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Stromal communication with cancer cells can influence treatment response. We show that stromal and breast cancer (BrCa) cells utilize paracrine and juxtacrine signaling to drive chemotherapy and radiation resistance. Upon heterotypic interaction, exosomes are transferred from stromal to BrCa cells. RNA within exosomes, which are largely noncoding transcripts and transposable elements, stimulates the pattern recognition receptor RIG-I to activate STAT1-dependent antiviral signaling. In parallel, stromal cells also activate NOTCH3 on BrCa cells. The paracrine antiviral and juxtacrine NOTCH3 pathways converge as STAT1 facilitates transcriptional responses to NOTCH3 and expands therapy-resistant tumor-initiating cells. Primary human and/or mouse BrCa analysis support the role of antiviral/NOTCH3 pathways in NOTCH signaling and stroma-mediated resistance, which is abrogated by combination therapy with gamma secretase inhibitors. Thus, stromal cells orchestrate an intricate crosstalk with BrCa cells by utilizing exosomes to instigate antiviral signaling. This expands BrCa subpopulations adept at resisting therapy and reinitiating tumor growth.
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Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/radioterapia , Exossomos/metabolismo , Comunicação Parácrina , Células Estromais/metabolismo , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Simulação por Computador , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Interferons/metabolismo , Camundongos Nus , Tolerância a Radiação , Receptores Notch/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
PURPOSE: Genome sequencing (GS) enables comprehensive molecular analysis of tumors and identification of hereditary cancer predisposition. According to guidelines, directly determining pathogenic germline variants (PGVs) requires pretest genetic counseling, which is cost-ineffective. Referral for genetic counseling based on tumor variants alone could miss relevant PGVs and/or result in unnecessary referrals. METHODS: We validated GS for detection of germline variants and simulated 3 strategies using paired tumor-normal GS data of 937 metastatic patients. In strategy-1, genetic counseling before tumor testing allowed direct PGV analysis. In strategy-2 and -3, germline testing and referral for post-test genetic counseling is based on tumor variants using Dutch (strategy-2) or Europen Society for Medical Oncology (ESMO) Precision Medicine Working Group (strategy-3) guidelines. RESULTS: In strategy-1, PGVs would be detected in 50 patients (number-needed-to counsel; NTC = 18.7). In strategy-2, 86 patients would have been referred for genetic counseling and 43 would have PGVs (NTC = 2). In strategy-3, 94 patients would have been referred for genetic counseling and 32 would have PGVs (NTC = 2.9). Hence, 43 and 62 patients, respectively, were unnecessarily referred based on a somatic variant. CONCLUSION: Both post-tumor test counseling strategies (2 and 3) had significantly lower NTC, and strategy-2 had the highest PGV yield. Combining pre-tumor test mainstreaming and post-tumor test counseling may maximize the clinically relevant PGV yield and minimize unnecessary referrals.
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Aconselhamento Genético , Neoplasias , Humanos , Testes Genéticos , Carga de Trabalho , Neoplasias/diagnóstico , Neoplasias/genética , Predisposição Genética para Doença , Mutação em Linhagem Germinativa/genéticaRESUMO
Large-scale sequencing studies are rapidly identifying putative oncogenic mutations in human tumors. However, discrimination between passenger and driver events in tumorigenesis remains challenging and requires in vivo validation studies in reliable animal models of human cancer. In this study, we describe a novel strategy for in vivo validation of candidate tumor suppressors implicated in invasive lobular breast carcinoma (ILC), which is hallmarked by loss of the cell-cell adhesion molecule E-cadherin. We describe an approach to model ILC by intraductal injection of lentiviral vectors encoding Cre recombinase, the CRISPR/Cas9 system, or both in female mice carrying conditional alleles of the Cdh1 gene, encoding for E-cadherin. Using this approach, we were able to target ILC-initiating cells and induce specific gene disruption of Pten by CRISPR/Cas9-mediated somatic gene editing. Whereas intraductal injection of Cas9-encoding lentiviruses induced Cas9-specific immune responses and development of tumors that did not resemble ILC, lentiviral delivery of a Pten targeting single-guide RNA (sgRNA) in mice with mammary gland-specific loss of E-cadherin and expression of Cas9 efficiently induced ILC development. This versatile platform can be used for rapid in vivo testing of putative tumor suppressor genes implicated in ILC, providing new opportunities for modeling invasive lobular breast carcinoma in mice.
