Relationship between MLH1, PMS2, MSH2 and MSH6 gene-specific alterations and tumor mutational burden in 1057 microsatellite instability-high solid tumors

Microsatellite instability-high (MSI-H) and tumor mutational burden (TMB) are predictive biomarkers for immune-checkpoint inhibitors (ICIs). Still, the relationship between the underlying cause(s) of MSI and TMB in tumors remains poorly defined. We investigated associations of TMB to mismatch repair (MMR) protein expression patterns by immunohistochemistry (IHC) and MMR mutations in a diverse sample of tumors. Hypothesized differences were identified by the protein/gene affected/mutated and the tumor histology/primary site. Overall, 1057 MSI-H tumors were identified from the 32 932 tested. MSI was examined by NGS using 7000+ target microsatellite loci. TMB was calculated using only nonsynonymous missense mutations sequenced with a 592-gene panel; a subset of MSI-H tumors also had MMR IHC performed. Analyses examined TMB by MMR protein heterodimer impacted (loss of MLH1/PMS2 vs. MSH2/MSH6 expression) and gene-specific mutations. The sample was 54.6% female; mean age was 63.5 years. Among IHC tested tumors, loss of co-expression of MLH1/PMS2 was more common (n = 544/705, 77.2%) than loss of MSH2/MSH6 (n = 81/705, 11.5%; P < .0001), and was associated with lower mean TMB (MLH1/PMS2: 25.03 mut/Mb vs MSH2/MSH6 46.83 mut/Mb; P < .0001). TMB also varied by tumor histology: colorectal cancers demonstrating MLH1/PMS2 loss had higher TMBs (33.14 mut/Mb) than endometrial cancers (20.60 mut/Mb) and other tumors (25.59 mut/Mb; P < .0001). MMR gene mutations were detected in 42.0% of tumors; among these, MSH6 mutations were most common (25.7%). MSH6 mutation patterns showed variability by tumor histology and TMB. TMB varies by underlying cause(s) of MSI and tumor histology; this heterogeneity may contribute to differences in response to ICI.     

Genetic toxicity assessment using liver cell models: past,present, and future

ABSTRACT

Genotoxic compounds may be detoxified to non-genotoxic metabolites while many pro-carcinogens require metabolic activation to exert their genotoxicity in vivo. Standard genotoxicity assays were developed and utilized for risk assessment for over 40 years. Most of these assays are conducted in metabolically incompetent rodent or human cell lines. Deficient in normal metabolism and relying on exogenous metabolic activation systems, the current in vitro genotoxicity assays often have yielded high false positive rates, which trigger unnecessary and costly in vivo studies. Metabolically active cells such as hepatocytes have been recognized as a promising cell model in predicting genotoxicity of carcinogens in vivo. In recent years, significant advances in tissue culture and biological technologies provided new opportunities for using hepatocytes in genetic toxicology. This review encompasses published studies (both in vitro and in vivo) using hepatocytes for genotoxicity assessment. Findings from both standard and newly developed genotoxicity assays are summarized. Various liver cell models used for genotoxicity assessment are described, including the potential application of advanced liver cell models such as 3D spheroids, organoids, and engineered hepatocytes. An integrated strategy, that includes the use of human-based cells with enhanced biological relevance and throughput, and applying the quantitative analysis of data, may provide an approach for future genotoxicity risk assessment.     

CDK12 and HER2 coamplification in two urothelial carcinomas with rapid and aggressive clinical progression

Cyclin‐dependent kinase 12 (CDK12), one of the key factors associated with DNA damage response pathways, is located on chromosome 17 proximal to Erb‐B2 receptor tyrosine kinase 2 (ERBB2). In this report, CDK12 and ERBB2 coamplification was detected by targeted next‐generation sequencing in two urothelial carcinomas. The staining intensity of the CDK12 and human epidermal growth factor receptor‐2 proteins was associated with the prognosis of each urothelial carcinoma case. Our results suggest that CDK12 coamplification with ERBB2 might be associated with tumor aggressiveness and contribution to cancer pathogenesis. Therapies targeting CDK12 should be developed for these patients.     

