RESUMO
The mechanisms underlying tumor dormancy have been elusive and not well characterized. We recently published an experimental model for the study of human tumor dormancy and the role of angiogenesis, and reported that the angiogenic switch was preceded by a local increase in VEGF-A and basic fibroblast growth factor. In this breast cancer xenograft model (MDA-MB-436 cells), analysis of differentially expressed genes revealed that heat shock protein 27 (HSP27) was significantly up-regulated in angiogenic cells compared with nonangiogenic cells. The effect of HSP27 down-regulation was further evaluated in cell lines, mouse models, and clinical datasets of human patients with breast cancer and melanoma. Stable down-regulation of HSP27 in angiogenic tumor cells was followed by long-term tumor dormancy in vivo. Strikingly, only 4 of 30 HSP27 knockdown xenograft tumors initiated rapid growth after day 70, in correlation with a regain of HSP27 protein expression. Significantly, no tumors escaped from dormancy without HSP27 expression. Down-regulation of HSP27 was associated with reduced endothelial cell proliferation and decreased secretion of VEGF-A, VEGF-C, and basic fibroblast growth factor. Conversely, overexpression of HSP27 in nonangiogenic cells resulted in expansive tumor growth in vivo. By clinical validation, strong HSP27 protein expression was associated with markers of aggressive tumors and decreased survival in patients with breast cancer and melanoma. An HSP27-associated gene expression signature was related to molecular subgroups and survival in breast cancer. Our findings suggest a role for HSP27 in the balance between tumor dormancy and tumor progression, mediated by tumor-vascular interactions. Targeting HSP27 might offer a useful strategy in cancer treatment.
Assuntos
Neoplasias da Mama/metabolismo , Regulação para Baixo , Proteínas de Choque Térmico HSP27/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Animais , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Feminino , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP27/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos SCID , Neovascularização Patológica/genética , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transplante Heterólogo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/metabolismoRESUMO
Although urothelial carcinoma (UC) of the urinary bladder generally portends a favorable prognosis, metastatic tumors often follow an aggressive clinical course. DNA was extracted from 40 µm of formalin-fixed, paraffin-embedded (FFPE) sections from 35 stage IV UCs that had relapsed and progressed after primary surgery and conventional chemotherapy. Next-generation sequencing (NGS) was performed on hybridization-captured, adaptor ligation-based libraries for 3320 exons of 182 cancer-related genes plus 37 introns from 14 genes frequently rearranged in cancer to at an average sequencing depth of 1164 × and evaluated for all classes of genomic alterations (GAs). Actionable GAs were defined as those impacting the selection of targeted anticancer therapies on the market or in registered clinical trials. A total of 139 GAs were identified, with an average of 4.0 GAs per tumor (range 0-10), of which 78 (56%) were considered actionable, with an average of 2.2 per tumor (range 0-7). Twenty-nine (83%) cases harbored at least one actionable GA including: PIK3CA (9 cases; 26%); CDKN2A/B (8 cases; 23%); CCND1 (5 cases; 14%); FGFR1 (5 cases; 14%); CCND3 (4 cases; 11%); FGFR3 (4 cases; 11%); MCL1 (4 cases; 11%); MDM2 (4 cases; 11%); EGFR (2 cases, 6%); ERBB2 (HER2/neu) (2 cases, 6%); NF1 (2 cases, 6%) and TSC1 (2 cases, 6%). Notable additional alterations included TP53 (19 cases, 54%) and RB1 (6 cases; 17%). Genes involved in chromatin modification were altered by nonsense mutation, splice site mutation or frameshift indel in a mutually exclusive manner in nearly half of all cases including KDM6A (10 cases; 29%) and ARID1A (7 cases; 20%). Comprehensive NGS of 35 UCs of the bladder revealed a diverse spectrum of actionable GAs in 83% of cases, which has the potential to inform treatment decisions for patients with relapsed and metastatic disease.
