RESUMO
The observation that BRCA1- and BRCA2-deficient cells are sensitive to inhibitors of poly(ADP-ribose) polymerase (PARP) has spurred the development of cancer therapies that use these inhibitors to target deficiencies in homologous recombination1. The cytotoxicity of PARP inhibitors depends on PARP trapping, the formation of non-covalent protein-DNA adducts composed of inhibited PARP1 bound to DNA lesions of unclear origins1-4. To address the nature of such lesions and the cellular consequences of PARP trapping, we undertook three CRISPR (clustered regularly interspersed palindromic repeats) screens to identify genes and pathways that mediate cellular resistance to olaparib, a clinically approved PARP inhibitor1. Here we present a high-confidence set of 73 genes, which when mutated cause increased sensitivity to PARP inhibitors. In addition to an expected enrichment for genes related to homologous recombination, we discovered that mutations in all three genes encoding ribonuclease H2 sensitized cells to PARP inhibition. We establish that the underlying cause of the PARP-inhibitor hypersensitivity of cells deficient in ribonuclease H2 is impaired ribonucleotide excision repair5. Embedded ribonucleotides, which are abundant in the genome of cells deficient in ribonucleotide excision repair, are substrates for cleavage by topoisomerase 1, resulting in PARP-trapping lesions that impede DNA replication and endanger genome integrity. We conclude that genomic ribonucleotides are a hitherto unappreciated source of PARP-trapping DNA lesions, and that the frequent deletion of RNASEH2B in metastatic prostate cancer and chronic lymphocytic leukaemia could provide an opportunity to exploit these findings therapeutically.
Assuntos
Sistemas CRISPR-Cas , Dano ao DNA , Edição de Genes , Neoplasias/genética , Neoplasias/patologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Ribonucleotídeos/genética , Animais , Proteína BRCA1/deficiência , Proteína BRCA1/genética , Linhagem Celular , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/genética , Replicação do DNA , DNA Topoisomerases Tipo I/metabolismo , Feminino , Genes BRCA1 , Genoma/genética , Células HeLa , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/enzimologia , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/deficiência , Poli(ADP-Ribose) Polimerase-1/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Ribonuclease H/deficiência , Ribonuclease H/genética , Ribonuclease H/metabolismo , Mutações Sintéticas Letais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Patients with prostate cancer frequently show resistance to androgen-deprivation therapy, a condition known as castration-resistant prostate cancer (CRPC). Acquiring a better understanding of the mechanisms that control the development of CRPC remains an unmet clinical need. The well-established dependency of cancer cells on the tumour microenvironment indicates that the microenvironment might control the emergence of CRPC. Here we identify IL-23 produced by myeloid-derived suppressor cells (MDSCs) as a driver of CRPC in mice and patients with CRPC. Mechanistically, IL-23 secreted by MDSCs can activate the androgen receptor pathway in prostate tumour cells, promoting cell survival and proliferation in androgen-deprived conditions. Intra-tumour MDSC infiltration and IL-23 concentration are increased in blood and tumour samples from patients with CRPC. Antibody-mediated inactivation of IL-23 restored sensitivity to androgen-deprivation therapy in mice. Taken together, these results reveal that MDSCs promote CRPC by acting in a non-cell autonomous manner. Treatments that block IL-23 can oppose MDSC-mediated resistance to castration in prostate cancer and synergize with standard therapies.
Assuntos
Interleucina-23/antagonistas & inibidores , Interleucina-23/metabolismo , Células Supressoras Mieloides/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Neoplasias de Próstata Resistentes à Castração/terapia , Antagonistas de Receptores de Andrógenos/farmacologia , Antagonistas de Receptores de Andrógenos/uso terapêutico , Androgênios/deficiência , Animais , Benzamidas , Proliferação de Células , Sobrevivência Celular , Humanos , Interleucina-23/sangue , Interleucina-23/imunologia , Masculino , Camundongos , Células Supressoras Mieloides/citologia , Células Supressoras Mieloides/imunologia , Nitrilas , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Feniltioidantoína/análogos & derivados , Feniltioidantoína/farmacologia , Feniltioidantoína/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/sangue , Neoplasias de Próstata Resistentes à Castração/metabolismo , Receptores Androgênicos/metabolismo , Receptores de Interleucina/metabolismo , Transdução de SinaisRESUMO
The classification of human cancers has traditionally relied on the tissue of origin, the histologic appearance and anatomical extent of disease, otherwise referred to as grade and stage. However, this system fails to explain the highly variable clinical behaviour seen for any one cancer. Molecular characterization through techniques such as next-generation sequencing (NGS) has led to an appreciation of the extreme genetic heterogeneity that underlies most human cancers. Because of the difficulties associated with fresh tissue biopsy, interest has increased in using circulating tumour material, such as circulating tumour cells (CTCs), as a non-invasive way to access tumour tissue. CTC enumeration has been demonstrated to have prognostic value in metastatic breast, colon and prostate cancers. Recent studies have also shown that CTCs are suitable material for molecular characterization, using techniques such as reverse transcription-polymerase chain reaction (RT-PCR), fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH) and NGS. Furthermore, genetic analysis of CTCs may be more suitable to study tumour heterogeneity and clonal evolution than fresh tissue biopsy. Whether blood-based biopsy techniques will be accepted as a replacement to fresh tissue biopsies remains to be seen, but there is reason for optimism. While significant barriers to this acceptance exist, blood-based biopsy techniques appear to be reliable and representative alternatives to fresh tissue biopsy.
