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AIM: The 5-year survival rate of pancreatic ductal adenocarcinoma (PDAC) is approximately 11% and has only improved marginally over the last three decades. For operable PDAC, resection and adjuvant FOLFIRINOX chemotherapy is standard of care. There is growing interest in perioperative regimens to improve outcomes. The non-randomized Phase II study "Gemcitabine and Abraxane for resectable Pancreatic cancer" (GAP) demonstrated the feasibility of perioperative gemcitabine/abraxane. Long-term survival in PDAC requires an effective immune response; hence, we undertook this translational study of the GAP trial cohort to identify immune-oncology biomarkers for clinical use. METHODS: We combined Nanostring nCounter technology with immunohistochemistry to investigate the correlation between gene expression and overall patient survival. Findings were investigated in samples from the International Cancer Genome Consortium (ICGC, n = 88) and the Australian Pancreatic Genome Initiative (APGI, n = 227). RESULTS: We confirmed that human equilibrative nucleoside transporter 1 (hENT1) expression was not a prognostic marker in PDAC but patients with high levels of hENT1 were more likely to live longer than 24 months post-surgery. Additionally, CD274 (PD-L1) and two novel biomarkers of survival, cathepsin W (CTSW) and C-reactive protein (CRP), were identified in the GAP cohort (n = 19). CRP expression was confirmed in data from the ICGC. Although PD-L1 and CTSW proteins were not significant across all three cohorts, results show that low CRP mRNA and protein expression are associated with longer overall survival in all three patient groups. CONCLUSION: PDAC patients with long survival have higher hENT1 expression levels. Furthermore, CRP expression is a biomarker of poor prognosis following perioperative chemotherapy and resection in PDAC patients and thus may be useful for identifying patients who could benefit from more aggressive adjuvant strategies.
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BACKGROUND: The risk of esophageal adenocarcinoma (EAC) is associated with gastro-esophageal reflux disease (GERD) and obesity. Lipid metabolism-targeted therapies decrease the risk of progressing from Barrett's esophagus (BE) to EAC, but the precise lipid metabolic changes and their roles in genotoxicity during EAC development are yet to be established. METHODS: Esophageal biopsies from the normal epithelium (NE), BE, and EAC, were analyzed using concurrent lipidomics and proteomics (n = 30) followed by orthogonal validation on independent samples using RNAseq transcriptomics (n = 22) and immunohistochemistry (IHC, n = 80). The EAC cell line FLO-1 was treated with FADS2 selective inhibitor SC26196, and/or bile acid cocktail, followed by immunofluorescence staining for γH2AX. RESULTS: Metabolism-focused Reactome analysis of the proteomics data revealed enrichment of fatty acid metabolism, ketone body metabolism, and biosynthesis of specialized pro-resolving mediators in EAC pathogenesis. Lipidomics revealed progressive alterations (NE-BE-EAC) in glycerophospholipid synthesis with decreasing triglycerides and increasing phosphatidylcholine and phosphatidylethanolamine, and sphingolipid synthesis with decreasing dihydroceramide and increasing ceramides. Furthermore, a progressive increase in lipids with C20 fatty acids and polyunsaturated lipids with ≥4 double bonds were also observed. Integration with transcriptome data identified candidate enzymes for IHC validation: Δ4-Desaturase, Sphingolipid 1 (DEGS1) which desaturates dihydroceramide to ceramide, and Δ5 and Δ6-Desaturases (fatty acid desaturases, FADS1 and FADS2), responsible for polyunsaturation. All three enzymes showed significant increases from BE through dysplasia to EAC, but transcript levels of DEGS1 were decreased suggesting post-translational regulation. Finally, the FADS2 selective inhibitor SC26196 significantly reduced polyunsaturated lipids with three and four double bonds and reduced bile acid-induced DNA double-strand breaks in FLO-1 cells in vitro. CONCLUSIONS: Integrated multiomics revealed sphingolipid and phospholipid metabolism rewiring during EAC development. FADS2 inhibition and reduction of the high polyunsaturated lipids effectively protected EAC cells from bile acid-induced DNA damage in vitro, potentially through reduced lipid peroxidation.
