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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.)

We profiled a large heterogenous cohort of matched diagnostic-relapse tumour tissue and paired plasma-derived cell free DNA (cfDNA) from patients with relapsed and progressive solid tumours of childhood. Tissue and cfDNA sequencing results were concordant, with a wider spectrum of mutant alleles and higher degree of intra-tumour heterogeneity captured by the latter, if sufficient circulating tumour-derived DNA (ctDNA) was present. Serial tumour sequencing identified putative drivers of relapse, with alterations in epigenetic drivers being a common feature. In keeping with epigenetic alterations being a common driver of many childhood cancers, fragmentomics analysis of cfDNA identified tumour-specific epigenetic states and transcription factor binding sites accessible in chromatin. This study leverages a large and well-annotated genomic dataset of aggressive childhood malignancies, identifies genomic and epigenetic drivers of childhood cancer relapse, and highlights the power and practicality of cfDNA analysis to capture both intra-tumoural heterogeneity and the epigenetic state of cancer cells.

Purpose: we tested whether ctDNA changes may be used to assess early response and clinical outcome in metastatic colorectal cancer (mCRC) patients undergoing front-line systemic anti-cancer therapy (SACT)., Experimental Design: 862 plasma samples were collected 4-weekly from baseline (BL) until disease progression in mCRC patients receiving front line SACT. ctDNA normalization was defined as ≥99% clearance after 1 month of therapy (Mo1) in the 3 variants with the highest allele frequency in BL ctDNA., Results: 83 paired samples from 75 patients were available for analysis. 12 pairs (14.4%) showed no variants in either BL or Mo1. In the remaining 71 comparisons (65 patients), 37 (52.1%) showed ctDNA normalization at Mo1. Patients that cleared ctDNA had significantly longer overall (45.6 months) and progression-free survival (13.9 months) compared to non-normalized patients [OS= 22.6 months (Log-rank p = 0.01) and PFS= 10.7 months (Log-rank p = 0.036) respectively]. In addition, higher response rate was observed in patients with ctDNA clearance (72.9%) compared to non-normalized cases (38.2%). Longitudinal sequencing of at least four timepoints in pts with a PFS>10 months showed emerging variants in 47.8% of cases; in all these patients the trajectory of these new "outlier" variants appeared in stark contrast with the clinical-radiological course of disease and the trend in other mutations., Conclusions: ctDNA clearance represents an early indicator of benefit from SACT in mCRC patients; serial tracking of multiple variants is warranted to improve specificity and to avoid misleading information due to the emergence of mutations of unknown clinical significance.

Malignant peripheral nerve sheath tumor (MPNST), an aggressive soft-tissue sarcoma, occurs in people with neurofibromatosis type 1 (NF1) and sporadically. Whole-genome and multiregional exome sequencing, transcriptomic, and methylation profiling of 95 tumor samples revealed the order of genomic events in tumor evolution. Following biallelic inactivation of NF1, loss of CDKN2A or TP53 with or without inactivation of polycomb repressive complex 2 (PRC2) leads to extensive somatic copy-number aberrations (SCNA). Distinct pathways of tumor evolution are associated with inactivation of PRC2 genes and H3K27 trimethylation (H3K27me3) status. Tumors with H3K27me3 loss evolve through extensive chromosomal losses followed by whole-genome doubling and chromosome 8 amplification, and show lower levels of immune cell infiltration. Retention of H3K27me3 leads to extensive genomic instability, but an immune cell-rich phenotype. Specific SCNAs detected in both tumor samples and cell-free DNA (cfDNA) act as a surrogate for H3K27me3 loss and immune infiltration, and predict prognosis., Significance: MPNST is the most common cause of death and morbidity for individuals with NF1, a relatively common tumor predisposition syndrome. Our results suggest that somatic copy-number and methylation profiling of tumor or cfDNA could serve as a biomarker for early diagnosis and to stratify patients into prognostic and treatment-related subgroups. This article is highlighted in the In This Issue feature, p. 517., (©2023 The Authors; Published by the American Association for Cancer Research.)

