German CLL Study Group Research Articles

Second-Line Therapies After Treatment with Fludarabine, Cyclophosphamide, and Rituximab (FCR) or Fludarabine and Cyclophosphamid Alone (FC) for Chronic Lymphocytic Leukemia (CLL) within the CLL8-Protocol of the German CLL Study Group (GCLLSG)

Abstract 2863 Introduction:The CLL8-trial is the first study that has shown not only an increase in complete remission rates and progression-free survival, but also an improved overall survival (OS) in physically fit, treatment-naúve CLL-patients (pts) with FCR-chemoimmunotherapy in comparison to FC alone [Hallek et al., Lancet 2010]. Despite this remarkable progress, CLL remains an incurable disease and virtually all pts will eventually relapse. So far, little is known about the efficacy of 2nd-line therapies of these pts. Patients and Methods:Between July 2003 and March 2006 817 pts in good physical fitness as defined by a cumulative illness rating scale (CIRS) score of ≤6 and creatinine clearance 70 ml/min were randomized within the trial and received 6 courses FC (n=409; F: 25mg/m2 i.v. d1–3 and C: 250 mg/m2 i.v. d1–3; q 28 days) without or with rituximab (n=408; 375 mg/m2 i.v. d0 cycle 1, 500 mg/m2 d1 of all subsequent cycles; q 28 days) for 1st-line treatment. Results:As of March 2009, 65% of the patients who had received FCR were free of progression compared to 45% of those who were treated with FC (p<0, 0001) [Hallek et al, Lancet, 2010]. Until July 2010, 232 pts were treated for relapsed CLL, among them 91 of 408 (22%) initially treated with FCR and 141 of 409 (35%) initially treated with FC. In 2nd-line treatment after FCR and FC, the drugs most frequently used either alone or in combination were rituximab (R, 52% of all 2nd-line therapies), fludarabine (F) and bendamustine (B) (21% each), as well as alemtuzumab (A, 12%). The combination of cyclophosphamide, doxorubicin, vincristine, prednisolone and rituximab (CHOP-R) was the most common treatment (35 pts, 15% of all 2nd-line therapies), applied mainly in cases with a relapse ≤24 months after FC/FCR, whereas FCR or BR were administered predominantly in case of relapse >24months (32 and 27 pts, 14% and 12%). Other prevalent 2nd-line therapies were single agent A (20 pts) or B (17 pts), CHOP and FC (11 pts respectively), chlorambucil (9 pts) as well as R monotherapy (7 pts). 9 pts underwent stem cell transplantations. Second-line therapies with FC+/−R and B+/−R were found to be more effective with regard to treatment-free survival (TFS, time to 2nd relapse) and OS when compared to A or CHOP-R and CHOP-like chemotherapies. However, the outcome of 2nd-line therapies seemed to be influenced by the 1st-line treatment. In pts initially treated with FC, FCR was found to be the most effective 2nd-line therapy (TFS: 23 months, OS: not reached), whereas in pts initially treated with FCR, a substitution of the chemotherapeutic agents FC by B seemed justified, as TFS was superior after 2nd-line treatment with B+/−R (16 and 18 months respectively) when compared to FC+/−R (11 and 8 months). Furthermore, in pts who had received FCR for 1st-line treatment, chemotherapy with FC or B was found to be at least equally or even more effective in prolonging OS than FCR or BR (OS calculated from beginning of 2nd-line therapy: FC: not reached, B: 45, FCR: 19, and BR 18 months). Conclusion:Second-line treatments of pts with a relapse after FC or FCR were found to be surprisingly heterogeneous even though the patient collective examined is comparatively homogenous due to the inclusion/exclusion criteria of a clinical trial. As the majority of CLL8-patients is still in remission and has not yet received a 2nd-line treatment, the therapies captured in this analysis are predominantly 2nd-line therapies for earlier relapses. Therefore and because of the short follow-up time, the results ought to be considered as preliminary and descriptive trends. The worse outcome of CHOP-like regimen and A-based therapies in comparison to more established CLL-therapies such as FC+/−R and B+/−R might be related to the fact, that these therapies were administered more often in case of an early relapse after FC/FCR, which is known to be related to other poor prognostic factors [Fink et al, ASH 2010]. Nevertheless, the observation of favorable TFS and OS times after 2nd-line treatment with FC+/−R and B+/−R supports the recommendation to repeat chemoimmunotherapy in case of a relapse >24 months after 1st-line treatment. Further analyses are needed to confirm the observation that chemotherapy (FC or B) without rituximab might be sufficient for for 2nd-line treatment after FCR. Disclosures:Cramer:Mundipharma: Travel Grants. Fink:F. Hoffmann La Roche:. Eichhorst:Hoffmann La Roche: Honoraria, Research Funding, Travel Grants; Mundipharma: Research Funding, Travel Grants; Gilead: Consultancy. Wendtner:Hoffmann La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Travel Grants. Pflug:Hoffmann La Roche:. Hallek:Roche: Consultancy, Honoraria, Research Funding; Mundipharma: Research Funding; Celgene: Honoraria. Fischer:Hoffmann La Roche:.

Prediction of Poor Outcome in CLL Patients Treated with FCR (Fludarabine, Cyclophosphamide, Rituximab) in the CLL8 Trial of the German CLL Study Group (GCLLSG)

Abstract 977 Introduction:For physically fit patients (pts) with chronic lymphocytic leukemia (CLL) the first-line treatment with fludarabine, cyclophosphamide and rituximab (FCR) is the new standard therapy. However, subgroup analyses in the CLL8 trial revealed that patients with a median progression free survival (PFS) of < 24 months after randomization showed a significantly shorter overall survival (OS) compared with pts achieving a PFS of ≥ 24 months. 15% of these patients were characterized by both, the presence of 17p deletions and TP53 gene mutations, another 7.5% by TP53 mutation alone. Interestingly, the majority of patients with a poor prognosis could not be defined by a mutation of p53 or del(17p). Therefore, an effort was made to further characterize the subgroup of patients with poor prognosis. Methods:In 143 patients out of 408 patients who received FCR in the CLL8 trial of the GCLLSG, an assessment of minimal residual disease (MRD) was available at final restaging. These patients were used for this analysis. Results for the primary endpoint PFS, the secondary endpoint OS, and central diagnostics performed for genomic aberrations by FISH and the IGHV gene status as well as for serum parameters before the start of therapy were available for all pts. MRD was determined at final restaging by multi-color flow cytometry from peripheral blood with a sensitivity of at least 10−4. The Kaplan-Meier method and the log-rank test were used to compare PFS and OS in pts with various combinations of risk factors. Results:This patient cohort used for the analysis was representative of the entire FCR population (n=408). There were no significant differences compared with the entire FCR population for age, ECOG status, B-symptoms, Binet or Rai stages, deletion of chromosome 17p, 11q, or 13q, trisomy 12, serum levels for s-TK or s-β2m. A combination of MRD levels of >10−2 or of MRD levels of >10−4 to <10−2plus at least one of the following three parameters (del(17p) or TP53 mutation or an unmutated IGHV-status) defined a group of patients at high risk of early progression (HR). The median PFS of HR pts was 22 months, the median PFS for patients defined as low risk (LR; n=103) was 69 months. HR patients had a 6.4 fold increased risk for progression (HR 6.4 95% CI: 3.970–10.347; p<0.0001) and a 5.7 fold increased risk for death, with a median OS of only 57 months (assessed from the beginning of FCR therapy). In contrast, median OS was not reached in the LR group at the time point of the analyses (HR 5.758, 95%CI:2.799–11.844, p<0.0001). Conclusion:The combined use of genetic markers and an MRD assessment at final restaging allows to identify CLL patients with a very poor outcome after FCR therapy. The high risk group identified by this approach should be treated within clinical trials using novel strategies including maintenance protocols or allogeneic stem cell transplantation. Disclosures:Fink:Hoffmann La Roche: Travel Grants. Pflug:Hoffmann La Roche: Travel Grants. Boettcher:Hoffmann La Roche: Honoraria, Travel Grants. Winkler:Hoffmann La Roche: Travel Grants. Ritgen:Hoffmann La Roche: Travel Grants. Fischer:Hoffmann La Roche: Travel Grants. Eichhorst:Hoffmann La Roche: Honoraria, Travel Grants. Wendtner:Hoffmann La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Travel Grants. Mendila:Hoffmann La Roche: Employment. Wenger:Hoffmann La Roche: Employment. Doehner:Hoffmann La Roche: Honoraria. Kneba:Hoffmann La Roche: Honoraria. Stilgenbauer:Hoffmann La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Travel Grants. Hallek:Hoffmann La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. [Display omitted] [Display omitted]

