Frameshift mutations in peripheral blood as a biomarker for surveillance of Lynch syndrome.

BACKGROUND: Lynch syndrome is a hereditary cancer predisposition syndrome caused by germline mutations in DNA mismatch repair genes, which lead to high microsatellite instability and frameshift mutations at coding mononucleotide repeats in the genome. Recurrent frameshift mutations in these regions are thought to play a central role in the increased risk of various cancers, but no biomarkers are currently available for the surveillance of high microsatellite instability-associated cancers. METHODS: A frameshift mutation-based biomarker panel was developed and validated by targeted next-generation sequencing of supernatant DNA from cultured high microsatellite instability colorectal cancer cells. This panel supported selection of 122 frameshift mutation targets as potential biomarkers. This biomarker panel was then tested using matched tumor, adjacent normal tissue, and buffy coat samples (53 samples) and blood-derived cell-free DNA (cfDNA) (38 samples) obtained from 45 high microsatellite instability and mismatch repair-deficient patients. We also sequenced cfDNA from 84 healthy participants to assess background noise. RESULTS: Recurrent frameshift mutations at coding mononucleotide repeats were detectable not only in tumors but also in cfDNA from high microsatellite instability and mismatch repair-deficient patients, including a Lynch syndrome carrier, with a varying range of target detection (up to 85.2%), whereas they were virtually undetectable in healthy participants. Receiver operating characteristic curve analysis showed high sensitivity and specificity (area under the curve = 0.94) of the investigated panel. CONCLUSIONS: We demonstrated that frameshift mutations can be detected in cfDNA from high microsatellite instability and mismatch repair-deficient patients and asymptomatic carriers. The 122-target frameshift mutation panel described here has promise as a tool for improved surveillance of high microsatellite instability and mismatch repair-deficient patients, with the potential to reduce the frequency of invasive screening methods for this high-cancer-risk cohort.

Organoids and metastatic orthotopic mouse model for mismatch repair-deficient colorectal cancer.

Background: Genome integrity is essential for the survival of an organism. DNA mismatch repair (MMR) genes (e.g., MLH1, MSH2, MSH6, and PMS2) play a critical role in the DNA damage response pathway for genome integrity maintenance. Germline mutations of MMR genes can lead to Lynch syndrome or constitutional mismatch repair deficiency syndrome, resulting in an increased lifetime risk of developing cancer characterized by high microsatellite instability (MSI-H) and high mutation burden. Although immunotherapy has been approved for MMR-deficient (MMRd) cancer patients, the overall response rate needs to be improved and other management options are needed. Methods: To better understand the biology of MMRd cancers, elucidate the resistance mechanisms to immune modulation, and develop vaccines and therapeutic testing platforms for this high-risk population, we generated organoids and an orthotopic mouse model from intestine tumors developed in a Msh2-deficient mouse model, and followed with a detailed characterization. Results: The organoids were shown to be of epithelial origin with stem cell features, to have a high frameshift mutation frequency with MSI-H and chromosome instability, and intra- and inter-tumor heterogeneity. An orthotopic model using intra-cecal implantation of tumor fragments derived from organoids showed progressive tumor growth, resulting in the development of adenocarcinomas mixed with mucinous features and distant metastasis in liver and lymph node. Conclusions: The established organoids with characteristics of MSI-H cancers can be used to study MMRd cancer biology. The orthotopic model, with its distant metastasis and expressing frameshift peptides, is suitable for evaluating the efficacy of neoantigen-based vaccines or anticancer drugs in combination with other therapies.

Mesothelioma Mouse Models with Mixed Genomic States of Chromosome and Microsatellite Instability.

Malignant mesothelioma (MMe) is a rare malignancy originating from the linings of the pleural, peritoneal and pericardial cavities. The best-defined risk factor is exposure to carcinogenic mineral fibers (e.g., asbestos). Genomic studies have revealed that the most frequent genetic lesions in human MMe are mutations in tumor suppressor genes. Several genetically engineered mouse models have been generated by introducing the same genetic lesions found in human MMe. However, most of these models require specialized breeding facilities and long-term exposure of mice to asbestos for MMe development. Thus, an alternative model with high tumor penetrance without asbestos is urgently needed. We characterized an orthotopic model using MMe cells derived from Cdkn2a+/-;Nf2+/- mice chronically injected with asbestos. These MMe cells were tumorigenic upon intraperitoneal injection. Moreover, MMe cells showed mixed chromosome and microsatellite instability, supporting the notion that genomic instability is relevant in MMe pathogenesis. In addition, microsatellite markers were detectable in the plasma of tumor-bearing mice, indicating a potential use for early cancer detection and monitoring the effects of interventions. This orthotopic model with rapid development of MMe without asbestos exposure represents genomic instability and specific molecular targets for therapeutic or preventive interventions to enable preclinical proof of concept for the intervention in an immunocompetent setting.