Genetic instability is a feature of chronic lymphocytic leukemia (CLL) with adverse prognosis. We hypothesized that chromosomal translocations or complex karyotypes and distinct somatic mutations may impact outcome after first-line chemoimmunotherapy of CLL patients. We performed metaphase karyotyping and next-generation sequencing (NGS) of 85 genes in pretreatment blood samples obtained from 161 patients registered for CLL11, a 3-arm phase 3 trial comparing frontline chlorambucil (Clb) vs Clb plus rituximab (Clb-R) or Clb plus obinutuzumab in CLL patients with significant comorbidity. Chromosomal aberrations as assessed by karyotyping were observed in 68.8% of 154 patients, 31.2% carried translocations, and 19.5% showed complex karyotypes. NGS revealed 198 missense/nonsense mutations and 76 small indels in 76.4% of patients. The most frequently mutated genes were NOTCH1, SF3B1, ATM, TP53, BIRC3, POT1, XPO1, and KRAS Sole chemotherapy, treatment with Clb-R, or genetic lesions in TP53 (9.9% of patients) and KRAS (6.2% of patients) were significantly associated with nonresponse to study therapy. In multivariate models, complex karyotypes and POT1 mutations (8.1% of patients) represented significant prognostic factors for an unfavorable survival, independently of IGHV mutation status, Binet stage, and serum β-2-microglobuline. Patients with the copresence of complex karyotypes and deletions/mutations involving TP53 demonstrated a particularly short survival. In summary, this is the first prospective, controlled study in CLL patients that shows a role of complex karyotype aberrations as an independent prognostic factor for survival after front-line therapy. Moreover, the study identifies mutations in KRAS and POT1 as novel determinants of outcome after chemoimmunotherapy using chlorambucil and anti-CD20 treatment.

Objective: The role of B cells and the subgroup of IL-10 producing B cells, known to have a regulatory function, in patients following a haematopoietic stem cell transplant (alloSCT) has not been clearly understood to date. Methods: We prospectively recruited 95 patients following an alloSCT and studied the B-cell reconstitution on days 30, 90 and 150. Regulatory B10 cells could be analysed in 22 consecutively recruited patients on day 30 post-transplant. Results: The total B-cell percentages in transplant recipients (median 0.33; range 0.01-5.9) were significantly reduced than the controls (P = 0.0001) and constituted predominantly of transitional CD24(high) CD38(high) B cells. Regulatory B10 cells could be analysed in 22 consecutively recruited patients on day 30 post-transplant. The percentages of B10 cells (median 1.35; 0.0-4.5) were significantly reduced in the transplant recipients in comparison with the control cohort (P < 0.0001). Interestingly, the percentages of B10 cells in patients with acute GvHD (median 1.7; 0.33-4.5) were significantly higher than those without GvHD (median 0.7; 0-1.9) (P = 0.0003). Conclusion: This is the first report demonstrating B10 cells in stem cell transplant recipients in the early post-alloSCT (30 d) period. Our data suggest a possible role for B10 cells in the pathophysiology of acute GvHD. Further longitudinal studies are warranted to understand the implications of our findings.

Reduced anticancer efficacy of cyclophosphamide and platinum salts has been reported in animals treated with anti-Gram-positive antibiotics. These effects were related to translocation of Gram-positive bacteria during mucositis with subsequent induction of cytotoxic oxygen reactive species and tumor invasion by pathogenic Th17 cells. To assess these hypotheses in a clinical setting, we identified patients receiving cyclophosphamide for chronic lymphocytic leukemia (CLL) and cisplatin for relapsed lymphoma. Data originated from the CLL8 trial (NCT00281918) and the Cologne Cohort of Neutropenic Patients (NCT01821456). Relevant antibiotics were defined as compounds with primary activity against Gram-positive bacteria. We evaluated their impact on response, progression-free survival (PFS) and overall survival (OS) by Kaplan-Meier methodology and Cox proportional hazards regression analysis. Among 800 available CLL patients, those receiving anti-Gram-positive antibiotics (n = 45/800) achieved a significantly lower overall response rate (OR 74.3% vs. 90.2%, p = 0.007). Patients with anti-Gram-positive antibiotics progressed significantly earlier, had a reduced OS (median PFS 14.1 vs. 44.1 mo, p < 0.001; median OS 56.1 vs. 91.7 mo, p < 0.001) and multivariate analysis showed that administration of anti-Gram-positive antibiotic treatment was independently associated with reduced PFS (Hazard ratio (HR) 2.090, p = 0.001) and OS (HR 2.966, p < 0.001). Of 122 patients with relapsed lymphoma, those treated with anti-Gram-positive antibiotics (n = 21/122) achieved a significantly lower OR rate (70.3% vs. 42.9%, p = 0.016). Patients with anti-Gram-positive antibiotics progressed significantly earlier than others (median PFS 2.3 vs. 11.5 mo, p = 0.001). As for multivariate analysis, the use of anti-Gram-positive antibiotics was independently associated with reduced PFS (HR 2.237, p = 0.012) and OS (HR 7.831, p < 0.001). Our data supports a potential negative impact of anti-Gram-positive antibiotics on the anticancer activity of cyclophosphamide and cisplatin in a clinical setting.

