Low frequency of mismatch repair deficiency in gallbladder cancer

The incidence of DNA mismatch repair (MMR)-deficient, microsatellite-unstable (MSI) tumors is of particular clinical relevance since it has been shown that MMR-deficient tumors are more responsive to immune checkpoint blockade, particularly using antibodies directed against the immune checkpoint PD-1 or PD-L1 [1], than MMR-proficient tumors. Histomorphologically, gallbladder cancer (GBC) is a diverse group of tumors, most often displaying phenotypes of an adenocarcinoma. Clinically, GBC is often treated together with cholangiocarcinomas (CCAs) as biliary tract cancers and thus as one disease, although genetic heterogeneity as well as differences in clinical behavior is nowadays widely acknowledged [2]. Most patients with GBC present with unresectable or metastatic disease. Despite systemic chemotherapy, prognosis remains poor and to date there are no established molecular targeted therapies tailored to GBC [3]. Molecular events occurring during the development of biliary tract cancers (BTC) are heterogeneous and likely follow a multistep process encompassing alterations of several oncogenes and tumor suppressor genes, such as KRAS, TP53, EGFR, and ERBB2/3. The genomic spectra of BTC have been previously nicely depicted [4], and we have recently demonstrated the presence of MSI in a small proportion (4/308; 1.3%) of CCAs [5].

So far, there are no systematic data available informing about the prevalence of DNA mismatch repair deficiency and MSI in GBC. Previous studies on MSI in biliary tract cancers have reported MMR deficiency and MSI in up to 30% of cases, particularly in liver-fluke-associated CCA specimens from endemic regions in Thailand [6]. For GBC, reported proportions of MSI tumors also varied greatly, because of a high diversity of marker panels used for determining MSI.

In the present study, we aimed to determine frequency and clinicopathological characteristics of MSI tumors in a well-characterized German cohort of GBC.

In summary, our study demonstrates that MMR deficiency-induced MSI is rare in this well-characterized, non-liver-fluke associated German GBC cohort (n = 69). The lower frequency of MSI in our study compared to several others may be explained, as previously demonstrated for CCA, but also for GBC [10‐16], by regional differences and associated pathogenetic mechanisms. In addition, in the present study we exclusively used mononucleotide repeats, which are more specific for detecting DNA mismatch repair deficiency-induced MSI than di- or tetranucleotide markers [17, 18], thereby avoiding false positive results due to incidental slippage events in MMR-proficient cells. Such events may explain the high rates of MSI in the literature, which in a recent retrospective literature analysis article was 18 out of 357 (5%) in GBC [19].

In our analysis, only one (1.4%) out of 69 tumors showed instability affecting all the sensitive mononucleotide markers BAT25, BAT26, and CAT25. As immunohistochemistry did not show loss of any of the four MMR proteins analyzed, MSI analysis was repeated twice from independent tissue blocks and TMA cores, and the MSI status was confirmed. MSI tumors presenting with retained expression of MMR proteins are rare, but have been previously reported in the literature [20, 21]. Possible reasons for such a discrepancy may be somatic missense mutations in DNA mismatch repair genes that result in the expression of a non-functional MMR protein, or, as reported recently, mutations in other MMR-related genes such as the exonuclease domain of the POLE gene [20, 21]. Further analysis such as exome or whole genome sequencing of tumor tissue would have been required to identify the molecular mechanisms leading to MSI and to inform about a possible hereditary background. Unfortunately, in our case complete somatic mutation analysis of MMR genes was not feasible due to limited amounts of available tumor DNA from FFPE tissue specimens.

The classical MSI phenotype including a special MSI histology with poor differentiation and dense lymphocyte infiltration, which is known from MSI colorectal cancers [22‐24], was not fully recapitulated in the detected MSI GBC. However, in line with the improved prognosis commonly reported to be associated with tumors of the MSI phenotype, the patient’s overall survival was 4.5 years, compared to a median overall survival of 1.6 years in the MSS group of the same stage (UICC 3). Due to the limited number of patients, a statistical analysis of potential survival benefits related to MSI in GBC was not feasible. A recent study, however, did not report a prolonged survival in MSI compared to non-MSI GBC patients [10].

Although the overall frequency of MSI in GBC is low with less than 2% in the present cohort, MSI testing of GBC should be considered if the patient has a chance to benefit from immune checkpoint blockade [25]. Recent studies demonstrated that patients with MSI BTC in fact may show significant clinical responses towards treatment with anti-PD-1 or anti-PD-L1 antibodies [1, 26]. Interestingly, even BTC patients with far progressed disease status that are therapy-refractory against chemotherapeutical agents commonly used in the treatment of BTC can show significant treatment responses upon immune checkpoint blockade using pembrolizumab [27]. Although we cannot state, whether MSI in GBC is associated with an increased immune infiltration due to limited patient numbers, considering the pronounced and sustainable benefit reported for advanced MSI cancer patients after treatment with immune checkpoint inhibitors, additional studies are strongly encouraged that use PCR-based MSI testing in parallel with MMR protein IHC prospectively in larger series of GBC.

Concerning MSI testing in GBC patients, this study has several clinical implications: i) it demonstrates that the frequency of MSI is low in Western-world GBC, and ii) PCR-based MSI analysis using mononucleotide markers should be considered due to their sensitivity and specificity for the detection of MMR deficiency-induced MSI, particularly in GBC patients who might be eligible for immune checkpoint blockade.

The frequency of MMR deficiency-induced MSI among Western-world gallbladder cancers is low. However, due to the significant clinical responses of MSI tumor patients towards immune checkpoint blockade, MSI analysis should be considered in this tumor type.