Assessment of branch point prediction tools to predict physiological branch points and their alteration by variants.

BACKGROUND: Branch points (BPs) map within short motifs upstream of acceptor splice sites (3'ss) and are essential for splicing of pre-mature mRNA. Several BP-dedicated bioinformatics tools, including HSF, SVM-BPfinder, BPP, Branchpointer, LaBranchoR and RNABPS were developed during the last decade. Here, we evaluated their capability to detect the position of BPs, and also to predict the impact on splicing of variants occurring upstream of 3'ss. RESULTS: We used a large set of constitutive and alternative human 3'ss collected from Ensembl (n = 264,787 3'ss) and from in-house RNAseq experiments (n = 51,986 3'ss). We also gathered an unprecedented collection of functional splicing data for 120 variants (62 unpublished) occurring in BP areas of disease-causing genes. Branchpointer showed the best performance to detect the relevant BPs upstream of constitutive and alternative 3'ss (99.48 and 65.84% accuracies, respectively). For variants occurring in a BP area, BPP emerged as having the best performance to predict effects on mRNA splicing, with an accuracy of 89.17%. CONCLUSIONS: Our investigations revealed that Branchpointer was optimal to detect BPs upstream of 3'ss, and that BPP was most relevant to predict splicing alteration due to variants in the BP area.

Novel diagnostic tool for prediction of variant spliceogenicity derived from a set of 395 combined in silico/in vitro studies: an international collaborative effort.

Variant interpretation is the key issue in molecular diagnosis. Spliceogenic variants exemplify this issue as each nucleotide variant can be deleterious via disruption or creation of splice site consensus sequences. Consequently, reliable in silico prediction of variant spliceogenicity would be a major improvement. Thanks to an international effort, a set of 395 variants studied at the mRNA level and occurring in 5' and 3' consensus regions (defined as the 11 and 14 bases surrounding the exon/intron junction, respectively) was collected for 11 different genes, including BRCA1, BRCA2, CFTR and RHD, and used to train and validate a new prediction protocol named Splicing Prediction in Consensus Elements (SPiCE). SPiCE combines in silico predictions from SpliceSiteFinder-like and MaxEntScan and uses logistic regression to define optimal decision thresholds. It revealed an unprecedented sensitivity and specificity of 99.5 and 95.2%, respectively, and the impact on splicing was correctly predicted for 98.8% of variants. We therefore propose SPiCE as the new tool for predicting variant spliceogenicity. It could be easily implemented in any diagnostic laboratory as a routine decision making tool to help geneticists to face the deluge of variants in the next-generation sequencing era. SPiCE is accessible at (https://sourceforge.net/projects/spicev2-1/).

Contribution of de novo and mosaic TP53 mutations to Li-Fraumeni syndrome.

BACKGROUND: Development of tumours such as adrenocortical carcinomas (ACC), choroid plexus tumours (CPT) or female breast cancers before age 31 or multiple primary cancers belonging to the Li-Fraumeni (LFS) spectrum is, independently of the familial history, highly suggestive of a germline TP53 mutation. The aim of this study was to determine the contribution of de novo and mosaic mutations to LFS. METHODS AND RESULTS: Among 328 unrelated patients harbouring a germline TP53 mutation identified by Sanger sequencing and/or QMPSF, we could show that the mutations had occurred de novo in 40 cases, without detectable parental age effect. Sanger sequencing revealed two mosaic mutations in a child with ACC and in an unaffected father of a child with medulloblastoma. Re-analysis of blood DNA by next-generation sequencing, performed at a depth above 500X, from 108 patients suggestive of LFS without detectable TP53 mutations, allowed us to identify 6 additional cases of mosaic TP53 mutations, in 2/49 children with ACC, 2/21 children with CPT, in 1/31 women with breast cancer before age 31 and in a patient who developed an osteosarcoma at age 12, a breast carcinoma and a breast sarcoma at age 35. CONCLUSIONS: This study performed on a large series of TP53 mutation carriers allows estimating the contribution to LFS of de novo mutations to at least 14% (48/336) and suggests that approximately one-fifth of these de novo mutations occur during embryonic development. Considering the medical impact of TP53 mutation identification, medical laboratories in charge of TP53 testing should ensure the detection of mosaic mutations.

