Center Diffuse Large B-cell Lymphoma Research Articles

New aspects in the biology of cutaneous B‐cell lymphomas

Immunologic and molecular genetic studies greatly contributed to a better understanding and interpretation of the distinct clinico-pathologic features of primary cutaneous B-cell lymphomas (CBCL), which are the basis for the consensus WHO-EORTC classification. There is increasingly accumulating evidence that these well defined clinico-pathologic entities of CBCL have specific immunologic and molecular features, which further support their nosologic categorization as well as either interesting similarities with other extranodal B-cell lymphomas or definite peculiarities as compared to nodal B-cell lymphomas of similar histotype (specifically, follicle center lymphoma and diffuse large B-cell lymphoma).

High throughput tissue microarray analysis of FHIT expression in diffuse large cell B-cell lymphoma from Saudi Arabia

Recent studies have suggested a potential prognostic role of alterations of the fragile histidine triad (FHIT) gene in diffuse large B-cell lymphoma. To evaluate possible mechanisms of FHIT inactivation and to further clarify its potential prognostic relevance, we analyzed a set of 114 diffuse large B-cell lymphoma with clinical follow-up information. Tissue microarrays were analyzed by immunohistochemistry for protein expression, and corresponding DNA samples were analyzed for FHIT promotor hypermethlyation. Reduced or absent FHIT expression was found in 75 of 114 diffuse large B-cell lymphoma (66%), but was unrelated to clinical tumor stage or patient prognosis. FHIT promotor hypermethylation was observed in 29 of 93 (23%) interpretable diffuse large B-cell lymphoma. Hypermethylation was not significantly correlated to protein expression loss, which could be explained by competing mechanisms for FHIT inactivation in a substantial fraction of non FHIT hypermethylated diffuse large B-cell lymphoma. Hypermethylation was significantly associated with poor prognosis of diffuse large B-cell lymphoma patients and predominantly seen in nongerminal center diffuse large B-cell lymphoma (27%), but less frequent (13%) in germinal center diffuse large B-cell lymphoma. In summary, these data suggest that promotor hypermethylation is responsible for reduced FHIT expression in a substantial subset of diffuse large B-cell lymphoma, which is primarily composed of nongerminal center subtype with poor patient prognosis.

Gene Expression Profile Associated with Survival in Patients with Diffuse Large B-Cell Lymphoma Treated by Chemotherapy and Rituximab.

Molecular profiles have been shown to correlate with survival in patients with diffuse large B-cell lymphoma (DLBCL) receiving conventional chemotherapy. However, most patients now receive monoclonal anti-CD20 antibodies in addition to chemotherapy, which might erase the poor prognosis associated with some markers (Mounier et al, Blood 2003; 101:4279) and reveal new prognostic factors related to rituximab mechanisms of action. This prompted us to undertake a gene expression study on the samples of patients (pts) included in the GELA LNH98-5 study that compared CHOP and rituximab-CHOP (R-CHOP) regimen. We collected all available frozen tissue samples from patients included into the LNH98-5 trial and completed the series with patients treated with the same regimens in participating centers during the same period. All those patients were between 60 and 80 years old, had histologically reviewed DLBCL, Ann Arbor stage II to IV disease and an ECOG Performance Status ≤3. High quality RNA from 53 patient (43 from the study itself and 10 from the additional cohort) treated with CHOP (30 pts) or R-CHOP (23 pts) were analyzed with Affymetrix HU133A micro-arrays.The main clinical characteristics of this subset of patients were similar to that of the remaining group of 346 patients involved in the LNH98-5 study. Two parameters were strongly correlated with disease progression: a high International Prognostic Index (IPI = 4 or 5) (relative risk (RR) 3.9 when compared to pts with an IPI = 2 or 3, p=0.002) and treatment regimen (CHOP: RR 4.2 when compared to R-CHOP, p=0.001). In both treatment arms, patients with a germinal center (GC) DLBCL profile had a better outcome than patients with an activated B-cell (ABC) one (ABC versus GC, RR of progression 2.6, p=0.06) and several transcripts previously associated with prognosis, such as BCL6, showed a strong correlation with survival (BCL6 low versus BCL6 high, RR of progression 3.1, p=0.004), further corroborating the clinical relevance of this series of patients. We then used Cox models adjusted for IPI and treatment regimen to test the association with outcome of each Affymetrix microarray probeset 1) in the entire population, 2) or with a differential effect according to the treatment regimen.This strategy allowed us to identify several transcripts that were highly correlated with survival, either in both groups or specifically in the R-CHOP group. Interestingly, some of these transcripts are related to apoptosis regulation, immune cell infiltration or B-cell signal transduction. We are currently investigating the prognostic significance of this series of transcripts, using Quantitative RT-PCR, in an independent set of 43 DLBCL patients who presented the same clinical characteristics than the initial test group and received R-CHOP in GELA centers after the closure of the LNH98-5 trial.

