non-GC Diffuse Large B-cell Lymphoma Research Articles

I. Pathological and clinical diversity in diffuse large B‐cell lymphoma

I. Pathological and clinical diversity in diffuse large B‐cell lymphoma

Cell cycle regulation score predicts relapse‐free survival in non‐germinal centre diffuse large B‐cell lymphoma patients treated by means of immunochemotherapy

The cell cycle is under strict regulation by the retinoblastoma, p53 and p27 pathways, and the disruption of these pathways is an important characteristic of diffuse large B-cell lymphoma (DLBCL). In this study, we wanted to assess the function and prognostic significance of these pathways in DLBCL patients. Tissue samples from 120 DLBCL patients treated by means of R-CHOP-type chemotherapy were stained for the cell cycle-regulating proteins p16, p21, p27 and p53, and the germinal centre (GC) phenotype was determined according to Hans' algorithm. Based on the number of impaired cell cycle-regulating pathways a predictive score was obtained, covering three different prognostic groups: a 'favourable' group with damage in 0-1 of the studied pathways, a 'poor' group with damage in all three pathways and an 'intermediate' group comprising the rest of the patients. The prognosis of non-GC DLBCL patients was significantly poorer vs. GC phenotype patients (P=0.015). The prognostic score proved especially useful among non-GC phenotype patients, with 3-yrs relapse-free survival of 100% vs. 62.6% vs. 24.3% in the 'favourable-', 'intermediate-' and 'poor prognosis' groups, respectively (P=0.003). The prognosis of non-GC DLBCL patients is progressively impaired with the accumulation of damage in different cell cycle-regulating pathways.

Fatty Acid Metabolism in Diffuse Large B-Cell Lymphoma (DLBCL): Interaction with Oncogenic Cell Signaling Pathways and the Identification of a Novel Treatment Paradigm.

AbstractAbstract 2711 Background:Fatty acid synthase (FASN) is a key enzyme of fatty acid synthesis and is upregulated in many cancers. Increased FASN in cancer is associated with poor prognosis, while inhibition of FASN results in cancer cell death. The MEK/ERK signal transduction is one of the primary pathways that activate tumor-related FASN. Lipoprotein lipase (LPL) is also involved in fatty acid metablishm as it releases free fatty acid (FFA) from circulating lipoproteins, making them available for cellular uptake. Notably, these concepts have emerged primarily from solid tumor studies; there is a comparative paucity of data in lymphoma. We examined the functional roles of FASN and LPL in DLBCL cells and their interaction with oncogenic signal transduction pathways including MEK/ERK and an upstream target, hypoxia inducible factor-1 alpha (HIF-1a). We also investigated potential therapeutic implications of targeting fatty acid metabolism for the treatment of DLBCL. Methods:We used the DLBCL cell lines OCI-LY3, OCI-LY19, SUDHL4, and