Combined tumor genomic profiling and exome sequencing in a breast cancer family implicates ATM in tumorigenesis: A proof of principle study.

Familial breast cancers (BCs) account for 10%-20% of all diagnosed BCs, yet only 20% of such tumors arise in the context of a germline mutation in known tumor suppressor genes such as BRCA1 or BRCA2. The vast genetic heterogeneity which characterizes non BRCA1 and non BRCA2 (or BRCAx) families makes grouped studies impossible to perform. Next generation sequencing techniques, however, allow individual families to be studied to identify rare and or private mutations but the high number of genetic variants identified need to be sorted using pathogenicity or recurrence criteria. An additional sorting criterion may be represented by the identification of candidate regions defined by tumor genomic rearrangements. Indeed, comparative genomic hybridization (CGH) using single nucleotide polymorphism (SNP) arrays allows the detection of conserved ancestral haplotypes within recurrent regions of loss of heterozygosity, common to several familial tumors, which can highlight genomic loci harboring a germline mutation in cancer predisposition genes. The combination of both exome sequencing and SNP array-CGH for a series of familial BC revealed a germline ATM mutation associated with a loss of the wild-type allele in two BC from a BRCAx family. The analysis of additional breast tumors from ten BC families in which a germline ATM mutation had been identified revealed a high frequency of wild-type allele loss. This result argues strongly in favor of the involvement of ATM in these tumors as a tumor suppressor gene and confirms that germline ATM mutations are involved in a subset of familial BC.

BRCA Share: A Collection of Clinical BRCA Gene Variants.

As next-generation sequencing increases access to human genetic variation, the challenge of determining clinical significance of variants becomes ever more acute. Germline variants in the BRCA1 and BRCA2 genes can confer substantial lifetime risk of breast and ovarian cancer. Assessment of variant pathogenicity is a vital part of clinical genetic testing for these genes. A database of clinical observations of BRCA variants is a critical resource in that process. This article describes BRCA Share™, a database created by a unique international alliance of academic centers and commercial testing laboratories. By integrating the content of the Universal Mutation Database generated by the French Unicancer Genetic Group with the testing results of two large commercial laboratories, Quest Diagnostics and Laboratory Corporation of America (LabCorp), BRCA Share™ has assembled one of the largest publicly accessible collections of BRCA variants currently available. Although access is available to academic researchers without charge, commercial participants in the project are required to pay a support fee and contribute their data. The fees fund the ongoing curation effort, as well as planned experiments to functionally characterize variants of uncertain significance. BRCA Share™ databases can therefore be considered as models of successful data sharing between private companies and the academic world.

Clinical, pathological, and molecular data on desmoplastic/nodular medulloblastoma: case studies and a review of the literature.

The aim of this study was to better define the clinical and biopathological features of patients with desmoplastic/nodular medulloblastoma (DNMB) and to further characterize this subgroup. 17 children aged < 5 years, with initial DNMB treated according to the HIT-SKK protocol, were evaluated. A retrospective central radiological review, a pathological and immunohistochemical study, and array-CGH and sequencing of germline SUFU and PTCH1 genes were performed. 15 histologically reviewed cases were confirmed as DNMB including three cases of medulloblastoma with extensive nodularity. Median age at diagnosis was 26 months. Radiology showed five cases with a vermis location and one with T2 hyperintensity. All cases showed a SHH immunoprofile. A 9q deletion was found in 6 cases, a MYCN-MYCL amplification in 1 case, and a SUFU germline mutation in 1 case (/9). The presence of SUFU and PTCH1 germline mutations agreed with previous reports. At 3 years, progression-free survival and overallsurvival rates were 72 ± 15% and 85 ± 10%, respectively. The rate of recurrence was relatively high (4 patients). This may have been because chemotherapy was delayed in two cases. Age > 3 years, and residual tumor may also have been an explanation for recurrence.

Validation of a yeast functional assay for p53 mutations using clonal sequencing.

