Early Stage Chronic Lymphocytic Leukemia Patients Research Articles

IGLV3-21 R110 Is a Prognostic Marker for Early Stage CLL Patients Under Ibrutinib Treatment or Watch & Wait: Results from the Double-Blind, Randomized, Placebo-Controlled GCLLSG CLL12 Trial

INTRODUCTION: IGLV3-21 R110 is a point mutation that can confer the ability for autonomous signaling to B cell receptors (BCR) using light chains encoded by the allele IGLV3-21 ∗ 01 or IGLV3-21 ∗ 04. Chronic lymphocytic leukemia (CLL) patients with IGLV3-21 R110 have a shorter time to first treatment and shorter overall survival, independent of their IGHV mutational status. However, outcomes of CLL patients using IGLV3-21 R110 have not been evaluated in clinical trials of BTK inhibitors. METHODS: The CLL12 trial was a prospective, double-blind, randomized phase 3 trial that compared ibrutinib (ibr, n=182) vs. placebo (pcb, n=181) in previously untreated Binet A CLL patients with intermediate to very high risk, while low risk patients were assigned to watch & wait (w&w, n=152). The primary endpoint of the study was event-free survival (EFS) (event defined as death, symptomatic progression or initiation of CLL treatment), while overall survival (OS) was a secondary endpoint. The IGLV3-21 locus was sequenced via amplicon-based targeted next generation sequencing (tNGS) in 514 of 515 samples collected at enrolment. IGLV rearrangements and point mutations were detected with ScanIndel as well as IgCaller and the results of the genetic analyses were correlated with outcome. RESULTS: IGHV was unmutated (U-IGHV) in 148 (28.3%) patients (w&w n=8, pcb n=70, ibr n=70) and 10 patients belonged to stereotyped B cell receptor (BCR) subset 2 (w&w n=2, pcb n=1, ibr n=7). IGLV3-21 rearrangement was found in 34 of 514 patients (6.6%) and was more frequent in the intermediate/high risk groups (pcb n=16, ibr n=13) than in the low risk group (w&w n=5). Twenty of 34 patients with IGLV3-21 rearrangement had an IGLV3-21 R110 mutation (w&w n=3/5, pcb n=10/16, ibr n=7/13). For 5 patients with IGLV3-21 (3 pcb, 2 ibr) the mutational status for IGLV3-21 R110 could not be determined by tNGS. In the total cohort, IGLV3-21 R110 often co-occurred with mutations in SF3B1 (odds ratio 8.54, p < 0.001), but not with mutations in TP53 or any of the other frequently mutated genes in CLL. Conversely, SF3B1 mutations were not enriched in subset 2 patients (p = 0.165). Also, there was no association of IGLV3-21 R110 or IGLV3-21 usage in general with IGHV mutational status. At a median follow-up of 69.3 months, there were 166 events for EFS and 32 for OS. CLL with IGLV3-21 had a shorter EFS in the pcb (HR 1.93, CI95% 1.05-3.55, p=0.033) and ibr (HR 3.71, CI95% 1.62-8.46, p=0.002) arm (Figure 1A). The estimated 5-year EFS rate was 51.0% without and 21.4% with IGLV3-21 rearrangementin the pcb group vs. 80.7% and 44.8% in the ibr group, respectively. Similarly, IGLV3-21 R110 associated with shorter EFS with ibr (HR 3.30, CI95% 1.17-9.31, p=0.024) while no impact was observed in the pcb group (HR 1.51, CI95% 0.66-3.46, p=0.332, Figure 1B). Patients with IGLV3-21 R110 mutation and ibr treatment had an estimated 5-year EFS rate similar to that of patients in the pcb group without IGLV3-21 R110 (50.0% vs. 49.8%). In low risk patients with w&w, both IGLV3-21 rearrangement and IGLV3-21 R110 were adverse markers for EFS (HR 4.99, CI95% 1.52-16.41, p=0.008 and HR 17.0, CI95% 4.99-58.13, p<0.001, respectively). For the full trial, we observed a prognostic impact of IGLV3-21 in the M-IGHV (HR 3.87, CI95% 2.14-6.99, p<0.001) but not in the U-IGHV (HR 1.61, CI95% 0.74-3.54, p=0.233) group. Multivariable analysis for the treatment arms including the IGLV3-21 R110 status and other variables associated significantly with EFS in univariate analyses (treatment arm, serum β2-microglobulin, IGHV status, del(17p), del(11q), +(12), mutations in XPO1, NOTCH1, SF3B1 and NFKBIE) revealed presence of IGLV3-21 R110 (HR 2.76, CI95% 1.32-5.80, p = 0.007) as an independent prognostic factor. OS was excellent in all genetic subgroups and in all arms of the trial. The estimated 5-year OS rate was between 92.9% and 95.8% for any of the 4 subgroups defined jointly by IGHV status and presence of IGLV3-21 R110. CONCLUSIONS: IGLV3-21 R110 was identified as an independent prognostic factor for shorter EFS in early stage CLL with intermediate/high risk score and was associated with reduced ibrutinib efficacy in the CLL12 trial.

