RESUMO
Intratumoral heterogeneity (ITH) provides the substrate for tumor evolution and treatment resistance, yet is remarkably understudied in lymphoma, due to the often limited amount of tissue that gets sampled during the routine diagnostic process, generally from a single nodal or extranodal site. Furthermore, the trajectory of how lymphoma, and especially non-Hodgkin lymphoma, spreads throughout the human body remains poorly understood. Here, we present a detailed characterization of ITH by applying whole-genome sequencing to spatially separated tumor samples harvested at the time of autopsy (n=24) and/or diagnosis (n=3) in three patients presenting with refractory B-cell non-Hodgkin lymphoma. Through deconvolution of bulk samples into clonal mixtures and inference of phylogenetic trees, we found evidence that polyclonal seeding underlies tumor dissemination in lymphoma. We identify mutation signatures associated with ancestral and descendant clones. In our series of patients with highly refractory lymphoma, the determinants of resistance were often harbored by founding clones, although there was also evidence of positive selection of driver mutations, likely under the influence of therapy. Lastly, we show that circulating tumor DNA is suitable for the detection of ancestral mutations but may miss a significant proportion of private mutations that can be detected in tissue. Our study clearly shows the existence of intricate patterns of regional and anatomical evolution that can only be disentangled through multi-regional tumor tissue profiling.
Assuntos
DNA Tumoral Circulante , Linfoma de Células B , Humanos , Filogenia , Autopsia , Mutação , Linfoma de Células B/genéticaRESUMO
Post-transplant lymphoproliferative disorder (PTLD) may arise after solid organ transplantation, and the most common subtype resembles diffuse large B cell lymphoma (DLBCL). In DLBCL-type PTLD, the anti-CD20 antibody rituximab (R) may be combined with chemotherapy (R-CHOP) or use a strategy (R-primary; similar to the PTLD-1 clinical trial) consisting of induction with four weekly doses of R-alone, without any chemotherapy or sequential R-CHOP follow-up. Here we report on a multicentre retrospective cohort of solid organ transplant patients with DLBCL-type PTLD that were treated with R. In 168 adults, two-year overall survival (OS) was 63·7% [95% CI (confidence interval) 56·6-71·7%]. No difference in OS was observed, whether patients were treated with R-CHOP versus the R-primary strategy. In the 109 patients treated with R-primary, multivariate analysis found that baseline IPI score and the response to R-induction predicted OS. Patients who responded to R-induction had durable remissions without the addition of chemotherapy. Conversely, of the 46 patients who had stable or progressive disease after R-induction (R-failure), those who received R-CHOP had an only marginally improved outcome, with a two-year OS of 45% (23·1-65·3%) vs. no R-CHOP at 32% (14·7-49·8%). In real-world patients, R-failure and high IPI scores predict a poor outcome in DLBCL-type PTLD.
