Rituximab and 17-allylamino-17-demethoxygeldanamycin induce synergistic apoptosis in B-cell chronic lymphocytic leukaemia

Treatment options for chronic lymphocytic leukaemia (CLL) are limited and eventually fail because of the development of toxicities or drug resistance. Thus, identification of new therapeutic strategies and targets is a high priority. The semisynthetic geldanamycin derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) inhibits heat shock protein 90 (Hsp90) binding to client proteins, thereby leading to their degradation. We demonstrate that at biologically active and clinically attainable levels (1 mumol/l), 17-AAG treatment of CLL B cells in vitro causes modest apoptosis as well as decreased AKT protein levels. Given the potential activation of AKT following antibody therapy in CLL, we evaluated the combination of 17-AAG and rituximab. These agents produced synergistic cytotoxicity of CLL cells in vitro. However, rituximab-mediated antibody-dependent cellular cytotoxicity was modestly reduced with 17-AAG, and complement-dependent cytotoxicity was not altered. We conclude that the combination of Hsp90 inhibitors with therapeutic antibodies, such as rituximab may represent a novel strategy to enhance therapeutic response in CLL. Furthermore, our data indicates that AKT and Hsp70 protein levels are relevant pharmacodynamic endpoints to monitor the in vivo effect of 17-AAG therapy.     

Humanized CD52 monoclonal antibody campath-1H as first-line treatment in chronic lymphocytic leukaemia

The humanized CD52 monoclonal antibody Campath-1H was used as first-line therapy in nine patients with progressive chronic lymphocytic leukaemia (CLL). Intravenous ( n  = 5) or subcutaneous ( n  = 4) injections (up to 30 mg/inj.) were given three times a week for a maximum of 18 weeks. Three patients achieved a complete remission (CR) and five patients a partial remission (PR) (response rate 89%). CLL cells were cleared from blood in all patients and from the bone marrow in seven patients. The response duration time was 8+–24+ months. Adverse events were mild except for one patient who developed CMV pneumonitis. All patients developed lymphocytopenia (B and T cells) but other haematological toxicities were negligible. Campath-1H is a highly effective and well-tolerated agent in patients with previously untreated CLL and further studies are warranted.     

Quantification improves the prognostic value of CD38 expression in B-cell chronic lymphocytic leukaemia

Recent studies have shown that CD38 expressed as a percentage of the antigen positivity can predict prognosis and disease progression in patients with B-cell chronic lymphocytic leukaemia (B-CLL). The present study showed that quantification of CD38 expressed as antibody-binding capacity (ABC) improves the prognostic value of the percentage of CD38 positivity in B-CLL. In a cohort of 81 patients with B-CLL, a level of CD38 expression of > or = 30% and an ABC value of 250 proved statistically valid cut-off points to predict disease progression (% CD38: P=0.0027; ABC: P < 0.0001). There was a positive and significant correlation between the percentage of CD38 expression and ABC (r=0.7; P < 0.0001). There was a better discrimination of survival using ABC rather than percentage CD38 positivity (P < 0.0001 compared with P=0.0027). Only ABC predicted for survival in patients under 60 years of age (P=0.0076) or with stage A disease (P=0.0195). Both percentage CD38 and ABC discriminated between time to first treatment for all patients but only ABC predicted time to treatment for stage A patients (P=0.0004). In conclusion, CD38 positivity is an important prognostic factor in B-CLL. However, quantification of CD38 is superior to the percentage positivity and should be used clinically in conjunction with other variables of predictive value to identify B-CLL patients that are likely to progress.     

Standard-dose anti-CD20 antibody rituximab has efficacy in chronic lymphocytic leukaemia: results from a Nordic multicentre study