ZMYM3 Mutations Cooperate with NOTCH1 Alterations, Reduce Histone H4 Acetylation and Promote Apoptosis Evasion in Chronic Lymphocytic Leukemia

Introduction: Recent next-generation sequencing (NGS) studies have identified up to 200 recurrently mutated genes in chronic lymphocytic leukemia (CLL), the vast majority of which appear mutated in <5% of CLL cases. Among the long list of CLL candidate driver genes is ZMYM3, a gene mutated in 2-4% of CLL patients. Since the impact of ZMYM3 mutations is unknown, we aimed to identify their clinical and biological consequences in CLL. Methods: 487 CLL patients were analyzed by targeted NGS using a customized panel of 54 CLL-related genes to characterize the mutational profile of ZMYM3 mutated patients ( ZMYM3MUT) and the clinical impact of these variants. In addition, we introduced ZMYM3 truncating mutations into the CLL-derived HG3 cell line with two genetic backgrounds: wild-type (WT), and NOTCH1 mutated cells ( NOTCH1MUT), to generate cells with either single ZMYM3MUT, NOTCH1MUT or combined ZMYM3MUTNOTCH1MUT. In these CLL models, we evaluated the impact of these alterations in gene expression, apoptosis, growth and DNA damage response (DDR). Results: A total of 32 ZMYM3 variants were identified in 30 CLL patients, most of which were loss-of-function mutations (24/32; 75%). These variants were distributed throughout the protein sequence, with a particular recurrent frameshift mutation occurring in 6 patients (p.P48fs) that was subsequently reproduced in our CLL model. Attending to their mutational profile, we identified that 70% (21/30) of ZMYM3MUT patients harbored mutations in the NOTCH pathway, either in NOTCH1 (60%) or in negative regulators such as MED12, FBXW7 and SPEN, suggesting a cooperation between ZMYM3 dysfunction and NOTCH1 signaling mutations in CLL evolution. Interestingly, ZMYM3MUT patients exhibited significantly shorter time to first treatment (TFT) than ZMYM3WT cases (median: 35 vs 52 months; p=0.010). Furthermore, ZMYM3 mutations shortened TFT of early stage CLL patients, including Binet A (48 vs 108 months, p=0.002) or Rai 0/1 (48 vs 91 months, p=0.016), as well as in low-risk cytogenetics del(13q) patients (median: 45 vs 93 months; p=0.033). We next addressed the biological implications of ZMYM3 mutations and their co-occurrence with NOTCH1 mutations in HG3 cells. First, we performed RNA-sequencing and analyzed the number of differentially expressed genes (DEGs) (|fold change|>2, adjusted p-value<0.05). Comparing with WT cells, we detected 155 DEGs in ZMYM3MUT and 83 DEGs in NOTCH1MUT, whereas ZMYM3MUTNOTCH1MUT cells showed 690 DEGs,indicating that the combination of both mutations profoundly dysregulate gene expression. Immune regulation, apoptosis and DNA damage response were some of the biological processes dysregulated by ZMYM3 mutations (p<0.05). Furthermore, considering the suggested role of ZMYM3 in chromatin remodeling (Puente et al, Nature. 2015) and that ZMYM3MUT cells showed altered expression of histone acetylation related genes (p<0.05), we identified that ZMYM3 mutations reduced global acetylation levels of histone H4 (p<0.05), which may underlie the ZMYM3-related changes in gene expression. To further define the functional effects of ZMYM3 mutations, we assessed in vitro their impact in the main dysregulated pathways identified by RNA-seq. First, we identified that ZMYM3 mutations impair DNA damage response, as reflected by the difficulties to arrest cell cycle in G2/M phase after irradiation. Moreover, we detected more γH2AX foci (p<0.05) in ZMYM3MUTcells, indicating persistent DNA damage; and fewer RAD51 and BRCA1 foci than WT cells (p<0.001) after irradiation, suggesting defective DNA damage response. In parallel, we identified that ZMYM3 mutations promote apoptosis evasion, especially in the context of NOTCH1 mutations (p<0.001), which led to enhanced growth of ZMYM3MUTNOTCH1MUT cells (p<0.05). Of note, ZMYM3MUT cells showed down-expression of caspases (-8, -7 and -3) and slightly higher levels of anti-apoptotic proteins (BCL2, MCL1 and BCL-XL), which may explain this apoptosis resistance. Conclusions: Mutations in ZMYM3 are mainly loss-of-function, associate with NOTCH1 signaling mutations and shorten TFT in CLL patients, suggesting that ZMYM3 mutational status may be a useful marker in the management of early stage CLL patients. Moreover, ZMYM3 mutations reduce histone H4 acetylation and cooperate with NOTCH1 mutations to dysregulate gene-expression, leading to impair DNA damage response and apoptosis evasion.