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Adenocarcinoma , Esófago de Barrett , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Esófago de Barrett/genética , Esófago de Barrett/metabolismo , Esófago de Barrett/patología , Ácidos y Sales Biliares , Daño del ADN/genética , Neoplasias Esofágicas , Ácido Graso Desaturasas/genética , Ácido Graso Desaturasas/metabolismo , Ácidos Grasos , Humanos , EsfingolípidosRESUMEN
BACKGROUND: Little is known about the association between signet ring cell (SRC) differentiation and response to neoadjuvant chemotherapy (nCT) or neoadjuvant chemoradiotherapy (nCRT) in patients with esophageal and junctional adenocarcinoma (EAC). We aimed to assess if SRC differentiation is associated with survival and response to nCT or nCRT in patients with EAC. METHODS: Patients who underwent nCT and nCRT followed by surgery for EAC from 2000 until 2016 were identified from two institutional prospectively maintained databases. The pretreatment biopsy report or surgical resection specimen was used to differentiate patients into an SRC or non-SRC group. RESULTS: Overall, 129 (19%) of 689 patients included had SRCs (nCT: n = 64; nCRT: n = 65). The SRC group had a more advanced ypT stage (p = 0.003), a higher number of positive lymph nodes in the resection specimen {median (interquartile range [IQR]) 2 [0-5] vs. 1 [0-3]; p = 0.002} and a higher rate of R1/R2 resections (19.4% vs. 12%; p = 0.026). SRC differentiation was not an independent prognostic factor for overall survival (OS) or disease-free survival (DFS). Following nCT, the SRC group had significantly shorter DFS (median [IQR] 12 [5-50] vs. 23 [8-164]; p = 0.013), but not OS, compared with the non-SRC group. In contrast, no differences according to SRC status for OS or DFS were found in patients who underwent nCRT. CONCLUSIONS: SRC differentiation was not independently associated with worse OS in patients with EAC who underwent neoadjuvant therapy and surgery. However, nCRT was associated with greater tumor downstaging and better DFS.
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Adenocarcinoma/mortalidad , Carcinoma de Células en Anillo de Sello/patología , Diferenciación Celular , Quimioradioterapia Adyuvante/mortalidad , Neoplasias Esofágicas/mortalidad , Unión Esofagogástrica/patología , Terapia Neoadyuvante/mortalidad , Adenocarcinoma/patología , Adenocarcinoma/terapia , Neoplasias Esofágicas/patología , Neoplasias Esofágicas/terapia , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Prospectivos , Estudios Retrospectivos , Tasa de SupervivenciaRESUMEN
BACKGROUND: Oesophageal adenocarcinoma (EAC) incidence is increasing and has a poor survival rate. Barrett's oesophagus (BE) is a precursor condition that is associated with EAC and often occurs in conjunction with chronic gastro-oesophageal reflux, however many individuals diagnosed with BE never progress to cancer. An understanding of the genomic features of BE and EAC may help with the early identification of at-risk individuals. METHODS: In this study, we assessed the genomic features of 16 BE samples using whole-genome sequencing. These included non-dysplastic samples collected at two time-points from two BE patients who had not progressed to EAC over several years. Seven other non-dysplastic samples and five dysplastic BE samples with high-grade dysplasia were also examined. We compared the genome profiles of these 16 BE samples with 22 EAC samples. RESULTS: We observed that samples from the two non-progressor individuals had low numbers of somatic single nucleotide variants, indels and structural variation events compared to dysplastic and the remaining non-dysplastic BE. EAC had the highest level of somatic genomic variations. Mutational signature 17, which is common in EAC, was also present in non-dysplastic and dysplastic BE, but was not present in the non-progressors. Many dysplastic samples had mutations in genes previously reported in EAC, whereas only mutations in CDKN2A or in the fragile site genes appeared common in non-dysplastic samples. Rearrangement signatures were used to identify a signature associated with localised complex events such as chromothripsis and breakage fusion-bridge that are characteristic of EACs. Two dysplastic BE samples had a high contribution of this signature and contained evidence of localised rearrangements. Two other dysplastic samples also had regions of localised structural rearrangements. There was no evidence for complex events in non-dysplastic samples. CONCLUSIONS: The presence of complex localised rearrangements in dysplastic samples indicates a need for further investigations into the role such events play in the progression from BE to EAC.
