RESUMO
Neuroblastoma is a malignant paediatric tumour of the sympathetic nervous system. Roughly half of these tumours regress spontaneously or are cured by limited therapy. By contrast, high-risk neuroblastomas have an unfavourable clinical course despite intensive multimodal treatment, and their molecular basis has remained largely elusive. Here we have performed whole-genome sequencing of 56 neuroblastomas (high-risk, n = 39; low-risk, n = 17) and discovered recurrent genomic rearrangements affecting a chromosomal region at 5p15.33 proximal of the telomerase reverse transcriptase gene (TERT). These rearrangements occurred only in high-risk neuroblastomas (12/39, 31%) in a mutually exclusive fashion with MYCN amplifications and ATRX mutations, which are known genetic events in this tumour type. In an extended case series (n = 217), TERT rearrangements defined a subgroup of high-risk tumours with particularly poor outcome. Despite a large structural diversity of these rearrangements, they all induced massive transcriptional upregulation of TERT. In the remaining high-risk tumours, TERT expression was also elevated in MYCN-amplified tumours, whereas alternative lengthening of telomeres was present in neuroblastomas without TERT or MYCN alterations, suggesting that telomere lengthening represents a central mechanism defining this subtype. The 5p15.33 rearrangements juxtapose the TERT coding sequence to strong enhancer elements, resulting in massive chromatin remodelling and DNA methylation of the affected region. Supporting a functional role of TERT, neuroblastoma cell lines bearing rearrangements or amplified MYCN exhibited both upregulated TERT expression and enzymatic telomerase activity. In summary, our findings show that remodelling of the genomic context abrogates transcriptional silencing of TERT in high-risk neuroblastoma and places telomerase activation in the centre of transformation in a large fraction of these tumours.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Genoma Humano/genética , Neuroblastoma/genética , Neuroblastoma/patologia , Recombinação Genética/genética , Telomerase/genética , Telomerase/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Cromatina/genética , Cromatina/metabolismo , Cromossomos Humanos Par 5/genética , DNA Helicases/genética , Metilação de DNA , Elementos Facilitadores Genéticos/genética , Ativação Enzimática/genética , Amplificação de Genes/genética , Inativação Gênica , Humanos , Lactente , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/classificação , Neuroblastoma/enzimologia , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Prognóstico , RNA Mensageiro/análise , RNA Mensageiro/genética , Risco , Translocação Genética/genética , Regulação para Cima/genética , Proteína Nuclear Ligada ao XRESUMO
We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Δex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer.
Assuntos
Genoma Humano/genética , Genômica , Neoplasias Pulmonares/genética , Mutação/genética , Carcinoma de Pequenas Células do Pulmão/genética , Alelos , Animais , Linhagem Celular Tumoral , Pontos de Quebra do Cromossomo , Ciclina D1/genética , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Sistemas Neurossecretores/metabolismo , Sistemas Neurossecretores/patologia , Proteínas Nucleares/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Proteína do Retinoblastoma/genética , Transdução de Sinais/genética , Carcinoma de Pequenas Células do Pulmão/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
The mediator complex is an evolutionary conserved key regulator of transcription of protein-coding genes and an integrative hub for diverse signaling pathways. In this study, we investigated whether the mediator subunit MED15 is implicated in castration-resistant prostate cancer (CRPC). MED15 expression and copy number/rearrangement status were assessed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), respectively on 718 prostate cancer (PCa) specimens and sequenced by Sanger on a subset. Furthermore, SMAD3 phosphorylation, androgen receptor (AR) and proliferation markers were evaluated by IHC. In PCa cells, siRNA/shRNA knockdown of MED15 was followed by proliferation assays with/without dihydrotestosterone (DHT), and treatments with recombinant TGF-ß3. Our results show that MED15 is overexpressed in 76% of distant metastatic CRPC (CRPC(MET) ) and 70% of local-recurrent CRPC (CRPC(LOC) ), in contrast to low frequencies in androgen-sensitive PCa, and no expression in benign prostatic tissue. Furthermore, MED15 overexpression correlates with worse clinical outcome thus defining a highly lethal phenotype. Moreover, TGF-ß signaling activation associates with MED15 overexpression in PCa tissues, and leads to increased expression of MED15 in PCa cells. MED15 knockdown effects phosphorylation and shuttling of p-SMAD3 to the nucleus as well as TGF-ß-enhanced proliferation. In PCa tissues, MED15 overexpression associates with AR overexpression/amplification and correlates with high proliferative activity. MED15 knockdown decreases both androgen-dependent and -independent proliferation in PCa cells. Taken together, these findings implicate MED15 in CRPC, and as MED15 is evolutionary conserved, it is likely to emerge as a lethal phenotype in other therapeutic-resistant diseases, and not restricted to our disease model.
