Clinical Chronic Lymphocytic Leukemia Research Articles

Autonomous BCR Signaling and Genetic Aberrations in CLL-Phenotype Monoclonal B Lymphocytosis in Siblings of CLL Patients

Clonal expansion of CD5-expressing B cells, termed monoclonal B lymphocytosis (MBL), is a precursor condition for chronic lymphocytic leukemia (CLL) with an increased incidence in CLL siblings. In app. 1% of MBL carriers, MBLprogresses eventually to clinically overt CLL. The mechanisms driving benign MBL expansion and subsequent malignant progression are unknown. Autonomous signaling of the clonotypic BCR of CLL cells, i.e. signaling without stimulation by cognate antigen, is an indispensable oncogenic signal in CLL. In order to investigate the early stages in CLL ontogeny, we sought to clarify whether MBL cells express BCR with autonomous signaling capacity and to compare the prevalence of inherited risk loci and acquired genetic aberrations in MBL-CLL sibling pairs. Peripheral blood of 191 siblings of CLL patients was screened with informed consent by six-color flow cytometry. A discrete CD19+CD5+CD20lowCD79low CLL-phenotype population of 0.2 to 1863 cells / µL blood was detected in 34 siblings (17.8%) of 26 CLL patients; 32 siblings (94%) had <500 clonal CLL-phenotype cells / µL. Clonal BCR were identified from highly purified MBL cells of 17 CLL siblings by ARTISAN PCR. Five MBL clones could be assigned to CLL stereotyped subsets, including two MBL BCR belonging to CLL subset #2 with expression of the IGLV3-21 gene with a G110R mutation and one IgG-expressing MBL belonging to CLL subset #4. Eleven MBL BCR, including all 5 assigned to a CLL stereotyped subset, were transduced into murine TKO cells arrested at the pro-B-cell stage by genetic deficiency of rag2, lambda5, and slp65. Slp65 function is reconstituted by a slp65-ERT2 fusion gene that enables BCR signaling in the presence of 4-hydroxytamoxifen. Calcium mobilization was measured in Indo-1 AM-loaded, live-gated TKO cells by flow cytometry as the ratio of signal intensities (SIR) at 485 and 405 nm prior and after tamoxifen supplementation and after BCR crosslinking with isotype-specific antibody. Robust calcium mobilization was observed in all MBL BCR-transduced TKO cells in the absence of external antigen and without experimental cross-linking, regardless of assignment to a CLL subtype. Calibrated quantification of this autonomous BCR signal, however, revealed lesser intensity in MBL BCR compared to the BCR of their CLL sibling (p=0.015, Wilcoxon's matched-pair signed rank test). BCR signaling strength was correlated to the degree of MBL clonal expansion (p=0.034). According to genotyping by single nucleotide polymorphism array, whole exome sequencing (WES), and targeted panel sequencing, a polygenic CLL risk score (PRS) of 24 risk loci was higher in both CLL and MBL siblings than in reference population data, but we found no evidence for a higher PRS in CLL cases in a matched-pair comparison to their MBL siblings. With respect to acquired genetic changes, quantitative analysis of SNP array data revealed recurrent CLL-associated copy number variations (CNV) at clonal levels in all of 11 informative CLL cases. In contrast, individual CNV were present in only a minor fraction of the highly purified MBL cells in six of ten samples (p=0.003; Fisher's exact test). Likewise, variant allele frequencies (VAF) of non-shared variants as detected by WES was significantly lower in MBL samples than in CLL cases (p<0.0001). In particular, subclonal variants defined by a VAF of 0.1-0.33 were more prevalent in MBL compared to CLL siblings (p=0.014), whereas clonal variants (VAF 0.43-0.55) were equally abundant (p=0.65) in both conditions. In this comprehensive comparison of MBL and CLL in siblings, CLL risk alleles were found with high and similar prevalence in CLL patients and MBL siblings, suggesting that CLL risk loci predispose to clonal expansion of CLL phenotype cells in both low-count MBL and CLL. Vice versa, we did not find evidence for a particular association of CLL risk alleles with malignant progression from MBL to clinical CLL. Autonomous BCR signaling operates in both MBL and CLL as predicted by BCR stereotype, albeit at lesser strength in MBL. Acquired CLL-genetic changes, both copy number and sequence variants, are frequently subclonal in MBL but not in CLL. These findings support a stepwise CLL pathogenetic model wherein autonomous BCR signaling causes a non-malignant expansion of clonal CD5+ B cells in genetically predisposed individuals, followed by malignant progression to CLL after gradual acquisition of pathogenic genetic variants.

NOTCH1 Aberrant Activation Is a Common Nonmutational Early Event in Chronic Lymphocytic Leukemia Hematopoietic Stem-Cells

Chronic lymphocytic leukemia (CLL) is a B cell disorder characterized by constitutive NOTCH1 activation. NOTCH1 mutations are recurrently associated with CLL providing a new clonal marker to track CLL origin. To investigate CLL-initiating cells, we assessed NOTCH1 mutational status and signaling in hematopoietic stem (HSCs) and progenitor cells from CLL bone marrow. We collected a total of 29 bone marrow (BM) samples including 22 CLL patients and 7 healthy donors (HDs). BM cells were sorted into CD34+CD38- HSCs (purity 94.23% ± 3.04% ) and CD34+CD38+ progenitor fraction (98.12% ± 1.34%). While Sanger sequencing analysis of the NOTCH1 failed to detect alterations in CD34+/CD38- HSCs, AS-PCR and Droplet digital PCR (ddPCR) indicated the presence of small HSCs mutated clones in 57% of cases. Altogether, these data confirm that NOTCH1 mutation is an early event in CLL hematopoiesis. The analysis of CD34+/CD38+ progenitors detected the NOTCH1 mutation in the majority of samples (66%). The NOTCH1 mutational burden progressively increased along specific stages of HSC differentiation (6.4%± 4.7 in CD34+CD38- to 14.9%±11.3 in CD34+CD38+CD10+CD19+ cells, 22.7%±6.5 in CD34-CD38+CD10+CD19+ cells and 40.5%±4.3 in neoplastic CD5+CD19+ cells). This suggests that the NOTCH1 lesion is selected and expands during HSC differentiation toward a B neoplastic cell, thus strengthening the hypothesis that the genetic alteration is an initial event associated with the stepwise malignant transformation of CLL. Conversely, CD34+ cells (1x106) from CLL patients carrying the mutation, when transplanted into NOD/SCID/IL2Rgnull (NSG) mice generated a mature CLL phenotype (the median percentage of CD5+CD19+ cells in hCD45+ cells was 38.5% and 43.8% in BM and spleen respectively) lacking NOTCH1 mutation (as showed by AS-PCR assay performed on enriched hCD45 cells). This data is more in line with the hypothesis that this genetic abnormality is acquired at the mature B cell stage as an additional leukemogenic event to transform into clinical CLL. Thus, we analyzed the NOTCH1 signaling status in HSCs and progenitor cells of NOTCH1-mutated and unmutated CLL samples. The flow-cytometric analysis of the active NOTCH1-ICN protein level showed unmutated and mutated CLL has a significantly higher NOTCH1-ICN level than HDs samples in both CD34+/CD38- HSCs and CD34+CD38+ (73.4%±22.9 and 83%±16.4 vs 33.3%±14.8; 94.4%±7.3 and 92.8%±4.3 vs 47.9%±13.8, p<0.01 and p<0.001, respectively). c-MYC expression was found significantly higher in HSCs cells from NOTCH1 mutated and unmutated CLL samples compared to HD (3.5±0.7 and 2.6±0.08 vs 1.3±0.1). Western blot analysis of the expression levels of the NOTCH1-TM subunit revealed that HSCs from mutated and unmutated CLL patients always expressed the NOTCH1-TM protein, compared to HDs where NOTCH1-TM was either absent or expressed at lower levels. The present study indicated that the pool of CD34+ cells, including HSC and progenitor compartments, have NOTCH1 aberrantly expressed and activated in CLL patients compared to HDs demonstrating a common nonmutational NOTCH1 activation occurring early in CLL hematopoiesis. This selective pressure might contribute to the onset of specific NOTCH1 mutations in a DNA context that is prone to spontaneous microdeletion. These data represent a rationale for the use of therapies targeting the NOTCH1 signaling in CLL aimed to inhibit the survival of CLL-initiating cells. DisclosuresNo relevant conflicts of interest to declare.

