Search

OBJECTIVE Chordomas are rare bone neoplasms characterized by a high recurrence rate and no benefit from any approved medical treatment to date. However, the investigation of molecular alterations in chordomas could be essential to prognosticate, guide clinical decision-making, and identify theranostic biomarkers. The aim of this study was to provide a detailed genomic landscape of a homogeneous series of 64 chordoma samples, revealing driver events, theranostic markers, and outcome-related genomic features. METHODS The authors conducted whole-exome sequencing (WES), targeted next-generation sequencing, and RNA sequencing of 64 skull base and spinal chordoma samples collected between December 2006 and September 2020. Clinical, histological, and radiological data were retrospectively analyzed and correlated to genetic findings. RESULTS The authors identified homozygous deletions of CDKN2A/2B, PIK3CA mutations, and alterations affecting genes of SWI/SNF chromatin remodeling complexes (PBRM1 and ARID1A) as potential theranostic biomarkers. Using matched germline WES, they observed a higher frequency of a common genetic variant (rs2305089; p.(Gly177Asp)) in TBXT (97.8%, p < 0.001) compared to its distribution in the general population. PIK3CA mutation was identified as an independent biomarker of short progression-free survival (HR 10.68, p = 0.0008). Loss of CDKN2A/2B was more frequently observed in spinal tumors and recurrent tumors. CONCLUSIONS In the current study, the authors identified driver events such as PBRM1 and PIK3CA mutations, TBXT alterations, or homozygous deletions of CDKN2A/2B, which could, for some, be considered potential theranostic markers and could allow for identifying novel therapeutic approaches. With the aim of a future biomolecular prognostication classification, alterations affecting PIK3CA and CDKN2A/2B could be considered as poor prognostic biomarkers.

DNA methylation-based classification of cancer provides a comprehensive molecular approach to diagnose tumours. In fact, DNA methylation profiling of human brain tumours already profoundly impacts clinical neuro-oncology. However, current implementation using hybridisation microarrays is time consuming and costly. We recently reported on shallow nanopore whole-genome sequencing for rapid and cost-effective generation of genome-wide 5-methylcytosine profiles as input to supervised classification. Here, we demonstrate that this approach allows us to discriminate a wide spectrum of primary brain tumours.Using public reference data of 82 distinct tumour entities, we performed nanopore genome sequencing on 382 tissue samples covering 46 brain tumour (sub)types. Using bootstrap sampling in a cohort of 55 cases, we found that a minimum set of 1000 random CpG features is sufficient for high-confidence classification by ad hoc random forests. We implemented score recalibration as a confidence measure for interpretation in a clinical context and empirically determined a platform-specific threshold in a randomly sampled discovery cohort (N = 185). Applying this cut-off to an independent validation series (n = 184) yielded 148 classifiable cases (sensitivity 80.4%) and demonstrated 100% specificity. Cross-lab validation demonstrated robustness with concordant results across four laboratories in 10/11 (90.9%) cases. In a prospective benchmarking (N = 15), the median time to results was 21.1 h.In conclusion, nanopore sequencing allows robust and rapid methylation-based classification across the full spectrum of brain tumours. Platform-specific confidence scores facilitate clinical implementation for which prospective evaluation is warranted and ongoing.

Uveal Melanoma (UM) is a rare cancer resulting from transformation of melanocytes residing in the uveal tract. Integrative analysis has identified four molecular and clinical subsets in UM. To improve our molecular understanding of UM we performed extensive multi-omics characterization comparing two aggressive UM patient-derived xenograft models with normal choroidal melanocytes, including DNA optical mapping, specific histone modifications, and DNA topology analysis by Hi-C. Our gene expression and cytogenetic analyses suggest that genomic instability is a hallmark of UM. We also identified a recurrent deletion in the BAP1 promoter resulting in loss of expression and associated with high risk of metastases in UM patients. Hi-C revealed chromatin topology changes associated with up-regulation of PRAME, an independent prognostic biomarker in UM and potential therapeutic target. Our findings illustrate how multi-omics approaches can improve the understanding of tumorigenesis and reveals two novel mechanisms of gene expression dysregulation in UM.

