and standard operating procedures for staining and interpretation, there was poor intercenter scoring agreement (kappa, 0.32; 95% confidence interval, 0.17-0.47). No significant associations between MRE11 scores and cause-specific survival were identified in BCON (n = 132) and BC2001 (n = 221) samples. Reoptimized staining improved agreement between scores from BCON tissue microarrays (n = 116), but MRE11 expression was not prognostic for cause-specific survival.
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Concordance of HER2 status between adequate biopsy samples and resection is good if adequate tumor fragments are included in the biopsy sample.23,26 A tissue microarray is an alternative to reduce the cost of antibodies in research setting, although they are not suitable for clinical testing.35 The main drawback of using tissue microarray is difficulty in avoiding false-negative results because of heterogeneity in HER2 amplification and protein expression.
Human epidermal growth factor receptor 2 (HER2) status determines gastric/gastroesophageal junction (GEJ) adenocarcinomas that benefit from targeted therapy; hence, HER2 testing has become a routine practice. Accurate HER2 testing is fundamental to select eligible patients who will benefit from HER2-targeted treatment. The reported HER2-positive rate in gastric/GEJ cancers ranges from 4.4% to 53.4%, and HER2-positive tumors are considered to have more-aggressive biologic behavior and tumor recurrence. Main modalities of HER2 testing in clinical practice include immunohistochemistry (IHC) for protein expression and in situ hybridization (ISH) for gene amplification. Many technical pitfalls affect the accuracy of HER2 result. Additionally, several issues in HER2 testing are related to the tumor biology, sample selection, interpretation of IHC and ISH results, and confirming HER2 status. Therefore, gastric/GEJ adenocarcinoma-specific HER2 testing protocols have been developed and standardized to minimize the impact of these preanalytical and analytical factors and to enhance reproducibility of HER2 testing results. This review provides up-to-date practical guidance to clinicians on accurate HER2 testing and interpretation of results in gastric/GEJ adenocarcinoma.
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: David Voduc and Torsten O. Nielsen are supported by the National Institutes of Health Strategic Partnering to Evaluate Cancer Signatures program. Torsten O. Nielsen is a scholar of the Michael Smith Foundation for Health Research. The Genetic Pathology Evaluation Centre is supported by an unrestricted educational grant from sanofi-aventis.

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Tissue Microarrays in Clinical Oncology

Section snippets

TMA Construction and Analysis

Advantages of TMAs

TMAs in the Study of Tumor Biology

TMAs for the Assessment of New Diagnostic Tools

TMAs for the Assessment of Prognostic and Predictive Values

TMAs in Quality Control and Clinical Practice

Validation of TMA Analysis

Conclusion and Future Directions

Mod Pathol

Cell

Hum Pathol

Am J Pathol

J Thorac Cardiovasc Surg

Blood

Mod Pathol

Hum Pathol

Pathology

Pathology

Exp Hematol

Am J Pathol

Lab Invest

Mod Pathol

Tissue microarrays for high-throughput molecular profiling of tumor specimens

Nat Med

AlphaB-crystallin is a novel oncoprotein that predicts poor clinical outcome in breast cancer

J Clin Invest

Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer

Science

Histopathological features of breast tumours in BRCA1BRCA2 and mutation-negative breast cancer families

Breast Cancer Res

Phenotypic characterization of BRCA1 and BRCA2 tumors based in a tissue microarray study with 37 immunohistochemical markers

Breast Cancer Res Treat

The androgen receptor is significantly associated with vascular endothelial growth factor and hypoxia sensing via hypoxia-inducible factors HIF-1aHIF-2a, and the prolyl hydroxylases in human prostate cancer

Clin Cancer Res

Colorectal cancer prevention

J Clin Oncol

Efficacy of screening mammographyA meta-analysis

JAMA

Comparison of annexin II, p63 and AMACR immunoreactivity in prostatic tissue: A tissue microarray study

J Clin Pathol

FLI-1 Expression in 4323 malignant and benign tumours: A multiple tumour tissue microarray analysis using polyclonal antibody

J Clin Pathol

Utility of syndecan-1 (CD138) expression in the diagnosis of undifferentiated malignant neoplasms: A tissue microarray study of 1,754 cases

Appl Immunohistochem Mol Morphol

Immunohistochemical staining for p63 is useful in the diagnosis of anal squamous cell carcinomas

Am J Surg Pathol

P63 expression in lung carcinoma: A tissue microarray study of 408 cases

Appl Immunohistochem Mol Morphol

TLE1 as a diagnostic immunohistochemical marker for synovial sarcoma emerging from gene expression profiling studies

Am J Surg Pathol