Prognostic Impact of Biallelic Versus Monoallelic Deletion 13q and the Proportion of Cells with Deletion 13q within the CLL8 Trial of the German CLL Study Group (GCLLSG)

Abstract▪2841▪This icon denotes a clinically relevant abstract Introduction:Genomic aberrations are widely used markers to predict survival of patients with chronic lymphocytic leukemia (CLL). Deletion of 13q as sole abnormality (del 13q) has been associated with a favorable prognosis. However, whether the clinical impact of monoallelic del 13q (mono-del 13q) is different as compared to biallelic del 13q (bi-del 13q), remains controversial. Similarly, it is unsettled, if there is an impact of clone size on outcome of patients with del 13q. Methods:The prognostic impact of mono-del 13q and bi-del 13q, as well as the proportion of cells with del 13q prior therapy was studied in the CLL8 trial of the GCLLSG (1st line FC vs FCR). Pretherapeutic variables, response rates (ORR, CR), progression free survival (PFS), and overall survival (OS) were compared between the groups. Results:224 patients with del 13q as single aberration were analyzed. 189 patients (84%) had a mono-del 13q, and 35 (16%) a bi-del 13q. B-symptoms were observed more frequently in patients with bi-del 13q (63% vs. 38%, P<.01). No significant differences were seen when comparing all other baseline characteristics between the two groups (Binet/Rai stage, ECOG, leukocyte count, sex, levels of thymidine kinase and ß2-mikroglobuline, IGHV mutation status, usage of V3-21 genes and TP53 mutation) and there was no significant difference in ORR (87% vs 89%) and CR (34% vs 37%) rates. However, PFS was significantly longer in patients with bi-del 13q (median not reached vs. 52 months, P=.04; Fig. 1a). When treated with FCR, patients with bi-del 13q had significantly higher CR rates than those with mono-del 13q (60% vs 47%), while there was no significant difference when treated with FC (25% vs 22%). Similarly, FCR resulted in longer PFS in patients with bi-del 13q as compared to mono-del 13q patients (P=.06). Interestingly, this difference was not observed for FC therapy. Regarding OS, there was a trend towards better outcome in patients with bi-del 13q when combining both treatment arms (P=.18, Fig. 1b). Comparison of treatment arms regarding OS was limited due to few events. However, it was of note that not a single death was observed in the group with bi-del 13q when treated with FCR. Interestingly, among bi-del 13q cases, PFS and OS was independent of the IGHV mutation status (P=.78 for PFS, P=.42 for OS). In contrast, patients with mono-del 13q with mutated IGHV genes had a significantly longer PFS and OS than those with unmutated IGHV genes (P<.01 for PFS, P<.01 for OS). [Display omitted] Regarding the clone size of del 13q, there were no significant differences in response rates, PFS and OS in relation to the % of cells with del 13q. This was irrespective of using clone size as a continuous variable, using the median value as cut-point, or separating clone size into quartiles (percentile 25: 42%, median: 76%, percentile 75: 88% del 13q). Median clone sizes in patients achieving a CR, PR, SD or PD were 74% (N=77), 78% (N=119), 74% (N=14) and 70% (N=1), respectively. Median PFS in months was 66, 59, 57 and 39 in quartiles 1–4 (P=.44) and median OS was not reached in either quartile. Conclusion:Although most baseline characteristics were not significantly different when comparing mono-del 13q and bi-del 13q, the presence of a bi-del 13q was associated with significantly longer PFS and a trend towards longer OS after FC-based 1st line treatment in CLL. Bi-del 13q appeared to predict for higher CR rates and longer PFS with FCR as compared to FC treatment. For patients with bi-del 13q, PFS and OS were independent of IGHV mutation status. The size of the aberrant del 13q clone did not impact response rate, PFS or OS. Disclosures:Fischer:Hoffmann La Roche:. Hallek:Hoffmann-la Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Stilgenbauer:Hoffmann La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Travel Grants.

Role of Microenvironment-Associated Chemokines and Cytokines for Binet Stage A CLL Patients Included in a Prospective Trial (CLL1 trial) of the German CLL Study Group (GCLLSG): sIl2Ralpha Is An Independent Predictor of Progression-Free Survival (PFS),

Abstract 3869 Background:In CLL, proliferation of the leukemic cell clone occurs in the bone marrow and lymphatic tissues rather than peripheral blood. In this microenvironment, CLL cells interact with accessory cells, such as T cells and CD68+ nurselike cells (NLCs) and display signs of B cell receptor (BCR) activation, suggesting that CLL proliferation is T cell- and BCR-driven. In addition, cytokines and chemokines secreted by leukemic cells, stromal cells and T cells are essential in forming the disease-specific microenvironment. However, the exact biological role of cytokines and chemokines needs to be defined, especially in the light of distinct prognostic and biological subgroups of patients (pts). Methods:In order to address this issue, we measured serum levels of chemokines and cytokines in a prospective cohort of 157 previously untreated Binet stage A pts., a small subgroup of the risk-stratified CLL1 trial of the GCLLSG. Median follow-up time of that subgroup was 50.3 months. Median time to progression was 61.3 months. Median time from diagnosis to study entry was 1 year. Serum samples had been centrally collected at study entry and stored at −80°C. Sera were analyzed on a luminex-based multiplex platform, allowing simultaneous screening of multiple serum parameters. In a pilot phase, sera of 21 pts were pre-analytically screened for a total of 62 different chemokines. Median serum levels of 27 different chemokines and cytokines were found to differ from healthy controls in a significant manner. Those 27 chemokines and cytokines were subsequently analyzed in 157 pts. For all parameters univariate and multivariate analyses were performed for progression-free survival. Results:Serum levels of CCL3 and CCL4, chemokines known to be secreted by CLL cells upon BCR engagement, were elevated compared to healthy controls. High CCL3 levels correlated strongly with high CCL4 levels (p<0.001) and tended to be associated with unmutated IgHV status (p=0.06), supporting the role of BCR triggering in biologically selected CLL pts. Concerning CCL3 and CCL4, no significant difference in PFS was detected. Serum levels of CCL2 and CCL17, both binding chemokine receptor CCR4, were concordantly elevated compared to healthy controls (p<0.001), suggesting a possible role of chemoattraction of CCR4+ T cells towards these chemokines in CLL. CXCL12, CCL21, and CXCL10, chemokines associated with chemotaxis of CLL cells, were concordantly elevated compared to healthy controls. Pts with serum levels of CCL21 beyond the median also had higher levels of CXCL10 (p<0.001) and CXCL12 (p=0.02). CLL pts have been found to have abnormal neovascularization in the bone marrow and lymph nodes. Elevated VEGF levels were strongly associated with elevated EGF level (p<0.001). High VEGF levels correlated with high white blood cell counts (p=0.008) and showed a trend towards association with del(11q) (p=0.07) and lymphadenopathy (p=0.165). Concerning VEGF, no significant difference in PFS was detected. In contrast, pts with high levels of sIl2R alpha showed a significant shorter PFS. When confirmed by conventional ELISA, median PFS in pts within the highest quartile of sIl2Ralpha levels were 22 months compared to 72 months in the lower three quartiles (p<0.001). When we analyzed sIl2R alpha together with genetic abnormalities like del(11q), trisomy 12, del(13q), del(17p), the risk stratification model used in CLL1 (high risk versus low risk for disease progression), the hierarchical model as published by Döhner et al., and IgHV status in a multivariate Cox regression, we found that sIl2R alpha (OR 2.5, 95% CI 1.4–4.8, p=0.004) and IgHV mutational status (OR 4.0, 95% CI 2.2–7.3, p<0.001) were both independent prognostic variables. Conclusion:The assessment of sera in a small subgroup of the CLL1 study cohort of the GCLLSG revealed that the levels of several chemokines and cytokines were elevated when compared to healthy controls. Median serum levels of different chemokines correlated with distinct biological characteristics of CLL pts, like genetic abnormalities or clinical parameters. In addition, sIl2R alpha could be identified as an independent prognostic variable for PFS in early CLL. Disclosures:Eichhorst:Hoffmann La Roche: Honoraria, Research Funding, Travel Grants; Mundipharma: Research Funding, Travel Grants; Gilead: Consultancy. Stilgenbauer:Hoffmann La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Travel Grants. Hallek:Hoffmann La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Bergmann:Celgene: Honoraria.

