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1.
Br J Haematol ; 173(1): 49-58, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26728869

RESUMO

Currently, comprehensive genetic testing of myeloid malignancies requires multiple testing strategies with high costs. Somatic mutations can be detected by next generation sequencing (NGS) but copy number variants (CNVs) require cytogenetic methods including karyotyping, fluorescence in situ hybidization and microarray. Here, we evaluated a new method for CNV detection using read depth data derived from a targeted NGS mutation panel. In a cohort of 270 samples, we detected pathogenic mutations in 208 samples and targeted CNVs in 68 cases. The most frequent CNVs were 7q deletion including LUC7L2 and EZH2, TP53 deletion, ETV6 deletion, gain of RAD21 on 8q, and 5q deletion, including NSD1 and NPM1. We were also able to detect exon-level duplications, including so-called KMT2A (MLL) partial tandem duplication, in 9 cases. In the 63 cases that were negative for mutations, targeted CNVs were observed in 4 cases. Targeted CNV detection by NGS had very high concordance with single nucleotide polymorphism microarray, the current gold standard. We found that ETV6 deletion was strongly associated with TP53 alterations and 7q deletion was associated with mutations in TP53, KRAS and IDH1. This proof-of-concept study demonstrates the feasibility of using the same NGS data to simultaneously detect both somatic mutations and targeted CNVs.


Assuntos
Variações do Número de Cópias de DNA , Neoplasias Hematológicas/genética , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Neoplasias/genética , Feminino , Humanos , Masculino , Nucleofosmina
2.
Ann Hematol ; 95(2): 201-10, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26555285

RESUMO

Detection of BCR-ABL1 mutations that confer resistance to tyrosine kinase inhibitors is important for management of patients with t(9;22);BCR-ABL1-positive (Ph+) leukemias. Testing is often performed using Sanger sequencing (SS) which has relatively poor sensitivity. Given the widespread adoption of next generation sequencing (NGS), we sought to reevaluate the testing in the context of NGS methods. We developed an NGS-based BCR-ABL1 mutation test on the Ion Torrent Personal Genome Machine (PGM) to test for resistance mutations, primarily in the kinase domain in BCR-ABL1. We analyzed 508 clinical samples from patients with Ph+ leukemias. In a subset of these samples (n = 97), we conducted a comparison of the NGS results to a classical SS-based test. NGS facilitated detection of low-level mutations (<20 % allele frequency) that were not detectable by SS. In a subset of cases with multiple mutations, NGS was also able to determine if two mutations were on the same molecule (compound) or on separate molecules (polyclonal) but this was limited by the distance between mutated positions and by the effects of apparent distance-dependent PCR recombination. We found 22 compound mutations that centered on one or two key residues including two novel compound mutants: Q252H/Y253H and F311Y/F359I. The advantages of NGS make it a superior method for inventorying BCR-ABL1 resistance mutations. However, data analysis may be complicated by short read lengths and the effects of PCR recombination.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fusão bcr-abl/genética , Mutação/genética , Inibidores de Proteínas Quinases , Análise de Sequência de DNA/métodos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Frequência do Gene/genética , Humanos , Inibidores de Proteínas Quinases/farmacologia , Reação em Cadeia da Polimerase em Tempo Real/métodos
3.
Exp Mol Pathol ; 99(1): 104-8, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26079545

RESUMO

Acute myeloid leukemia patients with recurrent cytogenetic abnormalities including inv(16);CBFB-MYH11 and t(15;17);PML-RARA may be assessed by monitoring the levels of the corresponding abnormal fusion transcripts by quantitative reverse transcription-PCR (qRT-PCR). Such testing is important for evaluating the response to therapy and for the detection of early relapse. Existing qRT-PCR methods are well established and in widespread use in clinical laboratories but they are laborious and require the generation of standard curves. Here, we describe a new method to quantitate fusion transcripts in acute myeloid leukemia by qRT-PCR without the need for standard curves. Our approach uses a plasmid calibrator containing both a fusion transcript sequence and a reference gene sequence, representing a perfect normalized copy number (fusion transcript copy number/reference gene transcript copy number; NCN) of 1.0. The NCN of patient specimens can be calculated relative to that of the single plasmid calibrator using experimentally derived PCR efficiency values. We compared the data obtained using the plasmid calibrator method to commercially available assays using standard curves and found that the results obtained by both methods are comparable over a broad range of values with similar sensitivities. Our method has the advantage of simplicity and is therefore lower in cost and may be less subject to errors that may be introduced during the generation of standard curves.