Assuntos
Linfoma Difuso de Grandes Células B , Transplante de Órgãos/efeitos adversos , Complicações Pós-Operatórias , Rituximab , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Ciclofosfamida/administração & dosagem , Ciclofosfamida/efeitos adversos , Intervalo Livre de Doença , Doxorrubicina/administração & dosagem , Doxorrubicina/efeitos adversos , Feminino , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/etiologia , Linfoma Difuso de Grandes Células B/mortalidade , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/tratamento farmacológico , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/mortalidade , Prednisona/administração & dosagem , Prednisona/efeitos adversos , Estudos Retrospectivos , Rituximab/administração & dosagem , Rituximab/efeitos adversos , Taxa de Sobrevida , Vincristina/administração & dosagem , Vincristina/efeitos adversosRESUMO
A significant proportion (up to 62%) of oral squamous cell carcinomas (OSCCs) may arise from oral potential malignant lesions (OPMLs), such as leukoplakia. Patient outcomes may thus be improved through detection of lesions at a risk for malignant transformation, by identifying and categorizing genetic changes in sequential, progressive OPMLs. We conducted array comparative genomic hybridization analysis of 25 sequential, progressive OPMLs and same-site OSCCs from five patients. Recurrent DNA copy number gains were identified on 1p in 20/25 cases (80%) with minimal, high-level amplification regions on 1p35 and 1p36. Other regions of gains were frequently observed: 11q13.4 (68%), 9q34.13 (64%), 21q22.3 (60%), 6p21 and 6q25 (56%) and 10q24, 19q13.2, 22q12, 5q31.2, 7p13, 10q24 and 14q22 (48%). DNA losses were observed in >20% of samples and mainly detected on 5q31.2 (35%), 16p13.2 (30%), 9q33.1 and 9q33.29 (25%) and 17q11.2, 3p26.2, 18q21.1, 4q34.1 and 8p23.2 (20%). Such copy number alterations (CNAs) were mapped in all grades of dysplasia that progressed, and their corresponding OSCCs, in 70% of patients, indicating that these CNAs may be associated with disease progression. Amplified genes mapping within recurrent CNAs (KHDRBS1, PARP1, RAB1A, HBEGF, PAIP2, BTBD7) were selected for validation, by quantitative real-time PCR, in an independent set of 32 progressive leukoplakia, 32 OSSCs and 21 non-progressive leukoplakia samples. Amplification of BTBD7, KHDRBS1, PARP1 and RAB1A was exclusively detected in progressive leukoplakia and corresponding OSCC. BTBD7, KHDRBS1, PARP1 and RAB1A may be associated with OSCC progression. Protein-protein interaction networks were created to identify possible pathways associated with OSCC progression.
Assuntos
Carcinogênese/genética , Carcinoma de Células Escamosas/genética , Leucoplasia Oral/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/diagnóstico , Análise por Conglomerados , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA , Feminino , Genoma Humano , Humanos , Leucoplasia Oral/diagnóstico , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
Deletion 7q is a common chromosomal abnormality in myeloid neoplasms. Detection of del(7q) in patients following cytotoxic therapies is highly suggestive of an emerging therapy-related myeloid neoplasm. In this study, we describe 39 patients who acquired del(7q) as a sole abnormality in their bone marrow following cytotoxic therapies for malignant neoplasms. The median interval from cytotoxic therapies to detection of del(7q) was 40 months (range, 4-190 months). Twenty-eight patients showed an interstitial and 11 showed a terminal 7q deletion. Fifteen patients (38%) had del(7q) as a large clone and 24 (62%) as a small clone. With a median follow-up of 21 months (range, 1-135 months), 18 (46%) patients developed therapy-related myeloid neoplasms, including all 15 patients with a large del(7q) clone and 3/24 (12.5%) with a small clone. Of the remaining 21 patients with a small del(7q) clone, 16 showed no evidence of therapy-related myeloid neoplasms and 5 had an inconclusive pathological diagnosis. We conclude that isolated del(7q) emerging in patients after cytotoxic therapy may not always be associated with therapy-related myeloid neoplasms in about half of patients. The clone size of del(7q) is critical; a large clone is almost always associated with therapy-related myeloid neoplasms, whereas a small clone can be a clinically indolent or transient finding.
Assuntos
Antineoplásicos/efeitos adversos , Deleção Cromossômica , Cromossomos Humanos Par 7 , Síndromes Mielodisplásicas/genética , Segunda Neoplasia Primária/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Bases de Dados Genéticas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/induzido quimicamenteRESUMO
Tumour-associated macrophages are linked with poor prognosis and resistance to therapy in Hodgkin lymphoma; however, there are no suitable preclinical models to identify macrophage-targeting therapeutics. We used primary human tumours to guide the development of a mimetic cryogel, wherein Hodgkin (but not Non-Hodgkin) lymphoma cells promoted primary human macrophage invasion. In an invasion inhibitor screen, we identified five drug hits that significantly reduced tumour-associated macrophage invasion: marimastat, batimastat, AS1517499, ruxolitinib, and PD-169316. Importantly, ruxolitinib has demonstrated recent success in Hodgkin lymphoma clinical trials. Both ruxolitinib and PD-169316 (a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor) decreased the percent of M2-like macrophages; however, only PD-169316 enhanced the percentage of M1-like macrophages. We validated p38 MAPK as an anti-invasion drug target with five additional drugs using a high-content imaging platform. With our biomimetic cryogel, we modeled macrophage invasion in Hodgkin lymphoma and then used it for target discovery and drug screening, ultimately identifying potential future therapeutics.