Proposal of A Prognostic Score for Previously Untreated Patients with Chronic Lymphocytic Leukemia Based on An Overall Survival Analysis of Three German CLL Study Group Phase III Trials

Abstract▪2831▪This icon denotes a clinically relevant abstract IntroductionPrognosis and need of treatment in CLL is currently determined by clinical staging systems of Binet and Rai. Recent research has focused on prognostic factors that may predict a poor prognosis independent of the clinical stage. Markers which have shown independent prognostic information are serum parameters and genetic factors (genomic aberrations, IgHV and p53 mutational status). To investigate the relevance of these different factors, we performed a pooled analysis using the data of three multicenter German CLL Study Group phase III trials (CLL1, CLL4 and CLL8). Based on this analysis we propose a prognostic score for previously untreated patients with early and advanced CLL. Material and MethodsPatients were recruited between 1997 and 2006 into three phase III trials: 715 in CLL1 (“watch and wait” versus fludarabine (F)), 362 in CLL4 (F versus F and cyclophosphamide (FC)) and 817 patients in the CLL8 trial (FC versus FC and rituximab (FCR)). Serum parameters and genetic factors were centrally analyzed prior to treatment. The main end point of all statistical analyses was overall survival. First, univariate analyses were performed including variables of different groups such as baseline characteristics, stage of disease, laboratory results, molecular cytogenetics, mutational status and serum parameters. Next, multivariate Cox regressions were applied including all parameters that showed a significant association with overall survival in univariate analyses. To create a prognostic score we developed a weighted grading algorithm for independent factors based on ranges of hazard ratios. Finally, a prognostic score was defined as the sum of single ratings of adverse factors. According to this score, four different risk groups for overall survival could be identified. ResultsIn total 1948 patients were eligible for the pooled analysis with a median age of 60 years (range, 30 to 81 years). After a median observation time of 63.4 months 485 deaths were reported. At study entry, 799 patients (42.4%) were at Binet stage A, 717 (38.0%) at Binet stage B and 370 (19.6%) at Binet stage C. Almost all considered variables were significantly associated with outcome and therefore included in the multivariate analysis. Based on the data of 1223 patients for whom all parameters were available, multivariate Cox regressions were performed and identified gender, age, ECOG score, del(17p), del(11q), IgHV mutational status, serum β2-microglobulin and serum thymidine kinase as independent factors for overall survival. Deletion 17p was the strongest adverse factor. Neither the clinical staging (Rai, Binet) nor the treatment modality were independent prognostic factors for overall survival. Similarly, the time interval between first diagnosis and study entry was not an independent prognostic factor. Due to the great differences between hazard ratios of independent factors, we developed a weighted grading system based on a simple algorithm to assign an individual grade to each adverse factor. By using this weighted grading, four different prognostic groups could be separated: low risk (score 0 – 2), intermediate risk (score 3 – 5), high risk (score 6 – 10) and very high risk (score 11 – 14) (figure 1). Overall survival rates were significantly different for these four groups with 95.2%, 86.9%, 67.7% and 18.7% survival after 5 years for the low, intermediate, high and very high risk group, respectively (p<0.0001). Moreover, within the group of patients showing a deletion 17p the score could distinguish patients of a high risk and a very high risk group (p<0.0001). Finally, the score could predict the individual risk for short overall survival independent of and within the different Binet or Rai stages (p<0.0001) (figure 2). ConclusionWhile Binet and Rai staging systems may remain important for the initial clinical assessment due to their simplicity, our prognostic score using a weighted combination of genetic and serum markers is superior to predict the overall survival of CLL patients. [Display omitted] [Display omitted] Disclosures:Pflug:Hoffmann-la Roche: Travel grant; Mundipharma: Travel grant. Eichhorst:Hoffmann La Roche: Honoraria, Research Funding, Travel Grants; Mundipharma: Research Funding, Travel Grants; Gilead: Consultancy. Bergmann:Celgene: Honoraria. Döhner:Hoffmann-la Roche: Research Funding. Stilgenbauer:Hoffmann La Roche: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Travel Grants. Fischer:Hoffmann La Roche: Travel Grants. Hallek:Hoffmann-la Roche: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees.

