Biology Of Chronic Lymphocytic Leukemia Research Articles

Pan-caspase peptide inhibitor to induce primary chronic lymphocytic leukemia (CLL) cell death through a non-apoptotic and non-necroptotic mechanism.

e13536 Background: It is thought that CLL cells are poised on the brink of apoptosis. Recent advances in the understanding of the balance of BCL2 family proteins in CLL biology have led to the deve...

Insulin growth factor 1 receptor expression is associated with NOTCH1 mutation, trisomy 12 and aggressive clinical course in chronic lymphocytic leukaemia.

IGF1R is emerging as an important gene in the pathogenesis of many solid and haematological cancers and its over-expression has been reported as frequently associated with aggressive disease and chemotherapy resistance. In this study we performed an investigation of the role of IGF1R expression in a large and representative prospective series of 217 chronic lymphocytic leukaemia (CLL) patients enrolled in the multicentre O-CLL1 protocol (clinicaltrial.gov #NCT00917540). High IGF1R gene expression was significantly associated with IGHV unmutated (IGHV-UM) status (p<0.0001), high CD38 expression (p<0.0001), trisomy 12 (p<0.0001), and del(11)(q23) (p=0.014). Interestingly, higher IGF1R expression (p=0.002) characterized patients with NOTCH1 mutation (c.7541_7542delCT), identified in 15.5% of cases of our series by next generation sequencing and ARMS-PCR. Furthermore, IGF1R expression has been proven as an independent prognostic factor associated with time to first treatment in our CLL prospective cohort. These data suggest that IGF1R may play an important role in CLL biology, in particular in aggressive CLL clones characterized by IGHV-UM, trisomy 12 and NOTCH1 mutation.

Three newly approved drugs for chronic lymphocytic leukemia: incorporating ibrutinib, idelalisib, and obinutuzumab into clinical practice.

Three agents have received Food and Drug Administration (FDA) approval for treatment of chronic lymphocytic leukemia (CLL) within the past year. Ibrutinib and idelalisib block B-cell receptor signaling through inhibition of Bruton tyrosine kinase and phosphatidylinositol 3-kinase δ molecules respectively, interfering with several pathways required for leukemia cell survival. Idelalisib has shown efficacy in the relapsed setting and is currently approved by the FDA for use in combination with rituximab. Ibrutinib has been studied in patients with relapsed CLL and as frontline therapy. In the relapsed setting, these agents produce durable remissions, and might be preferable to re-treatment with chemoimmunotherapy for many patients. Ibrutinib is also effective treatment for patients with deletion 17p and is approved by the FDA as frontline therapy in this patient group, although it does not appear to completely abrogate this adverse prognostic factor. These agents have a unique side effect profile and longer follow-up is required to further understand tolerability and rare adverse effects. Obinutuzumab is a type-2 monoclonal anti-CD20 antibody which results in direct and antibody-dependent cell-mediated cytotoxicity of leukemia cells. It is approved by the FDA for use in combination with chlorambucil, and has shown efficacy in the frontline setting in patients unfit for more intensive chemoimmunotherapy. It produces increased response rates and minimal residual disease negativity compared with chlorambucil/rituximab and is associated with an advantage in progression-free survival but not yet overall survival. These agents underscore our advancement in the understanding of the biology of CLL and will improve outcomes for many patients with CLL.

Shifting paradigms in the treatment of chronic lymphocytic leukemia.

The treatment of chronic lymphocytic leukemia (CLL) is evolving rapidly. Insight into the genetics and biology of the disease, including the importance of intracellular signaling pathways and interactions with the microenvironment has led to the development of rational targeted therapies which are having a major impact on the survival of patients with relapsed and high-risk disease. In addition, an exciting array of cellular therapies and immunotherapy options are in various stages of development. We review the current understanding of CLL genetics and biology, current treatment strategies in specific patient groups and opportunities for future treatment combinations which will bring the goal of cure or long-term disease control with minimal toxicity within reach for the majority of patients.

Targeted therapy for chronic lymphocytic leukemia: current status and future directions.

The majority of patients with chronic lymphocytic leukemia (CLL) respond to chemo-immunotherapy. However, long-term remission remains elusive and the majority of patients will die of complications related to CLL. In this review we discuss the recent developments in targeted therapy for CLL. Targeted therapy has evolved beyond the cell surface targeting of CD20 with rituximab. Our review focuses on the evolution of antibody therapy in CLL, strategies to target effector T cells to the tumor, inhibition of the B-cell receptor signaling pathway, and finally targeting the mediators of apoptosis. With our improved understanding of the biology of CLL, the evolution of targeted therapy has resulted in significant clinical responses in patients who are refractory to traditional treatment options and holds the potential for a future where we can manage this disease without chemotherapy.

TACI expression and signaling in chronic lymphocytic leukemia.

