RESUMO
PCR of TCR/Ig gene rearrangements is considered the method of choice for minimal residual disease (MRD) quantification in BCP-ALL, but flow cytometry analysis of leukemia-associated immunophenotypes (FCM-MRD) is faster and biologically more informative. FCM-MRD performed in 18 laboratories across seven countries was used for risk stratification of 1487 patients with BCP-ALL enrolled in the NOPHO ALL2008 protocol. When no informative FCM-marker was available, risk stratification was based on real-time quantitative PCR. An informative FCM-marker was found in 96.2% and only two patients (0.14%) had non-informative FCM and non-informative PCR-markers. The overall 5-year event-free survival was 86.1% with a cumulative incidence of relapse (CIR5y) of 9.5%. FCM-MRD levels on days 15 (HzR 4.0, p < 0.0001), 29 (HzR 2.7, p < 0.0001), and 79 (HzR 3.5, p < 0.0001) associated with hazard of relapse adjusted for age, cytogenetics, and WBC. The early (day 15) response associated with CIR5y adjusted for day 29 FCM-MRD, with higher levels in adults (median 2.4 × 10-2 versus 5.2 × 10-3, p < 0.0001). Undetectable FCM- and/or PCR-MRD on day 29 identified patients with a very good outcome (CIR5y = 3.2%). For patients who did not undergo transplantation, day 79 FCM-MRD > 10-4 associated with a CIR5y = 22.1%. In conclusion, FCM-MRD performed in a multicenter setting is a clinically useful method for MRD-based treatment stratification in BCP-ALL.
Assuntos
Neoplasia Residual/tratamento farmacológico , Neoplasia Residual/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Células Precursoras de Linfócitos B/efeitos dos fármacos , Células Precursoras de Linfócitos B/patologia , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Citometria de Fluxo/métodos , Humanos , Imunofenotipagem/métodos , Lactente , Masculino , Pessoa de Meia-Idade , Recidiva , Adulto JovemRESUMO
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) measured by PCR of clonal IgH/TCR rearrangements predicts relapse in T-cell acute lymphoblastic leukemia (T-ALL) and serves as risk stratification tool. Since 10% of patients have no suitable PCR-marker, we evaluated flowcytometry (FCM)-based MRD for risk stratification. We included 274 T-ALL patients treated in the NOPHO-ALL2008 protocol. MRD was measured by six-color FCM and real-time quantitative PCR. Day 29 PCR-MRD (cut-off 10-3) was used for risk stratification. At diagnosis, 93% had an FCM-marker for MRD monitoring, 84% a PCR-marker, and 99.3% (272/274) had a marker when combining the two. Adjusted for age and WBC, the hazard ratio for relapse was 3.55 (95% CI 1.4-9.0, p = 0.008) for day 29 FCM-MRD ≥ 10-3 and 5.6 (95% CI 2.0-16, p = 0.001) for PCR-MRD ≥ 10-3 compared with MRD < 10-3. Patients stratified to intermediate-risk therapy on day 29 with MRD 10-4-<10-3 had a 5-year event-free survival similar to intermediate-risk patients with MRD < 10-4 or undetectable, regardless of method for monitoring. Patients with day 15 FCM-MRD < 10-4 had a cumulative incidence of relapse of 2.3% (95% CI 0-6.8, n = 59). Thus, FCM-MRD allows early identification of patients eligible for reduced intensity therapy, but this needs further studies. In conclusion, FCM-MRD provides reliable risk prediction for T-ALL and can be used for stratification when no PCR-marker is available.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Citometria de Fluxo/métodos , Recidiva Local de Neoplasia/patologia , Neoplasia Residual/diagnóstico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Medição de Risco/métodos , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Prognóstico , Taxa de Sobrevida , Adulto JovemAssuntos
Cromossomos Humanos Par 16 , Cromossomos Humanos Par 1 , Sequenciamento de Nucleotídeos em Larga Escala , Leucemia Eritroblástica Aguda/genética , Fatores de Transcrição NFI/genética , Proteínas Repressoras/genética , Translocação Genética , Proteínas Supressoras de Tumor/genética , HumanosRESUMO
Monocytoid B cells are IgM(+) , IgD(-/+) , CD27(-) B cells, localized in the perisinusoidal area of the lymph node. These cells are especially prominent in infections such as those caused by toxoplasma and HIV. The ontogeny of monocytoid B cells with respect to B cell maturation is incompletely known. We analysed clonal expansion, somatic hypermutation and expression of activation-induced cytidine deaminase (AID) in monocytoid B cells. Sequence analysis of the rearranged immunoglobulin heavy chain genes amplified from microdissected monocytoid B cell zones with a high proportion of proliferating cells reveals the presence of multiple clones with low-level ongoing mutations (mean frequency: 0.46 × 10(-2) per bp). Mutation analysis of these ongoing mutations reveals strand bias, a preference of transitions over transversions as well as the occurrence of small deletions, as observed for somatically mutated immunoglobulin genes in the human germinal centre. Proliferation, ongoing mutation as well as expression of AID, combined, is evidence that monocytoid B cells acquire the mutations in the extrafollicular perisinusoidal area of the lymph node and pleads against a postgerminal centre B cell origin.
