RESUMEN
Although several studies have explored the molecular landscape of metastatic melanoma, the genetic determinants of therapy resistance are still largely unknown. Here, we aimed to determine the contribution of whole-exome sequencing and circulating free DNA (cfDNA) analysis in predicting response to therapy in a consecutive real-world cohort of 36 patients, undergoing fresh tissue biopsy and followed during treatment. Although the underpowered sample size limited statistical analysis, samples from non-responders had higher copy number variations and mutations in melanoma driver genes compared to responders in the BRAF V600+ subset. In the BRAF V600- subset, Tumor Mutational Burden (TMB) was twice that in responders vs. non-responders. Genomic layout revealed commonly known and novel potential intrinsic/acquired resistance driver gene variants. Among these, RAC1, FBXW7, GNAQ mutations, and BRAF/PTEN amplification/deletion were present in 42% and 67% of patients, respectively. Both Loss of Heterozygosity (LOH) load and tumor ploidy were inversely associated with TMB. In immunotherapy-treated patients, samples from responders showed higher TMB and lower LOH and were more frequently diploid compared to non-responders. Secondary germline testing and cfDNA analysis proved their efficacy in finding germline predisposing variants carriers (8.3%) and following dynamic changes during treatment as a surrogate of tissue biopsy, respectively.
Asunto(s)
Ácidos Nucleicos Libres de Células , Melanoma , Humanos , Variaciones en el Número de Copia de ADN , Secuenciación del Exoma , Melanoma/genética , Melanoma/terapia , Mutación , Proteínas Proto-Oncogénicas B-raf/genéticaRESUMEN
ATM germline pathogenic variants were recently found enriched in high-risk melanoma patients. However, ATM loss of heterozygosity (LOH) has never been investigated in melanoma and, therefore, a causal association with melanoma development has not been established yet. The purpose of this study was to functionally characterize 13 germline ATM variants found in high-risk melanoma patients-and classified by in silico tools as pathogenic, uncertain significance, or benign-using multiple assays evaluating ATM/pATM expression and/or LOH in melanoma tissues and cell lines. We assessed ATM status by Immunohistochemistry (IHC), Western Blot, Whole-Exome Sequencing/Copy Number Variation analysis, and RNA sequencing, supported by Sanger sequencing and microsatellite analyses. For most variants, IHC results matched those obtained with in silico classification and LOH analysis. Two pathogenic variants (p.Ser1135_Lys1192del and p.Ser1993ArgfsTer23) showed LOH and complete loss of ATM activation in melanoma. Two variants of unknown significance (p.Asn358Ile and p.Asn796His) showed reduced expression and LOH, suggestive of a deleterious effect. This study, showing a classic two-hit scenario in a well-known tumor suppressor gene, supports the inclusion of melanoma in the ATM-related cancer spectrum.
Asunto(s)
Ataxia Telangiectasia , Melanoma , Humanos , Ataxia Telangiectasia/genética , Proteínas de la Ataxia Telangiectasia Mutada/genética , Variaciones en el Número de Copia de ADN , Pérdida de Heterocigocidad , Melanoma/genéticaRESUMEN
Neuroblastoma (NB) and malignant cutaneous melanoma (CMM) are neural crest cells (NCC)-derived tumors and may have a shared genetic basis, but this has not been investigated systematically by genome-wide association studies (GWAS). We took a three-staged approach to conduct cross-disease meta-analysis of GWAS for NB and CMM (2101 NB cases and 4202 controls; 12 874 CMM cases and 23 203 controls) to identify shared loci. Findings were replicated in 1403 NB cases and 1403 controls of European ancestry and in 636 NB, 508 CMM cases and 2066 controls of Italian origin. We found a cross-association at locus 1p13.2 (rs2153977, odds ratio = 0.91, P = 5.36 × 10-8). We also detected a suggestive (P < 10-7) NB-CMM cross-association at 2q37.1 with opposite effect on cancer risk. Pathway analysis of 110 NB-CMM risk loci with P < 10-4 demonstrated enrichment of biological processes such as cell migration, cell cycle, metabolism and immune response, which are essential of human NCC development, underlying both tumors. In vitro and in silico analyses indicated that the rs2153977-T protective allele, located in an NB and CMM enhancer, decreased expression of SLC16A1 via long-range loop formation and altered a T-box protein binding site. Upon depletion of SLC16A1, we observed a decrease of cellular proliferation and invasion in both NB and CMM cell lines, suggesting its role as oncogene. This is the largest study to date examining pleiotropy across two NC cell-derived tumors identifying 1p13.2 as common susceptibility locus for NB and CMM risk. We demonstrate that combining genome-wide association studies results across cancers with same origins can identify new loci common to neuroblastoma and melanoma arising from tissues which originate from neural crest cells. Our results also show 1p13.2 confer risk to neuroblastoma and melanoma by regulating SLC16A1.