Assuntos
Análise Mutacional de DNA , Neoplasias/genética , Neoplasias/patologia , Células Neoplásicas Circulantes , Hibridização Genômica Comparativa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização in Situ Fluorescente , Neoplasias/sangue , Células Neoplásicas Circulantes/metabolismo , PrognósticoRESUMO
OBJECTIVE: Oesophageal cancer is the seventh most common cause of cancer-related death worldwide. Disease relapse is frequent and treatment options are limited. DESIGN: To identify new biomarker-defined therapeutic approaches for patients with oesophageal cancer, we integrated the genomic profiles of 17 oesophageal tumour-derived cell lines with drug sensitivity data from small molecule inhibitor profiling, identifying drug sensitivity effects associated with cancer driver gene alterations. We also interrogated recently described RNA interference screen data for these tumour cell lines to identify candidate genetic dependencies or vulnerabilities that could be exploited as therapeutic targets. RESULTS: By integrating the genomic features of oesophageal tumour cell lines with siRNA and drug screening data, we identified a series of candidate targets in oesophageal cancer, including a sensitivity to inhibition of the kinase BTK in MYC amplified oesophageal tumour cell lines. We found that this genetic dependency could be elicited with the clinical BTK/ERBB2 kinase inhibitor, ibrutinib. In both MYC and ERBB2 amplified tumour cells, ibrutinib downregulated ERK-mediated signal transduction, cMYC Ser-62 phosphorylation and levels of MYC protein, and elicited G1 cell cycle arrest and apoptosis, suggesting that this drug could be used to treat biomarker-selected groups of patients with oesophageal cancer. CONCLUSIONS: BTK represents a novel candidate therapeutic target in oesophageal cancer that can be targeted with ibrutinib. On the basis of this work, a proof-of-concept phase II clinical trial evaluating the efficacy of ibrutinib in patients with MYC and/or ERBB2 amplified advanced oesophageal cancer is currently underway (NCT02884453). TRIAL REGISTRATION NUMBER: NCT02884453; Pre-results.
Assuntos
Neoplasias Esofágicas , Proteínas Proto-Oncogênicas c-myc/genética , Pirazóis/farmacologia , Pirimidinas/farmacologia , Receptor ErbB-2/genética , Adenina/análogos & derivados , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Descoberta de Drogas/métodos , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/genética , Humanos , Farmacogenética , Testes Farmacogenômicos/métodos , Piperidinas , Interferência de RNA/efeitos dos fármacos , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Frequently, the number of circulating tumor cells (CTC) isolated in 7.5 mL of blood is too small to reliably determine tumor heterogeneity and to be representative as a "liquid biopsy". In the EU FP7 program CTCTrap, we aimed to validate and optimize the recently introduced Diagnostic LeukApheresis (DLA) to screen liters of blood. Here we present the results obtained from 34 metastatic cancer patients subjected to DLA in the participating institutions. About 7.5 mL blood processed with CellSearch® was used as "gold standard" reference. DLAs were obtained from 22 metastatic prostate and 12 metastatic breast cancer patients at four different institutions without any noticeable side effects. DLA samples were prepared and processed with different analysis techniques. Processing DLA using CellSearch resulted in a 0-32 fold increase in CTC yield compared to processing 7.5 mL blood. Filtration of DLA through 5 µm pores microsieves was accompanied by large CTC losses. Leukocyte depletion of 18 mL followed by CellSearch yielded an increase of the number of CTC but a relative decrease in yield (37%) versus CellSearch DLA. In four out of seven patients with 0 CTC detected in 7.5 mL of blood, CTC were detected in DLA (range 1-4 CTC). The CTC obtained through DLA enables molecular characterization of the tumor. CTC enrichment technologies however still need to be improved to isolate all the CTC present in the DLA.