Expression of TP73L is a helpful diagnostic marker of primary mediastinal large B-cell lymphomas

Primary mediastinal large B-cell lymphoma is a well-defined lymphoma entity whose molecular pathogenesis is incompletely understood and also lacking well-established diagnostic markers. Recently, the presence of overlapping features between classical Hodgkin’s lymphoma and primary mediastinal large B-cell lymphoma was highlighted by gene expression profiling as well as morphological studies. We investigated the expression of TP73L (commonly known as p63) isoforms in primary mediastinal large B-cell lymphoma at both protein and mRNA level, and demonstrated the exclusive presence of transactivating (TA) isoforms in all cases. We also demonstrated that TP73L is expressed in a subset of germinal center B-cells, as well as in some diffuse large B-cell lymphomas, but it is never present in classical Hodgkin lymphoma. Nodular lymphocyte predominant Hodgkin’s lymphoma also showed TP73L positivity by immunohistochemistry. Isoform analysis by real-time PCR showed that TA-TP73Lα is the most represented in primary mediastinal large B-cell lymphoma, but TA-TP73Lγ is the most differentially expressed in comparison to both germinal center B-cells and diffuse large B-cell lymphomas. TP73L expression proved a useful diagnostic marker of primary mediastinal large B-cell lymphoma, and gave new insights in to the molecular pathways playing a role in this lymphoma.

A novel diffuse large B-cell lymphoma-associated cancer testis antigen encoding a PAS domain protein.

Here we report that the OX-TES-1 SEREX antigen, which showed immunological reactivity with serum from four out of 10 diffuse large B-cell lymphoma (DLBCL) patients, is encoded by a novel gene, PAS domain containing 1 (PASD1). PASD1_v1 cDNA encodes a 639 amino-acid (aa) protein product, while an alternatively spliced variant (PASD1_v2), lacking intron 14, encodes a 773 aa protein, the first 638 aa of which are common to both proteins. The PASD1-predicted protein contains a PAS domain that, together with a putative leucine zipper and nuclear localisation signal, suggests it encodes a transcription factor. The expression of PASD1_v1 mRNA was confirmed by RT–PCR in seven DLBCL-derived cell lines, while PASD1_v2 mRNA appears to be preferentially expressed in cell lines derived from non-germinal centre DLBCL. Immunophenotyping studies of de novo DLBCL patients' tumours with antibodies to CD10, BCL-6 and MUM1 indicated that two patients mounting an immune response to PASD1 were of a poor prognosis non-germinal centre subtype. Expression of PASD1 mRNA was restricted to normal testis, while frequent expression was observed in solid tumours (25 out of 68), thus fulfilling the criteria for a novel cancer testis antigen. PASD1 has potential for lymphoma vaccine development that may also be widely applicable to other tumour types.

Mutation of the ATM Gene is Not Involved in the Pathogenesis of Either Follicle Center Lymphoma or its Transformation to Higher-grade Lymphoma