Asunto(s)
Neoplasias de las Glándulas Suprarrenales/genética , Melanoma/genética , Transportadores de Ácidos Monocarboxílicos/genética , Neuroblastoma/genética , Neoplasias Cutáneas/genética , Simportadores/genética , Neoplasias de las Glándulas Suprarrenales/patología , Diferenciación Celular/genética , Movimiento Celular/genética , Cromosomas Humanos Par 1/genética , Femenino , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Melanoma/patología , Cresta Neural/patología , Neuroblastoma/patología , Polimorfismo de Nucleótido Simple/genética , Neoplasias Cutáneas/patología , Melanoma Cutáneo MalignoRESUMEN
BACKGROUND: The p.E318K variant of the Melanocyte Inducing Transcription Factor (MITF) has been implicated in genetic predisposition to melanoma as an intermediate penetrance allele. However, the impact of this variant on clinico-phenotypic, as well as on dermoscopic patterns features of affected patients is not entirely defined. The purpose of our study was to assess the association between the p.E318K germline variant and clinic-phenotypical features of MITF+ compared to non-carriers (MITF-), including dermoscopic findings of melanomas and dysplastic nevi. METHODS: we retrospectively analyzed a consecutive series of 1386 patients recruited between 2000 and 2017 who underwent genetic testing for CDKN2A, CDK4, MC1R and MITF germline variants in our laboratory for diagnostic/research purposes. The patients were probands of melanoma-prone families and apparently sporadic single or multiple primary melanoma patients. For all, we collected clinical, pathological information and dermoscopic images of the histopathologically diagnosed melanomas and dysplastic nevi, when available. RESULTS: After excluding patients positive for CDKN2A/CDK4 pathogenic variants and those affected by non-cutaneous melanomas, our study cohort comprised 984 cutaneous melanoma patients, 22 MITF+ and 962 MITF-. MITF+ were more likely to develop dysplastic nevi and multiple primary melanomas. Nodular melanoma was more common in MITF+ patients (32% compared to 19% in MITF-). MITF+ patients showed more frequently dysplastic nevi and melanomas with uncommon dermoscopic patterns (unspecific), as opposed to MITF- patients, whose most prevalent pattern was the multicomponent. CONCLUSIONS: MITF+ patients tend to develop melanomas and dysplastic nevi with histopathological features, frequency and dermoscopic patterns often different from those prevalent in MITF- patients. Our results emphasize the importance of melanoma prevention programs for MITF+ patients, including dermatologic surveillance with digital follow-up.
