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OBJECTIVE: Somatic mutations in ASXL1 seem to have a negative prognostic impact in patients with several myeloid neoplasms, including myelofibrosis (MF). The aim of this work was to determine the prevalence and profile of ASXL1 mutations in MF. METHODS: We analyzed mutations in ASXL1 in 70 consecutive MF patients from 8 Spanish hospitals by means of Sanger sequencing, as well as JAK2, CALR, and MPL mutations. RESULTS: ASXL1 mutations were found in 16/70 (23%) of cases, most commonly p.Gly646TrpfsX12 (5/16). Most mutations (13/16) were frameshift mutations. Of 54 ASXL1- wild-type patients, 32 (59%) had at least one single nucleotide polymorphism (SNP), 27 of them had g.78128C>T, g.79017A>C, and g.79085T>C [triple SNP (TSNP) patients]. The 5-yr overall survival probability of TSNP patients was 67% (95% CI, 43-91%) vs. 90% (95% CI, 77-100%) in ASXL1-WT patients (P = 0.152). CONCLUSION: ASXL1 mutations were found in 23% of cases, p.Gly646TrpfsX12 being the most frequent. About 85% of mutations were found only in individual cases and 46% had not previously been reported, a pattern also seen in other series. Fifty percent of ASXL1-WT patients had a combination of three specific SNPs that might have a prognostic correlation that needs to be determined in larger series.
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Mutação , Mielofibrose Primária/genética , Proteínas Repressoras/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise Mutacional de DNA , Éxons , Feminino , Humanos , Janus Quinase 2/genética , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Mielofibrose Primária/diagnóstico , Mielofibrose Primária/mortalidade , Adulto JovemRESUMO
Tyrosine kinase inhibitors (TKI) have improved the management of patients with chronic myeloid leukemia (CML). However, a significant proportion of patients does not achieve the optimal response or are resistant to TKI. ABL1 kinase domain mutations have been extensively implicated in the pathogenesis of TKI resistance. Although deletion or insertion of nucleotides in BCR-ABL1 has rarely been described, we identified a CML patient with an already described 35 nucleotides insertion (BCR-ABL1(35INS)) of controversial significance, that confers resistance to imatinib but sensitivity to dasatinib.
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Benzamidas/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Mutagênese Insercional , Nucleotídeos/química , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , Adulto , Sequência de Bases , Dasatinibe , Éxons , Feminino , Humanos , Mesilato de Imatinib , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Tiazóis/uso terapêuticoAssuntos
Análise Mutacional de DNA/métodos , DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala , Mielofibrose Primária/genética , Receptores de Trombopoetina/genética , Trombocitemia Essencial/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Proteínas de Ligação a DNA/genética , Dioxigenases , Éxons/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/genética , Mielofibrose Primária/diagnóstico , Mielofibrose Primária/mortalidade , Prognóstico , Proteínas Proto-Oncogênicas/genética , Fatores de Processamento de RNA/genética , Trombocitemia Essencial/diagnóstico , Trombocitemia Essencial/mortalidadeRESUMO
e13a2 and e14a2 are the most frequent transcript types of the BCR::ABL1 fusion gene in chronic myeloid leukemia (CML). The current goal with tyrosine kinase inhibitors (TKI) is to achieve sustained deep molecular response (DMR) in order to discontinue TKI treatment and remain in the so-called treatment-free remission (TFR) phase, but biological factors associated with these goals are not well established. This study aimed to determine the effect of transcript type on TFR in patients receiving frontline treatment with imatinib (IM) or second-generation TKI (2G-TKI). Patients treated at least 119 months with IM presented less post-discontinuation relapse than those that discontinued IM before 119 months (p = 0.005). In addition, cases with the e14a2 transcript type treated at least 119 months with IM presented a better TFR (p = 0.024). On the other hand, the type of transcript did not affect the cytogenetic or molecular response in 2G-TKI treated patients; however, the use of 2G-TKI may be associated with higher and earlier DMR in patients with the e14a2 transcript.
