Thorsten Zenz

Addition of rituximab to fludarabine and cyclophosphamide in patients with chronic lymphocytic leukaemia: a randomised, open-label, phase 3 trial

BACKGROUND On the basis of promising results that were reported in several phase 2 trials, we investigated whether the addition of the monoclonal antibody rituximab to first-line chemotherapy with fludarabine and cyclophosphamide would improve the outcome of patients with chronic lymphocytic leukaemia. METHODS Treatment-naive, physically fit patients (aged 30-81 years) with CD20-positive chronic lymphocytic leukaemia were randomly assigned in a one-to-one ratio to receive six courses of intravenous fludarabine (25 mg/m(2) per day) and cyclophosphamide (250 mg/m(2) per day) for the first 3 days of each 28-day treatment course with or without rituximab (375 mg/m(2) on day 0 of first course, and 500 mg/m(2) on day 1 of second to sixth courses) in 190 centres in 11 countries. Investigators and patients were not masked to the computer-generated treatment assignment. The primary endpoint was progression-free survival (PFS). Analysis was by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00281918. FINDINGS 408 patients were assigned to fludarabine, cyclophosphamide, and rituximab (chemoimmunotherapy group) and 409 to fludarabine and cyclophosphamide (chemotherapy group); all patients were analysed. At 3 years after randomisation, 65% of patients in the chemoimmunotherapy group were free of progression compared with 45% in the chemotherapy group (hazard ratio 0·56 [95% CI 0·46-0·69], p<0·0001); 87% were alive versus 83%, respectively (0·67 [0·48-0·92]; p=0·01). Chemoimmunotherapy was more frequently associated with grade 3 and 4 neutropenia (136 [34%] of 404 vs 83 [21%] of 396; p<0·0001) and leucocytopenia (97 [24%] vs 48 [12%]; p<0·0001). Other side-effects, including severe infections, were not increased. There were eight (2%) treatment-related deaths in the chemoimmunotherapy group compared with ten (3%) in the chemotherapy group. INTERPRETATION Chemoimmunotherapy with fludarabine, cyclophosphamide, and rituximab improves progression-free survival and overall survival in patients with chronic lymphocytic leukaemia. Moreover, the results suggest that the choice of a specific first-line treatment changes the natural course of chronic lymphocytic leukaemia. FUNDING F Hoffmann-La Roche.

From pathogenesis to treatment of chronic lymphocytic leukaemia

Chronic lymphocytic leukaemia (CLL) has several unique features that distinguish it from other cancers. Most CLL tumour cells are inert and arrested in G0/G1 of the cell cycle and there is only a small proliferative compartment; however, the progressive accumulation of malignant cells will ultimately lead to symptomatic disease. Pathogenic mechanisms have been elucidated that involve multiple external (for example, microenvironmental stimuli and antigenic drive) and internal (genetic and epigenetic) events that are crucial in the transformation, progression and evolution of CLL. Our growing understanding of CLL biology is allowing the translation of targets and biological classifiers into clinical practice.

Allogeneic stem cell transplantation provides durable disease control in poor-risk chronic lymphocytic leukemia: long-term clinical and MRD results of the German CLL Study Group CLL3X trial

The purpose of this prospective multicenter phase 2 trial was to investigate the long-term outcome of reduced-intensity conditioning allogeneic stem cell transplantation (alloSCT) in patients with poor-risk chronic lymphocytic leukemia. Conditioning was fludarabine/ cyclophosphamide-based. Longitudinal quantitative monitoring of minimal residual disease (MRD) was performed centrally by MRD-flow or real-time quantitative polymerase chain reaction. One hundred eligible patients were enrolled, and 90 patients proceeded to alloSCT. With a median follow-up of 46 months (7-102 months), 4-year nonrelapse mortality, event-free survival (EFS) and overall survival (OS) were 23%, 42%, and 65%, respectively. Of 52 patients with MRD monitoring available, 27 (52%) were alive and MRD negative at 12 months after transplant. Four-year EFS of this subset was 89% with all event-free patients except for 2 being MRD negative at the most recent assessment. EFS was similar for all genetic subsets, including 17p deletion (17p-). In multivariate analyses, uncontrolled disease at alloSCT and in vivo T-cell depletion with alemtuzumab, but not 17p-, previous purine analogue refractoriness, or donor source (human leukocyte antigen-identical siblings or unrelated donors) had an adverse impact on EFS and OS. In conclusion, alloSCT for poor-risk chronic lymphocytic leukemia can result in long-term MRD-negative survival in up to one-half of the patients independent of the underlying genomic risk profile. This trial is registered at http://clinicaltrials.gov as NCT00281983.