Asunto(s)
Melanoma , Factor de Transcripción Asociado a Microftalmía , Neoplasias Cutáneas , Predisposición Genética a la Enfermedad , Humanos , Melanoma/genética , Factor de Transcripción Asociado a Microftalmía/genética , Fenotipo , Estudios Retrospectivos , Neoplasias Cutáneas/genéticaRESUMEN
BACKGROUND: Cyclin dependent kinase inhibitor 2A gene (CDKN2A) germline mutations have recently been associated with poor survival in patients with melanoma. Despite the high mutation rate in our cohort (up to 10% in patients with apparently sporadic melanoma), information on the impact of CDKN2A on survival in this cohort is lacking. OBJECTIVE: To investigate whether poor survival associated with CDKN2A germline mutations was confirmed in a high mutation-prevalence cohort of Italian patients with melanoma undergoing a mutation-based follow-up. METHODS: A total of 1239 patients with cutaneous melanoma were tested for CDKN2A mutational status and then assigned to a follow-up scheme according not only to family history but also to CDKN2A mutational status, as follow-up intervals were more frequent for CDKN2A germline mutation-positive (MUT+) patients. From this cohort, we selected 106 MUT+ patients (with familial melanoma or apparently sporadic melanoma) and 199 CDKN2A germline mutation-negative (MUT-) patients with sporadic melanoma who were matched by age and sex and had a similar tumor stage distribution. RESULTS: We found no difference in overall survival (hazard ratio, 0.85; 95% confidence interval, 0.48-1.52; P = .592,) or melanoma-specific survival (hazard ratio, 0.86; 95% confidence interval, 0.38-1.95; P = .718,) between MUT+ and MUT- patients. MUT+ patients were more likely to develop multiple melanomas and to undergo surgical excision of dysplastic nevi than were MUT- patients. LIMITATIONS: Retrospective study. CONCLUSION: CDKN2A mutations were not associated with survival in our cohort.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Síndrome del Nevo Displásico/genética , Melanoma/genética , Melanoma/mortalidad , Neoplasias Primarias Múltiples/genética , Neoplasias Primarias Múltiples/mortalidad , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/mortalidad , Adulto , Síndrome del Nevo Displásico/cirugía , Femenino , Estudios de Seguimiento , Mutación de Línea Germinal , Humanos , Italia/epidemiología , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Tasa de SupervivenciaRESUMEN
BACKGROUND: Multiple primary melanoma (MPM), in concert with a positive family history, is a predictor of cyclin-dependent kinase (CDK) inhibitor 2A (CDKN2A) germline mutations. A rule regarding the presence of either 2 or 3 or more cancer events (melanoma and pancreatic cancer) in low or high melanoma incidence populations, respectively, has been established to select patients for genetic referral. OBJECTIVE: We sought to determine the CDKN2A/CDK4/microphthalmia-associated transcription factor mutation rate among Italian patients with MPM to appropriately direct genetic counseling regardless of family history. METHODS: In all, 587 patients with MPM and an equal number with single primary melanomas and control subjects were consecutively enrolled at the participating centers and tested for CDKN2A, CDK4, and microphthalmia-associated transcription factor. RESULTS: CDKN2A germline mutations were found in 19% of patients with MPM versus 4.4% of patients with single primary melanoma. In familial MPM cases the mutation rate varied from 36.6% to 58.8%, whereas in sporadic MPM cases it varied from 8.2% to 17.6% in patients with 2 and 3 or more melanomas, respectively. The microphthalmia-associated transcription factor E318K mutation accounted for 3% of MPM cases altogether. LIMITATIONS: The study was hospital based, not population based. Rare novel susceptibility genes were not tested. CONCLUSION: Italian patients who developed 2 melanomas, even in situ, should be referred for genetic counseling even in the absence of family history.
Asunto(s)
Asesoramiento Genético , Melanoma/genética , Neoplasias Primarias Múltiples/genética , Selección de Paciente , Neoplasias Cutáneas/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Quinasa 4 Dependiente de la Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Mutación de Línea Germinal , Humanos , Italia , Factor de Transcripción Asociado a Microftalmía/genética , Persona de Mediana Edad , Tasa de Mutación , Adulto JovenRESUMEN
The Protection of Telomere 1 (POT1) gene was identified as a melanoma predisposition candidate nearly 10 years ago. Thereafter, various cancers have been proposed as associated with germline POT1 variants in the context of the so-called POT1 Predisposition Tumor Syndrome (POT1-TPD). While the key role, and related risks, of the alterations in POT1 in melanoma are established, the correlation between germline POT1 variants and the susceptibility to other cancers partially lacks evidence, due also to the rarity of POT1-TPD. Issues range from the absence of functional or segregation studies to biased datasets or the need for a revised classification of variants. Furthermore, a proposal of a surveillance protocol related to the cancers associated with POT1 pathogenic variants requires reliable data to avoid an excessive, possibly unjustified, burden for POT1 variant carriers. We propose a critical perspective regarding data published over the last 10 years that correlate POT1 variants to various types of cancer, other than cutaneous melanoma, to offer food for thought for the specialists who manage cancer predisposition syndromes and to stimulate a debate on the grey areas that have been exposed.