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BACKGROUND: High-quality data on bone marrow involvement (BMI) assessed by flow cytometry (FC) in follicular lymphoma (FL) is lacking. AIMS: We set up a prospective protocol with a 10-color tube and acquisition of 500.000 leukocytes on a Nav flow cytometer for evaluation of BMI in FL by FC. MATERIALS AND METHODS: FC was compared with a combination of histopathology and IGH gene rearrangement, which were considered the gold standard. We also compared BMI by FC with PET. RESULTS: Fifty-two patients were included (median 67 years, 54% female). BMI by FC was seen in 35 (67%), with a median involvement of 1.2% (interquartile range: 0.3%-7%) of leukocytes. Comparison with the gold standard revealed two false negatives and two false positives (potentially true involvement undetected by the gold standard). BMI by PET was seen in 14/46 (30%). Immunophenotype of FL in the bone marrow was highly heterogeneous. The most common phenotypic abnormality was dim expression of CD19 (>0.5 log loss in 30% of patients). CD10 was negative in 13 (37%) and incompletely positive (overlap with the negative population) in a further 8 (28%) while entirely positive only in 14 (48%). Other abnormalities (loss of CD20, gain or loss of CD79b, expression of CD43, and substantial loss of CD45) were rare. Computational analysis by means of FlowSOM confirmed the heterogeneous phenotype, with FL from different patients clustering in unrelated metaclusters. CONCLUSION: BMI by FL was frequent and immunophenotype was heterogeneous. However, this protocol enabled detection of FL in bone marrow in the vast majority of patients with bone marrow involvement by the gold standard.
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Linfoma Folicular , Feminino , Humanos , Masculino , Linfoma Folicular/genética , Citometria de Fluxo/métodos , Medula Óssea/patologia , Estudos Prospectivos , ImunofenotipagemRESUMO
Tyrosine kinase inhibitors have dramatically changed the outcome of chronic myeloid leukemia (CML), and nowadays, one of the main treatment goals is the achievement of deep molecular responses (DMRs), which can eventually lead to therapy discontinuation approaches. Few biological factors at diagnosis have been associated with this level of response. Telomere length (TL) in peripheral blood cells of patients with CML has been related to disease stage, response to therapy and disease progression, but little is known about its role on DMR. In this study, we analyzed if age-adjusted TL (referred as "delta-TL") at diagnosis of chronic phase (CP)-CML might correlate with the achievement of DMR under first-line imatinib treatment. TL from 96 CP-CML patients had been retrospectively analyzed at diagnosis by monochrome multiplex quantitative PCR. We observed that patients with longer age-adjusted telomeres at diagnosis had higher probabilities to achieve DMR with imatinib than those with shortened telomeres (P = 0.035 when delta-TL was studied as a continuous variable and P = 0.047 when categorized by the median). Moreover, patients carrying long telomeres also achieved major molecular response significantly earlier (P = 0.012). This study provides proof of concept that TL has a role in CML biology and when measured at diagnosis of CP-CML could help to identify patients likely to achieve DMR to first-line imatinib treatment.
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Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological diseases. Among them, the most well characterized subtype is MDS with isolated chromosome 5q deletion (MDS del(5q)), which is the only one defined by a cytogenetic abnormality that makes these patients candidates to be treated with lenalidomide. During the last decade, single cell (SC) analysis has emerged as a powerful tool to decipher clonal architecture and to further understand cancer and other diseases at higher resolution level compared to bulk sequencing techniques. In this study, a SC approach was used to analyze intratumoral heterogeneity in four patients with MDS del(5q). Single CD34+CD117+CD45+CD19- bone marrow hematopoietic stem progenitor cells were isolated using the C1 system (Fluidigm) from diagnosis or before receiving any treatment and from available follow-up samples. Selected somatic alterations were further analyzed in SC by high-throughput qPCR (Biomark HD, Fluidigm) using specific TaqMan assays. A median of 175 cells per sample were analyzed. Inferred clonal architectures were relatively simple and either linear or branching. Similar to previous studies based on bulk sequencing to infer clonal architecture, we were able to observe that an ancestral event in one patient can appear as a secondary hit in another one, thus reflecting the high intratumoral heterogeneity in MDS del(5q) and the importance of patient-specific molecular characterization.