Proteomic Profiling Based Classification of CLL Provides Prognostication for Modern Therapy and Identifies Novel Therapeutic Targets

The availability of targeted therapy and improved molecular characterization of Chronic Lymphocytic Leukemia (CLL) require a re-evaluation of treatment paradigms. As CLL heterogeneity is dependent on molecular and environmental factors, there is a need to create a new classification based on the integration of several factors. Here, we accomplish this goal by identifying CLL signatures using Reverse Phase Protein Array. Protein expression for 384 total and post translationally modified proteins was assessed in 871 CLL and Mature Small B Cell Leukemia (MSBL: HCL, HCLV, LGL-T, MCL, MZL, PLL, Richter's, T-Cell PLL) patients and was integrated with clinical data to identify strategies for improving diagnostics and therapy, making this the largest CLL proteomics study to date. Proteins were categorized into 40 protein functional groups (PFGs) based on literature and intra-dataset protein correlations and patients clustered based on PFG expression patterns into 6 recurrent protein expression signatures (PES) (Figure 1A). Individual protein expression (58/384 proteins), PFG expression (32/40) and overall PES were all highly prognostic of survival (OS) and time to first or second treatment (TTFT, TTST) (Figures 1B-C). The adhesion, apoptosis-occurring, apoptosis-regulating, heat shock, histone1 (marks), histone 2 (modifiers) and the STP-regulation PFGs were prognostic for all 3 outcome measures. Notably SG-A contained most of the MSBL and 15/16 cases of hairy cell leukemia, but the CLL cases within this SG fared very poorly. For OS, groups A and C had markedly inferior survival (P<0.0001) (10.3 and 20.3 median years) relative to the other 4 groups, which were statistically similar to each other. First treatment occurred sooner for Groups A and C (5.8 and 5.23 median years). Additionally, the TTST was also inferior for Group A (median 3.5 years). There were significant differences in age, hemoglobin, platelets, % BM and PB lymphocytes and β2M between the SG, but not for race (p = 0.84) or gender (p = 0.72). , Historically adverse cytogenetic aberrations del 11q and del17p events (23% overall) were less common in SG A, B, D and E (15, 14, 16, 17%) and overrepresented in SG C (32%), while historically favorable 13q changes were seen across all groups as was Trisomy 12 (14% overall), although SGs A and E were enriched (25%, 22%) while SG-F was low (5%) for Trisomy 12. SG membership superseded other traditional prognostic factors (Rai Staging, IGHV Status) and were prognostic for modern (BTK inhibition) and older CLL therapies. SGs A and C responded poorly to chemotherapy regimens compared to the other groups, whereas all groups responded well to BTK inhibitors except for SG-A. SGs and PFGs membership provided novel drug targets (see our other abstracts on TP53BP1 and ASNS) and defined optimal candidates for Watch and Wait (WaW) vs. early intervention. A model based on the accumulation of irregularities in ANXA1, TFRC, and SMAD2.p245 expression, optimally predicted TTFT overall and in early stage CLL patients. Patients with < 1 negative level of either of the 3 proteins, have a median TTFT of 14.59 years, whereas patients having 2-3 have a median of 5-6.27 years (P<0.0001). CHEK1.pS345, GAB2, IGFBP2, S100A4, WEE1.pS642, and ZAP70 were universally overexpressed by all SGs, suggesting them as ideal targets for inhibition. Collectively proteomics demonstrates promise for improving classification, therapy strategy determination, and identifying novel therapeutic targets. [Display omitted] DisclosuresFerrajoli: Janssen: Other: Advisory Board ; AstraZeneca: Other: Advisory Board, Research Funding; BeiGene: Other: Advisory Board, Research Funding. Thompson: Genentech: Other: Institution: Advisory/Consultancy, Honoraria, Research Grant/Funding; Amgen: Other: Institution: Honoraria, Research Grant/Funding; Adaptive Biotechnologies: Other: Institution: Advisory/Consultancy, Honoraria, Research Grant/Funding, Expert Testimony; Pharmacyclics: Other: Institution: Advisory/Consultancy, Honoraria, Research Grant/Funding; Janssen: Consultancy, Honoraria; Gilead: Other: Institution: Advisory/Consultancy, Honoraria; AbbVie: Other: Institution: Advisory/Consultancy, Honoraria, Research Grant/Funding. Burger: Pharmacyclics LLC: Consultancy, Other: Travel/Accommodations/Expenses, Research Funding, Speakers Bureau; Beigene: Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel/Accommodations/Expenses, Research Funding, Speakers Bureau; Gilead: Consultancy, Other: Travel/Accommodations/Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel/Accommodations/Expenses, Speakers Bureau; AstraZeneca: Consultancy; Janssen: Consultancy, Other: Travel/Accommodations/Expenses, Speakers Bureau. Wierda: Xencor: Research Funding; Karyopharm: Research Funding; Gilead Sciences: Research Funding; Acerta Pharma Inc.: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; AstraZeneca: Research Funding; Juno Therapeutics: Research Funding; KITE Pharma: Research Funding; Sunesis: Research Funding; Miragen: Research Funding; Oncternal Therapeutics, Inc.: Research Funding; Cyclacel: Research Funding; Loxo Oncology, Inc.: Research Funding; Janssen: Research Funding; Genentech: Research Funding; GSK/Novartis: Research Funding; Genzyme Corporation: Consultancy; AbbVie: Research Funding.

Abnormal Karyotype (Not Only Complex) at Diagnosis Is an Independent Prognostic Factor for Overall Survival in Early Stage Chronic Lymphocytic Leukemia Patients. Results of a Prospective Study with a Median Follow-up of More Than 7-Years