OBJECTIVES: This prospective multicentre study was conducted to assess the efficacy of the monoclonal anti-CD20 antibody rituximab in patients with chronic lymphocytic leukaemia (CLL). Secondary objectives were defined as the tolerability and feasibility of rituximab in patients with CLL. METHODS: Twenty-four heavily pretreated patients with CLL were treated with a standard dose of 375 mg m-2 of rituximab given once weekly for four doses. RESULTS: The overall response rate was 35% and all the responses were partial as defined by the revised NCI criteria. In 17 (85%) of 20 patients with initially measurable peripheral lymph nodes the size of lymph nodes decreased by at least 50%, while an improvement of the bone marrow infiltration was observed only in two (11%) of 18 evaluable patients. The median duration of the overall response was 12.5 wk. Rituximab was relatively well tolerated. Although side-effects were common (75%) they were usually mild or moderate. There was only one grade 3 adverse event and no grade 4 events. CONCLUSIONS: Standard-dose rituximab has activity in heavily pretreated patients with CLL, although the response is mainly limited to the lymph nodes and of short duration. Since rituximab has in vitro synergism with chemotherapeutic agents and is well tolerated by CLL patients, it is reasonable to investigate rituximab in combination with other treatments.     

Bendamustine + rituximab chemoimmunotherapy and maintenance lenalidomide in relapsed,refractory chronic lymphocytic leukaemia and small lymphocytic lymphoma: A Wisconsin Oncology Network Study

Bendamustine + rituximab (BR) has demonstrated high response rates in relapsed/refractory (R/R) chronic lymphocytic leukaemia (CLL) and small lymphocytic lymphoma (SLL). However, progression‐free survival (PFS) after BR is <18 months. This study was designed to determine if maintenance lenalidomide after BR induction could improve PFS in R/R CLL/SLL. Thirty‐four patients with R/R CLL/SLL who had received 1–5 prior chemotherapy regimens were treated with 6 cycles of BR induction. Patients achieving at least a minor response received twelve 28‐d cycles of lenalidomide 5–10 mg/d. The primary endpoint was PFS. The median age was 67 years, with a median of 2 prior therapies. Eleven patients had confirmed presence of 17p and/or 11q deletions. Twenty‐five (74%) completed 6 cycles of induction BR (response rate 56%). Nineteen (56%) patients received maintenance lenalidomide; only 6 patients completed the intended 12 cycles, highlighting the limited feasibility of lenalidomide in this setting, primarily due to haematological and infectious toxicities. The observed median PFS of 18·3 months is not significantly different from that of BR induction in R/R CLL/SLL without maintenance therapy (15·2 months). It is possible that lenalidomide maintenance may be more feasible and effective in the front‐line setting, which is being tested in an ongoing trial (NCT01754857).     

An Italian retrospective study on the routine clinical use of low-dose alemtuzumab in relapsed/refractory chronic lymphocytic leukaemia patients

Low‐dose alemtuzumab has shown a favourable toxicity profile coupled with good results in terms of efficacy in relapsed/refractory chronic lymphocytic leukaemia (CLL). We conducted a multicentre retrospective study on the routine clinical use of low‐dose alemtuzumab in this patient setting. One hundred and eight relapsed/refractory CLL patients from 11 Italian centres were included in the analysis. All patients had an Eastern Cooperative Oncology Group performance status ≤2 and the majority (84%) had adenopathies <5cm. Low‐dose alemtuzumab was defined as a total weekly dose ≤45 mg and a cumulative dose ≤600 mg given for up to 18 weeks. The overall response rate was 56% (22% complete remissions). After a median follow‐up of 42·2 months, the median overall survival and progression‐free survival were 39·0 and 19·4 months, respectively. In univariate analysis, response was inversely associated with lymph node (P = 0·01) and spleen (P = 0·02) size, fludarabine‐refractoriness (P = 0·01) and del(11q) (P = 0·009). Advanced age and del(17p) were not associated with a worse outcome. Cumulative dose of alemtuzumab was not associated to response. Toxicities were usually mild and manageable; severe infections occurred in seven patients (7%) during therapy. This retrospective analysis confirms that low‐dose alemtuzumab is a valid and currently used therapeutic option for the treatment of relapsed/refractory CLL.     

