Heterogeneous mechanisms of imparied lymphocyte responses in non- Hodgkin's lymphoma

Peripheral blood mononuclear cells (PBMC) from 18 untreated patients with non-Hodgkin's lymphoma (NHL) were studied to characterize the cellular mechanisms contributing to impaired in vitro lymphocyte responses after stimulation by the mitogen conconavalin A (Con-A). In vitro reactivity was quantitated by the 3H-thymidine incorporation in response to an optimal dose of Con-A. All patients demonstrated impaired in vitro reactivities compared to normal controls. These in vitro impairments were partially reversible since patient's cells precultured in media alone for 3 days demonstrated enhanced Con-A responses. In greater than half of the patients, the hyporeactive PBMC suppressed the enhanced reactivities of autologous precultured PBMC when assayed in cocultures. Suppressor activity was detected mainly in those untreated patients presenting with either constitutional symptoms or diffuse histology and in general was not marked compared to the severity of impairments. Adherent monocytes were shown to participate in the suppression of autologous lymphocyte reactivity but only appeared partially responsible for the in vitro impairments. In those patients lacking detectable suppressive activity, preculturing also enhanced Con-A reactivities and was compatible with the presence of a reversible, inhibitory mechanism differing from active suppression. Many patients' hyporeactive PBMC, however, failed to demonstrate normal responses after preculturing. This failure could not be directly attributed to aberrant regulatory populations, but rather appeared to possibly represent an additional intrinsic impairment of potentially reactive populations.     

How to define intermediate stage in Hodgkin's lymphoma?

Abstract:  Background : Intermediate or unfavourable stage Hodgkin's lymphoma (HL) definition relies upon at least three different scoring systems defined by cooperative groups (EORTC, GHSG and Canadian-ECOG). We aimed to investigate their efficacy and their correlation with International Prognostic Score (IPS) for advanced HL. Patients and methods : We studied a population of 1156 patients with localized stage HL treated prospectively within GELA centres in H8 (518 patients) and H9 (638 patients) protocols. Median age: 30 yr, 18%, Female 50%; stage I: 25%; stage II: 75%. According to scoring systems 70% had 0–1 EORTC factors; 60% 0–1 GHSG factors and 82% 0–1 Canadian factors. The IPS for advanced stages was available only in H9 study with 64% 0–1 factor. Results : Survival curves according to each of the different scoring systems could significantly discriminate the subgroup populations. When a multivariate Cox analysis was performed for overall survival (OS) including all the scoring system variables: age >45 yr, sex male, Haemoglobin <10.5 g/dL, lymphocytes <600/ μ L, B symptoms with elevated ESR, extra nodal sites did retain an independent significant value. Probability of OS was 99%, 98%, 92%, 82% and 73% for patients with 1–5 factors, respectively P  < 0.0001. Conclusion : These factors are similar for most of them with those described in the IPS when stages III and IV are replaced by extra nodal localization. This new score should be validated in other prospective trials, as it will simplify the Hodgkin prognostic scoring systems for localized and advanced stages.     

Prognostic relevance of thymidine kinase isozymes in adult non- Hodgkin's lymphoma

To determine whether kinase (TK) isozyme status adds clinically useful information in adult non-Hodgkin's lymphoma (NHL), we have analyzed peripheral blood plasma and lymphocytes of 44 patients with NHL for either TK1 or TK2 isozyme activity. On the basis of isozyme status, patients could be divided into two groups that did not differ significantly with respect to known determinants for survival. The median survival of patients exhibiting peripheral blood TK1 thymidine kinase activity was 40 wk and that of individuals with TK2 activity was in excess of 200 wk. These data suggest that peripheral blood TK1 isozyme is a useful independent biochemical marker for a subgroup of NHL who respond poorly to current therapy and thus require new therapeutic approaches.     

Deletions involving two distinct regions of 6q in B-cell non-Hodgkin lymphoma.