Assuntos
Doença de Hodgkin , Macrófagos Associados a Tumor , Humanos , Macrófagos Associados a Tumor/metabolismo , Macrófagos Associados a Tumor/patologia , Doença de Hodgkin/tratamento farmacológico , Doença de Hodgkin/patologia , Criogéis , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Matriz Extracelular/metabolismoRESUMO
In the two decades since Accreditation Council for Graduate Medical Education-accredited Molecular Genetic Pathology fellowships began, the field of clinical molecular pathology has evolved considerably. The American Board of Pathology gathered data from board-certified molecular genetic pathologists assessing the alignment of skills and knowledge gained during fellowship with current needs on the job. The Association of Molecular Pathology conducted a parallel survey of program directors, and included questions on how various topics were taught during fellowship, as well as ranking their importance. Both surveys showed that most training aligned well with the practice needs of former trainees. Genomic profiling of tumors by next-generation sequencing, bioinformatics, laboratory management, and regulatory issues were topics thought to require increased emphasis in training. Topics related to clinical genetics and microbiology were deemed less important by those in practice, perhaps reflecting the increasing subspecialization of molecular pathologists. Program directors still viewed these topics as important to provide foundational knowledge. Parentage, identity, and human leukocyte antigen testing were less important to both survey audiences. These data may be helpful in guiding future adjustments to the Molecular Genetic Pathology curriculum and Accreditation Council for Graduate Medical Education program requirements.
Assuntos
Bolsas de Estudo , Patologistas , Acreditação , Currículo , Educação de Pós-Graduação em Medicina , Humanos , Patologia Molecular , Estados UnidosRESUMO
INTRODUCTION: The tumor microenvironment (TME) in post-transplant lymphoproliferative disorders (PTLDs) remains unexplored. Tumor infiltrating lymphocytes (TILs) are prognostic in other lymphomas. We assessed the prognostic impact of TILs in monomorphic B-cell PTLD. METHODS: TIL density (CD3+ cells/mm2) was determined by CD3 immunohistochemistry in archived diagnostic biopsies from patients diagnosed with monomorphic B-cell PTLD. RESULTS: Amongst monomorphic PTLDs (N = 107), low TIL-count was associated with inferior 2-year progression-free survival (PFS) (41% versus 86%, P = .003) and 2-year overall survival (OS) (52% versus 93%, P = .003) by Kaplan-Meier analysis. Low TIL-count was significant on Cox univariate regression for inferior PFS (HR 4.5, 95% CI 2.0-9.9, P < .001) and OS (HR 4.6, 95% CI 1.8-11.8, P < .001). Multivariate analysis with clinical variables (age ≥60 years, high LDH, stage III/IV, CNS involvement) and TIL-count showed significance for PFS (HR 3.3, 95% CI 1.3-8.3, P = .010) and a non-significant trend for OS (HR 2.6, 95% CI 0.9-7.3, P = .064). A composite score including TILs and clinical variables (age ≥60 years, high LDH, stage III/IV, CNS involvement) effectively stratified monomorphic PTLD patients by PFS and OS (2-year OS: low-risk 93%, intermediate-risk 61%, high-risk 23%, P < .001). CONCLUSIONS: The TME and TILs are prognostically relevant in monomorphic PTLD. Prognostic models including measures of the TME may improve risk stratification for patients with monomorphic PTLDs.
