Heterogenous mismatch-repair status in colorectal cancer

Mismatch repair (MMR) defects characterize 2-4% of colorectal cancers linked to Lynch syndrome and 15% of sporadic colorectal cancers caused by epigenetic MLH1 promoter methylation. Various strategies can be used to preselect colorectal cancers for MMR protein testing, e.g. clinical guidelines for hereditary cancer, MMR prediction models that combine clinical and pathological information and potentially novel biomarker-based strategies [1‐5].

Universal assessment of immunohistochemical MMR staining is increasingly applied in colorectal cancer diagnostics in order to identify cases suspected of Lynch syndrome for further molecular diagnostics and to obtain treatment-predictive information linked to somatic methylation of MLH1[6].

The monoclonal antibodies used for immunohistochemical MMR protein staining generally result in stable and consistent staining patterns with retained staining or loss of staining. Functional interaction between the MLH1/PMS2 and the MSH2/MSH6 proteins implies that the expression pattern of the heterodimerizing protein partner may be used to direct mutation analysis. Aberrant MMR function typically leads to complete loss of nuclear staining in the tumor cells, particularly when linked to MLH1 promoter hypermethylation that leads to complete gene silencing [7]. In Lynch syndrome, the multitude of disease-predisposing mutations may have variable effects on epitope expression, from complete loss to weak or retained expression for one or both heterodimerizing proteins [8, 9]. Variable epitope expression may also result in alternative expression patterns, e.g. cytoplasmic staining and perinuclear staining, which are typically present throughout the tumor [10]. MMR protein immunostainings are generally stable and relatively easy to interpret; though challenges and pitfalls have been reported with false positive as well as false negative interpretations [10‐12]. Most commonly, these observations relate to technical artifacts caused by suboptimal fixation or paraffin-embedding, necrotic areas, sample storage, antibody specificity, clone selection or staining conditions [13, 14]. Also, neoadjuvant chemotherapy and radiotherapy may influence the results with a particular effect on MSH2/MSH6 staining [15, 16]. Heterogenous expression patterns with retained staining in the adenomatous part and loss of staining in a smaller, invasive part of the tumor have been reported but their relevance is uncertain [17]. We systematically collected colorectal cancers with heterogenous MMR protein staining patterns for detailed analysis with correlations e.g. to MSI status and MLH1 promoter methylation.

Immunohistochemical staining using alternative MMR protein antibodies confirmed heterogenous MMR protein expression in all 14 tumors. Heterogenous expression affected MLH1/PMS2 in 3 tumors, PMS2 in 2 tumors, MSH2/MSH6 in 10 tumors (of which two also expressed heterogeneity for MLH1/PMS2) and MSH6 only in 1 tumor (in which one block also expressed heterogeneity for PMS2). Areas with alternative expression patterns were well demarcated and appeared in three distinct patterns: “intraglandular” (retained/lost staining within or in between glandular formations), “clonal” (retained/lost staining in whole glands or groups of glands) and “compartmental” (retained/lost staining in larger tumor areas/compartments leading to retained/lost staining in between different tumor blocks) (Figure 1, Table 3). Various heterogenous expression patterns co-existed in 9/14 tumors, most commonly as intraglandular and clonal heterogeneity (figure 1c). The heterogenous staining patterns were present in 3-100% of the examined tumor area. In 4/14 cases, all tumor blocks showed heterogeneity, whereas the remaining tumors showed heterogeneity in a variable fraction of the tumor blocks (Table 3).

MSI was demonstrated in 13/14 tumors. Intra-tumor differences in MMR status, i.e.MSI versus MSS, in line with MMR protein staining expression was observed in 3 tumors (Table 3; case 1, 2 and 9). Non-consistent, homogenous MSI status in tumors with heterogenous MMR protein expression was observed in 2 cases (Table 3; cases 4 and 6). MLH1 promoter methylation was demonstrated in all 7 cases with complete (non-heterogenic) loss of MLH1/PMS2. In 2 cases (Table 3; cases 1 and 9) heterogenous MMR protein staining for MLH1/PMS2 correlated with heterogenous MLH1 promoter methylation, i.e. tumor areas with retained MLH1 expression did not show MLH1 methylation, whereas areas with loss of MLH1 expression showed MLH1 methylation. Concordant immunostaining and methylation status suggest functional intratumoral heterogeneity (Figure 2). DNA flow cytometric analysis was performed in one tumor (case 1) and demonstrated differences in DNA content within the heterogenous areas, which had DNA indices of 1.13 and 1.57, respectively (Figure 2).