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The most frequent BCR-ABL1-p210 transcripts in chronic myeloid leukemia (CML) are e14a2 and e13a2. Imatinib (IM) is the most common first-line tyrosine-kinase inhibitor (TKI) used to treat CML. Some studies suggest that BCR-ABL1 transcript types confer different responses to IM. The objective of this study was to correlate the expression of e14a2 or e13a2 to clinical characteristics, cumulative cytogenetic and molecular responses to IM, acquisition of deep molecular response (DMR) and its duration (sDMR), progression rate (CIP), overall survival (OS), and treatment-free remission (TFR) rate. We studied 202 CML patients, 76 expressing the e13a2 and 126 the e14a2, and correlated the differential transcript expression with the above-mentioned parameters. There were no differences in the cumulative incidence of cytogenetic responses nor in the acquisition of DMR and sDMR between the two groups, but the e14a2 transcript had a positive impact on molecular response during the first 6 months, whereas the e13a2 was associated with improved long-term OS. No correlation was observed between the transcript type and TFR rate.
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BACKGROUND: Within the hematopoietic compartment, fibromodulin (FMOD) is almost exclusively expressed in chronic lymphocytic leukemia (CLL) lymphocytes. We set out to determine whether FMOD could be of help in diagnosing borderline lymphoproliferative disorders (LPD). METHODS: We established 3 flow cytometry-defined groups (CLL [n = 65], borderline LPD [n = 28], broadly defined as those with CLLflow score between 35 and -20 or discordant CD43 and CLLflow, and non-CLL LPD [n = 40]). FMOD expression levels were determined by standard RT-PCR in whole-blood samples. Patients were included regardless of lymphocyte count but with tumor burden ≥40%. RESULTS: FMOD expression levels distinguished between CLL (median 98.5, interquartile range [IQR] 37.8-195.1) and non-CLL LPD (median 0.012, IQR 0.003-0.033) with a sensitivity and specificity of 1. Most borderline LPDs were CD5/CD23/CD200-positive with no loss of B-cell antigens and negative or partial expression of CD43. 16/22 patients with available cytogenetic analysis showed trisomy 12. In 25/28 (89%) of these patients, FMOD expression levels fell between CLL and non-CLL (median 3.58, IQR 1.06-6.21). DISCUSSION: This study could suggest that borderline LPDs may constitute a distinct group laying in the biological spectrum of chronic leukemic LPDs. Future studies will have to confirm these results with other biological data. Quantification of FMOD can potentially be of help in the diagnosis of phenotypically complex LPDs.
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Fibromodulina/sangue , Citometria de Fluxo/métodos , Leucemia Linfocítica Crônica de Células B/sangue , Transtornos Linfoproliferativos/sangue , Idoso , Idoso de 80 Anos ou mais , Citodiagnóstico/métodos , Diagnóstico Diferencial , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Contagem de Linfócitos , Transtornos Linfoproliferativos/patologia , MasculinoRESUMO
Myelodysplastic syndromes (MDS) are stem cell disorders caused by various gene abnormalities. We performed targeted deep sequencing in 39 patients with high-risk MDS and secondary acute myeloid leukemia (sAML) at diagnosis and follow-up (response and/or relapse), with the aim to define their mutational status, to establish if specific mutations are biomarkers of response to 5-azacytidine (AZA) and/or may have impact on survival. Overall, 95% of patients harbored at least one mutation. TP53, DNMT3A and SRSF2 were the most frequently altered genes. Mutations in TP53 correlated with higher risk features and shorter overall survival (OS) and progression free survival (PFS) in univariate analysis. Patients with SRSF2 mutations were associated with better OS and PFS. Response rate was 55%; but we could not correlate the presence of TET2 and TP53 mutations with AZA response. Patients with sAML presented more variations than patients with high-risk MDS, and usually at relapse the number of mutations increased, supporting the idea that in advanced stages of the disease there is a greater genomic complexity. These results confirm that mutation analysis can add prognostic value to high-risk MDS and sAML patients, not only at diagnosis but also at follow-up.