TACI is a membrane receptor of BAFF and APRIL, contributing to the differentiation and survival of normal B cells. Although malignant B cells are also subjected on TACI signaling, there is a remarkable intradisease and interindividual variability of TACI expression in B-cell malignancies. The aim of our study was to explore the possible role of TACI signaling in the biology of chronic lymphocytic leukemia (CLL), including its phenotypic and clinical characteristics and prognosis. Ninety-four patients and 19 healthy controls were studied. CLL patients exhibited variable TACI expression, with the majority of cases displaying low to undetectable TACI, along with low to undetectable BAFF and increased APRIL serum levels compared to healthy controls. CLL cells with high TACI expression displayed a better survival capacity in vitro, when cultured with BAFF and/or APRIL. Moreover, TACI expression was positively correlated with the presence of monoclonal gammopathy and inversely with CD11c expression. Therefore, our study provides further evidence for the contribution of BAFF/APRIL signaling to CLL biology, suggesting also that TACI detection might be useful in the selection of patients for novel targeting therapeutic approaches.

T-cell number and subtype influence the disease course of primary chronic lymphocytic leukaemia xenografts in alymphoid mice

ABSTRACTChronic lymphocytic leukaemia (CLL) cells require microenvironmental support for their proliferation. This can be recapitulated in highly immunocompromised hosts in the presence of T cells and other supporting cells. Current primary CLL xenograft models suffer from limited duration of tumour cell engraftment coupled with gradual T-cell outgrowth. Thus, a greater understanding of the interaction between CLL and T cells could improve their utility. In this study, using two distinct mouse xenograft models, we investigated whether xenografts recapitulate CLL biology, including natural environmental interactions with B-cell receptors and T cells, and whether manipulation of autologous T cells can expand the duration of CLL engraftment. We observed that primary CLL xenografts recapitulated both the tumour phenotype and T-cell repertoire observed in patients and that engraftment was significantly shorter for progressive tumours. A reduction in the number of patient T cells that were injected into the mice to 2-5% of the initial number or specific depletion of CD8+ cells extended the limited xenograft duration of progressive cases to that characteristic of indolent disease. We conclude that manipulation of T cells can enhance current CLL xenograft models and thus expand their utility for investigation of tumour biology and pre-clinical drug assessment.

The mystery of chronic lymphocytic leukemia (CLL): Why is it absent in Asians and what does this tell us about etiology, pathogenesis and biology?

Chronic lymphocytic leukemia/small lymphocytic lymphoma is common in persons of predominately European descent but rare in Asians. Why is unknown but is likely genetically-determined. Environmental factors may also operate but are likely to be less important. When CLL occurs in Asians it has different features than CLL in persons of predominately European descent. The reason(s) for this is also not understood. We reviewed data on CLL in Asians (mostly Han Chinese but also other ethnic groups) and compared these data with those from persons of predominately European descent with CLL. CLL incidence was about 5–10-fold less in Asians. Asians with CLL are younger, have atypical morphologic and immunologic features, an increased proportion of IGHV mutations and rearrangements and briefer freedom-from-progression than persons of predominately European descent with CLL. These observations provide clues to the etiology and biology of CLL. But the mystery continues; more research is needed.

Primary CLL Xenograft: A Model System to Study the Role of T-Cells in CLL Biology and Therapeutic Response

The interaction between chronic lymphocytic leukaemia (CLL) cells and T-cells is an important aspect of CLL biology. CLL cells require T-cell support for their proliferation and in addition induce proliferation of regulatory and cytotoxic (CD8+) T-cells. T-cell number and repertoire are both markedly affected by CLL therapy and there is considerable interest in how current treatments modulate the interaction between T-cells and the tumour clone. In this study we investigated whether this relationship was maintained in a xenotransplantation model.CLL engraftment in NOG mice was facilitated by humanisation of the murine microenvironment by allogeneic CD34+ umbilical cord cells or CD14+ monocytes. Accelerated engraftment of both CLL and T-cell compartments was observed in xenografts derived from patients with progressive CLL, suggesting that the biological properties of both subsets are maintained in the murine model. Furthermore, the distribution of helper (CD4+), cytotoxic (CD8+) and regulatory (CD4+CD25+FoxP3+) T-cells was maintained within the xenografts, including retention of the CD4:CD8 ratio. Interestingly, the anergic PD-1+CD160+CD244+TIM3+ T-cell phenotype reported in CLL patients was also evident in T-cells expanded in xenograft models. Consistent with an anergic T-cell phenotype, T-cells from CLL xenografts lacked anti-tumour activity in vitro. Importantly, such anergic cells were observed when T-cells were reconstituted from allogeneic cord blood cells as well as autologous cells, suggesting that CLL cells have the ability to shape T-cell populations of different origin in diverse microenvironments. Finally, to investigate the interaction between specific T-cell subsets and engrafted CLL cells, CD4+, CD8+, and CD25+ T-cells were depleted prior to generation of xenografts. CD8+ T-cell depletion significantly prolonged CLL engraftment (p≤0.01) whereas neither depletion of CD4+ nor CD25+cells had a significant impact.In summary, our results demonstrate that the relationship between CLL tumour cells and reactive T-cells is accurately maintained in a murine xenograft model. Such models will be of great value for investigation of aspects of T-cell function in CLL biology. DisclosuresNo relevant conflicts of interest to declare.