Assuntos
Subpopulações de Linfócitos B , Citidina Desaminase/biossíntese , Cadeias Pesadas de Imunoglobulinas/genética , Hipermutação Somática de Imunoglobulina , Sequência de Bases , Citidina Desaminase/genética , Análise Mutacional de DNA , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Genes de Imunoglobulinas , Humanos , Linfonodos/citologia , Linfonodos/inervação , Mutação , Análise de Sequência de DNAAssuntos
Cromossomos Humanos Par 16/genética , Cromossomos Humanos Par 1/genética , Rearranjo Gênico , Leucemia Eritroblástica Aguda/genética , Proteínas Repressoras/genética , Translocação Genética/genética , Proteínas Supressoras de Tumor/genética , Adolescente , Humanos , Cariotipagem , Leucemia Eritroblástica Aguda/diagnóstico , MasculinoRESUMO
With the aim of producing large quantities of mRNA-transfected monocyte-derived dendritic cells (DCs) to be used as cancer vaccines, a new clinical grade procedure has been developed. Peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis were enriched for monocytes by immunomagnetic depletion of CD19+ B cells and CD2+ T cells employing the ISOLEX 300i device. After 5 days of culture of enriched monocytes in gas permeable Teflon bags, using serum-free medium supplemented with granulocyte/macrophage-colony stimulating factor and interleukin-4 (IL-4), immature DCs were generated. Following transfection with mRNA from three human prostate cancer cell lines (DU145, LNCaP and PC-3), employing a newly developed square wave electroporation procedure, the immature DCs were immediately transferred to Teflon bags and matured for 48 h, using serum-free medium supplemented with IL-1alpha, IL-6, tumour necrosis factor-alpha and PGE2. The electroporation procedure efficiently transferred mRNA into the DCs with minor effect on the viability of the cells. The generated matured transfected DCs show high expression of the antigens CD83, CD80, CD86 and human leucocyte antigen-DR. Freezing and thawing of the transfected matured DCs had minor effect on cell viability and the phenotype. From 4 x 109 PBMCs, about 1 x 108 transfected matured DCs are produced. The thawed transfected DCs were able to elicit primary T-cell responses in vitro against antigens encoded by the prostate cancer mRNA as shown by enzyme-linked immunospot assay using mock-transfected DCs as control. Based on these results, clinical trials in cancer patients have been initiated.
Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Monócitos/citologia , RNA Mensageiro/genética , Transfecção , Adulto , Criopreservação , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Separação Imunomagnética , Imunofenotipagem , Depleção Linfocítica , Masculino , Linfócitos T/imunologiaRESUMO
Hodgkin's disease (HD) is a lymphoproliferative disease of predominantly B-cell origin. However, the reasons for the incomplete development of the B-cell phenotype and lack of immunoglobulin expression in classical HD (cHD) have not been fully explained. We examined the expression of PU.1 in HD, an Ets-family transcription factor, which regulates the expression of immunoglobulin and other genes that are important for B-cell development. Immunohistochemistry for PU.1 was performed on 35 cases of cHD and 15 cases of lymphocyte predominance HD as well as 67 non-Hodgkin's lymphomas (NHL). Expression of PU.1 was studied by Western blotting in four cHD-derived cell lines and in five NHL cell lines. We also studied the expression of two additional B-cell transcription factors, B-cell-specific activator protein and Oct-2. Our results show a striking lack of PU.1 expression by neoplastic cells in cHD but not in lymphocyte predominance HD. Our study also confirmed that B-cell-specific activator protein but not Oct-2 is not expressed by cHD. Western blotting showed no PU.1 protein expression in the cHD-derived cell lines, with the exception of one cell line of putative monocyte/histiocyte origin. The lack of PU.1 protein expression in cHD likely contributes to the lack of immunoglobulin expression and incomplete B-cell phenotype characteristic of the Reed-Sternberg cells in cHD.