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Neoplasias da Mama/genética , Neoplasias da Mama/fisiopatologia , Carcinoma Lobular/genética , Carcinoma Lobular/fisiopatologia , Edição de Genes , Glândulas Mamárias Humanas/fisiopatologia , Animais , Sistemas CRISPR-Cas , Caderinas/genética , Modelos Animais de Doenças , Feminino , Inativação Gênica , Genes Supressores de Tumor , Humanos , CamundongosRESUMO
Acquired mutations or altered gene expression patterns in brain metastases (BM) and/or leptomeningeal metastases (LM) of breast cancer may play a role in therapy-resistance and offer new molecular targets and treatment options. Despite expanding knowledge of genetic alterations in breast cancer and their metastases, clinical applications for patients with central nervous system (CNS) metastases are currently limited. An emerging tool are DNA-techniques that may detect genetic alterations of the CNS metastases in the cerebrospinal fluid (CSF). In this review we discuss genetic studies in breast cancer and CNS metastases and the role of liquid biopsies in CSF.
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Neoplasias Encefálicas , Neoplasias da Mama , Neoplasias do Sistema Nervoso Central , Humanos , Feminino , Neoplasias da Mama/patologia , Neoplasias do Sistema Nervoso Central/genética , Neoplasias do Sistema Nervoso Central/terapia , Neoplasias do Sistema Nervoso Central/líquido cefalorraquidiano , Biópsia Líquida/métodos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/líquido cefalorraquidiano , MutaçãoRESUMO
BACKGROUND: Treatment strategies inhibiting BRAF in combination with EGFR have been developed in patients with BRAFV600E mutant metastatic colorectal cancer, but intrinsic and secondary resistance remains a challenge. We aimed to investigate which genetic alterations cause intrinsic non-response and/or acquired resistance in these patients receiving therapies consisting of a backbone of BRAF and EGFR inhibition. METHODS: This was a cohort study on genetic alterations in patients with BRAFV600E mutant advanced colorectal cancer treated with inhibitors of the MAPK pathway. We examined tumour tissue for genetic alterations at baseline, during treatment and at progression. RESULTS: In total, 37 patients were included in this cohort. Genetic alterations in EGFR and in PIK3CA are associated with non-response. A greater fraction of non-responders (75%) versus responders (46%) had at least one genetic alteration in other genes than TP53, APC or BRAF. Secondary resistance mutations (n = 16 patients) were observed most frequently in the PI3K pathway (n = 6) and in receptor tyrosine kinases (n = 4), leading to increased upstream signalling. CONCLUSIONS: Genetic alterations in the PI3K and upstream receptor tyrosine kinases were mostly associated with intrinsic and acquired resistance. By understanding these alterations, simultaneous or alternating treatments with targeted inhibitors might improve response duration.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/genética , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Resistencia a Medicamentos Antineoplásicos/genética , Adulto , Idoso , Benzimidazóis/administração & dosagem , Carbamatos/administração & dosagem , Cetuximab/administração & dosagem , Estudos de Coortes , Neoplasias Colorretais/patologia , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Estudos Retrospectivos , Sulfonamidas/administração & dosagem , Tiazóis/administração & dosagemRESUMO
BACKGROUND: Molecular tumor boards (MTBs) provide rational, genomics-driven, patient-tailored treatment recommendations. Worldwide, MTBs differ in terms of scope, composition, methods, and recommendations. This study aimed to assess differences in methods and agreement in treatment recommendations among MTBs from tertiary cancer referral centers in The Netherlands. MATERIALS AND METHODS: MTBs from all tertiary cancer referral centers in The Netherlands were invited to participate. A survey assessing scope, value, logistics, composition, decision-making method, reporting, and registration of the MTBs was completed through on-site interviews with members from each MTB. Targeted therapy recommendations were compared using 10 anonymized cases. Participating MTBs were asked to provide a treatment recommendation in accordance with their own methods. Agreement was based on which molecular alteration(s) was considered actionable with the next line of targeted therapy. RESULTS: Interviews with 24 members of eight MTBs revealed that all participating MTBs focused on rare or complex mutational cancer profiles, operated independently of cancer type-specific multidisciplinary teams, and consisted of at least (thoracic and/or