PurposeA phase I/II trial to assess safety and efficacy of the combination bendamustine, rituximab, and lenalidomide (BRL) in patients with chronic lymphocytic leukemia (CLL).Patients and MethodsSeventeen relapsed or refractory (R/R) and five previously untreated (FL) CLL patients were enrolled in the trial. In the R/R cohort, four different dose levels of lenalidomide (maximum 15 mg/d) were used. In the FL cohort, lenalidomide was dose escalated from 5 mg/d to 15 mg/d. Bendamustine was used at doses of 50 or 90 mg/m(2) for R/R or FL treatment, respectively. 375 mg/m(2) Rituximab were used for the first and 500 mg/m(2) for subsequent treatment courses. Treatment consisted of up to six courses of 28 d.ResultsThe maximal tolerable dose of lenalidomide was 5 mg/d. The response rate was 47.1% in R/R and 60% in FL patients. Median progression-free survival was 8.0 months. Median overall survival was 22.9 and 12.3 months, respectively, in R/R and FL patients. Grade 3/4 hematological toxicity was observed in 71.4%, and severe infections in 47.6% of patients. Due to high toxicity and low response rate of BRL, the trial was closed prematurely.ConclusionBRL was associated with a high toxicity rate, a high number of treatment interruptions, and a low remission rate. Therefore, BRL cannot be considered an appropriate treatment option for patients with CLL.

Which genetic alterations drive tumorigenesis and how they evolve over the course of disease and therapy are central questions in cancer biology. Here we identify 44 recurrently mutated genes and 11 recurrent somatic copy number variations through whole-exome sequencing of 538 chronic lymphocytic leukaemia (CLL) and matched germline DNA samples, 278 of which were collected in a prospective clinical trial. These include previously unrecognized putative cancer drivers (RPS15, IKZF3), and collectively identify RNA processing and export, MYC activity, and MAPK signalling as central pathways involved in CLL. Clonality analysis of this large data set further enabled reconstruction of temporal relationships between driver events. Direct comparison between matched pre-treatment and relapse samples from 59 patients demonstrated highly frequent clonal evolution. Thus, large sequencing data sets of clinically informative samples enable the discovery of novel genes associated with cancer, the network of relationships between the driver events, and their impact on disease relapse and clinical outcome.