We have previously tested biopsies from 1469 breast tumours with a p53 functional assay in the context of a prospective clinical trial (EORTC 10994/BIG 1-00). The goal of the trial was to determine whether p53 status could be used to select patients who would benefit from inclusion of taxanes in anthracycline-based chemotherapy. The results of the trial were negative. To test whether this was because the functional assay misclassified the tumours, we have reanalysed two groups of biopsies by Sanger sequencing and Roche 454 next generation sequencing (NGS). Comparison of yeast data with pooled cDNA sequencing data in an initial cohort of 69 biopsies showed that conventional sequencing is insensitive when the mutant p53 content is low. A second cohort of 48 biopsies was used to compare directly the yeast assay with Sanger and NGS technology. The mutant sequence was difficult to detect in sequence chromatograms of pooled cDNA, whereas NGS unequivocally identified mutations in every case classified as mutant by the functional assay. The NGS data showed that small deletions, probably caused by PCR splicing, account for most of the unexplained background in the yeast assay. We conclude that mutation detection techniques that test multiple clones, such as the p53 functional assay and NGS, are more reliable than Sanger sequencing of pooled DNA; that the high p53 mutation rate (44%) seen with the yeast assay in the EORTC 10994/BIG 1-00 trial reflects this high sensitivity; and that NGS with Roche 454 technology could be used to identify the p53 mutations in the remaining tumours previously tested in yeast in the EORTC10994/BIG 1-00 trial.

High cumulative risks of cancer in patients with PTEN hamartoma tumour syndrome.

BACKGROUND: PTEN hamartoma tumour syndrome (PHTS) encompasses several clinical syndromes with germline mutations in the PTEN tumour suppressor gene, including Cowden syndrome which is characterised by an increased risk of breast and thyroid cancers. Because PHTS is rare, data regarding cancer risks and genotype-phenotype correlations are limited. The objective of this study was to better define cancer risks in this syndrome with respect to the type and location of PTEN mutations. METHODS: 154 PHTS individuals with a deleterious germline PTEN mutation were recruited from the activity of the Institut Bergonié genetic laboratory. Detailed phenotypic information was obtained for 146 of them. Age and sex adjusted standardised incidence ratio (SIR) calculations, cumulative cancer risk estimations, and genotype-phenotype analyses were performed. RESULTS: Elevated SIRs were found mainly for female breast cancer (39.1, 95% CI 24.8 to 58.6), thyroid cancer in women (43.2, 95% CI 19.7 to 82.1) and in men (199.5, 95% CI 106.39 to 342.03), melanoma in women (28.3, 95% CI 7.6 to 35.4) and in men (39.4, 95% CI 10.6 to 100.9), and endometrial cancer (48.7, 95% CI 9.8 to 142.3). Cumulative cancer risks at age 70 were 85% (95% CI 70% to 95%) for any cancer, 77% (95% CI 59% to 91%) for female breast cancer, and 38% (95% CI 25% to 56%) for thyroid cancer. The risk of cancer was two times greater in women with PHTS than in men with PHTS (p<0.05). CONCLUSIONS: This study shows a considerably high cumulative risk of cancer for patients with PHTS, mainly in women without clear genotype-phenotype correlation for this cancer risk. New recommendations for the management of PHTS patients are proposed.

Comprehensive analysis of PTEN status in breast carcinomas.

PTEN plays a well-established role in the negative regulation of the PI3K pathway, which is frequently activated in several cancer types, including breast cancer. A nuclear function in the maintenance of chromosomal stability has been proposed for PTEN but is yet to be clearly defined. In order to improve understanding of the role of PTEN in mammary tumorigenesis in terms of a possible gene dosage effect, its PI3K pathway function and its association with p53, we undertook comprehensive analysis of PTEN status in 135 sporadic invasive ductal carcinomas. Four PTEN status groups were defined; complete loss (19/135, 14%), reduced copy number (19/135, 14%), normal (86/135, 64%) and complex (11/135, 8%). Whereas the PTEN complete loss status was significantly associated with estrogen receptor (ER) negativity (p=0.006) and in particular the basal-like phenotype (p<0.0001), a reduced PTEN copy number was not associated with hormone receptor status or a particular breast cancer subtype. Overall, PI3K pathway alteration was suggested to be involved in 59% (79/134) of tumors as assessed by human epidermal growth factor receptor 2 overexpression, PIK3CA mutation or a complete loss of PTEN. A complex PTEN status was identified in a tumor subgroup which displayed a specific, complex DNA profile at the PTEN locus with a strikingly similar highly rearranged pan-genomic profile. All of these tumors had relapsed and were associated with a poorer prognosis in the context of node negative disease (p=1.4 × 10(-13) ) thus may represent a tumor subgroup with a common molecular alteration which could be targeted to improve clinical outcome.

