European Real-World Assessment of the Clinical Validity of a CE-IVD Panel for Ultra-Fast Next-Generation Sequencing in Solid Tumors.

Molecular profiling of solid tumors facilitates personalized, targeted therapeutic interventions. The ability to perform next-generation sequencing (NGS), especially from small tissue samples, in a short turnaround time (TAT) is essential to providing results that enable rapid clinical decisions. This multicenter study evaluated the performance of a CE in vitro diagnostic (IVD) assay, the Oncomine Dx Express Test, on the Ion Torrent Genexus System for detecting DNA and RNA variants in solid tumors. Eighty-two archived formalin-fixed paraffin embedded (FFPE) tissue samples from lung, colorectal, central nervous system, melanoma, breast, gastric, thyroid, and soft tissue cancers were used to assess the presence of single nucleotide variants (SNVs), insertions and deletions (indels), copy number variations (CNVs), gene fusions, and splice variants. These clinical samples were previously characterized at the various academic centers using orthogonal methods. The Oncomine Dx Express Test showed high performance with 100% concordance with previous characterization for SNVs, indels, CNVs, gene fusions, and splice variants. SNVs and indels with allele frequencies as low as 5% were correctly identified. The test detected all the expected ALK, RET, NTRK1, and ROS1 fusion isoforms and MET exon 14-skipping splice variants. The average TAT from extracted nucleic acids to the final variant report was 18.3 h. The Oncomine Dx Express Test in combination with the Ion Torrent Genexus System is a CE-IVD-compliant, performant, and multicenter reproducible method for NGS detection of actionable biomarkers from a range of tumor samples, providing results in a short TAT that could support timely decision- making for targeted therapeutic interventions.

Somatic mutations in mismatch repair genes in sporadic gastric carcinomas are not a cause but a consequence of the mutator phenotype.

In hereditary nonpolyposis colorectal cancer (HNPCC), patients' mismatch repair (MMR) gene mutations cause MMR deficiency, leading to microsatellite instability (MSI-H). MSI-H is also found in a substantial fraction of sporadic gastric carcinomas (SGC), mainly due to MLH1 promoter hypermethylation, although somatic mutations in MMR genes have been described. We aimed to investigate which MMR defects are present in SGC. Twenty-nine MSI-H SGC investigated previously for MLH1 promoter hypermethylation were screened for somatic mutations in MLH1, MSH2, MSH6, MLH3, and MBD4 by denaturing gradient gel electrophoresis and sequencing. Five truncating mutations (three in MSH6, one in MLH3, and one in MBD4) and one missense mutation (MLH1) were identified. Of these, three truncating mutations were in MSI-H cases that lack MLH1 hypermethylation. As all truncating mutations were found in the coding poly-A tracts, it seems likely that they result from the MSI phenotype rather than cause it. In summary, somatic mutations in MMR genes are rare in SGC and do not explain the development of these tumors reflecting, rather than causing, the mutator phenotype. Other MMR genes are probably involved in MSI-H gastric cancer without MLH1 hypermethylation.

Large Diversity of Functional Nanobodies from a Camelid Immune Library Revealed by an Alternative Analysis of Next-Generation Sequencing Data.

Next-generation sequencing (NGS) has been applied successfully to the field of therapeutic antibody discovery, often outperforming conventional screening campaigns which tend to identify only the more abundant selective antibody sequences. We used NGS to mine the functional nanobody repertoire from a phage-displayed camelid immune library directed to the recepteur d'origine nantais (RON) receptor kinase. Challenges to this application of NGS include accurate removal of read errors, correct identification of related sequences, and establishing meaningful inclusion criteria for sequences-of-interest. To this end, a sequence identity threshold was defined to separate unrelated full-length sequence clusters by exploring a large diverse set of publicly available nanobody sequences. When combined with majority-rule consensus building, applying this elegant clustering approach to the NGS data set revealed a wealth of >5,000-enriched candidate RON binders. The huge binding potential predicted by the NGS approach was explored through a set of randomly selected candidates: 90% were confirmed as RON binders, 50% of which functionally blocked RON in an ERK phosphorylation assay. Additional validation came from the correct prediction of all 35 RON binding nanobodies which were identified by a conventional screening campaign of the same immune library. More detailed characterization of a subset of RON binders revealed excellent functional potencies and a promising epitope diversity. In summary, our approach exposes the functional diversity and quality of the outbred camelid heavy chain-only immune response and confirms the power of NGS to identify large numbers of promising nanobodies.

MBD4 mutations are rare in gastric carcinomas with microsatellite instability.

MBD4 encodes a protein that interacts with the mismatch repair protein hMLH1. Therefore, it has been postulated that mutations in MBD4 may result in mismatch repair deficiency. Furthermore, it was shown that MBD4 is a target gene in mismatch repair-deficient tumors. We searched for mutations of MBD4 in sporadic gastric carcinomas (SGC) and compared them with the MSI status of the tumors by screening a series of 42 SGC [22 high microsatellte instability (MSI-H) and 20 microsatellite stable (MSS)] for mutations in both the poly(A)10 and (A)6 tracts of the gene as well as in its entire coding sequence. Mutations of the poly(A)10 tract of MBD4 were also screened in 126 MSI-H colon carcinomas. One of the 22 MSI-H gastric carcinomas and 12 of 126 MSI-H colon carcinomas showed MBD4 mutations in the poly(A)10 tract of exon 3. In addition, seven sequence variants were found in SGC, all of them having a polymorphic nature. We conclude that MBD4 mutations in the entire coding sequence, including the two poly(A) tracts, are rare in MSI gastric carcinomas. MBD4 is likely to be a bystander target gene in MSI-H tumors.

Molecular and clinical characteristics of MSH6 variants: an analysis of 25 index carriers of a germline variant.

The MSH6 gene is one of the mismatch-repair genes involved in hereditary nonpolyposis colorectal cancer (HNPCC). Three hundred sixteen individuals who were known or suspected to have HNPCC were analyzed for MSH6 germline mutations. For 25 index patients and 8 relatives with MSH6 variants, molecular and clinical features are described. For analysis of microsatellite instability (MSI), the five consensus markers were used. Immunohistochemical analysis of the MLH1, MSH2, and MSH6 proteins was performed. Five truncating MSH6 mutations, of which one was detected seven times, were found in 12 index patients, and 10 MSH6 variants with unknown pathogenicity were found in 13 index patients. Fourteen (54%) of 26 colorectal cancers (CRCs) and endometrial cancers showed no, or only weak, MSI. Twelve of 18 tumors of truncating-mutation carriers and 3 of 17 tumors of missense-mutation carriers showed loss of MSH6 staining. Six of the families that we studied fulfilled the original Amsterdam criteria; most families with MSH6, however, were only suspected to have HNPCC. In families that did not fulfill the revised Amsterdam criteria, the prevalence of MSH6 variants is about the same as the prevalence of those in MLH1/MSH2. Endometrial cancer and/or atypical hyperplasia were diagnosed in 8 of 12 female carriers of MSH6 truncating mutations. Most CRCs were localized distally in the colon. Although, molecularly, missense variants are labeled as doubtfully pathogenic, clinical data disclose a great resemblance between missense-variant carriers and truncating-mutation carriers. We conclude that, in all patients suspected to have HNPCC, MSH6-mutation analysis should be considered. Neither MSI nor immunohistochemistry should be a definitive selection criterion for MSH6-mutation analysis.