High‐throughput DNA analyses in cancers allow the detection of molecular abnormalities that can guide patients' treatment. The collection of a tumour DNA is mainly performed on a biopsy, an invasive procedure for the patients. Another less invasive method consists in using a fine needle. We showed that fine‐needle biopsies are as suitable as a classical biopsy for DNA analysis., High‐throughput molecular profiling of solid tumours using core needle biopsies (CNB) allows the identification of actionable molecular alterations, with around 70% success rate. Although several studies have demonstrated the utility of small biopsy specimens for molecular testing, there remains debate as to the sensitivity of the less invasive fine‐needle aspiration (FNA) compared to CNB to detect molecular alterations. We aimed to prospectively evaluate the potential of FNA to detect such alterations in various tumour types as compared to CNB in cancer patients included in the SHIVA02 trial. An in‐house amplicon‐based targeted sequencing panel (Illumina TSCA 99.3 kb panel covering 87 genes) was used to identify pathogenic variants and gene copy number variations (CNV) in concomitant CNB and FNA samples obtained from 61 patients enrolled in the SHIVA02 trial (NCT03084757). The main tumour types analysed were breast (38%), colon (15%), pancreas (11%), followed by cervix and stomach (7% each). We report 123 molecular alterations (85 variants, 23 amplifications and 15 homozygous deletions) among which 98 (80%) were concordant between CNB and FNA. The remaining discordances were mainly related to deletions status, yet undetected alterations were not exclusively specific to FNA. Comparative analysis of molecular alterations in CNB and FNA showed high concordance in terms of variants as well as CNVs identified. We conclude FNA could therefore be used in routine diagnostics workflow and clinical trials for tumour molecular profiling with the advantages of being minimally invasive and preserve tissue material needed for diagnostic, prognostic or theranostic purposes.

By 2030, pancreatic cancer will become the second leading cause of cancer-related deaths in the United States and in Europe. The management of patients with advanced pancreatic cancer relies on chemotherapy and poly (ADP-ribose) polymerase inhibitors for patients who carry BRCA1/2 inactivating alterations. Some variants, such as large insertion/deletions (Indels), inactivating BRCA1/2 and therefore of clinical relevance can be hard to detect by next-generation sequencing techniques. Here we report a 47-year-old patient presenting with pancreatic cancer whose tumour harbours a large somatic intra-exonic deletion of BRCA2 of 141 bp. This BRCA2 deletion, located in the C-terminal domain, can be considered as pathogenic and consequently affect tumorigenesis because it is involved in the interaction between the DSS1 protein and DNA. Thanks to the optimized bioinformatics algorithm, this intermediate size deletion in BRCA2 was identified, enabling personalized patient management via the inclusion of the patients in a clinical trial.

Objective Tumors harboring a POLE pathogenic variant, associated with high tumor mutational burden, are good candidates for immunotherapy. However, POLE pathogenic variants are not currently screened in routine clinical practice. Can these tumors be identified by means of an already available test? Methods We describe seven tumors harboring a POLE pathogenic variant, among eight patients with tumors harboring multiple BRCA1/2 variants (from 4 to 20). All patients were managed at Institut Curie, Paris. Five patients were selected because of unexpected tumor BRCA testing results with multiple variants and another three patients were selected because of a POLE pathogenic variant detected by large tumor testing. We looked for other tumor variants by Next-Generation Sequencing in tumors harboring multiple BRCA1/2 variants, and for multiple BRCA1/2 variants in tumors harboring a POLE pathogenic variant. Results Four of the five tumors selected because of multiple BRCA1/2 variants exhibited a POLE pathogenic variant, and all three tumors selected for POLE pathogenic variants exhibited multiple BRCA1/2 variants. Conclusions Tumor BRCA testing could be a way to detect tumors harboring a highly mutagenic POLE pathogenic variant.