Assuntos
Androgênios/genética , Glicina/análogos & derivados , Neoplasias de Próstata Resistentes à Castração/genética , Transdução de Sinais/genética , Idoso , Androgênios/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Glicina/biossíntese , Glicina/genética , Humanos , Hibridização in Situ Fluorescente , Masculino , Complexo Mediador/genética , Complexo Mediador/metabolismo , Pessoa de Meia-Idade , Neoplasias de Próstata Resistentes à Castração/patologia , Pirróis , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismoRESUMO
Castration-resistant prostate cancer (CRPC) is the most aggressive form of prostate cancer (PCa) and remains a significant therapeutic challenge. The key to the development of novel therapeutic targets for CRPC is to decipher the molecular alterations underlying this lethal disease. The aim of our study was to identify therapeutic targets for CRPC by assessing somatic copy number alterations (SCNAs) by whole-exome sequencing on five CRPC/normal paired formalin-fixed paraffin-embedded (FFPE) samples, using the SOLiD4 next-generation sequencing (NGS) platform. Data were validated using fluorescence in situ hybridization (FISH) on a PCa progression cohort. PTK2 and YWHAZ amplification, mRNA and protein expression were determined in selected PCa cell lines. Effects of PTK2 inhibition using TAE226 inhibitor and YWHAZ knock-down on cell proliferation and migration were tested in PC3 cells in vitro. In a larger validation cohort, the amplification frequency of YWHAZ was 3% in localized PCa and 48% in CRPC, whereas PTK2 was amplified in 1% of localized PCa and 35% in CRPC. YWHAZ knock-down and PTK2 inhibition significantly affected cell proliferation and migration in the PC3 cells. Our findings suggest that inhibition of YWHAZ and PTK2 could delay the progression of the disease in CRPC patients harbouring amplification of the latter genes. Furthermore, our validated whole-exome sequencing data show that FFPE tissue could be a promising alternative for SCNA screening using next-generation sequencing technologies.
Assuntos
Proteínas 14-3-3/genética , Variações do Número de Cópias de DNA/genética , Quinase 1 de Adesão Focal/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/cirurgia , Proteínas 14-3-3/metabolismo , Proliferação de Células/efeitos dos fármacos , Análise Mutacional de DNA/métodos , Exoma/genética , Quinase 1 de Adesão Focal/antagonistas & inibidores , Quinase 1 de Adesão Focal/metabolismo , Técnicas de Silenciamento de Genes , Estudos de Associação Genética/métodos , Humanos , Masculino , Terapia de Alvo Molecular/métodos , Morfolinas/farmacologia , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Orquiectomia , Inclusão em Parafina , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Análise de Sequência de DNA/métodos , Falha de Tratamento , Células Tumorais CultivadasRESUMO
The discovery of frequent 8p11-p12 amplifications in squamous cell lung cancer (SQLC) has fueled hopes that FGFR1, located inside this amplicon, might be a therapeutic target. In a clinical trial, only 11% of patients with 8p11 amplification (detected by FISH) responded to FGFR kinase inhibitor treatment. To understand the mechanism of FGFR1 dependency, we performed deep genomic characterization of 52 SQLCs with 8p11-p12 amplification, including 10 tumors obtained from patients who had been treated with FGFR inhibitors. We discovered somatically altered variants of FGFR1 with deletion of exons 1-8 that resulted from intragenic tail-to-tail rearrangements. These ectodomain-deficient FGFR1 variants (ΔEC-FGFR1) were expressed in the affected tumors and were tumorigenic in both in vitro and in vivo models of lung cancer. Mechanistically, breakage-fusion-bridges were the source of 8p11-p12 amplification, resulting from frequent head-to-head and tail-to-tail rearrangements. Generally, tail-to-tail rearrangements within or in close proximity upstream of FGFR1 were associated with FGFR1 dependency. Thus, the genomic events shaping the architecture of the 8p11-p12 amplicon provide a mechanistic explanation for the emergence of FGFR1-driven SQLC. Specifically, we believe that FGFR1 ectodomain-deficient and FGFR1-centered amplifications caused by tail-to-tail rearrangements are a novel somatic genomic event that might be predictive of therapeutically relevant FGFR1 dependency.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Amplificação de Genes , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Células Epiteliais/metabolismoRESUMO