The detection of minimal residual disease status in chronic lymphocytic leukemia

In this issue of Clinical Cytometry, Durrieu et al. on behalf of GEIL-GOELAMS present evidence that circulating levels of CD19+ CD5+ chronic lymphocytic leukemia (CLL)-like cells can be used to define a threshold for the detection of minimal residual disease (MRD) in CLL (this issue: page 346). This study was performed in 16 different hematology laboratories or services, and the data is analyzed centrally. The panel consisted of five tubes of four reagents each with CD19 and CD5 being present in each tube. They determined that the level of two CLL-like immunophenotypes CD19+/CD5+/CD43int/CD79blo and CD19+/ CD5+/CD81lo/CD22lo were less than 4 × 10−4 per WBC in unwashed, lyzed normal whole blood. Levels greater than this found in post treatment CLL samples were taken to signify the presence of MRD. The author further state that they are unable to determine monoclonality using light chain restriction due to their polyclonal nature in normal blood donors and the presence of cytophilic antibodies being present in the unwashed, lyzed method. And that bright CD5+ B cells are often lambda positive, and polyclonal. I have several preferences (biases) in this matter, and they include the use of CD20 rather than CD19 or preferably both CD20 and CD19 in combination to define B cells and the clone, demonstration of monoclonality or monotypia, and that reports be based on the absolute cell-count (WBC or absolute lymphocyte count ALC) and not just the percentage < or > 10−4. If the WBC is used, it should be used in conjunction with the ALC and even a B cell absolute count. I sympathize with the problem of cytophilic antibodies but in monoclonal B cell lymphocytosis studies, values of 5–50 light chain restricted B cells per microliter can be routinely detected (1). I was also surprised to see that the lower range of 1–3% CD5+ B cells seems to be the norm, whereas values of 10–25% seem to be more the rule. The European Research Initiative on CLL (ERIC) has published a standardized four-color approach to MRD detection in CLL (2). Both the authors of that study and this study find a WBC threshold of 10−4 below which is considered MRD negativity. The ERIC study allows for the primary role of kappa lambda tube for historical purposes but favors the combination of CD20/CD38/CD19/CD5 over the combinations of CD43/CD79b and CD81/CD22. They further point out the need for the evaluation of gate contamination, number of total events collected (500,000), and operator training. Certain precautions are required for differentiating between blood and marrow involvement in the setting of antibody therapies. However, due to the correlation of MRD between blood and marrow, blood should be the first site tested for MRD. The complete absence of B cells (B cell aplasia) might also be taken as a clinical measure of MRD. If ascertainment of marrow involvement in this setting were considered important, a bone marrow study would be indicated. Conversely, the timing of the blood MRD analysis could be postponed. The German CLL Study Group have also standardized three four tube panels of four-color reagents using a larger volume of blood, and where appropriate 2 million leucocytes were collected (3). This paper also shows that the overall ability of MRD flow is to detect and quantify residual CLL cells and is not affected by the addition of rituximab to FC chemotherapy (3). In a 2005 editorial, Montserrat (4) called for standardization and revision of 1996 NCI response guidelines to include MRD negative status as a goal. In 2008, Hallek et al. (5) revised the NCI guidelines to include not only a definition of MRD but also encouraged that the MRD detection become part of future CLL clinical trials because MRD negative status has prognostic impact. The GCLLSC, ERIC, and GEIL studies have all demonstrated that multicolor flow cytometry can meet the needs of a robust MRD assay. All three groups used a four-color, five tube panel and a sequential gating strategy. Newer clinical flow cytometers now allow for the use of 6–8 colors. The Salamanca group has developed an automated method for doing MRD detection by merging files on the same patient with the addition normal donor virtual 1:1 dilution as a control (6). Knowledge of the original CLL immunophenotype would be useful. One could envision an integration of the GCLLSC, ERIC, and GEIL methods combining CD19/CD20/CD5/CD38/CD79b/ CD22/CD41/CD81 and CD19/CD20/CD5/CD45/CD3/CD14/kappa/lambda. Del Poeta et al. (7) concluded that the addition of rituximab consolidation and maintenance therapy prolonged response duration significantly in patients with MRD + CLL. MRD detection is indicated in the investigation, care, and treatment of the CLL patient. Clinical trials will be required to answer the question of overall survival in CLL. Regardless, CLL remains a model disease for the appropriate treatment of the elderly with progressive disease.

5.17 Chemoimmunotherapy Improves Survival of Patients with Chronic Lymphocytic Leukemia

Abstract 3908 INTRODUCTION Chemoimmunotherapy, namely the combination of fludarabine, cyclophosphamide and rituximab (FCR), results in a higher response rate and a longer progression-free survival in patients with chronic lymphocytic leukemia (CLL). There is also evidence coming from several historical comparisons, observational, and epidemiological studies and, above all, a large randomized clinical trial (German CLL Study Group – Hallek M et al , Lancet 2010) that FCR prolongs survival. Since additional randomized studies to validate the superiority of FCR over older therapies in CLL are difficult to envisage, other approaches to confirm this observation are worth to be considered. AIM The aim of this study was to ascertain whether chemoimmunotherapy given at any time over the course of the disease, independently of the treatment phase, prolonged survival in a group of unselected patients with CLL from the Hospital Clinic of Barcelona. MATERIALS AND METHODS Out of 1042 consecutive patients diagnosed and followed-up from 1980 to December 2010, we selected 484 patients who received at some point of their disease evolution: (1) alkylating agents and no purine analogs (PA) nor rituximab (R) ( no PA no R ) (n=211; of which 67 PA ) (n=159). (3) PA plus rituximab ( PA+R ) (n=114). Clinical information (age, sex, Binet stage) and laboratory characteristics (β2-microglobulin, CD38, ZAP-70, genomic alterations, IGHV mutational status) at disease presentation, treatment, and follow-up was obtained from a database prospectively managed at our institution from the 1970s onwards. All treated patients were included in the analysis regardless of the number of cycles of therapy given and independently of whether they had participated in clinical trials or not. RESULTS The median age (range) of the whole series (n=340, 222M/118F) was 56 (24–84) years. The three groups were well balanced for key clinical characteristics such as age, sex, and Binet stage at treatment. It should be noted that patients older than 65 years were initially excluded from the analysis to avoid a bias in the results due to the general worse prognosis of older patients. On the other hand, patients from the PA and PA+R groups presented poorer risk prognosis factors such as a higher expression of CD38 (p=0.006), ZAP-70 (p=0.052), and adverse cytogenetic abnormalities (p=0.026) or unmutated IGHV genes (p=0.005) than those in the no PA no R group. After a median follow up of 9.4 years (range, 0.3–21), 148 (44%) patients remain alive. At 10 years, the overall survival of the PA+R group was 65% (95% CI, 53–77%) compared with 43% (35–51%) and 43% (31–54%) for the no PA no R and PA groups, respectively (p When all patients who did not receive PA or R were included in the study (n=211), the statistically significant difference was, not surprisingly, maintained (data not shown). In addition, patients from both the PA group and the PA+R group had poorer prognostic features. These data are in keeping with a conservative therapeutic approach in patients with low-risk disease and a poorer risk of patients treated with PA with or without R. This is also an additional argument in favor of the effectiveness of chemoimmunotherapy in high-risk patients. Interestingly, when survival of patients treated with PA+R was analyzed according to the time at which treatment was administered (first line, n=55 vs. ≥ second line, n=59), no differences were observed (p= 0.8) (Figure 2) CONCLUSION Chemoimmunotherapy prolongs survival of patients with CLL. Moreover, this effect could be independent of the phase of the disease at which chemoimmunotherapy is given. Disclosures: No relevant conflicts of interest to declare.

4.13 Proposal of a New Prognostic Score for Previously Untreated Patients with Chronic Lymphocytic Leukemia Based on an Overall Survival Analysis of Three German CLL Study Group Phase III Trials