Introduction: Only few studies have prospectively dealt with the prognostic impact on survival of both clinical and laboratory abnormalities in patients with early stage chronic lymphocytic leukemia (CLL) at diagnosis. In our previously published analyses including a prospective cohort of 307 CLL (Oliveira et al, Leuk Lymphoma. 2011; 52:429-35, Oliveira et al, Ann Hematol. 2015; 94:627-32), patients with atypical lymphocyte morphology, 11q deletion, and elevated beta-2 microglobulin had a shorter progression-free survival (PFS) and treatment-free survival (TFS). Abnormal karyotype (AK), 1 or more abnormalities at diagnosis, was not an independent prognostic factor at that time. Complex karyotype (CK), 3 or more abnormalities, despite having been described as an important prognostic marker in retrospective studies, was a rare finding at diagnosis in early stage CLL patients. The aim of this study is to evaluate prognostic factors at diagnosis in patients with early stage CLL for PFS, TFS and also OS, with a longer follow-up.Patients and methods: 307 patients were prospectively enrolled in the study from 2003 to 2010 in a single center. All clinical and laboratory data were homogeneously collected at diagnosis. Analysis of prognostic factors for survival was performed in 231 patients presenting early stage disease: lymphocytosis without other signs or symptoms of CLL (CLL Rai 0 and monoclonal B lymphocytosis, MBL). Survival curves were plotted using Kaplan-Meier's method and log-rank test was used to identify differences in survival between subgroups. Multivariate analysis was performed with Cox regression model. Variables included in the univariate analysis were: age, gender, LDH, beta-2 microglobulin, lymphocyte morphology, CD38, ZAP-70, deletions of 13q, 11q and 17p, trisomy 12, normal FISH and AK. The significant variables in the univariate analysis were included in the multivariate regression model.Results: Eighty-one patients had MBL and 150 were diagnosed with CLL Rai 0, 145 (62.8%) patients were male with a median age at diagnosis of 68 years (limits 27-93). One hundred sixteen patients presented a normal karyotype at diagnosis, 54 (23.3%) an AK [7 (3%) of them a CK], in 9 (3.9%) patients no metaphases were obtained for karyotype analysis and in 52 (22.5%) karyotype was not performed. With a median follow-up of 7.8 yrs (range 0.7-13.6), 37 (16%) patients died, 73 (31.6%) presented CLL progression and 60 (26%) required treatment. OS was 84%. AK was an independent prognostic factor for OS (OR 2.2, 95%CI: 1.0-4.6, p=0.038), TFS (OR 2.9, 95%CI: 1.4-6.0, p=0.003), and PFS (OR 2.4, 95%CI: 1.3-4.6, p=0.005). Atypical lymphocyte morphology was also an independent prognostic factor for OS (OR 2.7, 95%CI: 1.3-5.8, p=0.008) and elevated beta-2 microglobulin for PFS (OR 2.1, 95%CI:1.1-3.9, p=0.018).Conclusions: Abnormal karyotype (not only complex) at diagnosis in early stage CLL (CLL Rai 0 or MBL), is an independent prognostic factor for OS, PFS and TFS. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Tumor Microenvironment (TME) Regulation of Programmed Death-1 (PD1) Receptor and Its Ligands PDL1 and PDL2 in Chronic Lymphocytic Leukemia (CLL)