Assuntos
Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Proteínas de Fusão Oncogênica/genética , Reação em Cadeia da Polimerase/métodos , Aberrações Cromossômicas , Subunidade beta de Fator de Ligação ao Core/genética , Subunidade beta de Fator de Ligação ao Core/metabolismo , Análise Custo-Benefício , Fragmentação do DNA , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Plasmídeos/genética , Proteína da Leucemia Promielocítica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Manejo de Espécimes , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
4.
J Am Acad Dermatol ; 73(2): 228-36.e2, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26048061

RESUMO

BACKGROUND: T-cell receptor (TCR) clonality assessment is a principal diagnostic test in the management of mycosis fungoides (MF). However, current polymerase chain reaction-based methods may produce ambiguous results, often because of low abundance of clonal T lymphocytes, resulting in weak clonal peaks that cannot be size-resolved by contemporary capillary electrophoresis (CE). OBJECTIVE: We sought to determine if next-generation sequencing (NGS)-based detection has increased sensitivity for T-cell clonality over CE-based detection in MF. METHODS: Clonality was determined by an NGS-based method in which the TCR-γ variable region was polymerase chain reaction amplified and the products sequenced to establish the identity of rearranged variable and joining regions. RESULTS: Of the 35 MF cases tested, 29 (85%) showed a clonal T-cell rearrangement by NGS, compared with 15 (44%) by standard CE detection. Three patients with MF had follow-up testing that showed identical, clonal TCR sequences in subsequent skin biopsy specimens. LIMITATIONS: Clonal T-cell populations have been described in benign conditions; evidence of clonality alone, by any method, is not sufficient for diagnosis. CONCLUSION: TCR clonality assessment by NGS has superior sensitivity compared with CE-based detection. Further, NGS enables tracking of specific clones across multiple time points for more accurate identification of recurrent MF.


Assuntos
Predisposição Genética para Doença , Micose Fungoide/diagnóstico , Micose Fungoide/genética , Receptores de Antígenos de Linfócitos T/genética , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/genética , Adulto , Idoso , Clonagem Molecular/métodos , DNA de Neoplasias/genética , Bases de Dados Factuais , Eletroforese/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Reação em Cadeia da Polimerase/métodos , Prognóstico , Estudos Retrospectivos
5.
Cancer Immunol Immunother ; 61(7): 1113-24, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22198309

RESUMO

Cancer survivors often relapse due to evolving drug-resistant clones and repopulating tumor stem cells. Our preclinical study demonstrated that terminal cancer patient's lymphocytes can be converted from tolerant bystanders in vivo into effective cytotoxic T-lymphocytes in vitro killing patient's own tumor cells containing drug-resistant clones and tumor stem cells. We designed a clinical trial combining peginterferon α-2b with imatinib for treatment of stage III/IV gastrointestinal stromal tumor (GIST) with the rational that peginterferon α-2b serves as danger signals to promote antitumor immunity while imatinib's effective tumor killing undermines tumor-induced tolerance and supply tumor-specific antigens in vivo without leukopenia, thus allowing for proper dendritic cell and cytotoxic T-lymphocyte differentiation toward Th1 response. Interim analysis of eight patients demonstrated significant induction of IFN-γ-producing-CD8(+), -CD4(+), -NK cell, and IFN-γ-producing-tumor-infiltrating-lymphocytes, signifying significant Th1 response and NK cell activation. After a median follow-up of 3.6 years, complete response (CR) + partial response (PR) = 100%, overall survival = 100%, one patient died of unrelated illness while in remission, six of seven evaluable patients are either in continuing PR/CR (5 patients) or have progression-free survival (PFS, 1 patient) exceeding the upper limit of the 95% confidence level of the genotype-specific-PFS of the phase III imatinib-monotherapy (CALGB150105/SWOGS0033), demonstrating highly promising clinical outcomes. The current trial is closed in preparation for a larger future trial. We conclude that combination of targeted therapy and immunotherapy is safe and induced significant Th1 response and NK cell activation and demonstrated highly promising clinical efficacy in GIST, thus warranting development in other tumor types.


