Maurizio Rupolo

High-dose therapy and autologous peripheral blood stem cell transplantation as salvage treatment for AIDS-related lymphoma: long-term results of the Italian Cooperative Group on AIDS and Tumors (GICAT) study with analysis of prognostic factors.

After the introduction of highly active antiretroviral therapy (HAART), intensive treatment, including high-dose therapy (HDT) and peripheral blood stem cell transplantation (PBSCT), has become feasible in HIV-positive patients with Hodgkin (HL) and non-Hodgkin (NHL) lymphoma. Herein, we report the long-term results, on an intention-to-treat basis, of a prospective study on HDT and PBSCT in 50 HIV-positive HAART-responding patients with refractory/relapsed lymphoma. After debulking therapy, 2 patients had early toxic deaths, 10 had chemoresistant disease, 6 failed stem cell mobilization, 1 refused collection, and 4 progressed soon after PBSC harvest. Twenty-seven actually received transplant. Twenty-one patients are alive and disease-free after a median follow-up of 44 months (OS, 74.6%; PFS, 75.9%). Only lymphoma response significantly affected OS after transplantation. In multivariate analyses both lymphoma stage and low CD4 count negatively influenced the possibility to receive transplant. Median OS of all 50 eligible patients was 33 months (OS, 49.8%; PFS, 48.9%). Low CD4 count, marrow involvement, and poor performance status independently affected survival. PBSCT is a highly effective salvage treatment for chemosensitive AIDS-related lymphoma. It seems rational to explore its use earlier during the course of lymphoma to increase the proportion of patients who can actually receive transplant.

Rituximab (IDEC-C2B8): validation of a sensitive enzyme-linked immunoassay applied to a clinical pharmacokinetic study.

Rituximab is a chimeric monoclonal antibody (MAb) directed against the B-cell CD20 antigen that has been approved for therapy of relapsed and resistant follicular non-Hodgkins lymphoma (NHL). This study describes the development and validation of a highly sensitive, rapid, accurate, precise enzyme-linked immunosorbent assay (ELISA) to measure Rituximab serum concentrations. This study also describes the application of the ELISA method to a pharmacokinetic study in a homogeneous group of patients with follicular lymphoma who received 4 weekly doses of MAb at the standard dose of 375 mg/m2 as consolidation of chemotherapy. In the patients in this study, the median Rituximab serum concentrations increased during therapy, and showed a slow decline during the posttreatment period. The Rituximab elimination half-life of approximately 20 days accounts for the demonstrated accumulation of MAb in serum samples. Because previous pharmacokinetic studies showed a correlation between Rituximab serum levels and tumor response, the ELISA method used in this study, which allows a precise control of serum concentrations, could be useful for predicting the final response to the MAb and for selecting patients able to benefit from higher dosage or repeated drug administration.

Analysis of IgVH gene mutations in B cell chronic lymphocytic leukaemia according to antigen-driven selection identifies subgroups with different prognosis and usage of the canonical somatic hypermutation machinery

Cases of B‐cell chronic lymphocytic leukaemia (B‐CLL) with mutated (M) IgVH genes have a better prognosis than unmutated (UM) cases. We analysed the IgVH mutational status of B‐CLL according to the features of a canonical somatic hypermutation (SHM) process, correlating this data with survival. In a series of 141 B‐CLLs, 124 cases were examined for IgVH gene per cent mutations and skewing of replacement/silent mutations in the framework/complementarity‐determining regions as evidence of antigen‐driven selection; this identified three B‐CLL subsets: significantly mutated (sM), with evidence of antigen‐driven selection, not significantly mutated (nsM) and UM, without such evidence and IgVH gene per cent mutations above or below the 2% cut‐off. sM B‐CLL patients had longer survival within the good prognosis subgroup that had more than 2% mutations of IgVH genes. sM, nsM and UM B‐CLL were also characterized for the biased usage of IgVH families, intraclonal IgVH gene diversification, preference of mutations to target‐specific nucleotides or hotspots, and for the expression of enzymes involved in SHM (translesion DNA polymerase ζ and η and activation‐induced cytidine deaminase). These findings indicate the activation of a canonical SHM process in nsM and sM B‐CLLs and underscore the role of the antigen in defining the specific clinical and biological features of B‐CLL.