Loss of function of the ataxia-telangiectasia mutated (ATM) gene, located on human chromosome 11q22–23, is the cause of ataxia-telangiectasia (A–T), which is associated with an extremely high risk for lymphoma. Abnormalities in 11q22–23, including deletions and mutations of the ATM gene, have been reported in T-cell prolymphocytic leukemias, B-CLL and in mantle cell lymphoma. In a survey of gene expression in follicle center lymphomas (FCL) and diffuse large B-cell lymphomas (DLBCL), almost all FCL expressed significant levels of ATM and the majority of DLBCL expressed low levels of ATM. This finding raised the possibility that the transformation of some FCL to DLBCL might be associated with inactivation of the ATM gene. Therefore, we analyzed biopsy specimens of 17 patients with FCL obtained at the time of diagnosis, four subsequent biopsies obtained at the time of FCL relapse and seven subsequent biopsies at the time of transformation to DLBCL. A comprehensive analysis of the ATM gene was performed by denaturing high performance liquid chromatography and sequencing. The analysis covered all of the 66 exons including the 9168 base pairs of ATM coding sequence as well as 16,676 base pairs of non-coding sequence. Twenty-eight known polymorphisms and rare sequence variants were observed, but no classic A–T mutations were detected. In 11 tumors, both tumor B-cells and normal T-cells were sorted for separate examination, and in each case, polymorphisms and rare variants were present in both tumor and normal cells. No new ATM gene mutations were associated with transformation from FCL to DLBCL. Thus, ATM gene mutations do not play a pivotal role either in the pathogenesis of FCL or in its transformation to DLBCL.

Intensification of salvage treatment with high-dose sequential chemotherapy improves the outcome of patients with refractory or relapsed aggressive non-Hodgkin's lymphoma.

The aim of the present study was to retrospectively evaluate whether a high-dose sequential chemotherapy programme (HDS: cyclophosphamide, methotrexate, etoposide) administered prior to autologous transplantation could optimize the salvage of patients with refractory or relapsed aggressive non-Hodgkin's lymphoma. Between 1985 and 1999, 103 patients (median age 43 years, range 16-65) from eight Italian centres and one Swiss centre, with refractory (n = 38) or relapsed (n = 65) diffuse large B-cell and T-cell lymphoma, were treated using HDS followed by high-dose regimens with autologous haematopoietic stem cell transplantation. Eighty-three patients responded to the HDS regimen (81%, 95% C.I., 73- 88%) and 79 eventually achieved a complete response (CR) after autotransplantation (90%, 95% C.I., 81- 96%). None of 20 cases resistant to HDS attained CR. Treatment-related mortality was 4%. After a median follow-up of 24 months (range 6-174 months), 3-year estimates of overall survival, event-free survival and disease-free survival were 47% (95% C.I., 36-59%), 44% (95% C.I., 34-54%) and 64% (95% C.I., 50-74%) respectively. Multivariate analysis showed that chemosensitivity to HDS represented the strongest predictor of both CR and survival. This retrospective study shows that salvage treatment using HDS had relatively low toxicity and was associated with remarkable response rates, allowing further effective therapy with high-dose autograft programmes.

Immunohistochemical Detection of p53,bcl-2, and Retinoblastoma Proteins in Follicular Lymphoma

Mutations of p53 have been suggested to be involved in the histologic transformation of follicular lymphoma, but the role of the retinoblastoma (RB) gene, another tumor suppressor gene, in lymphomagenesis has not been established. To determine the roles of these tumor suppressor genes and their relationship with the anti-apoptotic bcl-2 gene in follicle center lymphoma, the immunohistochemical expression of p53, bcl-2, and RB proteins was correlated with cytologic grade in 50 cases of follicular lymphoma, and the results were compared to 23 cases of diffuse large B-cell lymphoma. The results showed that only 2 of 25 grade 1 follicular lymphoma were p53-positive compared to 16 of 25 grade 3 cases (P <.0001). A significantly lower number (13 of 25) of grade 3 follicular lymphomas expressed bcl-2 compared to grade 1 cases (23 of 25) (P <.004). Eight of 14 bcl-2-negative follicular lymphomas expressed p53, compared with 10 of 36 bcl-2-positive cases (P = .1). Twenty-four of 25 grade 3 follicular lymphomas showed 2+ to 3+ staining for RB protein compared to 9 of 21 grade 1 cases (P <.0002). Expression of p53 protein correlates significantly with higher cytologic grade in follicular lymphoma. Similar to earlier studies of breast cancer and lymphoma, there appears to be an inverse relationship between p53 and bcl-2 expression in follicular lymphoma. Inactivation of the retinoblastoma gene does not seem to be involved in the histogenesis of follicle center lymphoma or diffuse large B-cell lymphoma.