Assuntos
Neoplasias Gastrointestinais/terapia , Tumores do Estroma Gastrointestinal/terapia , Interferon-alfa/administração & dosagem , Piperazinas/administração & dosagem , Polietilenoglicóis/administração & dosagem , Pirimidinas/administração & dosagem , Idoso , Idoso de 80 Anos ou mais , Benzamidas , Intervalo Livre de Doença , Neoplasias Gastrointestinais/imunologia , Tumores do Estroma Gastrointestinal/imunologia , Humanos , Mesilato de Imatinib , Imunoterapia/métodos , Interferon alfa-2 , Interferon-alfa/imunologia , Interferon gama/biossíntese , Interferon gama/imunologia , Linfócitos/imunologia , Pessoa de Meia-Idade , Proteínas Recombinantes/administração & dosagem , Recidiva , Linfócitos T Citotóxicos/imunologia
6.
Int J Surg Case Rep ; 98: 107537, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36027833

RESUMO

INTRODUCTION AND IMPORTANCE: The management of large malignant tracheo-esophageal fistulas (TEF) is not standardized. Herein, we report a case with a malignant TEF associated with esophageal post-transplant lymphoproliferative disorder (PTLD) for whom we successfully performed a surgical repair. This contributes to the knowledge on how to treat large acquired malignant TEFs. CASE PRESENTATION: A 69-year old male presented with a one-week history of fever, productive cough and bilateral coarse crackles. In addition, he described a weight loss of 10 kg during the past three months. The patient's history included a kidney transplantation twenty years ago. Esophagogastroduodenoscopy with a biopsy of the esophagus was performed nine days before. Histopathology showed a PTLD of diffuse large B-cell lymphoma subtype. Subsequent diagnostics revealed a progressive TEF (approx. 2.0 × 1.5 cm) 3.0 cm above the carina. PET-CT scan showed an esophagus with slight tracer uptake in the middle third (approx. 11.5 cm length, SUV max 7.4). After decision against stenting, transthoracic subtotal esophagectomy with closure of the tracheal mouth of the fistula by a pedicled flap was performed. PTLD was treated with prednisone and rituximab. Tumor progression (brain metastasis) led to death 95 days after surgery. CLINICAL DISCUSSION: The treatment of a malignant TEF is complex and personalized while both the consequences of the esophago-tracheal connection and those of the underlying responsible diagnosis have to be considered concurrently. In this case, we considered surgery as the best treatment option due to a relatively good prognosis of the underlying diagnosis (PTLD) and a large fistula. Esophageal or dual stenting, the treatment of choice for small malignant TEF, would have been associated with a high risk of failure due to the wide trachea, extensively dilated esophagus, proximal location and large diameter of the fistula. CONCLUSION: Surgery can be considered for patients with a large acquired malignant TEF and positive long-term prognosis of the underlying diagnosis. Due to the complexity of TEF management, immediate pre-operative multidisciplinary discussion is advised.

7.
Am J Clin Pathol ; 153(2): 251-257, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31628845

RESUMO

OBJECTIVES: Many commonly used FLT3 mutational assay protocols require a tedious blast enrichment step. We investigated whether elimination of this step would still give equivalent results and compared the accuracy of variant allele fraction (VAF) between polymerase chain reaction/capillary electrophoresis (PCR/CE) vs next-generation sequencing (NGS) methods. METHODS: Total leukocyte vs blast-enriched whole-blood aliquots were tested for FLT3 internal tandem duplication (ITD) and tyrosine kinase domain mutations by PCR/CE. VAF of the ITD mutations was also compared with NGS VAF. RESULTS: Blast-enriched vs total leukocyte specimens showed 100% concordance in the 25 positive specimens. VAF was consistently lower by NGS, with poorer fidelity to PCR/CE VAF as the ITD size increased. CONCLUSIONS: Our study supports elimination of the blast enrichment step without compromising results or sensitivity. In addition, since NGS shows a loose correlation with PCR/CE quantitative results, NGS VAF should not be reported for FLT3 ITDs.


Assuntos
Crise Blástica/genética , Leucemia Mieloide Aguda/genética , Mutação , Sequências de Repetição em Tandem , Tirosina Quinase 3 Semelhante a fms/genética , Alelos , Eletroforese Capilar , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Reação em Cadeia da Polimerase
8.
Leukemia ; 34(11): 2981-2991, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32409689