medical) oncologists, pathologists, and clinical scientists in molecular pathology. Differences were the types of cancer discussed and the methods used to achieve a recommendation. Nevertheless, agreement among MTB recommendations, based on identified actionable molecular alteration(s), was high for the 10 evaluated cases (86%). CONCLUSION: MTBs associated with tertiary cancer referral centers in The Netherlands are similar in setup and reach a high agreement in recommendations for rare or complex mutational cancer profiles. We propose a "Dutch MTB model" for an optimal, collaborative, and nationally aligned MTB workflow. IMPLICATIONS FOR PRACTICE: Interpretation of genomic analyses for optimal choice of target therapy for patients with cancer is becoming increasingly complex. A molecular tumor board (MTB) supports oncologists in rationalizing therapy options. However, there is no consensus on the most optimal setup for an MTB, which can affect the quality of recommendations. This study reveals that the eight MTBs associated with tertiary cancer referral centers in The Netherlands are similar in setup and reach a high agreement in recommendations for rare or complex mutational profiles. The Dutch MTB model is based on a collaborative and nationally aligned workflow with interinstitutional collaboration and data sharing.
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Neoplasias , Médicos , Genômica , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Países Baixos , Patologia MolecularRESUMO
Tumour metastasis suppressors are inhibitors of metastasis but their mechanisms of action are generally not understood. We previously showed that the suppressor Raf kinase inhibitory protein (RKIP) inhibits breast tumour metastasis in part via let-7. Here, we demonstrate an integrated approach combining statistical analysis of breast tumour gene expression data and experimental validation to extend the signalling pathway for RKIP. We show that RKIP inhibits let-7 targets (HMGA2, BACH1) that in turn upregulate bone metastasis genes (MMP1, OPN, CXCR4). Our results reveal BACH1 as a novel let-7-regulated transcription factor that induces matrix metalloproteinase1 (MMP1) expression and promotes metastasis. An RKIP pathway metastasis signature (designated RPMS) derived from the complete signalling cascade predicts high metastatic risk better than the individual genes. These results highlight a powerful approach for identifying signalling pathways downstream of a key metastasis suppressor and indicate that analysis of genes in the context of their signalling environment is critical for understanding their predictive and therapeutic potential.
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Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Carcinoma/diagnóstico , Carcinoma/genética , MicroRNAs/fisiologia , Proteína de Ligação a Fosfatidiletanolamina/fisiologia , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/fisiologia , Neoplasias da Mama/patologia , Carcinoma/patologia , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Análise em Microsséries , Modelos Biológicos , Metástase Neoplásica , Proteína de Ligação a Fosfatidiletanolamina/genética , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Prognóstico , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Detection of peritoneal dissemination (PD) in gastric cancer (GC) patients remains challenging. The feasibility of tumor-guided cell-free DNA (cfDNA) detection in prospectively collected peritoneal fluid (ascites and peritoneal lavage) was investigated and compared to conventional cytology in 28 patients. Besides conventional cytology, next generation sequencing was performed on primary tumor DNA and cell-free DNA from peritoneal fluid. Patients were retrospectively grouped into: a positive group (with PD) and a negative group (without PD). Detectable mutations were found in the primary tumor of 68% (n = 19). Sensitivity of PD detection by tumor-guided cfDNA analysis was 91%, compared to 64% by conventional cytology. Within the positive group (n = 11), tumor-guided cfDNA was detected in all patients with ascites samples (4/4, 100%) and in 86% (6/7) of the lavage samples, opposed to 4/4 (100%) patients with ascites and 43% (3/7) with lavage by conventional cytology. Within the negative group (n = 8), conventional cytology was negative for all samples. In two patients, tumor-guided cfDNA was detected in peritoneal lavage fluid. Interestingly, these 2 patients developed PD within 6 months, suggesting a prognostic value of tumor-guided cfDNA detection. This study showed that tumor-guided cfDNA detection in peritoneal fluids of GC patients is feasible and superior to conventional cytology in detecting PD.