Clinical Impact of Telomere Shortening in Normal and Leukemia Cells in Chronic Lymphocytic Leukemia

Introduction: In chronic lymphocytic leukemia (CLL), short telomere length in the leukemia cells predicts poor prognosis. However, it is not known whether telomere length in normal tissues also predicts patient outcome and can improve the prognostic value of CLL telomere length. Prognosis in CLL is heterogeneous with the primary cause of death being cancer and infections, thought related to immunosuppression. The elderly, males and those with comorbidities have a particularly poor relative survival. Whether this reflects differences in the biology of CLL in these patient groups, or to extrinsic factors such as increased immunosuppression or fragility, is unknown. In normal individuals, the telomeres in somatic cells (ie, buccal cells (BC)) shorten with aging and prior comorbidities; short telomeres being predictive of early mortality related to infections, cancer and cardiovascular disease. In the present study, we have evaluated BC telomere length in patients with CLL, to determine if this is predictive of survival, and can enhance the predictive value of leukemia cell telomere length.Methods: The Manitoba CLL tumor bank contains 235 samples from newly diagnosed CLL patients between 2007-2011; with a median follow-up of 2 years. One-quarter of these patients required chemotherapy and one-tenth have died. Genomic DNA was extracted from purified CLL cells and buccal cells (BC) collected at diagnosis. Telomere length was established by multiplex quantitative real-time PCR. Telomere/standard (t/s) ratio was calculated using the beta-globulin gene as the standard. Statistical analysis was performed using Statistical Analysis Software (SAS) and Prism software.Results: The median adjusted telomere length was much shorter in CLL cells than in BCs being 0.53 and 2.01, respectively. In BCs, telomere length significantly shortened with increasing age (OR 1.04, CI 95% (1.00-1.078)) and at this short follow-up time, correlation with second malignancies was approaching significance (p=0.06). However, BC telomere length was not reflective of the number of comorbidities or survival. In contrast, telomere length in CLL cells was independent of age (p=0.44) and sex (p=0.75) confirming that these factors did not influence the cellular biology of the disease. Telomere length also correlated with other biological markers with short telomeres correlating with unmutated IgHV status (p<0.0001), Zap-70 positivity (p=0.05), and CD38 positivity (p=0.003). These patients with short telomeres also had clinical markers of poor prognosis including short lymphocyte doubling time (p=0.004), higher Rai stage (p=0.02) and an earlier time to treatment (p<0.0001). The prognostic value of CLL telomere length was not enhanced by the addition of BC telomere length.Conclusions: These results demonstrate that BC telomere length in CLL patients shorten with age, and short telomeres may predict subsequent second malignancies. Telomere length in CLL cells correlates with biological and clinical markers of aggressive disease, but it does not explain the poor prognosis seen in the elderly and male patients. While BC telomere length does not seem to influence the initial clinical course of CLL, ongoing studies are evaluating whether it is predictive of the long-term risk of infections and second malignancies. In addition, whether BC telomere length in CLL is shorter than in the normal population and whether shortening correlates with specific comorbidities is presently being determined. DisclosuresNo relevant conflicts of interest to declare.