Factors Associated with Non-Melanoma Skin Cancer (NMSC) Among Chronic Lymphocytic Leukemia (CLL) Patients

Background: The incidence of NMSC in patients with CLL is significantly higher compared to age- and sex- matched controls. Herein, we evaluate factors associated with risk of NMSC post-CLL diagnosis, including CLL clinical and prognostic characteristics, history of UV radiation exposure, severe sunburn, and combined genetic score of single nucleotide polymorphisms (SNPs) associated with NMSC.Methods: CLL patients newly diagnosed from 2002-2015 were enrolled and systematically followed by the Mayo Clinic/University of Iowa Lymphoma SPORE. Clinical and prognostic CLL data were obtained from the Mayo Clinic CLL database. NMSCs were abstracted both from medical records and from patient self-report. Self-reported history of the extent of mid-day sun exposure (h/week) at various ages (<13, 13-21, 22-40, >40 yrs) and severe sunburn were obtained from a risk factor questionnaire. The CLL International Prognostic Index (CLL-IPI) was computed as established using a weighted average of five independent CLL prognostic factors (IGHV mutational status, serum b2-microglobulin, Rai stage, age at CLL diagnosis and FISH 17p deletion/ TP53 status). CLL treatment was classified as positive if a person received T-cell immunosuppressive treatments that are purine analogue and/or alemtuzumab based treatments. All other therapies were combined into an “other category”. We computed a polygenetic risk score (PRS), a weighted average of the number of risk alleles across 13 SNPs that are known to be associated with NMSC, with the weights being the log of the odds ratio reported for each SNP. To evaluate associations with risk of NMSC post-CLL diagnosis we used Cox regression to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). CLL treatment was considered a time-dependent covariate in the analysis.Results: Among 1269 CLL patients, the median age at diagnosis was 63 years (range 24-91), 68% were male, and 6% had Rai stage III-IV at diagnosis. Based on the CLL-IPI, 44% were low risk, 34% intermediate risk, and 22% high/very-high risk. 9% of the CLL cases had prior history of NMSC and 44% reported history of severe sun burn. At a median follow-up of 7 years from CLL diagnosis, 196 patients had ≥1 NMSCs post-CLL diagnosis. Among these patients, 45 also had NMSC before CLL diagnosis. The most frequent NMSC was squamous cell carcinoma (63%), followed by basal cell (55%), and Merkel cell (1%). 264 patients received T-cell immunosuppressive treatments prior to NMSC diagnosis or prior to last follow up. Significant associations of clinical and prognostic characteristics with risk of NMSC post-CLL diagnosis were observed for age (HR=1.42 per 10 year increase, CI=1.24-1.62), male sex (HR=1.43, CI=1.04-1.97), prior history of NMSC (HR=3.85, CI=2.75-5.39), and CLL-IPI (HR=1.36, CI=1.13-1.64, adjusted for sex and prior NMSC). Mid-day sun exposures for each of the age groups considered showed no evidence of association with risk of NMSC post-CLL diagnosis; however, history of severe sunburn was significantly associated (HR=1.58, CI=1.06-2.36, adjusted for sex, CLL-IPI and prior NMSC). We observed an association between T-cell immunosuppressive treatments with risk of NMSC post-CLL diagnosis (HR=1.65, CI=1.14-2.40, adjusted for age, sex and prior NMSC). PRS was also associated with the risk of NMSC post-CLL diagnosis (HR=1.82, CI=1.21-2.72, adjusted for sex, CLL-IPI and prior NMSC).Conclusion: Screening for NMSC is important in CLL patients, and our data suggest that it is particularly important among patients with aggressive CLL according to CLL-IPI score, those receiving T-cell immunosuppressive treatments that are purine analogue and/or alemtuzumab based treatments, those who have a prior history of NMSC, or history of severe sunburn. PRS was also a significant factor in the risk of NMSC among CLL patients. Future studies should develop prediction models that include these factors. [Display omitted] DisclosuresKay:Tolero Corporation: Research Funding; Gilead: Research Funding; Agios: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Research Funding. Shanafelt:Genentech, Celgene, Pharamacyclics, Jansen, GlaxoSmithKline, AbbVie, Hospira, and Cephalon: Research Funding. Cerhan:Janssen: Other: Multiple Myeloma Registry Steering ; Janssen: Other: Scientific Advisory Board (REMICADELYM4001).

Delineation of clinical and biological factors associated with cutaneous squamous cell carcinoma among patients with chronic lymphocytic leukemia

The incidence of cutaneous squamous cell carcinoma (SCC) in patients with chronic lymphocytic leukemia (CLL) is significantly higher compared with age- and sex-matched controls. To evaluate the association of factors associated with SCC risk. Clinical CLL and SCC data were obtained from Mayo Clinic CLL Resource and self-reported questionnaires among patients with newly diagnosed CLL. We computed the CLL International Prognostic Index (CLL-IPI) from CLL prognostic factors, and a polygenic risk score from SCC susceptibility variants. We used Cox regression to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). Among 1269 patients with CLL, the median follow-up was 7years, and SCC subsequently developed in 124 patients. Significant associations with SCC risk were history of skin cancer (HR=4.80; 95% CI: 3.37-6.83), CLL-IPI (HR=1.42; 95% CI: 1.13-1.80), and polygenic risk score (HR=2.58; 95% CI: 1.50-4.43). In a multivariable model, these factors were independent predictors (C statistic=0.69; 95% CI: 0.62-0.76). T-cell immunosuppressive treatments were also associated with SCC risk (HR=2.29; 95% CI: 1.47-3.55; adjusted for age, sex, and prior SCC). The sample size decreases when combining all risk factors in a single model. SCC risk includes history of skin cancer, an aggressive disease at time of CLL diagnosis, receiving T-cell immunosuppressive treatments, and high polygenic risk score. Future studies should develop prediction models that include these factors to improved screening guidelines.