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Adenocarcinoma/genética , Adenocarcinoma/patología , Esófago de Barrett/complicaciones , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Reordenamiento Génico , Adenocarcinoma/complicaciones , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias Esofágicas/complicaciones , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación , Clasificación del Tumor , Secuenciación Completa del GenomaRESUMEN
Endometrial cancer is the most commonly diagnosed gynaecological malignancy. Unfortunately, 15-20% of women demonstrate persistent or recurrent tumours that are refractory to current chemotherapies. We previously identified activating mutations in fibroblast growth factor receptor 2 (FGFR2) in 12% (stage I/II) to 17% (stage III/IV) endometrioid ECs and found that these mutations are associated with shorter progression-free and cancer-specific survival. Although FGFR inhibitors are undergoing clinical trials for treatment of several cancer types, little is known about the mechanism by which they induce cell death. We show that treatment with BGJ398, AZD4547 and PD173074 causes mitochondrial depolarization, cytochrome c release and impaired mitochondrial respiration in two FGFR2-mutant EC cell lines (AN3CA and JHUEM2). Despite this mitochondrial dysfunction, we were unable to detect caspase activation following FGFR inhibition; in addition, the pan-caspase inhibitor Z-VAD-FMK was unable to prevent cell death, suggesting that the cell death is caspase-independent. Furthermore, while FGFR inhibition led to an increase in LC3 puncta, treatment with bafilomycin did not further increase lipidated LC3, suggesting that FGFR inhibition led to a block in autophagosome degradation. We confirmed that cell death is mitochondrial-dependent as it can be blocked by overexpression of Bcl-2 and/or Bcl-XL. Importantly, we show that combining FGFR inhibitors with the BH3 mimetics ABT737/ABT263 markedly increased cell death in vitro and is more effective than BGJ398 alone in vivo, where it leads to marked tumour regression. This work may have implications for the design of clinical trials to treat a wide range of patients with FGFR-dependent malignancies.
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Apoptosis , Neoplasias Endometriales/genética , Neoplasias Endometriales/patología , Mitocondrias/metabolismo , Mutación/genética , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Animales , Autofagosomas/metabolismo , Caspasas/metabolismo , Línea Celular Tumoral , Activación Enzimática , Femenino , Humanos , Concentración 50 Inhibidora , Ratones , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismoRESUMEN
Previous genome-wide association studies have identified a melanoma-associated locus at 1q42.1 that encompasses a â¼100-kb region spanning the PARP1 gene. Expression quantitative trait locus (eQTL) analysis in multiple cell types of the melanocytic lineage consistently demonstrated that the 1q42.1 melanoma risk allele (rs3219090[G]) is correlated with higher PARP1 levels. In silico fine-mapping and functional validation identified a common intronic indel, rs144361550 (-/GGGCCC; r2 = 0.947 with rs3219090), as displaying allele-specific transcriptional activity. A proteomic screen identified RECQL as binding to rs144361550 in an allele-preferential manner. In human primary melanocytes, PARP1 promoted cell proliferation and rescued BRAFV600E-induced senescence phenotypes in a PARylation-independent manner. PARP1 also transformed TERT-immortalized melanocytes expressing BRAFV600E. PARP1-mediated senescence rescue was accompanied by transcriptional activation of the melanocyte-lineage survival oncogene MITF, highlighting a new role for PARP1 in melanomagenesis.
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Proliferación Celular/genética , Intrones/genética , Melanocitos/metabolismo , Factor de Transcripción Asociado a Microftalmía/genética , Poli(ADP-Ribosa) Polimerasa-1/genética , Polimorfismo de Nucleótido Simple , Secuencia de Bases , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Células Cultivadas , Senescencia Celular/genética , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Humanos , Mutación INDEL , Immunoblotting , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Factor de Transcripción Asociado a Microftalmía/metabolismo , Microscopía Confocal , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Factores de Riesgo , Telomerasa/genética , Telomerasa/metabolismoRESUMEN
Germline mutations in the exonuclease domain of POLE have been shown to predispose to colorectal cancers and adenomas. POLE is an enzyme involved in DNA repair and chromosomal DNA replication. In order to assess whether such mutations might also predispose to cutaneous melanoma, we interrogated whole-genome and exome data from probands of 34 melanoma families lacking pathogenic mutations in known high penetrance melanoma susceptibility genes: CDKN2A, CDK4, BAP1, TERT, POT1, ACD and TERF2IP. We found a novel germline mutation, POLE p.(Trp347Cys), in a 7-case cutaneous melanoma family. Functional assays in S. pombe showed that this mutation led to an increased DNA mutation rate comparable to that seen with a Pol ε mutant with no exonuclease activity. We then performed targeted sequencing of POLE in 1243 cutaneous melanoma cases and found that a further ten probands had novel or rare variants in the exonuclease domain of POLE. Although this frequency is not significantly higher than that in unselected Caucasian controls, we observed multiple cancer types in the melanoma families, suggesting that some germline POLE mutations may predispose to a broad spectrum of cancers, including melanoma. In addition, we found the first mutation outside the exonuclease domain, p.(Gln520Arg), in a family with an extensive history of colorectal cancer.