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Líquido Ascítico , Ácidos Nucleicos Livres , Neoplasias Peritoneais , Neoplasias Gástricas , Humanos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Neoplasias Gástricas/diagnóstico , Neoplasias Peritoneais/secundário , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/diagnóstico , Feminino , Líquido Ascítico/patologia , Líquido Ascítico/metabolismo , Masculino , Pessoa de Meia-Idade , Idoso , Ácidos Nucleicos Livres/genética , Estudos Retrospectivos , DNA Tumoral Circulante/genética , Adulto , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Biomarcadores Tumorais/genética , Ascite/genética , Ascite/patologia , Ascite/diagnóstico , Mutação , Idoso de 80 Anos ou mais , Lavagem Peritoneal , DNA de Neoplasias/genética , DNA de Neoplasias/análiseRESUMO
BACKGROUND: Diagnosis and treatment of leptomeningeal metastases (LM) in epidermal growth factor receptor mutation positive (EGFRm+) NSCLC is challenging. We aimed to identify resistance mechanisms (RM) to osimertinib in cerebrospinal fluid (CSF) and plasma. METHODS: EGFRm+ patients with new or progressive LM during osimertinib were enrolled. NGS Ampliseq was performed on DNA isolated from CSF. Patients were prescribed osimertinib dose escalation (DE, 160mg QD) following lumbar puncture. Clinical and radiological response was evaluated four weeks after osimertinib DE. RESULTS: Twenty-eight patients were included. The driver mutation was identified in 93% of CSF samples (n=26). Seven (27%) harbored ≥1 RM. Twenty-five patients (89%) were prescribed osimertinib DE. Four weeks afterwards, symptoms improved in five patients, stabilized in nine and worsened in eleven patients. Twenty-one (84%) patients underwent MR imaging. Four showed radiological improvement, fourteen stabilization, and three worsening. CONCLUSIONS: In 27% of patients an RM was found in CSF ctDNA, none of which are targetable at time of writing, and clinical efficacy of osimertinib DE seems limited. There is much to gain in diagnostic as well as therapeutic strategies in EGFRm+ NSCLC LM.
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Two decades after the genomics revolution, oncology is rapidly transforming into a genome-driven discipline, yet routine cancer diagnostics is still mainly microscopy based, except for tumor type-specific predictive molecular tests. Pathology laboratories struggle to quickly validate and adopt biomarkers identified by genomics studies of new targeted therapies. Consequently, clinical implementation of newly approved biomarkers suffers substantial delays, leading to unequal patient access to these therapies. Whole-genome sequencing (WGS) can successfully address these challenges by providing a stable molecular diagnostic platform that allows detection of a multitude of genomic alterations in a single cost-efficient assay and facilitating rapid implementation, as well as by the development of new genomic biomarkers. Recently, the Whole-genome sequencing Implementation in standard Diagnostics for Every cancer patient (WIDE) study demonstrated that WGS is a feasible and clinically valid technique in routine clinical practice with a turnaround time of 11 workdays. As a result, WGS was successfully implemented at the Netherlands Cancer Institute as part of routine diagnostics in January 2021. The success of implementing WGS has relied on adhering to a comprehensive protocol including recording patient information, sample collection, shipment and storage logistics, sequencing data interpretation and reporting, integration into clinical decision-making and data usage. This protocol describes the use of fresh-frozen samples that are necessary for WGS but can be challenging to implement in pathology laboratories accustomed to using formalin-fixed paraffin-embedded samples. In addition, the protocol outlines key considerations to guide uptake of WGS in routine clinical care in hospitals worldwide.