Phase II study of subcutaneous alemtuzumab without dose escalation in patients with advanced-stage, relapsed chronic lymphocytic leukaemia

This phase II study ( n  = 20) aimed to evaluate type, severity and duration of side-effects and efficacy following subcutaneous (SC) alemtuzumab, without dose-escalation, in advanced-stage relapsed chronic lymphocytic leukaemia (CLL) patients. Alemtuzumab 30 and 3 mg was administered SC simultaneously day 1, followed by 30 mg three times per week. Injection-site-reactions were recorded every 6–24 h until resolved using National Cancer Institute criteria and a new skin toxicity subscale. The first doses of 30 mg and 3 mg produced injection-site-reactions (all but one were grade 1/2) in 13/20 and 9/20 patients, respectively. The second dose (on day 3) resulted in skin-reactions in 10/20 patients and the third, fourth, fifth and sixth injections produced reactions in 6/20, 1/20, 2/20 and 0/20 patients, respectively. Mild "flu-like" symptoms occurred during week 1 in 10/20 patients. All side-effects had subsided by the sixth dose. 15/20 patients (75%) responded (12 partial responses, three complete responses) with a median time-to-treatment-failure of 20 months. Symptomatic cytomegalovirus-reactivation occurred in 6/20 patients. Two deaths occurred: one bacterial pneumonia and one adenovirus-infection. The present study showed how to assess cutaneous-toxicity in detail and that 30 mg alemtuzumab SC administered upfront was well tolerated. Optimized alemtuzumab therapy in properly selected patients may result in high efficacy even in advanced CLL. Our results need to be confirmed in extended studies.     

The prognostic value of CD38 expression in chronic lymphocytic leukaemia patients studied prospectively at diagnosis: a single institute experience

The purpose of this study was to assess in chronic lymphocytic leukaemia (CLL) patients the prevalence and clinical impact of CD38 expression, evaluated prospectively at disease presentation, and to verify whether this parameter changes over time. In 242 consecutive and untreated CLL patients, the percentage of CD38+ cases, according to the 7%, 20% and 30% cut-off points, was 21%, 17% and 14%, respectively. Using the 7% threshold, CD38 positivity correlated with male sex, intermediate and high-risk (Rai I-IV) disease, lower Hb and platelet levels, and higher lymphocyte count. Furthermore, patients with a CD38 expression>or=7% showed a significantly lower 3-year probability of treatment-free survival (TFS) than CD38- patients (P<0.0001). At multivariate analysis, CD38 expression remained significantly associated to TFS, together with stage, lymphocyte count and morphology. Also, in the 146 patients with stage 0 CLL a CD38 expression>or=7% identified a subgroup of patients with a significantly lower 3-year probability of TFS (P=0.0005). Furthermore, this parameter did not change in 30 of 31 (97%) re-evaluated patients. In conclusion, this study indicates that, when tested at diagnosis and on fresh material, a CD38 expression>or=7% is an important parameter for the identification of early CLL patients with more aggressive disease and that its expression remains stable over time.     

CD20-positive T-cell chronic lymphocytic leukaemia

Although CD20 is considered to be a representative marker for B lymphocytes, the antigen is weakly expressed on a small subset of normal T lymphocytes. A 60-year-old man developed pancytopenia and hepatosplenomegaly due to clonal proliferation of atypical lymphocytes that were weakly positive for CD20. The leukaemic cells were also positive for T-cell antigens such as CD2, CD3, CD5, CD7, CD8 and T-cell receptor (TCR) Vβ8 and for activation antigens such as CD38 and HLA-DR, but were negative for CD19, CD21, CD22, CD25. Southern blot analysis revealed rearrangement of the TCR-β gene and a germline configuration of the immunoglobulin heavy chain gene. This is the first report of a case of clonal expansion of CD20dim Tlymphocytes     

Anticipation in familial chronic lymphocytic leukaemia

Anticipation, a phenomenon in which an inherited disease is diagnosed at an earlier age in each successive generation of a family, has been demonstrated in certain heritable neurological disorders and in multiple myeloma, non-Hodgkin's lymphoma and other haematological neoplasms. The present study was conducted to determine whether anticipation occurs in familial chronic lymphocytic leukaemia (CLL). Fourteen published reports of multigenerational familial CLL were analysed for anticipation, together with 10 previously unreported families with familial CLL, and the difference in disease-free survival between generations was determined. The difference between age at onset for each affected parent-child pair was tested against the null hypothesis that there was no difference in age at onset. The age at onset of the studied cases was also compared with that of the Surveillance Epidemiology and End Results (SEER) Program of the U.S. National Cancer Institute. The median ages at onset in the child and parent generations of all families (51.0 and 72.0 years respectively) were significantly different (P < 0.000001), and the null hypothesis was rejected (P < 0.000001). A significant difference was observed between the ages of onset of the child generation and the SEER population (P < 0.00001), but not between the parent generation and the SEER population. Anticipation characterizes familial CLL.     