Background: Epidermal growth factor receptor (EGFR) inhibitors, including monoclonal antibodies and tyrosine kinase inhibitors, have limited efficacy in head and neck squamous cell carcinoma (HNSCC) patients. In the randomized phase II PREDICTOR trial, we aimed at identifying predictive and pharmacodynamics biomarkers of 2-4 weeks afatinib (an irreversible pan-HER inhibitor) versus no treatment in the pre-operative setting (NCT01415674). We previously reported a 59% metabolic response rate on PET imaging in the afatinib arm. We report here the evaluation of predictive genomic and transcriptomic biomarkers of afatinib efficacy. Patients and Methods: All patients (41 in the afatinib arm and 20 in the no treatment arm) underwent a pre-treatment biopsy. We performed targeted DNA sequencing using an in-house NGS panel of 571 genes on baseline biopsies from 56 patients, and RNA-sequencing (RNAseq) in 54 patients. In the afatinib arm, 26 patients had paired pre- and post-treatment tumor samples. DNA and RNA alterations were correlated with metabolic response to afatinib using PET imaging, as well as overall survival (OS). Results: Most frequent molecular alterations, including known activating mutations and/or focal amplifications for oncogenes or homozygous deletions and inactivating mutations for tumor suppressor genes, involved genome integrity (TP53 [70%]), cell cycle (CCND1 [38%], CDKN2A [32%], CDKN2B [14%]), senescence (TERT [23%]), Wnt signaling (NOTCH1 [16%]), and the PI3K pathway (PIK3CA [14%]). In the afatinib arm, metabolic response was observed in 1 out of 7 patients (14%) and in 19 out of 28 patients (68%) in the Wnt altered and unaltered groups (p = 0.03, fisher exact test), respectively. In the whole cohort of patients, homozygous deletions of both CDKN2A and CDKN2B correlated with shorter OS, with 6-year survival of 22% in the CDKN2A/B altered group and 70% in the CDKN2A/B wild-type group (p = 0.004; log-rank test). In the afatinib treated patients, using a generalized linear mixed model with a patient as random effect and a quasi-binomial family, the ratio of B cells expression levels in the post-treated versus pre-treated samples was significantly higher in responder as compared to non-responder patients (p = 0.001). Conclusions: Wnt signaling pathway alterations and treatment-related dynamic changes in B cells proportions were identified as predictive and pharmacodynamics biomarkers of afatinib efficacy. CDKN2A/B homozygous deletions were associated with a poor prognosis in HNSCC patients treated with upfront surgery. Citation Format: Grégoire Marret, Maud Kamal, Jocelyn Gal, Stéphane Temam, Jerzy Klijianenko, Jean-Pierre Delord, Caroline Hoffmann, Gilles Dolivet, Olivier Malard, Jerôme Fayette, Olivier Capitain, Caroline Even, Sébastien Vergez, Lionel Geoffrois, Frédéric Rolland, Philippe Zrounba, Laurent Laccourreye, Joël Guigay, Nicolas Aide, Valérie Bénavent, Constance Lamy, Elodie Girard, Marta Jimenez, Ivan Bièche, Christophe Le Tourneau. Randomized phase II trial of pre-operative afatinib in non-metastatic head and neck squamous cell carcinoma patients: Identification of predictive biomarkers of response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1237.

The fast advances in molecular biology have led to the discovery of new genetic and epigenetic driver events that play a key role in the pathogenesis of central nervous system (CNS) tumors. These data showed the importance of molecular biology, and in particular methylation profiling and fusion detection, to complete the histological examination. Exploring all these complex genomic and epigenomic alterations with the technologies implemented in the routine diagnosis requires massive laboratory equipment. In addition, it could take weeks before getting data that could be important to adapt the diagnosis, prognosis, and to ultimately guide the treatment’s choice. We describe the innovative potential of a nanopore sequencing device to obtain fast and multiple information to characterize CNS tumors. Adaptive-sampling is a software-controlled enrichment unique method that allows simultaneous methylation profiling, copy number landscape assessment, and fusion gene detection. Thereby, this technology brings comprehensive molecular information that could be very important for CNS tumor classification in a very short delay (below one week) and with a relative low cost compared to the combined other molecular analysis needed to get the same information. We report ten cases of diverse CNS tumors with already known oncogenic structural variants such as YAP1-MAMLD1 fused ependymoma, CNS neuroblastoma with FOXR2 activation, CNS high-grade neuroepithelial tumor with BCOR and MEN1 alteration, and atypical teratoid rhabdoid tumors (ATRT). For these tumors, using adaptive sampling with the nanopore sequencing, we were able to detect fusion genes as driver events, while obtaining at the same time the methylation profile to corroborate the tumor classification. Our results with this combined approach, never described before, demonstrate its feasibility as a new extremely powerful tool and show remarkable perspectives for this technology to bring a fast and comprehensive tumor molecular characterization.

Bi-allelic loss-of-function (LoF) variants in the base excision repair (BER) gene NTHL1 cause a high-risk hereditary multi-tumor syndrome that includes breast cancer, but the contribution of heterozygous variants to hereditary breast cancer is unknown. An analysis of 4985 women with breast cancer, enriched for familial features, and 4786 cancer-free women revealed significant enrichment for NTHL1 LoF variants. Immunohistochemistry confirmed reduced NTHL1 expression in tumors from heterozygous carriers but the NTHL1 bi-allelic loss characteristic mutational signature (SBS 30) was not present. The analysis was extended to 27,421 breast cancer cases and 19,759 controls from 10 international studies revealing 138 cases and 93 controls with a heterozygous LoF variant (OR 1.06, 95% CI: 0.82–1.39) and 316 cases and 179 controls with a missense variant (OR 1.31, 95% CI: 1.09–1.57). Missense variants selected for deleterious features by a number of in silico bioinformatic prediction tools or located within the endonuclease III functional domain showed a stronger association with breast cancer. Somatic sequencing of breast cancers from carriers indicated that the risk associated with NTHL1 appears to operate through haploinsufficiency, consistent with other described low-penetrance breast cancer genes. Data from this very large international multicenter study suggests that heterozygous pathogenic germline coding variants in NTHL1 may be associated with low- to moderate- increased risk of breast cancer.