A scoring system based on the expression of six surface molecules allows the identification of three prognostic risk groups in B‐cell chronic lymphocytic leukemia

We have previously identified 12 surface antigens whose differential expression represented the signature of B‐cell chronic lymphocytic leukemia (B‐CLL) subsets with different prognosis. In the present study, expression data for these antigens, as determined in 137 B‐CLL cases, all with survivals, were utilized to devise a comprehensive immunophenotypic scoring system of prognostic relevance for B‐CLL patients. In particular, univariate z score was employed to identify the markers with greater prognostic impact, while maximally selected log‐rank statistics were chosen to define the optimal cut‐off points capable to split patients into two groups with different survivals. A weighted immunophenotypic scoring system was developed by integrating results from these analyses. Six antigens were selected: three positive prognosticators (CD62L, CD54, CD49c) and three negative prognosticators (CD49d, CD38, CD79b), with cut‐off values ranging from 30% to 50% of positive cells. By weighing the expression of each marker according to its statistical power, a complete scoring system, with point values comprised between 0 (complete absence of phenotypic conditions associated with good prognosis) and 9 (all the phenotypic conditions associated with good prognosis fulfilled), allowed to split the whole set of B‐CLL patients, into three distinctive prognostic groups (P = 4.78 × 10−11) with high‐ (score 0–3), intermediate‐ (score 4–6), and low‐ (score 7–9) risk of death. The three risk groups showed different distribution of cases as for Rais stages, IgVH mutations, and ZAP‐70 expression. The proposed immunophenotypic scoring system may be an additional useful tool in routine diagnostic/prognostic procedures for B‐CLL. J. Cell. Physiol. 207: 354–363, 2006.

Signature of B‐CLL with different prognosis by Shrunken centroids of surface antigen expression profiling

With the aim of identifying the immunophenotypic profile of B‐cell chronic lymphocytic leukemia (B‐CLL) subsets with different prognosis, we investigated by flow cytometry the expression of 36 surface antigens in 123 cases, all with survivals. By analyzing results with unsupervised (hierarchical and K‐means clustering) algorithms, three distinct immunophenotypic groups (I, II, and III) were identified, group I (51/123) with longer survivals, as compared to the group II (36/123) and III (36/123). The immunophenotypic signatures of these groups, as determined by applying the nearest Shrunken centroids method as class predictor, were characterized by the coordinated and differential expression of 12 surface markers, that is, group I: above‐average expression of CD62L, CD54, CD49c, and CD25, below‐average expression of CD38; group II: above‐average expression of CD38, CD49d, CD29, and CD49e; and group III: below‐average expression of the above markers, overexpression of CD23, CD20, SmIg, and CD79b. As opposed to groups II–III, group I B‐CLLs lacked expression of ZAP‐70 and activation‐induced cytidine deaminase in the majority of cases, while more frequently had mutated IgVH genes and IgVH mutations consistent with antigen‐driven selection. Our findings contribute to improve the immunophenotypical identification of disease subsets with different prognosis and suggest a set of surface antigens to be employed as prognosticators in routine diagnostic/prognostic procedures.