Introduction: Frontline treatment with full-dose FCR is considered standard of care for physically fit patients with CLL. In routine practice, however, adverse events during treatment often lead to reduction of the planned dose of FCR which might result in loss of treatment efficacy. The aim of our study was to evaluate the prognostic impact of reducing the prescribed dose of FCR and to identify risk factors that could help to predict such dose modification. Patients and Methods: Patients treated with FCR within two randomized phase 3 trials of the German CLL Study Group (GCLLSG) (CLL8 trial: FCR vs. FC; CLL10 trial: FCR vs. bendamustine plus rituximab) were pooled. In each patient, the planned dose of FCR (according to protocol) was compared with the actually applied dose (with deltas provided in %). Subjects with reductions of the planned dose of FCR due to early treatment discontinuation not caused by toxic adverse events, but refractory/progressive disease or other reasons were excluded from further analyses (Figure 1). Patients with ≤ 20% and with > 20% reduction of the planned dose of FCR were compared with regard to progression-free survival (PFS) and overall survival (OS). Logistic regression was used to identify risk factors of such dose modification. Results: A total of 635 patients treated with at least one dose of FCR were included in this study. Median age was 61 years, median CIRS score 1 (range 0-7), and median ECOG performance status 0 (range 0-2). The median follow up time was 57.3 months (range 1.4-96.4). 11% of patients had Binet stage A, 55% stage B, and 34% stage C disease. Among 540 patients with IGHV results, 59% had an unmutated status. In 550 cases with FISH results, del(17p) was found in 3% (exclusion criteria in CLL10 trial), del(11q) in 25%, 12+ in 10%, del(13q) in 38%, and normal karyotype in 24% (according to the hierarchical model by Dohner et al.). Of the 635 patients, 209 (33%) received FCR with > 20% reduction of the planned dose. These patients had significantly shorter PFS (median PFS 45.2 vs. 64.0 months, HR [hazard ratio]=1.627, 95%CI [confidence interval]=1.294-2.046, p 60 years (OR=1.601, 95%CI=1.105-2.320, p=0.013), Binet stage C (OR [odds ratio]=1.790, 95%CI=1.224-2.619, p=0.003), and serum β2-microglobuline > 3.5 mg/L (OR=1.495, 95%CI=1.009-2.215, p=0.045) were identified as independent risk factors for reducing the prescribed dose of FCR. Conclusions: This pooled analysis of patients receiving frontline therapy of CLL with FCR within two prospective GCLLSG trials for the first time indicates that reducing prescribed doses of FCR compromises not only PFS, but also reduces the benefit regarding OS. The adverse prognostic impact of a reduction of the planned dose of FCR by 20% or more is clinically meaningful and should be considered carefully, particularly if such dose reductions are made. Predictors of reducing the planned dose during FCR treatment (such as older age and advanced disease stage) may help to refine treatment decision-making in CLL. ![Figure 1.][1] Figure 1. Study population. ![Figure 2.][1] Figure 2. a) PFS and b) OS of patients with ≤ 20% (green) vs. > 20% (blue) reduction of the planned dose of FCR. Disclosures Kovacs: Mundipharma: Other: Travel grant. Cramer: Gilead: Other: Travel grant, Research Funding; Mundipharma: Other: Travel grant; Glaxo Smith Klein/Novartis: Research Funding; Hoffman LaRoche: Other: Travel grant, Research Funding, Speakers Bureau; Janssen: Other: Travel grant, Research Funding, Speakers Bureau; Astellas: Other: Travel grant. Fink: Roche: Honoraria, Other: travel grant. Fischer: Roche: Other: Travel Grants. Langerbeins: Janssen: Honoraria, Other: travel grants, Research Funding; Hoffmann-LaRoche: Honoraria, Other: travel grants, Research Funding; Mundipharma: Honoraria, Other: travel grants, Research Funding. Maurer: Mundipharma: Other: Travel grant. von Tresckow: Hoffman-LaRoche: Other: travel grants, Research Funding; Celgene: Other: travel grants; Janssen-Cilag: Honoraria, Research Funding. Wendtner: Genentech: Consultancy, Other: travel grants, Research Funding; AbbVie: Consultancy, Other: travel grants, Research Funding; Hoffmann-LaRoche: Consultancy, Other: travel grants, Research Funding; Gilead: Consultancy, Other: travel grants, Research Funding; Glaxo-SmithKline: Consultancy, Other: travel grants, Research Funding; Janssen-Cilag: Consultancy, Other: travel grants, Research Funding; Mundipharma: Consultancy, Other: travel grants, Research Funding; Celege: Consultancy, Other: Travel grants, Research Funding; Pharmacyclics: Consultancy, Other: travel grants, Research Funding. Stilgenbauer: Gilead: Consultancy, Other: travel grants, Research Funding; AbbVie: Consultancy, Other: travel grants, Research Funding; Celgene: Consultancy, Other: travel grants, Research Funding; Boehringer-Ingelheim: Consultancy, Other: travel grants, Research Funding; Amgen: Consultancy, Other: travel grants, Research Funding; Genzyme: Consultancy, Other: travel grants, Research Funding; Hoffman-LaRoche: Consultancy, Honoraria, Other: travel grants, Research Funding; Genentech: Consultancy, Other: travel grants, Research Funding; Janssen: Consultancy, Other: travel grants, Research Funding; GlaxoSmithKline: Consultancy, Other: travel grants, Research Funding; Mundipharma: Consultancy, Other: travel grants, Research Funding. Hallek: Celgene: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding; Boehringher Ingelheim: Honoraria, Other: Speakers Bureau and/or Advisory Board; Janssen: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding; Gilead: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding; AbbVie: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding; Roche: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding; Mundipharma: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding; Pharmacyclics: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding. Eichhorst: Roche: Consultancy, Research Funding, Speakers Bureau; AbbVie: Consultancy; MundiPharma: Consultancy, Research Funding, Speakers Bureau. Goede: Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; GSK: Honoraria; Mundipharma: Honoraria; Bristol-Myers Squibb: Honoraria. [1]: pending:yes