SUDHL10 in normoxic or hypoxic (0.2% O2) conditions. Cerulenin (FASN inhibitor) and Orlistat (FASN and LPL inhibitor) were utilized to examine the effect of fatty acid enzyme inhibition on cell signaling and cell death. We assessed cell viability with the MTT assay and apoptosis by flow cytometric analysis of Annexin-V/propidium iodide (PI). FASN and LPL mRNAs were quantified in DLBCL cell lines by RT-PCR as well as through gene expression profiling (GEP) analysis (by cell of origin) using the CaBIG dataset. Further, FASN and associated signaling pathways (MEK, ERK, and HIF-1a) were analyzed by Western blot. Finally, for investigation of potential interactions between FASN and HIF-1a, or MAPK signaling, we utilized short hairpin RNA interference (shRNA) to knock down (KD) pathways of interest. Results:FASN protein expression was readily detectable in all DLBCL cell lines in normoxia, while the expression of LPL was barely detectable in most cells, except in SUDHL10 and only in hypoxic conditions. RT-PCR showed that all DLBCL cell lines tested expressed high levels of FASN mRNA, while minimal levels of LPL could be detected; GEP showed that FASN was expressed more prominently in germinal center (GC) DLBCL (p=0.0006 vs GC control and p=0.0001 vs non-GC DLBCL), whereas LPL was preferentially expressed in non-GC DLBCL (p<0.0001 vs non-GC control and GC DLBCL). We next examined FASN expression following KD of MEK, ERK, or HIF-1a using shRNA in OCI-LY3 and SUDHL10 cells. HIF-1a KD significantly decreased FASN expression; this result was most prominent in OCI-LY3 cells, although it was also evident in SUDHL10. Interestingly, MEK and ERK KDs had minimal effect on FASN or LPL. Pharmacologic treatment with cerulenin, however, resulted in inhibition of MEK and ERK phosphorylation in OCI-LY3 cells. Additionally, treatment with Cerulenin or Orlistat (0.25–4 μg/mL for 48 hours) resulted in dose-dependent cytotoxicity across several DLBCL cell lines (OCI-LY3, SUDHL4, and SUDHL10) with an approximate IC50 of 1μg/mL in all lines. Furthermore, treatment with Cerulenin resulted in induction of apoptosis, which was mediated by caspase cleavage (caspases 3, 8 and 9) in SUDHL4 and OCI-LY3 cells. Conclusions:We demonstrated that FASN is constitutively activated in DLBCL with expression in part dependent on cell of origin, while LPL protein or message were mostly down-regulated. HIF-1a is a constitutively activated oncogenic pathway in DLBCL (Evens AM, et al. Br J Haematol 2008) and it appeared here to directly regulate FASN expression. In addition, we showed that targeting fatty acid metabolism may be harnessed as a potential therapeutic strategy. Further investigations are required to delineate the mechanisms through which MAPK and HIF-1a regulate FASN expression and to determine the in vivo implications of FASN inhibition on DLBCL tumor growth. Disclosures:No relevant conflicts of interest to declare.

