Introduction to Tissue Microarrays
Kononen first described the tissue microarray (TMA) in 1998, It represents a high-throughput technology that is used in the assessment of histology-based laboratory tests such as immunohistochemistry and fluorescent in-situ hybridization (FISH). To facilitate the rapid analysis of many patient samples, small cylindrical cores are taken from standard formalin-fixed and paraffin embedded tissue. It is then arranged in a matrix configuration in a recipient paraffin block. Since TMA has been introduced, this technique has been used in the study of tumor biology, assessment of novel molecular biomarkers, and quality assurance. This technique serves as a great validation and translation platform for different high-throughput molecular research.
TMA Construction and Analysis
- Identification and collection of samples represents the biggest portion of work associated with TMA construction.
- To address the scientific question proposed, the samples are required to be identified based on the availability.
- Archived tissue blocks are then retrieved.
- The best area to extract the core is determined.
- A sector map is used to guide the assembly and scoring.
- A "tissue microarrayer" is required for the physical construction of TMA.
- A core is then removed from a blank paraffin “recipient” block where core sizes range from 0.6-2.0mm.
- A tissue core is inserted in the hole created in the recipient block.
- TMA sectioning is performed using a traditional microtome.
- The number of sections available from each TMA block can very depending on the depth of donor blocks, histotechnologist skill, and thickness of each individual section.
- Although harsher techniques can cause tissue to detach from the TMA slide, immunohistochemistry or other applications can also be done on TMAs with minimum changes to standard protocols.
- The result of each scoring is recorded on the sector map.
- To reduce the potential for bias, TMA scoring is performed blinded to linked clinicopathological data.
- With the help of publicly available software, scores from the sector map can be merged with clinicopathological data.
- Savings – Some research groups are now using TMAs and this can be a great advantage as it represents more than a thousand tumors.
- Variability – there is less technical variability for the staining and interpretation process.
- Time – TMA is time-saving as an entire suite of biomarker studies can be finished in a few weeks compared to several months with whole sections.
- Material – Since TMA uses formalin fixed, paraffin embedded tissues, this is the most common method in the preservation of surgical specimens meaning source material is readily available and usually linked to long-term outcome data.
- Accuracy – In TMAs, the core selected is microscopic guided to ensure that the representative areas of tumors are sampled while avoiding areas of normal tissue and necrosis. Interpretation is also performed with morphologic correlation.
- Ethics – Since the technique does not require modified or additional procedures on patients, TMA technology raises minimal ethical concerns.
TMAs: Tumor Biology
- TMAs allow fast evaluations of individual molecular markers in big cohort studies.
- By checking and approving the results of large numbers in primary tumor cases, it complements molecular screening and discovery studies.
- The immunohistological profile for subtypes of cancer can be characterized using TMAs. For example, to find out the molecular profile of the BRCA ½ breast cancer using a large panel of antibodies, BRCA 1 tumors were found to be positive for hormone receptors, p53, negative for HER2, and expresses a particular set of cell cycle antigens.
- The insight of the biochemical aberrations leading to malignant progression was provided by identification of new molecular markers. This leads to the development of potential specific targeted therapies.
- Cores from progression TMAs demonstrates stages of neoplasia and are helpful in the development of diagnostic assays.
- For some cancers, accumulating evidence shows that screening can lower the incidence and mortality of cancers. For example, recent research reported that the expression of PCNA can be used to differentiate between esophageal adenocarcinoma and Barrett’s esophagus.
TMAs: Assessment of New Diagnostic Tools
- Treatment in modern oncology largely relies on the accuracy of diagnostic pathology by using molecular biomarkers and conventional histology.
- In primary unknown malignancies, it can be challenging as biopsies taken are usually poorly differentiated and hard to diagnose.
- The diagnostic accuracy of a biomarker can be estimated using large multi-tumor arrays.
- New diagnostic biomarkers that is identified using gene expression profiling can be rapidly tested through TMA technology to determine the practical diagnostic value of sarcomas and lymphomas. This is important as both sarcomas and lymphomas can be extremely difficult to subtype through purely morphologic approaches.