The survival of children with diffuse intrinsic pontine glioma (DIPG) remains dismal, with new treatments desperately needed. In a prospective biopsy-stratified clinical trial, we combined detailed molecular profiling and drug screening in newly established patient-derived models in vitro and in vivo. We identified in vitro sensitivity to MEK inhibitors in DIPGs harboring MAPK pathway alterations, but treatment of patient-derived xenograft models and a patient at relapse failed to elicit a significant response. We generated trametinib-resistant clones in a BRAFG469V model through continuous drug exposure and identified acquired mutations in MEK1/2 with sustained pathway upregulation. These cells showed hallmarks of mesenchymal transition and expression signatures overlapping with inherently trametinib-insensitive patient-derived cells, predicting sensitivity to dasatinib. Combined trametinib and dasatinib showed highly synergistic effects in vitro and on ex vivo brain slices. We highlight the MAPK pathway as a therapeutic target in DIPG and show the importance of parallel resistance modeling and combinatorial treatments for meaningful clinical translation., Significance: We report alterations in the MAPK pathway in DIPGs to confer initial sensitivity to targeted MEK inhibition. We further identify for the first time the mechanism of resistance to single-agent targeted therapy in these tumors and suggest a novel combinatorial treatment strategy to overcome it in the clinic. This article is highlighted in the In This Issue feature, p. 587., (©2021 The Authors; Published by the American Association for Cancer Research.)

Objective: Clinical diagnostic sequencing of circulating tumour DNA (ctDNA) is well advanced for adult patients, but application to paediatric cancer patients lags behind., Methods: To address this, we have developed a clinically relevant (67 gene) NGS capture panel and accompanying workflow that enables sensitive and reliable detection of low-frequency genetic variants in cell-free DNA (cfDNA) from children with solid tumours. We combined gene panel sequencing with low pass whole-genome sequencing of the same library to inform on genome-wide copy number changes in the blood., Results: Analytical validity was evaluated using control materials, and the method was found to be highly sensitive (0.96 for SNVs and 0.97 for INDEL), specific (0.82 for SNVs and 0.978 for INDEL), repeatable (>0.93 [95% CI: 0.89-0.95]) and reproducible (>0.87 [95% CI: 0.87-0.95]). Potential for clinical application was demonstrated in 39 childhood cancer patients with a spectrum of solid tumours in which the single nucleotide variants expected from tumour sequencing were detected in cfDNA in 94.4% (17/18) of cases with active extracranial disease. In 13 patients, where serial samples were available, we show a close correlation between events detected in cfDNA and treatment response, demonstrate that cfDNA analysis could be a useful tool to monitor disease progression, and show cfDNA sequencing has the potential to identify targetable variants that were not detected in tumour samples., Conclusions: This is the first pan-cancer DNA sequencing panel that we know to be optimised for cfDNA in children for blood-based molecular diagnostics in paediatric solid tumours., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Circulating tumor DNA (ctDNA) sequencing is being rapidly adopted in precision oncology, but the accuracy, sensitivity and reproducibility of ctDNA assays is poorly understood. Here we report the findings of a multi-site, cross-platform evaluation of the analytical performance of five industry-leading ctDNA assays. We evaluated each stage of the ctDNA sequencing workflow with simulations, synthetic DNA spike-in experiments and proficiency testing on standardized, cell-line-derived reference samples. Above 0.5% variant allele frequency, ctDNA mutations were detected with high sensitivity, precision and reproducibility by all five assays, whereas, below this limit, detection became unreliable and varied widely between assays, especially when input material was limited. Missed mutations (false negatives) were more common than erroneous candidates (false positives), indicating that the reliable sampling of rare ctDNA fragments is the key challenge for ctDNA assays. This comprehensive evaluation of the analytical performance of ctDNA assays serves to inform best practice guidelines and provides a resource for precision oncology., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Cyclin-dependent kinase 4/6 (CDK4/6) and PI3K inhibitors synergize in PIK3CA -mutant ER-positive HER2-negative breast cancer models. We conducted a phase Ib trial investigating the safety and efficacy of doublet CDK4/6 inhibitor palbociclib plus selective PI3K inhibitor taselisib in advanced solid tumors, and triplet palbociclib plus taselisib plus fulvestrant in 25 patients with PIK3CA -mutant, ER-positive HER2-negative advanced breast cancer. The triplet therapy response rate in PIK3CA- mutant, ER-positive HER2-negative cancer was 37.5% [95% confidence interval (CI), 18.8-59.4]. Durable disease control was observed in PIK3CA -mutant ER-negative breast cancer and other solid tumors with doublet therapy. Both combinations were well tolerated at pharmacodynamically active doses. In the triplet group, high baseline cyclin E1 expression associated with shorter progression-free survival (PFS; HR = 4.2; 95% CI, 1.3-13.1; P = 0.02). Early circulating tumor DNA (ctDNA) dynamics demonstrated high on-treatment ctDNA association with shorter PFS (HR = 5.2; 95% CI, 1.4-19.4; P = 0.04). Longitudinal plasma ctDNA sequencing provided genomic evolution evidence during triplet therapy. SIGNIFICANCE: The triplet of palbociclib, taselisib, and fulvestrant has promising efficacy in patients with heavily pretreated PIK3CA -mutant ER-positive HER2-negative advanced breast cancer. A subset of patients with PIK3CA -mutant triple-negative breast cancer derived clinical benefit from palbociclib and taselisib doublet, suggesting a potential nonchemotherapy targeted approach for this population. This article is highlighted in the In This Issue feature, p. 1 ., (©2020 American Association for Cancer Research.)