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BACKGROUND: The mutational status of immunoglobulin heavy-chain variable-region (IgVH) genes in the leukemic cells of chronic lymphocytic leukemia (CLL) is an important prognostic factor in the disease. We investigated whether the expression of ZAP-70 by CLL cells correlated with the IgVH mutational status, disease progression, and survival. METHODS: The expression of ZAP-70 was analyzed in T-cell and B-cell lines and in peripheral-blood samples from 56 patients with CLL with the use of flow cytometry, Western blotting, and immunohistochemistry. The results were correlated with the IgVH mutational status and clinical outcome. RESULTS: ZAP-70 was detected by flow-cytometric analysis in cells of T-cell lineage and in leukemic cells from 32 of 56 patients with CLL. In all patients in whom at least 20 percent of the leukemic cells were positive for ZAP-70, IgVH was unmutated, whereas IgVH mutations were found in 21 of 24 patients in whom less than 20 percent of the leukemic cells were positive for ZAP-70 (P<0.001). Concordant results were obtained when ZAP-70 expression was assessed by immunohistochemistry or Western blotting. The level of ZAP-70 expression did not change over time (median, 37 months) in sequential samples from 30 patients with CLL. Patients with Binet stage A CLL who had at least 20 percent ZAP-70-positive leukemic cells had more rapid progression and poorer survival than those with less than 20 percent ZAP-70-positive cells. CONCLUSIONS: Among patients with CLL, expression of ZAP-70, as detected by flow-cytometric analysis, correlated with IgVH mutational status, disease progression, and survival.
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Genes de Imunoglobulinas , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Proteínas Tirosina Quinases/genética , ADP-Ribosil Ciclase/análise , ADP-Ribosil Ciclase 1 , Antígenos CD/análise , Progressão da Doença , Citometria de Fluxo , Humanos , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/mortalidade , Glicoproteínas de Membrana , Prognóstico , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Análise de Sobrevida , Proteína-Tirosina Quinase ZAP-70RESUMO
PURPOSE: To determine the methylthioadenosine phosphorylase (MTAP) gene alterations in mantle cell lymphoma (MCL) and to investigate whether the targeted inactivation of the alternative de novo AMP synthesis pathway may be a useful therapeutic strategy in tumors with inactivation of this enzyme. EXPERIMENTAL DESIGN: MTAP gene deletion and protein expression were studied in 64 and 52 primary MCL, respectively, and the results were correlated with clinical behavior. Five MCL cell lines were analyzed for MTAP expression and for the in vitro sensitivity to L-alanosine, an inhibitor of adenylosuccinate synthetase, and hence de novo AMP synthesis. RESULTS: No protein expression was detected in 8 of 52 (15%) tumors and one cell line (Granta 519). Six of these MTAP negative tumors and Granta 519 cell line had a codeletion of MTAP and p16 genes; one case showed a deletion of MTAP, but not p16, and one tumor had no deletions in neither of these genes. Patients with MTAP deletions had a significant shorter overall survival (mean, 16.1 months) than patients with wild-type MTAP (mean, 63.6 months; P < 0.0001). L-Alanosine induced cytotoxicity and activation of the intrinsic mitochondrial-dependent apoptotic pathway in MCL cells. 9-beta-D-Erythrofuranosyladenine, an analogue of 5'-methylthioadenosine, selectively rescued MTAP-positive cells from L-alanosine toxicity. CONCLUSIONS: MTAP gene deletion and lack of protein expression are associated with poor prognosis in MCL and might identify patients who might benefit from treatment with de novo AMP synthesis pathway-targeted therapies.