The PD1 pathway is involved in the inactivation of immune effectors and is potentially reactive against neoplastic cells in the TME. T cells from CLL patients exhibit defective immunity, decreased proliferative capacity, and impaired effector function leading to T cell exhaustion. These functional defects coincide with higher CD244, CD160 and PD1 expression on T cells. In the Eμ-TCL1 transgenic CLL model, PDL1 checkpoint blockade restores immune dysfunction and impairs leukemia growth. PD1/PDL1 ligation also affects BCR signaling, and because PD1 is also expressed on CLL cells, PD1 interference might directly influence tumor growth and proliferation directly.We studied the 1) expression the PD1 receptor and its ligands and their correlation with TFTT; 2) role of the TME in controlling PD1 axis and miRNA expression; 3) role of the BCRi ibrutinib (IB) in the expression of the receptor and ligands.Early-stage CLL patients were prospectively enrolled at diagnosis (n=211, O-CLL1: clinicaltrial.govID:NCT00917540). Gene expression (GE) profiling was performed using the GeneChipVR Gene1.0 STArray (Affymetrix) and validated by qPCR (Thermofisher) and flow-cytometry (FC, BD Biosciences) in an independent subset of patients. CLL lymph node samples underwent in situ immunolocalization analyses. Autologous T-cells (AAT) were obtained by in vitro exposure of patient T-cells with anti CD3/CD28 Dynabeads (Thermofisher) and IL2 in co-culture with CLL cells. Cultures were monitored until substantial clumping was observed and tested for PD1 axis expression. HS5 stromal cells were used in co-culture experiments with B-CLL (ratio 1:100). MiR-424 expression was evaluated by qPCR using validated TaqMan® MicroRNA Assays (Thermofisher). In selected experiments IB was added to cell culture. PD1 GEP of circulating B-CLL cells was significantly higher than of ligands. No differences in GEP of PD1 or its ligands were observed stratifying cases based on CD38, ZAP-70, or FISH markers. Significantly higher PDL1 GE was detected in IGHV-M cases. GEP results were validated by qPCR on 27 independent CLL samples. The impact of GE of the PD1 axis on clinical outcomes in CLL cases (median follow-up 39 months, range 6−82 months) indicated TTFT was significantly shorter in cases with higher levels of PDL2, while no significant differences were detected based on higher or lower PD1 or PDL1 levels. Multivariate analysis indicated higher PDL2 gene expression retained an independent prognostic power (HR=1.9, 95%CI 1.1-3.4, P=.022) in predicting TTFT together with IGHV-UM status, B-lymphocytosis≥5000/mm3 (P=.020), and CD38 expression (P=.021). In situ immunolocalization of CLL tissue infiltrates showed variable expression of PD1 that characterized small lymphoid elements, while PDL1 and PDL2 (PDL2>PDL1) characterized larger medium-sized elements within proliferation centers. Co-localization studies revealed PD1 co-expression of CD20+CLL cells and in scattered CD3+ cells. Both PDL1 and PDL2 were variably expressed in CD20+CLL cells; few T-cells and macrophages showed expression of both ligands within CLL infiltrates. FC analysis of CLL cells after AAT co-culture showed a higher percentage expression of circulating PD1 axis members (n=27, PDL1>>PDL2>PD1) compared to baseline indicating that the changes