DNA vaccine encoding the moonlighting protein Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase (Ov-GAPDH) leads to partial protection in a mouse model of human filariasis

River blindness, caused by the filarial parasite Onchocerca volvulus, is a major socio-economic and public health problem in Sub-Saharan Africa. In January 2015, The Onchocerciasis Vaccine for Africa (TOVA) Initiative has been launched with the aim of providing new tools to complement mass drug administration (MDA) of ivermectin, thereby promoting elimination of onchocerciasis in Africa. In this context we here present Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase (Ov-GAPDH) as a possible DNA vaccine candidate. We report that in a laboratory model for filariasis, immunization with Ov-GAPDH led to a significant reduction of adult worm load and microfilaraemia in BALB/c mice after challenge infection with the filarial parasite Litomosoides sigmodontis. Mice were either vaccinated with Ov-GAPDH.DNA plasmid (Ov-pGAPDH.DNA) alone or in combination with recombinantly expressed Ov-GAPDH protein (Ov-rGAPDH). During the following challenge infection of immunized and control mice with L. sigmodontis, those formulations which included the DNA plasmid, led to a significant reduction of adult worm loads (up to 57% median reduction) and microfilaraemia (up to 94% reduction) in immunized animals. In a further experiment, immunization with a mixture of four overlapping, synthetic Ov-GAPDH peptides (Ov-GAPDHpept), with alum as adjuvant, did not significantly reduce worm loads. Our results indicate that DNA vaccination with Ov-GAPDH has protective potential against filarial challenge infection in the mouse model. This suggests a transfer of the approach into the cattle Onchocerca ochengi model, where it is possible to investigate the effects of this vaccination in the context of a natural host–parasite relationship.     

DNA methylation of the ELOVL2, FHL2, KLF14, C1orf132/MIR29B2C,and TRIM59 genes for age prediction from blood,saliva, and buccal swab samples

Many studies have reported age-associated DNA methylation changes and age-predictive models in various tissues and body fluids. Although age-associated DNA methylation changes can be tissue-specific, a multi-tissue age predictor that is applicable to various tissues and body fluids with considerable prediction accuracy might be valuable. In this study, DNA methylation at 5 CpG sites from the ELOVL2, FHL2, KLF14, C1orf132/MIR29B2C, and TRIM59 genes were investigated in 448 samples from blood, saliva, and buccal swabs. A multiplex methylation SNaPshot assay was developed to measure DNA methylation simultaneously at the 5 CpG sites. Among the 5 CpG sites, 3 CpG sites in the ELOVL2, KLF14 and TRIM59 genes demonstrated strong correlation between DNA methylation and age in all 3 sample types. Age prediction models built separately for each sample type using the DNA methylation values at the 5 CpG sites showed high prediction accuracy with a Mean Absolute Deviation from the chronological age (MAD) of 3.478 years in blood, 3.552 years in saliva and 4.293 years in buccal swab samples. A tissue-combined model constructed with 300 training samples including 100 samples from each blood, saliva and buccal swab samples demonstrated a very strong correlation between predicted and chronological ages (r = 0.937) and a high prediction accuracy with a MAD of 3.844 years in the 148 independent test set samples of 50 blood, 50 saliva and 48 buccal swab samples. Although more validation might be needed, the tissue-combined model’s prediction accuracies in each sample type were very much similar to those obtained from each tissue-specific model. The multiplex methylation SNaPshot assay and the age prediction models in our study would be useful in forensic analysis, which frequently involves DNA from blood, saliva, and buccal swab samples.     

STRmix™ put to the test: 300 000 non-contributor profiles compared to four-contributor DNA mixtures and the impact of replicates

Probabilistic genotyping approaches are increasingly used for the interpretation of DNA mixtures. To explore the specificity of one of these systems (STRmix^™), we conducted an extensive study using 24 complex mixtures: all were known or apparent 4-person mixtures with at least one contributor representing less than 20% of total DNA, and all mixtures had at least one contributor with suboptimal DNA quantity. Those mixtures were either generated in-house or from casework. All the mixtures were compared to 300,000 virtual non-contributors, resulting in a dataset of 7.2 million comparisons. The great majority of the non-contributor comparisons led to a LR lower than 1 for a specificity of 99.1%. The effect of using replicate amplifications to calculate the LR of non-contributors was also assessed as triplicates were used and led to an increased specificity of 99.8%. The very large extent of the analyzed data shows that STRmix^™ has an excellent ability to discriminate non-contributors from complex DNA mixtures.