In a (two-type) Wright–Fisher diffusion with directional selection and two-way mutation, let x denote today's frequency of the beneficial type, and given x, let h(x) be the probability that, among all individuals of today's population, the individual whose progeny will eventually take over in the population is of the beneficial type. Fearnhead (2002) and Taylor (2007) obtained a series representation for h(x). We develop a construction that contains elements of both the ancestral selection graph and the lookdown construction and includes pruning of certain lines upon mutation. Besides being interesting in its own right, this construction allows a transparent derivation of the series coefficients of h(x) and gives them a probabilistic meaning.

Clonal evolution in response to therapy is a central feature of disease relapse. This raises a fundamental question in cancer biology: what enables the relapse clone to replace the pre-treatment clone? In other words, is the increased fitness of the relapse clone due to a lower death rate during therapy (less sensitivity to therapy) or a higher growth rate following therapy (superior ability to compete during repopulation)?

Abstract Introduction: Within clinical trials in CLL, response assessment is based on the NCI 1996 guidelines or its update, the iwCLL 2008 guidelines, respectively. Whereas detailed response criteria with clear cut-off values are provided for the assessment of most parameters, a few parameters such as splenomegaly are not defined quantitatively in the guidelines. In addition, the detection of MRD, which was recommended for clinical trials but not formally included in the definition of response, is gaining increasing importance. Both MRD negativity (with a threshold of <10-4 leukemic cells per leukocytes) and the occurrence of a complete response (CR) predict long progression free survival (PFS). In order to investigate the value of MRD with respect to clinical response, the MRD status was explored in patients (pts) with CR and partial remission (PR) in two phase III trials of the GCLLSG. Furthermore, we evaluated the relevance of residual splenomegaly, lymphadenopathy or bone marrow involvement in MRD negative (-) pts with clinical PR. Patients and Methods: 542 pts from two prospective phase III trials of the GCLLSG (CLL8 trial: fludarabine and cyclophosphamide without (FC) or with rituximab (FCR); CLL10 trial: FCR vs bendamustine and rituximab (BR)) were included in the analysis (Figure 1). The comprehensive dataset included MRD results from peripheral blood at final restaging (RE) (2 months after the end of last treatment cycle), bone marrow (BM), clinical and radiological assessment for organomegaly and lymphadenopathy. Clinical response was defined according to the iwCLL 2008 guidelines. Splenomegaly was determined by physical and radiological examination. Moreover, different cut-off values defining splenomegaly by CT or ultrasound (longest diameter >12 cm and >14 cm) were investigated. PFS was analyzed using Kaplan-Meier methodology, and survival curves were compared using two-sided log-rank tests. Additionally hazard ratios (HR) and 95% confidence intervals (CI) were calculated. Significance was set at a p value<0.05. No adjustments for multiple testing were performed. Results: 542 pts were included in the analysis. Their median age was 61 years, median CIRS score 2 (range 0-6), median follow up time 45.9 months (mo) (range 5.5-96.1). 121 pts (22.3%) received FC, 283 (52.2%) FCR and 138 (25.5%) BR. 13.3% of pts had Binet stage A, 52.3 % stage B and 34.4% stage C disease. Among 514 pts with IGHV results, 63.0% had an unmutated status. In 524 pts FISH was available, del(17p) was only found in 1.3% (exclusion criterion in CLL10 trial), del(11q) in 25.0%, 12+ in 10.1%, normal in 25.4%, and del(13q) in 38.2% of the pts. MRD negativity was achieved in 81.8% (175/214) of pts with CR and in 47.9% (157/328) of the pts with PR, respectively. There was a statistically significant difference in PFS between MRD- CRs and MRD positive (+) CRs (69.2 mo vs 40.4 mo; HR 0.445, 95% CI=0.282-0.703, p=0.001). Additionally, there was a statistically significant difference between MRD- PRs and MRD+ CRs (61.7 mo vs 40.4 mo; HR 0.537, 95% CI=0.340-0.847, p=0.008). No statistically significant difference between MRD- CRs and MRD- PRs was detected (69.2 mo vs. 61.7 mo; HR 0.822, 95% CI=0.572-1.182, p=0.29) (Figure 2). Of the 157 pts presenting with an MRD- PR, 106 pts were evaluable for remaining CLL involvement: 48 pts (45.3%) had a splenomegaly, 12 (11.3%) lymphadenopathy, 19 (17.9%) bone marrow involvement as the sole abnormality. Only 25.5% (27) of the pts had more than one abnormality. There was no statistically significant difference in PFS between MRD- PRs with single splenomegaly and MRD- CRs (not reached (NR) vs 69.2 mo; HR 0.737, 95% CI=0.387-1.404, p=0.4). Moreover, patients with MRD- PRs and single splenomegaly had a statistically significant longer PFS than MRD+ CR pts (NR vs 40.4 mo; HR 0.348, 95% CI=0.172-0.701, p=0.003). (Figure 3) The difference was independent of the cut-off values used for splenomegaly (12 cm or 14 cm) (p=0.001 and p=0.03). Conclusion: MRD negativity determined in the peripheral blood after end of treatment is a potent predictor of treatment efficacy regardless of the clinical response assessment. The persistence of splenomegaly as sole abnormality post treatment in MRD- patients has no negative influence on PFS. More data are needed to prove the relevance of residual BM involvement and lymphadenopathy in MRD- PR pts. These results support the use of MRD for response evaluation. Figure 1 Figure 1. Disclosures Boettcher: Roche: Honoraria, Research Funding, Travel grant Other. Ritgen:Roche: Honoraria, Research Funding, Travel grant Other. Cramer:Mundipharma: Travel grant, Travel grant Other; Roche: Travel grant Other. Maurer:Mundipharma: Travel grant Other. Doehner:Roche: Research Funding. Stilgenbauer:Roche: Consultancy, Honoraria, Research Funding. Kneba:Mundipharma: Consultancy, Research Funding; Roche: Consultancy, Research Funding. Fischer:Roche: Travel grant Other. Hallek:Mundipharma: Consultancy, Research Funding; Roche: Consultancy, Research Funding. Eichhorst:Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding, Travel grant Other; Mundipharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Travel grant, Travel grant Other; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy. Off Label Use: The Combination of Bendamustine and Rituximab is not approved for frontline chemoimmunotherapy of CLL.