Assuntos
Neoplasias da Mama/sangue , Neoplasias da Mama/patologia , Células Neoplásicas Circulantes/patologia , Neoplasias de Próstata Resistentes à Castração/sangue , Neoplasias de Próstata Resistentes à Castração/patologia , Feminino , Humanos , Leucaférese/métodos , Biópsia Líquida/métodos , MasculinoRESUMO
Genome-wide association studies have identified more than 70 common variants that are associated with breast cancer risk. Most of these variants map to non-protein-coding regions and several map to gene deserts, regions of several hundred kilobases lacking protein-coding genes. We hypothesized that gene deserts harbor long-range regulatory elements that can physically interact with target genes to influence their expression. To test this, we developed Capture Hi-C (CHi-C), which, by incorporating a sequence capture step into a Hi-C protocol, allows high-resolution analysis of targeted regions of the genome. We used CHi-C to investigate long-range interactions at three breast cancer gene deserts mapping to 2q35, 8q24.21, and 9q31.2. We identified interaction peaks between putative regulatory elements ("bait fragments") within the captured regions and "targets" that included both protein-coding genes and long noncoding (lnc) RNAs over distances of 6.6 kb to 2.6 Mb. Target protein-coding genes were IGFBP5, KLF4, NSMCE2, and MYC; and target lncRNAs included DIRC3, PVT1, and CCDC26. For one gene desert, we were able to define two SNPs (rs12613955 and rs4442975) that were highly correlated with the published risk variant and that mapped within the bait end of an interaction peak. In vivo ChIP-qPCR data show that one of these, rs4442975, affects the binding of FOXA1 and implicate this SNP as a putative functional variant.
Assuntos
Neoplasias da Mama/genética , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla/métodos , Polimorfismo de Nucleotídeo Único , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Mapeamento Cromossômico , Cromossomos Humanos Par 2/genética , Cromossomos Humanos Par 8/genética , Cromossomos Humanos Par 9/genética , Genoma Humano/genética , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Fator 4 Semelhante a Kruppel , Células MCF-7 , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , Mapeamento de Interação de Proteínas , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Sequências Reguladoras de Ácido Nucleico/genética , Reprodutibilidade dos Testes , Análise de Sequência de DNARESUMO
Mitochondrial DNA (mtDNA) mutations leading to the disruption of respiratory complex I (CI) have been shown to exhibit anti-tumorigenic effects, at variance with those impairing only the function but not the assembly of the complex, which appear to contribute positively to cancer development. Owing to the challenges in the analysis of the multi-copy mitochondrial genome, it is yet to be determined whether tumour-associated mtDNA lesions occur as somatic modifying factors or as germ-line predisposing elements. Here we investigated the whole mitochondrial genome sequence of 20 pituitary adenomas with oncocytic phenotype and identified pathogenic and/or novel mtDNA mutations in 60% of the cases. Using highly sensitive techniques, namely fluorescent PCR and allele-specific locked nucleic acid quantitative PCR, we identified the most likely somatic nature of these mutations in our sample set, since none of the mutations was detected in the corresponding blood tissue of the patients analysed. Furthermore, we have subjected a series of 48 pituitary adenomas to a high-resolution array comparative genomic hybridization analysis, which revealed that CI disruptive mutations, and the oncocytic phenotype, significantly correlate with low number of chromosomal aberrations in the nuclear genome. We conclude that CI disruptive mutations in pituitary adenomas are somatic modifiers of tumorigenesis most likely contributing not only to the development of oncocytic change, but also to a less aggressive tumour phenotype, as indicated by a stable karyotype.
Assuntos
Adenoma/genética , Transformação Celular Neoplásica/genética , DNA Mitocondrial/genética , Complexo I de Transporte de Elétrons/genética , Instabilidade Genômica , Mutação , Neoplasias Hipofisárias/genética , Adenoma/patologia , Sequência de Aminoácidos , Transformação Celular Neoplásica/metabolismo , Variações do Número de Cópias de DNA , DNA Mitocondrial/química , DNA Mitocondrial/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fenótipo , Neoplasias Hipofisárias/patologia , Alinhamento de SequênciaRESUMO
BACKGROUND: Cutaneous squamous-cell carcinomas and keratoacanthomas are common findings in patients treated with BRAF inhibitors. METHODS: We performed a molecular analysis to identify oncogenic mutations (HRAS, KRAS, NRAS, CDKN2A, and TP53) in the lesions from patients treated with the BRAF inhibitor vemurafenib. An analysis of an independent validation set and functional studies with BRAF inhibitors in the presence of the prevalent RAS mutation was also performed. RESULTS: Among 21 tumor samples, 13 had RAS mutations (12 in HRAS). In a validation set of 14 samples, 8 had RAS mutations (4 in HRAS). Thus, 60% (21 of 35) of the specimens harbored RAS mutations, the most prevalent being HRAS Q61L. Increased proliferation of HRAS Q61L-mutant cell lines exposed to vemurafenib was associated with mitogen-activated protein kinase (MAPK)-pathway signaling and activation of ERK-mediated transcription. In a mouse model of HRAS Q61L-mediated skin carcinogenesis, the vemurafenib analogue PLX4720 was not an initiator or a promoter of carcinogenesis but accelerated growth of the lesions harboring HRAS mutations, and this growth was blocked by concomitant treatment with a MEK inhibitor. CONCLUSIONS: Mutations in RAS, particularly HRAS, are frequent in cutaneous squamous-cell carcinomas and keratoacanthomas that develop in patients treated with vemurafenib. The molecular mechanism is consistent with the paradoxical activation of MAPK signaling and leads to accelerated growth of these lesions. (Funded by Hoffmann-La Roche and others; ClinicalTrials.gov numbers, NCT00405587, NCT00949702, NCT01001299, and NCT01006980.).
