Punctate Nuclear MLH1 Staining With the VENTANA M1 Clone in Colorectal Carcinoma: Identification of 3 Patterns in...

In colorectal carcinoma (CRC), deficient DNA mismatch repair (dMMR) is an established predictive and prognostic biomarker and an essential screening marker for Lynch syndrome. Immunohistochemistry (IHC) for MLH1, PMS2, MSH2, and MSH6 is routinely used to assess expression of mismatch repair (MMR) proteins. Strong, homogeneous nuclear staining in tumor cells supports MMR proficiency (pMMR), whereas the absence of tumor nuclear staining in the presence of intact internal controls supports dMMR. Loss of protein expression typically occurs in heterodimeric pairs, most commonly MLH1/PMS2 or MSH2/MSH6, although isolated loss of PMS2 or MSH6 may also occur. A clinically important interpretive pitfall is the punctate or dot-like nuclear staining pattern observed with the VENTANA anti-MLH1 M1 clone. This aberrant pattern represents MLH1 loss but may be misinterpreted as retained MLH1 expression, particularly by pathologists unfamiliar with its significance. We evaluated 24 paired biopsy-resection CRC cases, including 11 MLH1/PMS2-deficient cases in which biopsy specimens showed punctate MLH1 staining with the M1 clone. In the corresponding resection specimens, 3 distinct MLH1 staining patterns were identified: pattern 1, complete loss of nuclear staining throughout the tumor; pattern 2, punctate staining in superficial tumor cells with complete loss in deeper tumor regions; and pattern 3, punctate staining throughout the tumor. Repeat MLH1 IHC using the BioCare Medical G168-15 clone confirmed that punctate nuclear staining with the VENTANA M1 clone corresponds to true MLH1 protein loss. Four cases were initially misinterpreted by the original pathologists, underscoring the clinical impact of this artifact. Our findings indicate that dMMR staining patterns, particularly aberrant MLH1 staining, are more susceptible to interpretive error than conventional retained staining patterns. Recognition of punctate MLH1 staining as a manifestation of MLH1 loss is essential to prevent false MMR-proficient interpretation and to ensure appropriate molecular workup, Lynch syndrome triage, and therapeutic decision-making.