Asunto(s)
Melanoma , Neoplasias Cutáneas , Humanos , Melanoma/genética , Neoplasias Cutáneas/genética , Alimentos , Síndrome , Telómero/genética , Complejo Shelterina , Proteínas de Unión a Telómeros/genéticaRESUMEN
Host, environmental and genetic factors differently modulate cutaneous melanoma (CM) risk across populations. Currently, the main genetic risk determinants are germline mutations in the major known high-risk susceptibility genes, CDKN2A and CDK4, and variants of the low-risk gene MC1R, which is key in the pigmentation process. This case-control study aimed at investigating the influence of the main host and environmental risk factors and of MC1R variation on CM risk in 390 CDKN2A-negative and 49 CDKN2A-positive Italian individuals. Multivariate analysis showed that MC1R variation, number of nevi and childhood sunburns doubled CM risk in CDKN2A-negative individuals. In CDKN2A-positive individuals, family history of CM and presence of atypical nevi, rather than MC1R status, modified risk (20.75- and 2.83-fold, respectively). Occupational sun exposure increased CM risk (three to sixfold) in both CDKN2A-negative and CDKN2A-positive individuals, reflecting the occupational habits of the Ligurian population and the geographical position of Liguria.
Asunto(s)
Genes p16 , Melanoma/genética , Exposición Profesional/efectos adversos , Receptor de Melanocortina Tipo 1/genética , Neoplasias Cutáneas/genética , Luz Solar/efectos adversos , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Intervalos de Confianza , Femenino , Humanos , Italia , Masculino , Melanoma/etiología , Persona de Mediana Edad , Análisis Multivariante , Mutación , Nevo/patología , Oportunidad Relativa , Factores de Riesgo , Neoplasias Cutáneas/etiología , Quemadura Solar/complicaciones , Adulto JovenRESUMEN
Birt-Hogg-Dubé syndrome (BHDS) is a rare autosomal dominant inherited disorder caused by a mutation in folliculin (FLCN) gene transmitted via germline autosomal dominant pattern. Patients with this syndrome have an increased susceptibility to renal cell carcinoma, lung cysts, spontaneous pneumothorax, and benign skin hamartomas, and its diagnosis is not easy and consequently underestimated. Several mutations have been identified in FLCN gene, among which the majority of alterations are frameshift (insertion/deletion), nonsense, or splice-site mutations that generally produce unfunctional truncated FLCN proteins. Our aim is to present a case of a BHDS family whose proband is a 56-year-old patient who has been experiencing multiple disorders, has an FLCN genetic mutation, and has also been identified to have a pathogenic variant in BRCA2 gene. Our further purpose is to emphasize the importance of the next-generation sequencing (NGS) approach to identify potential multiple germline mutations in complex and rare oncologic disorders, allowing strict and more targeted cancer screening programs.
RESUMEN
Multigene germline panel testing is recommended for Pancreatic Cancer (PC) patients; however, for non-BRCA1/2 genes, the clinical utility is unclear. A comprehensive multi-gene assessment in unselected Italian PC patients is missing. We evaluated the prevalence and impact of Pathogenic Variants (PV) in 51 PC susceptibility genes in a real-world series of 422 Italian PC patients unselected for Family History (FH), compared the clinical characteristics and conducted survival analyses. 17% of patients had PVs (70/422), mainly in BRCA1/2 (4.5%, all <70 y), CDKN2A (4.5%, all >50 y), ATM (2.1%). PV carriers were younger (64 vs. 67; p = 0.02) and had more frequent personal/FH of PC, melanoma and breast/ovarian cancer (all p < 0.05). The Overall Survival (OS) was longer in patients carrying PVs (HR 0.78; p = 0.090), comprising ATM carriers (HR 0.33; p = 0.054). In the oxaliplatin-treated subset, PV carriers showed better control of the disease, although this was not statistically significant (67% vs. 56%). CDKN2A, BRCA2 and ATM were the most frequently altered genes. ATM PVs were positively associated with OS in 41% of PV carriers, 60% of whom carried CDKN2A,BRCA2 or ATM PVs, had negative FH and would have been missed by traditional referral. Thus, CDKN2A and ATM should be added to BRCA1/2 testing regardless of FH.