in PD1 axis expression may be due to events triggered by activation of the TME. In contrast, T cell populations had higher expression of CD3+, CD4+ and CD8+ cells bearing both PD1 and PDL1, while no substantial increment for either CD3+, CD4+ or CD8+/PDL2+ cells following AAT co-culture. GE analysis also showed a similar increase in PDLs (n=15, PDL2>>PDL1>PD1) expression with respect to non-activated B-CLL cells. Interestingly, a 48h co-culture of B-CLL with HS5 stromal cells (n=6) showed a similar increase in PD1 axis GE pattern as AAT cultures. Expression of miR-424 also decreased significantly (<80%) after AAT co-cultures (n=6), indicating its potential involvement in PDL1 regulation. IB reduced PD1/PDL1 gene and protein expression on CLL B-cells; the IB inhibitory effect was also observed only on CD8+ cells.We indicate that PDL2 expression characterizes a subset of high-risk early stage CLL patients. PD1 axis expression is characteristic of the CLL TME whereby modulatory co-stimulatory signals may be counteracted by IB. Further studies will examine the role of miR-424 regulation with this cellular context. DisclosuresNo relevant conflicts of interest to declare.

Mutations of the Exportin 1 (XPO1) Gene Predict Shorter Time to First Treatment in 1092 Early Stage Chronic Lymphocytic Leukemia Patients. Α Training/Validation Study

Mutations of the Exportin 1 (XPO1) Gene Predict Shorter Time to First Treatment in 1092 Early Stage Chronic Lymphocytic Leukemia Patients. Α Training/Validation Study

IL-17 and IL-23 levels in patients with early-stage chronic lymphocytic leukemia.

OBJECTIVE:Cytokines produced by bone marrow mesenchymal stem cells are important components of the tumor microenvironment in chronic lymphocytic leukemia (CLL). The roles of IL-17 and IL-23 in both autoimmune diseases and tumor growth have been demonstrated. The role of the IL-17/23 axis in apoptosis has also been demonstrated in studies. Autoimmune cytopenias are common in CLL. In this study, we aimed to compare IL-17/IL-23 levels in early-stage CLL patients with healthy controls.METHODS:After obtaining ethical approval from the local ethics committee, 22 patients with early-stage chronic lymphocytic leukemia and 21 healthy control groups were included in this study. IL-17 and IL-23 were analyzed using the enzyme-linked immunosorbent assay method.RESULTS:The findings showed that the median IL-23 level was lower in men in the chronic lymphocytic leukemia group than women. There was a positive correlation between IL-17 and IL-23 levels in both the control group and the chronic lymphocytic leukemia group. There was no significant correlation between stage and IL-17 and IL-23 levels in chronic lymphocytic leukemia patients.CONCLUSION:Results of studies conducted on IL-17 and/or IL-23 in chronic lymphocytic leukemia in the literature are not consistent. These inconsistent results can be explained by the fact that the immune system develops differently in each individual due to environmental factors, past infections, intestinal flora, vaccines, ethnicity, and even gender. Therefore, it can be hypothesized that the development and application of personalized immunotherapy strategies instead of standard therapy in chronic lymphocytic leukemia may increase therapeutic success rates.