Role of Sprouty2 in the Pathogenesis of Chronic Lymphocytic Leukemia

INTRODUCTION: Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in adults representing about 35% of all adult leukemias in the United States. CLL is a B-cell neoplasm which is characterized by accumulation of long lived mature B-cells in peripheral blood (PB), bone marrow (BM) and lymph nodes (LN). We and others have reported that CLL cells in tissue microenvironments have constitutively activated MAPK-Erk and AKT signaling pathways, which promotes their proliferation and survival. However, the molecular mechanisms and the gene(s) responsible for the activation of these pathways in CLL are not clearly understood. We performed gene expression profiling of CLL cells from PB, BM and LN and transcriptome analyses of CLL cells from PB from good and poor prognosis patients. Our studies identified Sprouty 2 (Spry2), a negative feedback regulator as one of the key genes involved in the regulation of MAPK-Erk and Akt pathways in CLL. We observed that Spry2 levels were down-regulated in poor prognosis CLL cells. Interestingly, Spry2 is epigenetically silenced in most human and mouse lymphomas and ectopic expression of Spry2 induces apoptosis in mouse B cells. We categorized CLL patients into good and poor prognosis to further evaluate Spry2 in CLL biology. Poor prognosis patients were defined by unmutated immunoglobulin variable heavy chain (IgVH), 11q22 deletion, 17p deletion, trisomy 12 or high CD38 and Zap70 expression. Good prognosis patients were defined as having mutated IgVH, 13q14 deletions or low CD38 and Zap70 expression.RESULTS: We found that Spry2 expression was significantly decreased by 3.2 log2-fold, (p=0.0001) at transcript and protein levels, respectively, in CLL cells from patients with poor prognosis compared to patients with good prognosis. Spry2 is a negative feedback regulator which attenuates cell proliferation, migration and survival. To determine the effect of Spry2 on CLL cells we transfected Mec-1 cells (stable human CLL cell line) and primary human CLL cells with a Spry2 cDNA containing vector. Ectopic expression of Spry2 induced spontaneous apoptosis in Mec-1 cells and primary CLL cells from poor prognosis patients. These results indicate that Spry2 negatively regulates the survival of human CLL cells. CLL cells have been shown to have active B-cell receptor (BCR) signaling promoting proliferation in vivo. To study the effect of BCR stimulation on Spry2 we stimulated BCR on Mec-1 and primary CLL cells by BCR cross-linking. Unexpectedly, the rate of proliferation of Mec-1 cells decreased upon BCR stimulation. We were able to demonstrate a transient increase in Spry2 levels that peaked at 12 hours post BCR stimulation, which we suspected was mediating the decrease in proliferation observed. We therefore repeated the BCR stimulation experiment using siRNA to inhibit the transient increase in Spry2 in primary CLL cells from seven good prognosis patients and in the Mec-1 cells. As predicted, we observed a significant increase in proliferation in all primary CLL samples and in the Mec-1 cells upon Spry2 knock down following BCR stimulation. Spry2 attenuates ligand induced MAPK-Erk and AKT signaling leading to suppression of proliferation and survival. We also examined the effect of Spry2 on MAPK-Erk and Akt signaling in CLL cells. Upon induction of exogenous Spry2 expression in Mec-1 cells there was a significant decrease in p-Akt and p-Erk levels. Conversely, Spry2 knock down resulted in an increase in p-Erk and p-Akt and enhanced cell proliferation.CONCLUSIONS: Spry2 is significantly down-regulated in the B-cells from patients with poor prognosis CLL. Down-regulation of Spry2 in CLL cells leads to hyperactivation of MAPK-Erk and Akt signaling and exogenous and endogenous expression of Spry2 leads to spontaneous apoptosis in human CLL cells. These results identify Spry2 as a negative regulator of CLL cell survival and proliferation and provide a potential molecular target for therapeutic intervention. DisclosuresBociek:Seattle Genetics, Inc.: Research Funding.

Integrated Single-Cell Detection of Genotype and Phenotype in SF3B1-Mutated Chronic Lymphocytic Leukemia Cells

Cancer cell phenotype is controlled by both genetic composition and gene expression. Recent large-scale cancer sequencing studies have revealed extensive intratumoral genetic heterogeneity and have demonstrated its potential impact on clonal evolution and clinical outcome. The most direct approach to uncovering the impact of genetic heterogeneity on cellular phenotype requires integration of genetic and transcriptomic profiles of single cells. Currently, however, RNA and DNA cannot be reliably isolated from the same cell. Here, we demonstrate the feasibility for linking single-cell somatic mutation data with cellular transcriptional heterogeneity through a targeted RNA-based approach. By leveraging a microfluidic platform (Fluidigm BioMarkTMHD system) to perform multiplexed targeted amplification of RNA derived from hundreds of single cells, we have generated a versatile approach for the integrated detection of somatic mutations in relation to specific gene transcripts.We focused on a series of chronic lymphocytic leukemia (CLL) B cells that were previously characterized by bulk whole-exome (WES) and RNA-sequencing (RNA-Seq). We developed 2 classes of assays. First, we generated multiplexed nested quantitative RT-PCR assays of 96 genes with known involvement in CLL biology. Second, to simultaneously detect patient-specific somatic mutations in the same cell, we devised multiplexed pre-amplification primers targeting transcribed regions containing somatic point mutations. These regions were then amplified using paired nested primers, for detection of the wild-type or mutant alleles. We focused on those somatic mutations with detectable expression in bulk CLL RNA (> 5 FPKM by RNA-seq). When applied to either artificial oligonucleotide templates or bulk patient cDNA, these paired wild-type and mutant allele detection assays reliably demonstrated consistent differences in DCT values of >6 cycles.In total, we designed expression assays for 96 genes and 46 mutation detection applied to 5 CLL samples (median of 9 assays/sample, range 6-13). We examined up to 384 single cells from each of 5 samples and from normal CD19+ B cells. Based on expression of housekeeping genes ACTB and B2M, we observed viable expression in 1951 of 2112 cells (92.4%). We could clearly discern that expression of the 96 genes was heterogeneous across 354 single CLL-B cells and could discriminate CLL from 174 normal B cells by principal component analysis. 32 out of 46 (70%) mutation detection assays successfully distinguished between wild-type and mutant alleles and the mutant allele was consistently observed in the originating CLL cells, but not in unrelated CLL or non-leukemic B cells. Our RNA-based estimates of allele frequency agreed with single-cell targeted DNA-based detection of somatic mutations conducted for 3 of 5 CLL samples as well as with frequencies estimated from bulk WES-based cancer cell fraction (CCF) measurements.We applied our integrated assay design to 2 CLL samples known to harbor mutations in the putative CLL driver SF3B1: Patient 1 with bulk CCF of 17% (G742D) and Patient 2 with 87% (K700E). Mutation of this critical spliceosome component broadly changes RNA splicing profiles although the functional impact of these alternative splice variants on CLL biology remains unknown. We generated multiplex assays for SF3B1 mutation detection and for expression of mutation-associated alternative splice variants. Consistent with the bulk-sequencing results, we detected 50 of 373 (13.4%) single CLL cells from Patient 1 with SF3B1 mutation. Moreover, the subset of cells with SF3B1 mutation demonstrated high expression of splice variants relative to wild-type cells (GCC2 and MAP3K7, p< 0.000001). This SF3B1 mutated subclone also displayed reduced expression of RNA splicing factors (BTAF1, DDX17, SNW1, SRSF3, U2SURP; all p<0.05), cell cycle regulators (CDC27, PDS5A; p<0.015) and an inflammatory pathway gene (MALT1p=0.039), suggesting involvement of SF3B1 mutation in these biological processes. Analysis of Patient 2 is ongoing.Taken together, our study demonstrates the feasibility of linking genotype with gene expression at the RNA level. Furthermore, these analyses reveal the potential for single cell RNA-based analysis to directly uncover the effects of driver mutations on the leukemia cell phenotype. DisclosuresBrown:Sanofi, Onyx, Vertex, Novartis, Boehringer, GSK, Roche/Genentech, Emergent, Morphosys, Celgene, Janssen, Pharmacyclics, Gilead: Consultancy.

