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BACKGROUND: Both defects in mismatch repair (dMMR) and high microsatellite instability (MSI-H) have been recognised as crucial biomarkers that guide treatment strategies and disease management in colorectal cancer (CRC). As MMR and MSI tests are being widely conducted, an increasing number of MSI-H tumours have been identified in CRCs with mismatch repair proficiency (pMMR). The objective of this study was to assess the clinical features of patients with pMMR/MSI-H CRC and elucidate the underlying molecular mechanism in these cases. METHODS: From January 2015 to December 2018, 1684 cases of pMMR and 401 dMMR CRCs were enrolled. Of those patients, 93 pMMR/MSI-H were identified. The clinical phenotypes and prognosis were analysed. Frozen and paraffin-embedded tissue were available in 35 patients with pMMR/MSI-H, for which comprehensive genomic and transcriptomic analyses were performed. FINDINGS: In comparison to pMMR/MSS CRCs, pMMR/MSI-H CRCs exhibited significantly less tumour progression and better long-term prognosis. The pMMR/MSI-H cohorts displayed a higher presence of CD8+ T cells and NK cells when compared to the pMMR/MSS group. Mutational signature analysis revealed that nearly all samples exhibited deficiencies in MMR genes, and we also identified deleterious mutations in MSH3-K383fs. INTERPRETATION: This study revealed pMMR/MSI-H CRC as a distinct subgroup within CRC, which manifests diverse clinicopathological features and long-term prognostic outcomes. Distinct features in the tumour immune-microenvironment were observed in pMMR/MSI-H CRCs. Pathogenic deleterious mutations in MSH3-K383fs were frequently detected, suggesting another potential biomarker for identifying MSI-H. FUNDING: This work was supported by the Science and Technology Commission of Shanghai Municipality (20DZ1100101).

BackgroundUsing broad range cell-free DNA sequencing (BRcfDNA-Seq), a nontargeted next-generation sequencing (NGS) methodology, we previously identified a novel class of approximately 50 nt ultrashort single-stranded cell-free DNA (uscfDNA) in plasma that is distinctly different from 167 bp mononucleosomal cell-free DNA (mncfDNA). We hypothesize that uscfDNA possesses characteristics that are useful for disease detection.MethodsUsing BRcfDNA-Seq, we examined both cfDNA populations in the plasma of 18 noncancer controls and 14 patients with late-stage nonsmall cell lung carcinoma (NSCLC). In comparison to mncfDNA, we assessed whether functional element (FE) peaks, fragmentomics, end-motifs, and G-Quadruplex (G-Quad) signatures could be useful features of uscfDNA for NSCLC determination.ResultsIn noncancer participants, compared to mncfDNA, uscfDNA fragments showed a 45.2-fold increased tendency to form FE peaks (enriched in promoter, intronic, and exonic regions), demonstrated a distinct end-motif-frequency profile, and presented with a 4.9-fold increase in G-Quad signatures. Within NSCLC participants, only the uscfDNA population had discoverable FE peak candidates. Additionally, uscfDNA showcased different end-motif-frequency candidates distinct from mncfDNA. Although both cfDNA populations showed increased fragmentation in NSCLC, the G-Quad signatures were more discriminatory in uscfDNA. Compilation of cfDNA features using principal component analysis revealed that the first 5 principal components of both cfDNA subtypes had a cumulative explained variance of >80%.ConclusionsThese observations indicate that the distinct biological processes of uscfDNA and that FE peaks, fragmentomics, end-motifs, and G-Quad signatures are uscfDNA features with promising biomarker potential. These findings further justify its exploration as a distinct class of biomarker to augment pre-existing liquid biopsy approaches.