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Neoplasias , Humanos , Fluxo de Trabalho , Sequenciamento Completo do Genoma/métodos , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/patologia , Genômica , BiomarcadoresRESUMO
Cancer-associated fibroblasts (CAFs) are abundantly present in the microenvironment of virtually all tumors and strongly impact tumor progression. Despite increasing insight into their function and heterogeneity, little is known regarding the origin of CAFs. Understanding the origin of CAF heterogeneity is needed to develop successful CAF-based targeted therapies. Through various transplantation studies in mice, we show that CAFs in both invasive lobular breast cancer and triple-negative breast cancer originate from mammary tissue-resident normal fibroblasts (NFs). Single-cell transcriptomics, in vivo and in vitro studies reveal the transition of CD26+ and CD26- NF populations into inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myCAFs), respectively. Functional co-culture experiments show that CD26+ NFs transition into pro-tumorigenic iCAFs which recruit myeloid cells in a CXCL12-dependent manner and enhance tumor cell invasion via matrix-metalloproteinase (MMP) activity. Together, our data suggest that CD26+ and CD26- NFs transform into distinct CAF subpopulations in mouse models of breast cancer.
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Neoplasias da Mama , Fibroblastos Associados a Câncer , Neoplasias de Mama Triplo Negativas , Humanos , Animais , Camundongos , Feminino , Dipeptidil Peptidase 4/genética , Fibroblastos , Fibroblastos Associados a Câncer/patologia , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Miofibroblastos/patologia , Microambiente Tumoral , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular TumoralRESUMO
INTRODUCTION: With the approval of first-line osimertinib treatment in stage IV EGFR-mutated NSCLC, detection of resistance mechanisms will become increasingly important-and complex. Clear guidelines for analyses of these resistance mechanisms are currently lacking. Here, we provide our recommendations for optimal molecular diagnostics in the post-EGFR tyrosine kinase inhibitor (TKI) resistance setting. METHODS: We compared molecular workup strategies from three hospitals of 161 first- or second-generation EGFR TKI-treated cases and 159 osimertinib-treated cases. Laboratories used combinations of DNA next-generation sequencing (NGS), RNA NGS, in situ hybridization (ISH), and immunohistochemistry (IHC). RESULTS: Resistance mechanisms were identified in 72 first-generation TKI cases (51%) and 85 osimertinib cases (57%). RNA NGS, when performed, revealed fusions or exon-skipping events in 4% of early TKI cases and 10% of osimertinib cases. Of the 30 MET and HER2 amplifications, 10 were exclusively detected by ISH or IHC, and not detected by DNA NGS, mostly owing to low tumor cell percentage (<30%) and possibly tumor heterogeneity. CONCLUSIONS: Our real-world data support a method for molecular diagnostics, consisting of a parallel combination of DNA NGS, RNA NGS, MET ISH, and either HER2 ISH or IHC. Combining RNA and DNA isolation into one step limits dropout rates. In case of financial or tissue limitations, a sequential approach is justifiable, in which RNA NGS is only performed in case no resistance mechanisms are identified. Yet, this is suboptimal as-although rare-multiple acquired resistance mechanisms may occur.
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Whole genome sequencing (WGS) using fresh-frozen tissue and matched blood samples from cancer patients may become the most complete genetic tumor test. With the increasing availability of small biopsies and the need to screen more number of biomarkers, the use of a single all-inclusive test is preferable over multiple consecutive assays. To meet high-quality diagnostics standards, we optimized and clinically validated WGS sample and data processing procedures, resulting in a technical success rate of 95.6% for fresh-frozen samples with sufficient (≥20%) tumor content. Independent validation of identified biomarkers against commonly used diagnostic assays showed a high sensitivity (recall; 98.5%) and precision (positive predictive value; 97.8%) for detection of somatic single-nucleotide variants and insertions and deletions (across 22 genes), and high concordance for detection of gene amplification (97.0%; EGFR and MET) as well as somatic complete loss (100%; CDKN2A/p16). Gene fusion analysis showed a concordance of 91.3% between DNA-based WGS and an orthogonal RNA-based gene fusion assay. Microsatellite (in)stability assessment showed a sensitivity of 100% with a precision of 94%, and virus detection (human papillomavirus), an accuracy of 100% compared with standard testing. In conclusion, whole genome sequencing has a >95% sensitivity and precision compared with routinely used DNA techniques in diagnostics, and all relevant mutation types can be detected reliably in a single assay.