Intra-tumoral genetic heterogeneity and clonal evolution play a central role in disease relapse, and constitute one of the foremost obstacles to cancer cure. To measure intra-tumoral genetic heterogeneity, we recently developed an analytic framework which utilizes whole-exome sequencing (WES) to calculate the proportion of cancer cells within a sample harboring each mutation. Applying this approach to a clinically heterogeneous cohort of chronic lymphocytic leukemia (CLL), we previously discovered that the intra-tumoral genetic heterogeneity in primary patient samples prior to treatment initiation anticipates clonal evolution and is linked to adverse clinical outcome with therapy (Landau et al., Cell 2013).

We reconsider the Moran model in continuous time with population size N, two allelic types, and selection. We introduce a new particle representation, which we call the labelled Moran model, and which has the same distribution of type frequencies as the original Moran model, provided the initial values are chosen appropriately. In the new model, individuals are labelled 1,2,…,N; neutral resampling events may take place between arbitrary labels, whereas selective events only occur in the direction of increasing labels. With the help of elementary methods only, we not only recover fixation probabilities, but also obtain detailed insight into the number and nature of the selective events that play a role in the fixation process forward in time.

We consider the Moran model in continuous time with two types, mutation, and selection. We concentrate on the ancestral line and its stationary type distribution. Building on work by Fearnhead (J. Appl. Probab. 39 (2002), 38-54) and Taylor (Electron. J. Probab. 12 (2007), 808-847), we characterise this distribution via the fixation probability of the offspring of all individuals of favourable type (regardless of the offspring's types). We concentrate on a finite population and stay with the resulting discrete setting all the way through. This way, we extend previous results and gain new insight into the underlying particle picture.