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.

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.

TUMOSPEC: A Nation-Wide Study of Hereditary Breast and Ovarian Cancer Families with a Predicted Pathogenic Variant Identified through Multigene Panel Testing.

Assessment of age-dependent cancer risk for carriers of a predicted pathogenic variant (PPV) is often hampered by biases in data collection, with a frequent under-representation of cancer-free PPV carriers. TUMOSPEC was designed to estimate the cumulative risk of cancer for carriers of a PPV in a gene that is usually tested in a hereditary breast and ovarian cancer context. Index cases are enrolled consecutively among patients who undergo genetic testing as part of their care plan in France. First- and second-degree relatives and cousins of PPV carriers are invited to participate whether they are affected by cancer or not, and genotyped for the familial PPV. Clinical, family and epidemiological data are collected, and all data including sequencing data are centralized at the coordinating centre. The three-year feasibility study included 4431 prospective index cases, with 19.1% of them carrying a PPV. When invited by the coordinating centre, 65.3% of the relatives of index cases (5.7 relatives per family, on average) accepted the invitation to participate. The study logistics were well adapted to clinical and laboratory constraints, and collaboration between partners (clinicians, biologists, coordinating centre and participants) was smooth. Hence, TUMOSPEC is being pursued, with the aim of optimizing clinical management guidelines specific to each gene.

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.

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.

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.

Engineering Genetic Predisposition in Human Neuroepithelial Stem Cells Recapitulates Medulloblastoma Tumorigenesis.

Human neural stem cell cultures provide progenitor cells that are potential cells of origin for brain cancers. However, the extent to which genetic predisposition to tumor formation can be faithfully captured in stem cell lines is uncertain. Here, we evaluated neuroepithelial stem (NES) cells, representative of cerebellar progenitors. We transduced NES cells with MYCN, observing medulloblastoma upon orthotopic implantation in mice. Significantly, transcriptomes and patterns of DNA methylation from xenograft tumors were globally more representative of human medulloblastoma compared to a MYCN-driven genetically engineered mouse model. Orthotopic transplantation of NES cells generated from Gorlin syndrome patients, who are predisposed to medulloblastoma due to germline-mutated PTCH1, also generated medulloblastoma. We engineered candidate cooperating mutations in Gorlin NES cells, with mutation of DDX3X or loss of GSE1 both accelerating tumorigenesis. These findings demonstrate that human NES cells provide a potent experimental resource for dissecting genetic causation in medulloblastoma.

Severe PATCHED1 Deficiency in Cancer-Prone Gorlin Patient Cells Results in Intrinsic Radiosensitivity.

PURPOSE: Gorlin syndrome (or basal-cell nevus syndrome) is a cancer-prone genetic disease in which hypersusceptibility to secondary cancer and tissue reaction after radiation therapy is debated, as is increased radiosensitivity at cellular level. Gorlin syndrome results from heterozygous mutations in the PTCH1 gene for 60% of patients, and we therefore aimed to highlight correlations between intrinsic radiosensitivity and PTCH1 gene expression in fibroblasts from adult patients with Gorlin syndrome. METHODS AND MATERIALS: The radiosensitivity of fibroblasts from 6 patients with Gorlin syndrome was determined by cell-survival assay after high (0.5-3.5 Gy) and low (50-250 mGy) γ-ray doses. PTCH1 and DNA damage response gene expression was characterized by real-time polymerase chain reaction and Western blotting. DNA damage and repair were investigated by γH2AX and 53BP1 foci assay. PTCH1 knockdown was performed in cells from healthy donors by using stable RNA interference. Gorlin cells were genotyped by 2 complementary sequencing methods. RESULTS: Only cells from patients with Gorlin syndrome who presented severe deficiency in PATCHED1 protein exhibited a significant increase in cellular radiosensitivity, affecting cell responses to both high and low radiation doses. For 2 of the radiosensitive cell strains, heterozygous mutations in the 5' end of PTCH1 gene explain PATCHED1 protein deficiency. In all sensitive cells, DNA damage response pathways (ATM, CHK2, and P53 levels and activation by phosphorylation) were deregulated after irradiation, whereas DSB repair recognition was unimpaired. Furthermore, normal cells with RNA interference-mediated PTCH1 deficiency showed reduced survival after irradiation, directly linking this gene to high- and low-dose radiosensitivity. CONCLUSIONS: In the present study, we show an inverse correlation between PTCH1 expression level and cellular radiosensitivity, suggesting an explanation for the conflicting results previously reported for Gorlin syndrome and possibly providing a basis for prognostic screens for radiosensitive patients with Gorlin syndrome and PTCH1 mutations.

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/).

Germline SUFU mutation carriers and medulloblastoma: clinical characteristics, cancer risk, and prognosis.