Next generation sequencing (NGS) technologies have revolutionized cancer research allowing the comprehensive study of cancer using high throughput deep sequencing methodologies. These methods detect genomic alterations, nucleotide substitutions, insertions, deletions and copy number alterations. SOLiD (Sequencing by Oligonucleotide Ligation and Detection, Life Technologies) is a promising technology generating billions of 50 bp sequencing reads. This robust technique, successfully applied in gene identification, might be helpful in detecting novel genes associated with cancer initiation and progression using formalin fixed paraffin embedded (FFPE) tissue. This study's aim was to compare the validity of whole exome sequencing of fresh-frozen vs. FFPE tumor tissue by normalization to normal prostatic FFPE tissue, obtained from the same patient. One primary fresh-frozen sample, corresponding FFPE prostate cancer sample and matched adjacent normal prostatic tissue was subjected to exome sequencing. The sequenced reads were mapped and compared. Our study was the first to show comparable exome sequencing results between FFPE and corresponding fresh-frozen cancer tissues using SOLiD sequencing. A prior study has been conducted comparing the validity of sequencing of FFPE vs. fresh frozen samples using other NGS platforms. Our validation further proves that FFPE material is a reliable source of material for whole exome sequencing.
Assuntos
Criopreservação , Exoma , Sequenciamento de Nucleotídeos em Larga Escala , Inclusão em Parafina , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Formaldeído/química , Humanos , MasculinoRESUMO
Molecular and histopathological analysis of melanoma subtypes has revealed distinct epidemiological, genetic, and clinical features. However, immunotherapy for advanced metastatic melanoma patients does not differ based on subtype. Response to immune checkpoint inhibitors (ICI) has been shown to vary, therefore, predictive biomarkers are needed in the design of precision treatments. Targeted sequencing and histopathological analysis (CD8 and CD20 immunohistochemistry) were performed on subtypes of metastatic melanoma (cutaneous melanoma (CM, n = 10); head and neck melanoma (HNM, n = 7); uveal melanoma (UM, n = 4); acral lentiginous melanoma (AM, n = 1) and mucosal melanoma (MM, n = 1) treated with ICI). Progression-free survival (PFS) was significantly associated with high CD8 expression (p = 0.025) and mutations in DNA damage repair (DDR) pathway genes (p = 0.012) in all subtypes but not with CD20 expression. Our study identified that immune cell infiltration and DDR gene mutations may have an impact in response to ICI treatment in metastatic melanoma but differs among subtypes. Therefore, a comprehensive understanding of the immune infiltration cells' role and DDR gene mutations in metastatic melanoma may identify prognostic biomarkers.
Assuntos
Melanoma , Segunda Neoplasia Primária , Neoplasias Cutâneas , Dano ao DNA , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/genética , Mutação , Segunda Neoplasia Primária/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Melanoma Maligno CutâneoRESUMO
BACKGROUND: The present prospective study aimed at determining the impact of cell-free tumor DNA (ct-DNA), CA125 and HE4 from blood and ascites for quantification of tumor burden in patients with advanced high-grade serous epithelial ovarian cancer (EOC). METHODS: Genomic DNA was extracted from tumor FFPE and ct-DNA from plasma before surgery and on subsequent post-surgical days. Extracted DNA was subjected to hybrid-capture based next generation sequencing. Blood and ascites were sampled before surgery and on subsequent post-surgical days. 20 patients (10 undergoing complete resection (TR0), 10 undergoing incomplete resection (TR>0)) were included. RESULTS: The minor allele frequency (MAF) of TP53 mutations in ct-DNA of all patients with TR0 decreased significantly, compared to only one patient with TR>0. It was not possible to distinguish between patients with TR0 and patients with TR>0, using CA125 and HE4 from blood and ascites. CONCLUSIONS: Based upon the present findings, ct-DNA assessment in patients with high-grade serous EOC might help to better determine disease burden compared to standard tumor markers. Further studies should prospectively evaluate whether this enhancement of accuracy can help to optimize management of patients with EOC.