DNA methylation-based classification of cancer provides a comprehensive molecular approach to diagnose tumors. In fact, DNA methylation profiling of human brain tumors already profoundly impacts clinical neuro-oncology. However, current implementations using hybridization microarrays are time-consuming and costly. We recently reported on shallow nanopore whole-genome sequencing for rapid and cost-effective generation of genome-wide 5-methylcytosine profiles as input to supervised classification using random forests complemented by a medium-resolution copy number profile derived from the same raw data. Here, we demonstrate that this approach allows to discriminate a wide spectrum of primary brain tumors using public reference data of 82 distinct tumor entities. We developed a pseudo-probability score as a confidence score for interpretation in a clinical context. Using bootstrap sampling in a discovery cohort of N = 56 cases, we find that a minimum set of 1,000 random CpG features is sufficient for high-confidence classification by ad hoc random forests for most cases and demonstrate robustness across laboratories with matching results in 13/13 cases. When applying the confidence score threshold to an independent validation series (N = 111), the method demonstrated 100% specificity for the remaining 93 cases. In a prospective benchmarking (N = 15), median time to results was 21.1 hours. In conclusion, nanopore sequencing allows robust and rapid methylation-based classification across the full spectrum of brain tumors. The integrated confidence score facilitates possible clinical implementation, while requiring further prospective evaluation.

Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) are two cancer-derived blood biomarkers that inform on patient prognosis and treatment efficacy in breast cancer. We prospectively evaluated the clinical validity of quantifying both CTCs (CellSearch) and ctDNA (targeted next-generation sequencing). Their combined value as prognostic and early monitoring markers was assessed in 198 HER2-negative metastatic breast cancer patients. All patients were included in the prospective multicenter UCBG study COMET (NCT01745757) and treated by first-line chemotherapy with weekly paclitaxel and bevacizumab. Blood samples were obtained at baseline and before the second cycle of chemotherapy. At baseline, CTCs and ctDNA were respectively detected in 72 and 74% of patients and were moderately correlated (Kendall’s τ = 0.3). Only 26 (13%) patients had neither detectable ctDNA nor CTCs. Variants were most frequently observed in TP53 and PIK3CA genes. KMT2C/MLL3 variants detected in ctDNA were significantly associated with a lower CTC count, while the opposite trend was seen with GATA3 alterations. Both CTC and ctDNA levels at baseline and after four weeks of treatment were correlated with survival. For progression-free and overall survival, the best multivariate prognostic model included tumor subtype (triple negative vs other), grade (grade 3 vs other), ctDNA variant allele frequency (VAF) at baseline (per 10% increase), and CTC count at four weeks (≥5CTC/7.5 mL). Overall, this study demonstrates that CTCs and ctDNA have nonoverlapping detection profiles and complementary prognostic values in metastatic breast cancer patients. A comprehensive liquid-biopsy approach may involve simultaneous detection of ctDNA and CTCs.

BACKGROUND Cervical cancer (CC) remains a leading cause of gynaecological cancer-related mortality world wide and constitutes the third most common malignancy in women. The RAIDs consortium ( http://www.raids-fp7.eu/ ) conducted a prospective European study [BioRAIDs (NCT02428842)] with the objective to stratify CC patients for innovative treatments. A “metagene” of genomic markers in the PI3K pathway and epigenetic regulators had been previously associated with poor outcome [2] . METHODS To detect new, more specific, targets for treatment of patients who resist standard chemo-radiation, a high-dimensional Cox model was applied to define dominant molecular variants, copy number variations, and reverse phase protein arrays (RPPA). FINDINGS Survival analysis on 89 patients with all omics data available, suggested loss-of-function (LOF) or activating molecular alterations in nine genes to be candidate biomarkers for worse prognosis in patients treated by chemo-radiation while LOF of ATRX, MED13 as well as CASP8 were associated with better prognosis. When protein expression data by RPPA were factored in, the supposedly low molecular weight and nuclear form, of beta-catenin, phosphorylated in Ser552 (pβ-Cat552), ranked highest for good prognosis, while pβ-Cat675 was associated with worse prognosis. INTERPRETATION These findings call for molecularly targeted treatments involving p53, Wnt pathway, PI3K pathway, and epigenetic regulator genes. Pβ-Cat552 and pβ-Cat675 may be useful biomarkers to predict outcome to chemo-radiation, which targets the DNA repair axis. FUNDING European Union's Seventh Program for research, technological development and demonstration (agreement N°304,810), the Fondation ARC pour la recherche contre le cancer.

