RESUMO
The pathogenesis of COVID-19 warrants unravelling. Genetic polymorphism analysis may help answer the variability in disease outcome. To determine the role of KIR and HLA polymorphisms in susceptibility, progression, and severity of SARS-CoV-2 infection, 458 patients and 667 controls enrolled in this retrospective observational study from April to December 2020. Mild/moderate and severe/death study groups were established. HLA-A, -B, -C, and KIR genotyping were performed using the Lifecodes® HLA-SSO and KIR-SSO kits on the Luminex® 200™ xMAP fluoroanalyser. A probability score using multivariate binary logistic regression analysis was calculated to estimate the likelihood of severe COVID-19. ROC analysis was used to calculate the best cut-off point for predicting a worse clinical outcome with high sensitivity and specificity. A p ≤ 0.05 was considered statistically significant. KIR AA genotype protected positively against severity/death from COVID-19. Furthermore, KIR3DL1, KIR2DL3 and KIR2DS4 genes protected patients from severe forms of COVID-19. KIR Bx genotype, as well as KIR2DL2, KIR2DS2, KIR2DS3 and KIR3DS1 were identified as biomarkers of severe COVID-19. Our logistic regression model, which included clinical and KIR/HLA variables, categorised our cohort of patients as high/low risk for severe COVID-19 disease with high sensitivity and specificity (Se = 94.29%, 95% CI [80.84-99.30]; Sp = 84.55%, 95% CI [79.26-88.94]; OR = 47.58, 95%CI [11.73-193.12], p < 0.0001). These results illustrate an association between KIR/HLA ligand polymorphism and different COVID-19 outcomes and remarks the possibility of use them as a surrogate biomarkers to detect severe patients in possible future infectious outbreaks.
Assuntos
COVID-19 , Receptores KIR , SARS-CoV-2 , Humanos , COVID-19/genética , COVID-19/imunologia , COVID-19/virologia , Receptores KIR/genética , Masculino , Feminino , Pessoa de Meia-Idade , SARS-CoV-2/imunologia , Projetos Piloto , Estudos Retrospectivos , Polimorfismo Genético , Idoso , Genótipo , Predisposição Genética para Doença , Adulto , Índice de Gravidade de Doença , Antígenos HLA/genéticaRESUMO
Mixed acute rejection is a clinicopathological entity that is difficult to accurately diagnose, and so may be under-reported. Allografts are lost more often than in either humoral or cellular rejection. The diagnosis requires both histological and immunological studies on renal biopsy and blood specimens from the transplant recipient to provide the required rescue therapy to abolish the allogeneic response against the graft. We present a clinical case report of an active mixed acute rejection driven by a de novo donor-specific complement-binding anti-DQB1*03:01 antibody and intraepithelial CD8 T-cells in a patient with a kidney transplant. The patient was diagnosed, treated, and followed up as per the local institution's procedure with a full recovery of graft function. Our case emphasises the challenge of a mixed acute rejection and supports the need to improve the post-transplant outcome of recipients and their grafts.
Assuntos
Rejeição de Enxerto , Isoanticorpos , Linfócitos T CD8-Positivos , Antígenos HLA , Humanos , RimRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Minimal residual disease (MRD) is a powerful prognostic factor in acute lymphoblastic leukemia (ALL) and is used for patient stratification and treatment decisions, but its precise role in Philadelphia chromosome positive ALL is less clear. This uncertainty results largely from methodological differences relating to the use of real-time quantitative PCR (qRT-PCR) to measure BCR-ABL1 transcript levels for MRD analysis. We here describe the first results by the EURO-MRD consortium on standardization of qRT-PCR for the e1a2 BCR-ABL1 transcript in Ph + ALL, designed to overcome the lack of standardisation of laboratory procedures and data interpretation. Standardised use of EAC primer/probe sets and of centrally prepared plasmid standards had the greatest impact on reducing interlaboratory variability. In QC1 the proportion of analyses with BCR-ABL1/ABL1 ratios within half a log difference were 40/67 (60%) and 52/67 (78%) at 10-3 and 36/67 (53%) and 53/67 (79%) at 10-4BCR-ABL1/ABL1. Standardized RNA extraction, cDNA synthesis and cycler platforms did not improve results further, whereas stringent application of technical criteria for assay quality and uniform criteria for data interpretation and reporting were essential. We provide detailed laboratory recommendations for the standardized MRD analysis in routine diagnostic settings and in multicenter clinical trials for Ph + ALL.