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Biomarcadores de Tumor/genética , ADN Polimerasa II/genética , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal/genética , Melanoma/genética , Neoplasias Cutáneas/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Estudios de Seguimiento , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Melanoma/patología , Persona de Mediana Edad , Estadificación de Neoplasias , Linaje , Proteínas de Unión a Poli-ADP-Ribosa , Pronóstico , Neoplasias Cutáneas/patología , Adulto Joven , Melanoma Cutáneo MalignoRESUMEN
Mutations in Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A) and Cyclin-Dependent Kinase 4 (CDK4) contribute to susceptibility in approximately 40% of high-density cutaneous melanoma (CMM) families and about 2% of unselected CMM cases. BRCA-1 associated protein-1 (BAP1) has been more recently shown to predispose to CMM and uveal melanoma (UMM) in some families; however, its contribution to CMM development in the general population is unreported. We sought to determine the contribution of these genes to CMM susceptibility in a population-based sample of cases from Australia. We genotyped 1,109 probands from Queensland families and found that approximately 1.31% harbored mutations in CDKN2A, including some with novel missense mutations (p.R22W, p.G35R and p.I49F). BAP1 missense variants occurred in 0.63% of cases but no CDK4 variants were observed in the sample. This is the first estimate of the contribution of BAP1 and CDK4 to a population-based sample of CMM and supports the previously reported estimate of CDKN2A germline mutation prevalence.
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Quinasa 4 Dependiente de la Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Melanoma/genética , Mutación , Neoplasias Cutáneas/genética , Proteínas Supresoras de Tumor/genética , Ubiquitina Tiolesterasa/genética , Australia/epidemiología , Femenino , Estudios de Seguimiento , Humanos , Masculino , Melanoma/epidemiología , Prevalencia , Neoplasias Cutáneas/epidemiologíaRESUMEN
BACKGROUND: The shelterin complex protects chromosomal ends by regulating how the telomerase complex interacts with telomeres. Following the recent finding in familial melanoma of inactivating germline mutations in POT1, encoding a member of the shelterin complex, we searched for mutations in the other five components of the shelterin complex in melanoma families. METHODS: Next-generation sequencing techniques were used to screen 510 melanoma families (with unknown genetic etiology) and control cohorts for mutations in shelterin complex encoding genes: ACD, TERF2IP, TERF1, TERF2, and TINF 2. Maximum likelihood and LOD [logarithm (base 10) of odds] analyses were used. Mutation clustering was assessed with χ(2) and Fisher's exact tests. P values under .05 were considered statistically significant (one-tailed with Yates' correction). RESULTS: Six families had mutations in ACD and four families carried TERF2IP variants, which included nonsense mutations in both genes (p.Q320X and p.R364X, respectively) and point mutations that cosegregated with melanoma. Of five distinct mutations in ACD, four clustered in the POT1 binding domain, including p.Q320X. This clustering of novel mutations in the POT1 binding domain of ACD was statistically higher (P = .005) in melanoma probands compared with population control individuals (n = 6785), as were all novel and rare variants in both ACD (P = .040) and TERF2IP (P = .022). Families carrying ACD and TERF2IP mutations were also enriched with other cancer types, suggesting that these variants also predispose to a broader spectrum of cancers than just melanoma. Novel mutations were also observed in TERF1, TERF2, and TINF2, but these were not convincingly associated with melanoma. CONCLUSIONS: Our findings add to the growing support for telomere dysregulation as a key process associated with melanoma susceptibility.