BACKGROUND: There is a lack of information as to which molecular processes, present at diagnosis, favor tumour escape from standard-of-care treatments in cervical cancer (CC). RAIDs consortium (www.raids-fp7.eu), conducted a prospectively monitored trial, [BioRAIDs (NCT02428842)] with the objectives to generate high quality samples and molecular assessments to stratify patient populations and to identify molecular patterns associated with poor outcome. METHODS: Between 2013 and 2017, RAIDs collected a prospective CC sample and clinical dataset involving 419 participant patients from 18 centers in seven EU countries. Next Generation Sequencing has so far been carried out on a total of 182 samples from 377 evaluable (48%) patients, allowing to define dominant genetic alterations. Reverse phase protein expression arrays (RPPA) was applied to group patients into clusters. Activation of key genetic pathways and protein expression signatures were tested for associations with outcome. FINDINGS: At a median follow up (FU) of 22 months, progression-free survival rates of this FIGO stage IB1-IV population, treated predominantly (87%) by chemoradiation, were65•4% [CI95%: 60•2-71.1]. Dominant oncogenic alterations were seen in PIK3CA (40%), while dominant suppressor gene alterations were seen in KMT2D (15%) and KMT2C (16%). Cumulative frequency of loss-of-function (LOF) mutations in any epigenetic modulator gene alteration was 47% and it was associated with PIK3CA gene alterations in 32%. Patients with tumours harboring alterations in both pathways had a significantly poorer PFS. A new finding was the detection of a high frequency of gains of TLR4 gene amplifications (10%), as well as amplifications, mutations, and non-frame-shift deletions of Androgen receptor (AR) gene in 7% of patients. Finally, RPPA protein expression analysis defined three expression clusters. INTERPRETATION: Our data suggests that patient population may be stratified into four different treatment strategies based on molecular markers at the outset. FUND: European Union's Seventh Program grant agreement No 304810.

Numerous pan-genomic studies identified alterations in protein-coding genes and signaling pathways involved in bladder carcinogenesis, while non-coding somatic alterations remain weakly explored. The goal of this study was to identify clinical biomarkers in non-coding regions for bladder cancer patients. We have previously identified in bladder tumors two non-coding mutational hotspots occurring at high frequencies (&ge, 30%). These mutations are located close to the GPR126 and PLEKHS1 genes, at the guanine or the cytosine of a TGAACA core motif flanked, on both sides, by a stretch of palindromic sequences. Here, we hypothesize that such a pattern of recurrent non-coding mutations could be a signature of somatic genomic instability specifically involved in bladder cancer. We analyzed 26 additional mutable non-coding sites with the same core motif in a cohort of 103 bladder cancers composed of 44 NMIBC cases and 59 MIBC cases using high-resolution melting (HRM) and Sanger sequencing. Five bladder cancers were additionally analyzed for protein-coding gene mutations using a targeted NGS panel composed of 571 genes. Expression levels of three members of the APOBEC3 family genes were assessed using real-time quantitative RT-PCR. Non-coding somatic mutations were observed for at least one TGAACA core motif locus in 62.1% (64/103) of bladder tumor samples. These non-coding mutations co-occurred in the bladder tumors but were absent in prostate tumor, HPV-positive Head and Neck Squamous Cell Carcinoma, and high microsatellite instability (MSI-H) colorectal tumor series. This signature of palindromic non-coding somatic mutations, specific to bladder tumors, was not associated with patients&rsquo, outcome and was more frequent in females. Interestingly, this signature was associated with high tumor mutational burden (TMB) and high expression levels of APOBEC3B and interferon inducible genes. We identified a new type of somatic genomic instability targeting the TGAACA core motif loci flanked by palindromic sequences in bladder cancer. This mutational signature is a promising candidate clinical biomarker for the early detection of relapse and a major low-cost alternative to the TMB to monitor the response to immunotherapy for bladder cancer patients.

Genomic alterations of anal squamous cell carcinoma (ASCC) remain poorly understood due to the rarity of this tumor. Array comparative genomic hybridization and targeted gene sequencing were performed in 49 cases of ASCC. The most frequently altered regions (with a frequency greater than 25%) were 10 deleted regions (2q35, 2q36.3, 3p21.2, 4p16.3, 4p31.21, 7q36.1, 8p23.3, 10q23.2, 11q22.3, and 13q14.11) and 8 gained regions (1p36.33, 1q21.1, 3q26.32, 5p15.33, 8q24.3, 9q34.3, 16p13.3, and 19p13.3). The most frequent minimal regions of deletion (55%) encompassed the 11q22.3 region containing ATM, while the most frequent minimal regions of gain (57%) encompassed the 3q26.32 region containing PIK3CA. Recurrent homozygous deletions were observed for 5 loci (ie, TGFR2 in 4 cases), and recurrent focal amplifications were observed for 8 loci (ie, DDR2 and CCND1 in 3 cases, respectively). Several of the focal amplified genes are targets for specific therapies. Integrated analysis showed that the PI3K/Akt/mTOR signaling pathway was the pathway most extensively affected, particularly in recurrences compared to treatment‐naive tumors (64% vs 30%; P = .017). In patients with ASCC recurrences, poor overall survival (OS) was significantly correlated with a large number of altered regions (P = .024). These findings provide insight into the somatic genomic alterations in ASCC and highlight the key role of the druggable PI3K/Akt/mTOR signaling pathway.