Background: Germline mutations of suppressor of fused homolog (SUFU) predispose to sonic hedgehog (SHH) medulloblastoma. Germline SUFU mutations have been reported in nevoid basal cell carcinoma syndrome (NBCCS), but little is known about the cancer risk and clinical spectrum. Methods: We performed a retrospective review of all patients with medulloblastoma and a germline SUFU mutation in France. Results: Twenty-two patients from 17 families were identified with medulloblastoma and a germline SUFU mutation (median age at diagnosis: 16.5 mo). Macrocrania was present in 20 patients, but only 5 met the diagnostic criteria for NBCCS. Despite treatment with surgery and chemotherapy, to avoid radiotherapy in all patients except one, the outcome was worse than expected for SHH medulloblastoma, due to the high incidence of local relapses (8/22 patients) and second malignancies (n = 6 in 4/22 patients). The 5-year progression-free survival and overall survival rates were 42% and 66%. Mutations were inherited in 79% of patients, and 34 additional SUFU mutation carriers were identified within 14 families. Medulloblastoma penetrance was incomplete, but higher than in Patched 1 (PTCH1) mutation carriers. Besides medulloblastoma, 19 other tumors were recorded among the 56 SUFU mutation carriers, including basal cell carcinoma (BCC) in 2 patients and meningioma in 3 patients. Conclusion: Germline SUFU mutations strongly predispose to medulloblastoma in the first years of life, with worse prognosis than usually observed for SHH medulloblastoma. The clinical spectrum differs between SUFU and PTCH1 mutation carriers, and BCC incidence is much lower in SUFU mutation carriers. The optimal treatment of SUFU mutation-associated medulloblastoma has not been defined.

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.

Insertion of Alu elements at a PTEN hotspot in Cowden syndrome.

Cowden syndrome (CS) is an inherited autosomal dominant disorder associated with germline pathogenic variants of the PTEN tumor suppressor gene. Its phenotypical expression is highly variable and the existence of patients with a CS suggestive phenotype without pathogenic PTEN variant may be related to genetic heterogeneity. In order to explore this hypothesis through the detection of potentially deleterious variants enabling us to identify a new candidate gene, we performed whole-exome sequencing (WES) in a series of 22 CS patients without detectable PTEN pathogenic variant using conventional methods for mutation screening. We failed to identify a novel candidate gene, but interestingly in two patients WES revealed the presence of two distinct, previously undescribed Alu insertions with the same break points in exon 5. These insertions were not found in a series of 35 breast carcinomas that showed a loss of PTEN expression without a detectable alteration of this gene. This study reveals the presence of a PTEN Alu insertion hotspot involved in CS, and suggests that undetected PTEN pathogenic variants could contribute to CS.

Expression of Gastrin-Releasing Peptide Receptor in Breast Cancer and Its Association with Pathologic, Biologic, and Clinical Parameters: A Study of 1,432 Primary Tumors.

A growing body of evidence suggests that gastrin-releasing peptide receptor (GRPR) might be a valuable target in breast cancer. To understand which patients can be potential candidates for GRPR-based imaging or targeted therapy, we screened invasive breast cancers by immunohistochemistry for the presence and intensity of GRPR expression. Methods: We explored a tissue microarray of 1,432 primary breast tumors from patients who underwent surgery between 2000 and 2005 at Institut Bergonié, without prior neoadjuvant treatment. We studied associations between GRPR expression and clinical, pathologic, and biologic parameters. The association between GRPR expression and distant metastasis-free interval was also examined. Results: GRPR overexpression was found in 75.8% of the 1,432 tumors and was most strongly associated with estrogen receptor (ER) positivity (GRPR was high in 83.2% of ER-positive and 12% of ER-negative tumors; P < 0.00001). When molecular subtypes of breast cancer were considered, GRPR was overexpressed in 86.2% of luminal A-like tumors, 70.5% of luminal B-like human epidermal growth factor receptor 2 (HER2)-negative tumors, 82.8% of luminal B-like HER2-positive tumors, 21.3% of HER2-enriched tumors, and 7.8% of triple-negative tumors. Importantly, when breast tumors overexpressed GRPR, high GRPR expression was also found in metastatic lymph nodes in 94.6% of cases. Primary tumors with high GRPR expression were associated with lower risk of distant metastases at follow-up in univariate analysis (Log-rank P = 0.0084) but not in multivariate analysis. Hence, the prognostic impact of GRPR was lost when examined within specific molecular subtypes. Conclusion: Because GRPR is overexpressed in a high percentage of ER-positive tumors, GRPR targeting offers wide perspectives for imaging and treatment in patients with ER-positive breast cancer, using recently developed radiolabeled GRPR ligands.

The transfer of multigene panel testing for hereditary breast and ovarian cancer to healthcare: What are the implications for the management of patients and families?

Until recently, the molecular diagnosis of hereditary breast and ovarian cancer (HBOC) was mostly based on BRCA1/2 testing. Next generation sequencing and the recent discovery of new genes involved in HBOC now permit the transfer of genomic capture targeting multiple candidate genes from research to clinical use. However, the implications for the management of patients and their families have not been extensively studied, in particular since some of these genes are not well-established cancer predisposing genes. We studied 583 consecutive patients from Burgundy (France) fulfilling the criteria for BRCA testing using a next generation sequencing 25-genes panel including 20 well-established high-risk cancer genes as well as more recently identified predisposing HBOC cancer. A pathogenic BRCA1/2 mutation was found in 51 patients (9%). Besides, we found 37 pathogenic or likely pathogenic mutations in 10 different high to low-risk genes in 34 patients (6%). The most frequently mutated genes were CHEK2 (n = 12; 2%), ATM (n = 9; 1.5%), and PALB2 (n = 4; 0.6%). Three patients had a mutation in two different predisposing genes. The analysis of clinical actionability conducted in mutation-positive individuals revealed that additional disease-specific screening and/or prevention measures beyond those based on personal and family history alone had been recommended in 69% of cases. In conclusion, multigene panel testing is a powerful tool to identifying high to low-risk HBOC susceptibility genes. The penetrance and spectrum of cancers with these other genes are sometimes undefined, and further collaborative work is crucial to address this question.