Sporadic occurrence of non-diagnosed IgG4-related disease in lymphoma patients with a previous Sjögren's syndrome diagnosis.

BACKGROUND: IgG4-related disease (IgG4-RD) is a recently recognized fibro-inflammatory disorder, which may affect many organs, and often comes to clinical attention due to tumor-like organ swelling or is identified incidentally by specific biopsy findings. Typical histopathology of IgG4-RD is lymphoplasmacytic infiltration rich in IgG4 + plasma cells (PCs), storiform fibrosis, and obliterative phlebitis. Patients with sicca symptoms can be misdiagnosed as primary Sjögren's syndrome (pSS) instead of IgG4-RD because of clinical and histopathological similarities. Moreover, an association with lymphoma development is described in both diseases. This study investigated signs of IgG4-RD in a population-based cohort of patients diagnosed with pSS complicated by lymphoma. METHODS: Patients with pSS and lymphoma diagnoses and available lymphoma specimens were identified by linkage with the Swedish Patient Register 1964-2007 and the Cancer Register 1990-2007 (n = 79). Clinical data and lymphomas were reviewed and the diagnoses evaluated. All lymphoma tissues and available minor salivary gland biopsies (n = 11) were immunostained for IgG4 + PCs and evaluated for other histopathological signs of IgG4-RD. In a case with specific findings of IgG4-RD, other available tissue specimens of the same patient were investigated for IgG4-RD. RESULTS: Only one patient of 79 (1.3%) had >10 IgG4 + PCs/high power field (HPF) in the lymphoma tissue, an unspecified low-grade B-cell lymphoma localized in the submandibular gland. This patient also had other histopathological features of IgG4-RD in the lymphoma and a surgical lung biopsy taken five years before lymphoma diagnosis and, therefore, fulfilled the criteria for IgG4-RD. Occasional IgG4 + PCs (<10/HPF) without signs of IgG4-RD were observed in another six lymphomas. No IgG4 + PCs were identified in the minor salivary gland biopsies. CONCLUSION: Histopathological findings of IgG4-RD may co-exist with low malignant B-cell lymphoma in patients with initially suspected pSS and may be associated with an underlying IgG4-RD.

A population-based study of 135 lymphomas after solid organ transplantation: The role of Epstein-Barr virus, hepatitis C and diffuse large B-cell lymphoma subtype in clinical presentation and survival.

BACKGROUND: Epstein-Barr virus (EBV) plays a major role in the development of post-transplant lymphoproliferative disorder (PTLD), but there is an increasing awareness of EBV-negative PTLD. The clinical presentation of EBV-negative PTLD has not been as well characterised as EBV-positive cases. Further, there is limited knowledge on the clinical importance of diffuse large B-cell lymphoma (DLBCL) cell of origin subtype post-transplant. MATERIALS AND METHODS: We studied the role of EBV, hepatitis C (HCV) and DLBCL subtype in clinical presentation and survival in 135 post-transplant lymphomas diagnosed 1980-2006 in a population-based cohort of 10 010 Swedish solid organ transplant recipients. The lymphomas were re-evaluated according to WHO 2008, examined for EBV, and clinical data were collected from medical records. RESULTS: Lymphoma incidence rate was 159/100 000 person-years and is also reported by lymphoma subtype. EBV-negative lymphomas constituted 48% and were associated with HCV infection (p = 0.02), bone marrow involvement (p < 0.001), and T-cell phenotype (p = 0.002). Among DLBCL, 78% were of non-germinal centre subtype, which was associated with EBV-positivity (69%, p = 0.001), early occurrence (p = 0.03), heart/liver/lung/pancreas recipients (p = 0.02), anti-T-cell globulin (p = 0.001), and tacrolimus treatment (p = 0.02). DLBCL subtypes had similar overall survival. Five-year overall survival was 42% in all treated patients. Independent poor prognostic factors were older age, B symptoms, ECOG 2-4, kidney/pancreas/heart recipients, T-cell lymphoma, and HCV-infection. CONCLUSIONS: With long follow-up, a large part of PTLD is EBV-negative, due to a high proportion of T-cell lymphomas and low of polymorphic PTLD. EBV-negative PTLD have a different clinical presentation. HCV may play an aetiological role in late-onset PTLD and was revealed as a new prognostic factor for inferior survival that needs to be confirmed in larger studies. The heavier immunosuppression in non-kidney transplantations seems to play a role in the development of non-germinal centre DLBCL. DLBCL cell of origin subtype lacks prognostic importance in the transplant setting.