Assuntos
DNA Tumoral CirculanteRESUMO
AIMS: ERG rearrangements, mostly resulting in TMPRSS2-ERG fusions, are frequent alterations in prostate cancer (PCa), with a frequency ranging from 15% to 78%. As the reason for this variability is unknown, our aim was to investigate the ERG rearrangement frequency with a cohort design. METHODS AND RESULTS: We assessed three well-defined cohorts for ERG rearrangements, using fluorescence in situ hybridization (FISH). The first cohort comprised 119 prostatectomy specimens. The second and third cohorts included incidentally diagnosed PCa [71 cystoprostatectomy specimens, and 105 transurethral resection of the prostate (TURP) specimens]. Seventy of 119 (59%) cases of the prostatectomy cohort harboured ERG rearrangements. Regarding zonal origin, 2/11 (18%) transition zone (TZ) foci and 75/145 (52%) peripheral zone (PZ) foci harboured ERG rearrangements. Within the cystoprostatectomies, 24/71 (34%) cases harboured ERG rearrangements. Regarding zonal origin, 2/9 (22%) TZ foci and 26/86 (30%) PZ foci harboured ERG rearrangements. PCa incidentally identified by TURP harboured ERG rearrangements in 31/105 (29%) cases. CONCLUSIONS: ERG rearrangements occur in TZ PCa, although at a lower frequency than in PZ PCa. We confirmed that approximately half of all prostatectomies harbour ERG rearrangements. However, the frequency in incidentally diagnosed PCa cohorts was significantly lower, even if multifocality was considered. Consequently, zonal origin and cohort design are key for studying the clinical implications of ERG rearrangements.
Assuntos
Adenocarcinoma/genética , Rearranjo Gênico , Neoplasias da Próstata/genética , Transativadores/genética , Adenocarcinoma/patologia , Estudos de Coortes , Fusão Gênica , Humanos , Hibridização in Situ Fluorescente , Masculino , Proteínas de Fusão Oncogênica/genética , Prostatectomia , Neoplasias da Próstata/patologia , Projetos de Pesquisa , Serina Endopeptidases/genética , Regulador Transcricional ERGRESUMO
BACKGROUND: The availability of well-annotated prostate tissue samples through biobanks is key for research. Whereas fresh-frozen tissue is well suited for a broad spectrum of molecular analyses, its storage and handling is complex and cost-intensive. Formalin-fixed paraffin-embedded specimens (FFPE) are easy to handle and economic to store, but their applicability for molecular methods is restricted. The recently introduced Hepes-glutamic acid-buffer mediated Organic solvent Protection Effect (HOPE) is a promising alternative, which might have the potential to unite the benefits of FFPE and fresh-frozen specimen. Aim of the study was to compare HOPE-fixed, FFPE and fresh-frozen bio-specimens for their accessibility for diagnostic and research purposes. METHODS: 10 prostate cancer samples were each preserved with HOPE, formalin, and liquid nitrogen and studied with in-situ and molecular methods. Samples were H&E stained, and assessed by immunohistochemistry (i.e. PSA, GOLPH2, p63) and FISH (i.e. ERG rearrangement). We assessed DNA integrity by PCR, using control genes ranging from 100 to 600 bp amplicon size. RNA integrity was assessed through qRT-PCR on three housekeeping genes (TBP, GAPDH, ß-actin). Protein expression was analysed by performing western blot analysis using GOLPH2 and PSA antibodies. RESULTS: Of the HOPE samples, morphologic quality of H&E sections, immunohistochemical staining, and the FISH assay was at least equal to FFPE tissue, and significantly better than the fresh-frozen specimens. DNA, RNA, and protein analysis of HOPE samples provided similar results as compared to fresh-frozen specimens. As expected, FFPE-samples were inferior for most of the molecular analyses. CONCLUSIONS: This is the first study, comparatively assessing the suitability of these fixation methods for diagnostic and research utilization. Overall, HOPE-fixed bio-specimens combine the benefits of FFPE- and fresh-frozen samples. Results of this study have the potential to expand on contemporary prostate tissue biobanking approaches and can serve as a model for other organs and tumors.