Assuntos
Carcinoma de Células Escamosas/genética , Genes ras , Indóis/uso terapêutico , Mutação , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Neoplasias Cutâneas/genética , Sulfonamidas/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinoma de Células Escamosas/tratamento farmacológico , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Indóis/administração & dosagem , Masculino , Camundongos , Pessoa de Meia-Idade , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia , Sulfonamidas/administração & dosagem , VemurafenibRESUMO
Next generation sequencing has enabled systematic discovery of mutational spectra in cancer samples. Here, we used whole genome sequencing to characterize somatic mutations and structural variation in a primary acral melanoma and its lymph node metastasis. Our data show that the somatic mutational rates in this acral melanoma sample pair were more comparable to the rates reported in cancer genomes not associated with mutagenic exposure than in the genome of a melanoma cell line or the transcriptome of melanoma short-term cultures. Despite the perception that acral skin is sun-protected, the dominant mutational signature in these samples is compatible with damage due to ultraviolet light exposure. A nonsense mutation in ERCC5 discovered in both the primary and metastatic tumors could also have contributed to the mutational signature through accumulation of unrepaired dipyrimidine lesions. However, evidence of transcription-coupled repair was suggested by the lower mutational rate in the transcribed regions and expressed genes. The primary and the metastasis are highly similar at the level of global gene copy number alterations, loss of heterozygosity and single nucleotide variation (SNV). Furthermore, the majority of the SNVs in the primary tumor were propagated in the metastasis and one nonsynonymous coding SNV and one splice site mutation appeared to arise de novo in the metastatic lesion.
Assuntos
Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Melanoma/genética , Idoso , Variações do Número de Cópias de DNA , Análise Mutacional de DNA , Exoma , Humanos , Perda de Heterozigosidade , Masculino , Melanoma/patologia , Taxa de Mutação , Metástase Neoplásica , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Micropapillary carcinoma (MPC) is a rare histological special type of breast cancer, characterized by an aggressive clinical behaviour and a pattern of copy number aberrations (CNAs) distinct from that of grade- and oestrogen receptor (ER)-matched invasive carcinomas of no special type (IC-NSTs). The aims of this study were to determine whether MPCs are underpinned by a recurrent fusion gene(s) or mutations in 273 genes recurrently mutated in breast cancer. Sixteen MPCs were subjected to microarray-based comparative genomic hybridization (aCGH) analysis and Sequenom OncoCarta mutation analysis. Eight and five MPCs were subjected to targeted capture and RNA sequencing, respectively. aCGH analysis confirmed our previous observations about the repertoire of CNAs of MPCs. Sequencing analysis revealed a spectrum of mutations similar to those of luminal B IC-NSTs, and recurrent mutations affecting mitogen-activated protein kinase family genes and NBPF10. RNA-sequencing analysis identified 17 high-confidence fusion genes, eight of which were validated and two of which were in-frame. No recurrent fusions were identified in an independent series of MPCs and IC-NSTs. Forced expression of in-frame fusion genes (SLC2A1-FAF1 and BCAS4-AURKA) resulted in increased viability of breast cancer cells. In addition, genomic disruption of CDK12 caused by out-of-frame rearrangements was found in one MPC and in 13% of HER2-positive breast cancers, identified through a re-analysis of publicly available massively parallel sequencing data. In vitro analyses revealed that CDK12 gene disruption results in sensitivity to PARP inhibition, and forced expression of wild-type CDK12 in a CDK12-null cell line model resulted in relative resistance to PARP inhibition. Our findings demonstrate that MPCs are neither defined by highly recurrent mutations in the 273 genes tested, nor underpinned by a recurrent fusion gene. Although seemingly private genetic events, some of the fusion transcripts found in MPCs may play a role in maintenance of a malignant phenotype and potentially offer therapeutic opportunities.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Carcinoma Papilar/genética , Regulação Neoplásica da Expressão Gênica , Fusão Gênica , Mutação , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Proliferação de Células , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA , Análise Mutacional de DNA , Feminino , Dosagem de Genes , Predisposição Genética para Doença , Humanos , Invasividade Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Análise de Sequência de RNA , Fatores de TempoRESUMO
BACKGROUND: Taxanes such as paclitaxel and docetaxel are used successfully to treat breast cancer, usually in combination with other agents. They interfere with microtubules causing cell cycle arrest; however, the mechanisms underlying the clinical effects of taxanes are yet to be fully elucidated. METHODS: Isogenic paclitaxel resistant (PACR) MDAâMBâ231, paclitaxel resistant ZR75â1 and docetaxel resistant (DOCR) ZR75â1 cell lines were generated by incrementally increasing taxane dose in native cell lines in vitro. We used aCGH analysis to identify mechanisms driving taxane resistance. RESULTS: Taxane resistant cell lines exhibited an 18-170 fold increased resistance to taxanes, with the ZR75-1 resistant cell lines also demonstrating cross resistance to anthracyclines. Paclitaxel treatment of native cells resulted in a G2/M block and a decrease in the G1 phase of the cell cycle. However, in the resistant cell lines, minimal changes were present. Functional network analysis revealed that the mitotic prometaphase was lost in the resistant cell lines. CONCLUSION: This study established a model system for examining taxane resistance and demonstrated that both MDR and mitosis represent common mechanism of taxane resistance.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Paclitaxel/farmacologia , Taxoides/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Linhagem Celular Tumoral , Hibridização Genômica Comparativa , Docetaxel , Resistência a Múltiplos Medicamentos , Expressão Gênica , Humanos , Concentração Inibidora 50RESUMO