Abstract 2831 Introduction Prognosis and need of treatment in CLL is currently determined by clinical staging systems of Binet and Rai. Recent research has focused on prognostic factors that may predict a poor prognosis independent of the clinical stage. Markers which have shown independent prognostic information are serum parameters and genetic factors (genomic aberrations, IgHV and p53 mutational status). To investigate the relevance of these different factors, we performed a pooled analysis using the data of three multicenter German CLL Study Group phase III trials (CLL1, CLL4 and CLL8). Based on this analysis we propose a prognostic score for previously untreated patients with early and advanced CLL. Material and Methods Patients were recruited between 1997 and 2006 into three phase III trials: 715 in CLL1 (“watch and wait” versus fludarabine (F)), 362 in CLL4 (F versus F and cyclophosphamide (FC)) and 817 patients in the CLL8 trial (FC versus FC and rituximab (FCR)). Serum parameters and genetic factors were centrally analyzed prior to treatment. The main end point of all statistical analyses was overall survival. First, univariate analyses were performed including variables of different groups such as baseline characteristics, stage of disease, laboratory results, molecular cytogenetics, mutational status and serum parameters. Next, multivariate Cox regressions were applied including all parameters that showed a significant association with overall survival in univariate analyses. To create a prognostic score we developed a weighted grading algorithm for independent factors based on ranges of hazard ratios. Finally, a prognostic score was defined as the sum of single ratings of adverse factors. According to this score, four different risk groups for overall survival could be identified. Results In total 1948 patients were eligible for the pooled analysis with a median age of 60 years (range, 30 to 81 years). After a median observation time of 63.4 months 485 deaths were reported. At study entry, 799 patients (42.4%) were at Binet stage A, 717 (38.0%) at Binet stage B and 370 (19.6%) at Binet stage C. Almost all considered variables were significantly associated with outcome and therefore included in the multivariate analysis. Based on the data of 1223 patients for whom all parameters were available, multivariate Cox regressions were performed and identified gender, age, ECOG score, del(17p), del(11q), IgHV mutational status, serum β 2 -microglobulin and serum thymidine kinase as independent factors for overall survival. Deletion 17p was the strongest adverse factor. Neither the clinical staging (Rai, Binet) nor the treatment modality were independent prognostic factors for overall survival. Similarly, the time interval between first diagnosis and study entry was not an independent prognostic factor. Due to the great differences between hazard ratios of independent factors, we developed a weighted grading system based on a simple algorithm to assign an individual grade to each adverse factor. By using this weighted grading, four different prognostic groups could be separated: low risk (score 0 – 2), intermediate risk (score 3 – 5), high risk (score 6 – 10) and very high risk (score 11 – 14) (figure 1). Overall survival rates were significantly different for these four groups with 95.2%, 86.9%, 67.7% and 18.7% survival after 5 years for the low, intermediate, high and very high risk group, respectively (p Conclusion While Binet and Rai staging systems may remain important for the initial clinical assessment due to their simplicity, our prognostic score using a weighted combination of genetic and serum markers is superior to predict the overall survival of CLL patients. Disclosures: Pflug: Hoffmann-la Roche: Travel grant; Mundipharma: Travel grant. Eichhorst: Hoffmann La Roche: Honoraria, Research Funding, Travel Grants; Mundipharma: Research Funding, Travel Grants; Gilead: Consultancy. Bergmann: Celgene: Honoraria. Dohner: Hoffmann-la Roche: Research Funding. Stilgenbauer: Hoffmann La Roche: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Travel Grants. Fischer: Hoffmann La Roche: Travel Grants. Hallek: Hoffmann-la Roche: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees.

4.19 Prediction of Poor Outcome in Chronic Lymphocytic Leukemia Patients treated with FCR (Fludarabine, Cyclophosphamide, Rituximab) in the CLL8 Trial of the German CLL Study Group

4.19 Prediction of Poor Outcome in Chronic Lymphocytic Leukemia Patients treated with FCR (Fludarabine, Cyclophosphamide, Rituximab) in the CLL8 Trial of the German CLL Study Group

Progress in the treatment of chronic lymphocytic leukemia: results of the German CLL8 trial

Until now, no approach that is able to improve overall survival of chronic lymphocytic leukemia (CLL) patients has been available. In the German CLL Study Group (GCLLSG) CLL8 trial, treatment-naive, physically fit patients (aged 30–81 years) with CD20+ CLL were randomly assigned in a one-to-one ratio to receive six courses of intravenous fludarabine (25 mg/m2 per day) and cyclophosphamide (250 mg/m2 per day) for the first 3 days of each 28-day treatment course with or without rituximab (375 mg/m2 on day 0 of first course and 500 mg/m2 on day 1 of second to sixth courses). The two groups were well balanced with respect to baseline characteristics. The study was stopped at the preplanned interim analysis owing to an advantage in the median progression-free survival in the chemoimmunotherapy arm (51.8 vs 32.8 months; hazard ratio: 0.56; 95% CI: 0.46–0.69; p < 0.0001). Furthermore, at 3 years after randomization, 87% of patients in the chemoimmunotherapy group were alive compared with 83% in the chemotherapy group (hazard ratio: 0.67; 95% CI: 0.48–0.92; p = 0.01). In terms of toxicity, chemoimmunotherapy was more frequently associated with grade 3 and 4 neutropenia (p < 0.0001). There were eight (2%) treatment related-deaths in the chemoimmunotherapy arm compared with ten (3%) in the chemotherapy arm. The CLL8 trial has demonstrated that an association of rituximab, fludarabine and cyclophosphamide is effective in prolonging progression-free survival and overall survival of patients with symptomatic CLL, therefore establishing the new standard of treatment for physically fit patients.

Limited clinical relevance of imaging techniques in the follow-up of patients with advanced chronic lymphocytic leukemia: results of a meta-analysis

AbstractThe clinical value of imaging is well established for the follow-up of many lymphoid malignancies but not for chronic lymphocytic leukemia (CLL). A meta-analysis was performed with the dataset of 3 German CLL Study Group phase 3 trials (CLL4, CLL5, and CLL8) that included 1372 patients receiving first-line therapy for CLL. Response as well as progression during follow-up was reassessed according to the National Cancer Institute Working Group1996 criteria. A total of 481 events were counted as progressive disease during treatment or follow-up. Of these, 372 progressions (77%) were detected by clinical symptoms or blood counts. Computed tomography (CT) scans or ultrasound were relevant in 44 and 29 cases (9% and 6%), respectively. The decision for relapse treatment was determined by CT scan or ultrasound results in only 2 of 176 patients (1%). CT scan results had an impact on the prognosis of patients in complete remission only after the administration of conventional chemotherapy but not after chemoimmunotherapy. In conclusion, physical examination and blood count remain the methods of choice for staging and clinical follow-up of patients with CLL as recommended by the International Workshop on Chronic Lymphocytic Leukemia 2008 guidelines. These trials are registered at http://www.isrctn.org as ISRCTN 75653261 and ISRCTN 36294212 and at http://www.clinicaltrials.gov as NCT00281918.

IGHV-Mutation Status, IGHV-Gene Usage and Chromosomal Aberrations In CLL: Pooled Analysis within First-Line Clinical Trials of the German CLL Study Group (GCLLSG)

