RESUMO
Based on the profile of genetic alterations occurring in tumor samples from selected diffuse large B-cell lymphoma (DLBCL) patients, 2 recent whole-exome sequencing studies proposed partially overlapping classification systems. Using clustering techniques applied to targeted sequencing data derived from a large unselected population-based patient cohort with full clinical follow-up (n = 928), we investigated whether molecular subtypes can be robustly identified using methods potentially applicable in routine clinical practice. DNA extracted from DLBCL tumors diagnosed in patients residing in a catchment population of â¼4 million (14 centers) were sequenced with a targeted 293-gene hematological-malignancy panel. Bernoulli mixture-model clustering was applied and the resulting subtypes analyzed in relation to their clinical characteristics and outcomes. Five molecular subtypes were resolved, termed MYD88, BCL2, SOCS1/SGK1, TET2/SGK1, and NOTCH2, along with an unclassified group. The subtypes characterized by genetic alterations of BCL2, NOTCH2, and MYD88 recapitulated recent studies showing good, intermediate, and poor prognosis, respectively. The SOCS1/SGK1 subtype showed biological overlap with primary mediastinal B-cell lymphoma and conferred excellent prognosis. Although not identified as a distinct cluster, NOTCH1 mutation was associated with poor prognosis. The impact of TP53 mutation varied with genomic subtypes, conferring no effect in the NOTCH2 subtype and poor prognosis in the MYD88 subtype. Our findings confirm the existence of molecular subtypes of DLBCL, providing evidence that genomic tests have prognostic significance in non-selected DLBCL patients. The identification of both good and poor risk subtypes in patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) clearly show the clinical value of the approach, confirming the need for a consensus classification.
Assuntos
Análise Mutacional de DNA/métodos , Sequenciamento do Exoma , Linfoma Difuso de Grandes Células B/diagnóstico , Linfoma Difuso de Grandes Células B/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Pesquisa Biomédica/organização & administração , Criança , Pré-Escolar , Estudos de Coortes , Redes Comunitárias , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Hematológicas/classificação , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Humanos , Lactente , Linfoma Difuso de Grandes Células B/classificação , Linfoma Difuso de Grandes Células B/patologia , Masculino , Oncologia/organização & administração , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/métodos , Estadiamento de Neoplasias , Prognóstico , Transcriptoma , Reino Unido , Sequenciamento do Exoma/métodos , Adulto JovemRESUMO
Cell-of-origin subclassification of diffuse large B cell lymphoma (DLBCL) into activated B cell-like (ABC), germinal centre B cell-like (GCB) and unclassified (UNC) or type III by gene expression profiling is recommended in the latest update of the World Health Organization's classification of lymphoid neoplasms. There is, however, no accepted gold standard method or dataset for this classification. Here, we compare classification results using gene expression data for 68 formalin-fixed paraffin-embedded DLBCL samples measured on four different gene expression platforms (Illumina wG-DASLTM arrays, Affymetrix PrimeView arrays, Illumina TrueSeq RNA sequencing and the HTG EdgeSeq DLBCL Cell of Origin Assay EU using an established platform agnostic classification algorithm (DAC) and the classifier native to the HTG platform, which is CE marked for in vitro diagnostic use (CE-IVD). Classification methods and platforms show a high level of concordance, with agreement in at least 80% of cases and rising to much higher levels for classifications of high confidence. Our results demonstrate that cell-of-origin classification by gene expression profiling on different platforms is robust, and that the use of the confidence value alongside the classification result is important in clinical applications.