Assuntos
Proteínas de Fusão bcr-abl/genética , Cromossomo Filadélfia , Guias de Prática Clínica como Assunto , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Consenso , Humanos , Neoplasia Residual , RNA Mensageiro/análiseRESUMO
Transformation of Waldenström's macroglobulinemia (WM) to diffuse large B-cell lymphoma (DLBCL) occurs in up to 10% of patients and is associated with an adverse outcome. Here we performed the first whole-exome sequencing study of WM patients who evolved to DLBCL and report the genetic alterations that may drive this process. Our results demonstrate that transformation depends on the frequency and specificity of acquired variants, rather than on the duration of its evolution. We did not find a common pattern of mutations at diagnosis or transformation; however, there were certain abnormalities that were present in a high proportion of clonal tumor cells and conserved during this transition, suggesting that they have a key role as early drivers. In addition, recurrent mutations gained in some genes at transformation (for example, PIM1, FRYL and HNF1B) represent cooperating events in the selection of the clones responsible for disease progression. Detailed comparison reveals the gene abnormalities at diagnosis and transformation to be consistent with a branching model of evolution. Finally, the frequent mutation observed in the CD79B gene in this specific subset of patients implies that it is a potential biomarker predicting transformation in WM.
Assuntos
Biomarcadores Tumorais/genética , Antígenos CD79/genética , Transformação Celular Neoplásica/genética , Exoma , Linfoma Difuso de Grandes Células B/genética , Mutação , Proteínas de Neoplasias/genética , Macroglobulinemia de Waldenstrom/genética , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Chromosome 1q gains and 13q deletions are common cytogenetic aberrations in multiple myeloma (MM) that confer a poor prognosis. There are several techniques for the targeted study of these alterations, but interphase fluorescence in situ hybridization (FISH) is the current gold standard. The aim of the present study was to validate quantitative PCR (qPCR) as an alternative to FISH studies in CD138+-enriched plasma cells (PCs) from MM patients at diagnosis. We analyzed 1q gains and 13q deletions by qPCR in 57 and 60 MM patients, respectively. qPCR applicability was 84 and 88% for 1q and 13q, respectively. The qPCR and FISH methods had a sensitivity and specificity of 88 and 71% for 1q gains, and 79 and 100% for 13q deletions. A second qPCR assay for each region was carried out to confirm the previous results. Paired qPCR (two assays) and FISH results were available from 53 MM patients: 26 for 1q amplification and 27 for 13q deletion. qPCR assays gave concordant results (qPCR-consistent) in 20 of the 26 (77%) 1q gains and 25 of the 27 (93%) 13q deletions. Considering only the consistent data, the overall concordance among qPCR and FISH was 85 and 100% for 1q gains and 13q deletions, respectively. Our results show a substantial agreement between qPCR and the gold standard FISH technique, indicating the potential of qPCR as an alternative approach, particularly when the starting material is too scarce or cells are too damaged to obtain accurate results from FISH studies.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 13/genética , Cromossomos Humanos Par 1/genética , Mieloma Múltiplo/genética , Reação em Cadeia da Polimerase em Tempo Real , Feminino , Humanos , Hibridização in Situ Fluorescente , Masculino , Mieloma Múltiplo/patologiaAssuntos
Proteínas de Transporte/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Receptor alfa de Ácido Retinoico/metabolismo , Retinoides/metabolismo , Células-Tronco/metabolismo , Adulto , Cromossomos Humanos Par 15/metabolismo , Cromossomos Humanos Par 17/metabolismo , Proteínas de Ligação a DNA , Humanos , Masculino , Fatores de Transcrição , Translocação Genética/fisiologiaRESUMO
