Status In Chronic Lymphocytic Leukemia Research Articles

The evolving use of measurable residual disease in chronic lymphocytic leukemia clinical trials.

Measurable residual disease (MRD) status in chronic lymphocytic leukemia (CLL), assessed on and after treatment, correlates with increased progression-free and overall survival benefit. More recently, MRD assessment has been included in large clinical trials as a primary outcome and is increasingly used in routine practice as a prognostic tool, a therapeutic goal, and potentially a trigger for early intervention. Modern therapy for CLL delivers prolonged remissions, causing readout of traditional trial outcomes such as progression-free and overall survival to be inherently delayed. This represents a barrier for the rapid incorporation of novel drugs to the overall therapeutic armamentarium. MRD offers a dynamic and robust platform for the assessment of treatment efficacy in CLL, complementing traditional outcome measures and accelerating access to novel drugs. Here, we provide a comprehensive review of recent major clinical trials of CLL therapy, focusing on small-molecule inhibitors and monoclonal antibody combinations that have recently emerged as the standard frontline and relapse treatment options. We explore the assessment and reporting of MRD (including novel techniques) and the challenges of standardization and provide a comprehensive review of the relevance and adequacy of MRD as a clinical trial endpoint. We further discuss the impact that MRD data have on clinical decision-making and how it can influence a patient's experience. Finally, we evaluate how upcoming trial design and clinical practice are evolving in the face of MRD-driven outcomes.

Accuracy of predicting IgHV mutation status in chronic lymphocytic leukemia using RNA expression profiling and machine learning

Background: Immunoglobulin heavy chain (IgHV) mutation status is a unique prognostic indicator for predicting clinical course and response to targeted therapy in chronic lymphocytic leukemia (CLL). Most of the tests for the IgHV mutation status require extensive and complicated genomic evaluation. We explored the potential of using RNA expression data generated from routine targeted RNA sequencing by Next Generation Sequencing (NGS) along with machine learning for the prediction of the IgHV mutation status.

Clinical and economic burden of first-line chemoimmunotherapy by risk status in chronic lymphocytic leukemia

Objectives To evaluate the trend in cytogenetic/molecular testing rate in chronic lymphocytic leukemia (CLL) and assess the clinical and economic burden of first-line (1 L) treatment with chemoimmunotherapy (CIT) by risk status. Methods This retrospective cohort study identified patients with CLL from a U.S. managed care population. Medical records were obtained for eligible patients who initiated 1 L CIT between 1/1/2007 and 7/31/2019 and underwent prognostic testing to classify them as high risk (del(17p), TP53 mutation, del(11q), unmutated IGHV or complex karyotype) or as non-high risk by FISH only (non-del(17p) and non-del(11q)). Study outcomes included testing rate, time to next treatment (TTNT) or death, time to treatment failure (defined as time to change of therapy, non-chemotherapy intervention, hospice care or death), and total plan paid costs (medical + pharmacy) per patient per month (PPPM) in the 1 L period. Cox proportional hazard models and generalized linear models were used to calculate adjusted hazard ratio or rate ratio. Results Among the 1,808 patients with CLL, 612 were FISH or IGHV tested and the rate of testing increased from 30% to 44% from 2007–2019. High-risk patients (n = 119) had 65% higher risk of next treatment or death (median time: 2.4 vs 3.7 years), 65% higher risk of treatment failure (median time: 3.0 vs 4.9 years), and 33% higher costs ($12,194 vs $9,055, p = 0.027) during 1 L treatment than non-high risk patients (n = 134). Conclusions High-risk CLL patients treated with 1 L chemoimmunotherapy have poorer clinical and economic outcomes compared to non-high risk patients. Assessment of genetic risk remains suboptimal.

The pyroptosis-related gene signature predicts prognosis and indicates the immune microenvironment status of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world with great heterogeneity. Pyroptosis has recently been recognized as an inflammatory form of programmed cell death (PCD) and shares a close relationship with apoptosis. Although the role of apoptosis in CLL was comprehensively studied and successfully applied in clinical treatment, the relationship between pyroptosis genes and CLL remained largely unknown. In this study, eight differentially expressed pyroptosis-related genes (PRGs) were identified between CLL and normal B cells. In order to screen out the prognostic value of differentially expressed PRGs, univariate and multivariate Cox regression analyses were conducted and a risk model with three PRG signatures (GSDME, NLRP3, and PLCG1) was constructed. All CLL samples were stratified into high- and low-risk subgroups according to risk scores. The risk model showed high efficacy in predicting both overall survival (OS) and time to first treatment (TTFT). Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) showed the dysregulation of immune and inflammatory response in the high-risk group. Single-sample GSEA (ssGSEA) of immune cell infiltration and the activity of immune-related pathways also displayed decreased antitumor immunity in the high-risk group. In conclusion, PRGs are of prognostic value in CLL and may play important roles in tumor immunity, and the underlying relationship between PRGs and CLL needs to be explored further.