Clinical Impact of Stereotyped Antigen Receptors in Chronic Lymphocytic Leukemia

In chronic lymphocytic leukemia (CLL), the molecular features of the clonotypic B-cell receptor immunoglobulins (BcR IG) are set from the birth of the clone and in contrast to genetic aberrations, remain stable overtime, rendering the BcR IG a reference biomarker that is usually not significantly affected by clonal evolution. Approximately 30% of CLL cases carry quasi-identical BcR IGs and can be assigned to distinct stereotyped subsets. While preliminary evidence alludes to BcR IG stereotypy being relevant from a clinical viewpoint, this aspect has never been explored systematically or in a cohort of adequate size to enable meaningful conclusions. In order to assess the clinical implications of BcR IG stereotypy, we evaluated clinicobiological data from 8593 CLL patients, particularly focusing on 14 major stereotyped subsets of cases with unmutated (U-CLL) or mutated IGHV genes (M-CLL). The largest subset was #2 (n=254, 3%), within which 156 and 98 cases were M-CLL and U-CLL, respectively, followed by U-CLL subsets #1 (n=173, 2%) and #7 (n=123, 1.4%). Amongst M-CLL, the largest subsets were #4 (n=94, 1.1%) and #148 (n=92, 1%). Stereotyped subsets, even of the same mutational category i.e. U-CLL or M-CLL, exhibited significant clinicobiological differences regarding: age at diagnosis (median age ranging from 53-68 years, p<0.001); gender distribution (male/female ratios ranging from 0.85-4.4, p<0.001); the frequency of advanced stage disease (Binet C) at diagnosis (ranging from 9-21%, p<0.001); CD38 positivity (median percentage of positive cells ranging from 2-73%, p<0.05). Moving to cytogenetic findings, we detected subset-biased spectra of FISH-detected aberrations which can be summarized as follows: (i) isolated del(13q) varied from 10% in subset #8 (U-CLL), to 29% in subsets #59 (U-CLL) and #16 (M-CLL), and >75% in M-CLL subsets #77 and #148 (p<0.001); (ii) trisomy 12 was absent in subsets #31 (U-CLL) and #77 (M-CLL), in 20% of subset #201 (M-CLL) and, 60% in subset #8 (U-CLL) (p<0.001); (iii) del(11q) ranged from 69% in subset #31 (U-CLL) to 12% in subset #8 (U-CLL) and 0% in M-CLL subsets #16 and #201 (p<0.001); (iv) del(17p): absent from all M-CLL subsets except #148 (7.5%); absent from U-CLL subsets #5 and #59, and present in 19% and 17% of stereotyped subset #8 and #6 cases, respectively (p<0.05). We considered subset #2 separately since it included both M-CLL and U-CLL and identified a distinctive clinicobiological profile characterized by a constellation of features, including high incidence of Binet stage C disease (21%), CD38 positivity (41%) and certain cytogenetic abnormalities i.e. del(13q) (56%) and del(11q) (23%) and, in contrast, low frequency of del(17p) (4.5%). Notably, no differences were found between U-CLL versus M-CLL subset #2 cases regarding the incidence of these parameters. Survival analysis for time-to-first-treatment (TTFT) revealed that each stereotyped subset exhibited a distinct clinical course, different from that of other subsets even with similar IGHV gene mutational status and/or expressing the same IGHV gene. Interestingly, subsets #1 and #2 demonstrated a similar TTFT to that of del(17p) positive cases, while within subset #2 the clinical outcome was independent of IGHV mutational status. By integrating BcR IG stereotypy into the Döhner cytogenetic prognostication model, we found that subsets #1, #2 and #4, collectively accounting for ~7% of our series, and representing both U-CLL and M-CLL, constituted distinct clinical entities, with the latter exhibiting a particularly indolent disease even when compared to isolated del(13q) cases or cases with no cytogenetic aberrations. Finally, in multivariate analysis for TTFT, assignment to subset #2 emerged as a new independent unfavorable factor (p=0.002); furthermore, when restricting the analysis to Binet A cases, subset #2 exhibited the second highest hazard ratio after U-CLL. In conclusion, the molecular classification of CLL based on BcR IG stereotypy refines prognostication beyond IG mutational status and improves the Döhner hierarchical model. Hence, we suggest that a compartmentalized approach focusing on and comparing different subsets sheds light on CLL clinical behavior and biology and that BcR IG stereotypy represents a companion molecular diagnostic for personalized medicine in CLL. DisclosuresMontillo:Janssen: Honoraria.