BACKGROUND: Recent advances in circulating cell-free DNA (cfDNA) analysis from biofluids have opened new avenues for liquid biopsy (LB). However, current cfDNA LB assays are limited by the availability of existing information on established genotypes associated with tumor tissues. Certain cancers present with a limited list of established mutated cfDNA biomarkers, and thus, nonmutated cfDNA characteristics along with alternative biofluids are needed to broaden the available cfDNA targets for cancer detection. Saliva is an intriguing and accessible biofluid that has yet to be fully explored for its clinical utility for cancer detection. METHODS: In this report, we employed a low-coverage single stranded (ss) library NGS pipeline "Broad-Range cell-free DNA-Seq" (BRcfDNA-Seq) using saliva to comprehensively investigate the characteristics of salivary cfDNA (ScfDNA). The identification of cfDNA features has been made possible by applying novel cfDNA processing techniques that permit the incorporation of ultrashort, ss, and jagged DNA fragments. As a proof of concept using 10 gastric cancer (GC) and 10 noncancer samples, we examined whether ScfDNA characteristics, including fragmentomics, end motif profiles, microbial contribution, and human chromosomal mapping, could differentiate between these two groups. RESULTS: Individual and integrative analysis of these ScfDNA features demonstrated significant differences between the two cohorts, suggesting that disease state may affect the ScfDNA population by altering nuclear cleavage or the profile of contributory organism cfDNA to total ScfDNA. We report that principal component analysis integration of several aspects of salivary cell-free DNA fragmentomic profiles, genomic element profiles, end-motif sequence patterns, and distinct oral microbiome populations can differentiate the two populations with a p value of < 0.0001 (PC1). CONCLUSION: These novel features of ScfDNA characteristics could be clinically useful for improving saliva-based LB detection and the eventual monitoring of local or systemic diseases.

BackgroundGermline pathogenic variants in CDH1 are associated with increased risk of diffuse gastric cancer and lobular breast cancer. Risk reduction strategies include consideration of prophylactic surgery, thereby making accurate interpretation of germline CDH1 variants critical for physicians deciding on these procedures. The Clinical Genome Resource (ClinGen) CDH1 Variant Curation Expert Panel (VCEP) developed specifications for CDH1 variant curation with a goal to resolve variants of uncertain significance (VUS) and with ClinVar conflicting interpretations and continues to update these specifications.MethodsCDH1 variant classification specifications were modified based on updated genetic testing clinical criteria, new recommendations from ClinGen and expert knowledge from ongoing CDH1 variant curations. The CDH1 VCEP reviewed 273 variants using updated CDH1 specifications and incorporated published and unpublished data provided by diagnostic laboratories.ResultsUpdated CDH1-specific interpretation guidelines include 11 major modifications since the initial specifications from 2018. Using the refined guidelines, 97% (36 of 37) of variants with ClinVar conflicting interpretations were resolved to benign, likely benign, likely pathogenic or pathogenic, and 35% (15 of 43) of VUS were resolved to benign or likely benign. Overall, 88% (239 of 273) of curated variants had non-VUS classifications. To date, variants classified as pathogenic are either nonsense, frameshift, splicing, or affecting the translation initiation codon, and the only missense variants classified as pathogenic or likely pathogenic have been shown to affect splicing.ConclusionsThe development and evolution of CDH1-specific criteria by the expert panel resulted in decreased uncertain and conflicting interpretations of variants in this clinically actionable gene, which can ultimately lead to more effective clinical management recommendations.

BackgroundGermline variants in the DNA mismatch repair (MMR) genes (MLH1, MSH2, MSH6, and PMS2) cause Lynch syndrome, an autosomal dominant hereditary cancer susceptibility syndrome. The risk for endometrial cancer is significantly higher in women with MSH6 pathogenic/likely pathogenic (P/LP) variants compared with that for MLH1 or MSH2 variants.MethodsThe proband was tested via a clinical testing, Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT). RT-PCR was performed using patient's blood DNA and cDNA was analyzed by DNA sequencing and a cloning approach.ResultsWe report a 56-year-old female with endometrial cancer who carries a germline variant, MSH6 c.4001G > C, located at the last nucleotide of exon 9. While the pathogenicity of this variant was previously unknown, functional studies demonstrated that this variant completely abolished normal splicing and caused exon 9 skipping, which is expected to lead to a prematurely truncated or abnormal protein.ConclusionOur results indicate that this variant likely contributes to cancer predisposition through disruption of normal splicing, and is classified as likely pathogenic.