Rheumatoid arthritis, treatment with corticosteroids and risk of malignant lymphomas: results from a case-control study.

BACKGROUND: Benefits and risks of corticosteroid treatment in rheumatoid arthritis (RA) are debated. Patients with RA are at increased risk of malignant lymphomas. In a large case-control study of risk factors for lymphoma in RA, it was recently reported that steroid treatment was associated with decreased lymphoma risk. OBJECTIVE: To further assess the nature of the association between steroid treatment in RA and the risk of lymphoma. METHODS: In a cohort of 74 651 patients with RA, 378 patients with lymphoma and 378 matched RA controls were identified, and information on inflammatory activity and different aspects of steroid treatment (duration, therapeutic strategy and mode of administration) abstracted from their medical records. Lymphomas were reclassified (WHO classification) and examined for Epstein-Barr virus. Relative risks were assessed as adjusted odds ratios (ORs) through conditional logistic regression. RESULTS: A total duration of oral steroid treatment of <2 years was not associated with lymphoma risk (OR=0.87; 95% CI 0.51 to 1.5), whereas total treatment >2 years was associated with a lower lymphoma risk (OR=0.43; 95% CI 0.26 to 0.72). RA duration at the initiation of oral steroids did not affect lymphoma risk. Intra-articular steroids were associated with a reduced lymphoma risk, but only when used as swift flare treatment (OR=0.22; 95% CI 0.13 to 0.37). Analyses by lymphoma subtype showed a reduced risk of diffuse large B-cell lymphoma (crude OR=0.59; 95% CI 0.37 to 0.94). CONCLUSION: In this RA population, use of steroids was associated with reduced lymphoma risk. Whether this association is a generic effect of steroids or specific to the studied population remains unknown.

Myeloid leukaemia in systemic lupus erythematosus--a nested case-control study based on Swedish registers.

OBJECTIVE: To assess the risk factors for leukaemic transformation and myeloid leukaemia in patients with SLE. METHODS: A national SLE cohort identified through SLE discharge diagnoses in the Swedish hospital discharge register during 1964 to 1995 (n = 6438) was linked to the national cancer register. A nested case-control study in SLE patients who developed acute or chronic myeloid leukaemia was performed with SLE patients without malignancy as controls. Medical records from cases and controls were reviewed and bone marrow specimens were re-evaluated. A Medline search of previously published cases of SLE and myeloid leukaemia was performed. RESULTS: After confirmation of SLE diagnosis according to the ACR criteria, eight patients with SLE and myeloid leukaemia and 18 SLE controls were included in the study. Preceding leucopenia was significantly associated with leukaemia development, whereas other SLE manifestations were not. Two cases had a preceding bone marrow confirming myelodysplastic syndrome (MDS). Only two cases were significantly treated with cyclophosphamide or AZA. A Medline search resulted in only 15 previously published cases of coincident SLE and myeloid leukaemia. Preceding MDS was reported in five of these, whereas only eight had been treated with cytotoxic drugs. CONCLUSION: Low-dose chemotherapy was not a major cause of myeloid malignancy in our population-based cohort of SLE patients nor in the reported cases from literature. Leucopenia was a risk factor for myeloid leukaemia development and an MDS was frequently seen. Therefore bone marrow investigation should be considered in SLE patients with long-standing leucopenia and anaemia.

Expression of PD-1, PD-L1, and PD-L2 in posttransplant lymphoproliferative disorder after solid organ transplantation.

We studied the expression of programed death 1 (PD-1) receptor and its ligands (PD-L1/-L2) by immunohistochemistry and its association with clinicopathological features in 81 posttransplant lymphoproliferative disorders (PTLDs) following solid organ transplantation. Overall, 67% (54/81) of the PTLDs were positive in any of the three immunostainings. PD-1 was detected on tumor-infiltrating cells in 41% (33/81) of the PTLDs. PD-L1 was expressed on ≥5% of the tumor cells in 50% (40/80) and PD-L2 in 32% (23/72) of the PTLDs. All Burkitt lymphomas were PD-L1 negative. Expression of PD-L1 tended to be associated with non-germinal center-type of diffuse large B-cell lymphoma (63% vs. 33% in GC-type, p = .14) and latent membrane protein-1+ PTLD (76% vs. 44% in LPM1-, p = .09). Heart recipients had more frequent PTLDs with PD-1+ microenvironment (p = .01). The frequent expression of PD-1 or -L1/-L2 in PTLD warrants further clinical evaluation of the efficacy and safety of PD-(L)1 inhibitors for refractory PTLD.