Detailed analysis of p53 pathway defects in fludarabine-refractory chronic lymphocytic leukemia (CLL): dissecting the contribution of 17p deletion, TP53 mutation, p53-p21 dysfunction, and miR34a in a prospective clinical trial

The prognosis of fludarabine (F)-refractory chronic lymphocytic leukemia (CLL) is very poor, and underlying mechanisms are only partly understood. To assess the contribution of p53 abnormalities to F-refractory CLL, we studied TP53 mutations in the CLL2H trial (subcutaneous alemtuzumab; n = 99). We found TP53 mutations in 37% of patients. Twelve of 67 (18%) patients without the 17p deletion showed a TP53 mutation and 50% showed evidence of uniparental disomy. A total of 75% of cases with TP53 mutation (without 17p-) showed clonal evolution/expansion. TP53 mutations had no impact on overall survival (P = .48). CLL with the 17p deletion or TP53 mutation showed very low miR-34a expression. To investigate the mechanisms underlying refractory CLL beyond p53, we studied cases without 17p-/TP53 mutation in detail. In several paired samples before and after F-refractory disease, no change in p21/p53 induction was observed after DNA damage. Although TP53 mutations and 17p deletions are found in a high proportion of F-refractory CLL, more than half of the cases cannot be explained by p53 defects (deletion or mutation), and alternative mechanisms need to be investigated. Alemtuzumab is effective irrespective of genetic high-risk subgroups with TP53 mutations. These clinical trials are registered at www.clinicaltrials.gov as #NCT00274976.

miR-34a as part of the resistance network in chronic lymphocytic leukemia

17p (TP53) deletion identifies patients with chronic lymphocytic leukemia (CLL) who are resistant to chemotherapy. The members of the miR-34 family have been discovered to be direct p53 targets and mediate some of the p53-dependent effects. We studied miR-34a and miR-34b/c expression in a large cohort to define their potential role in refractory CLL. While no expression of miR-34b/c could be detected, we found variable expression levels of miR-34a. miR-34a levels were up-regulated after DNA damage in the presence of functional p53, but not in cases with 17p deletion (P < .001). We found a strong correlation of low miR-34a levels with impaired DNA damage response, TP53 mutations (without 17p deletion), and fludarabine-refractory disease (also in the absence of 17p deletion). Up-regulation of miR-34a after irradiation was associated with induction of Bax and p21, but not Puma. CLL cells with reduced miR-34a expression showed increased viability after DNA damage independently of 17p status. Therefore, low expression of miR-34a in CLL is associated with p53 inactivation but also chemotherapy-refractory disease, impaired DNA damage response, and apoptosis resistance irrespective of 17p deletion/TP53 mutation. The elucidation of mechanisms underlying miR-34a regulation and overcoming its role in chemotherapy resistance warrant further study.

Gene mutations and treatment outcome in chronic lymphocytic leukemia: results from the CLL8 trial

Mutations in TP53, NOTCH1, and SF3B1 were analyzed in the CLL8 study evaluating first-line therapy with fludarabine and cyclophosphamide (FC) or FC with rituximab (FCR) among patients with untreated chronic lymphocytic leukemia (CLL). TP53, NOTCH1, and SF3B1 were mutated in 11.5%, 10.0%, and 18.4% of patients, respectively. NOTCH1(mut) and SF3B1(mut) virtually showed mutual exclusivity (0.6% concurrence), but TP53(mut) was frequently found in NOTCH1(mut) (16.1%) and in SF3B1(mut) (14.0%) patients. There were few significant associations with clinical and laboratory characteristics, but genetic markers had a strong influence on response and survival. In multivariable analyses, an independent prognostic impact was found for FCR, thymidine kinase (TK) ≥10 U/L, unmutated IGHV, 11q deletion, 17p deletion, TP53(mut), and SF3B1(mut) on progression-free survival; and for FCR, age ≥65 years, Eastern Cooperative Oncology Group performance status ≥1, β2-microglobulin ≥3.5 mg/L, TK ≥10 U/L, unmutated IGHV, 17p deletion, and TP53(mut) on overall survival. Notably, predictive marker analysis identified an interaction of NOTCH1 mutational status and treatment in that rituximab failed to improve response and survival in patients with NOTCH1(mut). In conclusion, TP53 and SF3B1 mutations appear among the strongest prognostic markers in CLL patients receiving current-standard first-line therapy. NOTCH1(mut) was identified as a predictive marker for decreased benefit from the addition of rituximab to FC. This study is registered at www.clinicaltrials.gov as #NCT00281918.

