Abstract INTRODUCTION Medulloblastoma (MB) is one of the most prevalent embryonal malignant brain tumors, divided into four distinct molecular subgroups (WNT, SHH, group 3, and group 4) with its ...individual prognosis. A more extensive classification of MB has recently been provided, identifying numerous subtypes, some with poor prognosis, leading to a significant number of patients succumbing to the disease while many survivors experience long-term side effects, highlighting the need for efficient subgrouping methods. Distinct genome-wide DNA methylation profiles characterize each MB subgroup, and the advent of Nanopore DNA sequencing has positioned itself as a reliable technique for comprehensive copy number and methylation profiling. MATERIAL AND METHODS We evaluated the ability of Nanopore sequencing to provide clinically relevant methylation and copy number profiles of MB, on a discovery cohort of 45 frozen MB samples, benchmarked against the gold standard EPIC array, and subsequently validated on a cohort of 116 MB samples. RESULTS The majority of MB in both the discovery cohort (43/45; 95.6%) and the validation cohort (110/116; 94.8%) were accurately subgrouped by Nanopore sequencing. Flongle flow cells for 18 MB allowed for a more rapid and cost-effective analysis, with 94.4% (17/18) being correctly classified. Additionally, Nanopore sequencing allowed us to correctly subtype 24/30 (80.0%) within MB groups. DISCUSSION We provided proof of concept that Nanopore sequencing can subgroup MB using DNA extracted from formalin-fixed paraffin-embedded (FFPE) samples and cell-free DNA obtained from cerebrospinal fluid. This first large-scale study establishes the proof of concept that this modern and innovative technology is well-suited for MB classification. We confirm the use of Nanopore sequencing on circulating DNA and present an initial exploration of its application on DNA extracted from FFPE samples. Its ability to deliver quick and cost-effective results firmly establishes Nanopore sequencing as a game-changer in the field of MB classification.
BACKGROUND: Cervical cancer (CC) remains a leading cause of gynaecological cancer-related mortality with infection by human papilloma virus (HPV) being the most important risk factor. We analysed the ...association between different viral integration signatures, clinical parameters and outcome in pre-treated CCs. METHODS: Different integration signatures were identified using HPV double capture followed by next-generation sequencing (NGS) in 272 CC patients from the BioRAIDs study NCT02428842. Correlations between HPV integration signatures and clinical, biological and molecular features were assessed. RESULTS: Episomal HPV was much less frequent in CC as compared to anal carcinoma (p < 0.0001). We identified >300 different HPV-chromosomal junctions (inter-or intra-genic). The most frequent integration site in CC was in MACROD2 gene followed by MIPOL1/TTC6 and TP63. HPV integration signatures were not associated with histological subtype, FIGO staging, treatment or PFS. HPVs were more frequently episomal in PIK3CA mutated tumours (p = 0.023). Viral integration type was dependent on HPV genotype (p < 0.0001); HPV18 and HPV45 being always integrated. High HPV copy number was associated with longer PFS (p = 0.011). CONCLUSIONS: This is to our knowledge the first study assessing the prognostic value of HPV integration in a prospectively annotated CC cohort, which detects a hotspot of HPV integration at MACROD2; involved in impaired PARP1 activity and chromosome instability.
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.
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.
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.
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.
Aims
Low‐grade adenosquamous carcinoma of the breast (
LGASC
) is a rare variant of metaplastic carcinoma characterised by a favourable outcome and histologically composed of glandular and squamous ...elements in a spindle cell background typically associated with a lymphocytic stromal reaction. Because of its rarity, the immunophenotypic and genetic profile of
LGASC
has not been sufficiently characterised. The aim of this study was to gain insights into the molecular and phenotypic characteristics of
LGASC
.
Methods and results
We reviewed the clinical and morphological features and detailed the immunohistochemical characteristics of a retrospective series of 13
LGASC
s. Targeted sequencing of 50 genes was performed in 10 of 13 cases. Identified mutations were further assessed by Sanger sequencing in a validation series of 11 additional cases. All tumours showed a triple‐negative immunophenotype, expressed ‘basal’ keratins, showed variable levels of epidermal growth factor receptor expression, and did not express androgen receptor. Sequencing analysis of the screening set of
LGASC
s revealed a high rate (seven of 10 cases) of
PIK
3
CA
mutations, whereas no
TP
53
mutations were found. All
PIK
3
CA
mutations were missense mutations located either in exon 20 (
n
= 6) or in exon 9 (
n
= 1). The global
PIK
3
CA
mutation rate, including the validation series, was 52% (11 of 21 cases). No disease recurrences were observed. Correction added on 11 June 2018, after first online publication: The percentage of mutation rate was corrected to 52%
Conclusions
Our results indicate that
LGASC
of the breast is a low‐grade triple‐negative breast cancer that harbours a basal‐like phenotype with no androgen receptor expression, and shows a high rate of
PIK
3
CA
mutations but no
TP
53
mutations.