Assuntos
Carcinoma de Células de Transição/genética , Neoplasias da Bexiga Urinária/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência de DNARESUMO
The 17th Workshop on Recent Issues in Bioanalysis (17th WRIB) took place in Orlando, FL, USA on 19-23 June 2023. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 17th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines.Moreover, in-depth workshops on "EU IVDR 2017/746 Implementation and impact for the Global Biomarker Community: How to Comply with these NEW Regulations" and on "US FDA/OSIS Remote Regulatory Assessments (RRAs)" were the special features of the 17th edition.As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues.This 2023 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2023 edition of this comprehensive White Paper has been divided into three parts for editorial reasons.This publication (Part 2) covers the recommendations on Biomarkers, IVD/CDx, LBA and Cell-Based Assays. Part 1A (Mass Spectrometry Assays and Regulated Bioanalysis/BMV), P1B (Regulatory Inputs) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 16 of Bioanalysis, issues 9 and 7 (2024), respectively.
Assuntos
Biomarcadores , Terapia Baseada em Transplante de Células e Tecidos , Vacinas , Humanos , Biomarcadores/análise , Vacinas/imunologia , Citometria de Fluxo , Bioensaio/métodos , União Europeia , BrancosRESUMO
The 2022 16th Workshop on Recent Issues in Bioanalysis (WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity. Part 1 (Mass Spectrometry and ICH M10) and Part 2 (LBA, Biomarkers/CDx and Cytometry) are published in volume 15 of Bioanalysis, issues 16 and 15 (2023), respectively.
Assuntos
Medicamentos sob Prescrição , Tecnologia , Bioensaio/métodos , Biomarcadores/análise , Terapia Baseada em Transplante de Células e TecidosRESUMO
This phase 1 study evaluated the safety and tolerability of antiangiogenic therapy using vandetanib and metronomic cyclophosphamide and methotrexate in metastatic breast cancer. Eligible patients had metastatic breast cancer with 0-4 prior chemotherapy regimens. All received cyclophosphamide 50 mg daily, methotrexate 2.5 mg days 1-2 weekly, and vandetanib daily in 3 dose-escalation cohorts: 100 mg (C1), 200 mg (C2), and 300 mg (C3). The primary endpoint was safety and tolerability; secondary endpoints included response rate and evaluation of platelet-associated proteins. Twenty three patients were treated and evaluable for toxicity. Common mild toxicities included nausea, vomiting, LFTs abnormalities, fatigue, and rash. Three episodes of dose-limiting toxicity occurred in C3. In all cohorts, 1/3 of patients required vandetanib dose reduction, and 22 % ended therapy for toxicity. Of the 20 response-evaluable patients, 10 % demonstrated partial response and 15 % stable disease ≥24 weeks. Proteomic analyses demonstrated changes in platelet content of angiogenesis regulators, including vascular endothelial growth factor and platelet factor 4, with exposure to therapy. This regimen was tolerable at a maximum vandetanib dose of 200 mg; modest clinical activity was observed in this heavily pretreated population. Changes in the platelet proteome may serve as pharmacodynamic markers of angiogenesis inhibition. Metronomic chemotherapy is an attractive partner with biologics and deserves further study in metastatic breast cancer.
Assuntos
Plaquetas , Neoplasias da Mama/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Piperidinas/administração & dosagem , Quinazolinas/administração & dosagem , Administração Metronômica , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Biomarcadores Farmacológicos/metabolismo , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Neoplasias da Mama/patologia , Terapia Combinada , Ciclofosfamida/administração & dosagem , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/induzido quimicamente , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Feminino , Humanos , Metotrexato/administração & dosagem , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neovascularização Patológica/tratamento farmacológico , Piperidinas/efeitos adversos , Fator Plaquetário 4/metabolismo , Proteômica , Quinazolinas/efeitos adversos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
Multiplexed immunohistochemistry (mIHC) enables the detection, quantification, and localization of many markers within cell or tissue samples, leading to a better understanding of the architecture of a disease at the cellular level. Current mIHC techniques involve long staining and assay times, require dedicated and/or captive instrumentation, and entail tedious assay optimization, hindering their establishment as routine methods. Here, we demonstrate the use of the InSituPlex® method for spatial profiling of immuno-oncology targets in FFPE tumor tissue with the UltiMapper™ I/O PD-L1 multiplex assay. The panel consists of five protein markers to profile immune infiltration and PD-L1 expression and includes CD8, CD68, PD-L1, pan CK, and SOX10 markers. The assay shows benefits of high and low expression of markers, coexpression and colocalization of proteins in single cells, and completion of staining and image acquisition in 5.5 h. Through the combination of multiplexed characterization of protein expression in whole tissue sections, fast staining workflow, and compatibility with existing instrumentation, the InSituPlex method provides a robust modality for deep phenotyping of the tumor and its microenvironment.