AbstractAbstract 3609 Introduction:IGHV mutation status and genomic aberrations are of independent prognostic importance in CLL. Furthermore IGHV-gene usage showed prognostic value for distinct subgroups (e.g. VH3-21). While the introduction of chemoimmunotherapy (i.e. FCR) has led to remarkable improvement of outcome in CLL, it is unclear which of the known genetic markers retain their prognostic value. We therefore performed a pooled analysis of first-line CLL patients from 3 prospective clinical trials of the GCLLSG namely “CLL2M” (R-Bendamustine) “CLL4” (F vs. FC) and “CLL8” (FC vs. R-FC) to evaluate those genetic markers in a large, well characterized CLL cohort.Material and Methods: Genetic characterization was performed in a central laboratory (Ulm). IGHV data was available for 1063 patients. Genomic aberrations (FISH) were generated for 1053 patients. After pooling genetic data from the outlined GCLLSG first-line trials we investigated the impact of IGHV mutation status and VH gene usage within the hierarchical model of genomic abnormalities (deletion 13q (del(13q)) single (n=353), trisomy 12 (n=115), deletion 11q (del(11q)) (n=229), deletion 17p (del(17p)) (n=73)) concerning, progression free survival (PFS events= PD/death) and overall survival (OS). Median follow-up was 39.1 months. Results:For the del(13q) patients, with 56 % carrying a mutated IGHV gene, we found a significant difference in PFS and OS between the del(13q)/IGHV mutated and the IGHV unmutated subgroup (PFS: p-value 0.002; HR: 1.596; OS: p-value 0.002; HR: 2.15; median PFS mut./70.5 months vs. unmut./41.5 months).In the trisomy 12 subgroup 35 % of patients showed a mutated IGHV whereas 65 % showed an unmutated IGHV. For the trisomy12/IGHV unmutated subgroup we could detect a trend towards a shorter PFS and OS compared to the trisomy12/IGHV mutated group although this was not statistically significant (PFS: p-value 0.059; HR: 1.784; OS: p-value 0.079; HR: 3.05; median PFS not reached vs. 36.8 months).In the del(11q) subgroup 17 % were IGHV mutated, whereas 83 % were unmutated. For none of the clinical endpoints (PFS, OS) a significant difference between the 11q-/IGHV mutated group and the 11q-/IGHV unmutated group could be detected (PFS: p-value 0.451; OS: p-value 0.64; median PFS 32.7 months for both subgroups).Finally among the del(17p) pts. with 23 % IGHV-mutated cases we could not find any difference in PFS or OS among the IGHV-mutated or unmutated subgroup (PFS: p-value 0.995; HR: 0.998; OS: p-value 0.584; HR: 0.80, median PFS 8.8 vs. 9.6 months).In contrast to previous findings we detected no significant difference in clinical behaviour of the mutated VH3-23 subgroup in comparison to all non-VH3-23 mutated cases with regard to PFS and OS within the CLL8 cohort. (median PFS not reached vs. 59.7 months).For unmutated VH1-69 cases no significant differences concerning the clinical endpoints in comparison to all non-VH1-69 unmutated cases were detected. Finally the 66 VH3-21 patients in the pooled data set showed a PFS and OS which was surprisingly more comparable to that of IGHV-mutated cases (PFS: p-value 0.488; OS: p-value 0.467; median PFS 51.9 months for VH3-21 vs. 63.6 months for mutated non-VH3-21). However, stereotyped HCDR3 motifs have not been considered in this analysis. Conclusions:The current data were derived from pooled subgroup analyses based on genomic aberrations among patients enrolled in 1st line treatment trials (i.e. patients requiring treatment). It revealed a differential influence of IGHV-mutation status and gene usage in the different genomic subgroups. This might hint to different biological behaviour apart from postulated mechanisms like B cell receptor mediated antigen drive and could also be reflected by differences in clinical course. The impact of VH3-21 usage and potentially other VH genes appears to be less pronounced in this cohort. The prognostic role of distinct IGHV-genes must be further evaluated in different clinical situations also considering stereotyped B-cell receptors. [Display omitted] Disclosures:Bühler:Roche Pharma: Consultancy, Research Funding. Zenz:Roche: Honoraria; Boehringer: Honoraria; GSK: Honoraria; Celgene: Honoraria. Winkler:Roche Pharma AG: Consultancy, Research Funding. Fischer:Roche Pharma AG: Consultancy. Fink:Roche Pharma AG: Consultancy. Wendtner: Celgene, BayerSchering, Roche, Mundipharma: Consultancy, Honoraria. Eichhorst:Roche Pharma AG: Consultancy. Edelmann:Roche Pharma AG: Consultancy, Research Funding. Hallek:Roche Pharma AG: Consultancy. Stilgenbauer:Roche, Bayer, Celgene, GSK, Amgen, Mundipharma: Consultancy, Honoraria, Research Funding.

Chemoimmunotherapy with Chlorambucil and the Type II CD20-Antibody GA101 In Patients with Chronic Lymphocytic Leukemia and Comorbidity: Results of the Run-In Phase of the CLL11 (BO21004) Trial

AbstractAbstract 1387 Introduction:Chemoimmunotherapy with purine analogues and the CD20-antibody rituximab is the standard of care in younger and physically fit patients with chronic lymphocytic leukemia (CLL). However, the majority of CLL patients are of advanced age and many of those are afflicted with comorbidity. To date, there is no established standard treatment for such patients and the alkylating drug chlorambucil remains a valuable treatment option. The addition of CD20-antibodies (including the novel type II antibody GA101) to chlorambucil (CLB) has been proposed to improve alkylator-based therapy in CLL. Whether these regimens are beneficial in CLL patients with comorbidity remains to be explored. The German CLL Study Group (GCLLSG) in collaboration with F. Hoffmann-La Roche Ltd has initiated the multicenter international 3-armed CLL11 trial comparing CLB alone with CLB plus rituximab (R-CLB) or GA101 (G-CLB) in comorbid CLL patients. Here, we present the results of six elderly and comorbid CLL patients having received G-CLB treatment within the CLL11 safety run-in phase. Patients:Prior to the opening of the randomized phase of the CLL11 trial, six patients with previously untreated CLL in need of treatment and with a cumulative illness rating scale (CIRS) total score > 6 and/or a creatinine clearance (CrCl) < 70 ml/min were enrolled to receive 6 cycles of G-CLB. Chlorambucil was given orally at 0.5 mg/kgBW on d1 and d15 of each cycle (QW 28d). GA101 was administered intravenously on d1, d8 and d15 of cycle 1 and on d1 for cycles 2–6. Occurrence of (i) one treatment-related death or (ii) three episodes of febrile neutropenia/infections requiring antibiotic therapy during the first cycle were defined as stopping criteria for the trial. Results:All patients (2 females, 4 males) had CD20-positive CLL and active disease according to NCI/IWCLL criteria. The patients' age, CrCl and CIRS total score ranged between 71 and 79 years (median: 76 years), 38 and 83 ml/min (median: 51 ml/min), and from 5 to 9 (median: 8), respectively. So far, 4 patients completed therapy while 2 patients are still on treatment (both due to treatment delays). Therapy had to be delayed in 3 patients, mainly due to neutropenic episodes but also caused by a mechanical ileus unrelated to treatment and requiring surgical intervention and by a psychotic disorder. None of the patients have been withdrawn from treatment to date. Infusion-related reactions occurred in 5 patients, but were mild (grade 1–2) in all cases. NCI CTC grade 3 or 4 neutropenias were seen in 5 patients; 3 patients received G-CSF with immediate response. One patient had grade 3–4 thrombocytopenia while none of the patients developed grade 3–4 anemia. No grade 3–4 infections were observed and there were no febrile neutropenias or infections in the presence of neutropenia requiring antibiotic therapy. All 6 patients showed rapid clearing of lymphocytes from their peripheral blood within a few days of first dosing with G-CLB (response rates at the end-of-treatment will be reported). Conclusion:Chemoimmunotherapy with CLB and GA101 appears to be feasible in CLL patients of advanced age and with high burden of comorbidity. However, neutropenia as well as complications associated with comorbidity need special consideration in this very fragile patient population. None of the stopping criteria defined for the CLL11 run-in phase were met. The CLL11 trial therefore was opened for randomization in April 2010. Disclosures:Goede:Roche: Honoraria, Travel Grants. Fischer:Roche: Travel Grants. Fink:Roche: Travel Grants. Bieska:Roche: Employment. Humphrey:Roche: Employment. Bishop:Roche: Employment. Wenger:Roche: Employment. Hallek:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Serum Factors Predict Therapeutic Outcome In Patients with Chronic Lymphocytic Leukemia Treated In the CLL8 Trial of the German CLL Study Group (GCLLSG)