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Codón sin Sentido , Melanoma/genética , Neoplasias Cutáneas/genética , Proteínas de Unión a Telómeros/genética , Adulto , Anciano , ADN de Neoplasias/análisis , Femenino , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal , Humanos , Masculino , Persona de Mediana Edad , Linaje , Mutación Puntual , Análisis de Secuencia de ADN , Complejo Shelterina , Telómero/genética , Proteína 2 de Unión a Repeticiones Teloméricas/genéticaRESUMEN
RAF inhibitor therapy yields significant reductions in tumour burden in the majority of V600E-positive melanoma patients; however, resistance occurs within 2-18 months. Here we demonstrate that the mixed lineage kinases (MLK1-4) are MEK kinases that reactivate the MEK/ERK pathway in the presence of RAF inhibitors. Expression of MLK1-4 mediates resistance to RAF inhibitors and promotes survival in V600E-positive melanoma cell lines. Furthermore, we observe upregulation of the MLKs in 9 of 21 melanoma patients with acquired drug resistance. Consistent with this observation, MLKs promote resistance to RAF inhibitors in mouse models and contribute to acquired resistance in a cell line model. Lastly, we observe that a majority of MLK1 mutations identified in patients are gain-of-function mutations. In summary, our data demonstrate a role for MLKs as direct activators of the MEK/ERK pathway with implications for melanomagenesis and resistance to RAF inhibitors.
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Resistencia a Antineoplásicos/efectos de los fármacos , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Humanos , Indoles/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Mutación/genética , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas B-raf/metabolismo , Sulfonamidas/farmacología , Regulación hacia Arriba/efectos de los fármacos , VemurafenibRESUMEN
Deleterious germline variants in CDKN2A account for around 40% of familial melanoma cases, and rare variants in CDK4, BRCA2, BAP1 and the promoter of TERT have also been linked to the disease. Here we set out to identify new high-penetrance susceptibility genes by sequencing 184 melanoma cases from 105 pedigrees recruited in the UK, The Netherlands and Australia that were negative for variants in known predisposition genes. We identified families where melanoma cosegregates with loss-of-function variants in the protection of telomeres 1 gene (POT1), with a proportion of family members presenting with an early age of onset and multiple primary tumors. We show that these variants either affect POT1 mRNA splicing or alter key residues in the highly conserved oligonucleotide/oligosaccharide-binding (OB) domains of POT1, disrupting protein-telomere binding and leading to increased telomere length. These findings suggest that POT1 variants predispose to melanoma formation via a direct effect on telomeres.
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Predisposición Genética a la Enfermedad/genética , Melanoma/genética , Modelos Moleculares , Proteínas de Unión a Telómeros/genética , Telómero/metabolismo , Secuencia de Aminoácidos , Australia , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Países Bajos , Linaje , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADN , Complejo Shelterina , Neoplasias Cutáneas , Telómero/química , Telómero/genética , Proteínas de Unión a Telómeros/química , Proteínas de Unión a Telómeros/metabolismo , Reino Unido , Melanoma Cutáneo MalignoRESUMEN
We sought to identify fibroblast growth factor receptor 2 (FGFR2) kinase domain mutations that confer resistance to the pan-FGFR inhibitor, dovitinib, and explore the mechanism of action of the drug-resistant mutations. We cultured BaF3 cells overexpressing FGFR2 in high concentrations of dovitinib and identified 14 dovitinib-resistant mutations, including the N550K mutation observed in 25% of FGFR2(mutant) endometrial cancers (ECs). Structural and biochemical in vitro kinase analyses, together with BaF3 proliferation assays, showed that the resistance mutations elevate the intrinsic kinase activity of FGFR2. BaF3 lines were used to assess the ability of each mutation to confer cross-resistance to PD173074 and ponatinib. Unlike PD173074, ponatinib effectively inhibited all the dovitinib-resistant FGFR2 mutants except the V565I gatekeeper mutation, suggesting ponatinib but not dovitinib targets the active conformation of FGFR2 kinase. EC cell lines expressing wild-type FGFR2 were relatively resistant to all inhibitors, whereas EC cell lines expressing mutated FGFR2 showed differential sensitivity. Within the FGFR2(mutant) cell lines, three of seven showed marked resistance to PD173074 and relative resistance to dovitinib and ponatinib. This suggests that alternative mechanisms distinct from kinase domain mutations are responsible for intrinsic resistance in these three EC lines. Finally, overexpression of FGFR2(N550K) in JHUEM-2 cells (FGFR2(C383R)) conferred resistance (about five-fold) to PD173074, providing independent data that FGFR2(N550K) can be associated with drug resistance. Biochemical in vitro kinase analyses also show that ponatinib is more effective than dovitinib at inhibiting FGFR2(N550K). We propose that tumors harboring mutationally activated FGFRs should be treated with FGFR inhibitors that specifically bind the active kinase.