Assuntos
Infecções por Vírus Epstein-Barr , Linfoma , Transtornos Linfoproliferativos , Transplante de Órgãos , Infecções por Vírus Epstein-Barr/complicações , Humanos , Linfócitos do Interstício Tumoral/patologia , Linfoma/complicações , Transtornos Linfoproliferativos/diagnóstico , Transtornos Linfoproliferativos/etiologia , Pessoa de Meia-Idade , Transplante de Órgãos/efeitos adversos , Prognóstico , Estudos Retrospectivos , Microambiente TumoralRESUMO
BACKGROUND: A recently developed probe-based technology, the NanoString nCounter™ gene expression system, has been shown to allow accurate mRNA transcript quantification using low amounts of total RNA. We assessed the ability of this technology for mRNA expression quantification in archived formalin-fixed, paraffin-embedded (FFPE) oral carcinoma samples. RESULTS: We measured the mRNA transcript abundance of 20 genes (COL3A1, COL4A1, COL5A1, COL5A2, CTHRC1, CXCL1, CXCL13, MMP1, P4HA2, PDPN, PLOD2, POSTN, SDHA, SERPINE1, SERPINE2, SERPINH1, THBS2, TNC, GAPDH, RPS18) in 38 samples (19 paired fresh-frozen and FFPE oral carcinoma tissues, archived from 1997-2008) by both NanoString and SYBR Green I fluorescent dye-based quantitative real-time PCR (RQ-PCR). We compared gene expression data obtained by NanoString vs. RQ-PCR in both fresh-frozen and FFPE samples. Fresh-frozen samples showed a good overall Pearson correlation of 0.78, and FFPE samples showed a lower overall correlation coefficient of 0.59, which is likely due to sample quality. We found a higher correlation coefficient between fresh-frozen and FFPE samples analyzed by NanoString (r = 0.90) compared to fresh-frozen and FFPE samples analyzed by RQ-PCR (r = 0.50). In addition, NanoString data showed a higher mean correlation (r = 0.94) between individual fresh-frozen and FFPE sample pairs compared to RQ-PCR (r = 0.53). CONCLUSIONS: Based on our results, we conclude that both technologies are useful for gene expression quantification in fresh-frozen or FFPE tissues; however, the probe-based NanoString method achieved superior gene expression quantification results when compared to RQ-PCR in archived FFPE samples. We believe that this newly developed technique is optimal for large-scale validation studies using total RNA isolated from archived, FFPE samples.
Assuntos
Corantes Fluorescentes/química , Técnicas de Sonda Molecular , Sondas de Ácido Nucleico/química , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Benzotiazóis , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Cor , Diaminas , Perfilação da Expressão Gênica , Humanos , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Nanotecnologia/métodos , Compostos Orgânicos/química , Inclusão em Parafina , QuinolinasRESUMO
BACKGROUND: MicroRNAs (miRs) are non-coding RNA molecules involved in post-transcriptional regulation, with diverse functions in tissue development, differentiation, cell proliferation and apoptosis. miRs may be less prone to degradation during formalin fixation, facilitating miR expression studies in formalin-fixed paraffin-embedded (FFPE) tissue. RESULTS: Our study demonstrates that the TaqMan Human MicroRNA Array v1.0 (Early Access) platform is suitable for miR expression analysis in FFPE tissue with a high reproducibility (correlation coefficients of 0.95 between duplicates, p < 0.00001) and outlines the optimal performance conditions of this platform using clinical FFPE samples. We also outline a method of data analysis looking at differences in miR abundance between FFPE and fresh-frozen samples. By dividing the profiled miR into abundance strata of high (Ct<30), medium (30 < or = Ct < or = 35), and low (Ct>35), we show that reproducibility between technical replicates, equivalent dilutions, and FFPE vs. frozen samples is best in the high abundance stratum. We also demonstrate that the miR expression profiles of FFPE samples are comparable to those of fresh-frozen samples, with a correlation of up to 0.87 (p < 0.001), when examining all miRs, regardless of RNA extraction method used. Examining correlation coefficients between FFPE and fresh-frozen samples in terms of miR abundance reveals correlation coefficients of up to 0.32 (low abundance), 0.70 (medium abundance) and up to 0.97 (high abundance). CONCLUSION: Our study thus demonstrates the utility, reproducibility, and optimization steps needed in miR expression studies using FFPE samples on a high-throughput quantitative PCR-based miR platform, opening up a realm of research possibilities for retrospective studies.