RESUMO

FLT3-ITD mutations occur in 20-30% of AML patients and are associated with aggressive disease. Patients with relapsed FLT3-mutated disease respond well to 2nd generation FLT3 TKIs but inevitably relapse within a short timeframe. In this setting, until overt relapse occurs, the bone marrow microenvironment facilitates leukemia cell survival despite continued on-target inhibition. We demonstrate that human bone marrow derived conditioned medium (CM) protects FLT3-ITD+ AML cells from the 2nd generation FLT3 TKI quizartinib and activates STAT3 and STAT5 in leukemia cells. Extrinsic activation of STAT5 by CM is the primary mediator of leukemia cell resistance to FLT3 inhibition. Combination treatment with quizartinib and dasatinib abolishes STAT5 activation and significantly reduces the IC50 of quizartinib in FLT3-ITD+ AML cells cultured in CM. We demonstrate that CM protects FLT3-ITD+ AML cells from the inhibitory effects of quizartinib on glycolysis and that this is partially reversed by treating cells with the combination of quizartinib and dasatinib. Using a doxycycline-inducible STAT5 knockdown in the FLT3-ITD+ MOLM-13 cell line, we show that dasatinib-mediated suppression of leukemia cell glycolytic activity is STAT5-independent and provide a preclinical rationale for combination treatment with quizartinib and dasatinib in FLT3-ITD+ AML.


Assuntos
Benzotiazóis/farmacologia , Dasatinibe/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos/genética , Metabolismo Energético , Duplicação Gênica , Técnicas de Silenciamento de Genes , Glicólise , Humanos , Fosforilação , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Tirosina Quinase 3 Semelhante a fms/genética
10.
Arch Pathol Lab Med ; 143(1): 105-111, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29979613

RESUMO

CONTEXT.­: B-cell lymphomas exhibit balanced translocations that involve immunoglobulin loci and result from aberrant V(D)J recombination, class switch recombination, or somatic hypermutation. Although most of the breakpoints in the immunoglobulin loci occur in defined regions, those in the partner genes vary; therefore, it is unlikely that 2 independent clones would share identical breakpoints in both partners. Establishing whether a new lesion in a patient with history of lymphoma represents recurrence or a new process can be relevant. Polymerase chain reaction (PCR)-based clonality assays used in this setting rely only on evaluating the length of a given rearrangement. In contrast, next-generation sequencing (NGS) provides the exact translocation breakpoint at single-base resolution. OBJECTIVE.­: To determine if translocation breakpoint coordinates can serve as a molecular fingerprint unique to a distinct clonal population. DESIGN.­: Thirty-eight follicular lymphoma/diffuse large B-cell lymphoma samples collected from different anatomic sites and/or at different time points from 18 patients were analyzed by NGS. For comparison, PCR-based B-cell clonality and fluorescence in situ hybridization studies were performed on a subset of cases. RESULTS.­: IGH-BCL2 rearrangements were detected in all samples. The breakpoint coordinates on derivative chromosome(s) were identical in all samples from a given patient, but distinct between samples derived from different patients. Additionally, 5 patients carried a second rearrangement also with conserved breakpoint coordinates in the follow-up sample(s). CONCLUSIONS.­: Breakpoint coordinates in the immunoglobulin and partner genes can be used to establish clonal relatedness of anatomically/temporally distinct lesions. Additionally, an NGS-based approach has the potential to detect secondary translocations that may have prognostic and therapeutic significance.


Assuntos
Impressões Digitais de DNA , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Linfoma Folicular/genética , Linfoma Difuso de Grandes Células B/genética , Rearranjo Gênico , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Hibridização in Situ Fluorescente , Proteínas Proto-Oncogênicas c-bcl-2/genética , Translocação Genética
11.
Leuk Res ; 32(3): 383-93, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17720243

RESUMO

Anaplastic large cell lymphoma (ALCL) comprises a group of non-Hodgkin lymphomas characterized by the expression of the CD30/Ki-1 antigen. A subset of ALCL is characterized by chromosomal translocations involving the anaplastic lymphoma kinase (ALK) gene on chromosome 2. While the most common translocation is the t(2;5)(p23;q35) involving the nucleophosmin (NPM) gene on chromosome 5, up to 12 other translocations partners of the ALK gene have been identified. One of these is the t(1;2)(q25;p23) which results in the formation of the chimeric protein TPM3-ALK. While several of the signaling pathways induced by NPM-ALK have been elucidated, those involved in ALCLs harboring TPM3-ALK are largely unknown. In order to investigate the expression profiles of ALCLs carrying the NPM-ALK and TPM3-ALK fusions, we carried out cDNA microarray analysis of two ALCL tissue samples, one expressing the NPM-ALK fusion protein and the other the TPM3-ALK fusion protein. RNA was extracted from snap-frozen tissues, labeled with fluorescent dyes and analyzed using cDNAs microarray containing approximately 9,200 genes and expressed sequence tags (ESTs). Quantitative fluorescence RT-PCR was performed to validate the cDNA microarray data on nine selected gene targets. Our results show a significant overlap of genes deregulated in the NPM-ALK and TPM-ALK positive lymphomas. These deregulated genes are involved in diverse cellular functions, such as cell cycle regulation, apoptosis, proliferation, and adhesion. Interestingly, a subset of the genes was distinct in their expression pattern in the two types of lymphomas. More importantly, many genes that were not previously associated with ALK positive lymphomas were identified. Our results demonstrate the overlapping and unique transcriptional patterns associated with the NPM-ALK and TPM3-ALK fusions in ALCL.