Racial Variations in Disease Characteristics, Presentations, Treatments, and Outcomes in Chronic Lymphocytic Leukemia (CLL)

Introduction: Our current understanding of CLL biology and optimal therapies is based on clinical trials that have largely enrolled White patient populations. Whether these findings are applicable to Hispanic or non-White patients is unknown but critical to evaluate given the growing numbers of minority patients in the U.S. While retrospective studies have suggested inferior outcomes in non-White patients, prospective data on these disparities are lacking.Patients and Methods: We utilized the Mayo Clinic CLL database to better understand variations in disease characteristics, molecular differences, and treatment selections between non-Hispanic White patients and those not in this category (referred to hereafter as “other race”). After excluding 49 patients with missing diagnosis date and another 490 patients with missing race and/or ethnicity, the remaining 4104 cases within the registry were included in the analysis. Comparisons by race were made using Chi-square or Wilcoxon rank sum tests for qualitative or quantitative variables, respectively. Overall survival (OS) was calculated from diagnosis until death or last known alive date; time to first treatment (TTFT) was calculated from diagnosis until treatment date or date last known to be untreated. OS and TTFT were displayed via Kaplan-Meier plots, and differences in OS and TTFT by race were calculated using log-rank statistics. Individuals who were enrolled in early intervention trials (n=61) were excluded from analyses involving treatment.Results: In total, 4104 CLL patients were identified: 4011 (97.7%) non-Hispanic Whites and 93 (2.3%) other races. Median age at CLL diagnosis was younger for other race patients (59 vs. 63 years; p=0.002), while gender and stage distributions were comparable between both groups. Serum β-2-microglobulin and absolute lymphocyte counts at diagnosis were similar but more other race patients had elevated LDH (29.5% vs. 16.1%; p=0.02). Cytogenetic data by FISH were available for 1884 non-Hispanic Whites and 46 other race patients; poor-risk cytogenetics (17p and/or 11q deletions) were present in 23.9% of other race and 13.5% of non-Hispanic White patients, respectively (p=0.04). No statistically significant differences were observed for CD38, ZAP-70, or IGHV by raceTTFT was similar for both non-Hispanic Whites and other race patients (4.6 vs. 5.5 years, respectively; p=0.12). First-line treatment selection was similar for both groups (Table). . With a median follow-up of 12.1 years, median overall survival was 9.3 years for other race and 12.2 years in non-Hispanic White patients (P=0.15; Figure).Conclusions: This prospective data supports the notion that CLL might be biologically different between non-Hispanic White and other race patients with less favorable FISH present in the latter group. Despite the observation that TTFT and type of first therapy are similar between both groups, the trend towards shorter OS in other race patients argues that other factors might contribute to outcome differences, such as disease biology, access to care, co-morbidities, and socioeconomic considerations. Further studies with larger cohorts are warranted.Table:First-line treatment selection among treated individual by racialFirst-line TreatmentNon-Hispanic Whites(n=1875)Other race(n=41)Chemoimmunotherapy Purine analogue-based Alkylator-based675 (36.0) 495 (26.4) 180 (9.6)15 (35.6) 9 (22.0) 6 (14.6)Purine analogue -monotherapy157 (8.4)4 (9.8)Alkylator monotherapy610 (32.5)9 (22.0)PA+Alkylator41 (2.2)1 (2.4)Antibody alone190 (10.1)6 (14.6)Other202 (10.8)6 (14.6) [Display omitted] DisclosuresNabhan:Genentech: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Speakers Bureau. Kay:Celgene: Research Funding. Shanafelt:Genentech: Research Funding; GlaxoSmithKline: Research Funding; Celegene: Research Funding; Cephalon: Research Funding; Pharmacyclics/Jannsen: Research Funding; Hospira: Research Funding; Polyphenon E Int’l: Research Funding.

