Whole-genome landscapes of major melanoma subtypes

Melanoma of the skin is a common cancer only in Europeans, whereas it arises in internal body surfaces (mucosal sites) and on the hands and feet (acral sites) in people throughout the world. Here we report analysis of whole-genome sequences from cutaneous, acral and mucosal subtypes of melanoma. The heavily mutated landscape of coding and non-coding mutations in cutaneous melanoma resolved novel signatures of mutagenesis attributable to ultraviolet radiation. However, acral and mucosal melanomas were dominated by structural changes and mutation signatures of unknown aetiology, not previously identified in melanoma. The number of genes affected by recurrent mutations disrupting non-coding sequences was similar to that affected by recurrent mutations to coding sequences. Significantly mutated genes included BRAF, CDKN2A, NRAS and TP53 in cutaneous melanoma, BRAF, NRAS and NF1 in acral melanoma and SF3B1 in mucosal melanoma. Mutations affecting the TERT promoter were the most frequent of all; however, neither they nor ATRX mutations, which correlate with alternative telomere lengthening, were associated with greater telomere length. Most melanomas had potentially actionable mutations, most in components of the mitogen-activated protein kinase and phosphoinositol kinase pathways. The whole-genome mutation landscape of melanoma reveals diverse carcinogenic processes across its subtypes, some unrelated to sun exposure, and extends potential involvement of the non-coding genome in its pathogenesis

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Loss of Wild-Type ATRX Expression in Somatic Cell Hybrids Segregates with Activation of Alternative Lengthening of Telomeres

<div><p>Alternative Lengthening of Telomeres (ALT) is a non-telomerase mechanism of telomere lengthening that occurs in about 10% of cancers overall and is particularly common in astrocytic brain tumors and specific types of sarcomas. Somatic cell hybridization analyses have previously shown that normal telomerase-negative fibroblasts and telomerase-positive immortalized cell lines contain repressors of ALT activity, indicating that activation of ALT results from loss of one or more unidentified repressors. More recently, ATRX or DAXX was shown to be mutated both in tumors with telomere lengths suggestive of ALT activity and in ALT cell lines. Here, an ALT cell line was separately fused to each of four telomerase-positive cell lines, and four or five independent hybrid lines from each fusion were examined for expression of ATRX and DAXX and for telomere lengthening mechanism. The hybrid lines expressed either telomerase or ALT, with the other mechanism being repressed. DAXX was expressed normally in all parental cell lines and in all of the hybrids. ATRX was expressed normally in each of the four telomerase-positive parental cell lines and in every telomerase-positive hybrid line, and was abnormal in the ALT parental cells and in all but one of the ALT hybrids. This correlation between ALT activity and loss of ATRX expression is consistent with ATRX being a repressor of ALT.</p> </div

“Balancing Expectations with Actual Realities”: Conversations with Clinicians and Scientists in the First Year of a High-Risk Childhood Cancer Precision Medicine Trial

Precision medicine is changing cancer care and placing new demands on oncology professionals. Precision medicine trials for high-risk childhood cancer exemplify these complexities. We assessed clinicians&rsquo; (<i>n</i> = 39) and scientists&rsquo; (<i>n</i><i> </i>= 15) experiences in the first year of the PRecISion Medicine for Children with Cancer (PRISM) trial for children and adolescents with high-risk cancers, through an in-depth semi-structured interview. We thematically analysed participants&rsquo; responses regarding their professional challenges, and measured oncologists&rsquo; knowledge of genetics and confidence with somatic and germline molecular test results. Both groups described positive early experiences with PRISM but were cognisant of managing parents&rsquo; expectations. Key challenges for clinicians included understanding and communicating genomic results, balancing biopsy risks, and drug access. Most oncologists rated &lsquo;good&rsquo; knowledge of genetics, but a minority were &lsquo;very confident&rsquo; in interpreting (25%), explaining (34.4%) and making treatment recommendations (18.8%) based on somatic genetic test results. Challenges for scientists included greater emotional impact of their work and balancing translational outputs with academic productivity. Continued tracking of these challenges across the course of the trial, while assessing the perspectives of a wider range of stakeholders, is critical to drive the ongoing development of a workforce equipped to manage the demands of paediatric precision medicine

Optimization of a clofarabine-based drug combination regimen for the preclinical evaluation of pediatric acute lymphoblastic leukemia

