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In the HOVON68 CLL trial, patients 65 to 75 years of age had no survival benefit from the addition of low-dose alemtuzumab to fludarabine and cyclophosphamide (FC) in contrast to younger patients. The reasons are explored in this 5-year trial update using both survival analysis and competing risk analysis on non-CLL-related mortality. Elderly FCA patients died more frequently from causes not related to CLL, and more often related to comorbidity (mostly cardiovascular) than to infection. In a Cox multivariate analysis, del(17p), performance status >0, and comorbidity were associated with a higher non-CLL-related mortality in the elderly independent of the treatment modality. Thus, while the 'fit' elderly with no comorbidity or performance status of 0 might potentially benefit from chemo-immunotherapy with FC, caution is warranted, when considering alemtuzumab treatment in elderly patients with cardiovascular comorbidity.

A single-center, prospective, non-randomized clinical study was performed to examine the safety and feasibility of early discharge in patients undergoing consolidation chemotherapy for acute leukemia, or autologous stem cell transplant for lymphoma or multiple myeloma. Patients were discharged into ambulatory care the day after the last chemotherapy administration and were subsequently seen at the ambulatory care unit three times a week. One hundred and one of 224 patients were ineligible for the program, mostly because of their medical situation, the lack of a caregiver or the travel time to the hospital. The remaining 123 patients were able to spend more than 70% of the time at home. In 44% of cycles they were never readmitted. This study demonstrates the safety, feasibility and benefits of managing carefully selected patients. Patients and their caregivers felt safe and comfortable at home, and the vast majority preferred home care to in-hospital treatment.

In chronic lymphocytic leukemia (CLL), strategies to overcome drug resistance due to p53 dysfunction are highly needed. Platinum-based compounds such as cisplatinum (CDDP) are active in fludarabine-refractory CLL through a largely unknown mechanism. We analyzed the mechanism of action of CDDP in the context of p53 dysfunctionality. In vitro treatment with CDDP did not induce death in quiescent CLL cells, but did induce apoptosis in CD40-ligand (and CpG) stimulated and proliferating cells, irrespective of p53 function. In the p53 dysfunctional prolymphocytic cell-line MEC1, CDDP treatment resulted in apoptosis, cell cycle arrest and ABL1-dependent expression of TAp73, CDKN1A, PUMA and BID. TAp73 RNA-interference decreased sensitivity to CDDP. Finally, both in vitro stimulated CLL cells and lymph node (LN) derived CLL cells showed increased TAp73 expression in comparison with quiescent peripheral blood derived cells. Activity of CDDP may therefore be mediated by TAp73, especially in the context of activation such as occurs in the LN microenvironment.

The ATM-p53 DNA damage response pathway plays a crucial role in chemoresistance in chronic lymphocytic leukemia, as indicated by the adverse prognostic impact of deletions of 17p (locus of TP53) and 11q (locus of ATM) detected by fluorescence in situ hybridization (FISH) analysis. In addition to deletions, mutations in these respective genes are also associated with chemoresistance, and add to the prognostic information provided by FISH. In order to explore the possibility that dysfunction of the ATM-p53 pathway might also result from mechanisms other than ATM/TP53 deletion/mutation, assays have been developed that probe the functional integrity of the ATM-p53 pathway. Currently, four different p53 function assays have been developed that are based on the measurement of p53 and p53-dependent genes at the RNA (real-time polymerase chain reaction [RT-PCR]p21; RT-PCRmiR34a; reverse transcription-multiplex ligation-dependent probe amplification assay [RT-MLPA]p21, bax, puma and CD95) or protein (fluorescence activated cell sorting [FACS]p53-p21) level in untreated cells or following irradiation or drug treatment. Here we provide an overview of these assays based on the available literature.