The Outcome of CLL Patients According to IGHV Mutational Status: An Israeli Perspective

Introduction: The prognostic significance of immunoglobulin heavy-chain variable region gene (IGHV) mutational status in chronic lymphocytic leukemia (CLL) is well established. Previous studies have shown that CLL patients with mutated IGHV (M-IGHV) have a better prognosis, manifested in a longer time-to first treatment (TTFT) and overall survival (OS). Here we present an analysis on the impact of IGHV mutational status in an Israeli cohort of patients with CLL.Methods: A total of 254 patients with CLL (diagnosed from 1991 to 2020), followed at the Tel-Aviv Sourasky Medical Center were included. The IGHV mutational status has been determined by next-generation sequencing (NGS) or cDNA Sanger. The sequences with a germline homology 98% or higher were considered unmutated, and those with a homology less than 98% as mutated CLL. IGHV subsets were analyzed using the ARResT/AssignSubsets website. All data were statistically analyzed by IBM SPSS Statistics 27 (IBM Corporation, Armonk, NY, USA), P-values were two-sided, and the significance level was determined at a<0.05 were considered significant.Results: Out of 254 patients, 132 (52.0%) had an unmutated IGHV gene (UM-IGHV), and 122 (48.0%) were mutated (M-IGHV). At diagnosis, most patients (Table 1) were ≤65 years of age (n=157, 61.8%), males (n=160, 63.0%) and had an absolute lymphocytic count equal to or less than (≤)15.0 x10 9/L (n=115, 52.5%). Advanced Binet stage was more commonly associated with UM-IGHV (n=44, 57.9%) (P=0.033). Among 240 patients (94.1%), the most frequently used VH gene segments (Figure 1) included; VH1-69 (n=35, 14.6%), VH4-34 (n=24, 10.0%), VH1-2 (n=17, 7.1%), and VH3-23 (n=16, 6.7%). Six major B-cell receptors (BCR) subsets (Figure 2) were identified in 21/180 of patients (11.7%), which most commonly included: CLL#1 (n=6, 28.6%), CLL#4 (n=5, 23.8%), and CLL#2(n=3, 14.3%). Patients with M-IGHV had a longer time to first treatment (TTFT) (P<0.001, hazard ratio (HR)=2.5, 95% confidence interval [1.8-3.5], Figure 3A) and a better overall survival (OS) (P=0.002, HR=2.7, [1.4-5.1]) compared to those with UM-IGHV gene (Figure 3B).In multivariate analyses of the entire cohort; for TTFT (Table 2A), males (P=0.002, HR=1.9, [1.3-2.9]), >65 years of age at diagnosis (P=0.008, HR=1.8, [1.2-2.7]), advanced Binet stage (P<0.001, HR=2.4, [1.5-3.8]) and UM-IGHV mutational status (P=<0.001, HR=2.0, [1.3-2.9]) were found to be significant predictors for shorter TTFT, while in a multivariate analysis for OS (Table 2B), only >65 years of age at diagnosis (P=0.010, HR=2.6, [1.3-5.5]) and UM-IGHV mutational status (P=0.004, HR=2.9, [1.4-5.9]) were found to be significant factors for shorter OS.In multivariate analyses performed separately for each IGHV mutational status group; males (P=0.021, HR=2.1, [1.1-4.0]), age >65 years (P=0.002, HR=3.2, [1.5-6.4]) and advanced Binet stage (P<0.001, HR=4.1, [1.9-8.7]) were found as independent predictors for a shorter TTFT in M-IGHV patients, while only males retained a statistically significance in UM-IGHV patients (P=0.017 HR=2.0, [1.1-3.5]). Furthermore, in a multivariate analysis for OS; age>65 years at diagnosis (P=0.039, HR=4.3, [1.1-17.0]) was the only independent predictor in M-IGHV patients, while no variable maintained its statistical significance in UM-IGHV cases.Conclusion: As previously studied, our cohort demonstrates that M-IGHV CLL patients have better TTFT and OS. However, the rate of BCR subsets in Israel appears to be lower than expected. A separated multivariate analysis for M-IGHV and UM-IGHV patients revealed different independent predictors for TTFT and OS to each of the IGHV mutational status groups. Further studies with larger cohorts are required to deeply study these differences and provide further clinical insight into the pathophysiology of CLL. [Display omitted] DisclosuresJoffe: AstraZeneca: Consultancy; Epizyme: Consultancy. Herishanu: AbbVie: Honoraria, Research Funding; Janssen: Honoraria; Roche: Honoraria; Medison: Honoraria.