In vitro and in vivo effects of 2′‐deoxycoformycin (Pentostatin) on tumour cells from human γδ+ T‐cell malignancies

Hepatosplenic γδ+ T‐cell lymphoma represents a rare neoplasm of post‐thymic phenotype, characterized by an aggressive clinical course and a poor response to conventional chemotherapy. In the present study, we have examined the cytotoxic effects of the purine analogue 2′‐deoxycoformycin (dCF) on cultured mononuclear cells and purified γδ+ tumour cells from bone marrow or peripheral blood of four patients with hepatosplenic γδ+ T‐cell lymphoma. At a concentration of 10 µm, dCF, in the presence of 2′‐deoxyadenosine (dAdo), displayed an early and selective cytotoxic effect on γδ+ tumour T cells. After 48 h of in vitro exposure to dCF, the absolute number of viable CD3+/γδ+ tumour T cells was reduced by more than 90% in all samples with respect to control cultures, with absolute counts of viable CD3+/αβ+ lymphocytes being reduced only by 6–40% of the initial cell input. Analysis of cultures after 5 d of exposure to dCF plus dAdo revealed the persistence of normal CD3+/αβ+ T cells, which accounted, however, for only 20–25% of the initial cell input. Accordingly, the combination of dCF (10–100 µm) plus dAdo was able to induce a dose‐dependent inhibition of clonogenic growth and [3H]‐thymidine incorporation in purified CD3+/CD4−/CD8−γδ+ tumour cells. We also report that one patient with hepatosplenic γδ+ T‐cell lymphoma in terminal leukaemic phase showed a striking haematological response to single‐agent dCF given as fourth‐line treatment. In particular, the selective clearance of γδ+ tumour T cells in peripheral blood and bone marrow was observed starting after the second course of treatment. Our results suggest that dCF may represent a potentially active drug for the management of this aggressive form of T‐cell lymphoma.

Immune Recovery after Autologous Stem Cell Transplantation Is Not Different for HIV-Infected versus HIV-Uninfected Patients with Relapsed or Refractory Lymphoma

BACKGROUND High-dose chemotherapy (HDC) and autologous stem cell transplantation (ASCT) are feasible and effective salvage treatments for human immunodeficiency virus (HIV)-related relapse or refractory lymphoma. Among the main concerns with ASCT in HIV-infected persons is the additional immune depletion caused by treatment, which could amplify the preexisting immune deficit. The aims of our study were to assess the impact of conventional chemotherapy before salvage treatment was administered, in this population, and to evaluate immune reconstitution dynamics during ASCT. METHODS All 33 HIV-infected and HIV-uninfected patients who underwent comparable ASCT protocols at the National Cancer Institute (Aviano, Italy) who underwent 1 month of follow-up after transplantation were included in a prospective immunological study. Demographic, clinical, and immunovirological data were obtained before administration of induction therapy, during transplantation, and at 24 months of follow-up. RESULTS Before HDC, no significant differences were observed in CD4(+) cell subsets and signal joint T cell receptor excision circles (sjTRECs), although HIV-infected persons had inverted ratios of CD4(+) cells to CD8(+) cells because they had higher CD8(+) T cell counts, compared with HIV-uninfected persons. After ASCT, this inversion was also observed in HIV-uninfected patients up to 24 months. CD4(+) cell subsets had similar recoveries, with a temporary setback in HIV-infected persons 3 months after reinfusion, together with an increase in infections. sjTRECs demonstrated similar dynamics in both populations and serve as a useful predictive marker of recovery of CD4(+) cell subsets. No significant changes emerged in HIV DNA levels during the follow-up period, with values at 24 months significantly lower than those at baseline. CONCLUSIONS Our study demonstrated that ASCT in HIV-infected persons with lymphoma does not worsen the initial immune impairment and does not enhance viral replication or the peripheral HIV reservoir in the long term.

CD40L induces proliferation, self-renewal, rescue from apoptosis, and production of cytokines by CD40-expressing AML blasts