Assuntos
DNA de Neoplasias/análise , Fixadores , Proteínas de Neoplasias/análise , Próstata/química , RNA Neoplásico/análise , Fixação de Tecidos/métodos , Western Blotting/métodos , Criopreservação/métodos , DNA de Neoplasias/química , Formaldeído , Ácido Glutâmico , Humanos , Imuno-Histoquímica , Masculino , Proteínas de Neoplasias/química , Nitrogênio , RNA Neoplásico/químicaRESUMO
Salivary gland carcinomas represent a heterogeneous group of poorly characterized head and neck tumors. The purpose of this study was to evaluate ALK gene and protein aberrations in a large, well-characterized cohort of these tumors. A total of 182 salivary gland carcinomas were tested for anaplastic lymphoma kinase (ALK) positivity by immunohistochemistry (IHC) using the cut-off of 10% positive cells. ALK positive tumors were subjected to FISH analysis and followed by hybrid capture-based next generation sequencing (NGS). Of the 182 tumors, 8 were ALK positive by IHC. Further analysis using hybrid capture NGS analysis revealed a novel MYO18A (Exon1-40)-ALK (exon 20-29) gene fusion in one case of intraductal carcinoma. Additional genomic analyses resulted in the detection of inactivating mutations in BRAF and TP53, as well as amplifications of ERBB2 and ALK. ALK rearrangements are a rare entity in salivary gland carcinomas. We identified a potentially targetable novel ALK fusion in an intraductal carcinoma of minor salivary glands.
Assuntos
Quinase do Linfoma Anaplásico/genética , Biomarcadores Tumorais/genética , Carcinoma/genética , Neoplasias das Glândulas Salivares/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma/enzimologia , Carcinoma/patologia , Carcinoma Intraductal não Infiltrante/enzimologia , Carcinoma Intraductal não Infiltrante/genética , Carcinoma Intraductal não Infiltrante/patologia , Criança , Feminino , Amplificação de Genes , Fusão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Neoplasias das Glândulas Salivares/enzimologia , Neoplasias das Glândulas Salivares/patologia , Adulto JovemRESUMO
For many years, the risk-based therapy stratification of children with neuroblastoma has relied on clinical and molecular covariates. In recent years, genome analysis has revealed further alterations defining risk, tumor biology, and therapeutic targets. The implementation of a robust and scalable method for analyzing traditional and new molecular markers in routine diagnostics is an urgent clinical need. Here, we investigated targeted panel sequencing as a diagnostic approach to analyze all relevant genomic neuroblastoma risk markers in one assay. Our "neuroblastoma hybrid capture sequencing panel" (NB-HCSP) assay employs a technology for the high-coverage sequencing (>1000×) of 55 selected genes and neuroblastoma-relevant genomic regions, which allows for the detection of single nucleotide changes, structural rearrangements, and copy number alterations. We validated our assay by analyzing 15 neuroblastoma cell lines and a cohort of 20 neuroblastomas, for which reference routine diagnostic data and genome sequencing data were available. We observed a high concordance for risk markers identified by the NB-HSCP assay, clinical routine diagnostics, and genome sequencing. Subsequently, we demonstrated clinical applicability of the NB-HCSP assay by analyzing routine clinical samples. We conclude that the NB-HCSP assay may be implemented into routine diagnostics as a single assay that covers all essential covariates for initial neuroblastoma classification, extended risk stratification, and targeted therapy selection.