Assuntos
Doença de Hodgkin/metabolismo , Doença de Hodgkin/patologia , Linfócitos/metabolismo , Linfócitos/patologia , Proteínas Proto-Oncogênicas/fisiologia , Transativadores/fisiologia , Western Blotting , Proteínas de Ligação a DNA/metabolismo , Doença de Hodgkin/classificação , Humanos , Imuno-Histoquímica , Linfonodos/metabolismo , Linfonodos/patologia , Linfoma não Hodgkin/metabolismo , Fator 2 de Transcrição de Octâmero , Fenótipo , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
Gastric low grade MALT lymphomas show a pattern of somatic mutations in their rearranged immunoglobulin genes, indicative of antigen selection. This provides evidence for antigen stimulation in the lymphomagenesis. Gastric diffuse large B cell lymphomas develop secondary to low grade MALT lymphoma or de novo. To study whether antigen-selection is also a feature of primary diffuse large B cell lymphomas, we analysed somatic mutations in the rearranged immunoglobulin heavy chain (IgH) variable genes (VH). The rearranged VH genes of six cases of gastric primary diffuse large B cell lymphoma were amplified from genomic or complementary DNA by a VH gene family-specific polymerase chain reaction method. The PCR products were directly sequenced and were compared to published germline sequences to analyse somatic mutations. Similarly to low grade MALT lymphomas 5/6 primary diffuse large B cell lymphomas show a pattern of somatic mutation in their rearranged VH genes, indicative of antigen selection and suggesting a role for antigens in lymphomagenesis. One case showed bi-allelic VH gene rearrangements, which were non-functional due to extensive deletions. Antigen selection could not be demonstrated or excluded. Antigen selection is a common feature in most analysed primary diffuse large B cell lymphomas, although some heterogeneity in the mechanisms involved in the lymphomagenesis of gastric primary diffuse large B cell lymphomas has not been excluded entirely (case 4).
Assuntos
Rearranjo Gênico , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Linfoma de Células B/genética , Linfoma Difuso de Grandes Células B/genética , Neoplasias Gástricas/genética , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Antígenos de Neoplasias/genética , Sequência de Bases , Feminino , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Analysis of non-Hodgkin lymphoma (NHL) involvement of bone marrow trephine biopsy specimens by morphologic features and immunohistochemistry is often difficult, and the criteria for involvement are ill defined. We compared the morphologic and immunohistochemical analysis of B-cell NHL involvement with immunoglobulin heavy chain gene (IgH) rearrangement analysis by polymerase chain reaction (PCR) amplification of the complementarity determining region 3 (CDR3) in bone marrow biopsy specimens from patients with mantle cell lymphoma (n = 53) or hairy cell leukemia (n = 71). By combing morphologic features and phenotype, 54 specimens were considered positive, 62 negative, and 8 inconclusive. PCR analysis showed clonal IgH rearrangements in 46 positive and 6 inconclusive specimens. No clonal IgH rearrangements were present in 61 negative specimens. The 1 false-positive and most false-negative PCR results were likely due to sampling error or DNA degradation of the fixed tissues. In most cases, bone marrow involvement by NHL can be identified by histologic and immunohistochemical examination. Furthermore, clonality of the B-cell population can be detected by amplification of the IgH CDR3 on DNA extracted from bone marrow trephine biopsy sections, which can be helpful in cases diagnosed as inconclusive.