RESUMEN
Restoration of the p53 tumor suppressor for personalised cancer therapy is a promising treatment strategy. However, several high-affinity MDM2 inhibitors have shown substantial side effects in clinical trials. Thus, elucidation of the molecular mechanisms of action of p53 reactivating molecules with alternative functional principle is of the utmost importance. Here, we report a discovery of a novel allosteric mechanism of p53 reactivation through targeting the p53 N-terminus which promotes inhibition of both p53/MDM2 (murine double minute 2) and p53/MDM4 interactions. Using biochemical assays and molecular docking, we identified the binding site of two p53 reactivating molecules, RITA (reactivation of p53 and induction of tumor cell apoptosis) and protoporphyrin IX (PpIX). Ion mobility-mass spectrometry revealed that the binding of RITA to serine 33 and serine 37 is responsible for inducing the allosteric shift in p53, which shields the MDM2 binding residues of p53 and prevents its interactions with MDM2 and MDM4. Our results point to an alternative mechanism of blocking p53 interaction with MDM2 and MDM4 and may pave the way for the development of novel allosteric inhibitors of p53/MDM2 and p53/MDM4 interactions.
RESUMEN
The incidence of melanoma, among the most lethal cancers, is widespread and increasing. Metastatic melanoma has a poor prognosis, representing about 90% of skin cancer mortality. The increased knowledge of tumor biology and the greater understanding of the immune system role in the anti-tumor response has allowed us to develop a more rational approach to systemic therapies. The discovery of activating BRAF mutations in half of all melanomas has led to the development of molecularly targeted therapy with BRAF and MEK inhibitors, which dramatically improved outcomes of patients with stage IV BRAF-mutant melanoma. More recently, the results of clinical phase III studies conducted in the adjuvant setting led to the combined administration of BRAF and MEK inhibitors also in patients with resected high-risk melanoma (stage III). Therefore, BRAF mutation testing has become a priority to determine the oncologist's choice and course of therapy. In this review, we will report the molecular biology-based strategies used for BRAF mutation detection with the main advantages and disadvantages of the most commonly used diagnostic strategies. The timing of such molecular assessment in patients with cutaneous melanoma will be discussed, and we will also examine considerations and approaches for accurate and effective BRAF testing.
RESUMEN
Melanoma is one of the most aggressive tumors of the skin, and its incidence is growing worldwide. Historically considered a drug resistant disease, since 2011 the therapeutic landscape of melanoma has radically changed. Indeed, the improved knowledge of the immune system and its interactions with the tumor, and the ever more thorough molecular characterization of the disease, has allowed the development of immunotherapy on the one hand, and molecular target therapies on the other. The increased availability of more performing technologies like Next-Generation Sequencing (NGS), and the availability of increasingly large genetic panels, allows the identification of several potential therapeutic targets. In light of this, numerous clinical and preclinical trials are ongoing, to identify new molecular targets. Here, we review the landscape of mutated non-BRAF skin melanoma, in light of recent data deriving from Whole-Exome Sequencing (WES) or Whole-Genome Sequencing (WGS) studies on melanoma cohorts for which information on the mutation rate of each gene was available, for a total of 10 NGS studies and 992 samples, focusing on available, or in experimentation, targeted therapies beyond those targeting mutated BRAF. Namely, we describe 33 established and candidate driver genes altered with frequency greater than 1.5%, and the current status of targeted therapy for each gene. Only 1.1% of the samples showed no coding mutations, whereas 30% showed at least one mutation in the RAS genes (mostly NRAS) and 70% showed mutations outside of the RAS genes, suggesting potential new roads for targeted therapy. Ongoing clinical trials are available for 33.3% of the most frequently altered genes.
RESUMEN
Incidence of melanoma has been constantly growing during the last decades. Although most of the new diagnoses are represented by thin melanomas, the number of melanoma-related deaths in 2018 was 60,712 worldwide (Global Cancer Observatory, 2019). Until 2011, no systemic therapy showed to improve survival in patients with advanced or metastatic melanoma. At that time, standard of care was chemotherapy, with very limited results. The identification of BRAF V600 mutation, and the subsequent introduction of BRAF targeting drugs, radically changed the clinical practice and dramatically improved outcomes. In this review, we will retrace the development of molecular-target drugs and the current therapeutic scenario for patients with BRAF mutated melanoma, from the introduction of BRAF inhibitors as single agents to modern clinical practice. We will also discuss the resistance mechanisms identified so far, and the future therapeutic perspectives in BRAF mutated melanoma.