Assuntos
Linfoma Anaplásico de Células Grandes/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Tirosina Quinases/genética , Tropomiosina/genética , Adulto , Quinase do Linfoma Anaplásico , Criança , Perfilação da Expressão Gênica , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Receptores Proteína Tirosina Quinases , Translocação Genética
12.
J Clin Pathol ; 71(4): 372-378, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29197855

RESUMO

AIMS: Genetic abnormalities, including copy number variants (CNV), copy number neutral loss of heterozygosity (CN-LOH) and gene mutations, underlie the pathogenesis of myeloid malignancies and serve as important diagnostic, prognostic and/or therapeutic markers. Currently, multiple testing strategies are required for comprehensive genetic testing in myeloid malignancies. The aim of this proof-of-principle study was to investigate the feasibility of combining detection of genome-wide large CNVs, CN-LOH and targeted gene mutations into a single assay using next-generation sequencing (NGS). METHODS: For genome-wide CNV detection, we designed a single nucleotide polymorphism (SNP) sequencing backbone with 22 762 SNP regions evenly distributed across the entire genome. For targeted mutation detection, 62 frequently mutated genes in myeloid malignancies were targeted. We combined this SNP sequencing backbone with a targeted mutation panel, and sequenced 9 healthy individuals and 16 patients with myeloid malignancies using NGS. RESULTS: We detected 52 somatic CNVs, 11 instances of CN-LOH and 39 oncogenic mutations in the 16 patients with myeloid malignancies, and none in the 9 healthy individuals. All CNVs and CN-LOH were confirmed by SNP microarray analysis. CONCLUSIONS: We describe a genome-wide SNP sequencing backbone which allows for sensitive detection of genome-wide CNVs and CN-LOH using NGS. This proof-of-principle study has demonstrated that this strategy can provide more comprehensive genetic profiling for patients with myeloid malignancies using a single assay.


Assuntos
Variações do Número de Cópias de DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Transtornos Mieloproliferativos/genética , Feminino , Humanos , Masculino , Polimorfismo de Nucleotídeo Único/genética
13.
J Mol Diagn ; 9(2): 169-77, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17384208

RESUMO

Phosphorylation of tyrosine residues by protein tyrosine kinases mediates numerous cellular processes. Deregulated tyrosine phosphorylation underlies constitutive activation of signaling pathways leading to oncogenesis. Analytical techniques for evaluation of the global phosphoproteome level are challenging and can be improved on to enhance yields. Here, we evaluated several approaches to enrich for tyrosine phosphoproteins in cancer cells for subsequent liquid chromatography-tandem mass spectrometry analysis using lysates from SU-DHL-1 cells, which express the nucleophosmin-anaplastic lymphoma kinase tyrosine kinase as a model system. Cells were grown in the presence or absence of the phosphatase inhibitor sodium orthovanadate, and tyrosine phosphoproteins were subsequently enriched by immunoprecipitation or immunoaffinity chromatography and protein identification performed by liquid chromatography-tandem mass spectrometry. Our results show that sodium orthovanadate improves enrichment and thus detection of tyrosine phosphoproteins. Immunoprecipitation of tyrosine phosphoproteins using two different antiphosphotyrosine antibodies increased the number of protein identifications. Finally, peptides from proteins enriched by immunoprecipitation were more abundant (n=338) than those enriched by immunoaffinity chromatography (n=138), and relatively few proteins were found in common (n=43). Our data demonstrate the utility of an enrichment strategy for the mass spectrometry-based identification of tyrosine phosphoproteins and show the advantage of complementary techniques for greater protein identification.