Cell of origin in diffuse large B-cell lymphoma in systemic lupus erythematosus: molecular and clinical factors associated with survival.

BACKGROUND: SLE is associated with increased risk of diffuse large B-cell lymphoma (DLBCL). DLBCL is routinely classified by cell of origin (COO), with germinal centre B-cell (GCB) being more common and indicating better prognosis in the general population. We studied COO subtyping in patients with SLE diagnosed with DLBCL and their survival. PATIENTS AND METHODS: We evaluated 20 cases of SLE with DLBCL. Immunohistochemistry analysis was performed (BCL2, MYC, BCL6, CD10, CD20, FOXP1, GCET1, MUM1) in tissue microarrays. We examined associations between molecular and clinical features, including overall survival. RESULTS: Of the 20 DLBCL SLE cases, 12/20 cases (60%) were classified as non-GCB using Hans or Choi algorithms. MYC and BCL2 protein expression was positive in 6/20 (30%) and 8/20 (40%) SLE cases, respectively, with 2/20 (10%) co-expressing both markers. Seven (7/20) had only extranodal involvement at DLBCL diagnosis. As expected, non-GCB cases had worse survival. Cases presenting exclusively with extranodal disease were associated with shorter SLE duration and better survival despite higher BCL2 protein expression. CONCLUSIONS: We present novel data characterising DLBCL in SLE. Sixty per cent of the DLBCL in patients with SLE were non-GCB. The nodal and extranodal distribution in SLE was similar to what is known in the general population, but extranodal disease occurred more often with short SLE duration and was associated with longer overall survival. More research on cancer in SLE is the key to further understanding the complex interplay between cancer and the immune system.

Expression of the human germinal-centre-associated lymphoma protein in diffuse large B-cell lymphomas in patients with rheumatoid arthritis.

Diffuse large B-cell lymphomas (DLBCL) can be subdivided into germinal centre (GC)-like and non-GC-like subtypes by CD10, BCL6 and MUM1/IRF4 status. We previously reported that patients with severe rheumatoid arthritis (RA) are at increased risk of non-GC DLBCL. This study examined a new GC-marker, human germinal-centre-associated lymphoma (HGAL) protein, in RA-DLBCL. Of 111, 38 (34%) DLBCL were HGAL-positive and showed less disseminated disease and a tendency toward improved overall survival compared to HGAL-negative cases. This supports that a majority of RA-DLBCL are of non-GC origin, indicating a specific role for activated peripheral B cells in the pathogenesis of RA-DLBCL.

Expression of intratumoral forkhead box protein 3 in posttransplant lymphoproliferative disorders: clinical features and survival outcomes.

BACKGROUND: The infiltration of regulatory T cells (Tregs) in lymphomas is associated with better prognosis for some types of lymphomas, but knowledge of their role in posttransplant lymphoproliferative disorders (PTLDs) is limited. We therefore investigated the association between the expression of the Treg marker forkhead box protein 3 (FoxP3) in biopsies of PTLDs and survival, PTLD subtype, and clinical characteristics. METHODS: Seventy-four cases of PTLD after solid organ transplantation with sufficient material for further analysis were included from a population-based study of PTLDs in Sweden. The PTLD biopsies were reevaluated and stained with the 236A/E7 antibody to detect FoxP3 in lymphoma tissue. Detailed clinical data were collected retrospectively from medical records. RESULTS: Based on a cutoff level of 29 FoxP3 cells per mm, most (80%) of the PTLDs were FoxP3. Forty-seven of 74 PTLDs displayed no FoxP3 cells at all. The frequency of FoxP3 cells did not influence median overall survival. The FoxP3 PTLDs were more frequently of T-cell phenotype (P = 0.04), located at the graft (P = 0.03), occurred earlier after transplantation (P = 0.04), were more likely to develop in lung recipients (P = 0.04), and in patients that had received anti-T-cell globulin as induction therapy (P = 0.02). The FoxP3 PTLDs were associated with hepatitis C seropositivity (P = 0.03). In multivariate analysis, B-cell PTLD and hepatitis C infection were independent predictors of FoxP3 positivity. CONCLUSION: Our findings suggest that intratumoral FoxP3 Tregs do not influence survival in patients with PTLD. FoxP3 Tregs are rare in PTLD, possibly because of heavy immunosuppression.