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Alphapapillomavirus/genética , Neoplasias/diagnóstico , Neoplasias/genética , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/genética , Sequenciamento Completo do Genoma/métodos , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Variações do Número de Cópias de DNA , DNA Viral/genética , DNA Viral/isolamento & purificação , Confiabilidade dos Dados , Amplificação de Genes , Humanos , Mutação INDEL , Instabilidade de Microssatélites , Neoplasias/sangue , Neoplasias/patologia , Infecções por Papillomavirus/virologia , Polimorfismo de Nucleotídeo Único , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Estudos RetrospectivosRESUMO
INTRODUCTION: RET gene fusions are established oncogenic drivers in 1% of NSCLC. Accurate detection of advanced patients with RET fusions is essential to ensure optimal therapy choice. We investigated the performance of fluorescence in situ hybridization (FISH) as a diagnostic test for detecting functional RET fusions. METHODS: Between January 2016 and November 2019, a total of 4873 patients with NSCLC were routinely screened for RET fusions using either FISH (n = 2858) or targeted RNA next-generation sequencing (NGS) (n = 2015). If sufficient material was available, positive cases were analyzed by both methods (n = 39) and multiple FISH assays (n = 17). In an independent cohort of 520 patients with NSCLC, whole-genome sequencing data were investigated for disruptive structural variations and functional fusions in the RET and compared with ALK and ROS1 loci. RESULTS: FISH analysis revealed RET rearrangement in 48 of 2858 cases; of 30 rearranged cases double tested with NGS, only nine had a functional RET fusion. RNA NGS yielded RET fusions in 14 of 2015 cases; all nine cases double tested by FISH had RET locus rearrangement. Of these 18 verified RET fusion cases, 16 had a split signal and two a complex rearrangement by FISH. By whole-genome sequencing, the prevalence of functional fusions compared with all disruptive events was lower in the RET (4 of 9, 44%) than the ALK (27 of 34, 79%) and ROS1 (9 of 12, 75%) loci. CONCLUSIONS: FISH is a sensitive but unspecific technique for RET screening, always requiring a confirmation using an orthogonal technique, owing to frequently occurring RET rearrangements not resulting in functional fusions in NSCLC.
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Neoplasias Pulmonares , Proteínas Tirosina Quinases , Quinase do Linfoma Anaplásico/genética , Detecção Precoce de Câncer , Rearranjo Gênico , Humanos , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ret/genéticaRESUMO
Cigarette smoking is the main risk factor for the development of squamous cell lung carcinoma (SCC). However, the smoking-related molecular changes in SCC have not been studied. Gene expression studies in both histologically normal bronchial epithelium and SCC epithelial samples identified genes differentially expressed between current and ex-smokers. Subsequently, expression levels of the smoking-related genes in normal bronchial epithelium were compared with those in SCC cells, since we hypothesized that the smoking-induced changes would be also deregulated in SCC. Gene expression profiles were generated using Agilent whole human genome microarrays on laser-microdissected normal bronchial epithelium and SCC samples. Expression levels of 246 genes, mainly related to oxidative stress response, were significantly different between normal bronchial epithelium of current and ex-smokers. Such a differential gene expression profile did not exist in SCC cells of smokers and ex-smokers. Interestingly, when comparing SCC and normal bronchial epithelium from ex-smokers, the vast majority of these 246 genes were also deregulated in SCC. When comparing SCC with normal epithelium from smokers, 22% of the up-regulated genes showed a similar high expression in SCC whereas 79% of the down-regulated genes were even further reduced in SCC as compared to current smokers. The down-regulated genes included several tumour suppressor genes, such as C9orf9, INHBB, LRIG1, SCGB3A1, SERPINI2, STEAP3 and ZMYND10. Thus, our study shows that the majority of genes up-regulated in normal bronchial epithelium of current smokers show similar high expression levels in SCC, while down-regulated genes are even further repressed in SCC. Our data indicate that smoking-related changes in normal bronchial epithelial cells persist in malignant transformed squamous cells.