Assuntos
Espectrometria de Massas/métodos , Proteínas de Neoplasias/análise , Neoplasias/metabolismo , Fosfoproteínas/análise , Fosfotirosina/análise , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Cromatografia de Afinidade , Cromatografia Líquida , Humanos , Imunoprecipitação , Dados de Sequência Molecular , Proteínas de Neoplasias/química , Neoplasias/patologia , Fosfoproteínas/química , Fosfotirosina/química , Vanadatos/farmacologia
14.
Methods Mol Biol ; 1633: 151-161, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28735486

RESUMO

Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR)-based detection of abnormal fusion transcripts is an important strategy for the diagnosis and monitoring of patients with acute myeloid leukemia (AML) with t(8;21)(q22;q22); RUNX1-RUNX1T1, inv(16)(p13.1;q22); CBFB-MYH11 or t(15;17)(q22;q12); PML-RARA. In RT-qPCR assays, patient-derived cDNA is subjected to amplification using PCR primers directed against the fusion transcript of interest as well as a reference gene for normalization. Quantification is typically performed by constructing standard curves for each PCR run using a series of plasmid standards of known concentration that harbor the same fusion transcript or the same reference gene of interest. Fusion transcripts and reference gene copy numbers are then calculated in patient samples using these standard curves. The process of constructing standard curves is laborious and consumes additional reagents. In this chapter, we give the method details for a multiplex RT-qPCR strategy to detect and quantify the acute myeloid leukemia (AML)-associated fusion transcripts PML-RARA in patients with t(15;17) without the need for standard curves. This general method can also be applied to other AML-associated fusion transcripts such as CBFB-MYH11 and RUNX1-RUNX1T1.


Assuntos
Leucemia Mieloide Aguda/diagnóstico , Proteínas de Fusão Oncogênica/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Translocação Genética , Humanos , Leucemia Mieloide Aguda/genética , Plasmídeos , Padrões de Referência
15.
Leuk Res ; 56: 7-12, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28152414

RESUMO

NPM1 insertion mutations represent a common recurrent genetic abnormality in acute myeloid leukemia (AML) patients. The frequency of these mutations varies from approximately 30% overall up to 50% in patients with a normal karyotype. Several recent studies have exploited advances in massively parallel sequencing technology to shed light on the complex genomic landscape of AML. We hypothesize that variant allele fraction (VAF) data derived from massively parallel sequencing studies may provide further insights into the clonal architecture and pathogenesis of NPM1-driven leukemogenesis. Diagnostic peripheral blood or bone marrow samples from NPM1-mutated AML patients (n=120) were subjected to targeted sequencing using a panel of fifty-seven genes known to be commonly mutated in myeloid malignancies. NPM1 mutations were always accompanied by additional mutations and NPM1 had the highest VAF in only one case. Nearly all NPM1-mutated AML patients showed concurrent mutations in genes involved in regulation of DNA methylation (DNMT3A, TET2, IDH1, IDH2), RNA splicing (SRSF2, SF3B1), or in the cohesin complex (RAD21, SMC1A, SMC3, STAG2). Mutations in these genes had higher median VAFs that were higher (40% or greater) than the co-existing NPM1 mutations (median VAF 16.8%). Mutations associated with cell signaling pathways (FLT3, NRAS, and PTPN11) are also frequently encountered in NPM1-mutated AML cases, but had relatively low VAFs (7.0-11.9%). No cases of NPM1-mutated AML with a concurrent IDH2R172 mutation were observed, suggesting that these variants are mutually exclusive. Overall, these data suggest that NPM1 mutations are a secondary or late event in the pathogenesis of AML and are preceded by founder mutations in genes that may be associated with recently described preclinical states such as clonal hematopoiesis of indeterminate potential or clonal cytopenias of undetermined significance.


Assuntos
Leucemia Mieloide Aguda/genética , Mutação , Proteínas Nucleares/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Metilação de DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Nucleofosmina , Splicing de RNA/genética , Coesinas
16.
PLoS One ; 12(9): e0184154, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28880927

RESUMO

We demonstrate that SCF-KIT signaling induces synthesis and secretion of endothelin-3 (ET3) in human umbilical vein endothelial cells and melanoma cells in vitro, gastrointestinal stromal tumors, human sun-exposed skin, and myenteric plexus of human colon post-fasting in vivo. This is the first report of a physiological mechanism of ET3 induction. Integrating our finding with supporting data from literature leads us to discover a previously unreported pathway of nitric oxide (NO) generation derived from physiological endothelial NO synthase (eNOS) or neuronal NOS (nNOS) activation (referred to as the KIT-ET3-NO pathway). It involves: (1) SCF-expressing cells communicate with neighboring KIT-expressing cells directly or indirectly (cleaved soluble SCF). (2) SCF-KIT signaling induces timely local ET3 synthesis and secretion. (3) ET3 binds to ETBR on both sides of intercellular space. (4) ET3-binding-initiated-ETBR activation increases cytosolic Ca2+, activates cell-specific eNOS or nNOS. (5) Temporally- and spatially-precise NO generation. NO diffuses into neighboring cells, thus acts in both SCF- and KIT-expressing cells. (6) NO modulates diverse cell-specific functions by NO/cGMP pathway, controlling transcriptional factors, or other mechanisms. We demonstrate the critical physiological role of the KIT-ET3-NO pathway in fulfilling high demand (exceeding basal level) of endothelium-dependent NO generation for coping with atherosclerosis, pregnancy, and aging. The KIT-ET3-NO pathway most likely also play critical roles in other cell functions that involve dual requirement of SCF-KIT signaling and NO. New strategies (e.g. enhancing the KIT-ET3-NO pathway) to harness the benefit of endogenous eNOS and nNOS activation and precise NO generation for correcting pathophysiology and restoring functions warrant investigation.