PRIMA-1MET cytotoxic effect correlates with p53 protein reduction in TP53-mutated chronic lymphocytic leukemia cells

TP53 gene defects represent the most unfavorable prognostic factor in chronic lymphocytic leukemia (CLL). Although recently introduced small-molecule B-cell receptor signalling inhibitors have revolutionized CLL treatment, data for ibrutinib still point to impaired prognosis for TP53-affected patients. Among cancer-associated TP53 mutations, missense substitutions predominate and typically result in a high mutated-p53 protein level. Therefore, rescuing the p53 tumor suppressor function through specific small molecules restoring p53 wild-type (wt) conformation represents an attractive therapeutic strategy for cancer patients with TP53 missense mutations. We tested the effect of mutated-p53 reactivating molecule PRIMA-1MET in 62 clinical CLL samples characterized for TP53 mutations and p53 protein level. At the subtle PRIMA-1MET concentrations (1–4 μM), most samples manifested concentration-dependent viability decrease and, conversely, apoptosis induction, with the response being similar in both the TP53-mutated and TP53-wt groups, as well as in the TP53-mutated samples with p53 protein stabilization and without it. PRIMA-1MET was able to reduce mutated p53 protein in a proportion of TP53-mutated CLL samples, and this reduction correlated with a significantly stronger viability decrease and apoptosis induction than samples with stable p53 levels. CLL cells are mostly sensitive to PRIMA-1MET apart from those with stable mutated p53.

BTK and/or PLCG2 Mutations in Patients with Chronic Lymphocytic Leukemia (CLL) Treated with Ibrutinib: Characteristics and Outcomes at the Time of Progression

INTRODUCTION Mutations in BTK and PLCG2 have been reported to occur in ~80% of CLL patients (pts) who have progression of disease on ibrutinib therapy (Woyach, JCO 2017; Ahn, Blood 2017). These mutations are described as appearing months before actual relapse and thus considered as a potential predictive biomarker for future relapse (Quinquenel, Blood 2019). However, the outcomes of these pts after disease progression are not well described. In this study, we seek to investigate time to next therapy and overall survival (OS) following progression among CLL pts on ibrutinib therapy with and without these resistance mutations. METHODS Between 10/2012 and 6/2019, we identified 34 pts in the Mayo Clinic clinical CLL resource who progressed while receiving ibrutinib therapy and also had testing for BTK and PLCG2 mutation performed as part of routine clinical practice at either NeoGenomics Laboratories or The Ohio State University. OS was calculated from time of ibrutinib progression to last known alive or death date; OS was plotted using Kaplan Meier methods and was compared using the log-rank test between various groups (e.g., mutation positive vs negative; CLL progression vs Richter's). Cumulative incidence of time to next treatment in those who had a treatment after progression was adjusted for the competing risk of death. RESULTS Of 34 pts who progressed while receiving ibrutinib, 26 pts experienced CLL progression and 8 pts had Richter's transformation; baseline characteristics in Table 1A. The presence of a BTK or PLCG2 mutation was found in 20/34 (59%) pts (specific mutations in Table 1B). BTK mutation alone was present in 9 pts, 7 pts had PLCG2 mutation alone, and 4 pts had both mutations. Median time between a positive test and start of next therapy was 4 months (range 1-19 months) and did not vary between BTK vs PLCG2 mutations. Among the 26 pts with CLL progression, 18 (69%) pts had a mutation present: BTK alone (n=8), PLCG2 alone (n=6), both (n=4). Therapy following progression on ibrutinib in these pts was as follows: venetoclax (n=16; 11 pts who continued ibrutinib in combination), idelalisib (n=4), investigational treatments (n=2), continued ibrutinib alone (n=2), dose-adjusted EPOCH-R (etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab; n=1), and unknown (n=1). Twelve of the 26 pts with CLL progression on ibrutinib, including 8 pts with a prior resistance mutation detected, had subsequent progression of disease on the aforementioned next line therapy. Treatment of these patients consisted of the following: restarted ibrutinib in addition to current treatment of venetoclax (n=5), venetoclax (n=2), pembrolizumab (n=2; 1 pt with continued ibrutinib), obinutuzumab with continued ibrutinib (n=1), gemcitabine and vinorelbine with continued ibrutinib (n=1), and no further treatment (n=1). Among the 8 pts with Richter's transformation as the initial progression event on ibrutinib after mutation testing, 1 pt had a BTK mutation and 1 pt had a PLCG2 mutation. Treatments following progression on ibrutinib included multi-agent chemoimmunotherapy (n=3; 2 pts received rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone [R-CHOP] with continued ibrutinib and 1 pt received doxorubicin, bleomycin, vinblastine, dacarbazine [ABVD] alone), pembrolizumab (n=3; 1 pt in combination with continued ibrutinib), venetoclax in combination with continued ibrutinib (n=1), and venetoclax and obinutuzumab (n=1). The median time to next treatment (second line of treatment beyond ibrutinib) for the 31 pts who started another therapy following progression on ibrutinib was 16.7 months (95% CI 9.6-NE; Figure 1A) and was not significantly different for pts with or without a resistance mutation (p=0.57). Median OS for all 26 pts with CLL progression was 28.7 month and there was no difference according to presence or absence of a resistance mutation (median 28.7 months vs 18.2 months, p=0.53; Figure 1B). The 8 pts with Richter's transformation had a median OS of 7.1 months (95% CI 2.0-NE). CONCLUSION Approximately 60% of pts tested in this progression cohort had a BTK or PLCG2 mutation at time of or preceding progression on ibrutinib therapy. OS and time to next therapy did not differ statistically between pts with mutated vs non-mutated clones; however, caution should be applied with the conclusions given the limited sample size. Disclosures Ding: DTRM Biopharma: Research Funding; Merck: Research Funding. Kenderian:Novartis: Patents &amp; Royalties, Research Funding; Tolero: Research Funding; Humanigen: Other: Scientific advisory board , Patents &amp; Royalties, Research Funding; Lentigen: Research Funding; Morphosys: Research Funding; Kite/Gilead: Research Funding. Kay:MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB. Parikh:Ascentage Pharma: Research Funding; Genentech: Honoraria; Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding.

Complex karyotype as a predictor of high-risk chronic lymphocytic leukemia: A single center experience over 12 years

ObjectivesA complex karyotype (CK) is considered a poor prognostic marker in chronic lymphocytic leukemia (CLL). MethodsThe study analyzed 644 untreated CLL patients (pts) using conventional/molecular cytogenetics to reveal the presence of a CK and its composition and to assess its predictive value. The mutational status ofTP53 was detected by next generation sequencing. ResultsA CK was detected in 79 pts (12.3%). Patients with a CK showed shorter overall survival (OS) compared to those without a CK (77 months vs. 115 months, p < 0.0001). Chromosomes most frequently included in a CK were 13, 11, 17, 8, 2, and 6. The most common aberrations in a CK were translocations, numerical changes and dicentric chromosomes (with no effect on OS). Patients with aberrations ofTP53 and ATM were shown to have adverse prognosis comparable to patients with a CK without these abnormalities. A stronger impact of a CK on OS of female and older CLL patients was observed. ConclusionsThe determining of the presence of a CK is essential in modern clinical CLL practice. According to recent studies, the presence of a CK affects clinical and treatment decision-making.