RESUMEN
BACKGROUND: Identification of somatic mutations in key oncogenes in melanoma is important to lead the effective and efficient use of personalized anticancer treatment. Conventional methods focus on few genes per run and, therefore, are unable to screen for multiple genes simultaneously. The use of Next-Generation Sequencing (NGS) technologies enables sequencing of multiple cancer-driving genes in a single assay, with reduced costs and DNA quantity needed and increased mutation detection sensitivity. METHODS: We designed a customized IMI somatic gene panel for targeted sequencing of actionable melanoma mutations; this panel was tested on three different NGS platforms using 11 metastatic melanoma tissue samples in blinded manner between two EMQN quality certificated laboratory. RESULTS: The detection limit of our assay was set-up to a Variant Allele Frequency (VAF) of 10% with a coverage of at least 200x. All somatic variants detected by all NGS platforms with a VAF ≥ 10%, were also validated by an independent method. The IMI panel achieved a very good concordance among the three NGS platforms. CONCLUSION: This study demonstrated that, using the main sequencing platforms currently available in the diagnostic setting, the IMI panel can be adopted among different centers providing comparable results.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/normas , Melanoma/genética , Garantía de la Calidad de Atención de Salud , Análisis de Secuencia de ADN/normas , Neoplasias Cutáneas/genética , Análisis Mutacional de ADN/métodos , Análisis Mutacional de ADN/normas , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Italia , Masculino , Análisis de Secuencia de ADN/métodos , Melanoma Cutáneo MalignoRESUMEN
The contribution of recently established or candidate susceptibility genes to melanoma missing heritability has yet to be determined. Multigene panel testing could increase diagnostic yield and better define the role of candidate genes. We characterized 273 CDKN2A/ARF and CDK4-negative probands through a custom-designed targeted gene panel that included CDKN2A/ARF, CDK4, ACD, BAP1, MITF, POT1, TERF2IP, ATM, and PALB2. Co-segregation, loss of heterozygosity (LOH)/protein expression analysis, and splicing characterization were performed to improve variant classification. We identified 16 (5.9%) pathogenic and likely pathogenic variants in established high/medium penetrance cutaneous melanoma susceptibility genes (BAP1, POT1, ACD, MITF, and TERF2IP), including two novel variants in BAP1 and 4 in POT1. We also found four deleterious and five likely deleterious variants in ATM (3.3%). Thus, including potentially deleterious variants in ATM increased the diagnostic yield to about 9%. Inclusion of rare variants of uncertain significance would increase the overall detection yield to 14%. At least 10% of melanoma missing heritability may be explained through panel testing in our population. To our knowledge, this is the highest frequency of putative ATM deleterious variants reported in melanoma families, suggesting a possible role in melanoma susceptibility, which needs further investigation.
RESUMEN
The relative toxicity and mutagenicity of Me-lex, which selectively generates 3-methyladenine (3-MeA), is dependent on the nature of the DNA repair background. Base excision repair (BER)-defective S. cerevisiae strains mag1 and apn1apn2 were both significantly more sensitive to Me-lex toxicity, but only the latter is significantly more prone to Me-lex-induced mutagenesis. To examine the contribution of translesion synthesis (TLS) DNA polymerases in the bypass of Me-lex-induced lesions, the REV3 and REV1 genes were independently deleted in the parental yeast strain and in different DNA repair-deficient derivatives: the nucleotide excision repair (NER)-deficient rad14, and the BER-deficient mag1 or apn1apn2 strains. The strains contained an integrated ADE2 reporter gene under control of the transcription factor p53. A centromeric yeast expression vector containing the wild-type p53 cDNA was treated in vitro with increasing concentrations of Me-lex and transformed into the different yeast strains. The toxicity of Me-lex-induced lesions was evaluated based on the plasmid transformation efficiency compared to the untreated vector, while Me-lex mutagenicity was assessed using the p53 reporter assay. In the present study, we demonstrate that disruption of Polzeta (through deletion of its catalytic subunit coded by REV3) or Rev1 (by REV1 deletion) increased Me-lex lethality and decreased Me-lex mutagenicity in both the NER-defective (rad14) and BER-defective (mag1; apn1apn2) strains. Therefore, Polzeta and Rev1 contribute to resistance of the lethal effects of Me-lex-induced lesions (3-MeA and derived AP sites) by bypassing lesions and fixing some mutations.