Novel Putative Driver Gene Mutations in Chronic Lymphocytic Leukemia (CLL): Results from a Combined Analysis of Whole-Exome Sequencing of 262 Primary CLL Samples

Unbiased high-throughput massively parallel sequencing methods have transformed the process of discovery of novel putative driver gene mutations in cancer. In chronic lymphocytic leukemia (CLL), these methods have yielded several unexpected findings, including the driver genes SF3B1, NOTCH1 and POT1. Recent analysis, utilizing down-sampling of existing datasets, has shown that the discovery process of putative drivers is far from complete across cancer. In CLL, while driver gene mutations affecting >10% of patients were efficiently discovered with previously published CLL cohorts of up to 160 samples subjected to whole exome sequencing (WES), this sample size has only 0.78 power to detect drivers affecting 5% of patients, and only 0.12 power for drivers affecting 2% of patients. These calculations emphasize the need to apply unbiased WES to larger patient cohorts.To this end, we performed a combined analysis of CLL WES data joining together our previously published cohort of 159 CLLs with data from 103 CLLs collected by the International Cancer Genome Consortium (ICGC). The raw sequencing reads from these 262 primary tumor samples (102 CLL with unmutated IGHV, 147 with mutated IGHV, 13 with unknown IGHV status) were processed together and aligned to the hg19 reference genome. Somatic single nucleotide variations (sSNVs) and indels were detected using MuTect. Subsequently, inference of recurrently mutated genes was performed using the MutSig algorithm. This method combined several characteristics such as the overall mutation rate per sample, the gene specific background mutation rate, non-synonymous/synonymous ratio and mutation clustering to detect genes that are affected by mutations more than expected by chance.This analysis identified 40 recurrently mutated genes in this cohort. This included 22 of 25 previously identified recurrently mutated genes in CLL. In addition, 18 novel candidate CLL drivers were identified, mostly affecting 1-2% of patients. The novel candidates included two histone proteins HIST1H1D and HIST1H1C, in addition to the previously identified HIST1H1E. Another was IKZF3, affected by a recurrent sSNV resulting in a p.L162R change in its DNA binding domain, in close proximity to a region recently identified as critical for lenalidomide resistance in multiple myeloma (MM). An additional recurrently mutated gene was nuclear RNA export factor 1 (NXF1), which along with previously known recurrently mutated genes (SF3B1, XPO1, DDX3X), highlights the importance of RNA processing to CLL biology. Finally, this search for putative CLL driver genes also identified ASXL1 and TRAF3, already characterized as drivers in acute myeloid leukemia and MM, respectively. Of the 59 of 262 samples for which RNA-seq data were available, 76% of the identified driver mutations were detected and thereby validated. Validation using RNAseq detection of driver mutations and targeted sequencing within the entire cohort are ongoing.The larger size of our cohort enabled the separate application of the somatic mutation discovery process to samples with mutated or unmutated IGHV. Among the 147 samples with mutated IGHV, only 5 driver genes (TP53, SF3B1, MYD88, CHD2, RANBP2) retained significance. In contrast, analysis of the 102 IGHV unmutated samples revealed a distinct and more diverse pattern of recurrently mutated genes (lacking MYD88 and CHD2, and including NOTCH1, RPS15, POT1, NRAS, EGR2, BRAF, MED12, XPO1, BCOR, IKZF3, MAP2K1, FBXW7 and KRAS). This extended cohort also allowed for better resolution of the clinical impact of those genetic variants with greater than 4% prevalence in the cohort. For example, samples with POT1 mutations were found to be associated with shorter time from sample to therapy compared with those with wild-type POT1 (P= 0.02).Our study demonstrates that with larger cohort size, we can effectively detect putative driver genes with lower prevalence, but which may nonetheless have important biological and clinical impact. Moreover, our interrogation shows that subset analysis can reveal distinct driver patterns in different disease subsets. In particular, the marked clinical difference between CLLs with mutated and unmutated IGHV may reflect the higher likelihood of the latter group to harbor a broader spectrum of driver mutations with a more complex pattern of co-occurrence. DisclosuresBrown:Sanofi, Onyx, Vertex, Novartis, Boehringer, GSK, Roche/Genentech, Emergent, Morphosys, Celgene, Janssen, Pharmacyclics, Gilead: Consultancy.

Activation of the PI3K/AKT pathway by microRNA-22 results in CLL B-cell proliferation.

Chronic lymphocytic leukemia (CLL) is characterized by accumulation of clonal B cells arrested in G0/G1 stages that coexist, in different proportions, with proliferative B cells. Understanding the crosstalk between the proliferative subsets and their milieu could provide clues on CLL biology. We previously identified one of these subpopulations in the peripheral blood from unmutated patients that appears to be a hallmark of a progressive disease. Aiming to characterize the molecular mechanism underlying this proliferative behavior, we performed gene expression analysis comparing the global mRNA and microRNA expression of this leukemic subpopulation, and compared it with their quiescent counterparts. Our results suggest that proliferation of this fraction depend on microRNA-22 overexpression that induces phosphatase and tensin homolog downregulation and phosphoinositide 3-kinase (PI3K)/AKT pathway activation. Transfection experiments demonstrated that miR-22 overexpression in CLL B cells switches on PI3K/AKT, leading to downregulation of p27(-Kip1) and overexpression of Survivin and Ki-67 proteins. We also demonstrated that this pathway could be triggered by microenvironment signals like CD40 ligand/interleukin-4 and, more importantly, that this regulatory loop is also present in lymph nodes from progressive unmutated patients. Altogether, these results underline the key role of PI3K/AKT pathway in the generation of the CLL proliferative pool and provide additional rationale for the usage of PI3K inhibitors.