Assuntos
Neoplasias da Medula Óssea/patologia , Rearranjo Gênico , Cadeias Pesadas de Imunoglobulinas/genética , Leucemia de Células Pilosas/patologia , Linfoma não Hodgkin/patologia , Exame de Medula Óssea , Neoplasias da Medula Óssea/imunologia , DNA de Neoplasias/análise , Humanos , Região Variável de Imunoglobulina , Imunofenotipagem , Leucemia de Células Pilosas/imunologia , Linfoma não Hodgkin/imunologia , Estadiamento de Neoplasias , Reação em Cadeia da PolimeraseRESUMO
Splenic marginal-zone B cells, marginal-zone B cells of Peyer's patches in the gut, and nodal marginal-zone B cells (also identified as monocytoid B cells) share a similar morphology and immunophenotype. These cells likely represent a distinct subset of B cells in humans and rodents, but their precise ontogenetic relationship as well as their origin from B cells of the germinal center is still debated. To study this, we performed a mutation analysis of the rearranged immunoglobulin variable genes (VH) of microdissected single nodal and splenic marginal-zone cells. In addition, we investigated the presence of proliferating cells and B-cell clones in the human splenic and nodal marginal zone as well as adjacent germinal centers. This was performed by immunohistochemical staining for the Ki-67 antigen and denaturing gradient gel analysis of amplified immunoglobulin heavy chain genes' complementarity determining region 3 of microdissected cell clusters. A variable subset of nodal and splenic marginal-zone B cells showed somatic mutations in their rearranged VH genes, indicating that both virgin and memory B cells are present in the nodal and splenic marginal zone. Nodal and splenic marginal-zone B cells preferentially rearranged VH3 family genes such as DP47, DP49, DP54, and DP58. A preferential rearrangement of the same VH genes has been shown by others in the peripheral CD5(-) IgM+ B cells. These data suggest that the splenic and nodal marginal-zone B cells are closely related B-cell subsets. We also showed that marginal-zone B cells may cycle and that clones of B cells are frequently detected in the nodal as well as the splenic marginal zone. These clones are not related to those present in adjacent germinal centers. These data favor the hypothesis that clonal expansion occurs in the marginal zone. Whether the somatic hypermutation mechanism is activated during the clonal expansion in the marginal zone and which type of immune response triggers the clonal expansion need to be elucidated.
Assuntos
Linfócitos B/metabolismo , Linfonodos/citologia , Mutação , Baço/citologia , Sequência de Aminoácidos , Linfócitos B/citologia , Divisão Celular/imunologia , Separação Celular , Células Clonais , Análise Mutacional de DNA , Dissecação , Eletroforese em Gel de Poliacrilamida , Rearranjo Gênico/imunologia , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Genes de Imunoglobulinas , Centro Germinativo/química , Centro Germinativo/citologia , Humanos , Região Variável de Imunoglobulina/genética , Imuno-Histoquímica , Antígeno Ki-67/análise , Linfonodos/metabolismo , Dados de Sequência Molecular , Análise de Sequência de DNA , Baço/metabolismoRESUMO
Mantle cell lymphoma is a distinct clinicopathological entity associated with t(11;14) and cyclin D1 overexpression. The majority of cases show uniform morphological and phenotypic features characterized by a monotonous proliferation of small-to-medium-sized irregular B cells that express CD5 and bright surface immunoglobulin IgM and IgD. By sequence analysis of the rearranged immunoglobulin heavy chain variable genes (VH), it has been shown that these lymphoma cells carry little if no somatic mutations, as described for the fetal CD5+ cells or B1 cells. Besides mantle cell lymphoma with classic histological features, a morphological variant of mantle cell lymphoma with blastic features and a more aggressive clinical course has been described. To investigate whether this variant is closely related, by the cell of origin, to typical cases, we analysed the presence and the pattern of somatic mutations of the VH genes in a series of nine cases diagnosed as such. Our cases of blastic mantle cell lymphomas rearrange most frequently VH4 and VH3 family genes. In three cases there was a complete homology to published germline genes, and a near complete homology was documented in another three. In contrast, the remaining three cases showed somatic mutations in their rearranged VH genes. Mutation analysis revealed evidence for antigen selection in one of these three cases. Taken together, these data are similar to those of normal adult-type B1 cells and those described for chronic lymphocytic leukaemia (CLL) but slightly different to those reported for classic mantle cell lymphoma. It is likely that blastic mantle cell lymphoma as well as CLL originates from adult-type B1 cells. More cases will need to be studied to determine whether classic mantle cell lymphoma is different from the blastic subtype and if it arises from fetal-type B1 cells.