Assuntos
Endotelina-3/metabolismo , Óxido Nítrico/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptor de Endotelina B/metabolismo , Fator de Células-Tronco/metabolismo , Aterosclerose/patologia , Linhagem Celular Tumoral , Endotélio Vascular/metabolismo , Ensaio de Imunoadsorção Enzimática , Motilidade Gastrointestinal , Tumores do Estroma Gastrointestinal/metabolismo , Tumores do Estroma Gastrointestinal/patologia , Tumores do Estroma Gastrointestinal/fisiopatologia , Homeostase , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Imuno-Histoquímica , Melanoma/patologia , Plexo Mientérico/metabolismo , Invasividade Neoplásica , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais , Pele/metabolismo , Luz Solar , Fatores de Tempo , Regulação para Cima/genética , Vasodilatação
17.
J Mol Diagn ; 6(1): 16-21, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14736822

RESUMO

The requirement for sufficient quantities of starting RNA has limited the ability to evaluate multiple transcripts using reverse transcriptase-polymerase chain reaction (RT-PCR). In this study, we demonstrate the utility of linear RNA amplification for RT-PCR analysis of multiple gene transcripts including a chromosomal translocation, using the t(2;5)(p23;q35) as a model. RNA from the t(2;5)-positive cell line, SU-DHL-1, and the t(2;5)-negative cell line, HUT-78, was extracted and exposed to two rounds of linear amplification. RT-PCR using cDNA from the resultant amplified (a) RNA and total RNA resulted in the 177 bp NPM-ALK fusion gene product from the SU-DHL-1 cell line, but not from aRNA or total RNA from the HUT-78 cell line. DNA sequencing of the RT-PCR products from total and aRNA of SU-DHL-1 cells demonstrated identical sequences corresponding to the NPM-ALK fusion gene. Evaluation of 25 snap-frozen tissue samples, including eight NPM-ALK-positive ALCLs demonstrated 100% concordance of t(2;5) detection between cDNA from total RNA and that from aRNA. Our results show that linear amplification of RNA can enhance starting RNA greater than 200-fold and can be used for rapid and specific detection of multiplex gene expression from a variety of sources. This method can generate a renewable archive of representative cDNA, which can be used for retrospective screening of stored samples as well as positive controls for the clinical molecular diagnostic laboratory.


Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Translocação Genética , Linhagem Celular Tumoral , Cromossomos Humanos Par 2/genética , Cromossomos Humanos Par 5/genética , Humanos , Proteínas de Fusão Oncogênica/genética , Proteínas Tirosina Quinases/genética , Reprodutibilidade dos Testes
18.
Am J Clin Pathol ; 141(3): 348-59, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24515762

RESUMO

OBJECTIVES: To design and evaluate a next-generation sequencing (NGS)-based method for T-cell receptor γ (TCRG) gene-based T-cell clonality testing on the Ion Torrent Personal Genome Machine (Life Technologies, Carlsbad, CA) platform. METHODS: We analyzed a series of peripheral blood, bone marrow, and formalin-fixed paraffin-embedded tissue specimens with NGS vs traditional capillary electrophoresis methods. RESULTS: Using a custom analysis algorithm that we developed, our NGS assay identified between 2,215 and 48,222 unique TCRG rearrangements in a series of 48 samples. We established criteria for assigning clonality based on parameters derived from both the relative and absolute frequencies of reads. In a comparison with standard capillary electrophoresis, 19 of 19 polyclonal samples and 24 of 27 samples that appeared clonal were in agreement. The three discrepant samples demonstrated some of the pitfalls of amplicon length-based testing. Dilution studies with T-lymphoid cell lines demonstrated that a known clonal sequence could be routinely identified when present in as few as 0.1% of total cells demonstrating suitability in residual disease testing. A series of samples was also analyzed on a second NGS platform and yielded very similar results with respect to the frequency and sequence of the clonal rearrangement. CONCLUSIONS: In this proof-of-concept study, we describe an NGS-based T-cell clonality assay that is suitable for routine clinical testing either alone or as an adjunct to traditional methods.