Assuntos
Biomarcadores Tumorais/metabolismo , Perfilação da Expressão Gênica/métodos , Neoplasias/metabolismo , Coloração e Rotulagem/métodos , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Antígeno B7-H1/metabolismo , Antígenos CD8/metabolismo , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Humanos , Inclusão em Parafina , Fenótipo , Fatores de Transcrição SOXE/metabolismo , Análise Espacial , Fixação de TecidosRESUMO
Since the beginning of the COVID-19 pandemic, many therapeutic strategies have been tried, with mixed results, to prevent and treat adult multisystem inflammatory syndrome in COVID-19 (AMIS-COVID-19). The reason behind this may the complex web of highly intertwined pathophysiologic mechanisms involved in the SARS-CoV-2 infection and the corresponding human systemic response, leading to end-organ damage, disability, and death. Colchicine, high-dose aspirin, and montelukast are being investigated currently as potential modulators of AMIS-COVID-19 in patients who fail to improve with traditional therapeutic approaches. Here, we present a patient who presented with high fevers, extreme fatigue and dyspnea, and ongoing deterioration. As part of our clinical approach, we used the simultaneous combination of the three agents listed above, capitalizing on their different respective mechanisms of action against AMIS-COVID-19. Following the initiation of therapy, the patient showed symptomatic improvement within 24 h, with the ability to return to daily activities after 72 h of continued triple-agent approach. Based on this experience, we have reviewed the immunomodulatory basis of this regimen, including potential avenues in which it may prevent the development of cytokine release syndrome (CRS) and its clinical manifestation, AMIS-COVID-19. By blocking the early stages of an inflammatory response, via diverse mechanistic pathways, the regimen in question may prove effective in halting the escalation of CRS and AMIS-COVID-19 in acutely symptomatic, nonimproving COVID-19 patients.
RESUMO
MOTIVATION: Analysis of high-throughput proteomic/genomic data, in particular, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) data and microarray data, has led to a multitude of techniques aimed at identifying potential biomarkers. Most of the statistical techniques for comparing two groups are based on qualitative measures such as P-value. A quantitative way such as interval estimation for the contrasts of two groups is more appealing. RESULTS: We have devised a simultaneous confidence bands method capable of detecting potential biomarkers, while controlling for overall confidence coverage level, in high-dimensional datasets that discriminate two treatment groups using a permutation scheme. For example, for the SELDI-TOF MS data, we deal with the entire spectrum simultaneously and construct (1 - alpha) confidence bands for the mean differences between groups. Furthermore, peaks were identified based on the maximal differences between the groups as determined by the confidence bands. The analysis method herein described gives both qualitative (P-value) and quantitative data (magnitude of difference). The Clinical Proteomics Programs Databank's ovarian cancer dataset and data from in-house samples containing known spiked-in proteins were analyzed. We were able to identify potential biomarkers similar to those described in previous analysis of the ovarian cancer data, however, while these markers are highly significant between cancer and normal groups, our analysis indicated the absolute difference between the two groups was minimal. In addition, we found additional markers than those previously described with greater differences in average intensities. The proposed confidence bands method successfully detected the spiked-in peaks, as well as, secondary peaks generated by adducts and double-charged species. We also illustrate our method utilizing paired gene expression data from a prostate cancer microarray experiment by constructing confidence bands for the fold changes between cancer and normal samples. AVAILABILITY: R-package, 'seie.zip' (license: GNU GPL), is publiclly available at http://research2.dfci.harvard.edu/dfci/MS_spike-in_data/
Assuntos
Artefatos , Biomarcadores Tumorais/análise , Biologia Computacional/métodos , Espectrometria de Massas/métodos , Proteômica/métodos , Algoritmos , Biomarcadores/análise , Feminino , Humanos , Masculino , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/patologia , Neoplasias da Próstata/sangue , Neoplasias da Próstata/patologia , Análise Serial de Proteínas , Proteoma/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Several genetic loci are suspected to be involved in hereditary prostate cancer, including the hereditary prostate cancer 1 (HPC1) locus at chromosome 1q24-25. The ribonuclease L (RNase L) gene has been reported as the putative hereditary prostate cancer gene located at HPC1. If this is the case, mutations of RNase L should be found at a greater frequency in familial cancers than in sporadic prostate cancers. Examination of familial and sporadic cases of prostate cancer by polymerase chain reaction and DNA sequencing resulted in a mutational frequency rate that was not statistically different between the 2 forms of the disease. These results suggest that the mutations examined within this study are rare and may contribute to very few familial prostate cancers.