Assuntos
Rearranjo Gênico de Cadeia Pesada de Linfócito B , Linfoma não Hodgkin/genética , Mutação , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise de SequênciaAssuntos
Linfoma de Zona Marginal Tipo Células B/classificação , Linfoma de Zona Marginal Tipo Células B/patologia , Linfoma não Hodgkin/classificação , Linfoma não Hodgkin/patologia , Neoplasias Gástricas/classificação , Neoplasias Gástricas/patologia , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Humanos , Região Variável de Imunoglobulina , Linfoma de Zona Marginal Tipo Células B/imunologia , Linfoma não Hodgkin/imunologia , Reação em Cadeia da Polimerase , Neoplasias Gástricas/imunologia , Terminologia como AssuntoRESUMO
Marginal zone cell lymphoma is a recently described entity among the non-Hodgkin's lymphomas. It likely originates from the marginal zone B cells in the spleen and equivalent cells in the lymph node and extranodal tissues. Recent evidence indicates that marginal zone B cells are functionally heterogeneous and may differ with respect to the pattern of somatic hypermutation in their Ig variable genes. To test whether marginal zone lymphomas may originate from different subsets of marginal zone B cells, we performed a sequence and mutation analysis of the rearranged Ig heavy chain (IgH) variable genes (VH) of a series of 14 cases of marginal zone lymphoma, occurring in the spleen (4), the lymph node (4), the stomach (2), the orbit (2), the tongue (1), and the skin (1). Our data show that marginal zone cell lymphomas preferentially rearrange the VH4, VH3, and VH1 family genes, without preference for any particular VH gene. Somatic mutations are present in 13 cases; one case of marginal zone cell lymphoma of the skin showed a germline configuration of the rearranged VH gene. Mutation analysis shows evidence of antigen selection in three cases of marginal zone cell lymphoma, one of the spleen, stomach, and orbit, respectively. No evidence of antigen selection was present in the other cases. These data indicate that marginal zone cell lymphomas may arise from different subsets of marginal zone B cells. In addition, lymphomagenesis may not be triggered by antigen in all cases of marginal zone cell lymphoma.
Assuntos
Rearranjo Gênico , Genes de Imunoglobulinas , Cadeias Pesadas de Imunoglobulinas/genética , Subpopulações de Linfócitos/imunologia , Linfoma de Células B/genética , Linfoma de Células B/imunologia , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Linhagem da Célula/genética , Análise Mutacional de DNA , Feminino , Humanos , Linfonodos/imunologia , Linfonodos/patologia , Linfoma de Células B/patologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Baço/imunologia , Baço/patologiaRESUMO
According to the French-American-British (FAB) proposal on the classification of chronic lymphoid leukemia (CLL), the disorder can be subdivided into typical and atypical CLL. We recently demonstrated the prognostic significance of this subgrouping and based on these results we suggested that typical and atypical CLL represent two closely related, but different entities. These results prompted us to investigate 42 patients diagnosed with CLL based on the results of lymph node biopsy in order to identify the histologic counterpart of the CLL variants. A first group of 14 cases showed a monomorphic proliferation of small round lymphocytes associated with the occurrence of small pseudofollicles. All these cases were diagnosed as typical CLL on peripheral blood (13 cases) or bone marrow smear (1 case). The remaining 28 cases showed aberrant histologic features characterized by the presence of large numbers of paraimmunoblasts and prolymphocytes, forming very large pseudofollicles, and/or by nuclear irregularities of the neoplastic cells. Based on peripheral blood smears (22 cases) or bone marrow smears (six cases), two cases showed no peripheral blood involvement, 21 cases were diagnosed as atypical CLL, and five as typical CLL. From these data we can conclude that a histologic counterpart of the CLL variants recognized in the FAB proposal does exist; moreover, our data may explain reports on lymph node involvement by CLL composed of small cleaved cells and clarify the occurrence of pseudofollicles in cases described as mantle cell lymphomas.
Assuntos
Leucemia Linfocítica Crônica de Células B/patologia , Linfonodos/patologia , Adulto , Idoso , Antígenos CD/metabolismo , Biópsia , Medula Óssea/patologia , Aberrações Cromossômicas , Feminino , Seguimentos , Humanos , Antígeno Ki-67/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Linfonodos/metabolismo , Masculino , Pessoa de Meia-Idade , Taxa de SobrevidaRESUMO
The detection of clonality in B-cell lymphomas has been facilitated by polymerase chain reaction (PCR) analysis of the immunoglobulin heavy-chain gene (IgH) complementarity determining region 3 (CDR3) and size fractionation by polyacrylamide gel electrophoresis (PAGE). However, the detection of minor clonal populations and biallelic rearrangements and the isolation of monoclonal products from gels are sometimes problematic. This study evaluated whether denaturing gradient gel electrophoresis (DGGE), a technique that separates DNA based on nucleotide sequence rather than length, could alleviate these problems. A total of 32 selected cases was studied with a diagnosis of monoclonal (n = 10), polyclonal (n = 9), and indeterminate (n = 13) IgH gene rearrangements, which were determined by analysis of seminested IgH CDR3 PCR products in 8% PAGE. These cases were evaluated using DGGE of seminested IgH CDR3 PCR products that included a 40-bp GC clamp on the Jh primer. DGGE allowed the discrimination of monoclonal populations in 9 of 13 cases where 8% PAGE results were indeterminate. In addition, DGGE demonstrated biallelic IgH rearrangements in three cases where 8% PAGE revealed only one predominant product. DGGE facilitated the purification and isolation of clonal IgH CDR3 products for sequencing without prior cloning. As an adaptation of current IgH PCR protocols, DGGE can enhance the construction of tumor-specific CDR3 primers/probes for investigations of minimal residual disease.