The original article [1] contains errors in the attributions for the following co-authors: David Chia, David Elashoff, Liying Zhang, and Sung Kim. The corrected affiliations can be viewed in the authorship list of this Correction article.

BackgroundThe current methodology used to detect, diagnose, and monitor many types of cancers requires invasive tissue biopsy testing. Recently, liquid biopsy using blood, plasma, urine, saliva, and various other bodily fluids has shown utility to solve many issues associated with tissue biopsy. Blood/plasma has received most of the attention within the liquid biopsy field, however, obtaining blood samples from patients is still somewhat invasive and requires trained professionals. Using urine to detect cell-free DNA cancer biomarkers offers a truly non-invasive sampling method that can be easily and reproducibly conducted by patients.ContentNovel technologies and approaches have made the detection of small quantities of cell-free tumor DNA of varying lengths possible. Recent studies using urine circulating tumor DNA to detect cancer mutations and other biomarkers have shown sensitivity comparable to blood/plasma cell-free DNA liquid biopsy for many cancer types. Thus, urine cell-free DNA liquid biopsy may replace or provide supplementary information to tissue/blood biopsies. Further investigation with larger patient cohorts and standardization of pre-analytical factors is necessary to determine the utility of urine cell-free DNA liquid biopsy for cancer detection, diagnosis, and monitoring in a clinical setting.SummaryIn this mini-review we discuss the biological aspects of cell-free DNA in urine, numerous studies using urine cell-free DNA to detect urological cancers, and recent studies using urine cell-free DNA to detect and monitor non-urological cancers including lung, breast, colorectal, and other cancers.

Background and objectivePancreatic ductal adenocarcinoma (PDAC) is characterized by the occurrence of pathogenic variants in BRCA1/2 in 5-6% of patients. Biallelic loss of BRCA1/2 enriches for response to platinum agents and poly (ADP-ribose) polymerase 1 inhibitors. There is a dearth of evidence on the mechanism of inactivation of the wild-type BRCA1 allele in PDAC tumors with a germline BRCA1 (gBRCA1) pathogenic or likely pathogenic variant (P/LPV). Herein, we examine promotor hypermethylation as a "second hit" mechanism in patients with gBRCA1-PDAC.MethodsWe evaluated patients with PDAC who underwent Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) somatic and germline testing from an institutional database. DNA isolated from tumor tissue and matched normal peripheral blood were sequenced by MSK-IMPACT. In patients with gBRCA1-PDAC, we examined the somatic BRCA1 mutation status and promotor methylation status of the tumor BRCA1 allele via a methylation array analysis. In patients with sufficient remaining DNA, a second methylation analysis by pyrosequencing was performed.ResultsOf 1012 patients with PDAC, 19 (1.9%) were identified to harbor a gBRCA1 P/LPV. Fifteen patients underwent a methylation array and the mean percentage of BRCA1 promotor methylation was 3.62%. In seven patients in whom sufficient DNA was available, subsequent pyrosequencing confirmed an unmethylated BRCA1 promotor. Loss of heterozygosity was detected in 12 of 19 (63%, 95% confidence interval 38-84) patients, demonstrating loss of heterozygosity is the major molecular mechanism of BRCA1 inactivation in PDAC. Two (10.5%) cases had a somatic BRCA1 mutation.ConclusionsIn patients with gBRCA1-P/LPV-PDAC, loss of heterozygosity is the main inactivating mechanism of the wild-type BRCA1 allele in the tumor, and methylation of the BRCA1 promoter is a distinctly uncommon occurrence.