Diagnosis of PTEN mosaicism: the relevance of additional tumor DNA sequencing. A case report and review of the literature.

BACKGROUND: PTEN hamartoma syndrome (PHTS) is an autosomal dominant disorder characterized by pathogenic variants in the tumor suppressor gene phosphatase and tensin homolog (PTEN). It is associated with an increased risk of muco-cutaneous features, hamartomatous tumors, and cancers. Mosaicism has been found in a few cases of patients with de novo PHTS, identified from blood samples. We report a PHTS patient with no variant identified from blood sample. Constitutional PTEN mosaicism was detected through sequencing of DNA from different tumoral and non-tumoral samples. CASE PRESENTATION: Our patient presented clinical Cowden syndrome at 56 years of age, with three major criteria (macrocephaly, Lhermitte Duclos disease, oral papillomatosis), and two minor criteria (structural thyroid lesions, esophageal glycogenic acanthosis). Deep sequencing of PTEN of blood leukocytes did not reveal any pathogenic variants. Exploration of tumoral (colonic ganglioneuroma, esophageal papilloma, diapneusia fibroids) and non-tumoral stomach tissues found the same PTEN pathogenic variant (NM_000314.4 c.389G > A; p.(Arg130Gln)), with an allelic frequency of 12 to 59%, confirming genomic mosaicism for Cowden syndrome. CONCLUSIONS: This case report, and review of the literature, suggests that systematic tumor analysis is essential for patients presenting PTEN hamartoma syndrome in the absence of any causal variant identified in blood leukocytes, despite deep sequencing. In 65 to 70% of cases of clinical Cowden syndrome, no pathogenic variant in the PTEN is observed in blood samples: mosaicism may explain a significant number of these patients. Tumor analysis would improve our knowledge of the frequency of de novo variations in this syndrome. Finally, patients with mosaicism for PTEN may not have a mild phenotype; medical care identical to that of heterozygous carriers should be offered.

Association and performance of polygenic risk scores for breast cancer among French women presenting or not a familial predisposition to the disease.

BACKGROUND: Three partially overlapping breast cancer polygenic risk scores (PRS) comprising 77, 179 and 313 SNPs have been proposed for European-ancestry women by the Breast Cancer Association Consortium (BCAC) for improving risk prediction in the general population. However, the effect of these SNPs may vary from one country to another and within a country because of other factors. OBJECTIVE: To assess their associated risk and predictive performance in French women from (1) the CECILE population-based case-control study, (2) BRCA1 or BRCA2 (BRCA1/2) pathogenic variant (PV) carriers from the GEMO study, and (3) familial breast cancer cases with no BRCA1/2 PV and unrelated controls from the GENESIS study. RESULTS: All three PRS were associated with breast cancer in all studies, with odds ratios per standard deviation varying from 1.7 to 2.0 in CECILE and GENESIS, and hazard ratios varying from 1.1 to 1.4 in GEMO. The predictive performance of PRS313 in CECILE was similar to that reported in BCAC but lower than that in GENESIS (area under the receiver operating characteristic curve (AUC) = 0.67 and 0.75, respectively). PRS were less performant in BRCA2 and BRCA1 PV carriers (AUC = 0.58 and 0.54 respectively). CONCLUSION: Our results are in line with previous validation studies in the general population and in BRCA1/2 PV carriers. Additionally, we showed that PRS may be of clinical utility for women with a strong family history of breast cancer and no BRCA1/2 PV, and for those carrying a predicted PV in a moderate-risk gene like ATM, CHEK2 or PALB2.

Guidelines for splicing analysis in molecular diagnosis derived from a set of 327 combined in silico/in vitro studies on BRCA1 and BRCA2 variants.