Leukemia and myelodysplastic syndrome in granulomatosis with polyangiitis: subtypes, clinical characteristics, and outcome.

OBJECTIVE: Previous studies have shown that patients with granulomatosis with polyangiitis (GPA) have an increased risk of hematological malignancies, especially leukemia. Our aim was to assess clinical characteristics and treatment of patients with GPA complicated by hematological malignancies with focus on leukemia and to describe these malignancies in more detail. METHODS: From the Swedish population-based patient register, all individuals with a diagnosis of GPA from 1964-2012 were identified (n = 3224). Through linkage with the Swedish Cancer Register, we searched for all cases of leukemia [International Classification of Diseases (ICD) 7: 204-207 and corresponding codes ICD 8-10] registered after the first discharge listing GPA. The GPA diagnosis was evaluated using the European Medical Association classification algorithm. To confirm the hematological malignancy, all diagnostic bone marrow samples were reclassified. Clinical data of both the GPA and hematological malignancy were collected from medical files. RESULTS: Twenty-one cases were identified, all of myeloid origin, including 9 with myelodysplastic syndrome developing to acute myeloid leukemia (MDS-AML), 7 AML, 3 MDS, and 2 chronic myeloid leukemia. The median time from GPA diagnosis to hematological malignancy was 8 years (range 5-21). All patients had severe generalized GPA and had received high doses of cyclophosphamide (CYC; median cumulative dose 96.5 g). Cytopenia occurred in 76% of the patients prior to the hematological malignancy. CONCLUSION: The findings emphasize the longterm risk of leukemia and MDS in CYC-treated, severely ill patients with GPA. Cytopenia during the course of GPA may be a warning sign and warrants a liberal attitude toward bone marrow examination.

Expression of APRIL in diffuse large B cell lymphomas from patients with systemic lupus erythematosus and rheumatoid arthritis.

OBJECTIVE: Patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) have an increased risk of diffuse large B cell lymphoma (DLBCL). The cytokine A PRoliferating-Inducing Ligand (APRIL) is strongly expressed in DLBCL in the general population and is detected in high concentrations in sera from subgroups of patients with RA and SLE. To investigate a possible association between APRIL and DLBCL in RA and SLE, we examined APRIL expression in lymphoma biopsies from patients with RA and SLE and from DLBCL patients without inflammatory disease. METHODS: Lymphoma tissue from 95 RA, 12 SLE, and 63 comparator DLBCL cases were stained with anti-APRIL antibodies (Aprily-2). The percentage of positively stained cells of the comparator cases were divided into quartiles (1-4, where 4 = most stained) and compared with the results for the RA and SLE lymphomas. APRIL expression was correlated to clinical variables. RESULTS: The odds ratio for high expression of APRIL (quartiles 3 and 4) was elevated in the SLE DLBCL (OR 23.6, 95% CI 2.4-231.2), but not in the RA DLBCL (OR 0.8, 95% CI 0.3-2.0). RA patients in quartile 4 had higher cumulated RA disease activity than those in quartile 1 (p = 0.013). Epstein-Barr virus in the lymphoma tissue was associated with high APRIL expression (p = 0.009). CONCLUSION: The high expression of APRIL in DLBCL in SLE and in an RA subset might indicate an association between APRIL and lymphoma in these subsets of rheumatic diseases, but could also reflect a dysregulation of APRIL per se in these patient groups.

Characteristics of diffuse large B cell lymphomas in rheumatoid arthritis.

OBJECTIVE: Patients with rheumatoid arthritis (RA) are at increased risk of malignant lymphomas, with a correlation between RA disease severity and lymphoma risk, most pronounced for diffuse large B cell lymphomas (DLBCLs), which also constitute the majority of RA-associated lymphomas. DLBCLs can be further subdivided into germinal center (GC)-like and non-GC-like subtypes, with different cellular origins and prognoses. This study was undertaken to investigate whether RA displays a specific association with any of the DLBCL subtypes. METHODS: We identified 139 patients with DLBCLs within a population-based case-control study of 378 RA patients with lymphoma. The DLBCLs were examined for CD10, Bcl-6, and interferon regulatory factor 4 expression patterns, subclassified into GC and non-GC subtypes, and then correlated with clinical parameters. RESULTS: We found a statistically significant predominance of the non-GC subtype (97 patients; 70% of all DLBCLs). These patients more often had an advanced stage of lymphoma at diagnosis and had a worse 5-year overall survival rate (16% versus 33%) compared with patients with the GC subtype. There was a strong association with RA disease activity in both subtypes, with >70% of the GC and non-GC cases occurring in RA patients with the highest overall disease activity scores. CONCLUSION: These findings suggest that severe RA is particularly associated with the non-GC subtype of DLBCL, and indicate a critical role of activated peripheral B cells as the cells of origin in these lymphomas.