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Brônquios/metabolismo , Carcinoma de Células Escamosas/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Mucosa Respiratória/metabolismo , Fumar/efeitos adversos , Idoso , Estudos de Casos e Controles , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Abandono do Hábito de FumarRESUMO
BACKGROUND: Laser microdissection microscopy has become a rising tool to assess gene expression profiles of pure cell populations. Given the low yield of RNA, a second round of amplification is usually mandatory to yield sufficient amplified-RNA for microarray approaches. Since amplification induces truncation of RNA molecules, we studied the impact of a second round of amplification on identification of differentially expressed genes in relation to the probe - poly(A)-tail distances. RESULTS: Disagreement was observed between gene expression profiles acquired after a second round of amplification compared to a single round. Thirty percent of the differentially expressed genes identified after one round of amplification were not detected after two rounds. These inconsistent genes have a significant longer probe - poly(A)-tail distance. qRT-PCR on unamplified RNA confirmed differential expression of genes with a probe - poly(A)-tail distance >500 nucleotides appearing only after one round of amplification. CONCLUSION: Our data demonstrate a marked loss of 30% of truly differentially expressed genes after a second round of amplification. Therefore, we strongly recommend improvement of amplification procedures and importance of microarray probe design to allow detection of all differentially expressed genes in case of limited amounts of RNA.
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Artefatos , Perfilação da Expressão Gênica , Técnicas de Amplificação de Ácido Nucleico/métodos , Sondas de Ácido Nucleico/química , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Poli A/metabolismo , Humanos , Análise de Componente Principal , RNA Mensageiro/química , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Processamento de Sinais Assistido por Computador , Células Tumorais CultivadasRESUMO
Invasive lobular carcinoma (ILC) is an aggressive breast cancer subtype with poor response to chemotherapy. Besides loss of E-cadherin, a hallmark of ILC, genetic inactivation of PTEN is frequently observed in patients. Through concomitant Cre-mediated inactivation of E-cadherin and PTEN in mammary epithelium, we generated a mouse model of classical ILC (CLC), the main histological ILC subtype. While loss of E-cadherin induced cell dissemination and apoptosis, additional PTEN inactivation promoted cell survival and rapid formation of invasive mammary tumors that recapitulate the histological and molecular features, estrogen receptor (ER) status, growth kinetics, metastatic behavior, and tumor microenvironment of human CLC. Combined inactivation of E-cadherin and PTEN is sufficient to cause CLC development. These CLCs showed significant tumor regression upon BEZ235-mediated inhibition of PI3K signaling. In summary, this mouse model provides important insights into CLC development and suggests inhibition of phosphatidylinositol 3-kinase (PI3K) signaling as a potential therapeutic strategy for targeting CLC.
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Caderinas/genética , Carcinoma Lobular/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Neoplasias Mamárias Experimentais/genética , PTEN Fosfo-Hidrolase/genética , Inibidores de Fosfoinositídeo-3 Quinase , Animais , Antineoplásicos/farmacologia , Caderinas/deficiência , Carcinoma Lobular/tratamento farmacológico , Carcinoma Lobular/mortalidade , Carcinoma Lobular/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Perfilação da Expressão Gênica , Imidazóis/farmacologia , Integrases/genética , Integrases/metabolismo , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/mortalidade , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Knockout , Invasividade Neoplásica , PTEN Fosfo-Hidrolase/deficiência , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Quinolinas/farmacologia , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Transdução de Sinais , Análise de Sobrevida , Microambiente TumoralRESUMO
The epidemiological relationship between squamous cell lung cancer (SCC) and chronic obstructive pulmonary disease (COPD), both smoking-related diseases, suggests that they have also a genetic basis. We compared 35 SCC patients with and without COPD with whole-genome gene expression profiles of laser microdissected tissue. Validation of differential expression was performed for 25 genes using quantitative (q)RT-PCR. Subsequently, we performed array-based CGH on the same tumor samples. We found that 374 probes were differentially expressed in SCC from patients with and without COPD. Forty-four probes were derived from genes with mitochondrial functions and 34 probes were located on 5q. All these probes showed a lower expression level in SCC from non-COPD patients. For a random selection of 25 mitochondrial and 5q genes, we confirmed the differential expression by qRT-PCR. Loss of 3p, 5q and 9p was observed, via array-CGH, to be more frequent in SCC from non-COPD patients as compared to SCC from COPD patients. Combination of chromosomal aberrations and the location of the differentially expressed genes showed significant association for loss of the 5q31.2-31.3 region and reduced expression of the 5q genes. In conclusion, a more frequent loss of 5q and a low expression of genes located on 5q in SCC tumors of non-COPD patients compared to tumors from COPD patients was identified suggesting that different oncogenetic pathways are operational in patients with and without COPD.