DNA methylation profiling implicates exposure to PCBs in the pathogenesis of B-cell chronic lymphocytic leukemia

ObjectivesTo characterize the impact of PCB exposure on DNA methylation in peripheral blood leucocytes and to evaluate the corresponding changes in relation to possible health effects, with a focus on B-cell lymphoma. MethodsWe conducted an epigenome-wide association study on 611 adults free of diagnosed disease, living in Italy and Sweden, in whom we also measured plasma concentrations of 6 PCB congeners, DDE and hexachlorobenzene. ResultsWe identified 650 CpG sites whose methylation correlates strongly (FDR < 0.01) with plasma concentrations of at least one PCB congener. Stronger effects were observed in males and in Sweden. This epigenetic exposure profile shows extensive and highly statistically significant overlaps with published profiles associated with the risk of future B-cell chronic lymphocytic leukemia (CLL) as well as with clinical CLL (38 and 28 CpG sites, respectively). For all these sites, the methylation changes were in the same direction for increasing exposure and for higher disease risk or clinical disease status, suggesting an etiological link between exposure and CLL. Mediation analysis reinforced the suggestion of a causal link between exposure, changes in DNA methylation and disease.Disease connectivity analysis identified multiple additional diseases associated with differentially methylated genes, including melanoma for which an etiological link with PCB exposure is established, as well as developmental and neurological diseases for which there is corresponding epidemiological evidence. Differentially methylated genes include many homeobox genes, suggesting that PCBs target stem cells. Furthermore, numerous polycomb protein target genes were hypermethylated with increasing exposure, an effect known to constitute an early marker of carcinogenesis. ConclusionsThis study provides mechanistic evidence in support of a link between exposure to PCBs and the etiology of CLL and underlines the utility of omic profiling in the evaluation of the potential toxicity of environmental chemicals.

Discovery of a Novel Five Immune-Associated Fingerprints for Classification and Survival Prediction in Chronic Lymphocytic Leukemia

IntroductionCurrent staging methods do not accurately predict clinical outcome of patients with chronic lymphocytic leukemia (CLL) especially in the new era of immunotherapy. Recent studies suggested that immune-related gene signature predicted survival in glioblastoma, breast cancer and colorectal cancer. However, none related studies have been elucidated in CLL. Here, we hypothesized that risk-prediction model integrated with the immune signature could serve as an effective prognostic indicator in CLL. This study aimed to identify reliable and distinct immune-associated fingerprints for robust classification and survival prediction in patients with CLL.MethodsA total of 720 de novo CLL patients from multiple cohorts were enrolled with informed consents in the present study. LASSO Cox regression model was utilized to calculate immune-associated risk score (I-score) in R software. Principal Component Analysis (PCA) were performed to present the distribution of risk score. Moreover, the prognostic capability of the five immune-related fingerprints was demonstrated by PCR and ROC curve analysis with leave-one-out cross validation in the training set. Functional enrichment analyses of GO and KEGG in gene expression datasets were performed. Association between I-score and hallmark gene sets from the Molecular Signatures Database (MSigDB) were analyzed using GSEA software. Furthermore, preclinical experiments were conduct to examine the pathological mechanism of constitutive genes of I-score in CLL cell lines (MEC1, EHEB) and primary cells. Additionally, genomic regulatory network was displayed in Cytoscape software.ResultsIn the present study, we performed a comprehensive analysis to dissect the immune-associated fingerprints in CLL. A total of 305 clinical annotated CLL patients and 56 healthy donors with gene expression data were obtained from three independent cohorts. Two gene sets (immune system process, M13664 and immune response, M19817) were extracted from the MSigDB and combined to integrate the immune-related gene set containing 322 genes. Illustrated in the volcano plots, differentiated expressed genes of CLL cells comparing with normal B cells were calculated by limma test (|Log2Fold Change|>1, p<0.01; Figure 1A-C). Venn diagram was delineated to generate the specific CLL immune-associated gene expression panel, with 8 genes were identified (Figure 1D). PCA showed a different distribution pattern, confirming the enhanced immune phenotype in CLL.Then, we further investigate the association of the immuno-signature with clinical outcomes of CLL patients. By Lasso Cox regression analysis, the prognostic immune-related fingerprints were identified in the training set (Figure 2A). The risk score method was established: I-score = (-0.538)*CD3D expression+(-0.077)*CD83 expression+0.364*LAX1 expression+0.191*IL2RA expression+0.362*AIM2 expression, consisting of protective genes (CD3D, CD83) and risky genes (LAX1, IL2RA and AIM2; Figure 2B).Based on the median level of I-score as cut-off value, stratified high-risk patients were observed with significantly shorter overall survival compared with the low-risk group (Hazard Ratio, HR=5.493, p<0.001; Figure 3A). Univariate and multivariate cox regression analyses confirmed high I-score as an independent prognosis biomarker in CLL patients. To reduce disparity of diverse populations, we evaluated I-score in two independent centers of US and Germany with the same formula. The prognostic value of the immune fingerprints were corroborated in the internal validation series (HR=2.200, p=0.023; Figure 3B) and validation series (HR=1.769, p=0.004; Figure 3C). Intriguingly, we also observed the significant correlation between high I-score and 17p13 deletion (p=0.0316; Figure 3D), in accordance with patients' inferior outcome. Moreover, functional enrichment analyses of differentiated expressed genes stratified by I-score indicated that immune related BCR signaling pathway contributed to pathogenesis of CLL (Figure 4).ConclusionTo date, our study provides evidence for the first time that distinct immuno-related fingerprints predict survival in CLL. I-score is demonstrated as an efficient classification tool and robust method for prognosis evaluation, greatly facilitating risk stratification and individualized management of CLL patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Evolving DNA methylation and gene expression markers of B-cell chronic lymphocytic leukemia are present in pre-diagnostic blood samples more than 10\xa0years prior to diagnosis

BackgroundB-cell chronic lymphocytic leukemia (CLL) is a common type of adult leukemia. It often follows an indolent course and is preceded by monoclonal B-cell lymphocytosis, an asymptomatic condition, however it is not known what causes subjects with this condition to progress to CLL. Hence the discovery of prediagnostic markers has the potential to improve the identification of subjects likely to develop CLL and may also provide insights into the pathogenesis of the disease of potential clinical relevance.ResultsWe employed peripheral blood buffy coats of 347 apparently healthy subjects, of whom 28 were diagnosed with CLL 2.0–15.7 years after enrollment, to derive for the first time genome-wide DNA methylation, as well as gene and miRNA expression, profiles associated with the risk of future disease. After adjustment for white blood cell composition, we identified 722 differentially methylated CpG sites and 15 differentially expressed genes (Bonferroni-corrected p < 0.05) as well as 2 miRNAs (FDR < 0.05) which were associated with the risk of future CLL. The majority of these signals have also been observed in clinical CLL, suggesting the presence in prediagnostic blood of CLL-like cells. Future CLL cases who, at enrollment, had a relatively low B-cell fraction (<10%), and were therefore less likely to have been suffering from undiagnosed CLL or a precursor condition, showed profiles involving smaller numbers of the same differential signals with intensities, after adjusting for B-cell content, generally smaller than those observed in the full set of cases. A similar picture was obtained when the differential profiles of cases with time-to-diagnosis above the overall median period of 7.4 years were compared with those with shorted time-to-disease. Differentially methylated genes of major functional significance include numerous genes that encode for transcription factors, especially members of the homeobox family, while differentially expressed genes include, among others, multiple genes related to WNT signaling as well as the miRNAs miR-150-5p and miR-155-5p.ConclusionsOur findings demonstrate the presence in prediagnostic blood of future CLL patients, more than 10 years before diagnosis, of CLL-like cells which evolve as preclinical disease progresses, and point to early molecular alterations with a pathogenetic potential.