BACKGROUND: The aim of this study was to improve the predictive power of patient-derived xenografts (PDXs, also known as mouse avatars) to more accurately reflect outcomes of clofarabine-based treatment in pediatric acute lymphoblastic leukemia (ALL) patients. PROCEDURE: Pharmacokinetic (PK) studies were conducted using clofarabine at 3.5 to 15 mg/kg in mice. PDXs were established from relapsed/refractory ALL patients who exhibited good or poor responses to clofarabine. PDX engraftment and response to clofarabine (either as a single agent or in combinations) were assessed based on stringent objective response measures modeled after the clinical setting. RESULTS: In naïve immune-deficient NSG mice, we determined that a clofarabine dose of 3.5 mg/kg resulted in systemic exposures equivalent to those achieved in pediatric ALL patients treated with clofarabine-based regimens. This dose was markedly lower than the doses of clofarabine used in previously reported preclinical studies (typically 30-60 mg/kg) and, when scheduled consistent with the clinical regimen (daily × 5), resulted in 34-fold lower clofarabine exposures. Using a well-tolerated clofarabine/etoposide/cyclophosphamide combination regimen, we then found that the responses of PDXs better reflected the clinical responses of the patients from whom the PDXs were derived. CONCLUSIONS: This study has identified an in vivo clofarabine treatment regimen that reflects the clinical responses of relapsed/refractory pediatric ALL patients. This regimen could be used prospectively to identify patients who might benefit from clofarabine-based treatment. Our findings are an important step toward individualizing prospective patient selection for the use of clofarabine in relapsed/refractory pediatric ALL patients and highlight the need for detailed PK evaluation in murine PDX models

Growth curves of hybrid cell lines.

<p>GM847 cells were fused with J82, TE-85 or A549 telomerase-positive cell lines using polyethylene glycol. Proliferation of each hybrid clone was plotted as a function of population doubling and the number of days following fusion. Growth of MeT-5A/GM847 hybrid lines has been described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050062#pone.0050062-Duncan1" target="_blank">[21]</a>.</p

Detection of ALT activity by C-circle assay.

<p>A. Dot blot of C-circle assay products for each parental and hybrid line. Serial dilutions of genomic DNA from GM847 cells indicate linearity of the assay within this range. The presence of C-circles indicates the cell line utilizes the ALT mechanism. B. Quantitation of C-circle assay levels by densitometry. The results from two separate experiments are presented relative to the signal obtained by GM847 cells. Bars, average; error bars, range.</p

Agarose gel electrophoresis of ATRX exon 1 PCR products.

<p>ATRX exon 1 of each of the indicated samples was amplified by PCR as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050062#s2" target="_blank">Materials and Methods</a>. The PCR products (expected size 418bp) were subjected by agarose gel electrophoresis and verified by Sanger sequencing. GM02063 is the parent cell strain from which GM847 cells were derived by SV40-induced immortalization.</p

Correlation between ALT activity and lack of normal ATRX expression.

a<p>6/7 ALT hybrids underwent a period of growth arrest compared to 3/12 TEL hybrids (p = 0.0198, Fisher’s exact test);</p>b<p>TRAP assay: “+” indicates detectable telomerase activity (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050062#pone-0050062-g002" target="_blank">Figure 2</a>);</p>c<p>TRF: “+” indicates a terminal restriction fragment length pattern characteristic of ALT (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050062#pone-0050062-g003" target="_blank">Figure 3</a>);</p>d<p>CC assay: “+” indicates C-circle levels above the cut-off level for ALT activity (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050062#pone-0050062-g004" target="_blank">Figure 4</a>);</p>e<p>TLM: telomere lengthening mechanism deduced from TRAP, TRF and CC assay data; ALT and TEL indicate ALT-positive and telomerase-positive, respectively;</p>f<p>ATRX protein: “+:” indicates normal pattern of immunofluorescence (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050062#pone-0050062-g005" target="_blank">Figure 5</a>) and normal levels on Western (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050062#pone-0050062-g006" target="_blank">Figure 6</a>);</p>g<p>ATRX SNP: a single nucleotide polymorphism (rs3088074, c.3017C>G, p.Q929E) for which each of the parental lines is homozygous identified the parental origin(s) of the ATRX alleles in all hybrids except the TE-85/GM847 lines for which the SNP was not determined (nd), because TE-85 and GM847 both have G at this location;</p>h<p>The magnitude of the C peak was significantly smaller than that of the G peak;</p>i<p>ATRX exon 1: “+” indicates the presence of wild-type ATRX exon 1 as determined by PCR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050062#pone-0050062-g007" target="_blank">Figure 7</a>).</p