PurposeIdentification of inherited germline variants can guide personalized cancer screening, prevention, and treatment. Pathogenic and likely pathogenic (P/LP) germline variants in cancer predisposition genes are frequent among patients with locally advanced or metastatic urothelial carcinoma, but their prevalence and significance in patients with non-muscle-invasive bladder cancer (NMIBC), the most common form of urothelial carcinoma, is understudied.Experimental designGermline analysis was conducted on paired tumor/normal sequencing results from two distinct cohorts of patients initially diagnosed with NMIBC. Associations between clinicopathologic features and clinical outcomes with the presence of P/LP germline variants in ≥76 hereditary cancer predisposition genes were analyzed.ResultsA similar frequency of P/LP germline variants were seen in our two NMIBC cohorts [12% (12/99) vs. 8.7% (10/115), P = 0.4]. In the combined analysis, P/LP germline variants were found only in patients with high-grade NMIBC (22/163), but none of the 46 patients with low-grade NMIBC (13.5% vs. 0%, P = 0.005). Fifteen (9.2%) patients with high-grade NMIBC had P/LP variants in DNA damage response genes, most within the nucleotide excision repair (ERCC2/3) and homologous recombination repair (BRCA1, NBN, RAD50) pathways. Contrary to prior reports in patients with NMIBC not receiving Bacillus Calmette-Guerin (BCG), P/LP germline variants were not associated with worse recurrence-free or progression-free survival in patients treated with BCG or with risk of developing upper tract urothelial carcinoma.ConclusionsOur results support offering germline counseling and testing for all patients with high-grade bladder cancer, regardless of initial tumor stage. Therapeutic strategies that target impaired DNA repair may benefit patients with high-grade NMIBC.

Public access to genetic information is increasing, and community dermatologists may progressively encounter patients interested in genetic testing for melanoma risk. Clarifying potential utility will help plan for this inevitability. We determined interest and uptake of genetic risk feedback based on melanocortin receptor gene (MC1R) variants, immediate (two weeks) responses to risk feedback, and test utility at three months in patients (age ≥ 18, with a history of nonmelanoma skin cancer). Participants (N = 50) completed a baseline survey and were invited to consider MC1R testing via the study website. Testing interest and uptake were assessed through registration of test decision, request of a saliva test kit, and kit return (all yes/no). Immediate responses to risk feedback included feedback-relevant thoughts, emotions, communication, and information seeking after result receipt; test utility outcomes included family and physician communication and information seeking. Results indicated good retention at both time points (76%; 74%). Half (48%) logged onto the study website, and of these, most (92%) chose testing and (95%) returned a saliva sample. After two weeks, most (94%) had read all the risk feedback information and distress was low (M = 8.81, 7-28, SD = 2.23). Many (69%) had talked with their family about the results. By three months, most had spoken with family (92%) and physicians (80%) about skin cancer risk. Physician communication was higher (70%) in those tested versus those not tested (40%, p = 0.02). The substantial interest and promising outcomes associated with MC1R genetic testing in dermatology patients inform intervention strategies to enhance benefits and minimize risks of skin cancer genetic testing.

Gene fusions involving the neurotrophic receptor tyrosine kinase genes NTRK1, NTRK2, and NTRK3, are well established oncogenic drivers in a broad range of pediatric and adult tumors. These fusions are also important actionable markers, predicting often dramatic response to FDA approved kinase inhibitors. Accurate interpretation of the clinical significance of NTRK fusions is a high priority for diagnostic laboratories, but remains challenging and time consuming given the rapid pace of new data accumulation, the diversity of fusion partners and tumor types, and heterogeneous and incomplete information in variant databases and knowledgebases. The ClinGen NTRK Fusions Somatic Cancer Variant Curation Expert Panel (SC-VCEP) was formed to systematically address these challenges and create an expert-curated resource to support clinicians, researchers, patients and their families in making accurate interpretations and informed treatment decisions for NTRK fusion-driven tumors. We describe a system for NTRK fusion interpretation (including compilation of key elements and annotations) developed by the NTRK fusions SC-VCEP. We illustrate this stepwise process on examples of LMNA::NTRK1 and KANK1::NTRK2 fusions. Finally, we provide detailed analysis of current representation of NTRK fusions in public fusion databases and the CIViC knowledgebase, performed by the NTRK fusions SC-VCEP to determine existing gaps and prioritize future curation activities.