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Background: Patients with PAX3/7-FOXO1 fusion-negative rhabdomyosarcomas (fnRMS) harbouring the rare L122R MYOD1 mutation have significantly poorer prognosis than other fnRMS. We undertook a detailed clinicopathological evaluation of a cohort of patients with MYOD1 mutated fnRMS in order to improve risk stratification and treatment options., Procedure: Histological, mutational and clinical data from a cohort of patients with MYOD1 mutant RMS treated in Europe were analysed., Results: Thirty-two cases with mutant MYOD1 RMS were identified from patients enrolled in sequential European rhabdomyosarcoma clinical trials from 1992 to 2022 (n = 22) and non-trial cohorts (n = 10). Thirty cases had the recurrent L122R missense mutation, one case harboured a K124E mutation and one case had a truncating mutation (S63X). Increased MyoD1 and reduced MYF4 immunostaining were consistent features of MYOD1 L122R -mutated RMS. Applying the risk stratification of the European paediatric Soft tissue sarcoma Study Group (EpSSG) RMS2005 trial, among 20 localised RMS cases that could be assigned a risk category, one was Very High Risk, 13 were High Risk and six were Standard Risk. Eight patients had distant metastases at diagnosis. Of the 25 patients with adequate clinical follow-up data, 15/25 (60%) patients had an event at a median time of 9 months (12/15 included failure of local control) and 13/25 (52%) died of disease., Conclusion: This MYOD1 mutant cohort demonstrates increased MYOD and reduced MYF4 immunostaining, high risk of local failure and poor survival in agreement with other studies. Increased treatment intensity and improved local control should be considered for these patients., (© 2024 The Author(s). Pediatric Blood & Cancer published by Wiley Periodicals LLC.)

Background: Circulating free DNA sequencing (cfDNA-Seq) can portray cancer genome landscapes, but highly sensitive and specific technologies are necessary to accurately detect mutations with often low variant frequencies., Methods: We developed a customizable hybrid-capture cfDNA-Seq technology using off-the-shelf molecular barcodes and a novel duplex DNA molecule identification tool for enhanced error correction., Results: Modeling based on cfDNA yields from 58 patients showed that this technology, requiring 25 ng of cfDNA, could be applied to >95% of patients with metastatic colorectal cancer (mCRC). cfDNA-Seq of a 32-gene, 163.3-kbp target region detected 100% of single-nucleotide variants, with 0.15% variant frequency in spike-in experiments. Molecular barcode error correction reduced false-positive mutation calls by 97.5%. In 28 consecutively analyzed patients with mCRC, 80 out of 91 mutations previously detected by tumor tissue sequencing were called in the cfDNA. Call rates were similar for point mutations and indels. cfDNA-Seq identified typical mCRC driver mutations in patients in whom biopsy sequencing had failed or did not include key mCRC driver genes. Mutations only called in cfDNA but undetectable in matched biopsies included a subclonal resistance driver mutation to anti-EGFR antibodies in KRAS , parallel evolution of multiple PIK3CA mutations in 2 cases, and TP53 mutations originating from clonal hematopoiesis. Furthermore, cfDNA-Seq off-target read analysis allowed simultaneous genome-wide copy number profile reconstruction in 20 of 28 cases. Copy number profiles were validated by low-coverage whole-genome sequencing., Conclusions: This error-corrected, ultradeep cfDNA-Seq technology with a customizable target region and publicly available bioinformatics tools enables broad insights into cancer genomes and evolution., Clinicaltrialsgov Identifier: NCT02112357., (© 2018 American Association for Clinical Chemistry.)

Purpose: Epigenetic dysregulation is known to be an important contributor to myeloma pathogenesis but, unlike other B-cell malignancies, the full spectrum of somatic mutations in epigenetic modifiers has not been reported previously. We sought to address this using the results from whole-exome sequencing in the context of a large prospective clinical trial of newly diagnosed patients and targeted sequencing in a cohort of previously treated patients for comparison., Experimental Design: Whole-exome sequencing analysis of 463 presenting myeloma cases entered in the UK NCRI Myeloma XI study and targeted sequencing analysis of 156 previously treated cases from the University of Arkansas for Medical Sciences (Little Rock, AR). We correlated the presence of mutations with clinical outcome from diagnosis and compared the mutations found at diagnosis with later stages of disease., Results: In diagnostic myeloma patient samples, we identify significant mutations in genes encoding the histone 1 linker protein, previously identified in other B-cell malignancies. Our data suggest an adverse prognostic impact from the presence of lesions in genes encoding DNA methylation modifiers and the histone demethylase KDM6A/UTX The frequency of mutations in epigenetic modifiers appears to increase following treatment most notably in genes encoding histone methyltransferases and DNA methylation modifiers., Conclusions: Numerous mutations identified raise the possibility of targeted treatment strategies for patients either at diagnosis or relapse supporting the use of sequencing-based diagnostics in myeloma to help guide therapy as more epigenetic targeted agents become available. Clin Cancer Res; 22(23); 5783-94. ©2016 AACR., Competing Interests: Statement The authors submitted disclosures of potential conflicts of interest which appear on the final manuscript version as follows: C. Pawlyn is a consultant/advisory board member for Celgene and Takeda. M. Kaiser is a consultant/advisory board member for Amgen and Celgene. C. Heuck is an employee of Janssen R&D, reports receiving speakers bureau honoraria from Foundation Medicine, and is a consultant/advisory board member for Celgene and Millennium/Takeda. G. Cook reports receiving a commercial research grant from Celgene, speakers bureau honoraria from and is a consultant/advisory board member for Celgene and Janssen. R. Owen reports receiving a commercial research grant from Celgene, speakers bureau honoraria from Celgene and Takeda, support for attending academic meetings from Janssen and Takeda, and is a consultant/advisory board member for Celgene. W.M. Gregory reports receiving speakers bureau honoraria from Janssen and is a consultant/advisory board member for Celgene. G. Jackson reports receiving speakers bureau honoraria from Amgen, Celgene, Janssen, and Takeda and is a consultant/advisory board member for Amgen, Celgene, Janssen,Merck Sharp&Dohme, and Takeda. G.Morgan is a consultant/advisory board member for Bristol-Meyers Squibb, Celgene, Janssen, Kesios, Novartis, and Takeda. No potential conflicts of interest were disclosed by the other authors., (©2016 American Association for Cancer Research.)