Assessing the impact of variants of unknown significance (VUS) on splicing is a key issue in molecular diagnosis. This impact can be predicted by in silico tools, but proper evaluation and user guidelines are lacking. To fill this gap, we embarked upon the largest BRCA1 and BRCA2 splice study to date by testing 272 VUSs (327 analyses) within the BRCA splice network of Unicancer. All these VUSs were analyzed by using six tools (splice site prediction by neural network, splice site finder (SSF), MaxEntScan (MES), ESE finder, relative enhancer and silencer classification by unanimous enrichment, and human splicing finder) and the predictions obtained were compared with transcript analysis results. Combining MES and SSF gave 96% sensitivity and 83% specificity for VUSs occurring in the vicinity of consensus splice sites, that is, the surrounding 11 and 14 bases for the 5' and 3' sites, respectively. This study was also an opportunity to define guidelines for transcript analysis along with a tentative classification of splice variants. The guidelines drawn from this large series should be useful for the whole community, particularly in the context of growing sequencing capacities that require robust pipelines for variant interpretation.

Alterations of the p53 and PIK3CA/AKT/mTOR pathways in angiosarcomas: a pattern distinct from other sarcomas with complex genomics.

BACKGROUND: The p53 and phosphoinositide-3-kinase, catalytic, alpha polypeptide/v-akt murine thymoma viral oncogene homolog/mechanistic target of rapamycin (PIK3CA/AKT/mTOR) pathways frequently are altered in sarcoma with complex genomics, such as leiomyosarcoma (LMS) or undifferentiated pleomorphic sarcoma (UPS). The scale of genetic abnormalities in these pathways remains unknown in angiosarcoma (AS). METHODS: The authors investigated the status of critical genes involved in the p53 and PIK3CA/AKT/mTOR pathways in a series of 62 AS. RESULTS: The mutation and deletion rates of tumor protein 53 (TP53) were 4% and 0%, respectively. Overexpression of p53 was detected by immunohistochemistry in 49% of patients and was associated with inferior disease-free survival. Although p14 inactivation or overexpression of the human murine double minute homolog (HDM2) were frequent in LMS and UPS and could substitute for TP53 mutation or deletion, such alterations were rare in angiosarcomas. Phosphorylated ribosomal protein S6 kinase (p-S6K) and/or phosphorylated eukaryotic translation initiation factor 4E binding protein 1 (p-4eBP1) overexpression was observed in 42% of patients, suggesting frequent activation of the PIK3CA/AKT/mTOR pathway in angiosarcomas. Activation was not related to intragenic deletion of phosphatase and tensin homolog (PTEN), an aberration that is frequent in LMS and UPS but absent in angiosarcomas. CONCLUSIONS: The current results indicated that angiosarcomas constitute a distinct subgroup among sarcomas with complex genomics. Although TP53 mutation and PTEN deletion are frequent in LMS and UPS, these aberrations are rarely involved in the pathogenesis of angiosarcoma.

PTEN alterations in sporadic and BRCA1-associated triple negative breast carcinomas.

The similarities between sporadic basal-like breast cancer (BLBC) and BRCA1-mutated breast tumours raise the possibility that deregulation of the same pathway may underlie these tumour types. The aim of this study was to determine if PTEN aberrations are characteristic of both BRCA1 tumours and sporadic TN breast carcinomas with low BRCA1 expression, and can thus be used to identify sporadic tumours potentially sensitive to PARP inhibitors. Twelve BRCA1 tumours, 19 non-BRCA familial breast tumours and 71 unselected TN breast carcinomas were screened for PTEN mutations and assessed for PTEN expression and BRCA1 mRNA expression. Loss of PTEN expression was observed in 67% of BRCA1 tumours and more specifically in 89% of TN BRCA1 tumours highlighting the link between PTEN loss and BLBC in the context of germline BRCA1 mutations. Regarding unselected TN tumours, 56% showed PTEN expression loss and 35% displayed low BRCA1 mRNA expression. Unlike familial breast cancers with low BRCA1 mRNA expression, no significant correlation was observed between the loss of PTEN expression and low BRCA1 mRNA expression in this unselected TN tumours panel. Our data suggest that, unlike the germinal context, PTEN and BRCA1 alterations in sporadic TN breast tumours are independent events.