Assuntos
Perfilação da Expressão Gênica , Linfoma Difuso de Grandes Células B/genética , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Linfoma Difuso de Grandes Células B/classificação , Análise de Sequência com Séries de Oligonucleotídeos , RNA/genética , TranscriptomaRESUMO
The diagnosis of myelodysplastic syndromes (MDS) remains problematic due to the subjective nature of morphologic assessment. The reported high frequency of somatic mutations and increased structural variants by array-based cytogenetics have provided potential objective markers of disease; however, this has been complicated by reports of similar abnormalities in the healthy population. We aimed to identify distinguishing features between those with early MDS and reported healthy individuals by characterizing 69 patients who, following a nondiagnostic marrow, developed progressive dysplasia or acute myeloid leukemia. Targeted sequencing and array-based cytogenetics identified a driver mutation and/or structural variant in 91% (63/69) of prediagnostic samples with the mutational spectrum mirroring that in the MDS population. When compared with the reported healthy population, the mutations detected had significantly greater median variant allele fraction (40% vs 9% to 10%), and occurred more commonly with additional mutations (≥2 mutations, 64% vs 8%). Furthermore, mutational analysis identified a high-risk group of patients with a shorter time to disease progression and poorer overall survival. The mutational features in our cohort are distinct from those seen in the healthy population and, even in the absence of definitive disease, can predict outcome. Early detection may allow consideration of intervention in poor-risk patients.
Assuntos
Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Mutação , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/mortalidade , Análise Mutacional de DNA , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Estudos Retrospectivos , Fatores de Risco , Taxa de SobrevidaRESUMO
This study tested the validity of whole-genome expression profiling (GEP) using RNA from formalin-fixed, paraffin-embedded (FFPE) tissue to sub-classify Diffuse Large B-cell Lymphoma (DLBCL), in a population based cohort of 172 patients. GEP was performed using Illumina Whole Genome cDNA-mediated Annealing, Selection, extension & Ligation, and tumours were classified into germinal centre (GCB), activated B-cell (ABC) and Type-III subtypes. The method was highly reproducible and reliably classified cell lines of known phenotype. GCB and ABC subtypes were each characterized by unique gene expression signatures consistent with previously published data. A significant relationship between subtype and survival was observed, with ABC having the worst clinical outcome and in a multivariate survival model only age and GEP class remained significant. This effect was not seen when tumours were classified by immunohistochemistry. There was a significant association between age and subtype (mean ages ABC - 72·8 years, GC - 68·4 years, Type-III - 64·5 years). Older patients with ABC subtype were also over-represented in patients who died soon after diagnosis. The relationship between prognosis and subtype improved when only patients assigned to the three categories with the highest level of confidence were analysed. This study demonstrates that GEP-based classification of DLBCL can be applied to RNA extracted from routine FFPE samples and has potential for use in stratified medicine trials and clinical practice.
Assuntos
Genoma Humano , Linfoma Difuso de Grandes Células B/classificação , Idoso , Feminino , Perfilação da Expressão Gênica/métodos , Estudo de Associação Genômica Ampla , Humanos , Imuno-Histoquímica , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Masculino , Pessoa de Meia-Idade , Inclusão em Parafina , Prognóstico , Resultado do TratamentoRESUMO
Follicular lymphoma (FL) is morphologically and clinically diverse, with mutations in epigenetic regulators alongside t(14;18) identified as disease-initiating events. Identification of additional mutational entities confirms this cancer's heterogeneity, but whether mutational data can be resolved into mechanistically distinct subsets remains an open question. Targeted sequencing was applied to an unselected population-based FL cohort (n = 548) with full clinical follow-up (n = 538), which included 96 diffuse large B-cell lymphoma (DLBCL) transformations. We investigated whether molecular subclusters of FL can be identified and whether mutational data provide predictive information relating to transformation. DNA extracted from FL samples was sequenced with a 293-gene panel representing genes frequently mutated in DLBCL and FL. Three clusters were resolved using mutational data alone, independent of translocation status: FL_aSHM, with high burden of aberrant somatic hypermutation (aSHM) targets; FL_STAT6, with high STAT6 & CREBBP mutation and low aSHM; and FL_Com, with the absence of features of other subtypes and enriched KMT2D mutation. Analysis of mutation signatures demonstrated differential enrichment of predicted mutation signatures between subgroups and a dominant preference in the FL_aSHM subgroup for G(C>T)T and G(C>T)C transitions consistent with previously defined aSHM-like patterns. Of transformed cases with paired samples, 17 of 26 had evidence of branching evolution. Poorer overall survival (OS) in the aSHM group (P = .04) was associated with older age; however, overall tumor genetics provided limited information to predict individual patient risk. Our approach identifies 3 molecular subclusters of FL linked to differences in underlying mechanistic pathways. These clusters, which may be further resolved by the inclusion of translocation status and wider mutation profiles, have implications for understanding pathogenesis as well as improving treatment strategies in the future.