Assuntos
Mutação , Neoplasias da Próstata/genética , Ribonucleases/genética , Análise Mutacional de DNA , Frequência do Gene , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da PolimeraseRESUMO
INTRODUCTION: Mutations in the p53 tumor suppressor gene are generally believed to be a late event in the progression of prostate cancer, and are associated with androgen-independence, increased angiogenesis, metastasis, recurrence, and a worse prognosis. In this review, we examine the current literature available on p53 mutations found in prostate cancer and focus on stages A (T1) and B (T2) of the disease. The alteration of genes involved in p53 regulation are also examined, as well as animal models that support an early role for p53 in the initiation and development of prostate cancer. RESULTS: We report here that p53 mutations occur in approximately one third of early stage prostate cancers and that expression of HPV E6 or over-expression of mdm2 contributes to loss of p53 function in an additional 25% of organ-confined disease. High levels of p53 mutation are found in normal prostate tissue of prostate cancer patients and in the precursor lesion, prostatic intraepithelial neoplasia, further implicating p53 mutation or loss as an early event in prostate tumorigenesis. CONCLUSIONS: In contrast to popular opinion, p53 mutations are a common event in early stage, organ-confined prostate cancer and although more studies are needed, the loss of p53 function through expression of viral or cellular oncoproteins also appears quite common. Evidence from animal models of prostate cancer further supports the notion that loss of p53 function plays a critical role in the development of prostate cancer.
Assuntos
Genes p53/genética , Mutação/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Progressão da Doença , Expressão Gênica/genética , Humanos , Incidência , Masculino , Estadiamento de Neoplasias , Próstata/patologia , Neoplasia Prostática Intraepitelial/genética , Neoplasia Prostática Intraepitelial/patologia , Neoplasias da Próstata/epidemiologiaRESUMO
We hypothesized that next-generation sequencing could reveal actionable genomic alterations (GAs) and potentially expand treatment options for patients with advanced adenoid cystic carcinoma (ACC). Genomic profiling using next-generation sequencing was performed on hybridization-captured, adapter ligation libraries derived from 28 relapsed and metastatic formalin-fixed paraffin-embedded ACC. The 3230 exons of 182 cancer-related genes and 37 introns of 14 genes frequently rearranged in cancer were fully sequenced using the Illumina HiSeq 2000. All classes of GAs were evaluated. Actionable GAs were defined as those impacting targeted anticancer therapies on the market or in registered clinical trials. A total of 44 GAs were identified in the 28 ACC tumors, with 12 of 28 (42.9%) of tumors harboring at least 1 potentially actionable GA. The most common nonactionable GAs were identified in KD6MA (5 cases; 18%), ARID1A (4 cases; 14%), RUNX1 (2 cases; 7%), and MYC (2 cases; 7%). Actionable GAs included NOTCH1 (3 cases; 11%), MDM2 (2 cases; 7%), PDGFRA (2 cases; 7%), and CDKN2A/B (p16) (2 cases; 7%). Other potentially actionable GAs identified in a single case included: mutations in AKT1, BAP1, EGFR, and PIK3CA, homozygous deletion of FBXW7, and amplifications of CDK4, FGFR1, IGF1R, KDR, KIT, and MCL1. The frequency of GA in ACC is lower than that seen in the more common solid tumors. Comprehensive genomic profiling of ACC can identify actionable GAs in a subset of patients that could influence therapy for these difficult-to-treat progressive neoplasms.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma Adenoide Cístico/genética , Carcinoma Adenoide Cístico/secundário , Perfilação da Expressão Gênica/métodos , Testes Genéticos/métodos , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Antineoplásicos/uso terapêutico , Carcinoma Adenoide Cístico/tratamento farmacológico , Éxons , Feminino , Fixadores , Formaldeído , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Humanos , Íntrons , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Recidiva Local de Neoplasia/tratamento farmacológico , Inclusão em Parafina , Seleção de Pacientes , Fenótipo , Medicina de Precisão , Valor Preditivo dos Testes , Prognóstico , Fixação de TecidosRESUMO