The incidence of oesophagogastric junctional (OGJ) adenocarcinoma is rising rapidly in western countries, in contrast to the declining frequency of distal gastric carcinoma. Treatment options for adenocarcinomas involving the oesophagogastric junction are limited and the overall prognosis is extremely poor. To determine the genomic landscape of OGJ adenocarcinoma, exomes of eight tumours and matched germline DNA were subjected to massively parallel DNA sequencing. Microsatellite instability was observed in three tumours which coincided with an elevated number of somatic mutations. In total, 117 genes were identified that had predicted coding alterations in more than one tumour. Potentially actionable coding mutations were identified in 67 of these genes, including those in CR2, HGF , FGFR4, and ESRRB. Twenty-nine genes harbouring somatic coding mutations and copy number changes in the MSS OGJ dataset are also known to be altered with similar predicted functional consequence in other tumour types. Compared with the published mutational profile of gastric cancers, 49% (57/117) of recurrently mutated genes were unique to OGJ tumours. TP53, SYNE1, and ARID1A were amongst the most frequently mutated genes in a larger OGJ cohort. Our study provides an insight into the mutational landscape of OGJ adenocarcinomas and confirms that this is a highly mutated and heterogeneous disease. Furthermore, we have uncovered somatic mutations in therapeutically relevant genes which may represent candidate drug targets.
Assuntos
Adenocarcinoma/genética , DNA de Neoplasias/genética , Neoplasias Esofágicas/genética , Junção Esofagogástrica , Mutação , Neoplasias Gástricas/genética , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenocarcinoma/química , Adenocarcinoma/patologia , Adenosina Trifosfatases/análise , Adenosina Trifosfatases/genética , Adulto , Idoso , Variações do Número de Cópias de DNA/genética , Análise Mutacional de DNA , Enzimas Reparadoras do DNA/análise , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Neoplasias Esofágicas/química , Neoplasias Esofágicas/patologia , Junção Esofagogástrica/patologia , Exoma/genética , Feminino , Genoma Humano/genética , Humanos , Imuno-Histoquímica , Perda de Heterozigosidade/genética , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Endonuclease PMS2 de Reparo de Erro de Pareamento , Proteína 1 Homóloga a MutL , Proteínas MutL , Mutação/genética , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/genética , Estadiamento de Neoplasias , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Reação em Cadeia da Polimerase/métodos , Estudos Prospectivos , Neoplasias Gástricas/química , Neoplasias Gástricas/patologiaRESUMO
Histological special types of breast cancer have distinctive morphological features and account for up to 25 % of all invasive breast cancers. We sought to determine whether at the genomic level, histological special types of breast cancer are distinct from grade- and estrogen receptor (ER)-matched invasive carcinomas of no special type (IC-NSTs), and to define genes whose expression correlates with gene copy number in histological special types of breast cancer. We characterized 59 breast cancers of ten histological special types using array-based comparative genomic hybridization (aCGH). Hierarchical clustering revealed that the patterns of gene copy number aberrations segregated with ER-status and histological grade, and that samples from each of the breast cancer histological special types preferentially clustered together. We confirmed the patterns of gene copy number aberrations previously reported for lobular, micropapillary, metaplastic, and mucinous carcinomas. On the other hand, metaplastic and medullary carcinomas were found to have genomic profiles similar to those of grade- and ER-matched IC-NSTs. The genomic aberrations observed in invasive carcinomas with osteoclast-like stromal giant cells support its classification as IC-NST variant. Integrative aCGH and gene expression analysis led to the identification of 145 transcripts that were significantly overexpressed when amplified in histological special types of breast cancer. Our results illustrate that together with histological grade and ER-status, histological type is also associated with the patterns and complexity of gene copy number aberrations in breast cancer, with adenoid cystic and mucinous carcinomas being examples of ER-negative and ER-positive breast cancers with distinctive repertoires of gene copy number aberrations.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Adenocarcinoma Mucinoso/genética , Adenocarcinoma Mucinoso/patologia , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Genoma Humano , Humanos , Hibridização In Situ/métodos , Receptores de Estrogênio/metabolismoRESUMO
AIMS: The majority of adenoid cystic carcinomas (AdCCs), regardless of anatomical site, harbour the MYB-NFIB fusion gene. The aim of this study was to characterize the repertoire of somatic genetic events affecting known cancer genes in AdCCs. METHODS AND RESULTS: DNA was extracted from 13 microdissected breast AdCCs, and subjected to a mutation survey using the Sequenom OncoCarta Panel v1.0. Genes found to be mutated in any of the breast AdCCs and genes related to the same canonical molecular pathways, as well as KIT, a proto-oncogene whose protein product is expressed in AdCCs, were sequenced in an additional 68 AdCCs from various anatomical sites by Sanger sequencing. Using the Sequenom MassARRAY platform and Sanger sequencing, mutations in BRAF and HRAS were identified in three and one cases, respectively (breast, and head and neck). KIT, which has previously been reported to be mutated in AdCCs, was also investigated, but no mutations were identified. CONCLUSIONS: Our results demonstrate that mutations in genes pertaining to the canonical RAS pathway are found in a minority of AdCCs, and that activating KIT mutations are either absent or remarkably rare in these cancers, and unlikely to constitute a driver and therapeutic target for patients with AdCC.