RESUMO
In recent years, Non-small cell lung cancer (NSCLC) has evolved into a prime example for precision oncology with multiple FDA-approved "precision" drugs. For the majority of NSCLC lacking targetable genetic alterations, immune checkpoint inhibition (ICI) has become standard of care in first-line treatment or beyond. PD-L1 tumor expression represents the only approved predictive biomarker for PD-L1/PD-1 checkpoint inhibition by therapeutic antibodies. Since PD-L1-negative or low-expressing tumors may also respond to ICI, additional factors are likely to contribute in addition to PD-L1 expression. Tumor mutation burden (TMB) has emerged as a potential candidate; however, it is the most complex biomarker so far and might represent a challenge for routine diagnostics. We therefore established a hybrid capture (HC) next-generation sequencing (NGS) assay that covers all oncogenic driver alterations as well as TMB and validated TMB values by correlation with the assay (F1CDx) used for the CheckMate 227 study. Results of the first consecutive 417 patients analyzed in a routine clinical setting are presented. Data show that fast reliable comprehensive diagnostics including TMB and targetable alterations are obtained with a short turn-around time. Thus, even complex biomarkers can easily be implemented in routine practice to optimize treatment decisions for advanced NSCLC.
RESUMO
INTRODUCTION: ROS1 rearrangements are found in 1% of lung cancer patients. Therapeutic efficacy of crizotinib in this subset has been shown in early phase trials in the United States and East Asia. Here we present data on efficacy and safety of a prospective phase II trial evaluating crizotinib in European ROS1-positive patients (EUCROSS). PATIENTS AND METHODS: The trial was a multicenter, single-arm phase II trial (Clinicaltrial.gov identifier: NCT02183870). Key eligibility criteria included patients who were 18 years of age or older with advanced/metastatic lung cancer and centrally confirmed ROS1-rearranged lung cancer (fluorescence-in situ hybridization). Treatment included 250 mg crizotinib twice daily. The primary endpoint was investigator-assessed objective response rate (ORR) (Response Evaluation Criteria in Solid Tumors, version 1.1). Key secondary endpoints were progression-free survival (PFS), overall survival, efficacy by independent radiologic review, safety, health-related quality of life, and molecular characterization of tumor tissue. RESULTS: Thirty-four patients received treatment. Four patients were excluded from efficacy analysis. Investigator ORR was 70% (95% confidence interval [CI]: 51-85; 21 of 30 patients) and median PFS was 20.0 months (95% CI: 10.1-not reached). Two patients with ROS1 wild-type sequences assessed by DNA sequencing had progression as best response. CD74-ROS1-positive patients had a trend towards a higher ORR and longer median PFS. TP53-co-mutant patients had a significantly shorter median PFS than wild-type patients (7.0 months, 95% CI: 1.7-20.0 versus 24.1 months, 95% CI: 10.1-not reached; p = 0.022). Treatment-related adverse events were documented in 33 of 34 patients (97%). CONCLUSIONS: Crizotinib is highly effective and safe in patients with ROS1-rearranged lung cancer. ROS1-/TP53-co-aberrant patients had a significantly worse outcome compared to TP53 wild-type patients.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Crizotinibe/uso terapêutico , Rearranjo Gênico , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/secundário , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Feminino , Seguimentos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Critérios de Avaliação de Resposta em Tumores Sólidos , Taxa de SobrevidaRESUMO
Purpose: We sought to investigate the clinical response to MET inhibition in patients diagnosed with structural MET alterations and to characterize their functional relevance in cellular models.Experimental Design: Patients were selected for treatment with crizotinib upon results of hybrid capture-based next-generation sequencing. To confirm the clinical observations, we analyzed cellular models that express these MET kinase alterations.Results: Three individual patients were identified to harbor alterations within the MET receptor. Two patients showed genomic rearrangements, leading to a gene fusion of KIF5B or STARD3NL and MET One patient diagnosed with an EML4-ALK rearrangement developed a MET kinase domain duplication as a resistance mechanism to ceritinib. All 3 patients showed a partial response to crizotinib that effectively inhibits MET and ALK among other kinases. The results were further confirmed using orthogonal cellular models.Conclusions: Crizotinib leads to a clinical response in patients with MET rearrangements. Our functional analyses together with the clinical data suggest that these structural alterations may represent actionable targets in lung cancer patients. Clin Cancer Res; 24(6); 1337-43. ©2017 AACR.