Gene probe analysis demonstrates independent clonal evolution of co-existent chronic myeloid and chronic lymphocytic leukaemia

We describe a case of Philadelphia-positive chronic myeloid leukaemia occurring simultaneously with B-cell chronic lymphocytic leukaemia in a 69-yr-old male. Gene probe analysis of DNA from both peripheral blood and bone marrow provided evidence for the independent evolution of 2 clones in this case, with a predominant population showing immunoglobulin heavy chain gene rearrangement and a smaller population showing a rearrangement within the breakpoint cluster region of chromosome 22.     

Multidrug resistance mechanisms in chronic lymphocytic leukaemia

We evaluated the presence of P-glycoprotein (P-gp)-170, multidrug resistance protein (MRP), lung resistance protein (LRP)-56 and Bcl-2 in CD19-positive cells from 100 cases of chronic lymphocytic leukaemia (CLL). P-gp-170 was found in 73% of the CLL cases with no significant difference regarding stage or previous treatment. LRP-56 protein was homogeneously distributed with no differences for stage or treatment. MRP protein was detected at a low level of expression in 49.4% of CLL patients with no differences for stage or treatment. Bcl-2 protein was expressed at a high level in all CLL patients and higher levels were found in the advanced stage. This leads us to conclude that P-gp, MRP, LRP-56 and Bcl-2 are frequently expressed in CLL. P-gp, MRP and LRP are not correlated to stage or previous treatment. Bcl-2 is higher in advanced-stage patients. The clinical and biological significance of these zMDR mechanisms in CLL remains to be fully explained.     

CD49d associates with nodal presentation and subsequent development of lymphadenopathy in patients with chronic lymphocytic leukaemia

CD 49d is a surface integrin that is expressed on chronic lymphocytic leukaemia (CLL ) cells, and strongly correlates with more aggressive disease. Given its association with cell‐cell adhesion and leucocyte trafficking, we hypothesized that patients with high CD 49d expression would experience a clinical course dominated by lymphadenopathy. CD 49d expression was measured by flow cytometry and considered positive if expressed by ≥30% of CLL cells. The study included 797 newly diagnosed CLL /small lymphocytic leukaemia patients; 279 (35%) were CD 49d positive. CD 49d‐positive patients were more likely to present with lymphadenopathy (P  <  0·001); a finding that persisted after adjusting for fluorescence in situ hybridisation (FISH ) and IGHV mutation status [odds ratio (OR ) 2·51; 95% confidence interval (CI ) 1·64–3·83; P  <  0·001]. Among CLL Rai 0 patients, CD 49d positivity was associated with shorter time to development of lymphadenopathy (3·2 years vs not reached, P  <  0·01). This association was maintained after adjusting for either FISH [hazard ratio (HR ) 2·18; 95% CI 1·25–3·81; P  =  0·006) or IGHV status (HR 2·02; 95% CI 1·11–3·69; P  =  0·02) individually, but was attenuated when adjusting by both (HR 1·72; 95% CI 0·88–3·38; P  =  0·11).These data demonstrate that CD 49d‐positive CLL patients experience a disease course dominated by lymphadenopathy. These findings could have implications for therapy selection and disease monitoring.     

Biological effects and clinical significance of lenalidomide-induced tumour flare reaction in patients with chronic lymphocytic leukaemia: in vivo evidence of immune activation and antitumour response

Lenalidomide has demonstrated impressive antileukaemic effects in patients with chronic lymphocytic leukaemia (CLL). The mechanism(s) by which it mediates these effects remain unclear. Clinically, CLL patients treated with lenalidomide demonstrate an acute inflammatory reaction, the tumour flare reaction that is suggestive of an immune activation phenomenon. Samples from CLL patients treated with lenalidomide were used to evaluate its effect on the tumour cell and components of its microenvironment (immune cellular and cytokine). Lenalidomide was unable to directly induce apoptosis in CLL cells in vitro, however it modulated costimulatory (CD80, CD83, CD86) surface molecules on CLL cells in vitro and in vivo. Concurrently, we demonstrated that NK cell proliferation was induced by lenalidomide treatment in patients and correlated with clinical response. Cytokine analysis showed increase in levels of TNF-α post-lenalidomide treatment, consistent with acute inflammatory reaction. Furthermore, the basal cytokine profile (high IL-8, MIG, IP-10 and IL-4 levels and low IL-5, MIP1a, MIP1b, IL12/p70) was predictive of clinical response to lenalidomide. Collectively, our correlative studies provide further evidence that the antileukaemic effect of lenalidomide in CLL is mediated not only through modulation of the leukaemic clone but also through elements of the tumour microenvironment.     