PURPOSE: Micropapillary urothelial carcinoma (MPUC) is a rare and aggressive form of bladder cancer. We conducted genomic analyses [next-generation sequencing (NGS)] of MPUC and non-micropapillary urothelial bladder carcinomas (non-MPUC) to characterize the genomic landscape and identify targeted treatment options. EXPERIMENTAL DESIGN: DNA was extracted from 40 µm of formalin-fixed paraffin-embedded sections from 15 MPUC and 64 non-MPUC tumors. Sequencing (NGS) was performed on hybridization-captured, adaptor ligation-based libraries to high coverage for 3,230 exons of 182 cancer-related genes plus 37 introns from 14 genes frequently rearranged in cancer. The results were evaluated for all classes of genomic alteration. RESULTS: Mutations in the extracellular domain of ERBB2 were identified in 6 of 15 (40%) of MPUC: S310F (four cases), S310Y (one case), and R157W (one case). All six cases of MPUC with ERBB2 mutation were negative for ERBB2 amplification and Erbb2 overexpression. In contrast, 6 of 64 (9.4%) non-MPUC harbored an ERBB2 alteration, including base substitution (three cases), amplification (two cases), and gene fusion (one case), which is higher than the 2 of 159 (1.3%) protein-changing ERBB2 mutations reported for urinary tract cancer in COSMIC. The enrichment of ERBB2 alterations in MPUC compared with non-MPUC is significant both between this series (P < 0.0084) and for all types of urinary tract cancer in COSMIC (P < 0.001). CONCLUSIONS: NGS of MPUC revealed a high incidence of mutation in the extracellular domain of ERBB2, a gene for which there are five approved targeted therapies. NGS can identify genomic alteration, which inform treatment options for the majority of MPUC patients.
Assuntos
Receptor ErbB-2/genética , Neoplasias da Bexiga Urinária/genética , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Análise Mutacional de DNA , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Estrutura Terciária de Proteína , Análise de Sequência de DNARESUMO
PURPOSE: We queried whether comprehensive genomic profiling using a next-generation sequencing-based assay could identify novel and unanticipated targets of therapy for patients with relapsed invasive lobular carcinoma (ILC). EXPERIMENTAL DESIGN: DNA sequencing (Illumina HiSeq 2000) was conducted for 3,320 exons of 182 cancer-related genes and 37 introns of 14 genes frequently rearranged in cancer on indexed, adaptor-ligated, hybridization-captured libraries using DNA isolated from formalin-fixed paraffin-embedded sections from 22 histologically verified ILC. RESULTS: A total of 75 genomic alterations were identified with an average of 3.4 alterations per tumor (range, 1-6), of which 35 were actionable for an average of 1.59 actionable alterations per patient (range, 0-3). Nineteen of 22 (86%) of the ILC samples harbored at least one actionable alteration. Six (27%) cases featured alterations in ERRB2 including 4 (18%) with ERBB2 mutation, 1 (5%) with an ERBB2 gene fusion, and 1 (5%) with an ERBB2 copy number gain (amplification). The enrichment of ERBB2 mutations/fusion in CDH1-mutated ILC (5 of 22, 23%) compared with the 5 ERBB2 mutations in a series of 286 non-CDH1-mutated breast cancers from which the ILC cases were obtained (5 of 286, 2%) was significant (P = 0.0006). CONCLUSIONS: Comprehensive genomic profiling of relapsed CDH1-mutated ILC revealed actionable genomic alterations in 86% of cases, featured a high incidence of ERBB2 alterations, and can reveal actionable alterations that can inform treatment decisions for patients with ILC.