Assuntos
Neoplasias da Mama/genética , Carcinoma Adenoide Cístico/genética , Neoplasias de Cabeça e Pescoço/genética , Neoplasias Pulmonares/genética , Mutação/genética , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Adenoide Cístico/metabolismo , Carcinoma Adenoide Cístico/patologia , Análise Mutacional de DNA/métodos , Feminino , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismoRESUMO
The mechanisms underlying the progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) of the breast are yet to be fully elucidated. Several hypotheses have been put forward to explain the progression from DCIS to IDC, including the selection of a subpopulation of cancer cells with specific genetic aberrations, and the acquisition of new genetic aberrations or non-genetic mechanisms mediated by the tumour microenvironment. To determine whether synchronously diagnosed ipsilateral DCI and IDCs have modal populations with distinct repertoires of gene copy number aberrations and mutations in common oncogenes, matched frozen samples of DCIS and IDC were retrieved from 13 patients and subjected to microarray-based comparative genomic hybridization (aCGH) and Sequenom MassARRAY (Oncocarta v 1.0 panel). Fluorescence in situ hybridization and Sanger sequencing were employed to validate the aCGH and Sequenom findings, respectively. Although the genomic profiles of matched DCI and IDCs were similar, in three of 13 matched pairs amplification of distinct loci (ie 1q41, 2q24.2, 6q22.31, 7q11.21, 8q21.2 and 9p13.3) was either restricted to, or more prevalent in, the modal population of cancer cells of one of the components. Sequenom MassARRAY identified PIK3CA mutations restricted to the DCIS component in two cases, and in a third case the frequency of the PIK3CA mutant allele reduced from 49% in the DCIS to 25% in the IDC component. Despite the genomic similarities between synchronous DCIS and IDC, our data provide strong circumstantial evidence to suggest that in some cases the progression from DCIS to IDC is driven by the selection of non-modal clones that harbour a specific repertoire of genetic aberrations.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Carcinoma Intraductal não Infiltrante/genética , Análise Mutacional de DNA , Perfilação da Expressão Gênica , Heterogeneidade Genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Carcinoma Intraductal não Infiltrante/metabolismo , Carcinoma Intraductal não Infiltrante/patologia , Classe I de Fosfatidilinositol 3-Quinases , Evolução Clonal , Células Clonais , Hibridização Genômica Comparativa , DNA de Neoplasias/análise , Progressão da Doença , Feminino , Genômica/métodos , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Mutação , Neoplasias Primárias Múltiplas , Fosfatidilinositol 3-Quinases/genéticaRESUMO
Papillary carcinomas are a special histological type of breast cancer and have a relatively good outcome. We characterized the genomic and phenotypic characteristics of papillary carcinomas to determine whether they would constitute an entity distinct from grade- and oestrogen receptor (ER)-matched invasive ductal carcinomas of no special type (IDC-NSTs). The phenotype of 63 papillary carcinomas of the breast and grade- and ER-matched IDC-NSTs was determined by immunohistochemistry. DNA of sufficient quality was extracted from 49 microdissected papillary carcinomas and 49 microdissected grade- and ER-matched IDC-NSTs. These samples were subjected to high-resolution microarray-based comparative genomic hybridization (aCGH) and Sequenom MassARRAY sequencing analysis of 19 known oncogenes. Papillary carcinomas were predominantly of low histological grade, expressed immunohistochemical markers consistent with a luminal phenotype, and a lower rate of lymph node metastasis and p53 expression than grade- and ER-matched IDC-NSTs. Papillary carcinomas displayed less genomic aberrations than grade- and ER-matched IDC-NSTs; however, the patterns of gene copy number aberrations found in papillary carcinomas were similar to those of ER- and grade-matched IDC-NSTs, including 16q losses. Furthermore, PIK3CA mutations were found in 43% and 29% of papillary carcinomas and grade- and ER-matched IDC-NSTs, respectively. The genomic profiles of encapsulated, solid and invasive papillary carcinomas, the three morphological subtypes, were remarkably similar. Our results demonstrate that papillary carcinomas are a homogeneous special histological type of breast cancer. The similarities in the genomic profiles of papillary carcinomas and grade- and ER-matched IDC-NSTs suggest that papillary carcinomas may be best positioned as part of the spectrum of ER-positive breast cancers, rather than as a distinct entity. Furthermore, the good prognosis of papillary carcinomas may stem from the low rates of lymph node metastasis and p53 expression, low number of gene copy number aberrations and high prevalence of PIK3CA mutations.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Carcinoma Papilar/genética , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Carcinoma Papilar/patologia , Estudos de Casos e Controles , Classe I de Fosfatidilinositol 3-Quinases , Feminino , Humanos , Imunofenotipagem/métodos , Metástase Linfática , Mutação/genética , Fenótipo , Fosfatidilinositol 3-Quinases/genética , Receptores de Estrogênio/genéticaRESUMO
