Frequency and Genomic Aspects of Intrinsic Resistance to Vismodegib in Locally Advanced Basal Cell Carcinoma.

PURPOSE: Vismodegib is approved for the treatment of locally advanced basal cell carcinoma (laBCC), but some cases demonstrate intrinsic resistance (IR) to the drug. We sought to assess the frequency of IR to vismodegib in laBCC and its underlying genomic mechanisms. EXPERIMENTAL DESIGN: Response to vismodegib was evaluated in a cohort of 148 laBCC patients. Comprehensive genomic and transcriptomic profiling was performed in a subset of five intrinsically resistant BCC (IR-BCC). RESULTS: We identified that IR-BCC represents 6.1% of laBCC in the studied cohort. Prior treatment with chemotherapy was associated with IR. Genetic events that were previously associated with acquired resistance (AR) in BCC or medulloblastoma were observed in three out of five IR-BCC. However, IR-BCCs were distinct by highly rearranged polyploid genomes. Functional analyses identified hyperactivation of the HIPPO-YAP and WNT pathways at RNA and protein levels in IR-BCC. In vitro assay on the BCC cell line further confirmed that YAP1 overexpression increases the cell proliferation rate. CONCLUSIONS: IR to vismodegib is a rare event in laBCC. IR-BCCs frequently harbor resistance mutations in the Hh pathway, but also are characterized by hyperactivation of the HIPPO-YAP and WNT pathways.

Familial pancreatic adenocarcinoma: A retrospective analysis of germline genetic testing in a French multicentre cohort.

The rate of genetic diagnosis of French patients with familial pancreatic ductal adenocarcinoma (PDAC) is not known. We report germline genetic testing data from 133 index cases meeting criteria for familial pancreatic cancer (FPC) as well as 87 'FPC-like' index cases who did not fulfilled strict FPC definition but were evocative for a PDAC predisposition. The overall rate of genetic diagnosis (in BRCA1, BRCA2, CDKN2A, and ATM genes) was 8.3% in FPC patients and 4.6% in FPC-like patients, consistent with the literature in other populations. Genetic variants were also identified in FANCA and BAP1 genes, as well as in the CDKN2A p12 transcript. This pancreas-specific transcript is a known key player in driving pancreatic oncogenesis. This might be the first described case of a PDAC genetic predisposition due to a variant in this specific transcript.

Guidelines for reporting secondary findings of genome sequencing in cancer genes: the SFMPP recommendations.

In oncology, the expanding use of multi-gene panels to explore familial cancer predisposition and tumor genome analysis has led to increased secondary findings discoveries (SFs) and has given rise to important medical, ethical, and legal issues. The American College of Medical Genetics and Genomics published a policy statement for managing SFs for a list of genes, including 25 cancer-related genes. Currently, there are few recommendations in Europe. From June 2016 to May 2017, the French Society of Predictive and Personalized Medicine (SFMPP) established a working group of 47 experts to elaborate guidelines for managing information given on the SFs for genes related to cancers. A subgroup of ethicists, lawyers, patients' representatives, and psychologists provided ethical reflection, information guidelines, and materials (written consent form and video). A subgroup with medical expertise, including oncologists and clinical and molecular geneticists, provided independent evaluation and classification of 60 genes. The main criteria were the "actionability" of the genes (available screening or prevention strategies), the risk evaluation (severity, penetrance, and age of disease onset), and the level of evidence from published data. Genes were divided into three classes: for class 1 genes (n = 36), delivering the information on SFs was recommended; for class 2 genes (n = 5), delivering the information remained questionable because of insufficient data from the literature and/or level of evidence; and for class 3 genes (n = 19), delivering the information on SFs was not recommended. These guidelines for managing SFs for cancer-predisposing genes provide new insights for clinicians and laboratories to standardize clinical practices.

Truncating mutations of TP53AIP1 gene predispose to cutaneous melanoma.