Assuntos
Eletroforese Capilar , Rearranjo Gênico da Cadeia gama dos Receptores de Antígenos dos Linfócitos T/genética , Sequenciamento de Nucleotídeos em Larga Escala , Transtornos Linfoproliferativos/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Linhagem Celular , Humanos , Linfoma de Células T/genética
19.
J Clin Pathol ; 64(7): 618-25, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21486895

RESUMO

AIMS: The BCR-ABL1 T315I mutation imparts resistance to tyrosine kinase inhibitors currently available for treatment of chronic myelogenous leukaemia. Thus, quantitative monitoring of the emergence and expansion of T315I-positive subclones may be clinically useful. The goals of this study were to retrospectively review the authors' experience with Sanger sequencing-based BCR-ABL1 kinase domain mutation testing, paying particular attention to the T315I mutation, and to develop an alternative test for relative quantification of T315I using pyrosequencing. METHODS: The performance of a new T315I pyrosequencing assay was evaluated. Total RNA was isolated from whole blood and reverse-transcribed. The resulting cDNA was subjected to an initial round of PCR across the BCR-ABL1 breakpoint followed by a second round to amplify the sequence flanking ABL1 codon 315. The final PCR product was pyrosequenced to detect and quantify the T315I point mutation. Additional experiments were carried out to determine the effects of background untranslocated ABL1 on assay sensitivity in samples with low tumour burden. RESULTS: The results show that T315I was the most commonly detected kinase domain mutation and was persistent in follow-up testing. All 26 specimens that tested positive by Sanger sequencing for the T315I mutation were also positive using the pyrosequencing test. Relative quantification data derived from pyrosequencing matched the approximate wild-type/mutant ratios found by Sanger sequencing. Serial dilution experiments show sensitivity to 5% mutant allele. The authors also quantitatively assessed the influence of untranslocated ABL1 in the sample background on the assay and found that it occurred at levels not likely to influence performance. CONCLUSION: The described test is useful for detection and relative quantification of the T315I point mutation in chronic myelogenous leukaemia in a sensitive, specific and reproducible manner.


Assuntos
Análise Mutacional de DNA/métodos , Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Mutação Puntual , Sequência de Bases , Humanos , Dados de Sequência Molecular , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade
20.
Proteomics ; 7(15): 2603-16, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17610208

RESUMO

The molecular chaperone heat shock protein 90 (Hsp90) affects the function of many oncogenic signaling proteins including nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expressed in anaplastic large cell lymphoma (ALCL). While ALK-positive ALCL cells are sensitive to the Hsp90 inhibitor and the geldanamycin (GA) analog, 17-allylamino-17-demethoxygeldanamycin (17-AAG), the proteomic effects of these drugs on ALK-positive ALCL cells are unpublished. In this study, we investigated the cellular, biologic, and proteomic changes occurring in ALK-positive ALCL cells in response to GA treatment. GA induced G2/M cell cycle arrest and caspase-3-mediated apoptosis. Furthermore, quantitative proteomic changes analyzed by cleavable isotope-coded affinity tag-LC-MS/MS (cICAT-LC-MS/MS) identified 176 differentially expressed proteins. Out of these, 49 were upregulated 1.5-fold or greater and 70 were downregulated 1.5-fold or greater in GA-treated cells. Analysis of biological functions of differentially expressed proteins revealed diverse changes, including induction of proteins involved in the 26S proteasome as well as downregulation of proteins involved in signal transduction and protein and nucleic acid metabolism. Pathway analysis revealed changes in MAPK, WNT, NF-kappaB, TGFbeta, PPAR, and integrin signaling components. Our studies reveal some of the molecular and proteomic consequences of Hsp90 inhibition in ALK-positive ALCL cells and provide novel insights into the mechanisms of its diverse cellular effects.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Benzoquinonas/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lactamas Macrocíclicas/farmacologia , Linfoma Difuso de Grandes Células B/metabolismo , Proteoma/análise , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Caspase 3/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA de Neoplasias/análise , Proteínas de Choque Térmico HSP90/fisiologia , Humanos , Linfoma Difuso de Grandes Células B/enzimologia , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Modelos Biológicos , Reprodutibilidade dos Testes
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