Exploring the nature of prediagnostic blood transcriptome markers of chronic lymphocytic leukemia by assessing their overlap with the transcriptome at the clinical stage

BackgroundWe recently identified 700 genes whose expression levels were predictive of chronic lymphocytic leukemia (CLL) in a genome-wide gene expression analysis of prediagnostic blood from future cases and matched controls. We hypothesized that a large fraction of these markers were likely related to early disease manifestations. Here we aim to gain a better understanding of the natural history of the identified markers by comparing results from our prediagnostic analysis, the only prediagnostic analysis to date, to results obtained from a meta-analysis of a series of publically available transcriptomics profiles obtained in incident CLL cases and controls.ResultsWe observed considerable overlap between the results from our prediagnostic study and the clinical CLL signals (p-value for overlap Bonferroni significant markers 0.01; p-value for overlap nominal significant markers < 2.20e-16). We observed similar patterns with time to diagnosis and similar functional annotations for the markers that were identified in both settings compared to the markers that were only identified in the prediagnostic study. These results suggest that both gene sets operate in similar pathways.ConclusionAn overlap exists between expression levels of genes predictive of CLL identified in prediagnostic blood and expression levels of genes associated to CLL at the clinical stage. Our analysis provides insight in a set of genes for which expression levels can be used to follow the time-course of the disease; providing an opportunity to study CLL progression in more detail in future studies.

Skin Cancers Among Chronic Lymphocytic Leukemia (CLL) Patients - the Effect of UV Radiation and CLL Clinical Characteristics

Background: It is well established that the incidence of skin cancers in patients with CLL are significantly elevated compared to age- and sex- matched controls. However, little is known about the characteristics of CLL patients who develop skin cancer. Herein, we evaluate the associations of CLL clinical and prognostic characteristics, along with UV radiation exposure, with risk of first skin cancer following CLL diagnosis.Methods: Newly diagnosed CLL patients from Minnesota, Iowa, and Wisconsin were enrolled in the Mayo Clinic case-control study from 2002-2015 and systematically followed in the Iowa/Mayo Lymphoma SPORE. Clinical and prognostic CLL data were obtained from the Mayo Clinic CLL database, and skin cancer clinical data were abstracted from medical records using a standard protocol. The CLL international prognostic index (CLL-IPI) was computed using a weighted average of five independent CLL prognostic factors (IGHV mutational status, serum b2-microglobulin, Rai stage, age, and FISH 17p deletion/TP53 status). Self-reported history of midday sun exposure at various ages (birth to age 12; 13 to 21 years; 22 to 40 years, and 41+ years) was obtained from a risk factor questionnaire. For each age, we asked the extent of mid-day sun as: practically no exposure (under 3 hours per week), little exposure (4-7 hours per week), moderate exposure (8 to 14 hours per week) and extensive exposure (15+ hours per week). Midday sun exposure was modeled as an ordinal covariate. To evaluate associations with risk of skin cancer following CLL diagnosis, we calculated time from date of CLL diagnosis to date of first skin cancer, death, or last known follow-up. We used Cox regression analysis to estimate hazard ratios (HRs) and 95% CIs. CLL treatment was considered a time-dependent covariate.Results: Among 846 CLL patients enrolled, the median age at diagnosis was 63 years (range 28-91), 68% were male, 7% had Rai stage III-IV at diagnosis. Based on the CLL-IPI, 42% were categorized as low risk, 33% as intermediate risk, and 25% as high or very-high risk. 109 CLL cases (13%) had one or more reported skin cancers at or prior to CLL diagnosis. Melanoma was observed in 19 (2%) cases and non-melanoma was in 90 (11%) cases. At a median follow-up of 7 years from CLL diagnosis, 165 patients (20%) had one or more skin cancers after CLL diagnosis. Among these patients, 49 had skin cancer before CLL diagnosis. The most frequent skin cancer was squamous cell carcinoma (59%), followed by basal cell (31%), melanoma (5%), and Merkel cell (1%). 552 (65%) of the 846 patients returned a questionnaire. Significant associations of clinical and prognostic characteristics with risk of first skin cancer were observed for age (HR=1.35 per 10 year increase, 95% CI=1.17-1.56, P<0.001), male sex (HR=1.38, 95% CI 0.98-1.96, p=0.07), prior history of skin cancer (HR=4.19, 95% CI=2.98-5.88, P<0.001), and CLL-IPI (HR=1.26, 95% CI= 1.03-1.54, P=0.026, after adjusting for age, sex, and prior skin cancer). Of note, the risk of first skin cancer in those CLL patients categorized as very high via CLL-IPI had 2.28 fold risk (95% CI 1.02-5.11). Midday sun exposures for each of the ages considered showed no evidence of association with risk of first skin cancer (all P>0.05). 50 CLL patients were treated prior to first skin cancer following CLL diagnosis; we observed no evidence of association between treatment and risk of first skin cancer (HR=1.44, 95% CI= 0.93-1.92, P=0.12).Conclusion: CLL patients who are at an increased risk of skin cancer following CLL diagnosis are those who either have had a prior history of skin cancer or a more aggressive CLL disease at diagnosis, according to CLL IPI. Routine skin cancer screening is currently recommended for CLL patients. Our data suggest that more frequent screening would be particularly important among patients with aggressive CLL and who have a prior history of skin cancer. Unexpectedly, we found no evidence of association of skin cancer risk with UV radiation following CLL diagnosis or with CLL treatment. Further investigation is needed to determine whether other factors increase the risk of skin cancer following CLL diagnosis. DisclosuresShanafelt:Genentech: Research Funding; GlaxoSmithkKine: Research Funding; Janssen: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding.

Abstract 4737: Comprehensive profiling of immunoglobulin sequences using hybrid capture-based next generation sequencing in B-cell hematologic malignancies

Abstract Background: Sequencing the genes encoding immunoglobulins is critical in detecting clonal cell populations as well as determining prognosis and therapeutic decisions in patients with lymphoid malignancies including B-cell leukemias, lymphomas and multiple myeloma. Current assays for identifying the rearranged immunoglobulin sequence in B-cell malignancies rely on sequence specific PCR based amplification of conserved immunoglobulin (IG) regions. We have developed a novel, hybrid capture-based approach to sequencing the immunoglobulin chains that enables identification of the heavy and light chain variable domains, complementarity-determining region (CDR) sequences and somatic hypermutation (SHM) status. Method: RNA and DNA were successfully extracted from 60 specimens, including 7 mantle cell lymphoma cell (MCL) lines and 53 clinical chronic lymphocytic leukemia (CLL) bone marrow aspirates. Adaptor-ligated DNA and cDNA sequencing libraries were captured by solution hybridization using custom bait-sets targeting the immunoglobulin variable, joining and class segments. All captured libraries were sequenced to high depth (Illumina HiSeq) in a CLIA-certified laboratory (Foundation Medicine). Results: The capture-based approach was validated using 7 MCL cell lines and 53 CLL samples profiled using a CLIA-certified commercial PCR-based assay (Invivoscribe). The immunoglobulin sequences derived from the 7 MCL cell lines were 100% concordant with identifying the published heavy and light chain variable domain and percent SHM. Comparison to 53 previously clinically tested samples showed 98% (52/53) concordance for identifying the presence of a clonal population, and 100% (39/39) concordance for identifying the IGHV domain. Comparison to 50 CLL samples previously tested for SHM resulted in 96% (48/50) overall concordance with 94% (31/33) concordant for no SHM and 100% (17/17) concordant for the presence of SHM. Additionally, secondary clones were identified in 11 samples. Conclusions: We have demonstrated that hybrid capture-based targeted DNA and RNA sequencing can be used to comprehensively characterize the immunoglobulin sequence of clonal tumor B cell populations. This capability enables quantification of SHM and identification of the variable domain, CDR3 sequence and class restriction. Integration of this methodology with comprehensive genomic profiling approaches will expand the clinical utility of such assays in patients with hematological malignancies and may provide important insights in immune oncology and response of patients to immunotherapies, including patients with solid tumors. Citation Format: Michelle K. Nahas, Lauren E. Young, Jeff Gardner, Omar Abdel-Wahab, Jie He, Amy L. Donahue, Kristina M. Knapp, Geoff A. Otto, Doron Lipson, Vincent A. Miller, Ross L. Levine, Philip J. Stephens. Comprehensive profiling of immunoglobulin sequences using hybrid capture-based next generation sequencing in B-cell hematologic malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4737. doi:10.1158/1538-7445.AM2015-4737