Genetic predisposition to cutaneous malignant melanoma (CMM) involves highly penetrant predisposing genes and low and intermediate penetrant predisposing alleles. However, the missing heritability in (CMM) is still high. For such and in order to identify new genetic factors for CMM, we conducted an exome sequencing study in high-risk CMM patients. Two rounds of exome sequencing were successively performed in 33 and 27 high-risk patients. We focused on genes carrying rare nonsense, frameshift, and splice variants (allelic frequency <1%) that were present in both series of exomes. An extension study was then conducted in a large cohort (1 079 CMM patients and 1 230 Caucasian ethnically matched healthy controls), and the inactivating variants frequency was compared between groups using two-sided Fisher exact test. Two TP53AIP1 truncating mutations were identified in four patients: a frameshift c.63_64insG, p.Q22Afs*81 in two patients from the same family and in the proband of a second family; and a nonsense mutation c.95 C > A, p.Ser32Stop in a patient with multiple CMMs. In all patients, TP53AIP1 truncating variants were strongly associated with CMM risk (two-sided Fisher exact test = 0.004, OR = 3.3[1.3-8.5]). Additionally, we showed that TP53AIP1 mRNA was strongly down-regulated throughout different phases of melanoma progression. TP53AIP1 gene is a TP53 target which plays a key role by inducting apoptosis in response to UV-induced DNA damage. Constitutional mutations of TP53AIP1 had previously been involved in susceptibility to prostate cancer. Our results show that constitutional truncating TP53AIP1 mutations predispose to CMM in the French population. Replication studies in other populations should be performed.

Occurrence of BAP1 germline mutations in cutaneous melanocytic tumors with loss of BAP1-expression: A pilot study.

Melanocytic BAP1-associated intradermal tumors (MBAITs) can either be sporadic or associated with a cancer-predisposition syndrome. In this study we explored the clinical status of 136 patients in which at least one MBAIT was found. 49/136 (36%) of them gave their signed consent for an oncogenetic BAP1 blood test. 28/136 patients (20%) diagnosed with an MBAIT had other MBAITs and/or a personal or familial history of BAP1-related cancers that could clinically designate them as potential carriers of a BAP1 germline mutation. 17 of these 28 patients underwent oncogenetic testing. A deleterious mutation of BAP1 was confirmed in 12/17 cases. 4/17 cases were wild-type; all had a single MBAIT and a history of skin melanoma. A variant of unknown significance was found in one case with multiple MBAITs. Among the 12 mutated cases, multiple MBAITs were present in 10/12 cases and were the only clinical sign in 4/12 cases. The remaining 32/49 blood-tested cases with an isolated MBAIT were wild type for BAP1 in 25/32 cases or showed a variant of unknown significance in 7/32 cases. We recommend, following the diagnosis of a MBAIT, performing a BAP1 immunohistochemistry in all other cutaneous melanocytic tumors removed previously or simultaneously and all skin melanomas. This screening could help clinicians prioritize which patients would most benefit from oncogenetic testing.

Pancreatic ductal adenocarcinoma in BRCA2 mutation carriers.

Germline BRCA2 mutations are the first known cause of inherited (familial) pancreatic ductal adenocarcinoma (PDAC). This tumor is the third most frequent cancer in carriers of germline BRCA2 mutations, as it occurs in around 10% of BRCA2 families. PDAC is known as one of the most highly lethal cancers, mainly because of its chemoresistance and frequently late diagnosis. Based on recent developments in molecular biology, a subgroup of BRCA2-associated PDAC has been created, allowing screening, early surgical treatment and personalized systemic treatment. BRCA2 germline mutation carriers who have ≥1 first-degree relative, or ≥2 blood relatives with PDAC, should undergo screening and regular follow-up based on magnetic resonance imaging and endoscopic ultrasound. The goal of screening is to detect early invasive PDAC and advanced precancerous lesions suitable for a stepwise surgical complete (R0) resection. Increasing evidence on the molecular role of the BRCA2 protein in the homologous recombination of DNA damages suggest that BRCA2-related PDAC are sensitive to agents causing DNA cross-linking damage, such as platinum salts, and treatments targeting rescue DNA repair pathways, such as poly(ADP-ribose) polymerase inhibitors that are currently under investigation.

PARKIN Inactivation Links Parkinson's Disease to Melanoma.