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Linfoma de Célula do Manto/enzimologia , Purina-Núcleosídeo Fosforilase/deficiência , Purina-Núcleosídeo Fosforilase/genética , Sequência de Bases , Linhagem Celular Tumoral , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 14 , Primers do DNA , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/mortalidade , Linfoma de Célula do Manto/patologia , Estudos Retrospectivos , Análise de Sobrevida , Fatores de Tempo , Translocação GenéticaRESUMO
BACKGROUND AND OBJECTIVES: Nucleoside transporters might play a relevant role in the intracellular targeting of many nucleoside analogs used in anticancer therapy. Two gene families (SLC28 and SLC29) encode the two types of human nucleoside transporters, concentrative nucleoside transporter (CNT) and equilibrative nucleoside transporter (ENT) proteins. Chronic lymphocytic leukemia (CLL) cells express both SLC28- and SLC29-related mRNA, although transport function seems to be mostly related to ENT-type transporters. Here we have analyzed the role of nucleoside transporters in nucleoside-derived drug bioavailability and action in mantle cell lymphoma (MCL) cells. DESIGN AND METHODS: The relative amounts of hENT1 and hENT2-related mRNA and protein were analyzed in five MCL cell lines and 20 primary MCL tumors by real-time quantitative reverse transcriptase polymerase chain reaction and western blots. Cell viability, measured by annexin V-FITC staining, and nucleoside-derived drug transport were also studied. RESULTS: MCL cells express higher levels of hENT1 protein than do CLL cells, and a good correlation was found between protein and mRNA levels of hENT1, thus indirectly suggesting that hENT1 might be transcriptionally regulated in MCL cells. More importantly, a significant correlation between these two parameters, drug uptake and sensitivity to gemcitabine, was also observed. INTERPRETATION AND CONCLUSIONS: These results further support the concept that nucleoside transporters are implicated in the therapeutic response to nucleoside analogs, and suggest a particular and novel role for hENT1 in the genotoxic response to selected nucleoside analogs, such as gemcitabine, in MCL cells.
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Desoxicitidina/análogos & derivados , Transportador Equilibrativo 1 de Nucleosídeo/análise , Linfoma de Célula do Manto/tratamento farmacológico , Linhagem Celular Tumoral , Desoxicitidina/farmacocinética , Desoxicitidina/uso terapêutico , Transportador Equilibrativo 1 de Nucleosídeo/genética , Humanos , Linfoma de Célula do Manto/patologia , Nucleosídeos/farmacocinética , RNA Mensageiro/análise , GencitabinaRESUMO
Clonal cytogenetic abnormalities are found in 20-30% of patients with chronic myelomonocytic leukemia (CMML), while gene mutations are present in >90% of cases. Patients with low risk cytogenetic features account for 80% of CMML cases and often fall into the low risk categories of CMML prognostic scoring systems, but the outcome differs considerably among them. We performed targeted deep sequencing of 83 myeloid-related genes in 56 CMML patients with low risk cytogenetic features or uninformative conventional cytogenetics (CC) at diagnosis, with the aim to identify the genetic characteristics of patients with a more aggressive disease. Targeted sequencing was also performed in a subset of these patients at time of acute myeloid leukemia (AML) transformation. Overall, 98% of patients harbored at least one mutation. Mutations in cell signaling genes were acquired at time of AML progression. Mutations in ASXL1, EZH2 and NRAS correlated with higher risk features and shorter overall survival (OS) and progression free survival (PFS). Patients with SRSF2 mutations associated with poorer OS, while absence of TET2 mutations (TET2wt) was predictive of shorter PFS. A decrease in OS and PFS was observed as the number of adverse risk gene mutations (ASXL1, EZH2, NRAS and SRSF2) increased. On multivariate analyses, CMML-specific scoring system (CPSS) and presence of adverse risk gene mutations remained significant for OS, while CPSS and TET2wt were predictive of PFS. These results confirm that mutation analysis can add prognostic value to patients with CMML and low risk cytogenetic features or uninformative CC.