B cells have been shown to be refractory to reprogramming and B-cell-derived induced pluripotent stem cells (iPSC) have only been generated from murine B cells engineered to carry doxycycline-inducible Oct4, Sox2, Klf4 and Myc (OSKM) cassette in every tissue and from EBV/SV40LT-immortalized lymphoblastoid cell lines. Here, we show for the first time that freshly isolated non-cultured human cord blood (CB)- and peripheral blood (PB)-derived CD19+CD20+ B cells can be reprogrammed to iPSCs carrying complete VDJH immunoglobulin (Ig) gene monoclonal rearrangements using non-integrative tetracistronic, but not monocistronic, OSKM-expressing Sendai Virus. Co-expression of C/EBPα with OSKM facilitates iPSC generation from both CB- and PB-derived B cells. We also demonstrate that myeloid cells are much easier to reprogram than B and T lymphocytes. Differentiation potential back into the cell type of their origin of B-cell-, T-cell-, myeloid- and fibroblast-iPSCs is not skewed, suggesting that their differentiation does not seem influenced by 'epigenetic memory'. Our data reflect the actual cell-autonomous reprogramming capacity of human primary B cells because biased reprogramming was avoided by using freshly isolated primary cells, not exposed to cytokine cocktails favoring proliferation, differentiation or survival. The ability to reprogram CB/PB-derived primary human B cells offers an unprecedented opportunity for studying developmental B lymphopoiesis and modeling B-cell malignancies.
Assuntos
Linfócitos B/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Reprogramação Celular/genética , Sangue Fetal/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucócitos Mononucleares/metabolismo , Linfócitos B/citologia , Linfócitos B/imunologia , Sequência de Bases , Proteínas Estimuladoras de Ligação a CCAAT/imunologia , Diferenciação Celular , Separação Celular , Reprogramação Celular/imunologia , Sangue Fetal/citologia , Sangue Fetal/imunologia , Expressão Gênica , Vetores Genéticos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/imunologia , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/imunologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Dados de Sequência Molecular , Células Mieloides/citologia , Células Mieloides/imunologia , Células Mieloides/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/imunologia , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/imunologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/imunologia , Vírus Sendai/genética , Recombinação V(D)J/imunologiaRESUMO
We have analyzed the applicability, sensitivity and prognostic value of allele-specific oligonucleotide real-time quantitative PCR (ASO RQ-PCR) as a method for minimal residual disease (MRD) assessment in patients with multiple myeloma (MM), comparing the results with those of multiparameter flow cytometry (MFC). A total of 170 patients enrolled in three consecutive Spanish trials achieving at least partial response after treatment were included. Lack of clonality detection (n=31), unsuccessful sequencing (n=17) and suboptimal ASO performance (n=51) limited the applicability of PCR to 42% of cases. MRD was finally investigated in 103 patients (including 32 previously studied) with persistent disease identified by PCR and MFC in 54% and 46% of cases, respectively. A significant correlation in MRD quantitation by both the techniques was noted (r=0.881, P<0.001), being reflective of treatment intensity. Patients with <10(-4) residual tumor cells showed longer progression-free survival (PFS) compared with the rest (not reached (NR) vs 31 months, P=0.002), with similar results observed with MFC. Among complete responders (n=62), PCR discriminated two risk groups with different PFS (49 vs 26 months, P=0.001) and overall survival (NR vs 60 months, P=0.008). Thus, although less applicable than MFC, ASO RQ-PCR is a powerful technique to assess treatment efficacy and risk stratification in MM.