BACKGROUND: Melanoma incidence is higher in patients affected by Parkinson's disease (PD) and vice versa, but the genetic link shared by both diseases is unknown. As PARK2 is both a tumor suppressor gene and frequently mutated in young onset PD, we evaluated the role of PARK2 in melanoma predisposition and progression. METHODS: An in-depth PARK2 gene dosage analysis and sequencing was performed on 512 French case patients and 562 healthy control patients, as well as sporadic tumors and melanoma cell lines. The frequency of genetic alterations was compared between case patients and control patients using two-sided Fisher's exact tests and odds ratio (OR) calculations. We used western blotting to determine PARKIN expression in melanocytes and melanoma cell lines and transfection followed by clonogenic assays to evaluate the effect of PARKIN expression on cellular proliferation. All statistical tests were two-sided. RESULTS: Germline PARK2 mutations (including copy number variations, splicing, and putative deleterious missense mutations) were present in 25 case patients but only four control patients (OR = 3.95, 95% confidence interval = 1.34 to 15.75). Copy number variations (CNVs) and loss of heterozygosity were present in 60% and 74%, respectively, of primary tumors. PARKIN protein was expressed in melanocytes but not in most melanoma cell lines, and its expression decreased following melanocyte transformation by oncogenic NRAS. Re-expression of PARKIN in melanoma cell lines resulted in a drastic reduction of cell proliferation and inhibition of PARKIN in melanocytes stimulated their proliferation. CONCLUSION: Our results show an important role for PARK2 as a tumor suppressor both in melanoma predisposition and progression, which could explain the epidemiological association of these diseases.

Expanding the clinical spectrum of hereditary fibrosing poikiloderma with tendon contractures, myopathy and pulmonary fibrosis due to FAM111B mutations.

BACKGROUND: Hereditary Fibrosing Poikiloderma (HFP) with tendon contractures, myopathy and pulmonary fibrosis (POIKTMP [MIM 615704]) is a very recently described entity of syndromic inherited poikiloderma. Previously by using whole exome sequencing in five families, we identified the causative gene, FAM111B (NM_198947.3), the function of which is still unknown. Our objective in this study was to better define the specific features of POIKTMP through a larger series of patients. METHODS: Clinical and molecular data of two families and eight independent sporadic cases, including six new cases, were collected. RESULTS: Key features consist of: (i) early-onset poikiloderma, hypotrichosis and hypohidrosis; (ii) multiple contractures, in particular triceps surae muscle contractures; (iii) diffuse progressive muscular weakness; (iv) pulmonary fibrosis in adulthood and (v) other features including exocrine pancreatic insufficiency, liver impairment and growth retardation. Muscle magnetic resonance imaging was informative and showed muscle atrophy and fatty infiltration. Histological examination of skeletal muscle revealed extensive fibroadipose tissue infiltration. Microscopy of the skin showed a scleroderma-like aspect with fibrosis and alterations of the elastic network. FAM111B gene analysis identified five different missense variants (two recurrent mutations were found respectively in three and four independent families). All the mutations were predicted to localize in the trypsin-like cysteine/serine peptidase domain of the protein. We suggest gain-of-function or dominant-negative mutations resulting in FAM111B enzymatic activity changes. CONCLUSIONS: HFP with tendon contractures, myopathy and pulmonary fibrosis, is a multisystemic disorder due to autosomal dominant FAM111B mutations. Future functional studies will help in understanding the specific pathological process of this fibrosing disorder.

Genes involved in the WNT and vesicular trafficking pathways are associated with melanoma predisposition.