Assuntos
Neoplasias Hematológicas , Linfoma Folicular , Linfoma Difuso de Grandes Células B , Humanos , Linfoma Folicular/diagnóstico , Linfoma Folicular/genética , Linfoma Difuso de Grandes Células B/genética , Mutação , Translocação Genética , Neoplasias Hematológicas/genética , Reino UnidoRESUMO
This prospective study aimed to develop reproducible diagnostic criteria for sporadic Burkitt lymphoma (BL), applicable to routine practice, and to evaluate the efficacy of dose-modified (dm) CODOX-M/IVAC in patients diagnosed using these criteria. The study was open to patients with an aggressive B-cell lymphoma with an MKI67 fraction approaching 100%. Immunophenotype and fluorescent in situ hybridization (FISH) were used to separate BL from other aggressive B-cell lymphomas. BL was characterized by the presence of a cMYC rearrangement as a sole cytogenetic abnormality occurring in patients with a germinal center phenotype with absence of BCL-2 expression and abnormal TP53 expression. A total of 128 patients were eligible for the study, of whom 58 were considered to have BL and 70 to have diffuse large B-cell lymphoma (DLBCL). There were 110 clinically fit patients who received dmCODOX-M (methotrexate, dose 3 g/m(2)) with or without IVAC according to risk group. The 2-year progression-free survival was 64% (95% confidence interval [CI] 51%-77%) for BL, 55% (95% CI 42%-66%) for DLBCL, 85% (95% CI 73%-97%) for low risk, and 49% (95% CI 38%-60%) for high-risk patients. The observed differences in outcome and other clinical features validate the proposed diagnostic criteria. Compared with the previous trial LY06 with full-dose methotrexate (6.7 g/m(2)), there was a reduction in toxicity with comparable outcomes. This study was registered at www.clinicaltrials.gov as NCT00040690.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Linfoma de Burkitt/diagnóstico , Linfoma de Burkitt/tratamento farmacológico , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidade , Ciclofosfamida/administração & dosagem , Citarabina/administração & dosagem , Análise Citogenética , Intervalo Livre de Doença , Doxorrubicina/administração & dosagem , Etoposídeo/administração & dosagem , Humanos , Ifosfamida/administração & dosagem , Imunofenotipagem , Metotrexato/administração & dosagem , Pessoa de Meia-Idade , Resultado do Tratamento , Vincristina/administração & dosagemRESUMO
Fluorescent in situ hybridization (FISH) techniques can be used to identify a range of chromosome abnormalities that are clinically significant in many cancers. Multicolor FISH can be used to identify multiple targets, which can be simultaneously detected in individual cells using digital imaging microscopy. In an era of precision medicine there is a requirement to make a precise diagnosis and to have a molecular classification of the tumor that can guide therapy. Cancer genomics is now regarded as a sub-specialism in pathology and genomic testing needs to be robustly integrated into the routine diagnostic practice.The FISH techniques described in this chapter have been developed over many years in a busy hematopathology diagnostic laboratory. We describe robust in-house methods for both liquid samples (blood and bone marrow mainly) and formalin-fixed paraffin-embedded (FFPE) tissue biopsies that allow for large numbers of slides to be set up in batches. The techniques described are for interphase cells in tissues where metaphase chromosome techniques are generally not applicable. Some of the FISH tests need to be carried out as an "out-of-hours" emergency test to make a critical diagnosis while others provide prognostic information and are used to guide downstream patient management.
