“Tissue micro array technology for high-throughput molecular profiling of cancer.”

Kallioniemi, O. P., Wagner, U., Kononen, J., and Sauter, G. Hum Mol Genet, 10:
657-662, 2001.
· See this paper for a review of the technology and discussion of applications and
limitations.

Abstract: Tissue microarray (TMA) technology allows rapid visualization of moleculartargets in thousands of tissue specimens at a time, either at the DNA, RNA or proteinlevel. The technique facilitates rapid translation of molecular discoveries to clinical applications. By revealing the cellular localization, prevalence and clinical significance of candidate genes, TMAs are ideally suitable for genomics-based diagnostic and drug target discovery. TMAs have a number of advantages compared with conventional techniques. The speed of molecular analyses is increased by more than 100-fold, precious tissues are not destroyed and a very large number of molecular targets can be analyzed from consecutive TMA sections. The ability to study archival tissue specimens is an important advantage as such specimens are usually not applicable in other highthroughput genomic and proteomic surveys. Construction and analysis of TMAs can be automated, increasing the throughput even further. Most of the applications of the TMA technology have come from the field of cancer research. Examples include analysis of the frequency of molecular alterations in large tumor materials, exploration of tumor progression, identification of predictive or prognostic factors and validation of newly discovered genes as diagnostic and therapeutic targets.

“Delineation of prognostic biomarkers in prostate cancer”

Nature, 412: 822-826, 2001 Dhanasekaran, S. M., Barrette, T. R., Ghosh, D., Shah, R.,
Varambally, S., Kurachi, K., Pienta, K. J., Rubin, M. A., and Chinnaiyan, A. M.

Many researchers have used tissue micro arrays in combination with DNA micro arrays to validate the leads discovered by gene expression profiling. This is one of the most comprehensive studies describing this strategy.
Abstract: Prostate cancer is the most frequently diagnosed cancer in American men.Screening for prostate-specific antigen (PSA) has led to earlier detection of prostate cancer, but elevated serum PSA levels may be present in non-malignant conditions suchas benign prostatic hyperlasia (BPH). Characterization of gene-expression profiles that molecularly distinguish prostatic neoplasms may identify genes involved in prostate carcinogenesis, elucidate clinical biomarkers, and lead to an improved classification of prostate cancer. Using microarrays of complementary DNA, we examined geneexpression profiles of more than 50 normal and neoplastic prostate specimens and three common prostate-cancer cell lines. Signature expression profiles of normal adjacent prostate (NAP), BPH, localized prostate cancer, and metastatic, hormone-refractory prostate cancer were determined. Here we establish many associations between genes and prostate cancer. We assessed two of these genes-hepsin, a transmembrane serine protease, and pim-1, a serine/threonine kinase-at the protein level using tissue microarrays consisting of over 700 clinically stratified prostate-cancer specimens. Expression of hepsin and pim-1 proteins was significantly correlated with measures of clinical outcome. Thus, the integration of cDNA microarray, high-density tissue microarray, and linked clinical and pathology data is a powerful approach to molecular profiling of human cancer.

“Validation of tissue micro array technology in breast carcinoma.”
Camp, R. L., Charette, L. A., and Rimm, D. L. Lab Invest, 80: 1943-1949, 2000.

One of the early technology validation papers addressing the effects of tissue heterogeneity and age of the archival specimens on the utility of the technique.
Abstract: The recent development of tissue microarray technology has potentiated largescale retrospective cohort studies using archival formalin-fixed, paraffin-embedded tissues. A major obstacle to broad acceptance of microarrays is that they reduce the amount of tissue analyzed from a whole tissue section to a disk, 0.6 mm in diameter, that may not be representative of the protein expression patterns of the entire tumor. In this study, we examine the number to disks required to adequately represent the expression of three common antigens in invasive breast carcinoma--estrogen receptor, progesterone receptor, and the Her2/neu oncogene--in 38 cases of invasive breast carcinoma. We compared the staining of 2 to 10 microarray disks and the whole tissue sections from which they were derived and determined that analysis of two disks is comparable to analysis of a whole tissue section in more than 95% of cases. To evaluate the potential for using archival tissue in such arrays, we created a breast cancer microarray of 8 to 11 cases from each decade beginning in 1932 to the present day and evaluated the antigenicity of these markers and others. This array demonstrates that many proteins retain their antigenicity for more than 60 years, thus validating their study on archival tissues. We conclude that the tissue microarray technique, with 2-fold redundancy, is a valuable and accurate method for analysis of protein expression in large archival cohorts.

“Tissue micro arrays for high-throughput molecular profiling of tumor specimens.”
Kononen, J., Bubendorf, L., Kallioniemi, A., Barlund, M., Schraml, P., Leighton, S.,
Torhorst, J., Mihatsch, M. J., Sauter, G., and Kallioniemi, O. P.

This paper describes the construction and use of tissue microarrays for the first time.
Demonstrates the use of tissue microarrays in FISH, IHC, and mRNA ISH  applications and introduces the concept of using tissue microarrays to generate large
databases of marker data from tumor populations.
Abstract: Many genes and signalling pathways controlling cell proliferation, death and
differentiation, as well as genomic integrity, are involved in cancer development. New
techniques, such as serial analysis of gene expression and cDNA microarrays, have enabled measurement of the expression of thousands of genes in a single experiment,
revealing many new, potentially important cancer genes. These genome screening tools can comprehensively survey one tumor at a time; however, analysis of hundreds of specimens from patients in different stages of disease is needed to establish the
diagnostic, prognostic and therapeutic importance of each of the emerging cancer gene candidates. Here we have developed an array-based high-throughput technique that facilitates gene expression and copy number surveys of very large numbers of tumors. As many as 1000 cylindrical tissue biopsies from individual tumors can be distributed in a single tumor tissue microarray. Sections of the microarray provide targets for parallel in situ detection of DNA, RNA and protein targets in each specimen on the array, and consecutive sections allow the rapid analysis of hundreds of molecular markers in the same set of specimens. Our detection of six gene amplifications as well as p53 and estrogen receptor expression in breast cancer demonstrates the power of this technique for defining new subgroups of tumors.