Multifactorial predisposition to melanoma includes genes involved in pigmentation, immunity and DNA repair. Nonetheless, missing heritability in melanoma is still important. We studied the role of 335 candidate SNPs in melanoma susceptibility by using a dedicated chip and investigating 110 genes involved in different pathways. A discovery set was comprised of 1069 melanoma patients and 925 controls from France. Data were replicated using validation phases II (1085 cases and 801 controls from Spain) and III (1808 cases and 1894 controls from Germany and a second set of Spanish samples). In addition, an exome sequencing study was performed in three high-risk French melanoma families. Nineteen SNPs in 17 genes were initially associated with melanoma in the French population. Six SNPs were replicated in phase II, including two new SNPs in the WNT3 (rs199524) and VPS41 (rs11773094) genes. The role of VPS41 and WNT3 was confirmed in a meta-analysis (3940 melanoma cases and 3620 controls) with two-side p values of 0.002, (OR = 0.86) and 4.07 × 10(-10) (OR = 0.80), respectively. Exome sequencing revealed a non-synonymous VPS41 variant in one family that was shown to be strongly associated with familial melanoma (OR = 4.46, p = 0.001) in an independent sample of 178 melanoma families. WNT3 belongs to WNT pathway known to play a crucial role in melanoma, whereas VPS41 regulates vesicular trafficking and is thought to play a role in pigmentation. Our work identified two new pathways involved in melanoma predisposition. These results may be useful in the future for identifying individuals highly predisposed to melanoma.

High prevalence of deficient mismatch repair phenotype and the V600E BRAF mutation in elderly patients with colorectal cancer.

AIMS: Colorectal cancer (CRC) occurs mostly in the elderly. However, the biology of CRC in elderly has been poorly studied. This study examined the prevalence of deficient mismatch repair phenotype (dMMR) and BRAF mutations according to age. PATIENTS AND METHODS: MMR phenotype was prospectively determined by molecular analysis in patients of all ages undergoing surgery for CRC. BRAF V600E mutation status was analysed in a subset of dMMR tumours. RESULTS: A total of 754 patients who underwent surgery between 2005 and 2008 were included in the study. Amongst them, 272 (36%) were ≥75years old. The proportion of women <75 was 38% and that ≥75 was 53% (p<0.0001). The prevalence of dMMR was 19.4% in patients ≥75 and 10.7% in patients <75 (p=0.0017). For patients ≥75, the prevalence of dMMR was significantly higher in women than in men (27% vs 10.2%, respectively; p=0.003) but was similar in women and men <75 (12.5% vs 9.7%, respectively; p=0.4). We examined BRAF mutation status in 80 patients with dMMR tumours. The V600E BRAF mutation was significantly more frequent in patients ≥75 than in patients <75 (72.2% vs 11.4%, respectively; p<0.001). In patients ≥75, there was no difference in the prevalence of the BRAF V600E mutation according to sex (78% in women and 70% in men, p=0.9). CONCLUSIONS: The prevalence of dMMR in CRC is high in patients over 75. In elderly patients, dMMR tumours are significantly more frequent in women than in men. The BRAF mutation is frequent in elderly patients with CRC.

A large French case-control study emphasizes the role of rare Mc1R variants in melanoma risk.

BACKGROUND: The MC1R gene implicated in melanogenesis and skin pigmentation is highly polymorphic. Several alleles are associated with red hair and fair skin phenotypes and contribute to melanoma risk. OBJECTIVE: This work aims to assess the effect of different classes of MC1R variants, notably rare variants, on melanoma risk. Methods. MC1R coding region was sequenced in 1131 melanoma patients and 869 healthy controls. MC1R variants were classified as RHC (R) and non-RHC (r). Rare variants (frequency < 1%) were subdivided into two subgroups, predicted to be damaging (D) or not (nD). RESULTS: Both R and r alleles were associated with melanoma (OR = 2.66 [2.20-3.23] and 1.51 [1.32-1.73]) and had similar population attributable risks (15.8% and 16.6%). We also identified 69 rare variants, of which 25 were novel. D variants were strongly associated with melanoma (OR = 2.38 [1.38-4.15]) and clustered in the same MC1R domains as R alleles (intracellular 2, transmembrane 2 and 7). CONCLUSION: This work confirms the role of R and r alleles in melanoma risk in the French population and proposes a novel class of rare D variants as important melanoma risk factors. These findings may improve the definition of high-risk subjects that could be targeted for melanoma prevention and screening.

Telomerase reverse transcriptase promoter mutations in primary cutaneous melanoma.