Assuntos
Citodiagnóstico/métodos , Sondas de DNA/genética , Hibridização in Situ Fluorescente/métodos , Neoplasias/diagnóstico , Aberrações Cromossômicas , Sondas de DNA/uso terapêutico , Humanos , Neoplasias/genética , Neoplasias/patologia , PrognósticoRESUMO
Strong uniform expression of FOXP1 protein occurs in a subgroup of non-germinal centre (GC) diffuse large B-cell lymphomas (DLBCL). We have investigated gene rearrangement as a potential mechanism for deregulated expression of FOXP1 however, using FISH FOXP1 translocations were not found in any case with over-expression of the protein.
Assuntos
Fatores de Transcrição Forkhead/genética , Linfoma Difuso de Grandes Células B/genética , Proteínas Repressoras/genética , Fatores de Transcrição Forkhead/análise , Regulação Neoplásica da Expressão Gênica , Rearranjo Gênico , Humanos , Hibridização in Situ Fluorescente , Linfoma de Células B/genética , Proteínas Repressoras/análiseRESUMO
The t(14;18) is present in a significant proportion of diffuse large B-cell lymphomas (DLBCLs), however, the prognostic effect of the translocation and the relationship with transformed follicular lymphoma remains controversial. To clarify these uncertainties, interphase fluorescence in situ hybridization (FISH) was used to determine the incidence of the t(14;18) in nodal DLBCL, and this was correlated with BCL2 expression, germinal center (GC) immunophenotype, and patient outcome. FISH was performed on paraffin-extracted nuclei from 137 de novo nodal DLBCLs. Eighteen of 137 de novo DLBCLs were t(14;18) positive. The t(14;18) was most commonly associated with the subset of DLBCLs that expressed a GC phenotype, defined as CD10+, BCL6+ (GC-type DLBCL): positive in 14 of 47 (30%) cases, compared with 4 of 89 (5%) in the non-GC group (Pearson's chi(2) = 28.4; P < 0.0001). All cases with a translocation expressed BCL2 protein, however, 40 expressed BCL2 protein without a t(14;18). GC-type DLBCL patients with a t(14;18) had a significantly worse survival compared with GC-type DLBCL patients without the translocation (2-year survivals were 29 and 63%, respectively; P = 0.006). Of the cases without the translocation, BCL2 protein expression did not affect survival. In contrast, in the non-GC group of DLBCLs, BCL2 protein expression reduced the 2-year overall survival from 64% in the BCL2-negative group to 38%, with a median survival of 15.0 months (P = 0.02). In conclusion, the t(14;18) is common in DLBCLs, particularly in GC-type DLBCLs, where the presence of the translocation has a poor prognostic effect. BCL2 protein expression defines a group of non-GC DLBCL patients with a poor prognosis.
Assuntos
Cromossomos Humanos Par 14 , Cromossomos Humanos Par 18 , Centro Germinativo/patologia , Linfoma de Células B/genética , Linfoma Difuso de Grandes Células B/genética , Translocação Genética , Humanos , Hibridização in Situ Fluorescente , Linfoma de Células B/mortalidade , Linfoma Difuso de Grandes Células B/mortalidade , Proteínas Proto-Oncogênicas c-bcl-2/análise , Estudos RetrospectivosRESUMO
Indolent lymphoproliferative disorders such as Waldenstrom's macroglobulinemia (WM) are characterized by defective apoptosis, which leads to progressive accumulation of slowly dividing neoplastic lymphocytes. Activation of nuclear factor kappa B (NFkappaB) is considered to have a central pathogenic role in some hematological malignancies, including multiple myeloma, Hodgkin's disease, and extranodal marginal zone lymphoma (ENMZL). NFkappaB activation may inhibit apoptosis through the transactivation of genes such as Bcl-2 and may therefore be an important mechanism in indolent lymphoproliferative disorders, including WM. In order to assess this potential mechanism, we used immunohistochemistry to determine the presence and subcellular localisation of the major NFkappaB subunits p50 and p65. Nuclear staining of NFkappaB subunits (indicative of activation) was not seen in any of the 40 cases examined. Thirty-seven (95%) cases showed cytoplasmic positivity for both p50 and p65 and one case demonstrated cytoplasmic staining for p65 alone, while the two remaining cases showed complete absence of staining. We would therefore conclude that NFkappaB activation is not a feature of WM and that alternative mechanisms of apoptosis inhibition should be investigated in this disorder.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Apoptose , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Macroglobulinemia de Waldenstrom/metabolismo , Macroglobulinemia de Waldenstrom/patologia , Idoso , Proteína 10 de Linfoma CCL de Células B , Feminino , Humanos , Imuno-Histoquímica , Masculino , Subunidade p50 de NF-kappa B , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fator de Transcrição RelA , Macroglobulinemia de Waldenstrom/genéticaRESUMO