Assuntos
Perfilação da Expressão Gênica/métodos , MicroRNAs/análise , Reação em Cadeia da Polimerase/métodos , Formaldeído , Humanos , Microfluídica , Inclusão em Parafina , Análise de Regressão , Reprodutibilidade dos TestesRESUMO
Our understanding of genetic disease(s) has increased exponentially since the completion of human genome sequencing and the development of numerous techniques to detect genetic variants. These techniques have not only allowed us to diagnose genetic disease, but in so doing, also provide increased understanding of the pathogenesis of these diseases to aid in developing appropriate therapeutic options. Additionally, the advent of next-generation or massively parallel sequencing (NGS/MPS) is increasingly being used in the clinical setting, as it can detect a number of abnormalities from point mutations to chromosomal rearrangements as well as aberrations within the transcriptome. In this article, we will discuss the use of multiple techniques that are used in genetic diagnosis. © 2020 by John Wiley & Sons, Inc.
Assuntos
Aberrações Cromossômicas , Transtornos Cromossômicos/diagnóstico , Testes Genéticos/métodos , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Técnicas de Diagnóstico Molecular/métodos , Análise de Sequência de DNA/métodos , Transtornos Cromossômicos/genética , Humanos , Técnicas de Diagnóstico Molecular/tendências , TranscriptomaRESUMO
OBJECTIVES: At a discussion on molecular/cytogenetic education for hematopathology fellows at the 2018 Society for Hematopathology Program Directors Meeting, consensus was that fellows should understand basic principles and indications for and limitations of molecular/cytogenetic testing used in routine practice. Fellows should also be adept at integrating results of such testing for rendering a final diagnosis. To aid these consensus goals, representatives from the Society for Hematopathology and the Association for Molecular Pathology formed a working group to devise a molecular/cytogenetic curriculum for hematopathology fellow education. CURRICULUM SUMMARY: The curriculum includes a primer on cytogenetics and molecular techniques. The bulk of the curriculum reviews the molecular pathology of individual malignant hematologic disorders, with applicable molecular/cytogenetic testing for each and following the 2017 World Health Organization classification of hematologic neoplasms. Benign hematologic disorders and bone marrow failure syndromes are also discussed briefly. Extensive tables are used to summarize genetics of individual disorders and appropriate methodologies. CONCLUSIONS: This curriculum provides an overview of the current understanding of the molecular biology of hematologic disorders and appropriate ancillary testing for their evaluation. The curriculum may be used by program directors for training hematopathology fellows or by practicing hematopathologists.
Assuntos
Currículo , Educação de Pós-Graduação em Medicina , Patologia Clínica/educação , Patologia Molecular/educação , Bolsas de Estudo , Hematologia , HumanosRESUMO
Prognostic biomarkers for recurrence of Oral Squamous Cell Carcinoma (OSCC) are urgently needed. We aimed to independently validate a 4-gene expression signature (MMP1, COL4A1, P4HA2, THBS2) predictive of OSCC recurrence risk. Gene expression was measured using Nanostring nCounter® in 245 histologically normal surgical resection margins from 62 patients. Association between risk scores for individual patients and recurrence was assessed by Kaplan-Meier analysis. Signature performance was quantified by concordance index (CI), hazard ratio (HR) and the area under receiver operating characteristics (AUC). Risk scores for recurrence were significantly higher than recurrence-free patients (p = 9.58e-7, Welch's t-test). A solid performance of the 4-gene signature was determined: CI = 0.64, HR = 3.38 (p = 1.4E-4; log-rank test), AUC = 0.71. We showed that three margins per patient are sufficient to preserve predictive performance (CI = 0.65; HR = 2.92; p = 2.94e-3; AUC = 0.71). Association between the predicted risk scores and recurrence was assessed and showed HR = 2.44 (p = 9.6E-3; log-rank test, N = 62). Signature performance analysis was repeated using an optimized threshold (70th percentile of risks), resulting in HR = 3.38 (p = 1.4E-4; log-rank test, N = 62). The 4-gene signature was validated as predictive of recurrence risk in an independent cohort of patients with resected OSCC and histologically negative margins, and is potentially applicable for clinical decision making on adjuvant treatment and disease monitoring.