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Sequenciamento de Nucleotídeos em Larga Escala , Leucemia Mielomonocítica Crônica/genética , Idoso , Transformação Celular Neoplásica , Aberrações Cromossômicas , Análise Mutacional de DNA , Progressão da Doença , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Cariotipagem , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Leucemia Mielomonocítica Crônica/diagnóstico , Perda de Heterozigosidade , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Mutação , Prognóstico , Resultado do TratamentoRESUMO
Mantle cell lymphoma (MCL) is a mature B-cell proliferation characterized by the presence of translocation t(11;14)(q13;q32), an aggressive clinical course, and poor response to chemotherapy. The majority of drugs currently used in the treatment of lymphoproliferative disorders induce cell death by triggering apoptosis, but few data concerning drug-induced apoptosis in MCL have been reported. We have analysed the mechanisms of drug-induced cell death in four cell lines with the t(11;14) and in primary cells from 10 patients with MCL. Mitoxantrone, a topoisomerase II inhibitor, induced a strong cytotoxic effect in three cell lines (JVM-2, REC-1, and Granta 519), and in primary MCL cells. This cytotoxic effect due to apoptosis induction was observed despite the presence of either p53 or ATM abnormalities. However, no cytotoxic effect was detected after incubation with DNA-damaging agents in the NCEB-1 cell line, carrying p53 and ATM alterations, despite the presence of functional mitochondrial machinery. These results support that mitoxantrone can be effective in the treatment of MCL but that this activity requires the integrity of functional DNA-damage response genes.
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Apoptose , Ciclofosfamida/análogos & derivados , Linfoma de Célula do Manto/patologia , Mitocôndrias/patologia , Mitoxantrona/farmacologia , Vidarabina/análogos & derivados , Anexina A5/farmacologia , Antineoplásicos/farmacologia , Ciclo Celular , Morte Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Corantes/farmacologia , Ciclofosfamida/farmacologia , Dano ao DNA , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Genes p53 , Humanos , Hibridização in Situ Fluorescente , Leucócitos Mononucleares/metabolismo , Potenciais da Membrana , Mitocôndrias/metabolismo , Proteínas/química , Espécies Reativas de Oxigênio , Estaurosporina/farmacologia , Fatores de Tempo , Translocação Genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Vidarabina/farmacologiaRESUMO
In up to 5-15% of studies of lymphoproliferative disorders (LPD), flow cytometry (FCM) or immunomorphologic methods cannot discriminate malignant from reactive processes. The aim of this work was to determine the usefulness of PCR for solving these diagnostic uncertainties. We analyzed IGH and TCRγ genes by PCR in 106 samples with inconclusive FCM results. A clonal result was registered in 36/106 studies, with a LPD being confirmed in 27 (75%) of these cases. Specifically, 9/9 IGH clonal and 16/25 TCRγ clonal results were finally diagnosed with LPD. Additionally, two clonal TCRγ samples with suspicion of undefined LPD were finally diagnosed with T LPD. Although polyclonal results were obtained in 47 of the cases studied (38 IGH and nine TCRγ), hematologic neoplasms were diagnosed in 4/38 IGH polyclonal and in 1/9 TCRγ polyclonal studies. There were also 14 PCR polyclonal results (four IGH, 10 TCRγ), albeit nonconclusive. Of these, 2/4 were eventually diagnosed with B-cell lymphoma and 3/10 with T-cell LPD. In eight IGH samples, the results of PCR techniques were noninformative but in 3/8 cases a B lymphoma was finally confirmed. We concluded that PCR is a useful technique to identify LPD when FCM is inconclusive. A PCR clonal B result is indicative of malignancy but IGH polyclonal and nonconclusive results do not exclude lymphoid neoplasms. Interpretation of T-cell clonality should be based on all the available clinical and analytical data.