Fluorescence in situ hybridization (FISH) has been used to demonstrate the t(14;18) in up to 100% of follicular lymphoma (FL) cases, however, there is little reproducible data using fixed tissue. The aim was therefore to develop a robust FISH method for the demonstration of translocations in archival tissue. The technique was evaluated by comparison with multiplex polymerase chain reaction (PCR), capable of detecting the majority of known breakpoints. Twenty-eight paired frozen and fixed cases of FL and 20 reactive controls were analyzed. The t(14;18) was detected in 23 of 28 cases using PCR on frozen material and 8 of 20 in paraffin. Using FISH, 24 of 26 frozen and 26 of 28 paraffin cases had a demonstrable translocation. All 20 reactive nodes were negative for the t(14;18) by PCR. Using FISH, one of the reactive cases had occasional cells with a translocation FISH pattern, demonstrable in frozen and paraffin samples. This is consistent with the presence of the t(14;18), which is well described in normal individuals. Both PCR and FISH are highly effective for t(14;18) analysis in unfixed tissue. When only paraffin blocks are available, FISH is the method of choice, and a result was achieved in 100% of cases. The method is applicable to the retrospective analysis of a range of translocations.
Assuntos
Cromossomos Humanos Par 14 , Cromossomos Humanos Par 18 , Hibridização in Situ Fluorescente/métodos , Linfonodos/ultraestrutura , Reação em Cadeia da Polimerase/métodos , Translocação Genética , HumanosAssuntos
Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 9/genética , Linfoma de Células B/genética , Recidiva Local de Neoplasia/genética , Neoplasias Esplênicas/genética , Translocação Genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Feminino , Humanos , Hibridização in Situ Fluorescente , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/patologia , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Neoplasias Esplênicas/tratamento farmacológico , Neoplasias Esplênicas/patologiaRESUMO
Strong expression of Forkhead box-P1 (FOXP1), a winged-helix transcription factor, has been identified as an independent prognostic factor in diffuse large B-cell lymphoma (DLBCL). However, possible mechanisms of deregulation of this gene, on 3p14.1, have yet to be elucidated. We have identified a breakpoint at the IGA1 gene in the immunoglobulin heavy chain (IGH) locus at 14q32 that was juxtaposed to the FOXP1 gene locus in a gastric DLBCL that showed strong expression of FOXP1. This may be one possible mechanism of deregulating FOXP1 expression by placing it under the control of IGH enhancers.
Assuntos
Cromossomos Humanos Par 14 , Cromossomos Humanos Par 3 , Fatores de Transcrição Forkhead/genética , Linfoma de Células B/genética , Linfoma Difuso de Grandes Células B/genética , Proteínas Repressoras/genética , Translocação Genética , Sequência de Bases , Primers do DNA , Humanos , Imuno-Histoquímica , Hibridização in Situ FluorescenteRESUMO
FOXP1 (Forkhead box-P1) is a winged-helix transcription factor that is differentially expressed in resting and activated B cells. FOXP1 expression has been demonstrated in a subset of diffuse large B-cell lymphomas (DLBCLs) and is more common in the nongerminal center (non-GC), activated B-cell type; however, its prognostic significance is uncertain. We analyzed presentation lymph nodes from 126 patients with nodal DLBCL, previously classified according to GC and BCL2 status, for FOXP1 protein expression using standard immunocytochemistry. Uniform high FOXP1 expression was demonstrated in 23 of 126 patients with DLBCL. This high level of expression was almost exclusively confined to patients who lacked the GC phenotype, who expressed MUM-1 and BCL2 in the absence of t(14; 18), and who were identified as a subgroup of patients with particularly poor outcomes in a group with already poor prognoses. The data presented suggest that high FOXP1 expression is an independent prognostic factor in DLBCL.