TP53 mutation profile in Chronic Lymphocytic Leukemia: Evidence for a disease specific profile from a comprehensive analysis of 268 mutations

The TP53 mutation profile in chronic lymphocytic leukemia (CLL) and the correlation of TP53 mutations with allele status or associated molecular genetics are currently unknown. We performed a large mutation analysis of TP53 at four centers and characterized the pattern of TP53 mutations in CLL. We report on 268 mutations in 254 patients with CLL. Missense mutations appeared in 74% of cases compared with deletions and insertions (20%), nonsense (4%) and splice site (2%) mutations. The majority (243 of 268) of mutations were located in the DNA-binding domain. Transitions were found in 131 of 268 mutations, with only 41 occurring at methylated CpG sites (15%), suggesting that transitions at CpGs are uncommon. The codons most frequently mutated were at positions 175, 179, 248 and 273; in addition, we detected a common 2-nt deletion in the codon 209. Most mutations (199 of 259) were accompanied by deletion of the other allele (17p–). Interestingly, trisomy 12 (without 17p–) was only found in one of 60 cases with TP53 mutation (without 17p–) compared with 60 of 16 in the cohort without mutation (P=0.006). The mutational profile was not different in the cohorts with and without previous therapy, suggesting that the mechanism underlying the development of mutations may be similar, independent of treatment.

A novel Fc-engineered monoclonal antibody to CD37 with enhanced ADCC and high proapoptotic activity for treatment of B-cell malignancies

The tetraspanin CD37 is widely expressed in B-cell malignancies and represents an attractive target for immunotherapy with mAbs. We have chimerized a high-affinity mouse Ab to CD37 and engineered the CH2 domain for improved binding to human Fcγ receptors. The resulting mAb 37.1 showed high intrinsic proapoptotic activity on malignant B cells accompanied by homotypic aggregation. Furthermore, the Ab-mediated high Ab-dependent cell-mediated cytotoxicity (ADCC) on lymphoma and primary CLL cells. mAb 37.1 strongly depleted normal B cells as well as spiked B-lymphoma cells in blood samples from healthy donors as well as malignant B cells in blood from CLL patients. In all assays, mAb 37.1 was superior to rituximab in terms of potency and maximal cell lysis. A single dose of mAb CD37.1 administered to human CD37-transgenic mice resulted in a reversible, dose-dependent reduction of peripheral B cells. In a Ramos mouse model of human B-cell lymphoma, administration of mAb 37.1 strongly suppressed tumor growth. Finally, a surrogate Fc-engineered Ab to macaque CD37, with in vitro proapoptotic and ADCC activities very similar to those of mAb 37.1, induced dose-dependent, reversible B-cell depletion in cynomolgus monkeys. In conclusion, the remarkable preclinical pharmacodynamic and antitumor effects of mAb 37.1 warrant clinical development for B-cell malignancies.

Inflammatory cytokines and signaling pathways are associated with survival of primary chronic lymphocytic leukemia cells in vitro: a dominant role of CCL2