Prostate cancer and PARP inhibitors: progress and challenges.

Despite survival improvements achieved over the last two decades, prostate cancer remains lethal at the metastatic castration-resistant stage (mCRPC) and new therapeutic approaches are needed. Germinal and/or somatic alterations of DNA-damage response pathway genes are found in a substantial number of patients with advanced prostate cancers, mainly of poor prognosis. Such alterations induce a dependency for single strand break reparation through the poly(adenosine diphosphate-ribose) polymerase (PARP) system, providing the rationale to develop PARP inhibitors. In solid tumors, the first demonstration of an improvement in overall survival was provided by olaparib in patients with mCRPC harboring homologous recombination repair deficiencies. Although this represents a major milestone, a number of issues relating to PARP inhibitors remain. This timely review synthesizes and discusses the rationale and development of PARP inhibitors, biomarker-based approaches associated and the future challenges related to their prescription as well as patient pathways.

Concordance Between Tumor and Germline BRCA Status in High-Grade Ovarian Carcinoma Patients in the Phase III PAOLA-1/ENGOT-ov25 Trial.

BACKGROUND: PAOLA1 is a phase III study assessing olaparib maintenance therapy in advanced high-grade ovarian carcinoma patients responding to first-line platinum-taxane-based chemotherapy plus bevacizumab as standard of care. Randomization was stratified by treatment outcome and tumor BRCA1/2 status (tBRCA) at screening. METHODS: tBRCA was tested on formalin-fixed, paraffin-embedded tumor blocks on 5 French platforms using 2 next-generation sequencing methods based either on hybrid capture or amplicon technology. One of the exploratory objectives was to assess the concordance between germline (gBRCA) and tBRCA testing in French patients. gBRCA testing was performed on blood samples on the same platforms. RESULTS: From May 2015 to July 2017, tBRCA tests were performed for 1176 screened patients. Only 52 (4.4%) tumor samples were noncontributive. The median interval between reception of the tumor sample and availability of the tBRCA status result was 37 days (range = 8-260). A pathogenic variant was reported in 27.1% tumor samples (319 of 1176 screened patients). tBRCA and gBRCA testing were performed for 451 French patients with negative results for both tests in 306 patients (67.8%) and positive results for both tests in 85 patients (18.8%). Only 1 large genomic rearrangement of BRCA1 was detected, exclusively in the blood sample. Interestingly, tBRCA testing revealed 6.4% of pathogenic variant (29 of 451) not detected by gBRCA testing. CONCLUSIONS: tBRCA testing is an appropriate tool with an acceptable turnaround time for clinical practice and a low failure rate, ensuring reliable identification of patients likely to benefit from poly(ADP-ribose) polymerase inhibitor therapy.

5' Region Large Genomic Rearrangements in the BRCA1 Gene in French Families: Identification of a Tandem Triplication and Nine Distinct Deletions with Five Recurrent Breakpoints.

BACKGROUND: Large genomic rearrangements (LGR) in BRCA1 consisting of deletions/duplications of one or several exons have been found throughout the gene with a large proportion occurring in the 5' region from the promoter to exon 2. The aim of this study was to better characterize those LGR in French high-risk breast/ovarian cancer families. METHODS: DNA from 20 families with one apparent duplication and nine deletions was analyzed with a dedicated comparative genomic hybridization (CGH) array, high-resolution BRCA1 Genomic Morse Codes analysis and Sanger sequencing. RESULTS: The apparent duplication was in fact a tandem triplication of exons 1 and 2 and part of intron 2 of BRCA1, fully characterized here for the first time. We calculated a causality score with the multifactorial model from data obtained from six families, classifying this variant as benign. Among the nine deletions detected in this region, eight have never been identified. The breakpoints fell in six recurrent regions and could confirm some specific conformation of the chromatin. CONCLUSIONS: Taken together, our results firmly establish that the BRCA1 5' region is a frequent site of different LGRs and highlight the importance of the segmental duplication and Alu sequences, particularly the very high homologous region, in the mechanism of a recombination event. This also confirmed that those events are not systematically deleterious.