Richter Syndrome (RS): Genome-Wide Promoter Methylation Profile Differs From De Novo Diffuse Large B-Cell Lymphoma (DLBCL) and Affects Genes Involved in Stem-Cell Maintenance and TP53 Pathway

Abstract 1359RS represents the development of DLBCL in the context of chronic lymphocytic leukemia (CLL). The pathogenesis of RS is still largely unknown. We have previously shown that RS lacks many of the typical genetic lesions recurrently observed in de novo DLBCL (Rossi et al, Blood 2011). Here, we report genome-wide promoter methylation profiling of RS and compare it with the methylation profile of de novo DLBCL, and non-transformed CLL. Methods.The study included 21 RS, 10 clonally related CLL phases (seven paired with RS samples), 9 de novo non-GC DLBCL, 6 non-transformed CLL and 6 normal peripheral blood CD19+ B-cells. All RS were classified as DLBCL, showed a non-germinal center (GC) phenotype, and lacked EBV infection. Both RS and DLBCL DNA was extracted from diagnostic frozen lymph node biopsies. DNA samples underwent bisulfite treatment with the EZ DNA methylation kit and were hybridized on Illumina Infinium HumanMethylation27 arrays, that enables the interrogation of more than 27,000 CpG island over the entire human genome. Probes were discarded if they: i) mapped outside CpG islands; ii) presented a standard deviation <0.10 across all the samples; or iii) were always non-methylated, always fully methylated, or always partially methylated. For clustering and for unpaired t-tests, probes mapped on sex chromosomes were excluded. Supervised analysis of differential methylation between groups was performed using a t-test on the continuous beta values, followed by multiple test correction (MTC). A paired t-test was utilized for comparing the methylation profile between the RS phase and the matched CLL-phase. Probes showing a q-value <0.1 were defined differentially methylated. Results.By unsupervised clustering, RS samples separated from both de novo DLBCL, that instead clustered in a single group, and non-transformed CLL.There were over 3,000 probes differentially methylated between RS and de novo DLBCL. The 443 probes showing higher methylation in RS were significantly enriched of promoter regions of genes involved in TP53 signaling (P=6E-03) and cell cycle regulation (P=5E-02) pathways. The 2,687 probes showing higher methylation in de novo DLBCL were significantly enriched of promoter regions of genes involved in histone modification, including genes harboring the H3K27me3 mark (P < 1.0E–16), and of genes that are targets of the EED and SUZ12 Polycomb proteins (P < 1.0E–16).Analysis of paired CLL/RS sequential samples revealed an extensive overlap between the methylation profile of the CLL phase and the transformed RS phase, suggesting that changes in the methylation pattern are an early event in RS pathogenesis that occurred already at the time of the initial CLL clone. Two single probes in the promoter regions of the OSM and S1PR4/EDG6 genes were differentially methylated in CLL/RS sequential samples and, therefore, might have contributed to the acquisition of the aggressive clinico-pathologic phenotype of RS. OSM codes for oncostatin M, which inhibits cell proliferation and induces cell differentiation and apoptosis. S1PR4/EDG6 is a gene specifically expressed in the lymphoid tissue and involved in cell migration. Both OSM and S1PR4/EDG6 promoter regions were unmethylated in the chronic CLL phase, and became methylated in the paired aggressive RS phase, suggesting the acquisition of methylation during transformation.CLL that subsequently transformed to RS differed from CLL that never transformed to RS for 2 probes showing a higher and 2 a lower degree of methylation. Since changes in the methylation pattern are an early event in RS, this observation prompts the investigation of the methylation status of DLX5 and GBGT1, the two genes with promoter regions showing a higher degree of methylation, as biomarkers allowing the early identification of CLL that will transform to RS. Conclusions.RS clearly differ from de novo DLBCL in terms of methylation profile. The overrepresentation of epigenetic changes affecting genes of the TP53 pathway, along with the significantly higher prevalence of TP53 structural abnormalities, might explain the differences in chemosensitivity between RS and de novo DLBCL. Epigenetic changes are an early events in RS pathogenesis, suggesting that, similarly to what shown in transformed follicular lymphoma, lesions initiating transformation are acquired by a cell belonging to the initial tumor clone, rather being progressively accumulated during disease course. Disclosures:No relevant conflicts of interest to declare.

Translocations Involving MUM1 are Rare in Diffuse Large B-cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) comprises a diverse group of neoplasms that have recently been subdivided by gene expression profiling and immunohistochemical studies into at least 2 subgroups [germinal center (GC) type and non-GC type]. The non-GC subtype has a post-GC activated phenotype and typically expresses MUM1 by immunohistochemistry. We hypothesized that MUM1 may be dysregulated/up-regulated in these tumors by a chromosomal translocation, as is seen in many cases of plasma cell myeloma [where MUM1 is juxtaposed with the immunoglobulin heavy chain gene (IgH)]. Therefore, using a novel MUM1 break-apart probe constructed in our laboratory, we performed fluorescence in situ hybridization on 33 cases of DLBCL (17 GC type and 16 non-GC type) for a MUM1 translocation. We identified 1 case of a MUM1 translocation out of 31 cases with successful fluorescence in situ hybridization. This case was a non-GC DLBCL (1/15). We conclude that genetic abnormalities involving MUM1 are rare in DLBCL and that a mechanism of deregulation of the MUM1 protein other than by a translocation event is involved in the majority of non-GC cases.