Background Oral cavity is the most prevalent site of head and neck squamous cell carcinomas (HNSCCs). Most often diagnosed at a locally advanced stage, treatment is multimodal with surgery as the cornerstone. The aim of this study was to explore the molecular landscape of a homogenous cohort of oral cavity squamous cell carcinomas (OCSCCs), and to assess the prognostic value of tumor mutational burden (TMB), along with classical molecular and clinical parameters. Patients and methods One hundred and fifty-one consecutive patients with OCSCC treated with upfront surgery at the Institut Curie were analyzed. Sequencing of tumor DNA from frozen specimens was carried out using an in-house targeted next-generation sequencing panel (571 genes). The impact of molecular alterations and TMB on disease-free survival (DFS) and overall survival (OS) was evaluated in univariate and multivariate analyses. Results Pathological tumor stage, extranodal spread, vascular emboli, and perineural invasion were associated with both DFS and OS. TP53 was the most mutated gene (71%). Other frequent molecular alterations included the TERT promoter (50%), CDKN2A (25%), FAT1 (17%), PIK3CA (14%), and NOTCH1 (15%) genes. Transforming growth factor-β pathway alterations (4%) were associated with poor OS (P = 0.01) and DFS (P = 0.02) in univariate and multivariate analyses. High TMB was associated with prolonged OS (P = 0.01 and P = 0.02, in the highest 10% and 20% TMB values, respectively), but not with DFS. Correlation of TMB with OS remained significant in multivariate analysis (P = 0.01 and P = 0.005 in the highest 10% and 20% TMB values, respectively). Pathological tumor stage combined with high TMB was associated with good prognosis. Conclusion Our results suggest that a high TMB is associated with a favorable prognosis in patients with OCSCC treated with upfront surgery., Highlights • High TMB is associated with a favorable prognosis in patients with OCSCC treated with upfront surgery • Pathological tumor stage combined with high TMB is associated with good prognosis • TP53 was the most mutated gene (71%). Other frequent molecular alterations included the TERT promoter (50%) • TGFβ pathway alterations were associated with poor outcomes, although it was only observed in 4% of the patients

The American College of Medical Genetics and Genomics (ACMG) guidelines for sequence variant classification include two criteria, PP3 and BP4, for combining computational data with other evidence types contributing to sequence variant classification. PP3 and BP4 assert that computational modeling can provide “Supporting” evidence for or against pathogenicity within the ACMG framework. Here, leveraging a meta-analysis of ATM and CHEK2 breast cancer case-control mutation screening data, we evaluate the strength of evidence determined from the relatively simple computational tool Align-GVGD. Importantly, application of Align-GVGD to these ATM and CHEK2 data is free of logical circularities, hidden multiple testing, and use of other ACMG evidence types. For both genes, rare missense substitutions that are assigned the most severe Align-GVGD grade exceed a “Moderate pathogenic” evidence threshold when analyzed in a Bayesian framework; accordingly, we argue that the ACMG classification rules be updated for well-calibrated computational tools. Additionally, congruent with previous analyses of ATM and CHEK2 case-control mutation screening data, we find that both genes have a considerable burden of pathogenic missense substitutions, and that severe ATM rare missense have increased odds ratios compared to truncating and splice junction variants, indicative of a potential dominant-negative effect for those missense substitutions.

International audience

In this article, a synthesis of data from the literature on postpartum post-traumatic stress disorder (PTSD) related to childbirth is presented, knowing that the psychological suffering in this period of life is frequently associated with a certain taboo. The criteria to diagnose psychotraumatism, as well as postpartum PTSD just after the birth are here recalled as well as the importance of evaluating the subjective experience of the mother wich is a central issue. The clinical specificity and key symptoms of postpartum PTSD are described along with the risk and protective factors. In the light of scientific knowledge in the field of psychotraumatology, we present the prevention strategies of this disorder that can be applied, but which remain to be evaluated and tailored to this specific context.Dans cet article, nous proposons une synthèse des données de la littérature sur le trouble de stress post-traumatique (TSPT) du postpartum en lien avec l’accouchement, en sachant que ce trouble continue d’être entouré d’un certain tabou. Les critères diagnostiques du psychotraumatisme, ainsi que du TSPT du postpartum (TSPTPP) en lien avec l’accouchement sont présentés. L’importance de l’évaluation du vécu subjectif de la mère est centrale. Les spécificités cliniques du TSPTPP en lien avec l’accouchement, à l’instar de la dépression du postpartum, sont présentées, de même que les facteurs de risque et de protection. A la lumière des connaissances scientifiques dans le champ de la psychotraumatologie, nous présentons les stratégies de prévention de ce trouble envisageables, mais qui restent à évaluer dans ce contexte spécifique.