In patients with chronic lymphocytic leukemia (CLL), effector and memory CD4 + and CD8 + T cells are expanded. We investigated whether these CLL specific T-cell expansions also occur in monoclonal B lymphocytosis (MBL), the pre-leukemic state of CLL, which is currently distinguished from CLL by an arbitrarily chosen cut-off value of CD19 of 5.0 × 10(9)/L. Whereas an increase in effector and memory CD4 + and CD8 + T cells was found in CLL, these expansions could not be found in MBL. Although a significant correlation was found between absolute B cell count (CD19) and T cell numbers, correlation coefficients were rather low. Therefore, we analyzed whether an optimal threshold for CD19 number could be defined which best related to an expansion of T cells. The B-cell threshold that best predicted expansion of CD3 +, CD4 + and CD8 + T cells, respectively, was 10 × 10(9)/L. Our study indicates that a higher cut-off value than the current 5.0 × 10(9)/L relates better to the biological impact of CLL.

In patients with chronic lymphocytic leukemia (CLL), numbers of CD8 + CD45RA +/- CD27- effector T cells are expanded. We investigated whether this expansion is also present in other B cell malignancies and the possible mechanism underlying these changes. Whereas an increase in total CD4+and CD8+ T cell numbers was found only in CLL, numbers of CD4+ and CD8+ effector T cells were significantly increased in both CLL and indolent lymphoma, but not aggressive lymphoma and myeloma. Interestingly, PD-1 expression was decreased on effector T cells and inversely correlated with effector T cell numbers, suggesting a functional role for PD-1 in regulating T cell homeostasis. In vitro experiments revealed impaired up-regulation of PD-1 upon T cell activation in the presence of malignant but also healthy B cells. Our data suggest that in CLL and indolent lymphoma, the malignant B cells affect PD-1 expression on effector T cells, resulting in an expansion of these subsets.

Recently, it has been described that patients with chronic lymphocytic leukemia (CLL) have increased numbers of regulatory T (T(reg)) cells. In the present study, we analysed the mechanism behind T(reg) cells expansion in CLL. Neither analysis of the T-cell receptor repertoire nor CD45 isoform expression of T(reg) cells from patients with CLL provided evidence for chronic (tumor) antigenic stimulation as a possible cause for T(reg) cells expansion in CLL. We found evidence however for increased formation of T(reg) cells via CD70 costimulation, because we observed that CD40 ligand activated CLL cells (which might be considered a model of lymph node CLL cells) strongly induced CD70-dependent formation of T(reg) cells. Reverse transcription-multiplex ligation-dependent probe amplification assay expression analysis of 34 apoptosis-regulating genes showed that in comparison with other CD4(+) T-cells, T(reg) cells from both healthy individuals (HD) and patients with CLL had a high expression of pro-apoptotic Noxa and a low expression of anti-apoptotic Bcl-2. Strikingly, Bcl-2 levels of T(reg) cells in patients with CLL were significantly higher than in HD. Finally, the different apoptotic profile resulted in differences at the functional level, because T(reg) cells from patients with CLL were more resistant to drug-induced apoptosis than T(reg) cells from HD. In conclusion, T(reg) cells in CLL may accumulate both by increased formation, facilitated by CD27-CD70 interaction in the lymph node proliferation centres, and decreased sensitivity to apoptosis because of a shifted Noxa-Bcl-2 balance.

Artificial T cell receptor (TCR) ligands can be used to direct virus-specific cytotoxic T lymphocytes (CTL) towards tumor cells. Because of their size, these constructs may differ from cognate peptides in their ability to induce T cell activation. We here analysed signalling outcomes upon synapse formation between human cytomegalovirus (CMV)-specific CTL and chronic lymphocytic leukemia (CLL) cells through targeted complexes (TC) containing anti-CD20 single-chain variable fragment and biotinylated major histocompatibility complex class I molecules presenting peptides from CMVpp65. TC coated CLL cells were effectively lysed by CMVpp65-specific CTL but induced less interferon gamma (IFN-gamma) production than peptide loaded targets. Confocal microscopy revealed that TC induced a redistribution of TCR/CD3 but not CD2 towards the immunological synapse. Furthermore, morphological examination of immunological synapses showed smaller and 'patchy' interactions between TC coated B cells and CTL as compared with peptide coated targets. Finally, pre-incubation of CTL with CD2 antibodies led to an Fc-dependent redistribution of CD2 into TC-induced synapses and restored IFN-gamma production by CMV-specific CTL. Thus, redistribution of CD2 towards the immunological synapse appears to be essential for full T cell activation. However, CD2 triggering is not required for efficient lysis of tumor cells, demonstrating that CTL require only minimal stimulation to release their cytotoxic content.