Assuntos
Neoplasias da Mama/genética , Caderinas/genética , Carcinoma Ductal de Mama/genética , Carcinoma Lobular/genética , Mutação , Receptor ErbB-2/genética , Adulto , Idoso , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caderinas/metabolismo , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/metabolismo , Carcinoma Lobular/patologia , Éxons/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Histocitoquímica , Humanos , Pessoa de Meia-Idade , Taxa de Mutação , Metástase Neoplásica , Recidiva Local de Neoplasia , Inclusão em Parafina , Receptor ErbB-2/metabolismo , Fixação de TecidosRESUMO
BACKGROUND: Most personalized cancer care strategies involving DNA sequencing are highly reliant on acquiring sufficient fresh or frozen tissue. It has been challenging to comprehensively evaluate the genome of advanced prostate cancer (PCa) because of limited access to metastatic tissue. OBJECTIVE: To demonstrate the feasibility of a novel next-generation sequencing (NGS)-based platform that can be used with archival formalin-fixed paraffin-embedded (FFPE) biopsy tissue to evaluate the spectrum of DNA alterations seen in advanced PCa. DESIGN, SETTING, AND PARTICIPANTS: FFPE samples (including archival prostatectomies and prostate needle biopsies) were obtained from 45 patients representing the spectrum of disease: localized PCa, metastatic hormone-naive PCa, and metastatic castration-resistant PCa (CRPC). We also assessed paired primaries and metastases to understand disease heterogeneity and disease progression. INTERVENTION: At least 50 ng of tumor DNA was extracted from FFPE samples and used for hybridization capture and NGS using the Illumina HiSeq 2000 platform. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: A total of 3320 exons of 182 cancer-associated genes and 37 introns of 14 commonly rearranged genes were evaluated for genomic alterations. RESULTS AND LIMITATIONS: We obtained an average sequencing depth of >900X. Overall, 44% of CRPCs harbored genomic alterations involving the androgen receptor gene (AR), including AR copy number gain (24% of CRPCs) or AR point mutation (20% of CRPCs). Other recurrent mutations included transmembrane protease, serine 2 gene (TMPRSS2):v-ets erythroblastosis virus E26 oncogene homolog (avian) gene (ERG) fusion (44%); phosphatase and tensin homolog gene (PTEN) loss (44%); tumor protein p53 gene (TP53) mutation (40%); retinoblastoma gene (RB) loss (28%); v-myc myelocytomatosis viral oncogene homolog (avian) gene (MYC) gain (12%); and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α gene (PIK3CA) mutation (4%). There was a high incidence of genomic alterations involving key genes important for DNA repair, including breast cancer 2, early onset gene (BRCA2) loss (12%) and ataxia telangiectasia mutated gene (ATM) mutations (8%); these alterations are potentially targetable with poly(adenosine diphosphate-ribose)polymerase inhibitors. A novel and actionable rearrangement involving the v-raf murine sarcoma viral oncogene homolog B1 gene (BRAF) was also detected. CONCLUSIONS: This first-in-principle study demonstrates the feasibility of performing in-depth DNA analyses using FFPE tissue and brings new insight toward understanding the genomic landscape within advanced PCa.
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Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias da Próstata/genética , Análise de Sequência de DNA/métodos , Antagonistas de Androgênios/uso terapêutico , Antineoplásicos Hormonais , Biópsia por Agulha , Resistencia a Medicamentos Antineoplásicos , Éxons , Estudos de Viabilidade , Fixadores , Formaldeído , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Humanos , Íntrons , Masculino , Gradação de Tumores , Invasividade Neoplásica , Orquiectomia , Inclusão em Parafina , Seleção de Pacientes , Fenótipo , Medicina de Precisão , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Fixação de TecidosRESUMO
As more clinically relevant cancer genes are identified, comprehensive diagnostic approaches are needed to match patients to therapies, raising the challenge of optimization and analytical validation of assays that interrogate millions of bases of cancer genomes altered by multiple mechanisms. Here we describe a test based on massively parallel DNA sequencing to characterize base substitutions, short insertions and deletions (indels), copy number alterations and selected fusions across 287 cancer-related genes from routine formalin-fixed and paraffin-embedded (FFPE) clinical specimens. We implemented a practical validation strategy with reference samples of pooled cell lines that model key determinants of accuracy, including mutant allele frequency, indel length and amplitude of copy change. Test sensitivity achieved was 95-99% across alteration types, with high specificity (positive predictive value >99%). We confirmed accuracy using 249 FFPE cancer specimens characterized by established assays. Application of the test to 2,221 clinical cases revealed clinically actionable alterations in 76% of tumors, three times the number of actionable alterations detected by current diagnostic tests.