Influence of low absolute lymphocyte count of patients with nongerminal center type diffuse large B-cell lymphoma with R-CHOP therapy

BackgroundRituximab has dramatic impact on outcome of patients with diffuse large B-cell lymphoma (DLBCL), especially nongerminal center (non-GC) type. A low absolute lymphocyte count (ALC) before rituximab, cyclophosphamide, vincristine, adriamycin, and prednisone (R-CHOP) therapy as a surrogate marker of immune status is associated with poor clinical outcome in DLBCL. Therefore, we hypothesized that low ALC before R-CHOP would have effect on the survival in non-GC type. Patients and methodsOne hundred and thirty-six DLBCL patients who were treated with R-CHOP from 2003 to 2007 were analyzed in the present study. ResultsALC ≥1.0×109/l predicted a longer 3-year progression-free survival (PFS) and 3-year overall survival (OS) versus ALC <1.0×109/l (82.6% versus 60.0%, P=0.005 and 87.2% versus 62.0%, P<0.001, respectively). Non-GC type had similar PFS and OS to germinal center type (68.2% versus 80.0%, P=0.074 and 72.7% versus 82.9%, P=0.111, respectively). However, considering clinical influence of the ALC according to immunophenotype, low ALC in non-GC type DLBCL was associated with lower PFS and OS compared with others (PFS, P=0.002; OS, P<0.001). Multivariate analysis revealed that low ALC in non-GC type had lower PFS [hazard ratio (HR)=3.324, P=0.001] and OS (HR=4.318, P<0.001), independent of international prognostic index. ConclusionA low ALC in non-GC type DLBCL counteracted the beneficial effect of rituximab on survival.

NIK and the Alternative NF-κB Pathway in Human Lymphomas.

Abstract 3943Poster Board III-879The nuclear factor κB (NF-κB) family of transcription factors is required for the development of T and B lymphocytes and the regulation of the innate and adaptive immune response. Deregulated NF-κB activity has been associated with a number of malignancies and several types of lymphomas depend on NF-κB activity for cell proliferation and survival. A number of studies have been published in the last few years demonstrating the importance of the alternative NF-κB pathway in lymphomas. The NF-κB-Inducing Kinase (NIK or MAP3K14) is a serine/threonine kinase that is essential for the activation of the alternative NF-κB pathway. NIK induces the phosphorylation of the NF-κB member p100, which is followed by the processing of p100 to p52 and its subsequent nuclear translocation. Gene expression data from 106 lymphoma samples with different diagnosis (diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), mucosa-associated lymphoid tissue (MALT), nodal marginal zone lymphoma (NMZL) and chronic lymphocytic leukemia (CLL)) were previously generated using Agilent oligonucleotide microarrays. Data analysis indicated variability in NIK expression among the different lymphoma samples, showing a higher expression in CLL and FL samples. NIK expression was closely associated with the expression of other members of the alternative NF-κB pathway, such as NFKB2, RELB and CD40. To investigate the correlation between NIK gene expression and functional pathways, we performed a gene set enrichment analysis (GSEA). We found that the expression of NIK was positively and significantly correlated with several biologically important pathways in both lymphomagenesis and normal leukocyte development and function, such as B- and T-cell receptor pathways, CD40 signaling pathway, and the classical and alternative NF-κB pathways. Twenty seven lymphoma-derived cell lines were examined by Western blot for expression of NIK and p100/p52. The pathway was found to be frequently activated in these cell lines since high levels of p52 were detected in the majority of MCL (5/9 cell lines), Hodgkin lymphoma (3/3), CLL (1/1), DLBCL (7/9) and T-cell lymphoma (5/5) cell lines. Two-thirds of these p52-positive cell lines also expressed NIK and three of them expressed a truncated form of p100. The expression of NIK and p52 was also associated with Epstein-Barr virus (EBV) infection, given that four out of five EBV-positive cell lines showed elevated levels of these proteins. p100/p52 expression was immunohistochemically analyzed using tissue microarrays including paraffin-embedded tissues from totally 356 DLBCL patients. Examination of these samples indicated that the alternative pathway is activated in a subset of these tumors, with nuclear p52 expressed in 17% of the cases. A significant positive correlation between EBV-infection and p52 expression was established and the majority of the p52-positive cases also expressed nuclear p50, suggesting that both the alternative and classical NF-κB pathways are frequently activated in the same tumors. Alternative and classical NF-κB activation was more frequently present in the non-GC DLBCL type, but also observed in a significant proportion of the GC-DLBCL cases. Furthermore, array based comparative genomic hybridization (aCGH) revealed mono-allelic loss of TRAF3, a negative regulator of NIK, in 5 out of 22 DLBCL cases. Taken together, our results show that the alternative NF-κB pathway is activated in a subset of human lymphoma samples and cell lines. This highlights the relevance of NIK as a potential therapeutic target in lymphoid malignancies. Disclosures:No relevant conflicts of interest to declare.