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Bencimidazoles/farmacología , Imidazoles/farmacología , Mutación , Piridazinas/farmacología , Pirimidinas/farmacología , Quinolonas/farmacología , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión/genética , Biocatálisis/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Electroforesis en Gel de Poliacrilamida , Neoplasias Endometriales/genética , Neoplasias Endometriales/metabolismo , Neoplasias Endometriales/patología , Femenino , Humanos , Concentración 50 Inhibidora , Modelos Moleculares , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Estructura Terciaria de Proteína , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/química , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismoRESUMEN
Melanoma of unknown primary (MUP) is an uncommon phenomenon whereby patients present with metastatic disease without an evident primary site. To determine their likely site of origin, we combined exome sequencing from 33 MUPs to assess the total rate of somatic mutations and degree of UV mutagenesis. An independent cohort of 91 archival MUPs was also screened for 46 hot spot mutations highly prevalent in melanoma including BRAF, NRAS, KIT, GNAQ, and GNA11. Results showed that the majority of MUPs exhibited high somatic mutation rates, high ratios of C>T/G>A transitions, and a high rate of BRAF (45 of 101, 45%) and NRAS (32 of 101, 32%) mutations, collectively indicating a mutation profile consistent with cutaneous sun-exposed melanomas. These data suggest that a significant proportion of MUPs arise from regressed or unrecognized primary cutaneous melanomas or arise de novo in lymph nodes from nevus cells that have migrated from the skin.
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Melanoma/genética , Melanoma/patología , Mutación/genética , Neoplasias Primarias Desconocidas/genética , Neoplasias Primarias Desconocidas/patología , Neoplasias Cutáneas/genética , Luz Solar , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Estudios de Cohortes , Análisis Mutacional de ADN , Exoma/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Cutáneas/patología , Melanoma Cutáneo MalignoRESUMEN
Mutations in multiple oncogenes including KRAS, CTNNB1, PIK3CA and FGFR2 have been identified in endometrial cancer. The aim of this study was to provide insight into the clinicopathological features associated with patterns of mutation in these genes, a necessary step in planning targeted therapies for endometrial cancer. 466 endometrioid endometrial tumors were tested for mutations in FGFR2, KRAS, CTNNB1, and PIK3CA. The relationships between mutation status, tumor microsatellite instability (MSI) and clinicopathological features including overall survival (OS) and disease-free survival (DFS) were evaluated using Kaplan-Meier survival analysis and Cox proportional hazard models. Mutations were identified in FGFR2 (48/466); KRAS (87/464); CTNNB1 (88/454) and PIK3CA (104/464). KRAS and FGFR2 mutations were significantly more common, and CTNNB1 mutations less common, in MSI positive tumors. KRAS and FGFR2 occurred in a near mutually exclusive pattern (p = 0.05) and, surprisingly, mutations in KRAS and CTNNB1 also occurred in a near mutually exclusive pattern (p = 0.0002). Multivariate analysis revealed that mutation in KRAS and FGFR2 showed a trend (p = 0.06) towards longer and shorter DFS, respectively. In the 386 patients with early stage disease (stage I and II), FGFR2 mutation was significantly associated with shorter DFS (HR = 3.24; 95% confidence interval, CI, 1.35-7.77; p = 0.008) and OS (HR = 2.00; 95% CI 1.09-3.65; p = 0.025) and KRAS was associated with longer DFS (HR = 0.23; 95% CI 0.05-0.97; p = 0.045). In conclusion, although KRAS and FGFR2 mutations share similar activation of the MAPK pathway, our data suggest very different roles in tumor biology. This has implications for the implementation of anti-FGFR or anti-MEK biologic therapies.
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Neoplasias Endometriales/metabolismo , Regulación Neoplásica de la Expresión Génica , Mutación , Fosfatidilinositol 3-Quinasas/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , beta Catenina/genética , Proteínas ras/genética , Anciano , Fosfatidilinositol 3-Quinasa Clase I , Supervivencia sin Enfermedad , Neoplasias Endometriales/genética , Femenino , Perfilación de la Expresión Génica , Genes ras , Humanos , Inestabilidad de Microsatélites , Persona de Mediana Edad , Mutación Puntual , Modelos de Riesgos Proporcionales , Resultado del TratamientoRESUMEN
We sequenced eight melanoma exomes to identify new somatic mutations in metastatic melanoma. Focusing on the mitogen-activated protein (MAP) kinase kinase kinase (MAP3K) family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9. Structural modeling predicted that mutations in the kinase domain may affect the activity and regulation of these protein kinases. The position of the mutations and the loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely to be inactivating. In in vitro kinase assays, MAP3K5 I780F and MAP3K9 W333* variants had reduced kinase activity. Overexpression of MAP3K5 or MAP3K9 mutants in HEK293T cells reduced the phosphorylation of downstream MAP kinases. Attenuation of MAP3K9 function in melanoma cells using siRNA led to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.