The recurrent loss of genetic material from a specific chromosomal region in a given tumor type suggests the presence of a tumor-suppressor gene, the loss or inactivation of which may be relevant for tumorigenesis. In this study, we provide molecular evidence for the recurrent association between deletions on the long arm of chromosome 6 and B-cell non-Hodgkin lymphoma (B-NHL). Normal and tumor DNAs from 71 cases of B-NHL were studied for loss of constitutional heterozygosity (LOH) at 19 loci on chromosome 6 using a panel of restriction fragment length polymorphism (RFLP) probes. LOH, indicating deletion of all or part of 6q, was detected in 16 of 71 cases (22.5%), ranging from low-grade to high-grade B-NHL. The isolated loss of 6p or the loss of other chromosomes (8, 17, 22) tested as controls for specificity was not observed in any case. Comparison of the extent of the deletions among different cases allowed the identification of two distinct regions of minimal deletion (RMD) at 6q25 to 6q27 (RMD-1) and at 6q21 to 6q23 (RMD-2), respectively, suggesting the existence of two tumor-suppressor genes. These data support a role for 6q deletions in B-NHL pathogenesis and provide a basis for identifying the corresponding tumor-suppressor genes.     

t(3;22)(q27;q11): a novel translocation associated with diffuse non- Hodgkin's lymphoma [see comments]

Of 187 specimens of non-Hodgkin's lymphoma and four hyperplastic lymphoid proliferations with clonal chromosome abnormalities ascertained serially over a 4 1/2-year period, nine cases with t(3;22)(q27;q11) were identified. Seven of the lymphomas were diffuse tumors, predominantly large cell type. The eighth tumor, a follicular small cleaved cell lymphoma, exhibited a t(3;22) and a t(14;18)(q32;q21). The ninth case was a lymph node from a human immunodeficiency virus-positive patient which showed atypical hyperplasia. Overall survival of t(3;22) diffuse lymphoma patients was not different from that of patients with abnormal karyotypes without t(3;22). The t(3;22) diffuse tumors studied showed a disproportionate frequency of lambda light chain on their cell surfaces, a finding similar to that observed in t(8;22)(q24;q11) Burkitt's lymphomas. Our results indicate that the t(3;22)(q27;q11) is the third most common recurring translocation in diffuse non-Hodgkin's lymphoma.     

Pathogenesis of Hodgkin's lymphoma

Abstract:  In Hodgkin's lymphoma (HL), the B cell origin of the tumour cells, the Hodgkin and Reed-Sternberg (HRS) cells, has been disclosed by molecular single cell analysis about 10 yr ago. This finding formed the basis for various studies aimed to better understand the pathogenesis of this peculiar malignancy and the pathophysiology of the HRS cells. Work of our groups in this regard was focussed recently on two main topics, namely the study of differential gene expression in HRS cells and the pathogenesis of composite lymphomas. Composite lymphomas are combinations of HL and B cell non-Hodgkin lymphomas, that turned out to be often clonally related. By molecular analysis of several composite lymphomas for potential transforming events, we identified examples of both shared as well as distinct transforming events. Comparing gene expression profiles of HL-derived cell lines with the corresponding profiles from other B cell lymphomas and normal B cell subsets revealed a global down-regulation of the B cell-specific gene expression signature in HRS cells. Moreover, we identifed aberrant expression and activity of multiple receptor tyrosine kinases in HRS cells of classical and to a lesser extend lymphocyte predominant HL, which appears to be a unique feature of HL, and may offer novel strategies for treatment.     

Immunotherapy of Hodgkin's lymphoma

Abstract:  Many new treatment approaches have given promising results in experimental Hodgkin's lymphoma (HL) models. Early clinical trials evaluating antibody based compounds as immunotoxins (ITs), radioimmunotherapy (RIT), bispecific molecules (BSMs), and recently monoclonal antibodies (MAbs), have demonstrated some clinical efficacy in patients with advanced refractory or relapsed HL. In addition, cellular immunotherapy is evolving. Although it seems unlikely to cure chemotherapy resistant patients with larger tumor masses by either of these approaches alone, the combination with conventional chemotherapy might help to overcome resistance of Hodgkin-/Reed-Sternberg (H-RS) cells. Another rationale for the development of these immunotherapies is to eliminate residual disease and thereby to prevent relapses from the disease. Currently, several clinical studies are running. A murine MAb (Ki-4) based 131 Iodine conjugate has shown efficacy in refractory HL patients in a phase II study, but less toxic constructs using alternate MAbs or isotopes should be developed. A humanized as well as a fully human anti-CD30 MAb are being tested in clinical phase I/II studies. These MAbs could engage the human immune system against the H-RS cells. In addition, these MAbs could be then combined with conventional chemotherapy in order to improve the treatment of HL.     