Assuntos
Eletroforese em Gel de Ágar , Rearranjo Gênico do Linfócito B/genética , Genes de Imunoglobulinas , Cadeias Pesadas de Imunoglobulinas/genética , Anticorpos Monoclonais/análise , Sequência de Bases , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Sensibilidade e EspecificidadeRESUMO
The genotype and clonality of Reed-Sternberg (RS) cells in Hodgkin's disease (HD) has remained a controversial issue, largely due to the scarcity of RS cells in tissues and the limitations of the techniques used to resolve this issue. Southern hybridization and polymerase chain reaction (PCR) assays using DNA extracted from tissues can only document clonal gene rearrangements, but do not indicate which cellular population is responsible for such rearrangements. To overcome the limitations of these previous techniques for studying the genotype and clonality of RS cells, we analysed single RS cells with a single-cell PCR assay to detect immunoglobulin heavy chain gene (IgH) rearrangements and X-chromosome inactivation. Six cases of nodular sclerosis (NS) HD were studied. The RS cells displayed a B-cell phenotype in three cases and a null-cell phenotype in the other three cases. IgH rearrangements were detected in the RS cells of the three cases with a B-cell phenotype, but not in the other cases. In these three cases the IgH rearrangements in the RS cells were polyclonal, although a subpopulation of clonal RS cells was documented in one case. The finding that the RS cells with IgH rearrangements were not monoclonal was supported in one case by studying the pattern of X-chromosome inactivation in single RS cells by a single-cell human androgen receptor gene (HUMARA) PCR assay. Our results indicate that NSHD begins as a polyclonal process in which a clonal RS cell population may arise; and that the RS cells in a subset of NSHD show evidence of B-lineage differentiation.
Assuntos
Doença de Hodgkin/genética , Células de Reed-Sternberg/patologia , Sequência de Bases , Células Clonais , Eletroforese em Gel de Poliacrilamida , Genótipo , Humanos , Dados de Sequência Molecular , Fenótipo , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: Polymerase chain reaction (PCR) based assays are becoming more reliable, simpler, and faster alternatives to traditional Southern blot hybridization (SBH) analysis for the detection of clonal immunoglobulin heavy chain (IgH) gene rearrangements. However, a variety of technical approaches have been reported with markedly different results. METHODS: We analyzed the frozen tissue of 147 neoplastic and hyperplastic lesions on which SBH had previously been performed. Semi-nested and single-step PCR methods were compared. Consensus primers to the joining segments and the framework region (FR) III of the variable segments of the IgH gene were used. All PCR products were analyzed by polyacrylamide gel electrophoresis (PAGE). Thirteen samples were re-analyzed using a denaturing gradient gel electrophoresis (DGGE) system. RESULTS: The overall concordance between SBH and semi-nested PCR assays was 80.2%. In the non-Hodgkin's lymphoma (NHL) group, 75% of the cases with IgH rearrangements by SBH were found to be monoclonal by PCR. Regardless of type of lesion, 71.7% of the cases with IgH rearrangements by SBH were found to be clonal by PCR. The concordance between the semi-nested and single-step procedures was 87.1%. DGGE was helpful in clarifying the results for cases in which the PAGE analysis was difficult to interpret. CONCLUSIONS: PCR analysis of IgH gene rearrangements was found to be an efficient technique for the initial determination of clonality in lymphoid proliferations. The single-step method had an advantage over the semi-nested method because of its simplicity and speed. The DGGE system was useful for the assessment of clonality in cases with equivocal results after PAGE. However, a combination of these techniques in specific cases may achieve higher specificity and sensitivity.