Assuntos
Alelos , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Neoplasia Residual/genética , Citometria de Fluxo , Rearranjo Gênico do Linfócito B , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias kappa de Imunoglobulina/genética , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/mortalidade , Neoplasia Residual/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNARESUMO
We evaluated the MYD88 L265P mutation in Waldenström's macroglobulinemia (WM) and B-cell lymphoproliferative disorders by specific polymerase chain reaction (PCR) (sensitivity â¼10(-3)). No mutation was seen in normal donors, while it was present in 101/117 (86%) WM patients, 27/31 (87%) IgM monoclonal gammapathies of uncertain significance (MGUS), 3/14 (21%) splenic marginal zone lymphomas and 9/48 (19%) non-germinal center (GC) diffuse large B-cell lymphomas (DLBCLs). The mutation was absent in all 28 GC-DLBCLs, 13 DLBCLs not subclassified, 35 hairy cell leukemias, 39 chronic lymphocytic leukemias (16 with M-component), 25 IgA or IgG-MGUS, 24 multiple myeloma (3 with an IgM isotype), 6 amyloidosis, 9 lymphoplasmacytic lymphomas and 1 IgM-related neuropathy. Among WM and IgM-MGUS, MYD88 L265P mutation was associated with some differences in clinical and biological characteristics, although usually minor; wild-type MYD88 cases had smaller M-component (1.77 vs 2.72 g/dl, P=0.022), more lymphocytosis (24 vs 5%, P=0.006), higher lactate dehydrogenase level (371 vs 265 UI/L, P=0.002), atypical immunophenotype (CD23-CD27+ +FMC7+ +), less Immunoglobulin Heavy Chain Variable gene (IGHV) somatic hypermutation (57 vs 97%, P=0.012) and less IGHV3-23 gene selection (9 vs 27%, P=0.014). These small differences did not lead to different time to first therapy, response to treatment or progression-free or overall survival.
Assuntos
Mutação , Fator 88 de Diferenciação Mieloide/genética , Macroglobulinemia de Waldenstrom/genética , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Progressão da Doença , Humanos , Imunoglobulina M/metabolismo , Transtornos Linfoproliferativos/diagnóstico , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/metabolismo , Pessoa de Meia-Idade , Fator 88 de Diferenciação Mieloide/metabolismo , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , Macroglobulinemia de Waldenstrom/diagnóstico , Macroglobulinemia de Waldenstrom/metabolismo , Macroglobulinemia de Waldenstrom/mortalidadeRESUMO
The PML-RARA fusion protein is found in approximately 97% of patients with acute promyelocytic leukemia (APL). APL can be associated with life-threatening bleeding complications when undiagnosed and not treated expeditiously. The PML-RARA fusion protein arrests maturation of myeloid cells at the promyelocytic stage, leading to the accumulation of neoplastic promyelocytes. Complete remission can be obtained by treatment with all-trans-retinoic acid (ATRA) in combination with chemotherapy. Diagnosis of APL is based on the detection of t(15;17) by karyotyping, fluorescence in situ hybridization or PCR. These techniques are laborious and demand specialized laboratories. We developed a fast (performed within 4-5 h) and sensitive (detection of at least 10% malignant cells in normal background) flow cytometric immunobead assay for the detection of PML-RARA fusion proteins in cell lysates using a bead-bound anti-RARA capture antibody and a phycoerythrin-conjugated anti-PML detection antibody. Testing of 163 newly diagnosed patients (including 46 APL cases) with the PML-RARA immunobead assay showed full concordance with the PML-RARA PCR results. As the applied antibodies recognize outer domains of the fusion protein, the assay appeared to work independently of the PML gene break point region. Importantly, the assay can be used in parallel with routine immunophenotyping for fast and easy diagnosis of APL.