Evaluation of Somatic Hypermutation Status in Chronic Lymphocytic Leukemia (CLL) in the Era of Next Generation Sequencing.

Somatic hypermutation (SHM) status provides an important prognostic indicator for chronic lymphocytic leukemia (CLL), a very common type of mature B-cell leukemia. Owing to the adverse prognosis associated with an unmutated immunoglobulin heavy chain variable (IGHV) status, SHM testing is performed as a standard of care in CLL. Conventionally, SHM testing has been performed using labor intensive and primarily analog Sanger sequencing method following PCR amplification of the clonal immunoglobulin heavy chain gene rearrangements in CLL cells. In comparison, recent availability of next generation sequencing (NGS) allows more versatile detection and direct identification of clonal immunoglobulin gene rearrangements in neoplastic B-cell populations. The ability to identify specific clonal IGHV signature(s) in both baseline (diagnostic) and post-treatment settings enables unique clinical applications of NGS such as determination of SHM status, minimal residual disease (MRD) monitoring, clonal heterogeneity and B cell receptor IG stereotypy. We provide a review of current practices and recommendations for SHM determination using NGS including examples of difficult cases.

Disparities in survival by insurance status in chronic lymphocytic leukemia.

e18134 Background: Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in western world. It is the disease of older adults with a median age at diagnosis of approximately 70 years. However, significant proportion of younger adults are diagnosed with CLL and they usually have an adverse clinical and molecular risk profile necessitating early interventional strategies compared to older patients. Socioeconomic disparities may affect survival for some patient populations. This study examined survival by insurance status for younger patients with CLL. Methods: The Surveillance, Epidemiology, and End Results database was used to identify patients, aged 15 - 64 years, who were diagnosed with CLL between 2007 and 2014 and whose insurance status (no insurance, Medicaid, and other insurance) was documented at diagnosis. Unadjusted associations between insurance status and overall survival (OS) were plotted using Kaplan-Meier curves. The association between insurance status and CLL-specific mortality was assessed using multivariate Cox proportional hazards regression models, adjusting for sociodemographic covariates. Results: A total of 10,913 patients with CLL were included with a median follow up of 44 months. Five-year survival estimates were 87.3%, 87.2%, and 94.0% for patients with no insurance, Medicaid, and other insurance, respectively. In multivariate analysis, compared to patients with other insurance, patients with no insurance (hazard ratio [HR] = 2.33, 95% confidence interval [CI] = 1.73 – 3.140) and Medicaid (HR = 1.95, 95% CI = 1.49 – 2.54) had significantly worse OS survival after adjusting for sociodemographic factors. Conclusions: Younger patients with no insurance or Medicaid have lower survival outcomes after diagnosis with CLL compared to patients with other insurance. Addressing barriers to care, providing resources, and expanding access to care is paramount to reducing the gap in survival for the uninsured patients and those with lower socioeconomic status. Further studies are needed to address poor survival outcomes among Medicaid patients.

Dynamic BH3 Profiling Predicts Patient Response and MRD Status in Chronic Lymphocytic Leukemia (CLL) Patients Undergoing Frontline Treatment with Kinase Inhibitor Plus FCR (KI+FCR)

Dynamic BH3 Profiling Predicts Patient Response and MRD Status in Chronic Lymphocytic Leukemia (CLL) Patients Undergoing Frontline Treatment with Kinase Inhibitor Plus FCR (KI+FCR)

Comprehensive Characterization of NOTCH1 Mutational Status in Chronic Lymphocytic Leukemia: Clinical Relevance of Subclonal Mutations and Mutation Types