Assuntos
Carcinoma de Células Escamosas/diagnóstico , Colágeno Tipo IV/genética , Metaloproteinase 1 da Matriz/genética , Neoplasias Bucais/diagnóstico , Prolil Hidroxilases/genética , Trombospondinas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/genética , Feminino , Seguimentos , Humanos , Masculino , Margens de Excisão , Pessoa de Meia-Idade , Neoplasias Bucais/genética , Recidiva Local de Neoplasia , Prognóstico , TranscriptomaRESUMO
Mantle cell lymphoma (MCL) shows a clinical aggressiveness that varies from patient to patient. Despite major advances in outcomes with current immunochemotherapy, the future development of therapies requires risk stratification to tailor therapy intensity. Within the group of reference pathologists for the ongoing trials of the European MCL Network, we performed a round robin test on a tissue microarray to evaluate the reproducibility in assessing the biomarkers of outcome in MCL. Cytological subtype, Ki67-index and expression of p53 and SOX11 were evaluated on 20 diagnostic tumour samples by eight participating labs independently. We demonstrate that the assessment of the proliferation index by counting the Ki67 positive cells as well as assessment of SOX11 and p53 expression status is reproducible between labs. For the most established prognostic biomarker, Ki67, the intra-class correlation coefficient was very good when assessed as a continuous parameter (0.87). The agreement was lower when the values were analysed in a dichotomized way applying the commonly used cutoff of 30% (kappa = 0.65, complete concordance of all labs in 13/20 (65%)). Cases with discrepant results between labs in the dichotomized analysis showed mean values close to the cutoff of 30%. Centralised scoring and digital image analysis revealed results in line with the scores from individual labs. All cases in our cohort were additionally assessed for gene expression signatures and of TP53 gene alterations. Given the good reproducibility when guidelines of assessment are applied, the biomarker studied in this inter-laboratory test presents potential candidates to be enhanced for risk-stratification in the future clinical trials.
Assuntos
Antígeno Ki-67/metabolismo , Linfoma de Célula do Manto/patologia , Fatores de Transcrição SOXC/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Biomarcadores Tumorais/análise , Citodiagnóstico/métodos , Feminino , Humanos , Linfoma de Célula do Manto/metabolismo , Masculino , Prognóstico , Reprodutibilidade dos TestesRESUMO
Unknown primary malignancy in the head and neck is not an infrequent diagnosis for patients with metastatic cervical lymph nodes. Although linked with a relatively good prognosis following radiation treatment, widespread radiation is coupled with significant morbidity. Altered microRNA (miRNA) expression has been associated with both cancer progression and metastasis. We sought to determine whether miRNA expression analysis could be used as a diagnostic tool to discover the primary site of malignancy, within the head and neck. We used quantitative real-time PCR to identify miRNA expression profiles of squamous cell carcinoma of the tonsil, base of tongue and post-nasal space, as well as their corresponding metastatic lymph nodes, from 6 patients. Our results revealed that each cancer maintained its expression profile between the primary site and the nodal metastasis (r = 0.82, p < 0.0001). In addition, each anatomical sub-site maintained a distinct miRNA profile between individual patients (r = 0.79, p < 0.0001). Finally, between sub-sites, the miRNA profiles were distinct (p < 0.0001). As proof of principle, our study provides an indication that miRNA expression analysis may be useful to compare the primary lesion and local metastatic disease. This may be clinically relevant to predict the primary site of origin of metastatic disease, when the primary site remains obscure.