Adhesion molecule expression on B-cell chronic lymphocytic leukemia cells: malignant cell phenotypes define distinct disease subsets

Expression of surface adhesion molecules of the Ig superfamily (CD54 and CD58), of the integrin family (beta 1, beta 2, and beta 3 chains), of the selectin family (L-selectin), and of the lymphocyte homing receptor (CD44) was analyzed on B-cell chronic lymphocytic leukemia (B- CLL) cells from 74 patients. The aim of the study was the definition of phenotypically distinct disease subsets and the correlation of adhesion molecule phenotypes with clinical parameters. Expression of CD58 on B- CLL cells defined more advanced disease stages. In comparison with beta chain-positive cases, patients whose cells did not express beta 1, beta 2, and beta 3 integrin chains fell into the most favorable prognostic group, with lower lymphocytosis and the absence of splenomegaly, diffuse bone marrow infiltration, and therapy requirement. A novel finding was the expression of beta 3 chains on cells from a minority (12 of 74) of B-CLL cases. beta 3 chains were always coexpressed with beta 1 and beta 2 chains. Two-color immunofluorescence analyses of adhesion molecules such as alpha x beta 2 integrin (LeuM5) and L- selectin (Leu8) showed that these markers were detectable on variable proportions of leukemic cells, thus confirming the intraclonal phenotypic heterogeneity of B-CLL. Differences in the intensity of CD44 expression were also shown among the various B-CLL clones. Finally, no major variations were shown by comparison of adhesion molecule phenotypes of leukemic cells simultaneously obtained from blood and bone marrow, and of CD5+ versus CD5- clones.     

Mutated VH genes and preferential VH3-21 use define new subsets of mantle cell lymphoma

Mantle cell lymphoma (MCL) is believed to originate from a naive B cell. However, we recently demonstrated that a subset of MCL displayed mutated V(H) genes. We also reported restricted use of certain V(H) genes. To assess the prognostic impact of these new findings, we performed V(H) gene analysis of 110 patients, revealing that 18 (16%) patients had mutated and 92 (84%) patients had unmutated V(H) genes. Because the mutation rate was low in the mutated group (2.2%-6.7%), further investigation of the germline V(H) gene in T cells from 5 patients with mutated V(H) genes was carried out; results showed that the unrearranged V(H) gene was identical to the published sequence. These data confirm that the base pair substitutions within the rearranged V(H) genes represent hypermutations, and indicate germinal center exposure. However, V(H) gene mutation status did not correlate with prognosis because there was no difference in clinical outcome between the unmutated and mutated groups. The most frequently used V(H) genes were V(H)3-21 (21 patients) and V(H)4-34 (19 patients). A novel finding was that V(H)3-21(+) MCL almost exclusively expressed lambda light chains and displayed highly restricted use of the V(lambda)3-19 gene. V(H)3-21(+) patients had longer median survival than the remaining patients (53 vs 34 months; P =.03), but they tended to be younger at diagnosis. The combined use of V(H)3-21/V(lambda)3-19 suggests a possible role for antigen(s) in the pathogenesis of these tumors and indicates that V(H)3-21(+) patients constitute a new MCL entity.     

Analysis of internal deletions within the BCL6 gene in B-cell non-Hodgkin's lymphoma

The BCL6 gene is frequently altered by chromosomal translocations and/or point mutations at its 5' non-coding portion in B-cell non-Hodgkin's lymphoma (B-NHL). We analysed submicroscopic structural alterations of the BCL6 gene which had arisen from internal deletion in four cases with B-NHL and found that these deletions overlapped at the 280 bp region in the first intron. In electrophoretic mobility shift assay, nuclear extracts prepared from various cell lines were shown to bind to a fragment from this commonly deleted region. Our results suggest that deregulation of BCL6 expression would be caused by loss of this putative protein-binding sequence in some B-NHL cases.     

First-line treatment of Hodgkin's lymphoma

Substantial clinical progress over the last decades has made Hodgkin's lymphoma (HL) into one of the most curable human cancers in adults. About 80% of patients in all stages and of all histological subtypes experience long-term disease-free survival. Modern treatment strategies aim to improve chemotherapy and radiotherapy, while minimizing therapy-related toxicities. Ongoing trials investigate a reduction of chemotherapy doses or cycles and the application of lower-radiation doses and smaller radiation field sizes. For patients with a specific high-risk profile, novel approaches with more intense drug combinations are currently being investigated in clinical trials. Here, we review recent approaches in the first-line treatment of early-favorable, early-unfavorable, and advanced-stage HL.     