Background.NOTCH1 mutations have been associated with an aggressive clinical course in chronic lymphocytic leukemia (CLL), and may predict anti-CD20 immunotherapy failure. Although about 80% of NOTCH1 mutations are 2-bp deletions (delCT) causing a sequence frameshift within the PEST domain, other mutations may occur involving either the PEST domain, i.e. frameshift mutations other than delCT (FS), missense (MS) and nonsense (NS) mutations, or, more recently described, the 3'UTR NOTCH1 sequence (UTR).Aim. To evaluate the clinical impact of the different types of NOTCH1 mutations in terms of time-to-first-treatment (TTFT) and overall survival (OS).Methods.NOTCH1 mutations were screened in 1003 CLL cases by NGS in exon 34 and part of 3'UTR with at least 1000X coverage and of 1% sensitivity. In selected cases, somatic mutations were confirmed by sequencing of germ line DNA from purified T cells. In our cohort, 529 patients were treated and 151 died. Cytogenetics distribution (n=970) was: 276, normal karyotype; 110, del11; 132, tris12; 369, del13; 83, del17. IGHV gene status (n=891) was: 529, (UM); 362, mutated (M).Results.i) Clinical impact of NOTCH1 mutations and correlation with cytogenetics and IGHV gene status. Collectively, NOTCH1 mutations were detected in 190 cases (18.9%): 167 cases bore a single mutation, while 19, 3 and 1 cases bore 2, 3 or 4 mutations, respectively. According to previous studies, cases with NOTCH1 mutations presented a significant shorter TTFT and OS respect to NOTCH1-wild-type (NOTCH1-wt) cases (p<0.0001, and p<0.0001, respectively), and NOTCH1 mutations were more frequent in tris12 CLL cases (p<0.0056) and IGHV UM cases (p<0.0001; Figure1a).ii) Subclonal analysis of NOTCH1 mutations. Considering the threshold of variant allele frequency of 12% for clonal and subclonal definition, NOTCH1 mutated patients were divided into 118 clonal and 72 subclonal cases. Again in agreement with literature data: a) cases with subclonal NOTCH1 mutations experienced shorter TTFT than NOTCH1-wt cases (p=0.0184), but significantly longer than cases with clonal mutations (p<0.0001); b) no significant difference was observed between cases with subclonal NOTCH1 mutations and NOTCH1-wt in terms of OS (p=0.8246), while statistically significant shorter OS intervals were experienced by cases bearing clonal NOTCH1 mutations (p=0.0001).iii) Clinical impact of different types of NOTCH1 mutations. The different types of NOTCH1 mutations (delCT, FS, MS, NS, UTR) were evaluated for their putative different prognostic impact in CLL. In order to assign a patient to a given NOTCH1 mutation type, cases presenting more than one mutations were classified using the mutation type presented at the higher percentage. Therefore, the NOTCH1 mutated cases were reclassified as follows: 123 delCT, 12 FS, 8 MS, 17 NS, and 30 UTR. According to Kaplan-Meyer curves, cases belonging to the five categories revealed a significant shorter TTFT respect to NOTCH1-wt cases (delCT p<0.0001; UTR p=0.0001; FS p<0.0001; MS p=0.0274; NS p=0.0108). On the other hand, OS intervals were significantly shorter compared to NOTCH1-wt CLL only in cases bearing delCT or FS mutations (p=0.0003, and p<0.0001, respectively). Notably, when clinical courses of CLL carrying different NOTCH1 mutation types were compared each other, patients carrying FS mutations experienced both shorter TTFT and OS compared to cases belonging to the other mutation groups (p=0.0169, and p=0.0149, respectively; Figura1b). A possible explanation for this phenomenon could be the fact that, in our cohort, FS mutations other than delCT were all located upstream the NOTCH1 region involved in the canonical delCT mutations (Figure1c), allegedly determining a more effective disruption of the PEST domain, and a more pronounced deregulation of NOTCH1 protein.Conclusion. Our data confirm the capacity of NOTCH1 mutations to predict both TTFT and OS, although subclonal NOTCH1 mutations seem to have a prognostic relevance more for TTFT than OS. Moreover, we demonstrated for the first time that frameshift mutations different from the canonical delCT NOTCH1 mutation predicted for significantly shorter TTFT and OS compared to the other types of NOTCH1 mutations. A screening of NOTCH1 mutations in CLL, also outside the canonical region involved in delCT mutations, is therefore warranted for a correct identification of NOTCH1 mutated cases. [Display omitted] DisclosuresRossi:Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janseen: Honoraria. Gaidano:Janssen: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Morphosys: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Speakers Bureau.

Telomere status in chronic lymphocytic leukemia with TP53 disruption.

In chronic lymphocytic leukemia (CLL), telomere dysfunction is associated with poor outcomes. TP53 is involved in cellular responses to dysfunctional telomeres, and its inactivation is the strongest adverse prognostic factor for CLL. Given the biological relationship between TP53 and telomeres, and their prognostic value, it is important to improve our understanding of the impact of TP53 alterations on telomeres. We performed a comprehensive study of the deletions and mutations of the TP53 gene and telomere parameters, including hTERT and the shelterin complex, in 115 CLL patients. We found that any type of TP53 alteration was associated with very short telomeres and high hTERT expression, independently of other biological CLL features. Patients with disrupted TP53 showed telomere deletions and chromosomal end-to-end fusions in cells with complex karyotypes. TP53 disruption was characterized by downregulation of shelterin genes. Interestingly, low expression of POT1, TPP1 and TIN2 was also found in some patients with wild-type TP53 and had an adverse impact on progression-free survival after standard genotoxic therapy. In conclusion, we have demonstrated that patients with disrupted TP53 have severe telomere dysfunction and high genomic instability. Thus, the telomeric profile could be tested as a biomarker in CLL patients treated with new therapeutic agents.