Assuntos
Adenocarcinoma de Pulmão/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/genética , Adenocarcinoma de Pulmão/diagnóstico , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/metabolismo , Adulto , Quinase do Linfoma Anaplásico/antagonistas & inibidores , Crizotinibe/farmacologia , Crizotinibe/uso terapêutico , Feminino , Duplicação Gênica , Rearranjo Gênico , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Estadiamento de Neoplasias , Proteínas de Fusão Oncogênica/química , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-met/química , Tomografia Computadorizada por Raios XRESUMO
Neuroblastoma is a pediatric tumor of the sympathetic nervous system. Its clinical course ranges from spontaneous tumor regression to fatal progression. To investigate the molecular features of the divergent tumor subtypes, we performed genome sequencing on 416 pretreatment neuroblastomas and assessed telomere maintenance mechanisms in 208 of these tumors. We found that patients whose tumors lacked telomere maintenance mechanisms had an excellent prognosis, whereas the prognosis of patients whose tumors harbored telomere maintenance mechanisms was substantially worse. Survival rates were lowest for neuroblastoma patients whose tumors harbored telomere maintenance mechanisms in combination with RAS and/or p53 pathway mutations. Spontaneous tumor regression occurred both in the presence and absence of these mutations in patients with telomere maintenance-negative tumors. On the basis of these data, we propose a mechanistic classification of neuroblastoma that may benefit the clinical management of patients.
Assuntos
Neuroblastoma/classificação , Neuroblastoma/mortalidade , Homeostase do Telômero/genética , Criança , Pré-Escolar , Intervalo Livre de Doença , Exoma/genética , Genoma Humano , Humanos , Redes e Vias Metabólicas/genética , Mutação , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Prognóstico , Análise de Sequência de DNA , Proteína Supressora de Tumor p53/genética , Proteínas ras/genéticaRESUMO
Small cell lung cancer (SCLC) patient-derived xenografts (PDX) can be generated from biopsies or circulating tumor cells (CTC), though scarcity of tissue and low efficiency of tumor growth have previously limited these approaches. Applying an established clinical-translational pipeline for tissue collection and an automated microfluidic platform for CTC enrichment, we generated 17 biopsy-derived PDXs and 17 CTC-derived PDXs in a 2-year timeframe, at 89% and 38% efficiency, respectively. Whole-exome sequencing showed that somatic alterations are stably maintained between patient tumors and PDXs. Early-passage PDXs maintain the genomic and transcriptional profiles of the founder PDX. In vivo treatment with etoposide and platinum (EP) in 30 PDX models demonstrated greater sensitivity in PDXs from EP-naïve patients, and resistance to EP corresponded to increased expression of a MYC gene signature. Finally, serial CTC-derived PDXs generated from an individual patient at multiple time points accurately recapitulated the evolving drug sensitivities of that patient's disease. Collectively, this work highlights the translational potential of this strategy.Significance: Effective translational research utilizing SCLC PDX models requires both efficient generation of models from patients and fidelity of those models in representing patient tumor characteristics. We present approaches for efficient generation of PDXs from both biopsies and CTCs, and demonstrate that these models capture the mutational landscape and functional features of the donor tumors. Cancer Discov; 8(5); 600-15. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 517.
Assuntos
Genômica , Neoplasias Pulmonares/genética , Carcinoma de Pequenas Células do Pulmão/genética , Animais , Biópsia , Modelos Animais de Doenças , Genômica/métodos , Humanos , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Carcinoma de Pequenas Células do Pulmão/diagnóstico , Carcinoma de Pequenas Células do Pulmão/terapia , Tomografia Computadorizada por Raios X , Sequenciamento do Exoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pulmonary large-cell neuroendocrine carcinomas (LCNECs) have similarities with other lung cancers, but their precise relationship has remained unclear. Here we perform a comprehensive genomic (n = 60) and transcriptomic (n = 69) analysis of 75 LCNECs and identify two molecular subgroups: "type I LCNECs" with bi-allelic TP53 and STK11/KEAP1 alterations (37%), and "type II LCNECs" enriched for bi-allelic inactivation of TP53 and RB1 (42%). Despite sharing genomic alterations with adenocarcinomas and squamous cell carcinomas, no transcriptional relationship was found; instead LCNECs form distinct transcriptional subgroups with closest similarity to SCLC. While type I LCNECs and SCLCs exhibit a neuroendocrine profile with ASCL1high/DLL3high/NOTCHlow, type II LCNECs bear TP53 and RB1 alterations and differ from most SCLC tumors with reduced neuroendocrine markers, a pattern of ASCL1low/DLL3low/NOTCHhigh, and an upregulation of immune-related pathways. In conclusion, LCNECs comprise two molecularly defined subgroups, and distinguishing them from SCLC may allow stratified targeted treatment of high-grade neuroendocrine lung tumors.