Assuntos
Neoplasias de Cabeça e Pescoço/genética , MicroRNAs/genética , Neoplasias Primárias Desconhecidas/genética , Adulto , Idoso , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-IdadeRESUMO
INTRODUCTION: It has been shown that the quantity of circulating tumor cells (CTCs) in breast cancer patients is an independent predictor of survival and treatment response. Real time quantitative reverse transcriptase PCR (Q-RT-PCR) is a sensitive technique for detection of CTCs. Our aim was to investigate whether the technique can be used also to quantitate these CTCs. METHODS: We tested cytokeratin 19 (CK19), maspin, mammaglobin, GAPDH and RPL19 genes for their level of expression and linearity of amplification in serial dilutions of RNA extracted from the MDA-MB-231, UACC-812, T47D and HS578T breast cancer cell lines. To simulate CTCs, serial dilutions of cultured T47D and HS578T cells were added to peripheral blood from healthy volunteers. The samples were subjected to enrichment, RNA extraction and Q-RT-PCR. RESULTS: CK19 was reliably expressed in all four cell lines with a linear relationship between the quantity of added cells and the amount of CK19 RNA. The lower limit of reliable detection was 5 cells per sample, which corresponds to a concentration of 0.7 cell/ml in 7.5 ml of blood or would translate to a lower CTC concentration in a larger volume of blood. CONCLUSION: This technique may prove useful for high throughput comparative quantification of CTCs in individual patients during treatment and subsequent follow up for research and clinical management purposes.
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Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Células Epiteliais/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Reações Falso-Positivas , Marcadores Genéticos , Técnicas Genéticas , Gliceraldeído 3-Fosfato Desidrogenase (NADP+)/metabolismo , Humanos , Queratina-19/biossíntese , Mamoglobina A , Proteínas de Neoplasias/biossíntese , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Proteínas Ribossômicas/metabolismo , Serpinas/biossíntese , Uteroglobina/biossínteseRESUMO
Follicular lymphoma (FL) is presented as a germinal centre B cell lymphoma that is characterized by an indolent clinical course, but remains - paradoxically - largely incurable to date. The last years have seen significant progress in our understanding of FL lymphomagenesis, which is a multi-step process beginning in the bone marrow with the hallmark t(14;18)(q32;q21) translocation. The pathobiology of FL is complex and combines broad somatic changes at the level of both the genome and the epigenome, the latter evidenced by highly recurrent mutations in chromatin-modifying genes such as KMT2D and CREBBP. While the importance of the FL microenvironment has since long been well understood, it has become evident that somatic lesions within tumour cells re-educate normal immune and stromal cells to their advantage. Enhanced understanding of FL pathogenesis is currently leading to refined therapeutic targeting of perturbed biology, paving the way for precision medicine in this lymphoma subtype.
Assuntos
Cromossomos Humanos Par 14 , Epigênese Genética , Linfoma Folicular , Proteínas de Neoplasias , Translocação Genética , Microambiente Tumoral/genética , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 14/metabolismo , Cromossomos Humanos Par 18/genética , Cromossomos Humanos Par 18/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Linfoma Folicular/genética , Linfoma Folicular/metabolismo , Linfoma Folicular/patologia , Linfoma Folicular/fisiopatologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismoRESUMO
With the advent of next-generation sequencing and its prolific use in the clinical realm, it would appear that techniques such as PCR would not be in high demand. This is not the case however, as PCR techniques play an important role in the success of NGS technology. Although NGS has rapidly become an important part of clinical molecular diagnostics, whole genome sequencing is still difficult to implement in a clinical laboratory due to high costs of sequencing, as well as issues surrounding data processing, analysis, and data storage, which can reduce efficiency and increase turnaround times. As a result, targeted sequencing is often used in clinical diagnostics, due to its increased efficiency. PCR techniques play an integral role in targeted NGS sequencing, allowing for the generation of multiple NGS libraries and the sequencing of multiple targeted regions simultaneously. We will outline the methods we employ in PCR amplification of targeted genomic regions for cancer mutation hotspots using the Ampliseq Cancer Hotspot v2 panel (Life Technologies, Carlsbad, CA).