Mutations in Multiple Genes Cause Deregulation of the NFkB Pathway in Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease comprising multiple biologically and clinically distinct subgroups, including germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. Gene expression profile studies have shown that a key feature of its most aggressive subtype, ABC-DLBCL, is the constitutive activation of the NF-kB transcription complex. However, except for a small fraction of cases (Lenz et al., Science 2008), it remains unclear whether NF-kB activation in these tumors reflects an intrinsic program of the cell of origin or represents a primary pathogenetic event. To address this question, we first characterized 165 DLBCL samples (18 cell lines and 147 primary biopsies, including 26 ABC, 28 GCB, 10 unclassified and 83 not profiled) for the presence of active, nuclear NF-kB complexes by using immunohistochemical/immunofluorescence staining of NFKB1 p105/p50 (as a readout for the canonical pathway) and NFKB2 p100/p52 (as a readout for the non-canonical pathway). Nuclear localization of NF-kB, indicative of constitutive activity, was observed in 14/26 (54%) ABC-DLBCL and 8/28 (28%) GCB-DLBCL primary biopsies, as well as in 3/10 (30%) unclassified and 48/83 (58%) non-profiled cases, and correlated with significant enrichment in expression of NF-kB target genes, as assessed by gene set enrichment analysis (GSEA) of transcriptionally profiled cases. In addition, the more sensitive GSEA approach detected a gene expression signature of NF-kB activity in >90% ABC-DLBCLs and 53% GCB-DLBCLs, indicating that constitutive activation of this key signaling pathway is a common feature of ABC-DLBCL but can also be observed in a smaller fraction of GCB-DLBCL. To investigate whether NF-kB activity represents a primary pathogenetic event, we then screened for mutations the complete coding sequences of 31 genes encoding for NF-kB pathway components in a panel of 14 ABC-DLBCLs, which was expanded to 48 samples (12 ABC, 26 GCB and 10 not profiled) for validation of the mutated genes. The results showed that >50% of ABC-DLBCL (n=15/26) and a smaller fraction of GCB-DLBCL (n=8/26, 31%) carry somatic mutations in multiple genes, including negative (TNFAIP3/A20) and positive (CARD11, TRAF2, TRAF5, MAP3K7/TAK1 and TNFRSF11A/RANK) regulators of NF-kB. Of these, the A20 gene, which encodes for a ubiquitin-editing enzyme involved in termination of NF-kB responses, is the most commonly affected, with ~27% ABC-DLBCLs and 25% (8/32) immunohistochemically classified non-GC DLBCL displaying biallelic A20 inactivation by somatic mutations and/or deletions. Sequence changes include premature nonsense mutations, frameshift deletions/insertions and splice site mutations, leading to severely truncated proteins that lack functionally relevant domains and have thus lost their enzymatic activity. In virtually all mutated cases, FISH analysis revealed loss of the second allele, while 4 additional samples showed biallelic deletion of the gene. Thus, A20 is inactivated by a classic “two-hit” mechanism, suggesting a tumor suppressor role. Less frequently, missense mutations of CARD11 (10%) and TRAF2 (4%) produce molecules with significantly enhanced ability to activate NF-kB in transient transfection/reporter gene assays. Our results demonstrate that NF-kB activation in DLBCL is caused by genetic lesions affecting multiple genes, whose loss or activation may promote lymphomagenesis by leading to abnormally prolonged NF-kB responses. These findings provide the rationale and the assays for the identification of patients amenable to NF-kB targeted therapeutic intervention.