We previously reported a disease segregating causal germline mutation in a melanoma family and recurrent somatic mutations in metastasized tumours from unrelated patients in the core promoter region of the telomerase reverse transcriptase (TERT) gene. Here we show that the TERT promoter mutations, besides causing an increased gene expression, associate with increased patient age, increased Breslow thickness and tumour ulceration in 287 primary melanomas. The mutations are more frequent at both intermittently and chronically sun-exposed sites than non-exposed sites and tend to co-occur with BRAF and CDKN2A alterations. The association with parameters generally connected with poor outcome, coupled with high recurrence and mechanistic relevance, raises the possibility of the eventual use of TERT promoter mutations in the disease management.

Correlation of phenotype/genotype in a cohort of 23 xeroderma pigmentosum-variant patients reveals 12 new disease-causing POLH mutations.

Xeroderma pigmentosum variant (XP-V) is a rare genetic disease, characterized by some sunlight sensitivity and predisposition to cutaneous malignancies. We described clinical and genetic features of the largest collection ever published of 23 XPV patients (ages between 21 and 86) from 20 unrelated families. Primary fibroblasts from patients showed normal nucleotide excision repair but UV-hypersensitivity in the presence of caffeine, a signature of the XP-V syndrome. 87% of patients developed skin tumors with a median age of 21 for the first occurrence. The median numbers of basal-cell carcinoma was 13 per patient, six for squamous-cell carcinoma, and five for melanoma. XP-V is due to defects in the translesion-synthesis DNA polymerase Polη coded by the POLH gene. DNA sequencing of POLH revealed 29 mutations, where 12 have not been previously identified, leading to truncated polymerases in 69% of patients. Four missense mutations are correlated with the protein stability by structural modeling of the Polη polymerase domain. There is a clear relationship between the types of missense mutations and clinical severity. For truncating mutations, which lead to an absence of or to inactive proteins, the life-cumulated UV exposure is probably the best predictor of cancer incidence, reinforcing the necessity to protect XP-Vs from sun exposure.

Germline BAP1 mutations predispose to renal cell carcinomas.

The genetic cause of some familial nonsyndromic renal cell carcinomas (RCC) defined by at least two affected first-degree relatives is unknown. By combining whole-exome sequencing and tumor profiling in a family prone to cases of RCC, we identified a germline BAP1 mutation c.277A>G (p.Thr93Ala) as the probable genetic basis of RCC predisposition. This mutation segregated with all four RCC-affected relatives. Furthermore, BAP1 was found to be inactivated in RCC-affected individuals from this family. No BAP1 mutations were identified in 32 familial cases presenting with only RCC. We then screened for germline BAP1 deleterious mutations in familial aggregations of cancers within the spectrum of the recently described BAP1-associated tumor predisposition syndrome, including uveal melanoma, malignant pleural mesothelioma, and cutaneous melanoma. Among the 11 families that included individuals identified as carrying germline deleterious BAP1 mutations, 6 families presented with 9 RCC-affected individuals, demonstrating a significantly increased risk for RCC. This strongly argues that RCC belongs to the BAP1 syndrome and that BAP1 is a RCC-predisposition gene.

Melanoma prone families with CDK4 germline mutation: phenotypic profile and associations with MC1R variants.

BACKGROUND: CDKN2A and CDK4 are high risk susceptibility genes for cutaneous malignant melanoma. Melanoma families with CDKN2A germline mutations have been extensively characterised, whereas CDK4 families are rare and lack a systematic investigation of their phenotype. METHODS: All known families with CDK4 germline mutations (n=17) were recruited for the study by contacting the authors of published papers or by requests via the Melanoma Genetics Consortium (GenoMEL). Phenotypic data related to primary melanoma and pigmentation characteristics were collected. The CDK4 exon 2 and the complete coding region of the MC1R gene were sequenced. RESULTS: Eleven families carried the CDK4 R24H mutation whereas six families had the R24C mutation. The total number of subjects with verified melanoma was 103, with a median age at first melanoma diagnosis of 39 years. Forty-three (41.7%) subjects had developed multiple primary melanomas (MPM). A CDK4 mutation was found in 89 (including 62 melanoma cases) of 209 tested subjects. CDK4 positive family members (both melanoma cases and unaffected subjects) were more likely to have clinically atypical nevi than CDK4 negative family members (p<0.001). MPM subjects had a higher frequency of MC1R red hair colour variants compared with subjects with one tumour (p=0.010). CONCLUSION: Our study shows that families with CDK4 germline mutations cannot be distinguished phenotypically from CDKN2A melanoma families, which are characterised by early onset of disease, increased occurrence of clinically atypical nevi, and development of MPM. In a clinical setting, the CDK4 gene should therefore always be examined when a melanoma family tests negative for CDKN2A mutation.