Asunto(s)
Adenina/análogos & derivados , Antimutagênicos/farmacología , Metilación de ADN/efectos de los fármacos , Mutágenos/toxicidad , Netropsina/análogos & derivados , Nucleotidiltransferasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenina/química , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , Reparación del ADN/fisiología , Enzimas Reparadoras del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Netropsina/toxicidad , Nucleotidiltransferasas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteína p53 Supresora de TumorRESUMEN
[This corrects the article DOI: 10.18632/oncotarget.23204.].
RESUMEN
Due to the high mutational somatic burden of Cutaneous Malignant Melanoma (CMM) a thorough profiling of the driver mutations and their interplay is necessary to explain the timing of tumorigenesis or for the identification of actionable genetic events. The aim of this study was to establish the mutation rate of some of the key drivers in melanoma tumorigenesis combining molecular analyses and/or immunohistochemistry in 93 primary CMMs from an Italian cohort also characterized for germline status, and to investigate an interplay between germline and somatic variants. BRAF mutations were present in 68% of cases, while CDKN2A germline mutations were found in 16 % and p16 loss in tissue was found in 63%. TERT promoter somatic mutations were detected in 38% of cases while the TERT -245T>C polymorphism was found in 51% of cases. NRAS mutations were found in 39% of BRAF negative or undetermined cases. NF1 was expressed in all cases analysed. MC1R variations were both considered as a dichotomous variable or scored. While a positive, although not significant association between CDKN2A germline mutations, but not MC1R variants, and BRAF somatic mutation was found, we did not observe other associations between germline and somatic events. A yet undescribed inverse correlation between TERT -245T>C polymorphism and the presence of BRAF mutation was found. It is possible to hypothesize that -245T>C polymorphism could be included in those genotypes which may influence the occurrence of BRAF mutations. Further studies are needed to investigate the role of -245T>C polymorphism as a germline predictor of BRAF somatic mutation status.
RESUMEN
Background: The BRCA1-associated protein-1 (BAP1) tumor predisposition syndrome (BAP1-TPDS) is a hereditary tumor syndrome caused by germline pathogenic variants in BAP1 encoding a tumor suppressor associated with uveal melanoma, mesothelioma, cutaneous melanoma, renal cell carcinoma, and cutaneous BAP1-inactivated melanocytic tumors. However, the full spectrum of tumors associated with the syndrome is yet to be determined. Improved understanding of the BAP1-TPDS is crucial for appropriate clinical management of BAP1 germline variant carriers and their families, including genetic counseling and surveillance for new tumors. Methods: We collated germline variant status, tumor diagnoses, and information on BAP1 immunohistochemistry or loss of somatic heterozygosity on 106 published and 75 unpublished BAP1 germline variant-positive families worldwide to better characterize the genotypes and phenotypes associated with the BAP1-TPDS. Tumor spectrum and ages of onset were compared between missense and null variants. All statistical tests were two-sided. Results: The 181 families carried 140 unique BAP1 germline variants. The collated data confirmed the core tumor spectrum associated with the BAP1-TPDS and showed that some families carrying missense variants can exhibit this phenotype. A variety of noncore BAP1-TPDS -associated tumors were found in families of variant carriers. Median ages of onset of core tumor types were lower in null than missense variant carriers for all tumors combined (P < .001), mesothelioma (P < .001), cutaneous melanoma (P < .001), and nonmelanoma skin cancer (P < .001). Conclusions: This analysis substantially increases the number of pathogenic BAP1 germline variants and refines the phenotype. It highlights the need for a curated registry of germline variant carriers for proper assessment of the clinical phenotype of the BAP1-TPDS and pathogenicity of new variants, thus guiding management of patients and informing areas requiring further research.