Asunto(s)
MAP Quinasa Quinasa Quinasa 5/genética , Quinasas Quinasa Quinasa PAM/genética , Melanoma/genética , Mutación , Neoplasias Cutáneas/genética , Antineoplásicos/farmacología , Secuencia de Bases , Línea Celular Tumoral , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Exoma , Humanos , Pérdida de Heterocigocidad , Melanoma/tratamiento farmacológico , Melanoma/secundario , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/patología , Temozolomida , Células Tumorales CultivadasRESUMEN
So far, two genes associated with familial melanoma have been identified, accounting for a minority of genetic risk in families. Mutations in CDKN2A account for approximately 40% of familial cases, and predisposing mutations in CDK4 have been reported in a very small number of melanoma kindreds. Here we report the whole-genome sequencing of probands from several melanoma families, which we performed in order to identify other genes associated with familial melanoma. We identify one individual carrying a novel germline variant (coding DNA sequence c.G1075A; protein sequence p.E318K; rs149617956) in the melanoma-lineage-specific oncogene microphthalmia-associated transcription factor (MITF). Although the variant co-segregated with melanoma in some but not all cases in the family, linkage analysis of 31 families subsequently identified to carry the variant generated a log of odds (lod) score of 2.7 under a dominant model, indicating E318K as a possible intermediate risk variant. Consistent with this, the E318K variant was significantly associated with melanoma in a large Australian case-control sample. Likewise, it was similarly associated in an independent case-control sample from the United Kingdom. In the Australian sample, the variant allele was significantly over-represented in cases with a family history of melanoma, multiple primary melanomas, or both. The variant allele was also associated with increased naevus count and non-blue eye colour. Functional analysis of E318K showed that MITF encoded by the variant allele had impaired sumoylation and differentially regulated several MITF targets. These data indicate that MITF is a melanoma-predisposition gene and highlight the utility of whole-genome sequencing to identify novel rare variants associated with disease susceptibility.
Asunto(s)
Predisposición Genética a la Enfermedad , Melanoma/genética , Factor de Transcripción Asociado a Microftalmía/genética , Mutación , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Sumoilación/genética , Adulto JovenRESUMEN
OBJECTIVE: Ovarian cancer is the leading cause of death from gynecologic malignancies in the Western world. Fibroblast growth factor receptor (FGFR) signaling has been implicated to play a role in ovarian tumorigenesis. Mutational activation of one member of this receptor family, FGFR2, is a frequent event in endometrioid endometrial cancer. Given the similarities in the histologic and molecular genetics of ovarian and endometrial cancers, we hypothesized that activating FGFR2 mutations may occur in a subset of endometrioid ovarian tumors, and possibly other histotypes. METHODS: Six FGFR2 exons were sequenced in 120 primary ovarian tumors representing the major histologic subtypes. RESULTS: FGFR2 mutation was detected at low frequency in endometrioid (1/46, 2.2%) and serous (1/41, 2.4%) ovarian cancer. No mutations were detected in clear cell, mucinous, or mixed histology tumors or in the ovarian cancer cell lines tested. Functional characterization of the FGFR2 mutations confirmed that the mutations detected in ovarian cancer result in receptor activation. CONCLUSIONS: Despite the low incidence of FGFR2 mutations in ovarian cancer, the two FGFR2 mutations identified in ovarian tumors (S252W, Y376C) overlap with the oncogenic mutations previously identified in endometrial tumors, suggesting activated FGFR2 may contribute to ovarian cancer pathogenesis in a small subset of ovarian tumors.