AbstractAbstract 918 Introduction:Various factors are used to predict the clinical course of chronic lymphocytic leukemia (CLL). Here we evaluated the importance of the serum markers soluble CD23 (sCD23), β2-microglobulin (s-β2m) and thymidine kinase (s-TK) compared to other prognostic markers in patients (pts) receiving a first-line therapy with fludarabine and cyclophosphamide (FC) or FC plus rituximab (FCR) in the CLL8 trial of the GCLLSG. Methods:The analysis of the serum factors was centrally performed prior to treatment. Kaplan-Meier method and the log-rank test were used to compare progression-free survival (PFS) and overall survival (OS). A Cox regression model was applied for the multivariate analysis and included the following parameters: age, sex, disease stage, time from first diagnosis, ECOG performance status, presence of B-symptoms, white blood cell count, sCD23, s-TK and s-β2m genomic aberrations, IGHV mutational status, and type of therapy (FC versus FCR). Cut-off values for s-β2m and for s-TK were 3.5 mg/l and 10 U/l, respectively. Results:Since exploratory analyses suggested a particular role for sCD23, the contribution of sCD23 was examined in more detail. Data for sCD23 was available for 616 pts. This cohort was representative of the full trial population (n=817) with respect to baseline characteristics. Results for response rates, PFS, OS and sCD23 were available for 574 pts. Based on their sCD23 levels, pts were categorized by quartiles: 139 pts were assigned to group A (≤2807.5 U/mL), 145 to group B (>2807.5 U/mL -≤5507 U/mL), 146 to group C (>5507 U/mL-≤10920.5 U/mL) and 144 to group D (>10920.5 U/mL). Significant differences were observed with respect to complete remissions (CR): Pts of group A achieved a double fold CR rate (48.2%) compared to pts of group D (23.6%; p<0.001). Correspondingly, group D had the highest rate of non-responses (11.8%). PFS and OS were longest for pts with low serum levels for sCD23: The median PFS in group A was 51.9 months (mo) compared to 29.8 mo in group D (p<0.001). The median OS for group D was 62.5 mo, but was not reached in the other groups.Next multivariate analyses (Table1) were performed and showed that s-TK, 17p-deletion (del(17p)) and the type of therapy were independent predictors of PFS and OS. del(17p) was the strongest adverse factor for both PFS and OS. sCD23 and IGHV were independent prognostic factors for PFS but not for OS. s-ß2m, age and ECOG status independently predicted OS.Finally, we investigated whether combinations of different serum markers would define distinct risk groups. Using the three serum markers, four risk categories were defined: Low risk (LR) = no serum factor elevation, intermediate risk (IR) = either s-ß2m or s-TK high, but sCD23 low, high risk (HR) = high s-ß2m and s-TK, but low sCD23, and very high risk (VHR) = high sCD23, regardless of s-ß2m and s-TK. Median PFS was significantly different for the four groups, with 59.7, 49.3, 35 and 29.8 months for LR, IR, HR, and VHR patients respectively (p<0.001). However, the difference between HR and VHR was not statistically significant (p=0.2). Conclusion:Elevated serum levels of s-ß2m, s-TK, and sCD23 predict poor outcome after first-line therapy with FC or FCR in pts with CLL, independently of other features such as IGHV status or del(17p). Measurement of sCD23 in addition to s-TK and s-ß2m seems to allow a more precise prediction of PFS.Table 1Multivariate analysis of the effects of various prognostic parameters on PFS and OSPFS n=504OS n=515ParameterHazard ratio95% CIp-valueHazard ratio95% CIp valuesCD23 (>10920.5 U/mL)1.3401.020–1.7610.036–––s-TK ≥10.0 U/L1.4541.052–2.0090.0232.0071.085–3,7110.026s-β2m ≥ 3.5mg/L–––1.7261.123–2.6520.013del(17p)6.4304.154–9.952<0.0018.6604.841–15.490<0.001IGHV unmutated1.4111.035–1.9220.029–––Age–––0.6460.419–0.9950.047ECOG pre ≥ 1–––1.8681.239–2.8160.003FCR treatment0.5060.393–0.651<0.0010.6710.447–1.0080.05 Disclosures:Fink:Roche: . Pflug:Roche: Travel grants. Eichhorst:Roche: Research Funding, Travel grants. Wendtner:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Travel grants. Mendila:Roche: Employment. Wenger:Roche: Employment. Stilgenbauer:Amgen: Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Sanofi Aventis: Research Funding. Hallek:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Fischer:Roche: Research Funding, Travel grants.

High-Resolution SNP-Array Profiling of Chronic Lymphocytic Leukemia

AbstractAbstract 50Genomic aberrations are important prognostic factors in chronic lymphocytic leukemia (CLL) [Döhner et al., 2000]. However, known genomic aberrations fail to fully explain the biologic and clinical heterogeneity of the disease. We sought to precisely map copy number alterations (CNA) and copy number neutral losses of heterozygocity (LOH) to better characterize known recurrent aberrations and to identify new genetic lesions.We used Affymetrix 6.0 single nucleotide polymorphism (SNP) array analyses on CD19 sorted CLL cells. Data were analyzed using dChipSNP, a modified array normalization algorithm guided by cytogenetic abnormalities and a circular binary segmentation. We studied samples from 346 patients enrolled on the CLL8 trial of the German CLL Study Group. Data of 145 samples were analyzed against intraindividual reference DNA (paired), data of 201 samples against a pool of reference DNA (unpaired). FISH data were available for all samples, the distribution of genomic aberrations was as follows: del(13q14) in 59.8%, del(11q23) in 26.3%, trisomy 12 in 11.6%, and del(17p13) in 8.4%. IGHV was mutated in 32.9%, unmutated in 63.3%, and unknown in 3.8%.In total, 261 tumor-specific CNA could be discovered among the 145 paired samples. Genomic aberrations were found in 85.5% of these cases. The average number of aberrations per case was 1.8; according to the hierarchical model of genomic aberrations, it was 3.5 in del(17p), 2.4 in del(11q23), 1.7 in del(13q14) single, and 0.5 in normal karyotype CLL. The minimally deleted region (MDR) on 13q14 was 277.25 kb in size and contained mir15a and mir16, DLEU1 and DLEU2; RFP2 was not part of the MDR. Deletions on 13q were highly heterogeneous in size, ranging from 294 kb to 68 Mb. On 11q23 the MDR only contained ATM, the smallest lesion of 78.5 kb being intragenic; in two of theses cases, the deletion size was too small to be detected by FISH analysis. TP53 was affected in all del(17p13) cases except two; one tumor-specific deletion of 635.7 kb was detected in cytoband 17p13.2 harboring 30 genes and a second deletion of 780 kb in 17p13.3 containing – among 15 other genes – MNT, a tumor suppressor acting as an antagonist of MYC. A partial trisomy on chromosome 12 was not detected. Of the 261 CNA, 95 were located in genomic regions that are not evaluated by our routine FISH probe panel; 17 regions were affected recurrently: del(1p35.3) [2/145], del(1q23.3) [2/145], del(1q42.12) [2/145], +2p [5/145], del(3p21.31) [2/145], del(6p25.3) [3/145], +(6p25.3) [2/145], del(6q) [11/145], del(7q23.1) [2/145], +(8q24.21) [3/145], del(9q13-q21.13) [2/145], del(10q24) [2/145], del(14q24.3) [2/145], del(14q12.3) [2/145], del(15q15.1) [2/145], +18 [3/145] and +19 [7/145]. The frequency of these CNA was subsequently evaluated within the cohort of 201 (unpaired) samples. Five of 17 regions were affected in more than 2% in the whole cohort: +2p, del(6q), +8q24.21, del(15q15.1), and +19. Gain of 2p was found in 6.9% of cases, the minimally amplified region was 1.9 Mb in size and contained e.g. BCL11A and REL. Del(6q) was detected in 6,4%, the deletions were heterogeneous, an MDR could not be identified. 16 cases had 8q24.21 gains, the minimally amplified region was delineated by three intragenic gains in MYC. 14 cases had loss in 15q15.1 focussing on MGA, a potential suppressor of transcriptional activation by MYC. 8 cases had total or partial gains of chromosome 19, among those two overlapping partial gains with a minimally amplified region of 2.17 Mb in 19p13.2. Tumor-specific LOH were identified in 6.0% (9/145) located on 13q in three cases and in one case each on 17p, 12q, 11p, 1p, 3 and 22q. The LOH on chromosomes one and three overlapped with recurrent losses in 1p35.3 and 3p21.31. Essential members of the ATR-pathway were located in these regions: ATRIP and RPA2. However, mutational analyses of the two candidate genes in 48 cases revealed no mutations.SNP array analysis is a reliable tool to identify and further characterize genomic aberrations in CLL. MDR on 13q14 was delineated to a 277.25 kb segment affecting mir15a, mir16, DLEU1 and DLEU2 but not RFP2; the MDR on 11q23 to a segment only containing ATM. Cases with del(11q23) and del(17p) showed a higher genomic complexity than those with normal karyotype or del(13q14) as single abnormality. Relatively few novel genetic lesions were identified. Although occurring at low frequency, they may lead to the discovery of new genes involved in CLL pathogenesis. Disclosures:Stilgenbauer:Amgen: Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Sanofi Aventis: Research Funding.

