Uncertainty in the Utility of Immunohistochemistry in Mismatch Repair Protein Expression in Epithelial Ovarian Cancer

Discussion

In this study we evaluated MLH1, MSH2, and MSH6 protein expression through IHC in 472 samples of ovarian carcinomas of diverse histological subtypes. Our findings indicate an overall frequency of loss of MMR protein expression of 12.7%. Furthermore, the frequency of LoE observed varied by specimen type, with a lower overall frequency of LoE in full-section slides (0.7%) compared to TMA slides (15.9% and 21.4% for 1 mm and 0.6 mm cores, respectively), which suggest that specimen type is important to consider when assessing IHC in ovarian samples. Finally, our data suggest that LoE may be more common in ovarian epithelial tumors with non-serous histologies.

Our overall TMA-based estimates (17.9%) are higher than that reported in the few studies, to date, that have evaluated loss of MMR protein expression based on IHC in unselected invasive epithelial ovarian carcinomas, whereas our full-section estimates (0.7%) are lower (8-10) The study by Domanska et al. (8) represents the only report of IHC on full section ovarian cancer slides; loss of MLH1, MSH2, MSH6 and PMS2 expression was 6.1% in patients age 40 or below at diagnosis. Two studies have performed IHC on TMA sections. Malander et al. (9) evaluated MLH1, MSH2, MSH6 and PMS2 and noted loss of protein expression in 2.3% (3/128) of cases. Rosen et al. (10) reported a loss of expression of MLH1 and MSH2 based on TMA sections in 2.2% (7/322) of unselected ovarian cancers. More recently, a meta-analysis estimated a frequency of MMR deficiency of approximately 10% based on IHC and/or MSI results in ovarian cancers (3).

Although our study is the largest, to date, of MMR protein deficiency in ovarian cancer based on IHC, it is also the first multi-center study to include both TMAs and full-sections, thus the lack of consistency in MMR frequency found in our study could not have been detected in prior efforts. Furthermore, the limited details on IHC methodology (such as detailed protocol, equipment and antibodies, and the scoring system) in prior reports preclude our ability to fully compare our results with prior studies. Of note, the LoE frequency varied based on whether a cut-off score of 3 versus 4 was used, suggesting it is imperative to report scoring methodologies in published reports to allow cross comparisons between studies.

Validation of the use of TMA for MMR protein expression has been evaluated in a single colorectal cancer study in which IHC for MLH1 and MSH2 was conducted in 263 colorectal cases through TMA (3 cores per case) and full-section. Results indicated comparable frequencies of 98.8% and 99.2%, respectively (20). In ovarian cancers, validation of TMA technology has been performed on several markers, including p53, Ki-67, estrogen and progesterone receptors, among others, (21, 22) however to our knowledge there are no published studies that have validated IHC for MMR proteins expression in ovarian cancer. Specifically, the study by Rosen et al. represents the only published report in which full-sections and TMAs were compared in high-grade serous ovarian cancers through IHC studies of Ki-67, estrogen receptors and p53 and correlation co-efficients were reported as 0.86, 0.93, and 0.82, respectively (23). In general, correlation co-efficients of greater than 0.8 are considered evidence of excellent correlation. In a subsequent study, Hecht et al. performed IHC for Ki-67, estrogen receptors, and p53 among others using triplicate core samples from 174 epithelial ovarian cancers and demonstrated high intraclass correlation between the cores (21). Ultimately, there have been studies to validate the use of TMAs for MMR protein expression in colorectal cancer as well as studies to validate the use of TMAs to evaluate protein expression in ovarian cancers (which have not included MMR proteins). Consequently, studies to evaluate MMR protein expression in ovarian cancers using either TMA or full-section slides have been conducted. Our study differs from prior efforts as we used a combination of TMAs and full-section slides to evaluate MMR protein expression in ovarian cancers. Based on published reports, we presumed TMA and full-section cases were equivalent, however our results pertaining to MMR expression suggest they may not be directly comparable due to differences observed by specimen type. These findings suggest that validity of TMA may vary by antigen, and clearly demonstrate the need for large scale studies to validate the use of IHC for MMR protein expression specifically in ovarian cancers, prior to their use in the clinical setting. Moreover, in an effort to further clarify these differences, tumor samples on 19 participants in the study population were evaluated through both full-section and TMA slides for expression of three MMR proteins based on IHC. Results demonstrated poor correlation of full-section and TMA-based samples, with a LIN correlation coefficient of 0.4, 0.3 and 0.7 for MLH1, MSH2 and MSH6, respectively (data not shown).

