br Analysis of mismatch repair function and results Analysis
Analysis of mismatch repair function and results Analysis of microsatellite instability: Multiplex PCR amplification of 5 mononucleotide microsatellite loci was performed using the MSI Analysis System (Promega). Amplification products were analyzed using the ABI 3500Dx capillary electrophoresis instrument (Applied Biosystems, Foster City, CA, USA). The neoplasm is designated as having high MSI (MSI-H) when novel allele lengths are identified in the neoplastic bosentan compared with normal or germline cells at 2 or more microsatellite loci. Immunohistochemical staining of DNA mismatch repair proteins: IHC staining of MLH1 (clone ES05, 1:100; Dako), MSH2 (clone FE11, 1:100, Dako), PMS2 (clone EP51, 1:50, Dako), and MSH6 (clone EP49, 1:200, Dako) was performed using an automated immunostainer (BENCHMARK® XT, Ventana Medical System, Tucson, AZ, USA) and reviewed by 2 board-certified pathologists. Results: MSI was observed in all 5 mononucleotide microsatellite loci compared with tumor tissues and peripheral blood, consistent with MSI-H (Fig. 3). IHC staining revealed that the tumor was negative for MLH1 and PMS2 (Fig. 4).
Discussion Cancer cells can evade immune surveillance through several mechanisms. One of the most crucial mechanisms is suppressing immune responses by expressing immune checkpoints. Immune checkpoints have been extensively studied in the past few years, resulting in the development of immune checkpoint blockade. Pembrolizumab (formerly MK-3475), a humanized monoclonal IgG4 antibody that targets immune checkpoint molecule: PD-1, has shown durable responses in melanoma, non-small- cell lung cancer, and several other cancer types. Emerging data suggest that approximately 20% of patients also respond to PD-1 blockade. However, identify a useful predictive biomarker for anti-PD-1 blockade in gastric cancer is challenging. Programmed cell death ligand 1 (PD-L1) expression in tumor and invasive borders may potentially predict the response to PD-1 blockade; however, the predictability and optimal cutoff threshold remain controversial and inconclusive. Muro et al reported the efficacy of pembrolizumab in pretreated patients with PD-L1 expressing (distinctive stromal or ≥1% tumor nest cell PD-L1 staining) recurrent or metastatic adenocarcinoma of the stomach or gastroesophageal junction. The objective response rate was 22%, and the median response duration was 24 weeks among the 36 enrolled patients who were treated with pembrolizumab. PD-L1 appears to be a suboptimal predictive biomarker for PD-1 blockade in gastric cancer. Virus-associated malignancies more favorably respond to immune checkpoint blockade. Higher expression of PD-L1 induced by viral infection and more tumor infiltrating lymphocytes in the microenvironment are hypothetic mechanisms underlying the more favorable responses. Pembrolizumab is effective against virus-associated malignancies, such as nasopharyngeal carcinoma, Merkel cell carcinoma, and human papillomavirus-associated oropharyngeal cancer. A small fraction of gastric adenocarcinoma is associated with Epstein–Barr virus (EBV). However, the response to immune checkpoint blockade in EBV-associated gastric adenocarcinoma is unclear, and EBV-encoded small RNA is not established as a biomarker for immune checkpoint blockade in gastric cancer. Deficient MMR (dMMR) causes MSI-H that results in a 10–100-fold increase in mutational load. Studies have reported that tumors with a high mutational load more favorably respond to immune checkpoint blockade. Moreover, Le et al revealed that metastatic colon cancers harboring MSI-H favorably respond to PD-1 blockade, with an objective response rate of 40%. Approximately 15%–20% of patients in all stages of gastric cancer demonstrate the MSI-H phenotype. MSI-H is associated with favorable prognosis in gastric cancer, whereas its predictability of the response to immune checkpoint blockade is controversial. At the annual meeting of the American Society of Clinical Oncology in 2016, Le et al reported the results of a subsequent cohort of various cancer types other than CRC with dMMR treated with pembrolizumab. Two of the three enrolled patients with advanced gastric cancer responded to PD-1 blockade; one had a complete response and one had a partial response. In contrast, Chen et al reported a patient with MMR-proficient (pMMR) and microsatellite-stable (MSS) gastric cancer who exhibited a partial response to pembrolizumab. This result indicates that pMMR and an MSS status may not fully predict resistance to PD-1 blockade in gastric cancer, as suggested by a previous study on CRC by Le et al, in which none of the 18 MSS patients responded to PD-1 blockade.