Heterogeneous MMR protein expression is a rare phenomenon, but corresponds to differences in MMR status within the tumor and is therefore important to recognize to prevent false-positive or false-negative evaluations. We identified three distinct patterns of heterogeneity, i.e. intraglandular, clonal and compartmental heterogenous MMR protein expression. The different patterns co-existed within the same tumor and the extent of the tumor involved varied. In-depth analysis suggests that multiple causes may apply, e.g. variable epitope expression, expression related to variable differentiation, second hit mutations or methylation in selected tumor clones and possibly influence from factors linked to the tumor microenvironment such as hypoxia and oxidative stress [25].

Intraglandular and/or clonal heterogeneity throughout the tumor, which may be caused by variable epitope expression, was identified in 4 tumors (cases 4, 5, 15 and 16, Table 3). Homogenous loss of MLH1/PMS2 and heterogenous expression of MSH2/MSH6 was identified in 7 tumors that were consistently MSI and showed MLH1 promoter methylation (Table 3). This expression pattern has previously been observed and may relate either to a germline MSH2/MSH6 mutation that allows for partial epitope binding in the presence of somatic MLH1 methylation or to secondary MSH2/MSH6 inactivation [25‐27]. Heterogenous MLH1 and/or PMS2 expression, suggestive of variable MLH1 methylation/second hit mutations was observed in 2 tumors (case 2 and 6, Table 3). Case 1 showed a more complex pattern of MMR protein expression and intra-tumor differences in MSI, MLH1 promoter methylation and DNA content, suggestive of a tumor composed of two distinct clones (case 1, Figure 2).

Variable MMR status did in some tumors correspond to variable differentiation, e.g. mucinous areas (cases 5 and 15), poor differentiation (cases 16 and 17) or adenomatous components (case 2, Figure 3). Correlation between MSI status and expression also of other molecular markers has been described [28, 29]. Different, through homogenous, MMR protein expression patterns in distinct tumor compartments were observed in a mucinous adenocarcinoma (case 9) with loss of MLH1/PMS2 expression, MSI and MLH1 methylation in 1/7 tumor blocks that corresponded to an adenomatous tumor component (Figure 1d). Sample mix was excluded through histologic review and penta-D marker fragment analysis (data not shown). Homogenous loss/reduced staining of MSH2/MSH6 throughout the tumor and additional loss of PMS2 in 7/10 tumor blocks was observed in a mucinous adenocarcinoma (case 5). This case most likely reflects how the mucinous tumor component progressed in another line than the non-mucinous tumor component. Though compartmental loss of MMR protein expression is rare, this observation motivates thorough evaluation of different tumor compartments, particularly when areas with variable expression are identified.

Limitations to our study include analysis based on surgical specimens though biopsy material may produce stainings of better technical quality [30‐33]. At the same time use of biopsy material implies analysis of a restricted tumor area that may not capture areas with alternative expression. Also, information on MMR gene mutation status was not available in all cases. The 4 tumors from Lynch syndrome mutation carriers, however, expressed heterogeneity in between different tumor blocks, which showed homogenous as well as heterogenous loss in clonal and intraglandular patterns. Recognition of so-called “patchy” MMR protein staining has been reported and were also considered herein (Table 3). This phenomenon differs from the heterogenous staining patterns described herein in that it primarily relates to MSH6 stainings, neoadjuvant treatment [15, 16] or represents a weak or cytoplasmic staining rather than the distinct and well-demarcated areas of retained staining and loss of staining described herein.

Our study verifies heterogenous MMR status in a subset of colorectal cancer. Heterogenous MMR protein expression appears in three major forms, which frequently co-exist and correlate to differences in MMR status. We suggest that variable MMR protein staining patterns should be considered and when observed linked to extended analysis in order to ensure correct classification of MMR status.