Uveal melanoma (UM) is a rare cancer resulting from the transformation of melanocytes in the uveal tract. Integrative analysis has identified four molecular and clinical subsets of 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 using 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 the upregulation of PRAME, an independent prognostic biomarker in UM, and a potential therapeutic target. Our findings illustrate how multi-omics approaches can improve our understanding of tumorigenesis and reveal two distinct mechanisms of gene expression dysregulation in UM.
Display omitted
•Aggressive uveal melanomas display genomic instability•Promoter deletions are a recurrent mechanism of BAP1 deficiency•Looping and chromatin marks identify distal regulatory elements for PRAME expression
Gentien et al. perform extensive multi-omics of PDX derived from highly aggressive uveal melanomas (UMs) and normal uveal melanocytes to identify genomic, epigenomic, and transcriptomic patterns associated with tumorigenesis. Integrative analyses reveal genomic instability and identify mechanisms of dysregulation for two highly important genes, BAP1 and PRAME, in aggressive UMs.
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 two powerful and widely used tools: git and GitLab. 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.
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 two powerful and widely used tools: git and GitLab. 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.
Abstract Objective Fibromyalgia (FM) is a chronic musculoskeletal pain disorder characterized by widespread pain. This study focuses on patients’ attributions of illness and of symptom onset. Methods ...Semi-structured interviews were conducted with 56 women to elicit patients’ views on what triggered their FM. The transcripts of the interviews were analyzed using a classical indexing technique to identify key themes. Content analysis was performed by two independent coders. Results Primary causal attributions fell into five categories: psychological problems (28 respondents); somatic concerns (N = 12); violence/abuse during childhood (N = 7), gynaecological/obstetrical problems (N = 6), and fatigue (N = 3). Patients’ attributions were internal and external in the same proportions, more frequently unstable than stable, and more often described uncontrollable than controllable. Participants expressed decrements in self-esteem and feelings such as self-blame or despair; global perceptions of persistent pain and long-lasting problems, evoking chronicity and hopelessness; and low perceived control over their lives as well as beliefs that nothing can be done, thus increasing a feeling of guilt and vulnerability. Patients’ narratives emphasized disruptive circumstances surrounding symptom onset. Conclusion Attributions often referred to the psychological dimension of the events surrounding FM onset, even though some of them also had a clear somatic dimension. Many narratives mentioned successive disruptive events and suggested an increasing loss of control. Addressing these illness representations may contribute to tailor the treatment and to help patients gain self-coherency by providing means to understand pain onset but also to guide future behaviours, particularly in terms of adjustment and help-seeking.
Anal squamous cell carcinomas (ASCC) are rare tumours in humans. The etiological role of HPV infection is now well established but little is known about the molecular landscape and signalling ...pathways involved in the pathogenesis of this cancer. Here we report the results from a whole exome sequencing of a homogeneous group of 20 treatment-naive ASCC. A total of 2422 somatic single nucleotide variations (SNV) were found, with an overall moderate rate of somatic mutations per tumour (median: 105 relevant SNV per tumour) but a high mutational load in 3 tumours. The mutational signatures associated with age and APOBEC were observed in 100% and 60% of tumours respectively. The most frequently mutated genes were
(25%) followed by
(15%),
(15%)
and
(15%), the two last ones having never been described in ASCC. The main copy number alterations were gains of chromosome 3q (affecting
) and losses of chromosome 11q (affecting
. The combined analysis of somatic mutations and copy number alterations show that recurrent alterations of the PI3K/AKT/mTOR pathway are frequent (60%) in these tumours, as well as potentially targetable alterations of other signalling pathways that have never been described in ASCC such as chromatin remodelling (45%) and ubiquitin mediated proteolysis (35%). These results highlight the possible implication of these aberrant signalling pathways in anal carcinogenesis and suggest promising new therapeutic approaches in ASCC. The high somatic mutation burden found in some tumours, suggesting an elevated neoantigen load could also predict sensitivity of ASCC to immunotherapy.
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.
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