Interleukin-6 (IL6) plays a major role in the pathogenesis of multiple myeloma. In patients with monoclonal gammopathy serum levels of sIL6R have been found to be increased. The role of IL6 in the regulation of soluble receptors is still unclear. In a phase I/II study we treated 12 myeloma patients with high-affinity chimeric anti-IL6 monoclonal antibodies. This treatment resulted in a total in vivo blockage of IL6 activity and as a result we had an unique opportunity to gain insight into the possible regulation effects of IL6 on these soluble IL6 receptors. Pre-treatment sIL6R levels were elevated in 9 of the 12 patients; pre-treatment sgp130 levels were significantly increased in all patients. Total blockage of IL6 activity by the high-affinity cMab did not influence sIL6R in 10 of these 12 patients and sgp130 levels remained stable in all patients. Of the 2 patients whose sIL6R levels increased during therapy, one had progressive disease and the other developed an acute infection. We conclude that in most end-stage myeloma patients sIL6R and sgp130 serum levels are elevated, but that there is no relation between IL6 activity and sIL6R or sgp130 levels.

In patients with the acquired immunodeficiency syndrome, the incidence of non-Hodgkin's lymphoma is increased. Two major subgroups of AIDS-related NHL (ARL) have been defined: Burkitt-type NHL (BL) and polymorphic centroblastic/immunoblast-rich large cell lymphomas (CB/IB LCL). These subgroups differ in their association with the Epstein-Barr virus (EBV) and thus possibly in their pathogenesis. We studied the expression of EBER (EBV small RNA's), and EBV latent antigens LMP-1 and EBNA-2 in 43 cases of ARL and related this to histology and immune status (CD4-cell count). In addition, in 19 cases the expression of adhesion molecules (LFA-1 (CD18), ICAM-1 (CD54), alpha4beta1 integrin (CD49d/CD29), L-selectin (CD62L) and CD44) was studied. We found major differences between the two subgroups. Patients with BL had significantly higher CD4-cell counts; only 40% of their lymphomas were EBV-positive, and when EBV-positive, were of the type I latency phenotype. Expression of adhesion molecules important for immune recognition was absent or low in all BL. In contrast, the majority of CB/IB LCL were EBER-positive (79%). 58% of EBV-positive LCL (particularly those in patients with CD4-cell counts below 0.2 x 10(9)/1) had a type II or III latency phenotype. Most LCL showed expression of LFA-1, ICAM-1 and alpha4beta1 integrin. CD44s expression was restricted to CB/IB LCL, in whom high expression of the metastasis-associated exon v6-containing CD44 variant was also observed. The observed EBV-latency types and full expression of adhesion molecules suggest that defective Epstein-Barr virus immunity is important in the pathogenesis of CB/IB large cell lymphomas.

In this review we discuss the possible value of additional information concerning growth characteristics of NHL's for the prognosis of the individual patient. Techniques for measuring cell cycle kinetic parameters (e.g. S-phase assessment) and proliferation related antigens (e.g. anti-transferrin receptor and Ki67) are shortly reviewed. This is followed by an overview of proliferation studies in NHL. Until now in most of these studies overall measurements of tumour tissue have been performed. Moreover the parameters that have been assessed are related to proliferation state rather than proliferation rate. None of the methods used has a convincing superiority with regard to clinical outcome. In the future, tumour specific assessment (by means of double-labeling techniques) of proliferation rate parameters might provide information, more relevant to the prognosis of the individual patient with NHL.