Activation of the NF‐κB signalling pathway in diffuse large B‐cell lymphoma: clinical implications

To characterize the activation of the nuclear factor (NF)-kappaB pathway in diffuse large B-cell lymphoma (DLBCL) by immunohistochemistry. Sixty-six DLBCLs treated with anthracycline-containing chemotherapy were evaluated with antibodies against phosphorylated p65 (P-p65), p65, p50, p52, IKK alpha, and phosphorylated I kappaB (P-I kappaB). NF-kappaB activation was based on the expression of P-p65, P-I kappaB, and nuclear expression of p65 or p52 in the tumour cells. P-p65 and P-I kappaB were expressed in 13 (20%) and 17 cases (26%), respectively. p65, p52 and IKK alpha were found in the cytoplasm. A correlation was found between expression of P-p65 and P-I kappaB (P < 0.0001), but not between the two subtypes of DLBCL [germinal centre B cell and non-germinal centre (GC)]. P-p65+ tumours showed a better response to chemotherapy (P = 0.025) and a trend to increased event-free survival (P = 0.08). However, P-I kappaB expression was not associated with either clinical response or survival. Bcl-2 was not preferentially expressed on DLBCL tumours with NF-kappaB activation, as determined by expression of P-p65 and P-I kappaB proteins. NF-kappaB activation in DLBCL is preferentially mediated through the classical pathway and a novel mechanism involving phosphorylation of p65. Activation of NF-kappaB by P-p65 is associated with good prognosis. NF-kappaB activation is not confined to non-GC DLBCL exclusively.

Age related Epstein Barr virus associated B cell lymphoproliferative disorder in Peru

19514 Background: Age related Epstein Barr virus (EBV) associated B cell lymphoproliferative disorder is a recent new entity described in Japanese patients with diffuse large B cell lymphoma (DLBCL) and infection with EBV. It usually affects elderly patients with poor survival. Methods: In order to establish the correlation of EBV with DLBCL, we investigated the EBV status by RNA- in situ hybridization (ISH) in newly diagnosed nodal DLBCL.Between January 2002 and December 2004, seventy-four newly diagnosed nodal DLBCL patients were included in the analysis. Clinical data were reviewed retrospectively and patient’s biopsies were analyzed for the presence of EBV encoded RNA (EBER) by ISH and the immunohistochemical expression of BCL6, CD10 and MUM-1/IRF4 by tissue microarray (TMA) technique. Results: In this cohort the mean age was 66.1 years old (range 23–84) at diagnostic; male/female ratio 39/35; 46 patients (65.7%) with advanced stage (III/IV); 53 cases (71.6%) were of the non Germinal Center DLBCL type (non GC). This group was associated with a higher IPI than the Germinal Center DLBCL type (GC). Also there was a trend that the non GC type had a poorer overall survival than the GC type (p=0.171). Eleven cases of the cohort (14.9%) were identified as EBER-positive and were called as Age related EBV associated B cell lymphoproliferative disorder. Most of the cases EBER (+) were of the non GC group (10/11), showing a trend between EBER+ and non-GC DLBCL (OR=0.21, p=0.11 fisher 2-tails). EBER positive cases were associated with an advanced age (> 60 years), poorer status performance, more advanced stage, higher IPI risk group and poorer outcome to initial treatment. Overall survival in the EBER positive cases and negative were 1.1 vs. 15.5 months respectively (p=0.001). At the multivariable level de EBER status is an independent variable compared with the International Prognostic Index (IPI) (p=0.001) with a 3.2-fold (95% CI, 1.5 - 7.2) risk for death for positive cases Conclusions: Age related EBV associated B cell lymphoproliferative disorder is frequent between DLBCL in Peru. It was related to more advanced age, poor status performance, non-GC group and very short overall survival in comparison with DLBCL. New treatment strategies should be defined in this subgroup of poor prognostic. No significant financial relationships to disclose.