Measurement of lymphocyte aggregation by flow cytometry-physiological implications in chronic lymphocytic leukemia.

Cellular aggregation is a physiological response of lymphocytes to various extracellular stimuli. Currently, lymphocytes aggregation is only evaluated qualitatively or by semiquantitative methods. In this study, we assessed the capacity of flow cytometry to measure lymphocytes aggregation in a quantitative, accurate, and reproducible manner, and examined the significance of aggregation responses in various lymphoproliferative diseases. Extracellular triggers such as anti-CD19 antibodies or phorbol ester were utilized to induce lymphoid cells aggregation in a concentration dependent manner. Aggregation was quantified by flow cytometry based on the forward or side scatter (SSC), or by dark-field SSC of aggregates measured by ImageStreamX. Accuracy, reproducibility, and limitations of the methodology were evaluated. Aggregation responses were measured in various types of lymphoproliferative diseases, and correlated with immunophenotyping and IGHV mutational status in chronic lymphocytic leukemia. Lymphoid aggregates provoked by extracellular stimuli elevate the forward and SSC signals relatively to the number of cells in each event. Aggregation responses vary among different types of lymphoproliferative diseases. Moreover, elevated levels of CD19-induced aggregation are associated with aberrant chronic lymphocytic leukemia characteristics, but not with IGHV mutational status of the disease We have demonstrated that flow cytometry can provide accurate and reproducible measurement of both primary as well as T and B cell lines aggregation in response to extracellular stimuli. The use of quantitative evaluation of activation driven or other cellular aggregation may provide an analytical tool to elucidate biochemical and molecular mechanisms associated with lymphoproliferative diseases. © 2015 International Clinical Cytometry Society.

Proteins interaction network and modeling of IGVH mutational status in chronic lymphocytic leukemia.

BackgroundChronic lymphocytic leukemia (CLL) is an incurable malignancy of mature B-lymphocytes, characterized as being a heterogeneous disease with variable clinical manifestation and survival. Mutational statuses of rearranged immunoglobulin heavy chain variable (IGVH) genes has been consider one of the most important prognostic factors in CLL, but despite of its proven value to predict the course of the disease, the regulatory programs and biological mechanisms responsible for the differences in clinical behavior are poorly understood.MethodsIn this study, (i) we performed differential gene expression analysis between the IGVH statuses using multiple and independent CLL cohorts in microarrays platforms, based on this information, (ii) we constructed a simplified protein-protein interaction (PPI) network and (iii) investigated its structure and critical genes. This provided the basis to (iv) develop a Boolean model, (v) infer biological regulatory mechanism and (vi) performed perturbation simulations in order to analyze the network in dynamic state.ResultsThe result of topological analysis and the Boolean model showed that the transcriptional relationships of IGVH mutational status were determined by specific regulatory proteins (PTEN, FOS, EGR1, TNF, TGFBR3, IFGR2 and LPL). The dynamics of the network was controlled by attractors whose genes were involved in multiple and diverse signaling pathways, which may suggest a variety of mechanisms related with progression occurring over time in the disease. The overexpression of FOS and TNF fixed the fate of the system as they can activate important genes implicated in the regulation of process of adhesion, apoptosis, immune response, cell proliferation and other signaling pathways related with cancer.ConclusionThe differences in prognosis prediction of the IGVH mutational status are related with several regulatory hubs that determine the dynamic of the system.Electronic supplementary materialThe online version of this article (doi:10.1186/s12976-015-0008-z) contains supplementary material, which is available to authorized users.

Surrogate molecular markers for IGHV mutational status in chronic lymphocytic leukemia for predicting time to first treatment