Infant high-grade gliomas appear clinically distinct from their counterparts in older children, indicating that histopathologic grading may not accurately reflect the biology of these tumors. We have collected 241 cases under 4 years of age, and carried out histologic review, methylation profiling, and custom panel, genome, or exome sequencing. After excluding tumors representing other established entities or subgroups, we identified 130 cases to be part of an "intrinsic" spectrum of disease specific to the infant population. These included those with targetable MAPK alterations, and a large proportion of remaining cases harboring gene fusions targeting ALK ( n = 31), NTRK1/2/3 ( n = 21), ROS1 ( n = 9), and MET ( n = 4) as their driving alterations, with evidence of efficacy of targeted agents in the clinic. These data strongly support the concept that infant gliomas require a change in diagnostic practice and management. SIGNIFICANCE: Infant high-grade gliomas in the cerebral hemispheres comprise novel subgroups, with a prevalence of ALK, NTRK1/2/3, ROS1 , or MET gene fusions. Kinase fusion-positive tumors have better outcome and respond to targeted therapy clinically. Other subgroups have poor outcome, with fusion-negative cases possibly representing an epigenetically driven pluripotent stem cell phenotype. See related commentary by Szulzewsky and Cimino, p. 904 . This article is highlighted in the In This Issue feature, p. 890 ., (©2020 American Association for Cancer Research.)

While most testicular germ cell tumours (TGCTs) exhibit exquisite sensitivity to platinum chemotherapy, ~10% are platinum resistant. To gain insight into the underlying mechanisms, we undertake whole exome sequencing and copy number analysis in 40 tumours from 26 cases with platinum-resistant TGCT, and combine this with published genomic data on an additional 624 TGCTs. We integrate analyses for driver mutations, mutational burden, global, arm-level and focal copy number (CN) events, and SNV and CN signatures. Albeit preliminary and observational in nature, these analyses provide support for a possible mechanistic link between early driver mutations in RAS and KIT and the widespread copy number events by which TGCT is characterised.

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.)