Adenoid cystic carcinoma (AdCC) is a rare form of triple-negative and basal-like breast cancer that has an indolent clinical behaviour. Four breast AdCCs were recently shown to harbour the recurrent chromosomal translocation t(6;9)(q22-23;p23-24), which leads to the formation of the MYB-NFIB fusion gene. Our aims were (i) to determine the prevalence of the MYB-NFIB fusion gene in AdCCs of the breast; (ii) to characterize the gene copy number aberrations found in AdCCs; and (iii) to determine whether AdCCs are genomically distinct from histological grade-matched or triple-negative and basal-like invasive ductal carcinomas of no special type (IDC-NSTs). The presence of the MYB-NFIB fusion gene was investigated in 13 AdCCs of the breast by fluorescence in situ hybridization (FISH) and reverse transcriptase-PCR (RT-PCR), and MYB and BRCA1 RNA expression was determined by quantitative RT-PCR. Fourteen AdCCs, 14 histological grade-matched IDC-NSTs, and 14 IDC-NSTs of triple-negative and basal-like phenotype were microdissected and subjected to high-resolution microarray-based comparative genomic hybridization (aCGH). The MYB-NFIB fusion gene was detected in all but one AdCC. aCGH analysis demonstrated a relatively low number of copy number aberrations and a lack of recurrent amplifications in breast AdCCs. Contrary to grade-matched IDC-NSTs, AdCCs lacked 1q gains and 16q losses, and in contrast with basal-like IDC-NSTs, AdCCs displayed fewer gene copy number aberrations and expressed MYB and BRCA1 at significantly higher levels. Breast AdCCs constitute an entity distinct from grade-matched and triple-negative and basal-like IDC-NSTs, emphasizing the importance of histological subtyping of triple-negative and basal-like breast carcinomas.
Assuntos
Neoplasias da Mama/genética , Carcinoma Adenoide Cístico/genética , Proteínas de Fusão Oncogênica/genética , Neoplasias da Mama/patologia , Carcinoma Adenoide Cístico/patologia , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patologia , Feminino , Dosagem de Genes , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Microdissecção , Receptor ErbB-3/biossíntese , Receptor ErbB-3/genética , Receptores de Estrogênio/biossíntese , Receptores de Estrogênio/genética , Receptores de Progesterona/biossíntese , Receptores de Progesterona/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise Serial de TecidosRESUMO
BRCA1 encodes a tumour suppressor protein that plays pivotal roles in homologous recombination (HR) DNA repair, cell-cycle checkpoints, and transcriptional regulation. BRCA1 germline mutations confer a high risk of early-onset breast and ovarian cancer. In more than 80% of cases, tumours arising in BRCA1 germline mutation carriers are oestrogen receptor (ER)-negative; however, up to 15% are ER-positive. It has been suggested that BRCA1 ER-positive breast cancers constitute sporadic cancers arising in the context of a BRCA1 germline mutation rather than being causally related to BRCA1 loss-of-function. Whole-genome massively parallel sequencing of ER-positive and ER-negative BRCA1 breast cancers, and their respective germline DNAs, was used to characterize the genetic landscape of BRCA1 cancers at base-pair resolution. Only BRCA1 germline mutations, somatic loss of the wild-type allele, and TP53 somatic mutations were recurrently found in the index cases. BRCA1 breast cancers displayed a mutational signature consistent with that caused by lack of HR DNA repair in both ER-positive and ER-negative cases. Sequencing analysis of independent cohorts of hereditary BRCA1 and sporadic non-BRCA1 breast cancers for the presence of recurrent pathogenic mutations and/or homozygous deletions found in the index cases revealed that DAPK3, TMEM135, KIAA1797, PDE4D, and GATA4 are potential additional drivers of breast cancers. This study demonstrates that BRCA1 pathogenic germline mutations coupled with somatic loss of the wild-type allele are not sufficient for hereditary breast cancers to display an ER-negative phenotype, and has led to the identification of three potential novel breast cancer genes (ie DAPK3, TMEM135, and GATA4).
Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Mutação em Linhagem Germinativa , Receptores de Estrogênio/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteína BRCA1/metabolismo , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Carcinoma Ductal de Mama/diagnóstico , Carcinoma Ductal de Mama/metabolismo , Análise Mutacional de DNA , Distúrbios no Reparo do DNA , DNA de Neoplasias/análise , Proteínas Quinases Associadas com Morte Celular , Feminino , Fator de Transcrição GATA4/genética , Genômica , Humanos , Perda de Heterozigosidade , Pessoa de Meia-Idade , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMO
INTRODUCTION: The 19q12 locus is amplified in a subgroup of oestrogen receptor (ER)-negative grade III breast cancers. This amplicon comprises nine genes, including cyclin E1 (CCNE1), which has been proposed as its 'driver'. The aim of this study was to identify the genes within the 19q12 amplicon whose expression is required for the survival of cancer cells harbouring their amplification. METHODS: We investigated the presence of 19q12 amplification in a series of 313 frozen primary breast cancers and 56 breast cancer cell lines using microarray comparative genomic hybridisation (aCGH). The nine genes mapping to the smallest region of amplification on 19q12 were silenced using RNA interference in phenotypically matched breast cancer cell lines with (MDA-MB-157 and HCC1569) and without (Hs578T, MCF7, MDA-MB-231, ZR75.1, JIMT1 and BT474) amplification of this locus. Genes whose silencing was selectively lethal in amplified cells were taken forward for further validation. The effects of cyclin-dependent kinase 2 (CDK2) silencing and chemical inhibition were tested in cancer cells with and without CCNE1 amplification. RESULTS: 19q12 amplification was identified in 7.8% of ER-negative grade III breast cancer. Of the nine genes mapping to this amplicon, UQCRFS1, POP4, PLEKHF1, C19ORF12, CCNE1 and C19ORF2 were significantly over-expressed when amplified in primary breast cancers and/or breast cancer cell lines. Silencing of POP4, PLEKHF1, CCNE1 and TSZH3 selectively reduced cell viability in cancer cells harbouring their amplification. Cancer cells with CCNE1 amplification were shown to be dependent on CDK2 expression and kinase activity for their survival. CONCLUSIONS: The 19q12 amplicon may harbour more than a single 'driver', given that expression of POP4, PLEKHF1, CCNE1 and TSZH3 is required for the survival of cancer cells displaying their amplification. The observation that cancer cells harbouring CCNE1 gene amplification are sensitive to CDK2 inhibitors provides a rationale for the testing of these chemical inhibitors in a subgroup of patients with ER-negative grade III breast cancers.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Cromossomos Humanos Par 19 , Regulação Neoplásica da Expressão Gênica , Proteínas Reguladoras de Apoptose/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Ciclina E/genética , Quinase 2 Dependente de Ciclina/genética , Feminino , Amplificação de Genes , Proteínas de Homeodomínio/genética , Humanos , Gradação de Tumores , Proteínas Oncogênicas/genética , Interferência de RNA , Receptores de Estrogênio/metabolismo , Ribonucleases/genética , Ribonucleoproteínas/genéticaRESUMO
Oncocytic carcinomas are composed of mitochondrion-rich cells. Though recognised by the WHO classification as a histological special type of breast cancer, their status as a discrete pathological entity remains a matter of contention. Given that oncocytic tumours of other anatomical sites display distinct clinico-pathological and molecular features, we sought to define the molecular genetic features of mitochondrion-rich breast tumours and to compare them with a series of histological grade- and oestrogen receptor status-matched invasive ductal carcinomas of no special type. Seventeen mitochondrion-rich breast carcinomas, including nine bona fide oncocytic carcinomas, were profiled with antibodies against oestrogen, progesterone and androgen receptors, HER2, Ki67, GCDFP-15, chromogranin, epithelial membrane antigen, cytokeratin 7, cytokeratin 14, CD68 and mitochondria antigen. These tumours were microdissected and DNA extracted from samples with >70% of tumour cells. Fourteen cases yielded DNA of sufficient quality/quantity and were subjected to high-resolution microarray comparative genomic hybridisation analysis. The genomic profiles were compared to those of 28 grade- and oestrogen receptor status-matched invasive ductal carcinomas of no special type. Oncocytic and other mitochondrion-rich tumours did not differ significantly between themselves. As a group, mitochondrion-rich carcinomas were immunophenotypically heterogenous. Recurrent copy number changes were similar to those described in unselected breast cancers. However, unsupervised and supervised analysis identified a subset of mitochondrion-rich cancers, which often displayed gains of 11q13.1-q13.2 and 19p13. Changes in the latter two chromosomal regions have been shown to be associated with oncocytic tumours of the kidney and thyroid, respectively, and host several nuclear genes with specific mitochondrial function. Our results indicate that in a way akin to oncocytic tumours of other anatomical sites, at least a subset of mitochondrion-rich breast carcinomas may be underpinned by a distinct pattern of chromosomal changes potentially relevant for mitochondria accumulation and constitute a discrete molecular entity.