Possible reasons for the discrepancy observed by specimen type include the large size at diagnosis of ovarian tumors compared to colorectal tumors, which may contribute to their heterogeneity as they may be composed of different clonal populations exhibiting different MMR reactivity. Another possible explanation is that the fixative does not adequately penetrate the large blocks of cancer, typical of ovarian cancer, which may impact IHC for some proteins more than others. Interestingly, the frequency of LoE inversely correlated with the size of surface area of the tissue examined (0.7% in full-sections (usually several mm in diameter) vs. 15.9% in TMA cores of 1 mm in diameter vs. 21.4% in TMA cores measuring 0.6 mm in diameter). It is possible that when evaluating larger size tissues, there is a higher likelihood of identifying even a small area strongly staining for the evaluated marker, thus potentially leading to a lower frequency of LoE. Conversely, when a small size sample of a large tumor is evaluated, a negative stain will not completely exclude the possibility that a non-sampled area of the same tumor be positive. Finally, another possibility considered was whether discrepancy in results may have occurred due to considerable differences between the three study sites. However, comparison of demographic variables between study participants did not suggest any underlying differences in the study populations at the three sites to account for our findings. In fact, all three sites encompassed primarily a Caucasian population and each study was population-based, thus collected specimens from multiple hospitals within their study area. Ultimately, future efforts focused on differences between full section and TMA slides are needed on a larger range of antibodies, to determine their validity in each antibody prior to use in the clinical setting.

The expected proportions of histopathologic subtypes for epithelial ovarian cancers in the general population are: approximately 55-70% with serous histology, followed by the mucinous (3-9%), endometrioid (8-15%), clear cell (7-13%), mixed (4-6%) and undifferentiated subtypes (24). Our finding of overrepresentation of non-serous histologies, particularly mucinous, clear cell, and endometrioid subtypes, in MMR-deficient ovarian tumors is consistent with prior studies (3, 8-10, 15).

There were a number of strengths in the current study, including the large sample size, population-based design of the parent studies, as well as the comprehensive collection of clinical and demographic data on study participants. Despite these strengths, it is important to acknowledge that our study was designed to evaluate MMR protein deficiency in ovarian tumors, not validate the use of TMA specific to MMR proteins. Nevertheless, our findings that MMR LoE frequency varies by specimen type (i.e., full section slides versus TMA) is of broad clinical relevance as it serves to demonstrate the limitation for the use of IHC as a reliable methodology for the assessment of MMR deficiency in ovarian cancer. Given our findings, it is critical to evaluate the validity of results of MMR deficiency in ovarian cancers, prior to its use as a predictive or prognostic marker. Other factors that may affect IHC results performed on paraffin-embedded tissues include type and time of fixation, thickness of tissue sections, age of the parental tissue block, and tumor heterogeneity. In fact, multiple studies to evaluate HER2 in breast cancer have demonstrated the multitude of factors that may significantly affect the results of immunostains (25). As a result of how this may significantly impact patient care, both American Society of Clinical Oncology (ASCO) and College of American Pathology (CAP) have provided guidelines for the processing of breast specimens to be stained for Her2 (26). Ultimately, the implementation of similar guidelines for ovarian cancer specimens will improve the immunohistochemical evaluation of these tumors, and will improve our ability to better determine the basis of discrepant results, as they arise. Finally, IHC was limited to the three more commonly stained proteins, MLH1, MSH2, and MSH6, at the time at which testing was done. PMS2 was not done, as it has more recently been included in routine IHC analyses.

In conclusion, our study suggests that a significant portion of human ovarian cancers exhibit defects in the mismatch repair pathway, proposing this pathway as a potential contributor to the development of epithelial ovarian cancers. The results also indicate that immunohistochemical techniques for the assessment of MMR proteins deficiency may have limited diagnostic accuracy in ovarian cancer and require further validation studies to assess their utility. Future efforts focused on inter and intra-institutional standardization of IHC methodologies is crucial for the successful utilization of this technique for clinical usage in ovarian tumors.