Assuntos
Carcinoma Neuroendócrino/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Tumores Neuroendócrinos/genética , Carcinoma de Pequenas Células do Pulmão/genética , Análise Mutacional de DNA , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Técnicas In Vitro , Neoplasias Pulmonares/genéticaRESUMO
INTRODUCTION: Detection of somatic genomic alterations in the plasma of patients with cancer ("liquid biopsy") are increasingly being used in the clinic. However, the concordance of alterations identified in liquid biopsies with those detected in cancer specimens is not routinely being determined. METHODS: We sought to systematically compare alterations found by a massively parallel sequencing liquid biopsy assay covering 39 genes (NEOliquid [NEO New Oncology GmbH, Köln, Germany]) with those identified through routine diagnostic testing in a certified central pathology laboratory in a cohort of patients with nonsquamous NSCLC. NEOliquid is based on enrichment of the genomic territory of interest by hybrid capture and is thus capable of detecting point mutations, small insertions and deletions, copy number alterations, and gene rearrangements/fusions in a single assay. RESULTS: In a cohort of 82 patients with matched blood/tissue samples, the concordance between NEOliquid and tissue-based routine testing was 98%, the sensitivity of NEOliquid was higher than 70%, and the specificity was 100%. Discordant cases included those with insufficient amounts of circulaating tumor DNA in plasma and cases in which known driver mutations (e.g., isocitrate dehydrogenase (NADP(+)), 1 systolic gene [IDH1] R132H, kinesin family member 5B gene [KIF5b-ret proto-oncogene [RET], or MNNG HOS Transforming gene [MET] exon 14) were found in the plasma but were not interrogated by routine tissue analyses. CONCLUSIONS: In summary, NEOliquid offers accurate and reliable detection of clinically relevant driver alterations in plasma of patients with cancer.
Assuntos
Genômica/métodos , Biópsia Líquida/métodos , Neoplasias/sangue , Neoplasias/diagnóstico , Feminino , Humanos , Masculino , Neoplasias/genética , Proto-Oncogene MasRESUMO
Purpose: The 8p12-p11 locus is frequently amplified in squamous cell lung cancer (SQLC); the receptor tyrosine kinase fibroblast growth factor receptor 1 (FGFR1) being one of the most prominent targets of this amplification. Thus, small molecules inhibiting FGFRs have been employed to treat FGFR1-amplified SQLC. However, only about 11% of such FGFR1-amplified tumors respond to single-agent FGFR inhibition and several tumors exhibited insufficient tumor shrinkage, compatible with the existence of drug-resistant tumor cells.Experimental Design: To investigate possible mechanisms of resistance to FGFR inhibition, we studied the lung cancer cell lines DMS114 and H1581. Both cell lines are highly sensitive to three different FGFR inhibitors, but exhibit sustained residual cellular viability under treatment, indicating a subpopulation of existing drug-resistant cells. We isolated these subpopulations by treating the cells with constant high doses of FGFR inhibitors.Results: The FGFR inhibitor-resistant cells were cross-resistant and characterized by sustained MAPK pathway activation. In drug-resistant H1581 cells, we identified NRAS amplification and DUSP6 deletion, leading to MAPK pathway reactivation. Furthermore, we detected subclonal NRAS amplifications in 3 of 20 (15%) primary human FGFR1-amplified SQLC specimens. In contrast, drug-resistant DMS114 cells exhibited transcriptional upregulation of MET that drove MAPK pathway reactivation. As a consequence, we demonstrate that rational combination therapies resensitize resistant cells to treatment with FGFR inhibitors.Conclusions: We provide evidence for the existence of diverse mechanisms of primary drug resistance in FGFR1-amplified lung cancer and provide a rational strategy to improve FGFR inhibitor therapies by combination treatment. Clin Cancer Res; 23(18); 5527-36. ©2017 AACR.