Personalizing therapy for older adults with lymphoid malignancies: options and obstacles.

Increasing age is both a risk factor for and a negative prognostic factor in lymphoid malignancies. The disparities in outcomes between older and younger adults with lymphoid malignancies may reflect age-related differences in treatment and in biology of disease. Lymphomas in older adults are biologically more aggressive. Only small age-related differences in the frequency of cytogenetic abnormalities are seen in multiple myeloma. No major differences in the biology of chronic lymphocytic leukemia (CCL) are seen across the age spectrum. Chemotherapy and immunotherapy in older adults with lymphoid malignancies are marked by greater vulnerability to toxicity of therapy. Excessive toxicity can result in poorer outcomes, either directly through treatment-related mortality, or through decreased dose intensity. Thus, new approaches to predict toxicity of therapy and stratified treatment algorithms based on risk of toxicity are needed. Herein we detail some of the promising approaches to predicting toxicity and tailoring treatment for older adults with lymphoid malignancies.

How will B-cell-receptor–targeted therapies change future CLL therapy?

AbstractFor many years there has been considerable disassociation between the understood biology of chronic lymphocytic leukemia (CLL) and the therapeutics used to treat this disease. With the introduction of the first targeted CD20 antibody rituximab and its addition to chemotherapy came the first observation that minimal residual disease–negative (MRD-negative) complete responses (CRs) could be obtained with dramatically improved progression-free survival and overall survival. This advance was soon to be surpassed by the introduction of therapeutics that target B-cell receptor (BCR) signaling. New data show that BCR-inhibiting agents are very active for the treatment of relapsed CLL, despite the lack of MRD-negative CR, with durability of response being considerably more impressive than previously observed with other agents not producing MRD-negative CRs. This perspective provides a view of where these agents may take us in the future as CLL therapy evolves with this exciting new class of drugs.

The investigational agent MLN2238 induces apoptosis and is cytotoxic to CLL cellsin vitro, as a single agent and in combination with other drugs

Chronic lymphocytic leukaemia (CLL) is the most common haematological malignancy in the U.S. The course of the disease has been shown to be negatively impacted by increased levels of BCL2. Strategies to downregulate BCL2 and shift the balance towards cellular demise are actively being explored. Therefore, we examined whether the investigational agent MLN2238 could inhibit the proteasomal machinery and induce CLL cell death while also downregulating BCL2. MLN2238-induced cell death was studied in peripheral blood mononuclear cells from 28 CLL patients. MLN2238 produced a dose-dependent reduction in BCL2 and CLL cell viability with maximum cell death observed at a 50nmol/l concentration by 48h. Annexin-V staining, PARP1 and caspase-3 cleavage along with an increase in mitochondrial membrane permeability were noted after cells were treated with MLN2238; however, apoptosis was only partially blocked by the pan-caspase inhibitor z-VAD.fmk. Furthermore, we observed enhanced anti-CLL effects in tumour cells treated with either a combination of MLN2238 and the BH3 mimetic AT-101 or MLN2238 and fludarabine. Together, our data suggest the potential for proteasome inhibitor based therapy in CLL and the rationale design of drug combination strategies based on CLL biology.

Prognostic relevance of angiopoietin-2, fibroblast growth factor-2 and endoglin mRNA expressions in chronic lymphocytic leukemia.

Elevated levels of circulating angiogenic cytokines and increased expression of genes encoding angiogenic factors have been reported in recent years in patients with chronic lymphocytic leukemia (CLL) but data regarding prognostic and predictive significance are still limited. Therefore, in the present study based upon our prior pilot results, we measured mRNA expressions of angiopoietin-2 (Ang-2), fibroblast growth factor-2 (FGF-2) and endoglin (CD105) by reverse transcription quantitative PCR in purified CD19+ cells from 70 untreated CLL patients (median age, 63 years; males, 64%; Rai III/IV stages, 29 %; unmutated IgVH genes, 60 %) and evaluated their possible association with established prognostic factors and clinical course of the disease. Higher expression of Ang-2 was significantly associated with unmutated IgVH genes (n = 55, p = 0.003). Higher CD105 expression was significantly associated with unmutated IgVH genes (n = 55, p < 0.001), high CD38 expression (n = 66, p = 0.022), high ZAP-70 expression (n = 66, p = 0.010), Rai stage I-IV (n = 70, p < 0.001), progressive clinical course of CLL (n = 70, p = 0.001) and shorter time to treatment (n = 70; p < 0.001). Expression of FGF-2 was not significantly associated with any of the prognostic markers. These results indicate that elevated expression of Ang-2 and in particular CD105 by CLL cells is associated with unfavorable prognostic features and clinical outcome; thus, both cytokines appear to play an important role in biology and progression of CLL and warrant further investigation.