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Genes Codificadores da Cadeia gama de Receptores de Linfócitos T/genética , Cadeias Pesadas de Imunoglobulinas/genética , Linfoma de Células B/diagnóstico , Linfoma de Células T/diagnóstico , Reação em Cadeia da Polimerase/métodos , Linfócitos B/citologia , DNA/análise , Citometria de Fluxo , Imunofluorescência , Humanos , Imunofenotipagem , Linfócitos T/citologiaRESUMO
In up to 5-15% of studies of lymphoproliferative disorders (LPD) flow cytometry (FCM) or immunomorphologic methods cannot discriminate malignant from reactive processes. The aim of this work was to determine the usefulness of PCR for solving these diagnostic uncertainties. We analyzed IGH and TCRγ genes by PCR in 106 samples with inconclusive FCM results. A clonal result was registered in 36/106 studies, with a LPD being confirmed in 27 (75%) of these cases. Specifically, 9/9 IGH clonal and 16/25 TCRγ clonal results were finally diagnosed with LPD. Additionally, 2 clonal TCRγ samples with suspicion of undefined LPD were finally diagnosed with T LPD. Although polyclonal results were obtained in 47 of the cases studied (38 IGH and 9 TCRγ), hematologic neoplasms were diagnosed in 4/38 IGH polyclonal and in 1/9 TCRγ polyclonal studies. There were also 14 PCR polyclonal results (4 IGH, 10 TCRγ), albeit non-conclusive. Of these, 2/4 were eventually diagnosed with B-cell lymphoma and 3/10 with T-cell LPD. In 8 IGH samples the results of PCR techniques were non-informative but in 3/8 cases a B lymphoma was finally confirmed. We concluded that PCR is a useful technique to identify LPD when FCM is inconclusive. A PCR clonal B result is indicative of malignancy but IGH polyclonal and non-conclusive results do not exclude lymphoid neoplasms. Interpretation of T-cell clonality should be based on all the available clinical and analytical data. © 2013 Clinical Cytometry Society.
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BACKGROUND AND OBJECTIVES: Tyrosine kinase inhibitors (TKI) have improved the management of patients with chronic myeloid leukemia (CML). However, a significant proportion of patients do not achieve the optimal response or are resistant to TKI. ABL kinase domain mutations have been extensively implicated in the pathogenesis of TKI resistance. Treatment with second-generation TKI has produced high rates of hematologic and cytogenetic responses in mutated ABL patients. The aim of this study was to determine the type and frequency of ABL mutations in patients who were resistant to imatinib or had lost the response, and to analyze the effect of second-generation TKI on their outcome. PATIENTS AND METHODS: The presence of ABL mutations in 45 CML patients resistant to imatinib was evaluated by direct sequencing and was correlated with the results of the cytogenetic study (performed in 39 cases). The outcome of these patients after therapy with nilotinib or dasatinib was analyzed. RESULTS: ABL mutations were detected in 14 out of 45 resistant patients. Patients with clonal cytogenetic evolution tended to develop mutations more frequently than those without clonal evolution. Nine out of the 15 patients with ABL mutation responded to a treatment switch to nilotinib (n=4), dasatinib (n=2), interferon (n=1) or hematopoietic stem cell transplantation (n=2). CONCLUSION: The frequency of ABL mutations in CML patients resistant to imatinib is high and is more frequent among those with clonal cytogenetic evolution. The change to second-generation TKI can overcome imatinib resistance in most of the mutated patients.