ZAP-70 is a marker of clinical outcome in chronic lymphocytic leukemia (CLL), however its assessment suffers from a lack of standardization consensus. To identify novel markers able to surrogate IGHV mutational status, CD19(+)CD5(+)-B-lymphocytes from 216 patients enrolled in a prospective study (ClinicalTrial.gov Identifier:NCT00917540), underwent gene expression profiling. Samples were split into CLL-Training (n=102) and CLL-Validation (n=114) sets, and an independent supervised analysis for IGHV mutational status was performed considering all genes with gene expression equal or above that of ZAP-70. Thirty-one genes (23 up- and 8 down-regulated) and 23 genes (18 up- and 5 down-regulated) satisfied these criteria in the CLL-Training and CLL-Validation sets, respectively, and 20 common genes (15 up and 5 down) were found to be differentially regulated in both sets. Two (SNORA70F, NRIP1) of the down-regulated and 6 (SEPT10, ZNF667, TGFBR3, MBOAT1, LPL, CRY1) of the up-regulated genes were significantly associated with a reduced risk of disease progression in both sets. Forcing the afore-mentioned genes in a Cox multivariate model together with IGHV mutational status, only CRY1 (HR=2.3, 95% CI: 1.1–4.9, P=.027) and MBOAT1 (HR=2.1, 95% CI: 1.1–3.7, P=.018) retained their independent prognostic impact, supporting the hypothesis that these genes may potentially act as surrogates for predicting IGHV mutational status.

PERIPHERAL BLOOD LYMPHOCYTE PHENOTYPE OF ZAP-70+ AND ZAP-70− PATIENTS WITH B-CELL CHRONIC LYMPHOCYTIC LEUKAEMIA

Up to now, the immune status of chronic lymphocytic leukemia (CLL) patients in association with the expression of zeta-chain-associated protein kinase 70 (ZAP-70) in leukemic cells has not been evaluated. The aim of this work was the study of the peripheral blood (PB) T-lymphocyte phenotypes in ZAP-70-positive (ZAP-70(+)) and ZAP-70-negative (ZAP-70(-)) untreated patients with CLL. ZAP-70-, CD25-, CD3-, CD4-, and CD8-positive lymphocytes were enumerated by flow cytometry in PB of 120 untreated CLL patients. CD8+, CD3+CD4+ and CD3+CD25+ cells were counted for the non-leukemic lymphocytes. The patients were distributed into two groups: the ZAP-70(+) group of high CLL progression (n = 61), and the ZAP-70(-) group of low CLL progression (n = 59). In the ZAP-70(+) group, the ratio CD4/CD8 (0.33 ± 0.62; p = 0.001) and the numbers of the CD3+ (34.8 ± 8.1%; p = 0.01), CD3+CD4+ (24.4% ± 4.8; p = 0.001), and CD3+CD25+ (6.2 ± 0.91%; p = 0.001) lymphocytes were reduced and the percentage of the CD8+ cells (73.1 ± 4.6%; p = 0.0001) was above the norm. In the ZAP-70(-) group, the number of the CD3+CD4+ cells (36.9 ± 6.1%; p = 0.001) was within the norm, but the numbers of the CD8+ (11.3 ± 1.1%; p = 0.0001) and CD3+ (41.2 ± 5.3%; p = 0.05) lymphocytes were reduced; the ratio CD4/CD8 (3.26 ± 0.88; p = 0.001) and the percentage of the CD3+CD25+ cells (27.1 ± 3.4%; p = 0.0001) were above the norm. Our data show that the increased CD4/CD8 ratio, caused by the reduced number of the CD8+ lymphocytes, and the increased number of CD3+CD25+ cells are characteristic for the ZAP-70(-) group (slow progressing) of untreated CLL patients. In ZAP-70(+) patients, the CD4/CD8 ratio was significantly below the norm indicating an active disease process. Results of our study contribute to identification of CLL patients with different prognosis in routine diagnostic/prognostic procedures.

CD14+HLA-DRlow/- expression: A novel prognostic factor in chronic lymphocytic leukemia.

Currently, no prognostic factors exist for determining the host immune status of chronic lymphocytic leukemia (CLL) patients. Therefore, the present report analyzed cluster of differentiation 14 (CD14)+ human leukocyte antigen (HLA)-DRlow/− myeloid-derived suppressor cells (MDSC) from 49 CLL patients and demonstrated that these cells were significantly expanded in all CLL patients when compared with monoclonal B cell lymphocytosis patients and healthy volunteers. Furthermore, upregulation of CD14+HLA-DRlow/− MDSCs was correlated with CLL tumor progression and a poor prognosis for CLL patients, and CD14+HLA-DRlow/− MDSCs were significantly correlated with the presence of CD4+ T and CD5+CD19+ cells in CLL patients, which could significantly inhibit the CD4+ T-cell immune response, contributing to CLL cell progression in CLL patients.