Association of chronic inflammation, not its treatment, with increased lymphoma risk in rheumatoid arthritis.

OBJECTIVE: Chronic inflammatory conditions such as rheumatoid arthritis (RA) have been associated with malignant lymphomas. This study was undertaken to investigate which patients are at highest risk, and whether antirheumatic treatment is hazardous or protective. METHODS: We performed a matched case-control study of 378 consecutive Swedish RA patients in whom malignant lymphoma occurred between 1964 and 1995 (from a population-based RA cohort of 74,651 RA patients), and 378 controls. Information on disease characteristics and treatment from onset of RA until lymphoma diagnosis was abstracted from medical records. Lymphoma specimens were reclassified and tested for Epstein-Barr virus (EBV). Relative risks (odds ratios [ORs]) for lymphomas (by subtype) associated with deciles of cumulative disease activity were assessed, as were ORs associated with drug treatments. RESULTS: The relative risks of lymphoma were only modestly elevated up to the seventh decile of cumulative disease activity. Thereafter, the relative risk increased dramatically (OR ninth decile 9.4 [95% confidence interval 3.1-28.0], OR tenth decile 61.6 [95% confidence interval 21.0-181.0]). Most lymphomas (48%) were of the diffuse large B cell type, but other lymphoma subtypes also displayed an association with cumulative disease activity. Standard nonbiologic treatments did not increase lymphoma risk. EBV was present in 12% of lymphomas. CONCLUSION: Risk of lymphoma is substantially increased in a subset of patients with RA, those with very severe disease. High inflammatory activity, rather than its treatment, is a major risk determinant.

Evaluation of immunophenotype in diffuse large B-cell lymphoma and its impact on prognosis.

Diffuse large B-cell lymphoma (DLBCL) has been shown to be comprised of at least two prognostic entities, depending on its resemblance to normal germinal center or activated B cells, using global gene expression profiling. Also, the expression patterns of bcl-6, CD10 and IRF-4 (also known as MUM1) have been suggested as alternative means of identifying the germinal- and nongerminal center (activated B-cell like) groups. In the present study, we evaluated by immunohistochemistry the expression patterns of CD10, bcl-6, IRF-4 and bcl-2 in a large material of 161 DLBCL patients. Using two different approaches, patients with germinal center phenotype displayed a significantly better survival than the nongerminal center group. Positive staining for bcl-6 or CD10 predicted for superior survival, while expression of IRF-4 alone showed no association with prognosis. Furthermore, expression of bcl-2 was associated with worse event-free survival and overall survival. In a multivariate analysis, a high international prognostic index score (3-5), non-GC phenotype and bcl-2 were independent adverse prognostic factors. Here we confirm the prognostic importance of determining the germinal- or nongerminal center phenotype in patients with DLBCL.

Monoclonal Antibodies Recognizing Normal and Neoplastic Human Adrenal Cortex.

Four monoclonal antibodies (MAb) were generated by immunization of mice with dispersed cells from normal human adrenal gland (Na) and adrenocortical adenoma causing cortisol excess (Ac). Immunohistochemically reacted cryosections revealed differential labeling of the normal cortical parenchyma, and immunofluorescence on dispersed cells displayed that Ac5 alone labeled the cell surface. Immunoprecipitation demonstrated that the antibodies recognized apparently different structures of 51-88 kDa. Immunohistochemical examination of several normal human tissues substantiated restricted reactivity, especially for the Na2 and Na7 antibodies, and that the adrenal medulla was not stained by any of the antibodies. The antibodies recognized the vast majority of the parenchymal cells of cortical adenomas (n = 21). Each antibody also reacted with all adrenocortical carcinomas (n = 17), and the staining generally was most intense and extensive with Na7. Analysis of other pathological human tissues revealed highly restricted reactivity for the Na2 antibody. Na2 and Na5 failed to stain 17 renal cell carcinomas. None of the antibodies recognized pheochromocytomas. These antibodies may lead to improved histological recognition and characterization of human adrenal lesions.

Expression of CCL5/RANTES by Hodgkin and Reed-Sternberg cells and its possible role in the recruitment of mast cells into lymphomatous tissue.