Assuntos
Análise Mutacional de DNA/métodos , Técnicas de Diagnóstico Molecular/métodos , Neoplasias/genética , Análise de Sequência de DNA/métodos , Variações do Número de Cópias de DNA , Frequência do Gene , Humanos , Neoplasias/diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Many growth factors, leukotrines, and biological ligands are not circulating free in plasma or serum, except in the case of late or disseminated disease. During early tumor growth and angiogenesis, platelets actively and selectively sequester regulators of angiogenesis and, as such, the platelet protein content can be used as a marker of early tumor growth or angiogenesis. With the recent increase in the clinical use of biologic modifiers in cancer and chronic disease therapy, the search for markers of early disease, therapeutic response, and/or recurrence has suggested that analysis of platelet proteins may be more relevant and accurate. We provide a guideline for the proteomic analysis of platelet proteome, placing specific emphasis on angiogenesis regulators, even though other platelet proteins may serve as markers of disease in the future. The analysis of serum/plasma has been fraught with difficulties because of the extraordinarily large number of proteins and because some of the proteins are contained in extraordinarily large amounts, masking the less abundant proteins. Thus, platelets may provide a much more biologically relevant analyte for biomarker discovery.
Assuntos
Plaquetas/citologia , Plaquetas/metabolismo , Proteínas Sanguíneas/metabolismo , Separação Celular/métodos , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Proteínas Sanguíneas/isolamento & purificação , Coleta de Amostras Sanguíneas , Fracionamento Químico , Humanos , Camundongos , Análise Serial de ProteínasRESUMO
Applying a next-generation sequencing assay targeting 145 cancer-relevant genes in 40 colorectal cancer and 24 non-small cell lung cancer formalin-fixed paraffin-embedded tissue specimens identified at least one clinically relevant genomic alteration in 59% of the samples and revealed two gene fusions, C2orf44-ALK in a colorectal cancer sample and KIF5B-RET in a lung adenocarcinoma. Further screening of 561 lung adenocarcinomas identified 11 additional tumors with KIF5B-RET gene fusions (2.0%; 95% CI 0.8-3.1%). Cells expressing oncogenic KIF5B-RET are sensitive to multi-kinase inhibitors that inhibit RET.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Colorretais/genética , Cinesinas/genética , Neoplasias Pulmonares/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas c-ret/genética , Receptores Proteína Tirosina Quinases/genética , Quinase do Linfoma Anaplásico , Animais , Biópsia , Transformação Celular Neoplásica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Cinesinas/antagonistas & inibidores , Neoplasias Pulmonares/patologia , Camundongos , Células NIH 3T3 , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidoresAssuntos
Neoplasias Inflamatórias Mamárias/tratamento farmacológico , Neoplasias Inflamatórias Mamárias/genética , Receptor ErbB-2/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Ductal de Mama/tratamento farmacológico , Carcinoma Ductal de Mama/enzimologia , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patologia , Feminino , Humanos , Neoplasias Inflamatórias Mamárias/enzimologia , Neoplasias Inflamatórias Mamárias/patologia , Lapatinib , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Quinazolinas/administração & dosagem , Receptor ErbB-2/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/enzimologia , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
The underlying basis for rising levels of prostate-specific antigen (PSA) in prostate cancer is not fully understood, but attention has turned to the possibility that loss of normal p53 function might be directly involved. We have investigated the relationship between p53 function and PSA expression using in vitro and in vivo approaches. Three prostate cancer-derived p53 mutants (F134L, M237L, R273H) were introduced into LNCaP prostate cancer cells and stable transfectants established. Expression of mutant p53 was demonstrated by Western blot analysis, inactivation of wtp53 function, and a loss of p53-dependent responses to DNA damage induced by UV-irradiation and cisplatin. Levels of PSA mRNA and secreted protein were determined by RT-PCR and Western blotting, respectively. Serine protease activity was assessed using an esterase assay. In vivo effects of mutant p53 expression were examined after orthotopic implantation into prostates of nude mice. Expression of all p53 mutants was associated with elevated PSA mRNA and secreted PSA protein. In a representative line, mutant p53 was also associated with increased PSA protease-like activity compared with a control line expressing wildtype p53. Overall PSA levels, and PSA levels in serum from mice bearing tumors derived from cells expressing mutant p53, were increased compared with levels in mice bearing tumors derived from control cells. In addition, the tumors derived from cells with mutant p53 had increased vascularization and induced lymph node metastases. These data provide in vitro and in vivo support for the notion that p53 mutations directly contribute to increased levels of serum PSA, and are associated with more aggressive tumors.