P53 Functional Assessment and Correlation With 17p Deletion and/Or TP53 Mutation Status In Chronic Lymphocytic Leukemia (CLL). A Preliminary Report Of The ICLL001 Bomp Trial On Behalf Of The French CLL Intergroup (GCFLLC/MW - GOELAMS)

Despite improvement in treatment strategies, virtually all chronic lymphocytic leukemia (CLL) patients will relapse and experience tumor resistance. The 17p deletion resulting in loss of the TP53 gene, found in up to 20-40% of relapsing patients, is strongly associated with impaired response to genotoxic agents, reduced progression free survival and poor overall survival. The 17p deletion usually coincides with TP53 mutation, leading to the impairment of the p53-associated pathway. In addition, sole TP53 mutations (without 17p deletion) appear also associated with poor outcome in prospective trials. However, TP53 mutation screening is time consuming, can be not exhaustive, and the respective impact of different patterns of TP53 gene impairment on p53 function and prognostic remains unclear. We previously developed a functional assay to detect p53 dysfunction (Le Garff-Tavernier, 2011) and we aim to validate this analysis on a large prospective trial.Clinical and laboratory data were collected from CLL patients (pts) enrolled in the ICLL001 – BOMP phase II trial of the French CLL intergroup (NCT01612988) evaluating a prephase of ofatumumab (300 mg) followed by 6 monthly courses of BOMP including bendamustine (70 mg/m2 d1-2), ofatumumab 1000 mg TD (d1 and d15 on 1st and 2nd courses) and high dose methylprednisolone (1 g/m2 d1-3) in fit patients with relapsing CLL and IWCLL treatment criteria. In addition to conventional screening, we focused on p53 evaluation. FISH analysis for 17p deletion was done with a 10% cut-off for positive result, TP53 gene mutation screening was performed using Sanger sequencing of the entire coding region (exons 2–11) and the p53 functional status in CLL cells was determined by a flow cytometry assay based on induction of p53 and p21 protein expression after etoposide and nutlin-3a exposition.Data from the first 55 enrolled pts are available. Sex ratio M/F was 3.3 and median age was 63.8 yrs (44.6-76.4). CLL diagnostic had been done 7,2 (1,9-16,8) years before inclusion. All patients had according to IWCLL criteria an active disease of Binet stage of A (11%), B (57%) and C (32%) respectively. Patients had been previously pretreated with a median of 1 (1-3) lines, including FCR (or FCR-like) in 51 (93%) pts and 22 (42%) pts had experienced high-risk relapses within 24 months post-FCR, with 7 (13%) pts being fludarabine refractory (less than PR after fludarabine regiment and/or response lasting less than 6 months). IGVH gene status was unmutated in 90%, elevated β2-microglobulin (>4) was found in 52%. Karyotypes were complex (≥ 3 abnormalities) in 18/46 (39%) successful cases. Using FISH, we found 15/55 (27%) del17p (median of positive cells 71%, range 10-98), 6/55 (11%) tri12, 18/55 (33%) del11q, 35/55 (64%) del13q. Results of p53 functional assay was available for 52 pts with the following results: normal in 31 pts and abnormal in 21 pts including type A (n=4), type B (n=13) and type C (n=4) dysfunction. Mutation screening was available in 55 pts. No mutation were detected in 38 pts, one significant mutation was detected in 14 pts within exon 5 (n=1), exon 6 (n=2), exon 7 (n=2), exon 8 (n=6), exon 10 (n=1) and intronic splice site (n=2) ; 3 pts had 2 mutations within exons 7 and 8 (n=1), exons 7 and 10 (n=1), exons 5 and 7 (n=1). Among the 52 pts with available functional results we found the 7 following groups (Table). In this study, the sensitivity and specificity of the p53 functional test to detect patient with 17p deletion and/or TP53 mutation was 89.5% (66.9 –99.7) and 87.9% (71.8 – 96.6) respectively.Response to p53/21 functional assay17p deletionTP53 mutationn%Group 1NormalNoNo2956Group 2AbnormalYesYes1325Group 3AbnormalYesNo12Group 4AbnormalNoYes35.5Group 5AbnormalNoNo47.5Group 6NormalYesNo12Group 7NormalNoYes12This study shows that an in vitro p53 functional analysis can predict with an acceptable sensitivity the presence of TP53 gene disruption and could be useful to identify pts with TP53 mutation without 17p deletion. Interestingly, this functional assay coupled with cytogenetic and mutational screening could reveal 3 sub-groups of pts with potential clinical consequences: i) normal p53 function despite a del17p deletion (group 6) ii) normal p53 function despite a TP53 mutation (group 7) and in contrast iii) abnormal p53 function without any TP53 gene disruption (group 5) allowing to describe alternative alterations of p53 pathway. Disclosures:Dilhuydy:Roche: Honoraria. Leblond:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Mundipharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Feugier:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees. Tournilhac:MUNDIPHARMA: Consultancy, travel funding Other; GSK: Consultancy, travel funding, travel funding Other; Celgene: Consultancy, teaching, teaching Other.