Robust establishment of survival in multiple myeloma (MM) and its relationship to recurrent genetic aberrations is required as outcomes are variable despite apparent similar staging. We assayed copy number alterations (CNA) and translocations in 1036 patients from the NCRI Myeloma XI trial and linked these to overall survival (OS) and progression-free survival. Through a meta-anlysis of these data with data from MRC Myeloma IX trial, totalling 1905 newly diagnosed MM patients (NDMM), we confirm the association of t(4;14), t(14;16), t(14;20), del(17p) and gain(1q21) with poor prognosis with hazard ratios (HRs) for OS of 1.60 (P=4.77 × 10 -7 ), 1.74 (P=0.0005), 1.90 (P=0.0089), 2.10 (P=8.86 × 10 -14 ) and 1.68 (P=2.18 × 10 -14 ), respectively. Patients with 'double-hit' defined by co-occurrence of at least two adverse lesions have an especially poor prognosis with HRs for OS of 2.67 (P=8.13 × 10 -27 ) for all patients and 3.19 (P=1.23 × 10 -18 ) for intensively treated patients. Using comprehensive CNA and translocation profiling in Myeloma XI we also demonstrate a strong association between t(4;14) and BIRC2/BIRC3 deletion (P=8.7 × 10 -15 ), including homozygous deletion. Finally, we define distinct sub-groups of hyperdiploid MM, with either gain(1q21) and CCND2 overexpression (P<0.0001) or gain(11q25) and CCND1 overexpression (P<0.0001). Profiling multiple genetic lesions can identify MM patients likely to relapse early allowing stratification of treatment.

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.)

Purpose: At the molecular level, myeloma is characterized by copy number abnormalities and recurrent translocations into the immunoglobulin heavy chain locus. Novel methods, such as massively parallel sequencing, have begun to describe the pattern of tumor-acquired mutations, but their clinical relevance has yet to be established., Methods: We performed whole-exome sequencing for 463 patients who presented with myeloma and were enrolled onto the National Cancer Research Institute Myeloma XI trial, for whom complete molecular cytogenetic and clinical outcome data were available., Results: We identified 15 significantly mutated genes: IRF4, KRAS, NRAS, MAX, HIST1H1E, RB1, EGR1, TP53, TRAF3, FAM46C, DIS3, BRAF, LTB, CYLD, and FGFR3. The mutational spectrum is dominated by mutations in the RAS (43%) and nuclear factor-κB (17%) pathways, but although they are prognostically neutral, they could be targeted therapeutically. Mutations in CCND1 and DNA repair pathway alterations (TP53, ATM, ATR, and ZNFHX4 mutations) are associated with a negative impact on survival. In contrast, those in IRF4 and EGR1 are associated with a favorable overall survival. We combined these novel mutation risk factors with the recurrent molecular adverse features and international staging system to generate an international staging system mutation score that can identify a high-risk population of patients who experience relapse and die prematurely., Conclusion: We have refined our understanding of genetic events in myeloma and identified clinically relevant mutations that may be used to better stratify patients at presentation., (© 2015 by American Society of Clinical Oncology.)

We have sequenced 463 presenting cases of myeloma entered into the UK Myeloma XI study using whole exome sequencing. Here we identify mutations induced as a consequence of misdirected AID in the partner oncogenes of IGH translocations, which are activating and associated with impaired clinical outcome. An APOBEC mutational signature is seen in 3.8% of cases and is linked to the translocation-mediated deregulation of MAF and MAFB, a known poor prognostic factor. Patients with this signature have an increased mutational load and a poor prognosis. Loss of MAF or MAFB expression results in decreased APOBEC3B and APOBEC4 expression, indicating a transcriptional control mechanism. Kataegis, a further mutational pattern associated with APOBEC deregulation, is seen at the sites of the MYC translocation. The APOBEC mutational signature seen in myeloma is, therefore, associated with poor prognosis primary and secondary translocations and the molecular mechanisms involved in generating them.

Risk stratification in myeloma requires an accurate assessment of the presence of a range of molecular abnormalities including the differing IGH translocations and the recurrent copy number abnormalities that can impact clinical behavior. Currently, interphase fluorescence in situ hybridization is used to detect these abnormalities. High failure rates, slow turnaround, cost, and labor intensiveness make it difficult and expensive to use in routine clinical practice. Multiplex ligation-dependent probe amplification (MLPA), a molecular approach based on a multiplex polymerase chain reaction method, offers an alternative for the assessment of copy number changes present in the myeloma genome. Here, we provide evidence showing that MLPA is a powerful tool for the efficient detection of copy number abnormalities and when combined with expression assays, MLPA can detect all of the prognostically relevant molecular events which characterize presenting myeloma. This approach opens the way for a molecular diagnostic strategy that is efficient, high throughput, and cost effective., (© 2014 The Authors. Genes, Chromosomes & Cancer Published by Wiley Periodicals, Inc.)