CD44 standard isoform expression is associated with diffuse large B cell lymphoma of non-germinal center B cell-like types

18531 Background: Diffuse large B cell lymphoma (DLBCL) can be subdivided into germinal center B cell-like (GCB) and non- germinal center B cell-like (Non-GC) types by immunohistochemical profiling. Previous studies showed better survival rate for the GCB groups. CD44 is necessary for tumor spread and metastasis and its expression is generally associated with unfavorable prognosis. We analyzed the expression and prognostic significance of standard isoform CD44s and its variant isoform CD44v6 in DLBCL types. Methods: Tissue microarray blocks were created from 52 nodal DLBCL with control tissue. Immunohistochemical staining for CD10, Bcl-6, MUM-1, CD44s, and CD44v6 were performed. The median follow-up period was 44 months. Results: Nodal DLBCLs were subclassified into GCB [CD10+ or CD10-/Bcl6+/MUM1+, n=17 (33%)] and non-GC subgroups [CD10-/Bcl6- or CD10-/Bcl6+/MUM1+, n=35 (67%)]. CD44s expression appeared more on non-GC cases of DLBCL (p=0.04). CD44s and CD44v6 did not result in any difference according to tumor stage, IPI scores, LDH levels. Upon survival analysis, CD44s and CD44v6 expression did not show any statistical correlation. Conclusions: CD44s expression may play a role during lymphomatogenesis of non-GC type DLBCL. No significant financial relationships to disclose.

Immunophenotype as prognostic factor for diffuse large B-cell lymphoma in patients undergoing clinical risk-adapted therapy

For patients with diffuse large B-cell lymphoma (DLBCL), the International Prognostic Index (IPI) predicts the likelihood for cure with chemotherapy. Biological parameters, including expression of Bcl-6, Bcl-2, CD10, major histocompatibility complex class II, and categorization as germinal center (GC) type have been described as IPI-independent prognostic factors. Biological parameters were evaluated retrospectively by immunohistochemistry in 60 consecutive DLBCL patients of the prerituximab era. Forty-one of 60 patients underwent a risk-adapted treatment strategy including autologous stem-cell transplantation for high-risk patients (age-adjusted IPI = 2-3; slow response to chemotherapy). Bcl-6 expression was associated with superior overall survival (OS) independently of the IPI. Inferior progression-free survival (PFS) was independently correlated with high expression of Bcl-2 and low positivity for HLA-DR and CD10. Distinction into GC and non-GC DLBCL on the basis of Bcl-6, CD10, and IRF-4 expression had no independent prognostic value. Within the risk-adapted treatment strategy, only HLA-DR retained a prognostic impact on OS (P = 0.0058) and PFS (P = 0.0002). In 60 patients with DLBCL treated with risk-adapted therapy, immunohistochemical subcategorization of DLBCL into GC and non-GC type has little clinical value. The IPI-associated risk appears to be mitigated by intensified upfront therapy. Low HLA-DR expression is associated with poor outcome after intensified upfront therapy. Therefore, additional treatment modalities appear to be required.

Addition of Rituximab to CHOP Regimen Improves Clinical Outcome in Non-Germinal Center Type Diffuse Large B-Cell Lymphoma.