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Reação em Cadeia da Polimerase , Código de Barras de DNA Taxonômico , Biblioteca Genômica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normasRESUMO
OBJECTIVES: Clinical laboratories are rapidly implementing next-generation sequencing (NGS) tests for mutation analysis, but there are few guidelines regarding sample quality for successful results. METHODS: We aimed to establish tissue quality parameters for successful NGS in solid tumors and to improve NGS performance. RESULTS: Analysis of 614 clinical cases tested in 2013 using a 50-gene hotspot mutation panel identified the major cause for unsuccessful NGS analysis was DNA less than 10 ng (91%, 67/74) associated with extremely small and low cellularity samples. High success rates were associated with resection procedures (333/342, 97%) and biopsied tumor larger than 10 mm(2) (77/77, 100%). NGS can be successfully performed on bone specimens processed with formic acid-based decalcification procedures (8/11, 73%). Tumor type and paraffin block age did not affect success. We demonstrated that NGS can be carried out on samples with less than 10 ng DNA. Analysis of 408 cases tested in 2014 using an optimized workflow showed improved NGS success rates from 88% to 95% (387/408) with pronounced improvement among tiny (<10 mm(2)) samples (from 76% to 94%) as well as cytology samples (from 58% to 87%). CONCLUSIONS: Identifying preanalytical tissue factors allows us to improve NGS performance and to successfully test tumors obtained from minimally invasive procedures.
Assuntos
Biomarcadores Tumorais/genética , DNA de Neoplasias/análise , Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias/diagnóstico , Manejo de Espécimes , Análise Mutacional de DNA , DNA de Neoplasias/isolamento & purificação , Humanos , Mutação , Neoplasias/genética , Inclusão em Parafina , Análise de Sequência de DNA , Fixação de Tecidos , Fluxo de TrabalhoRESUMO
Application of next-generation sequencing (NGS) technology to routine clinical practice has enabled characterization of personalized cancer genomes to identify patients likely to have a response to targeted therapy. The proper selection of tumor sample for downstream NGS based mutational analysis is critical to generate accurate results and to guide therapeutic intervention. However, multiple pre-analytic factors come into play in determining the success of NGS testing. In this review, we discuss pre-analytic requirements for AmpliSeq PCR-based sequencing using Ion Torrent Personal Genome Machine (PGM) (Life Technologies), a NGS sequencing platform that is often used by clinical laboratories for sequencing solid tumors because of its low input DNA requirement from formalin fixed and paraffin embedded tissue. The success of NGS mutational analysis is affected not only by the input DNA quantity but also by several other factors, including the specimen type, the DNA quality, and the tumor cellularity. Here, we review tissue requirements for solid tumor NGS based mutational analysis, including procedure types, tissue types, tumor volume and fraction, decalcification, and treatment effects.
RESUMO
OBJECTIVES: Deletion 5q is a common chromosomal abnormality in both de novo and therapy-related myeloid neoplasms (t-MNs). The detection of isolated del(5q) in patients following therapies for various malignancies raises serious concern for an emerging t-MN. METHODS: We identified 25 patients who developed isolated del(5q) following cytotoxic therapy (n = 21) or tyrosine kinase inhibitor (TKI; n = 4) therapy. Twenty-four patients had an interstitial and one had a terminal 5q deletion. The 5q31/EGR1 gene was deleted in 20 patients and intact in five patients. The clone size as assessed by metaphase analysis was minor (10%-30%) in 12 patients and large (45%-100%) in 13 patients. After a median follow-up of 17 months, none of the 12 patients with a minor del(5q) clone developed t-MN; del(5q) disappeared in four patients and persisted in eight patients. By contrast, 12 of 13 patients with a large del(5q) clone developed t-MN, and del(5q) was persistent in all patients who had follow-up cytogenetic testing. CONCLUSIONS: Development of del(5q) in patients following cytotoxic therapies or TKI may not always be associated with t-MN. A close follow-up seems an appropriate approach for patients who had a minor del(5q) clone.