HL is a malignant lymphoma characterized by a small number of malignant HRS cells among a major population of infiltrating reactive cells, e.g., lymphocytes and eosinophils. We previously reported that mast cells are present in HL-affected lymph nodes and therein are the predominant CD30L-expressing cells. The CD30L expressed on mast cells is functionally active and can provide stimulatory signals to HRS cells. Thus, mast cells constitute an important portion of the infiltrating reactive cells that contribute to tumor progression in HL. Control of the recruitment of this previously unrecognized cell and its interactions with tumor cells are essentially unknown. To elucidate if mast cells might be specifically attracted to the tumor area by chemokines produced by HRS cells, we investigated chemokine expression in HL cell lines and in vivo. By RNase protection assay, mRNA expression of several chemokines could be detected in the cell lines. Despite the heterogeneous expression profile exhibited by the cell lines, 4 of 5 expressed CCL5 (RANTES) mRNA. RT-PCR and immunohistochemistry confirmed expression of CCL5 in vivo. Furthermore, secreted CCL5 was detected in conditioned media from 3 of the cell lines. In a migration assay, we found that CCL5 present in conditioned medium could induce mast cell migration. Taken together, our results suggest that CCL5 produced by HRS cells is one mechanism by which mast cells can be attracted into the tumor tissue in HL.

Somatic hypermutation and V(H) gene usage in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is considered to derive from naïve, pregerminal center (GC) CD5+ B-cells. However, the cell of origin has been questioned in recent studies that showed somatic hypermutations in the immunoglobulin (Ig) variable heavy chain (V(H)) genes in subsets of MCL. To clarify this issue, we analyzed the IgV(H) genes for the presence of somatic hypermutations in 51 MCL cases. Twenty percent of the MCL cases displayed somatically mutated V(H) genes (defined as >2% mutated), whereas 80% showed unmutated V(H) genes. This finding suggests that MCL is a genetically heterogeneous disease, with the majority of cases originating from unmutated pre-GC B-cells and a subset deriving from more mature B-cells which have been exposed to the GC environment and have undergone somatic hypermutation. A biased V(H) gene usage has been demonstrated in several B-cell malignancies; however, this has not yet been investigated in MCL, although V(H)4-34 overusage has been indicated by small studies. Interestingly, we found a restricted usage of three individual V(H) genes in our MCL material; V(H)4-34 (22%), V(H)3-21 (16%) and V(H)5-51 (12%). This novel finding of preferential V(H) gene usage in half of the MCL cases may suggest an antigen driven process occurring in B-cells expressing specific VH genes, thus implicating that Ig specificity could be involved in mantle cell lymphoma development.

Chromosomal imbalances in diffuse large B-cell lymphoma detected by comparative genomic hybridization.

Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma. In contrast to many other hematological malignancies, no chromosomal abnormalities with a diagnostic or prognostic value have been identified in DLBCL. Numerical chromosomal imbalances were characterized by comparative genomic hybridization (CGH) performed on 54 DLBCL tumors from a total of 40 patients. The clonal relatedness was demonstrated in 9 of 11 pairs of matched diagnostic tumors and their relapses as determined by IGH gene rearrangement analysis and/or the CGH profiles. Furthermore, immunohistochemical expression analyses of BCL2 and BCL6/LAZ3 were performed on all cases. Copy number changes were detected in 94% of the diagnostic tumor samples and in all of the relapses. Chromosomal losses in diagnostic tumors were preferentially observed at 8p22-pter (29%), 1p34-pter (26%), 6q23-qter (20%), 17p12-pter (17%) and 22q (17%), 9p23-pter (14%), whereas gains were mainly seen in Xq25-26 (43%), 13q22 (26%), 12cen-q14 (20%), 3q24-25 (11%), 7 (11%), and 18q12-21 (11%). Loss of 22q was significantly more commonly seen in the diagnostic tumor samples with more advanced clinical stage in other words, Stage III-IV compared with Stage I-II, and band 18q21 was significantly more often gained in relapses as compared to diagnostic tumors. None of the recurrent alterations were detected as a single abnormality, suggesting that other genetic lesions below the detection level of CGH may be the initiating event in the tumorigenesis of DLBCL. However, the distribution of CGH alterations support the idea of a progression of genetic events where loss of 8p and 9p and gain of 3q, 13q, and 18q would represent relatively early events because they were distributed in tumors with only two abnormalities.