Assuntos
Biomarcadores Tumorais/genética , Linfoma Difuso de Grandes Células B/diagnóstico , Proteínas Repressoras/genética , Biomarcadores Tumorais/análise , Distribuição de Qui-Quadrado , Proteínas de Ligação a DNA/análise , Fatores de Transcrição Forkhead , Regulação Neoplásica da Expressão Gênica , Humanos , Fatores Reguladores de Interferon , Linfonodos/patologia , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/mortalidade , Prognóstico , Proteínas Proto-Oncogênicas c-bcl-2/análise , Proteínas Repressoras/análise , Análise de Sobrevida , Fatores de Transcrição/análiseRESUMO
The International Prognostic Index (IPI) identifies poor- and good-risk patients with diffuse large B cell lymphoma (DLBCL); however, the majority of patients have an intermediate IPI, with an uncertain prognosis. To determine whether cellular factors can be combined with the IPI to more accurately predict outcome, we have analyzed 177 presentation nodal DLBCLs for the expression of bcl-2 and a germinal center (GC) phenotype (defined by expression of bcl-6 and CD10). P53 gene band shifts were detected using single-stranded conformational polymorphism polymerase chain reaction analysis of exons 5-9 and were correlated with protein expression. In a Cox regression analysis, IPI (R = 0.22, P <.0001) and bcl-2 (R = 0.14, P =.0001) were independent poor prognostic factors and a GC phenotype predicted a favorable outcome (R = -0.025, P =.02). Neither p53 expression nor band shifts had a significant effect on survival. Using the IPI alone, 8% of patients were identified as high risk. Expression of bcl-2 in the intermediate IPI group identified a further 28% of patients with an overall survival comparable to the high IPI group. In the intermediate IPI, bcl-2(-) group, the presence of a GC phenotype improved overall survival to levels approaching the IPI low group. Following this analysis only 15% of patients failed to be assigned to a favorable- or poor-risk group. Sequential addition of bcl-2 expression and GC phenotype into the IPI significantly improves risk stratification in DLBCL. For the 36% of high-risk patients with a 2-year overall survival of 19%, alternative treatment strategies should be considered in future trials.
Assuntos
Centro Germinativo/metabolismo , Linfoma de Células B/patologia , Linfoma Difuso de Grandes Células B/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Feminino , Seguimentos , Humanos , Linfoma de Células B/diagnóstico , Linfoma de Células B/metabolismo , Linfoma Difuso de Grandes Células B/diagnóstico , Linfoma Difuso de Grandes Células B/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação , Fenótipo , Polimorfismo Conformacional de Fita Simples , Prognóstico , Medição de Risco , Análise de Sobrevida , Resultado do Tratamento , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Diffuse large B-cell lymphomas (DLBCL) are a heterogeneous group of tumours, varying in clinical features, immunophenotype and cytogenetics. The aim of this study was to investigate the prognostic significance of BCL6 gene rearrangement at the 3q27 locus in patients with primary nodal disease, and to examine interrelationships with immunophenotype and International Prognostic Index (IPI). We have developed a fluorescent in situ hybridization (FISH)-based technique for the retrospective analysis of the effect of BCL6 gene rearrangements on survival, using nuclei extracted from paraffin-embedded tissue. FISH results were obtained in 111 presentation cases of nodal DLBCL. The IPI was calculated and each case was stained immunocytochemically for BCL6, BCL2 and CD10. 3q27 rearrangements were detected in 25% of cases. BCL2 protein and a germinal centre (GC) phenotype (defined as CD10+, BCL6+) were expressed in 56% and 41% of cases respectively. In multivariate analysis, rearrangement of 3q27 and BCL2 expression and the absence of a GC phenotype were associated with a poor prognosis. These factors can be used in conjunction with the IPI to improve risk stratification in nodal DLBCL.