Assuntos
Citometria de Fluxo , Imunoensaio , Leucemia Promielocítica Aguda/diagnóstico , Leucemia Promielocítica Aguda/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Adulto , Estudos de Casos e Controles , Criança , Cromossomos Humanos Par 15/genética , Cromossomos Humanos Par 17/genética , Feminino , Humanos , Leucemia Promielocítica Aguda/imunologia , Masculino , Proteínas de Fusão Oncogênica/imunologia , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Células Tumorais CultivadasRESUMO
There is barely any information about the prognostic significance of FLT3 expression and mutational status in cytogenetically distinct subgroups of acute lymphoblastic leukemia (ALL). We analyzed the presence of FLT3-tyrosine kinase domain (TKD) and FLT3-internal tandem duplication (ITD) mutations as well as FLT3 expression levels in 54 newly diagnosed patients with B-ALL (n=49) or T-ALL (n=5). All B/T-ALL samples tested negative for the presence of FLT3-TKD or FLT3-ITD. None of the T-ALL and E2A-PBX1+ B-ALL overexpressed FLT3. In contrast, mainly MLL-AF4+ B-ALL but also ETV6-RUNX1+, BCR-ABL+ or B-ALL displaying normal cytogenetics exhibited significantly higher FLT3 expression levels than normal bone marrow, supporting that aberrantly increased transcription of FLT3, rather than activating FLT3 mutations, contributes to the pathogenesis of these B-ALL. Using the median FLT3 expression as cut-off value we found that high-level FLT3 expression is associated with an extremely poor 1-year overall survival (OS; 0 vs 71%; P=0.002) and disease-free survival (DFS; 0 vs 43%; P=0.03) in MLL-AF4+ B-ALL but not in MLL-germline B-ALL. Cox regression analysis with OS/DFS as end points showed that age>14 years and high-level FLT3 expression were independent prognostic factors when all ALL patients were analyzed together. Importantly, when the MLL-AF4+ B-ALL subgroup was analyzed separately, high-level FLT3 expression was the only independent prognostic factor for OS and treatment outcome. These findings indicate that high FLT3 expression identifies MLL-AF4+ ALL patients at very high risk of treatment failure and poor survival, emphasizing the value of ongoing/future clinical trials for FLT3 inhibitors.
Assuntos
Mutação , Proteína de Leucina Linfoide-Mieloide/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Tirosina Quinase 3 Semelhante a fms/fisiologia , Histona-Lisina N-Metiltransferase , Humanos , Prognóstico , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
The frequencies of human leukocyte antigen (HLA) class I and class II specificities and haplotypic associations were determined in 1940 unrelated donors from Castilla y León and compared with other Iberian, Mediterranean and European populations. Specificities were determined using polymerase chain reaction reverse sequence-specific oligonucleotide or polymerase chain reaction sequence-specific primer techniques. In the analysis, 19, 29 and 13 specificities were found for HLA-A, -B and -DRB1, respectively, with HLA-A*02 (26%), -A*01 (11%), -B*44 (16%), -B*35 (10%), -DRB1*07 (16%) and -DRB1*13 (14%) showing the highest frequencies. In addition, 10 common HLA-A-B-DRB1 haplotypic associations were observed, A*01-B*08-DRB1*03 (3%) and A*29-B*44-DRB1*07 (3%) being the most frequent ones. These findings indicate that the population of Castilla y León is genetically equidistant from the Portuguese and other Spanish populations and shares a common origin with other Iberian populations, in which European, Mediterranean and North African genetic components are present; this is in agreement with the historical and genetic background of the population. These data contribute to a better understanding of the genetic structure of the Iberian Peninsula and provide a healthy control population from our region that should be useful for the study of disease associations.