TMAs: Assessment of Prognostic and Predictive Value
In clinical oncology, treatment decisions are based on the probable risk of recurrence and potential benefits from a specific treatment. Now, clinicians are using disposal prognostic models based on retrospective analysis of large outcome databases. However, these models rely on standard clinic-pathological features that were used during initial data collection. Therefore, cohorts that have mature outcome data used in data building usually do not contain important biomarker information that is now routinely collected. For example, HER2 is currently known as an important prognostic factor in breast cancer but in the SEER data, there is absence of HER2 status. Since TMA construction utilizes standard archived tissues, it can be used to reanalyze the SEER data, making it a great and valuable technique.
The number of potentially useful biomarkers are increasing, and the expression of countless genes can be studied using DNA microarray technology. However, due to high cost and low tissue availability, the application of DNA microarray technology is limited in archival tissue libraries. TMA is a great substitute as it only requires a small core from the archival block where each core can be used to evaluate multiple biomarkers. Data acquired can be interpreted and its outcome applied in large patient cohorts. Results from TMA can also be easily validated using readily available IHC and in-situ hybridization methods. The sole disadvantage of TMA is the necessity of assessing each biomarker on its own, thus making it unsuitable as a discovery tool.
- To find clinically useful biomarkers, 2 steps are used:
- Discovery: This phase screens many molecular markers and to seek out biomarkers linked to clinically relevant endpoints.
- Validation: Protein products in the genes of top candidates can be assayed in patient cohorts using TMAs.
TMAs can be used to validate gene expression profiles and has great potential in prognostic models for less common malignancies and diseases that lack good prognostic models. Biomarker research can also be used to develop predictive tests such as predicting response to conventional adjuvant treatments.
TMA: Quality Control and Clinical Practice
The majority of epidemiology studies are obtained from one institution where the sample processing, data collection, and analysis are performed centrally. Many research have reported contradictory results on the prognostic or predictive value of a novel biomarker. Inter-institution variations in laboratory technique may be the reason for this difference. The test has to be consistent and reproducible to be effectively used in a clinical setting. TMAs can be used in this case to apply quality assurance by assessing inter-laboratory variations. Unstained sections from the TMA can be sent to multiple labs where each lab processes and scores it independently. Results are then assessed for concordance and compared. TMAs can be more cost-efficient for routine testing but is not usually used in clinical laboratory testing.
The highest criticism of TMA is due to the small tissue core as there is concern that small cores may not accurately reflect scores from whole sections. Although whole sections are considered to be the gold standard for in-situ tests, it represents only a small part of the tumor and may not contain more diagnostic tissue compared to a TMA core. This issue is addressed by assessing the tumor region where the core is extracted. By utilizing one to four cores per source block, most studies show good concordance of both scores from TMA and the whole section. Generally, TMAs are considered to be adequate.
Another significant issue is due to tissue loss during the process of sectioning, transfer, and staining. It has been reported that the estimated core loss to be 10-30% due to technical causes. The tissue quality can be affected by preparation and storage of source blocks causing it to affect biomarker results. All TMAs can be assumed to have some non-reactive cores that may be uninterpretable of have false negatives during analysis. Missing biomarker data and underestimation of the incidence of the molecular marker can be attributed to the combination of sampling error during core extraction and core loss during slide preparation. For studies involving multiple biomarkers, missing biomarkers might require the exclusion of cases and consequently, loss of statistical power in analysis. This issue can be resolved by extracting multiple cores from the source block.
The number of cores extracted per source block has to be decided during TMA construction. It is also crucial to remember that the workload increases with the use of multiple cores, especially in large series. Although the use of multiple cores may improve data retention, it has been reported that the analysis of a single core can reproduce the same effect.
For a TMA study, the most vital step is to identify the appropriate cohort that has accessible archived tissue. Prognostic studies need thorough, complete, and long-term follow up data for each patient. Diagnostic studies need source blocks from numerous tumor types and tissues in different stages of malignant progression. In laboratory medicine, TMA assures the quality and characterization of new antibodies for IHC. Many big molecular epidemiological studies now use TMAs, but so far has limited impact in clinical oncology. Biomarkers need to be validated before using it in clinical application. Although lengthy, it can significantly affect estimates for prognosis and clinical practice. TMA is now widely recognized with source blocks collected during clinical studies. A planned construction of TMAs using surgical materials from cancer trials can help future projects. The TMA is an amazing tool as it is accessible, cost-effective, and reliable in the assessment of molecular markers, and plays an important role in translational research and oncology.