Triple-negative breast cancer (TNBC) represents 10% of all breast cancers and is a very heterogeneous disease. Globally, women with TNBC have a poor prognosis, and the development of effective targeted therapies remains a real challenge. Patient-derived xenografts (PDX) are clinically relevant models that have emerged as important tools for the analysis of drug activity and predictive biomarker discovery. The purpose of this work was to analyze the molecular heterogeneity of a large panel of TNBC PDX (n = 61) in order to test targeted therapies and identify biomarkers of response. At the gene expression level, TNBC PDX represent all of the various TNBC subtypes identified by the Lehmann classification except for immunomodulatory subtype, which is underrepresented in PDX. NGS and copy number data showed a similar diversity of significantly mutated gene and somatic copy number alteration in PDX and the Cancer Genome Atlas TNBC patients. The genes most commonly altered were TP53 and oncogenes and tumor suppressors of the PI3K/AKT/mTOR and MAPK pathways. PDX showed similar morphology and immunohistochemistry markers to those of the original tumors. Efficacy experiments with PI3K and MAPK inhibitor monotherapy or combination therapy showed an antitumor activity in PDX carrying genomic mutations of PIK3CA and NRAS genes. TNBC PDX reproduce the molecular heterogeneity of TNBC patients. This large collection of PDX is a clinically relevant platform for drug testing, biomarker discovery and translational research.

Background: There is a lack of information as to which molecular processes, present at diagnosis, favor tumour escape from standard-of-care treatments in cervical cancer (CC). RAIDs consortium (www.raids-fp7.eu), conducted a prospectively monitored trial, [BioRAIDs (NCT02428842)] with the objectives to generate high quality samples and molecular assessments to stratify patient populations and to identify molecular patterns associated with poor outcome. Methods: Between 2013-2017, RAIDs collected a prospective CC sample and clinical dataset involving 419 participant patients from 18 centers in seven EU countries. Next Generation Sequencing has so far been carried out on a total of 182 samples from 377 evaluable (48%) patients, allowing to define dominant genetic alterations. Reverse phase protein expression arrays (RPPA) was applied to group patients into clusters. Activation of key genetic pathways and protein expression signatures were tested for associations with outcome. Findings: At a median FU of 22 months, progression-free survival rates of this FIGO stage IB1-IV population, treated predominantly (87%) by chemoradiation, were 65*4% [CI95%: 60*2-71.1]. Dominant oncogenic alterations were seen in PIK3CA (40%), while dominant suppressor gene alterations were seen in KMT2D (15%) and KMT2C (16%). Cumulative frequency of loss-of-function (LOF) mutations in any epigenetic modulatorgene alteration was 47% and it was associated with PIK3CA gene alterations in 32%. Patients with tumours harbouring alterations in both pathways had a significantly poorer PFS. A new finding was the detection of a high frequency of gains of TLR4 gene amplifications (10%), as well as amplifications, mutations, and non-frame-shift deletions of Androgen receptor (AR) gene in 7% of patients. Finally, RPPA protein expression analysis defined three expression clusters. Interpretation: Our data suggests that patient population may be stratified into four different treatment strategies based on molecular markers at the outset. Funding: European Union's Seventh Program grant agreement No 304810. Declaration of Interest: All authors declared: No conflict of interest. Ethical Approval: Ethical review was conducted according to national requirements and in accordance with the Declaration of Helsinki. All tumour and serum samples and clinical data were registered in a common database using a unique barcode system assuring data privacy.