OBJECTIVE Conflicting experimental and clinical results have been reported regarding the role of CD40 in acute myeloid leukemia (AML). In the present study, we analyzed the capability of CD40L/CD154 to modulate several functional aspects of CD40-expressing AML blasts. METHODS After defining the constitutive expression levels of CD40 in a wide panel (n = 67) of AMLs and evaluating the capability of cytokines to modulate its expression, we investigated the effects of CD40 engagement by soluble (s) CD40L on proliferation, self-renewal capacity, apoptosis, homotypic adhesion, and cytokine production of leukemia cells. RESULTS CD40 was detected in blast cells from about 37% of AMLs, the highest frequency being documented in monocytic subtypes, and its expression was upregulated or de novo induced by treatment with interleukin (IL)-1alpha, IL-3, IL-4, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma, and tumor necrosis factor-alpha. Exposure of CD40(+) AML blasts to sCD40L resulted in a dose-dependent proliferative response, enhancement of clonogenic growth and self-renewal capacity, and a striking increase in colony size. CD40 engagement was able to rescue AML blasts from apoptosis induced by serum deprivation, as demonstrated by reduced expression of APO2.7 and annexin-V binding, as well as upregulation of the anti-apoptotic protein bcl-x(L). CD40 triggering upregulated cell surface expression of the adhesion molecules CD54, CD58, and CD15 and resulted in homotypic aggregation of leukemia cells at least in part CD54-dependent. An increased production of IL-6 and GM-CSF by CD40(+) AML blasts was also documented upon sCD40L exposure. CONCLUSIONS This study indicates a possible involvement of CD40 in the interactions of AML blasts with other growth-sustaining microenvironmental accessory cells and immune effectors, in turn expressing CD40L. Caution in the use of CD40 triggering in immunotherapy of AMLs is also suggested.

CD90/Thy-1 is preferentially expressed on blast cells of high risk acute myeloid leukaemias*

Different transformation mechanisms have been proposed for elderly acute myeloid leukaemia (AML) and secondary AML (sAML) when compared with de novo AML or AML of younger patients. However, little is known regarding differences in the immunophenotypic profile of blast cells in these diseases. We systematically analysed, by flow cytometry, 148 patients affected by de novo (100 cases) or sAML (48 cases). By defining a cut‐off level of 20% of CD34+ cells co‐expressing CD90, the frequency of CD90+ cases was higher in sAML (40%) versusde novo AML (6%, P < 0·001), elderly AML (>60 years) (24%) versus AML of younger patients (10%, P = 0·010) and poor‐ versus good‐risk karyotypes (according to the Medical Research Council classification, P < 0·001). The correlation between CD90 expression, sAML and unfavourable karyotypes was confirmed by analysing the subset of CD34+ AML cases alone (91/148). Consistently, univariate analysis showed that expression of CD90 was statistically relevant in predicting a shorter survival in CD90+ AML patients (P = 0·042). Our results, demonstrating CD90 expression in AML with unfavourable clinical and biological features, suggest an origin of these diseases from a CD90‐expressing haemopoietic progenitor and indicate the use of CD90 as an additional marker of prognostic value in AML.

Hodgkin's disease: A disorder of dysregulated cellular cross-talk

Hodgkin’s disease (HD) is a peculiar type of human malignant lymphoma characterized by a very low frequency of tumor cells, the so called Hodgkin and Reed-Sternberg (H-RS) cells, embedded in a hyperplastic background of non-neoplastic (reactive) cells recruited and activated by H-RS cells-derived cytokines. H-RS cells can be functionally regarded as antigen-presenting cells (APC) able to elicit an intense, but anergic and ineffective, T-cell mediated immune response along with a hyperplastic inflammatory reaction which involves several cell types including T- and B-cells, neutrophils, eosinophils, plasma cells, fibroblasts and stromal cells. In tissues involved by HD, malignant H-RS cells and their reactive neighboring cells are able to cross-talk via a complex network of cytokine- and cell contact-dependent interactions. As a result of such interactions, mediated by specific surface receptors and adhesion molecules on both tumor and non-neoplastic cells, H-RS cells may receive several proliferative and anti-apoptotic signals favoring the cellular expansion and tumor cell survival in HD. The ineffective T-cell immune response elicited by the abnormal APC function of H-RS cells may further contribute to the biologic and clinical progression of HD. Innovative therapeutic strategies aimed at blocking the pathways of dysregulated cellular cross-talk among H-RS cells and bystander reactive cell populations might be beneficial in the treatment of HD patients.