Somatic Hypermutation and V-J Gene Usage for CLL Prognosis: Evaluating Data from Miseq NGS Vs PCR-Sanger Sequencing Approaches

Introduction: Somatic hypermutation (SHM), an important process that evolved to increase the affinity of immunoglobulin molecules, occurs in the germinal center during B-cell maturation. The presence of SHM, defined as ≥ 2% difference from the germline sequence of the immunoglobulin heavy chain variable (IGHV) region genes, provides important prognostic information for patients with chronic lymphocytic leukemia (CLL), as leukemic clones that fail to undergo SHM are more aggressive tumors. In addition, presence of the V3-21 variable segment portends a worse prognosis independent of mutation status or other prognostic indicators. The gold standard method used to determine SHM status and presence of V3-21 rearrangements requires two steps: a PCR/capillary electrophoresis (CE) based method to detect clonality, followed by Sanger sequencing. This multistep approach is labor intensive and time consuming.In order to perform a clinical evaluation of the relative merits of NGS and Sanger sequencing approaches, DNA was tested from anonymized, blinded clinical samples obtained from the University of Bologna (n=62).Methods: We examined a next-generation sequencing (NGS) approach for determining SHM status for IGHV region and compared these results to results from PCR-based clonality testing using the BIOMED-2 IGH FR1 master mixes, followed by gel extraction and Sanger sequencing. The MiSeq SHM Assay employs two master mixes. One amplifies genomic DNA between the leader (VHL) region and the joining (J) region. The other amplifies from the framework1 (FR1) to J region. The NGS assay design of V and J consensus primers allowed PCR products to be sequenced directly on the MiSeq platform. PCR was followed by amplicon purification using the AMPureXP PCR system. Purified equimolar amounts of amplicons were pooled to form a library. A portion of the library was sequenced using the MiSeq v3 Reagent kit (600 cycles). Sequencing data were analyzed using LymphoTrack™ bioinformatics software.Results: Prior to testing clinical samples the analytical performance of the SHM assay was evaluated using diluted contrived samples, which exhibit known V-J rearrangement and SHM status. For limit of detection (LOD), limit of blank (LOB), linearity, and precision and reproducibility (P/R) the assay demonstrated excellent linearity (R2>0.99), sensitivity (1%), repeatability (<2% variance) and reproducibility (<2% variance). In addition, good concordance (R2>0.99) was demonstrated between the NGS assay and the traditional method of using gel extraction and Sanger sequencing in evaluating the SHM status of cell line DNAs.The clinical performance of the NGS SHM assay was evaluated by testing over 62 CLL samples that had also been tested using the traditional CE assay for clonality. The overall diagnostic accuracy was greater than 96%, sensitivity (ST) was 94%, specificity (SP) was 73%, positive predictive value (PPV) was 94%, and negative predictive value (NPV) was 72%. Forty-six samples that were tested for SHM status by both the MiSeq SHM assay and traditional Sanger sequencing had a concordance of 96%. Twenty-five samples were tested for SHM status by both MiSeq VHL/J and FR1/J assays with a concordance of 99%. Furthermore, the VHL/J mix detected clonality in SHM samples where mutation rate appeared to interfere with IGH FR1 priming. It was also observed that the CE assay could give a false impression of clonality if multiple clones were of very similar size – an occurrence quickly resolved by NGS sequencing showing the different DNA sequences of the same size from multiple rearranged clones thus indicating a polyclonal situation.Conclusion: Both the NGS SHM MiSeq assay and PCR capillary electrophoresis-based clonality assays identified a majority of clonal IGH V-J rearrangements in clinical CLL samples. However, the BIOMED-2 IGH FR1 assay missed certain of the clonal rearrangements and determination of SHM status was a labor intensive, multistep process, whereas data output from the NGS assay included the DNA sequence, V-J usage and SHM status of CLL patient samples. The NGS assay also provided frequency distributions of V region and J region segments important in detecting IGH V3-21, a determinant of poor prognosis in CLL. The MiSeq SHM assay and traditional PCR-based clonality-Sanger sequencing assays demonstrated excellent analytical performance and good clinical concordance. DisclosuresHuang:Invivoscribe, Inc.: Employment. Hutt:Invivoscribe, Inc.: Employment. Panganiban:Invivoscribe, Inc.: Employment. Miller:Invivoscribe, Inc.: Employment, Equity Ownership. Thornes:Invivoscribe, Inc.: Employment. Klass:Invivoscribe, Inc.: Employment.

Chimeric antigen receptors for the adoptive T cell therapy of hematologic malignancies

The genetic modification of autologous T cells with chimeric antigen receptors (CARs) represents a breakthrough for gene engineering as a cancer therapy for hematologic malignancies. By targeting the CD19 antigen, we have demonstrated robust and rapid anti-leukemia activity in patients with heavily pre-treated and chemotherapy-refractory B cell acute lymphoblastic leukemia (B-ALL). We demonstrated rapid induction of deep molecular remissions in adults, which has been recently confirmed in a case report involving a child with B-ALL. In contrast to the results when treating B-ALL, outcomes have been more modest in patients with chronic lymphocytic leukemia (CLL) or other non-hodgkin's lymphoma (NHL). We review the clinical trial experience targeting B-ALL and CLL and speculate on the possible reasons for the different outcomes and propose potential optimization to CAR T cell therapy when targeting CLL or other indolent NHL. Lastly, we discuss the pre-clinical development and potential for clinical translation for using CAR T cells against multiple myeloma and acute myeloid leukemia. We highlight the potential risks and benefits by targeting these poor outcome hematologic malignancies.

Combined Patterns of IGHV Repertoire and Cytogenetic/Molecular Alterations in Monoclonal B Lymphocytosis versus Chronic Lymphocytic Leukemia

BackgroundChronic lymphocytic leukemia (CLL)-like monoclonal B lymphocytosis (MBL) with (MBLhi) or without (MBLlo) absolute B-lymphocytosis precedes most CLL cases,the specific determinants for malignant progression remaining unknown.Methodology/Principal FindingsFor this purpose, simultaneous iFISH and molecular analysis of well-established cytogenetic alterations of chromosomes 11, 12, 13, 14 and 17 together with the pattern of rearrangement of the IGHV genes were performed in CLL-like cells from MBL and CLL cases. Our results based on 78 CLL-like MBL and 117 CLL clones from 166 subjects living in the same geographical area, show the existence of three major groups of clones with distinct but partially overlapping patterns of IGHV gene usage, IGHV mutational status and cytogenetic alterations. These included a group enriched in MBLlo clones expressing specific IGHV subgroups (e.g. VH3-23) with no or isolated good-prognosis cytogenetic alterations, a second group which mainly consisted of clinical MBLhi and advanced stage CLL with a skewed but different CLL-associated IGHV gene repertoire (e.g. VH1-69), frequently associated with complex karyotypes and poor-prognosis cytogenetic alterations, and a third group of clones with intermediate features, with prevalence of mutated IGHV genes, and higher numbers of del(13q)+ clonal B-cells.Conclusions/SignificanceThese findings suggest that the specific IGHV repertoire and IGHV mutational status of CLL-like B-cell clones may modulate the type of cytogenetic alterations acquired, their rate of acquisition and/or potentially also their clinical consequences. Further long-term follow-up studies investigating the IGHV gene repertoire of MBLlo clones in distinct geographic areas and microenvironments are required to confirm our findings and shed light on the potential role of some antigen-binding BCR specificities contributing to clonal evolution.