Assuntos
Frequência do Gene , Antígenos HLA-A/genética , Antígenos HLA-B/genética , Cadeias HLA-DRB1/genética , Haplótipos , Feminino , Humanos , Masculino , Espanha/etnologiaRESUMO
The human leukocyte antigen (HLA) system could play an essential role in multiple myeloma (MM) disease control. This report describes the results comparing HLA-DRB1 phenotypic frequencies in 181 MM patients (53 smoldering/indolent MM and 128 symptomatic MM patients) and healthy individuals. Higher DRB1*01 phenotypic frequencies were found in the smoldering patients compared with symptomatic MM patients (38% vs 14%, P = 0.001) and with the healthy individuals (38% vs 22%, P = 0.01). Additionally, higher DRB1*07 phenotypic frequencies were found in symptomatic MM compared with control population (38% vs 28%, P = 0.01). The present data suggest that HLA-DRB1*01 individuals may have a better ability to efficiently present myeloma-related antigens to immunocompetent cells, which could favor a better immune response against the tumor. This would translate into a more appropriate disease control associated with more indolent disease and prolonged survival.
Assuntos
Antígenos HLA-DR/genética , Mieloma Múltiplo/genética , Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Feminino , Antígenos HLA-DR/imunologia , Cadeias HLA-DRB1 , Humanos , Masculino , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/mortalidade , FenótipoRESUMO
RAN, ZHX2 and RCBTB2 (CHC1L) expression was evaluated by quantitative real time reverse transcription polymerase chain reaction in plasma cells from 85 monoclonal gammopathies: 58 symptomatic multiple myeloma (MM) (52 untreated, six relapsed), eight smouldering MM, five monoclonal gammopathy of undetermined significance, four plasma cell leukaemias and 10 myeloid cell lines. ZHX2 was weakly expressed in high-risk/proliferative disease compared to low-risk or indolent disease. High ZHX2 expression was associated with better response and longer survival after high-dose therapy. RCBTB2 expression was weaker in hyperdiploid versus non-hyperdiploid cases while RAN was more expressed in symptomatic MM and cell lines.
Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas de Homeodomínio/metabolismo , Mieloma Múltiplo/metabolismo , Proteínas de Neoplasias/metabolismo , Fatores de Transcrição/metabolismo , Fatores Etários , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/genética , Células da Medula Óssea/metabolismo , Feminino , Expressão Gênica , Perfilação da Expressão Gênica/métodos , Proteínas de Homeodomínio/genética , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/tratamento farmacológico , Proteínas de Neoplasias/genética , Paraproteinemias/tratamento farmacológico , Paraproteinemias/metabolismo , Plasmócitos/metabolismo , Prognóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de Transcrição/genética , Resultado do Tratamento , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismoRESUMO
The hypervariable regions of immunoglobulin heavy-chain (IgH) rearrangements provide a specific tumor marker in multiple myeloma (MM). Recently, real-time PCR assays have been developed in order to quantify the number of tumor cells after treatment. However, these strategies are hampered by the presence of somatic hypermutation (SH) in VDJH rearrangements from multiple myeloma (MM) patients, which causes mismatches between primers and/or probes and the target, leading to a nonaccurate quantification of tumor cells. Our group has recently described a 60% incidence of incomplete DJH rearrangements in MM patients, with no or very low rates of SH. In this study, we compare the efficiency of a real-time PCR approach for the analysis of both complete and incomplete IgH rearrangements in eight MM patients using only three JH consensus probes. We were able to design an allele-specific oligonucleotide for both the complete and incomplete rearrangement in all patients. DJH rearrangements fulfilled the criteria of effectiveness for real-time PCR in all samples (ie no unspecific amplification, detection of less than 10 tumor cells within 10(5) polyclonal background and correlation coefficients of standard curves higher than 0.98). By contrast, only three out of eight VDJH rearrangements fulfilled these criteria. Further analyses showed that the remaining five VDJH rearrangements carried three or more somatic mutations in the probe and primer sites, leading to a dramatic decrease in the melting temperature. These results support the use of incomplete DJH rearrangements instead of complete somatically mutated VDJH rearrangements for investigation of minimal residual disease in multiple myeloma.