Background: In recent years, diffuse large B-cell lymphoma (DLBCL) has been classified into the germinal center B-cell (GC) type, the activated B-cell (ABC) type, and the type 3 using global gene expression profiling or immunohistochemical staining. It has been reported that the GC type DLBCL showed significantly longer survival than the non-GC (ABC and the type 3) type DLBCL treated with CHOP or CHOP like regimen not using rituximab.Methods: We analyzed retrospectively the prognosis between the GC and non-GC types of DLBCL treated with R-CHOP regimen. All 50 patients with DLBCL, diagnosed between July 2003 and July 2005 were included in this study. The pathology was reviewed by hematopathologist and confirmed to be de novo DLBCL according to the WHO classification. Patients with primary CNS- and post-transplant lymphomas were excluded. GC type or non-GC type DLBCL was determined by immunohistochemistry such as the expression patterns of CD10, BCL-6, and IRF-4 (MUM1). All patients were initially treated with six cycles of R-CHOP regimen consisting of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone. If we evaluated partial response after six cycles of R-CHOP, the patients have added radiation therapy.Results: The patients consisted of 30 GC type and 20 non-GC type DLBCL with a median age of 61.0 yr (range 31–83 yr). The median follow up of surviving patients was 24 months. CR rate between GC and non-GC types were 57.0% vs. 75.0%, p=0.186, and overall response rate were 87.0% vs. 90.0%, p=0.929, respectively. The median of progression free survival was 17.3 months vs. 19.6 months, p=0.80. There is no statistical significance difference between two groups.Conclusion: These results suggest that addition of rituximab to CHOP regimen improves clinical outcome of non-GC type DLBCL as well as GC type DLBCL.

Treatment Effect of Rituximab Plus Chemotherapy Is Obtained by Improving Survival from Non-Germinal Center-Type Untreated Diffuse Large B-Cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disorder that can be classified into at least three subgroups according to DNA microarray-based expression profiles: germinal center B-cell-like (GC) type, activated B-cell-like type, and type 3. Immunohistochemistry used to determine the GC and non-GC subtypes of DLBCL and to predict survival show results similar to those of cDNA microarray analysis. There are a few reviews of the treatment effect of rituximab in relation to immunohistological protein expression, but there are no reports comparing clinical outcomes of GC-type DLBCL with non-GC-type DLBCL for patients treated with rituximab plus chemotherapy. We analyzed relations between the treatment effect of rituximab plus conventional chemotherapy and immunohistochemically determined subtypes in cases of untreated DLBCL. Immunohistochemical stain results for CD10, bcl-6 and MUM1 were used to subclassify the case as same as previous reported studies. Sixty-nine patients were divided into 2 treatment groups, rituximab plus TCOP (cyclophosphamide, therarubicin, vincristine, and prednisolone) (R-TCOP) and TCOP alone. The R-TCOP group included 38 patients (12 women, 26 men; age range, 53–89 years; mean age, 68.1 years); median follow-up of surviving patients was 13 months. The TCOP group included 31 patients (15 women, 16 men; age range, 45–88 years; mean age, 66.4 years); median follow-up of surviving patients was 25 months. Of the 69 DLBCLs, 26 (38%) were considered GC type and 43 (65%) were considered non-GC type by immunohistochemical analysis. In the R-TCOP group, 18 (48%) DLBCLs were classified as GC type and 20 (52%) were classified as non-GC type. In the TCOP group, 8 (26%) DBLCLs were classified as GC type and 23 (74%) cases were classified as non-GC type. Overall survival (OS) was significantly longer for patients treated with R-TCOP than for those treated with TCOP alone (P=0.002). In both the R-TCOP and TCOP groups, no significant difference in OS was observed between GC-type and non-GC-type DLBCLs (P=0.36 and P=0.16, respectively). Among GC-type cases, OS did not differ significantly between the two treatments, but among non-GC DLBCLs, OS was significantly longer for patients treated with R-TCOP than for those treated with TCOP alone (P=0.005). Furthermore, among the low IPI, non-GC-type cases, OS did not differ significantly between the two treatments (P=0.8), but among the high IPI, non-GC-type cases, a significant difference was observed between the two treatments (P=0.008). In prior studies, the addition of rituximab to conventional chemotherapy was shown to improve outcome in patients with DLBCL. Our findings suggest that these improved outcomes may be due primarily to the beneficial effect of rituximab added to chemotherapy on non-GC DLBCLs, especially when the IPI is high. The non-GC type includes activated B-cell-like DLBCL, which is characterized by activation of NF-kappa B target genes. Rituximab inhibits the constitutive nuclear factor-kappa B signaling pathway in non-Hodgkin lymphoma cell lines; therefore, we believe our results point to the clinical importance of NF-kappa B as a therapeutic target for DLBCL.