Distinct IGHV Repertoire Features In Chinese Chronic Lymphocytic Leukemia Patients

Mounting evidence indicates that immunoglobulin variable heavy-chain (IGHV) repertoire and mutational status in chronic lymphocytic leukemia (CLL) patients are prognostically relevant. However, rare data is available in Chinese CLL population. Our group investigated 270 Chinese CLL patients for their IGHV sequences and discovered significant differences between Chinese and European CLL patients. First of all, 169 (62.6%) patients in our group got mutated IGHV and 101 (37.4%) were unmutated, rendering a considerable higher percentage of mutated subgroup compared with European patients (55%) (Figure 1). While IGVH3 is still the most frequently used IGVH gene in Chinese CLL patients (135/270, 50%), discrepancy occurs in the usage of IGVH1 gene, which only presents in 13.7% (37/274) patients in our cohort whereas 23.79% for European (Figure 2). Regarding IGHV subgroups, IGHV3-23 and IGHV4-34 are more often used in Chinese CLL patients (10.7% and 10.4%, respectively). Remarkably, IGHV1-69, the most prevalent IGHV subgroup in European CLL patients (12.81%), only accounts for 5.2% (14/270) Chinese cases. [Display omitted] [Display omitted] [Display omitted] We further studied the distribution of stereotyped BCR in our cohort. Thirty-eight patients (14.07%) with stereotyped BCR that belonged to 21 subsets were identified, with 1 to 7 sequences contained each. Among them, subset 1 and subset 8 are the most common types with 6 and 7 cases respectively. Three new subsets were discovered (Table 1). Notably, only 1 case belonged to subset 2, the subset with largest group size in western world. Hence, we conclude that Chinese CLL patients show unique IGHV repertoire features compared to patients from western countries. While the mechanism within remains unknown, the discrepancy might due to antigenic difference in geographically remote areas.Table 1Three new subsets of BCR stereotypy in Chinese CLL patientsNO.IGHVIGHDIGHJM/UMIdentityHCDR3 AA sequenceLengthNovel 1NJ-15IGHV4-59*083-22*016*03UM100,00%ARGNYYDSSGYYYVGYYYYYMDV23NJ-31IGHV4-59*013-22*016*03UM99,65%ARGDYYDSSGYYYVGYYYYYMDV23Novel 2NJ-186IGHV3-23*013-22*014*02M96.60%AKGYRDNYDGDQSSVFDS18NJ-23IGHV3-23*012-21*014*02M96,53%AKGYRDNYDGDQSSVFDS18Novel 3NJ-36IGHV4-34*016-6*015*02M93,33%AKLMAGRPNWFDP13NJ-123IGHV4-34*016-6*015*02M91,67%AKLMAGRPNWFDP13 Disclosures:No relevant conflicts of interest to declare.

The detection of minimal residual disease status in chronic lymphocytic leukemia

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

Peptide vaccination induces profound changes in the immune system in patients with B-cell chronic lymphocytic leukemia

Although the immune status of chronic lymphocytic leukemia (CLL) patients is mostly characterized by immunosuppression, there is an accumulation of in vivo (graft-versus-leukemia effect) and in vitro (spontaneous remissions after infections) data that indicates that CLL might be effectively targeted by T-cell based immunotherapy. Recently, we characterized receptor for hyaluronic acid mediated motility (RHAMM) as a preferential target for immunotherapy of CLL. We also completed a RHAMM-derived peptide vaccination phase I/II clinical trial in CLL. Here, we present a detailed immunological analysis of six CLL patients vaccinated with HLA-A2 restricted RHAMM-derived epitope R3 (ILSLELMKL). Beside effective induction of R3-specific cytotoxic T-cells, peptide vaccination caused profound changes in different T-cell subsets as well as cytokines. We present longitudinal analyses of Th17, CD8(+)CD103(+), CD8(+)CD137(+) and IL-17 producing CD8(+) T cells (CD8(+)IL- -17(+)) as well as important cytokines involved in regulation of immune response such as TGF-β, IL-10, IL-2 and TNF throughout the peptide vaccination period.

Chronic Lymphocytic Leukemia: “Cinderella” Is Becoming a Star

It has taken time for the status of chronic lymphocytic leukemia (CLL) to change within the scientific community. CLL, characterized by the accumulation of seemingly innocent long-lived monoclonal B cells exhibiting mature morphologies, has long been considered the "Cinderella" of blood cancers. CLL is receiving increasing attention from biologists and clinicians, however, because understanding of this disease may elucidate the association between lymphoid tumous and autoimmunity as well as help to define the relationships between antigen stimulation and malignant transformation.