Targeted Gene Profiling Identifies Differential Genetic Make-up Depending On Chronic Lymphocytic Leukaemia Subtype

Abstract 1383▪▪This icon denotes a clinically relevant abstractExplorative genome-wide next-generation sequencing of leukaemias and lymphomas has revealed a wide spectrum of acquired mutations and considerable tumour heterogeneity that might be responsible for disease initiation, resistance to treatments and relapse. There is, therefore, a clinical need to identify these genetic abnormalities in a diagnostic setting.Here, we present the development and validation of a targeted next generation mutation analysis tool. To compare the distribution pattern of genetic abnormalities in chronic lymphocytic leukemia (CLL), we performed targeted deep sequencing on CLL samples using a TruSeq custom designed targeted amplicon assay (TSCA, Illumina). We reveal differential mutation distribution patterns depending on clinical CLL subgroups.The TSCA panel was designed to amplify 21 genes (table 1) with known or suspected links to either the development of CLL or as response predictors, including TP53, SF3B1 (Puente, Nature, 2011; Quesada et al, 2012) and NOTCH1 (Rossi, Blood, 2012). Where genes have known mutational hotspots in CLL, only these regions were included in our panel, for example exons 5–8 of TP53. For genes such as MAP2K1, where mutations are distributed throughout the coding region, every exon was targeted. In total, we were able to design an amplicon panel able to cover 99% of our desired 36,035bp target region.Table 1List of genes included in CLL custom amplicon panelASXL1ATMCHD2DDX3XFBXW7HMCN1IRF4KLHL6LRP1BMAP2K1MAPK1MED12NOTCH1PCLOPOT1SAMHD1SF3B1TP53XPO1ZFPM2ZMYM3In order to validate our approach, we used samples previously subjected to whole genome sequencing as controls. Of the 13 individual mutations in the control cohort, we were successfully able to detect 10 (77%) with our custom assay to an average depth of 1380x. A 19bp deletion in TP53 failed to be picked up by the variant calling software, and 2 point mutations in ATM were not detected due to the targeted nature of the assay. There was a single false positive mutation across all samples in ZFPM2, caused by a sequencing error in a homopolymer region.The sample group consisted of 45 representative CLL cases, split into two cohorts. The first cohort consisted of 11 cases that have yet to receive any treatment, whilst the second cohort comprised 34 relapsed/refractory cases. Analysis of further samples is in progress.We performed library preparation according to the manufacturers instructions. Each sample was dual indexed with two 8bp “barcodes” prior to equimolar pooling, and the final pooled library was processed on an Illumina MiSeq instrument using the TruSeq 2×150bp paired end sequencing protocol. The run produced 1.6Gb of passed filter sequence data, with 92.8% of above the quality threshold of Q30. The average depth of coverage across all samples was 849x.Primary analysis of the sequencing data was performed using the cloud based data analysis package from Illumina, which carried out the alignment and variant calling. A conservative quality score threshold of >99 was set, with all variants above this carried forward for further analysis.Our custom amplicon panel detected mutations in 35 of the samples, comprising 8 indels and 45 point mutations. Of the 54 mutations, 40 were missense, 8 were frame-shifts, 1 was a nonsense mutation and 5 are predicted to have functional effects on splicing domains. The most frequently mutated gene was TP53, followed by SF3B1, PCLO and NOTCH1 (figure 1). [Display omitted] Importantly, there was good correlation between mutation allele frequencies from whole genome sequencing, targeted deep sequencing and TSCA, demonstrating that the high sensitivity of large-scale genome sequencers can be reliably applied in a diagnostic setting.We describe mutation hotspots and mutation distribution patterns and link them to clinical behaviour. For example: SF3B1 mutations occurred in 15% of patients and were linked to reduced progression free survival.In conclusion, our technique allows for rapid mutation detection of the most frequently mutated genes in CLL. Further refinements in amplicon design and variant calling will lead to added precision. TSCA design and validation for other haematological diseases is in progress. Disclosures:No relevant conflicts of interest to declare.

Disruption of BIRC3 associates with Fludarabine Chemorefractoriness in TP53 Wild Type Chronic Lymphocytic Leukemia

Abstract 466The genetic lesions identified to date do not fully recapitulate the molecular pathogenesis of chronic lymphocytic leukemia (CLL) and do not entirely explain the development of severe complications, such as chemorefractoriness. BIRC3, along with TRAF2 and TRAF3, cooperate in the same protein complex that negatively regulates MAP3K14, the central activator of non-canonical NF-kB signaling. Following the initial observation of recurrent BIRC3 mutations in splenic marginal zone lymphomas, we performed targeted re-sequencing of the BIRC3 coding sequence and splicing sites across the spectrum of B-cell neoplasia. By this screening, BIRC3 mutations were identified only in CLL (2/20), while were absent in diffuse large B-cell lymphoma (0/30), Burkitt lymphoma (0/38), follicular lymphoma (0/20), extranodal marginal zone lymphoma (0/65), hairy cell leukemia (0/19), and multiple myeloma (0/22). This observation prompted the investigation of prevalence and clinical impact of BIRC3 genetic lesion in CLL. The study population included 4 clinical CLL panels representative of different disease phases: i) a cohort of fludarabine-refractory CLL (n=49); ii) a cohort of fludarabine-sensitive CLL (n=62); iii) clonally related Richter syndrome (RS) (n=33; all diffuse large B cell lymphomas); and iv) a consecutive series of newly diagnosed and previously untreated CLL (n=308). BIRC3 was analyzed for mutations by Sanger sequencing (exons 2–9) and for copy number abnormalities by FISH (probe RP11-177O8). BIRC3 was affected in 12/49 (24%) fludarabine-refractory CLL by inactivating mutations (7 frameshift and 1 non-sense) and gene deletions (n=7) that distributed in a mutually exclusive fashion with TP53 disruption (p=.004) (Fig 1A and 1B). Two fludarabine-refractory CLL carried a biallelic inactivation of the BIRC3 gene by mutation of one allele and deletion of the second allele. All inactivating mutations were somatically acquired, were predicted to generate aberrant transcripts carrying premature stop codons, and caused elimination or truncation of the C-terminal RING domain, whose E3 ubiquitin ligase activity is essential for MAP3K14 proteosomal degradation by BIRC3. Western blot analysis confirmed the expression of aberrant bands corresponding in size to the predicted truncated BIRC3, and revealed constitutive non-canonical NF-kB activation in fludarabine-refractory CLL harboring BIRC3 disruption by mutations or deletion, as documented by NFkB2 processing from p100 to p52 (Fig. 1). BIRC3 was the sole gene of the TRAF3/MAP3K14-TRAF2/BIRC3 complex targeted by molecular lesions after extensive mutation and FISH analysis of TRAF2, TRAF3 and MAP3K14 in the fludarabine-refractory CLL cohort (n=49). To investigate whether BIRC3 genetic lesions were restricted to chemorefractory cases, we analyzed BIRC3 for mutations and deletions in other CLL subsets. Fludarabine-sensitive CLL were consistently devoid of BIRC3 disruption in all cases (n=62), suggesting that BIRC3 genetic lesions specifically associate with a chemorefractory phenotype among CLL requiring treatment (Fig. 1A). BIRC3 disruption was restricted to 1/33 (3.0%) clonally-related RS, corroborating the notion that RS is molecularly distinct from chemorefractory progression without transformation (Fig. 1A). In a consecutive series evaluated at CLL diagnosis, BIRC3 disruption was rare (13/308, 4%, all TP53 wild type), associated with primary chemorefractoriness among cases requiring treatment, and predicted poor outcome (Fig. 1A). By univariate analysis, the crude impact of BIRC3 disruption on survival was a ∼5.3 fold increase in the hazard of death (HR: 5.31; 95% CI: 2.62–10.73) and a significant overall survival (OS) shortening (median OS in BIRC3 disrupted cases: 3.1 years vs not reached in BIRC3 wild type cases; p<.001) (Fig. 1D). Multivariate analysis selected BIRC3 disruption as an independent risk factor of OS (HR: 4.25; 95% CI: 1.74–10.36; p=.001) after controlling for confounding clinical (age and Rai stage) and genetic (IGHV mutation status, 11q22-q23 deletion, TP53 disruption, NOTCH1 mutations) variables at diagnosis. These data document that disruption of BIRC3, a critical regulator of non-canonical NF-kB signaling, associates with fludarabine-refractoriness among TP53 wild type CLL and identifies a subgroup of patients showing poor outcome. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Unmutated IGHV1-69/D3-16/J3 Stereotyped HCDR3 Rearrangements (Subset 6) Are Associated with Indolent Disease Course and Have Outcome Independent of Mutational Status In Early Stage CLL (Rai 0)