Chronic lymphocytic leukaemia cells are efficiently killed by an anti-CD20 monoclonal antibody selected for improved engagement of FcgammaRIIIA/CD16

Patients with chronic lymphocytic leukaemia (CLL) treated with a combination of fludarabine, cyclophosphamide and rituximab show a high response rate. However, only a poor response is observed following rituximab monotherapy. The use of chemo-immunotherapy is often associated with haematological and infectious complications. Thus, an antibody with an enhanced ability to kill CLL cells could lead to better clinical responses to antibody monotherapy and the possibility of lowering drug doses during chemo-immunotherapy. We generated a chimeric anti-CD20 monoclonal antibody (mAb), EMAB-6, which has a low fucose content. Apoptosis and complement activities for EMAB-6 were similar to those seen for rituximab. By contrast, EMAB-6 mAb showed improved Fcγ receptor IIIA (FcγRIIIA)/CD16 binding and FcγRIIIA-dependent effector functions. It induced a higher in vitro antibody-dependent cellular cytotoxicity against CLL cells and a higher FcγRIIIA-mediated interleukin-2 production by FcγRIIIA⁺ Jurkat cells in the presence of CLL cells at both low and maximally saturating concentrations. Comparative studies between CLL and lymphoma cells coated with EMAB-6 or rituximab indicated that the difference of efficacy was more pronounced at low doses and when target cells expressed fewer CD20 molecules. Thus, EMAB-6 mAb represents a promising drug candidate for the treatment of CLL by inducing a strong cytotoxicity against tumour cells that express low CD20 levels.     

Ofatumumab retreatment and maintenance in fludarabine‐refractory chronic lymphocytic leukaemia patients

There are limited data on retreatment with monoclonal antibodies (mAb) in patients with chronic lymphocytic leukaemia (CLL). In a pivotal study, ofatumumab (human anti‐CD20 mAb) monotherapy demonstrated a 47% objective response rate (ORR) in fludarabine refractory CLL patients. From this study, a subset of 29 patients who had at least stable disease and then progressed were retreated with eight weekly ofatumumab infusions (induction treatment period), followed by monthly infusions for up to 2 years (maintenance treatment period). The ORR after 8 weeks of induction retreatment was 45% and 24% had continued disease control after maintenance at 52 weeks. Efficacy and safety of the retreated patients were compared with their initial results in the pivotal study. Response duration was 24·1 months vs. 6·8 months; time to next therapy was 14·8 months vs. 12·3 months; and progression‐free survival was 7·4 months vs. 7·9 months (medians). Upon retreatment, 72% had infusion reactions, mostly Grade 1–2. Three patients had fatal infections. In summary, ofatumumab retreatment and maintenance therapy was feasible in patients with heavily pretreated CLL and appeared to result in more durable disease control than initial ofatumumab treatment in this subset of patients who may have a more favourable disease profile.     

Idiotype-pulsed dendritic cells are able to induce antitumoral cytotoxic CD8 cells in chronic lymphocytic leukaemia

Idiotypic structures of immunoglobulins from malignant B cells constitute tumour-specific antigens, though the function of immunoglobulin-specific CD8+ T cells in disease control and rejection remains unclear. We have studied five cases of B chronic lymphocytic leukaemia patients affected with indolent (three patients) or aggressive (two patients) disease. We showed that CD8+ T cells with major histocompatibility complex class I-restricted cytotoxicity against autologous tumour B cells could be generated following repeated stimulations with idiotype-pulsed dendritic cells in vitro. CD8+ T-cell lines were able to upregulate CD69 expression and to release interferon (IFN)-gamma upon contact with the autologous B cells, though cytolytic activity was only substantiated for patients with indolent disease. The failure of cytolytic activity in patients with aggressive disease may be explained by a skewed maturation of memory CD8 cells.