Assuntos
Rearranjo Gênico , Genes de Imunoglobulinas , Cadeias Pesadas de Imunoglobulinas/genética , Região de Junção de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Mieloma Múltiplo/diagnóstico , Primers do DNA/química , Humanos , Mieloma Múltiplo/genética , Neoplasia Residual , Reação em Cadeia da Polimerase/métodos , Sensibilidade e EspecificidadeRESUMO
Interphase fluorescence in situ hybridization (iFISH) is increasingly used for the identification of BCR/ABL gene rearrangements in chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). In the present study, we have explored the incidence of both typical and atypical iFISH patterns of BCR/ABL gene rearrangements in a series of 168 consecutive BCR/ABL+ patients--135 CML, 31 precursor B-ALL and two acute myeloblastic leukemia (AML) cases--and established their underlying genetic alterations through further molecular and chromosome analyses. Two different FISH probes (Vysis Inc., Downers Grove, IL, USA) were used: the LSI BCR/ABL dual color extra signal (ES) and the dual color dual fusion BCR/ABL probe (D-FISH). Our results show that most BCR/ABL+ patients (83%, including 88% of all CML, 61% of ALL and one of two AML) displayed typical iFISH patterns of either Major (M) BCR/ABL (87% of CML, 13% of ALL and one of the two AML) or minor (m) BCR/ABL gene rearrangements (1% of all CML and 48% of ALL cases) with the two probes. Further molecular and cytogenetic studies confirmed the presence of such typical rearrangements in all except one of these ALL cases who had coexistence of an MBCR/ABL and an mBCR/ABL gene rearrangement together with monosomy 9. In the remaining 29 cases (17%), up to five different atypical iFISH patterns were detected with the ES probe. Atypical iFISH patterns were most frequently due to additional numerical changes--most often supernumerary Philadelphia (Ph) chromosome (7%) but also gain or loss of chromosome 9 (1%) or 22 (1%). Deletion of 9q sequences proximal to the breakpoint were also frequently observed with the ES probe (8%). Application of the D-FISH probe showed that in most of these latter cases (5%) deletion of 22q sequences distal to the breakpoint also occurred. The remaining cases with atypical iFISH had cryptic insertion of BCR in 9q34 (1%). Exact interpretation of each iFISH pattern was supported by FISH on metaphases and molecular determination of the BCR breakpoint. In summary, our results indicate that despite the high incidence of typical iFISH patterns of BCR/ABL gene rearrangements, atypical patterns are also found in BCR/ABL+ acute leukemias; the precise definition of the alteration present in individual cases is dependent on metaphase studies and molecular definition of the breakpoint.
Assuntos
Aberrações Cromossômicas , Proteínas de Fusão bcr-abl/genética , Rearranjo Gênico , Hibridização in Situ Fluorescente , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Medula Óssea/patologia , Cromossomos Humanos Par 22/genética , Cromossomos Humanos Par 9/genética , Deleção de Genes , Humanos , Incidência , Interfase , Cariotipagem , MonossomiaRESUMO
Contaminating tumour cells in apheresis products have proved to influence the outcome of patients with multiple myeloma (MM) undergoing autologous stem cell transplantation (APBSCT). The gene scanning of clonally rearranged VDJ segments of the heavy chain immunoglobulin gene (VDJH) is a reproducible and easy to perform technique that can be optimised for clinical laboratories. We used it to analyse the aphereses of 27 MM patients undergoing APBSCT with clonally detectable VDJH segments, and 14 of them yielded monoclonal peaks in at least one apheresis product. The presence of positive results was not related to any pre-transplant characteristics, except the age at diagnosis (lower in patients with negative products, P = 0.04). Moreover, a better pre-transplant response trended to associate with a negative result (P = 0.069). Patients with clonally free products were more likely to obtain a better response to transplant (complete remission, 54% vs 28%; >90% reduction in the M-component, 93% vs 43% P = 0.028). In addition, patients transplanted with polyclonal products had longer progression-free survival, (39 vs 19 months, P = 0.037) and overall survival (81% vs 28% at 5 years, P = 0.045) than those transplanted with monoclonal apheresis. In summary, the gene scanning of apheresis products is a useful and clinically relevant technique in MM transplanted patients.