PTCH1 mutation and local aggressiveness of odontogenic keratocystic tumors in children: is there a relationship?

Keratocystic odontogenic tumors (KCOTs) are locally aggressive jaw lesions that may be related to PTCH1 mutations in isolation or in association with nevoid basal cell carcinoma syndrome. We sought to clarify the role of PTCH1 mutation in KCOT aggressiveness. We assessed cyst pathological characteristics, Ki-67 immunostaining, and somatic and germinal PTCH1 mutation in 16 KCOTs from 10 unrelated patients. Ten PTCH1 mutations were identified in 16 tumors. All tumors with PTCH1 mutations presented the criteria of pathological aggressiveness. We also noted the presence of a chorionic epithelial structure apparently acting as a secondary germinal center in these same tumors. Ki-67 immunostaining was not associated with PTCH1 mutation. KCOTs harboring the mutation display a chorionic epithelial structure that acts as a secondary germinal center. Genetic and microenvironmental factors might interact to propel tumor development.

Deficient mismatch repair phenotype is a prognostic factor for colorectal cancer in elderly patients.

OBJECTIVE: About 15% of colorectal adenocarcinomas have a deficient DNA mismatch repair phenotype. The frequency of deficient DNA mismatch repair tumours increases with age due to the hypermethylation of hMLH1 promoter. The study aimed to determine the prognostic value of deficient DNA mismatch repair phenotype in elderly patients. DESIGN: Mismatch repair phenotype was retrospectively determined by molecular analysis in consecutive resected colorectal adenocarcinoma specimens from patients over 75 years of age from 4 Oncology centres. RESULTS: 231 patients (median age: 81, range: 75-100) were enrolled from 2005 to 2008. Mean prevalence of deficient DNA mismatch repair phenotype was 22.5%, and 36% for patients over 85 years. Deficient DNA mismatch repair status was significantly associated with older age, female sex, proximal colon primary and high grade tumour. For stage II tumours no deficient DNA mismatch repair tumours had a recurrence at end of follow-up compared to 17% for tumours with proficient phenotype. The proficient phenotype status was significantly associated with worse age-adjusted overall survival [HR 2.60; 95% CI 1.05-6.44; p=0.039]. For stage III tumours a trend for less recurrence was observed for deficient DNA mismatch repair phenotype (16%) compared to proficient phenotype (36%). CONCLUSION: deficient DNA mismatch repair phenotype is a prognostic factor in stage II colorectal tumour in elderly patients. Our results suggest that mismatch repair phenotype should be taken in consideration for adjuvant chemotherapy decision in elderly patients.

Genetic variation at KIT locus may predispose to melanoma.

As loss of KIT frequently occurs in melanoma progression, we hypothesized that KIT is implicated in predisposition to melanoma (MM). Thus, we sequenced the KIT coding region in 112 familial MM cases and 143 matched controls and genotyped tag single-nucleotide polymorphisms (SNPs) in two cohorts of melanoma patients and matched controls. Five rare KIT substitutions, all predicted possibly or probably deleterious, were identified in five patients, but none in controls [RR = 2.26 (1.26-2.26)]. Expressed in melanocyte lines, three substitutions inhibited KIT signaling. Comparison with exomes database (7020 alleles) confirmed a significant excess of rare deleterious KIT substitutions in patients. Additionally, a common SNP, rs2237028, was associated with MM risk, and 6 KIT variants were associated with nevus count. Our data strongly suggest that rare KIT substitutions predispose to melanoma and that common variants at KIT locus may also impact nevus count and melanoma risk.