Background The ataxia telangiectasia mutated (ATM) gene is a moderate-risk breast cancer susceptibility gene; germline loss-of-function variants are found in up to 3% of hereditary breast and ovarian cancer (HBOC) families who undergo genetic testing. So far, no clear histopathological and molecular features of breast tumours occurring in ATM deleterious variant carriers have been described, but identification of an ATM-associated tumour signature may help in patient management. Methods To characterise hallmarks of ATM-associated tumours, we performed systematic pathology review of tumours from 21 participants from ataxia-telangiectasia families and 18 participants from HBOC families, as well as copy number profiling on a subset of 23 tumours. Morphology of ATM-associated tumours was compared with that of 599 patients with no BRCA1 and BRCA2 mutations from a hospital-based series, as well as with data from The Cancer Genome Atlas. Absolute copy number and loss of heterozygosity (LOH) profiles were obtained from the OncoScan SNP array. In addition, we performed whole-genome sequencing on four tumours from ATM loss-of-function variant carriers with available frozen material. Results We found that ATM-associated tumours belong mostly to the luminal B subtype, are tetraploid and show LOH at the ATM locus at 11q22–23. Unlike tumours in which BRCA1 or BRCA2 is inactivated, tumours arising in ATM deleterious variant carriers are not associated with increased large-scale genomic instability as measured by the large-scale state transitions signature. Losses at 13q14.11-q14.3, 17p13.2-p12, 21p11.2-p11.1 and 22q11.23 were observed. Somatic alterations at these loci may therefore represent biomarkers for ATM testing and harbour driver mutations in potentially ‘druggable’ genes that would allow patients to be directed towards tailored therapeutic strategies. Conclusions Although ATM is involved in the DNA damage response, ATM-associated tumours are distinct from BRCA1-associated tumours in terms of morphological characteristics and genomic alterations, and they are also distinguishable from sporadic breast tumours, thus opening up the possibility to identify ATM variant carriers outside the ataxia-telangiectasia disorder and direct them towards effective cancer risk management and therapeutic strategies. Electronic supplementary material The online version of this article (10.1186/s13058-018-0951-9) contains supplementary material, which is available to authorized users.

The use of a bioinformatics pipeline as a tool to support diagnostic and theranostic decisions in the healthcare process requires the definition of detailed development workflow guidelines. Therefore, we implemented protocols that describe step-by-step all the command lines and actions that the developers have to follow. Our protocols capitalized on the two powerful and widely used tools git and GitLab, and are based on gitflow, a well-established workflow in the software engineering community. They address two use cases: a nominal mode to develop a new feature in the bioinformatics pipeline and a hotfix mode to correct a bug that occurred in the production environment. The protocols are available as a comprehensive documentation at https://biogitflow.readthedocs.io and the main concepts, steps and principles are presented in this report.

Medulloblastoma (MB) can be classified into four molecular subgroups (WNT group, SHH group, group 3, and group 4). The gold standard of assignment of molecular subgroup through DNA methylation profiling uses Illumina EPIC array. However, this tool has some limitation in terms of cost and timing, in order to get the results soon enough for clinical use. We present an alternative DNA methylation assay based on nanopore sequencing efficient for rapid, cheaper, and reliable subgrouping of clinical MB samples. Low-depth whole genome with long-read single-molecule nanopore sequencing was used to simultaneously assess copy number profile and MB subgrouping based on DNA methylation. The DNA methylation data generated by Nanopore sequencing were compared to a publicly available reference cohort comprising over 2,800 brain tumors including the four subgroups of MB (Capper et al. Nature; 2018) to generate a score that estimates a confidence with a tumor group assignment. Among the 24 MB analyzed with nanopore sequencing (six WNT, nine SHH, five group 3, and four group 4), all of them were classified in the appropriate subgroup established by expression-based Nanostring subgrouping. In addition to the subgrouping, we also examine the genomic profile. Furthermore, all previously identified clinically relevant genomic rearrangements (mostly MYC and MYCN amplifications) were also detected with our assay. In conclusion, we are confirming the full reliability of nanopore sequencing as a novel rapid and cheap assay for methylation-based MB subgrouping. We now plan to implement this technology to other embryonal tumors of the central nervous system.

Panel sequencing is a practical option in genetic diagnosis. Enrichment and library preparation steps are critical in the diagnostic setting. In order to test the value of HaloPlex technology in diagnosis, we designed a custom oncogenetic panel including 62 genes. The procedure was tested on a training set of 71 controls and then blindly validated on 48 consecutive hereditary breast/ovarian cancer (HBOC) patients tested negative for BRCA1/2 mutation. Libraries were sequenced on HiSeq2500 and data were analysed with our academic bioinformatics pipeline. Point mutations were detected using Varscan2, median size indels were detected using Pindel and large genomic rearrangements (LGR) were detected by DESeq. Proper coverage was obtained. However, highly variable read depth was observed within genes. Excluding pseudogene analysis, all point mutations were detected on the training set. All indels were also detected using Pindel. On the other hand, DESeq allowed LGR detection but with poor specificity, preventing its use in diagnostics. Mutations were detected in 8% of BRCA1/2-negative HBOC cases. HaloPlex technology appears to be an efficient and promising solution for gene panel diagnostics. Data analysis remains a major challenge and geneticists should enhance their bioinformatics knowledge in order to ensure good quality diagnostic results.