Adjuvant immunotoxin therapy with anti-B4-blocked ricin after autologous bone marrow transplantation for patients with B-cell non- Hodgkin's lymphoma

Anti-B-blocked ricin (anti-B4-bR) combines the specificity of the anti- B4 (CD19) monoclonal antibody with the protein toxin "blocked ricin." In blocked ricin, affinity ligands are attached to the ricin B-chain to attenuate its lectin binding capacity. In a phase I trial, Anti-B4-bR was administered by 7-day continuous infusion to 12 patients in complete remission after autologous bone marrow transplantation (ABMT) for relapsed B-cell non-Hodgkin's lymphoma (NHL). Patients were treated at 20, 40, and 50 micrograms/kg/d for 7 days. Potentially therapeutic serum levels could be sustained for 3 to 4 days. The maximum tolerated dose was 40 micrograms/kg/d for 7 days (total 280 micrograms/kg). The dose-limiting toxicities were reversible grade IV thrombocytopenia and elevation of hepatic transaminases. Mild capillary leak syndrome was manifested by hypoalbuminemia, peripheral edema (4 patients), and dyspnea (1 patient). Anti-immunotoxin antibodies developed in 7 patients. Eleven patients remain in complete remission between 13 and 26 months post-ABMT (median 17 months). These results show that Anti-B4- bR can be administered with tolerable, reversible toxicities to patients with B-cell NHL in complete remission following ABMT.     

Effect of IL-6 promoter polymorphism on incidence and outcome in Hodgkin's lymphoma

A single nucleotide polymorphism (SNP) is present at position -174 of the human interleukin-6 gene. The risk of developing Hodgkin's lymphoma (HL) in young adults decreases with an increasing number of C alleles at this position. We analysed the effect of this SNP on incidence and outcome in HL. DNA samples from 408 cases and 349 controls were screened and analysed following stratification by age, histological subtype and Epstein-Barr virus status. Although the risk of classical HL in young adults decreased with increasing C alleles, case-control differences were not significant. An excess of G alleles was observed for nodular lymphocyte predominant HL in young adults (n = 21), which was significant.     

Treatment of pediatric Hodgkin's lymphoma.

Today the majority of children and adolescents diagnosed with Hodgkin's lymphoma will enjoy long-term disease-free survival. As a result, contemporary treatment strategies have focused on reducing therapy for patients with favorable disease presentations and reserved aggressive treatment modalities for patients with relapsed or refractory disease. The desire to avoid late treatment toxicity has prompted refinements in therapy designed to reduce growth impairment, second malignancy, and life-threatening organ dysfunction in long-term survivors. Treatment with radiation therapy alone is recommended only for older patients with localized disease who have achieved skeletal maturity, but requires surgical staging and places greater volumes of normal tissues at risk for late carcinogenesis. Treatment with chemotherapy alone avoids the long-term growth, organ dysfunction, and solid tumor induction associated with high-dose, extended-field radiation. However, these protocols prescribe higher cumulative doses of alkylating agent chemotherapy, which may increase the risk of treatment complications from myelosuppression, gonadal injury, and secondary leukemia. Combined modality therapy regimens have resulted in excellent treatment outcomes and reduced the incidence of treatment sequelae by utilizing lower doses and smaller volumes of radiation therapy and fewer cycles of less toxic chemotherapy in clinically staged children. Risk-adapted therapies using two to four cycles of multiagent chemotherapy and lower radiation doses and volumes have maintained excellent disease-free survival rates in clinically staged patients with localized favorable disease presentations. Novel approaches including compacted dose-intensive multiagent chemotherapy are currently under investigation with the objectives of improving outcome and reducing treatment sequelae in patients with advanced and unfavorable disease.     

Biallelic DNA rearrangements and deletions within the BCL-6 gene in B-cell non-Hodgkin's lymphoma

Summary. Chromosomal translocations involving band 3q27 are recently described common specific cytogenetic abnormalities in B-cell neoplasms, and the BCL-6 gene, identified on 3q27, was shown to be disrupted and over-expressed in lymphoma cells having these chromosomal translocations. In the present study we found rearrangements within the BCL-6 gene in seven out of 3 5 cases with B-cell non-Hodgkin's lymphoma (NHL). Further analysis revealed that three of these patients with BCL-6 abnormality had multiple rearranged bands hybridized with probes from a single restriction fragment within the major translocation cluster (MTC). suggesting that independent DNA rearrangements would occur on both alleles. Additionally, Southern blot analysis indicated that three patients carry deletions encompassing the area containing the first exon of the BCL-6 gene. Our results suggest that biallelic DNA rearrangements and deletions would occasionally occur in NHL patients with BCL-6 abnormality.