Background Chronic lymphocytic leukemia cells show prolonged survival in vivo, but rapidly die by spontaneous apoptosis in vitro, unless they are co-cultured with stromal cells or non-malignant leukocytes. The objective of this study was to characterize the survival-inducing cross-talk of chronic lymphocytic leukemia cells with their microenvironment to identify novel therapeutic targets. Design and Methods We analyzed and compared microarray-based expression profiles of chronic lymphocytic leukemia cells before and after three different survival-inducing culture conditions: (i) stromal cell co-culture, (ii) stromal cell conditioned medium and (iii) high cell density cultures of unsorted peripheral blood mononuclear cells. Cytokine antibody arrays were applied to study the composition of soluble factors present in these cultures. Results The different survival-supportive culture conditions induced distinct gene expression changes, the majority of which were common to all three conditions. Pathway analyses identified – in addition to known signaling networks in chronic lymphocytic leukemia – novel pathways, of which Toll-like receptor signaling, nuclear respiratory factor-2 (NRF2)-mediated oxidative stress response, and signaling via triggering receptor expressed on myeloid cells-1 (TREM1) were the most relevant. A high proportion of up-regulated genes were inflammatory cytokines, of which chemokine (C-C motif) ligand 2 (CCL2) was shown to be induced in monocytes by the presence of chronic lymphocytic leukemia cells in vitro. In addition, increased serum levels of this chemokine were detected in patients with chronic lymphocytic leukemia. Conclusions Our data provide several lines of evidence that an inflammatory microenvironment is induced in survival-supportive cultures of chronic lymphocytic leukemia cells which might be directly or indirectly involved in the prolonged survival of the malignant cells.

Lenalidomide treatment of chronic lymphocytic leukaemia patients reduces regulatory T cells and induces Th17 T helper cells

Chronic lymphocytic leukaemia (CLL) remains an incurable disease, and the development of novel treatment options is therefore imperative. The immunomodulatory drug lenalidomide has shown promise in early clinical trials (Chanan-Khan & Porter, 2006; Chanan-Khan & Cheson, 2008; Ferrajoli et al, 2008). However, the exact mode of action of lenalidomide is unclear, and development of a ‘tumour flare reaction’ (TFR) is a critical side effect (Andritsos et al, 2008). In order to understand the mode of action of lenalidomide in CLL, we performed an analysis of intra-patient follow-up samples to assess the potential in vivo target cell population that contributes to effects and side effects. We initially investigated whether correlations that were previously reported (Andritsos et al, 2008) between in vitro changes of surface markers on CLL cells and development of TFR could also be found in vivo. We analysed peripheral blood lymphocytes (PBL) from three patients (Table SI) that were treated with lenalidomide (5 mg/d starting dose, escalated by 5 mg/d every 28 d). Two of the patients developed TFR upon therapy (Fig 1). However, the most pronounced upregulation of CD86 and CD40 in vivo was detected in Patient 2 who did not experience a TFR (Fig 2A), while Patient 1 (with a TFR) showed no upregulation of CD40. These findings would suggest that there is no strict correlation of development of a TFR with in vivo induction of CD40 and/or CD86 on CLL cells. This is in contrast to effects of lenalidomide in vitro, which have also been reported previously (Andritsos et al, 2008): treatment resulted in upregulation of CD40 and CD86 in the majority of samples (9/12 and 11/12, respectively, Figure S1). Interestingly this effect was also observed directly on pure CD19 sorted cells (8/12 and 11/12), excluding indirect induction by non-malignant bystander cells (Figure S1). In contrast, only 30–58% of patients experience a TFR after treatment in vivo, depending on lenalidomide dosage (Chanan-Khan & Porter, 2006; Ferrajoli et al, 2008). This makes it unlikely that in vitro upregulation of CD40 and CD86 predicts TFR. Also, in contrast to previous in vitro analyses (Lapalombella et al, 2008), in vivo only one of the patients presented with more CD38 cells during treatment (Patient 1, Fig 2A). A lymph node (LN) aspirate obtained from Patient 3 during TFR however showed twofold more CD38 CLL cells than cells drawn from PBL (data not shown), but no LN aspirate from a timepoint before initiation of treatment was available for comparison. Thus, our findings uncovered interesting differences between the in vitro and in vivo effects of lenalidomide on CLL cells in a small number of patients. Similar to thalidomide, lenalidomide is postulated to impact not only on CLL cells, but also activate and induce proliferation of T cells and natural killer (NK) cells in a mouse model (Hernandez-Ilizaliturri et al, 2005), in healthy volunteers and in patients suffering from multiple myeloma (Chang et al, 2006) and activate them for antibody-dependent cellular cytotoxicity (ADCC) against CLL cells in vitro (Lapalombella et al, 2008). Similarly, we found induction of CD16CD56NK cell numbers in 2/3 CLL patients after treatment with lenalidomide in vivo (Fig 2B). At the same time, the activation status of NK cells decreased as measured by CD69 and CD25 levels. However, it has to be kept in mind that these are early