Assuntos
Carcinoma de Células Escamosas/genética , Aberrações Cromossômicas , Cromossomos Humanos Par 5 , Neoplasias Pulmonares/genética , Doença Pulmonar Obstrutiva Crônica/genética , Idoso , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/etiologia , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/fisiopatologia , Cromossomos Humanos Par 5/genética , Regulação para Baixo/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/fisiopatologia , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Mitocôndrias/genética , Estadiamento de Neoplasias , Doença Pulmonar Obstrutiva Crônica/complicações , Doença Pulmonar Obstrutiva Crônica/patologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologiaRESUMO
About 50% of patients presenting with resectable lung cancer develop distant metastases within 5 years. Genomic markers predicting metastatic behaviour of squamous cell lung carcinoma (SCC) are currently underexposed. We analyzed a cohort of patients with primary SCC using array-based comparative genomic hybridization (aCGH) to identify which genomic aberrations are related to metastatic behaviour. The cohort consisted of 34 patients with a follow-up of at least 5 years, 8 with metastases in regional lymph nodes only and 26 patients without any metastases at the time of surgery. Eleven of the latter 26 developed metastases in distant organs within 3 years after surgery. Copy number changes observed in at least 40% of all SCC included gains at chromosomal arms 3q, 5p, 8q, 19q, 20p, 22q and losses at 3p, 4p, 4q, 5q, 8p and 9p. High copy number amplifications were observed at 2p15-p16, 3q24-q29, 8p11-p12, 8q23-q24, and 12p12, containing candidate oncogenes such as BCL11A, REL, ECT2, PIK3CA, ADAM9, MYC and KRAS. Amplification of 2p15-p16 is a novel finding in SCC. Another novel finding is the homozygous deletion observed at 4q33-34.1 in 15% of the SCC cases. Gains at 7q36, 8p12, 10q22, 12p12, loss at 4p14 and the homozygous deletions at 4q occurred significantly more frequent in SCC from patients with lymph node metastases only. SCC from patients with distant metastases showed a significantly higher gain frequency at 8q22-q24 and loss at 8p23 and 13q21, and a significantly lower gain frequency at 2p12 and 2p16 and loss at 11q25 compared with SCC from patients without metastases. Of these, gains at 7q, 8p and 10q were restricted to SCC with lymph node metastasis and gain at 8q was restricted to patients with distant metastasis. Two genomic aberrations, i.e. loss of 4p and gain of 19q12 were observed more frequently in SCC with only lymph node metastases as compared to SCC with distant metastases. In conclusion, we identified genomic aberrations in primary SCC that were related to lymph node or distant metastases.
Assuntos
Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/genética , Aberrações Cromossômicas , Marcadores Genéticos/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Metástase Linfática , Idoso , Idoso de 80 Anos ou mais , Carcinógenos , Carcinoma de Células Escamosas/patologia , Hibridização Genômica Comparativa , Feminino , Seguimentos , Amplificação de Genes , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
To improve current angiogenic gene therapy with a vascular endothelial growth factor (VEGF)-encoding plasmid (Baumgartner et al. Circulation 1998; 97: 1114-23 [1]; Kusumanto et al. Fifth Annual Meeting of the American Society of Gene Therapy, Boston, 2002, Abstr. 621 [2]), we have generated a combination plasmid, encoding the VEGF gene and the thymidine phosphorylase (TP, also known as platelet-derived endothelial growth factor (PD-ECGF) or gliostatin (GLS)) gene: phVEGF165-TP.MB. Upon transfection in COS-7 cells both gene products were expressed and functional as shown by Western blots, ELISAs and bioassays. Culture supernatants of COS-7 cells transfected with this plasmid were able to induce endothelial proliferation. In an in vitro angiogenesis assay with recombinant proteins, TP was able to increase VEGF-induced tube formation. The phVEGF165-TP.MB plasmid is therefore a promising candidate for in vivo angiogenesis studies.