Asunto(s)
Mutación , Neoplasias Ováricas/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Adenocarcinoma Mucinoso/genética , Carcinoma Endometrioide/genética , Cistadenocarcinoma Seroso/genética , Exones , Femenino , HumanosRESUMEN
We report that 10% of melanoma tumors and cell lines harbor mutations in the fibroblast growth factor receptor 2 (FGFR2) gene. These novel mutations include three truncating mutations and 20 missense mutations occurring at evolutionary conserved residues in FGFR2 as well as among all four FGFRs. The mutation spectrum is characteristic of those induced by UV radiation. Mapping of these mutations onto the known crystal structures of FGFR2 followed by in vitro and in vivo studies show that these mutations result in receptor loss of function through several distinct mechanisms, including loss of ligand binding affinity, impaired receptor dimerization, destabilization of the extracellular domains, and reduced kinase activity. To our knowledge, this is the first demonstration of loss-of-function mutations in a class IV receptor tyrosine kinase in cancer. Taken into account with our recent discovery of activating FGFR2 mutations in endometrial cancer, we suggest that FGFR2 may join the list of genes that play context-dependent opposing roles in cancer.
Asunto(s)
Melanoma/genética , Mutación , Polimorfismo de Nucleótido Simple , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/química , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Neoplasias Cutáneas/genética , División Celular , Línea Celular Tumoral , Secuencia Conservada , Humanos , Melanoma/patología , Modelos Moleculares , Conformación Proteica , Neoplasias Cutáneas/patologíaRESUMEN
Heterozygous disruption of the Men1 gene predisposes mice to the development of multiple endocrine tumors, accurately mimicking the human MEN1 cancer predisposition syndrome. Additionally, Men1(+/-) mice frequently develop sex cord adenomas. The mechanism underlying the susceptibility of these mice to sex cord tumor development has not been fully determined, but data suggest it may involve transcriptional regulation of key growth promoting/repressing genes. To identify potential menin-regulated genes that may be important for tumor suppression in sex cord cells, we compared the global gene expression profiles of testis and ovary adenomas with other endocrine tumors of the pancreas and pituitary from Men1 heterozygous mice and with control tissues. Gonadal tumors clustered separately from pancreas and pituitary tumors with only a few genes (e.g., Cdkn2c) commonly dysregulated in all tumor types. Testis and ovary tumors displayed a higher level of transcriptional similarity to each other than they did to their respective control tissues. Among genes that had decreased expression in tumors was significant over-representation of genes associated with the TGF-beta, hedgehog and Wnt signaling, indicating that loss of menin function affects these pathways at the level of transcription. Aberrant protein expression in Leydig and granulosa cells of 2 transcriptionally dysregulated gene products, Gata6 and Csf1r were confirmed by immunohistochemistry. We propose that sex cord tumor susceptibility in Men1(+/-) mice involves deregulated cell proliferation due to dysregulation of multiple cell growth regulating genes including: reduced Cdkn2c transcription, loss of TGF-beta pathway tumor suppressor function (e.g., Gata6) and transcriptional activation of Csf1r.
Asunto(s)
Factor de Transcripción GATA6/análisis , Perfilación de la Expresión Génica , Proteínas Proto-Oncogénicas/fisiología , Receptor de Factor Estimulante de Colonias de Macrófagos/análisis , Tumores de los Cordones Sexuales y Estroma de las Gónadas/etiología , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/fisiología , Animales , Análisis por Conglomerados , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas/genéticaRESUMEN
KRAS activation and PTEN inactivation are frequent events in endometrial tumorigenesis, occurring in 10% to 30% and 26% to 80% of endometrial cancers, respectively. Because we have recently shown activating mutations in fibroblast growth factor receptor 2 (FGFR2) in 16% of endometrioid endometrial cancers, we sought to determine the genetic context in which FGFR2 mutations occur. Analysis of 116 primary endometrioid endometrial cancers revealed that FGFR2 and KRAS mutations were mutually exclusive, whereas FGFR2 mutations were seen concomitantly with PTEN mutations. Here, we show that shRNA knockdown of FGFR2 or treatment with a pan-FGFR inhibitor, PD173074, resulted in cell cycle arrest and induction of cell death in endometrial cancer cells with activating mutations in FGFR2. This cell death in response to FGFR2 inhibition occurred within the context of loss-of-function mutations in PTEN and constitutive AKT phosphorylation, and was associated with a marked reduction in extracellular signal-regulated kinase 1/2 activation. Together, these data suggest that inhibition of FGFR2 may be a viable therapeutic option in endometrial tumors possessing activating mutations in FGFR2, despite the frequent abrogation of PTEN in this cancer type.