Allogeneic Stem Cell Transplantation Can Overcome the Adverse Prognostic Impact of TP53 Mutation In Chronic Lymphocytic Leukemia (CLL): Results From the GCLLSG CLL3x Trial

AbstractAbstract 2357There is ample evidence that poor-risk CLL, as defined by fludarabine refractoriness or the presence of deletion 17p-, can be successfully treated by allogeneic stem cell transplantation (alloSCT). It is unknown, however, whether alloSCT can also overcome the treatment resistance associated with TP53 mutations seen under conventional fludarabine combination therapy. Therefore we have assessed the impact of TP53 mutations on the outcome of alloSCT with the patient cohort enrolled on the CLL3X trial of the German CLL Study Group. Patients and Methods:The CLL3X trial included 90 patients with poor-risk CLL who were allografted with unmanipulated blood stem cells from related or unrelated donors after nonmyeloablative conditioning. With a median follow-up of 46 months, 4-year event-free (EFS) and overall survival (OS) was 42% and 65%, respectively (Blood July 1, 2010). PFS and OS of 13 patients with deletion 17p- were similar to that of patients without this abnormality. TP53 mutations were identified by denaturating high-performance liquid chromatography (DHLPC) (exons 4–10). In addition, cases with deletion 17p- where no TP53 mutation was detected were also directly sequenced. Results:The TP53 mutational status could be obtained in 72 of 90 patients who had informative DNA samples from the time of study entry available. Of these, 19 (26%) showed TP53 mutations; 7 (10%) with a concurrent deletion 17p-, and 9 (12%) in the absence of deletion 17p-. 17p- status was not available in three TP53-mutated patients (4%). Three additional patients (4%) had 17p- without TP53 mutation. Four-year EFS and OS was 46% and 56% with TP53 mutation vs 38% and 66% without TP53 mutation (Figure). Within the TP53-mutated group, 4-year EFS and OS was 44% and 56% for patients without deletion 17p- vs 38% and 50% for patients with concurrent deletion 17p-. None of these differences were statistically significant. Among the patients who were event-free 12 months post alloSCT and had results of minimal residual disease (MRD) assessment available, the probability of being MRD-negative at this landmark was 71% with TP53 mutations and 63% without (p = 1.0). Finally, multivariate analysis using Cox regression modeling (adjusting for age, deletion 17p-, remission status at alloSCT, and T cell depletion) did not show a significant impact of TP53 mutations on EFS (Hazard ratio (HR) 0.71; 95%CI 0.31–1.61) and OS (HR 1.13; 95%CI 0.41–3.12). [Display omitted] Conclusions:AlloSCT can provide long-term EFS in about 40% of patients with poor-risk CLL with TP53 mutation independent from the presence of concurrent deletion 17p-. Disease control appears to be similar in patients with and without TP53 mutation, suggesting that alloSCT can overcome the treatment resistance associated with this abnormality. Disclosures:Stilgenbauer: Amgen: Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Sanofi Aventis: Research Funding.

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

AbstractThe 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.

Rituximab plus fludarabine and cyclophosphamide or other agents in chronic lymphocytic leukemia

Over the last few years, several monoclonal antibodies have been investigated in patients with B-cell lymphoid malignancies. Rituximab is the most important monoclonal antibody of clinical value in these disorders. Rituximab is an IgG1 chimeric antibody containing murine light- and heavy-chain variable region sequences and human constant region sequences. Since approval in 1997, rituximab has become the standard of care in follicular B-cell lymphoma, chronic lymphocytic leukemia (CLL) and aggressive lymphoma when combined with chemotherapy. Higher clinical benefits of rituximab can be seen in patients with CLL when it is added to other chemotherapeutic agents. Several recent reports have suggested that in patients with CLL, rituximab combined with purine nucleoside analogs (PNAs) or PNAs and cyclophosphamide may improve the results with acceptable toxicity, both in previously untreated and refractory/relapsed patients. The randomized, multinational Phase III study (REACH trial) has shown that rituximab combined with fludarabine and cyclophosphamide (R-FC regimen) results in 10 months longer progression-free survival, and higher overall response and complete response rates than fludarabine and cyclophosphamide (FC regimen) in previously treated patients. The German CLL study group initiated a multicenter, multinational Phase III trial, CLL8, to evaluate the efficacy and tolerability of R-FC versus FC for the first-line treatment of patients with advanced CLL. The overall response rate was significantly higher in the R-FC arm (95%) compared with FC (88%). The complete response rate in the R-FC arm was 44% compared with 27% in the FC arm. The recently updated analysis has demonstrated longer overall survival in the R-FC group. Recent clinical observations have revealed that combinations of rituximab with pentostatin and cyclophosphamide, or cladribine and cyclophosphamide are also highly active regimens in previously untreated CLL. In addition, the results of treatment with high-dose methylprednisolone in combination with rituximab in advanced CLL resistant to fludarabine have been reported recently by several groups. However, available therapies are only partially effective in CLL, exposing an obvious need to develop new, more specific and active drugs. Recently, several new anti-CD20 monoclonal antibodies have been developed and are now being evaluated in clinical trials.

TP53 Mutation and Survival in Chronic Lymphocytic Leukemia

The precise prognostic impact of TP53 mutation and its incorporation into treatment algorithms in chronic lymphocytic leukemia (CLL) is unclear. We set out to define the impact of TP53 mutations in CLL. We assessed TP53 mutations by denaturing high-performance liquid chromatography (exons 2 to 11) in a randomized prospective trial (n = 375) with a follow-up of 52.8 months (German CLL Study Group CLL4 trial; fludarabine [F] v F + cyclophosphamide [FC]). We found TP53 mutations in 8.5% of patients (28 of 328 patients). None of the patients with TP53 mutation showed a complete response. In patients with TP53 mutation, compared with patients without TP53 mutation, median progression-free survival (PFS; 23.3 v 62.2 months, respectively) and overall survival (OS; 29.2 v 84.6 months, respectively) were significantly decreased (both P < .001). TP53 mutations in the absence of 17p deletions were found in 4.5% of patients. PFS and OS for patients with 17p deletion and patients with TP53 mutation in the absence of 17p deletion were similar. Multivariate analysis identified TP53 mutation as the strongest prognostic marker regarding PFS (hazard ratio [HR] = 3.8; P < .001) and OS (HR = 7.2; P < .001). Other independent predictors of OS were IGHV mutation status (HR = 1.9), 11q deletion (HR = 1.9), 17p deletion (HR = 2.3), and FC treatment arm (HR = 0.6). CLL with TP53 mutation carries a poor prognosis regardless of the presence of 17p deletion when treated with F-based chemotherapy. Thus, TP53 mutation analysis should be incorporated into the evaluation of patients with CLL before treatment initiation. Patients with TP53 mutation should be considered for alternative treatment approaches.

Bendamustine in B-Cell Malignancies: The New 46-Year-Old Kid on the Block

On March 20, 2008, bendamustine HCl (Treanda, Cephalon, Inc.) was approved by the U.S. Food and Drug Administration (FDA) for treatment of chronic lymphocytic leukemia (CLL). This approval was based on a randomized, multicenter trial comparing the test drug ( n = 162) to chlorambucil ( n = 157) as a