AbstractAbstract 1371 Background:IGHV1-69 gene identifies the most frequent IGHV gene in chronic lymphocytic leukemia (CLL) and identifies the paradigm of unmutated CLL (U-CLL), being used in roughly 1/3 U-CLL. It is often rearranged to form subsets of stereotyped HCDR3 patterns, likely selected and transformed from the natural naïve B-cell repertoire (Blood. 2010; 115:71-7). Being unmutated, IGHV1-69 CLL are hypothetically expected to have competent tumor B-cell receptors (BCR) and to progress more rapidly. However, it has not been investigated if progression occurs similarly in all the subsets. Aims:we aimed to investigate the prognostic significance of mutational status and of stereotypic B-cell receptors in IGHV1-69+ CLL. Methods:Nucleotide sequences of the tumor IGHV1-69/D/J rearrangement, clinical and molecular prognostic parameters at diagnosis and clinical status at follow-up of 294 IGHV1-69+ CLL patients were obtained from 22 hematological Institutions in Italy. CLL B-cell derived IGHV1-69 rearrangements were scanned for HCDR3 stereotypic patterns and assigned to subsets according to the criteria by Murray et al (Blood. 2008; 111: 1524–1533). Enpoint of outcome was time to progression requiring first treatment according to NCI criteria (TTFT) in Rai stage 0 CLL. Results:Of 294 IGHV1-69+ CLL, 264 (89,8%) were unmutated, 168 (57,1%) were assigned to subsets, of which subsets 7 (n=23, 7,8%), 6 (17, 5,8%), 3 (13, 4,4%), 5 (10, 3,4%) and 9 (10, 3,4%) were the most frequent. CD38, ZAP70, normal or sole del13, +12, del11 and del17p scored positive in 109/264 (58,7%), 139/245 (56,7%), 128/248 (50,5%), 51/248 (20,6%), 43/248 (17,3%) and 34/248 (13,7%). CLLs were reclassified as 18 (6,1%) clinical MBL, 101 (34,4%) Rai 0, 155 (52,7%) Rai I-II and 20 (6,8%) Rai III-IV CLL. Subset 6 was also UM in 16/17 (94,1%) cases. Prevalence of CD38 (p<.001), ZAP70 (p=.016), normal or sole del13 (p<.001), +12 (p=.026), del11 (p=.011), and clinical high risk CLL (p=.025) were lower in IGHV1-69 M-CLL than in IGHV1-69 U-CLL. TTFT was significantly shorter in stage 0 IGHV1-69 U-CLL than in IGHV1-69 M-CLL (49 vs 144 months, p<.001, while it was not different between CLL assigned or not to subsets (65 vs 55 months, p=.346). However, specific analysis of individual subsets revealed differential outcomes (p=.005). Among all, it emerged that subset 6 had a TTFT equivalent to IGHV1-69 M-CLL (p=.29) and significantly longer than stage 0 IGHV1-69 U-CLL (median not reached vs 48 months, p=.017). Conclusions:our analysis documents and confirms that unmutated status of IGHV, and not stereotypy, is a relevant prognosticator of outcome (TTFT) in CLL. In the IGHV1-69 CLL it exclude a role of IGHV gene use for CLL progression. However, the good prognosis of Rai 0 U-CLL assigned to subset 6 suggests a differential clinical benign course of this particular subset, irrelevant of unmutated status. One possibility is that the IGHV1-69/D3-16/J3 rearrangements of subset 6 produce a tumor-specific BCR with stereotypic HCDR3 patterns that are anergized by antigen while circulating in the peripheral blood in early stage (Rai 0) CLL. Disclosures:No relevant conflicts of interest to declare.

Evaluation of circulating endothelial cells as noninvasive marker of angiogenesis in patients with chronic lymphocytic leukemia

An increased angiogenesis has been documented in bone marrow and lymph nodes of patients with chronic lymphocytic leukemia (CLL). There is accumulating evidence that circulating endothelial cells (CECs) play an important role in angiogenesis. The aim of our study was to compare the number of CECs in peripheral blood of CLL patients and healthy donors and to correlate these numbers with bone marrow microvessel density (MVD) and known prognostic factors in CLL. Proportions of resting CECs (rCECs), activated CECs (aCECs), apoptotic CECs (apoCECs) and circulating precursor endothelial cells (CPECs) were estimated by flow cytometry in 104 untreated CLL patients and 29 healthy blood donors. The MVD was analysed in the bone marrow biopsy in 21 CLL patients and 11 controls using the ‘hot spot analysis’ of vessels' density (×200 HPS). We found significantly higher numbers of CPECs, rCECs, aCECs and apoCECs in CLL patients than in healthy controls (p < 0.001 for all comparisons). Furthermore, the rCECs number was higher in advanced versus low clinical stage CLL (median 17.5 cells/µL vs. 13.5 cells/µL, p = 0.05). The MVD was significantly higher in the bone marrow of CLL patients when compared with controls (p = 0.016). However, we did not find any correlation between MVD and different CECs populations. In conclusion, the number of CECs is increased in CLL and correlates with stage of the disease, but does not seem to directly reflect the intensity of neovascularisaton in the bone marrow.