XPO1 Mutations May Identify Binet Α Chronic Lymphocytic Leukemia Patients with Shorter Time to First Treatment

Background. Chronic lymphocytic leukemia (CLL) is the most common leukemia of adults in western countries. CLL is a highly heterogeneous disease; some patients may never require treatment, whereas other relapse early after frontline therapy. In approximately 70% of newly diagnosed cases, CLL presents at an early clinical stage and is managed with a watch &amp; wait strategy. Until now, few clinical and molecular predictors inform on the risk of treatment requirement and the impact of CLL gene mutations is not completely understood. Purpose. We aimed at identifying new molecular markers that may predict early treatment requirement and may help clinicians to better plan the watch &amp; wait strategy in asymptomatic early stage CLL patients. Methods. This study includes 295 Binet A CLL patients referring at our institution who did not require treatment for at least 3 months after diagnosis. Tumor genomic DNA (gDNA) was isolated from peripheral blood mononuclear cells at the time of diagnosis. gDNA was analyzed in the coding exons plus splice sites of the most frequently mutated genes in CLL with a next-generation-sequencing (NGS) approach. NGS analysis was performed on the Illumina MiSeq instrument (coverage &gt;2000x in &gt;80% of the target region). The somatic function of VarScan2 was used for variant calling and a stringent bioinformatic pipeline was developed to protect against the false call of polymorphisms and sequencing errors. A threshold of 5% of variant allele frequency was set for variant calling. The primary endpoint was time to first treatment (TTFT) defined as the timeinterval between the date of CLL diagnosis and the date of first CLL treatment. Statistical analysis was performed using SPSS version 24.0. Results. The median age of the study cohort was 70.8 years old, 136 (46.1%) patients were female, 71 (24.1%) harbored unmutated IGHV genes, 44 (14.9%) had trisomy 12, 12 (4.1%) had 17p deletion, 15 (5.1%) had 11q deletion and 150 (50.8%) had 13q deletion. NGS mutational analysis showed that NOTCH1 was the most frequently mutated gene occurring in 25 (8.5%) patients, followed by ATM in 18 (6.1%), TP53 in 17 (5.8%), MYD88 in 12 (4.1%), SF3B1 in 10 (3.4%), XPO1 in 7 (2.4%), EGR2 in 6 (2.0%), NFKBIE in 4 (1.4%), POT1 in 2 (0.7%), and BIRC3 in 1 (0.3%) patients. After a median follow-up of 9.5 years, 80 (27.1%) patients required treatment. In univariate analysis, molecular characteristics associated with a shorter TTFT were trisomy 12 (HR: 2.42; 95% CI 1.43-4.15; p=0.001), unmutated IGHV genes (HR: 4.51; 95% CI 2.83-7.05; p&lt;0.0001) and mutations of XPO1 (HR: 8.88; 95% CI 3.77-20.95; p&lt;0.0001), NOTCH1 (HR: 3.02; 95% CI 1.66-5.51; p&lt;0.001) and SF3B1 (HR: 2.65; 95% CI 1.15-6.10; p=0.022). Interestingly, neither 17p deletion nor TP53 mutations associated with a shorter TTFT, in line with the notion that TP53 disruption interacts with treatment but not with a watch &amp; wait strategy. By multivariate analysis, XPO1 mutations (HR: 4.24; 95% CI 1.72-10.44; p=0.002) maintained an independent association with a shorter TTFT (Table 1). At 10 years, XPO1 mutated patients had a probability of remaining free from treatment of 0% compared to 69.3% for wild type cases (p&lt;0.0001) (Figure 1). Conclusions. Mutations of the XPO1 gene, encoding for exportin 1 which mediates the nuclear export of proteins and RNA, are an independent predictor of shorter TTFT and, if validated, might help clinicians in a better management of the watch &amp; wait strategy for Binet A CLL patients. Moreover, since most of mutations, approximately 90%, affect the glutamic acid in position 571, polymerase chain reaction based methods may be used to identify XPO1 mutations in a simple and time effective manner. Disclosures Rossi: Abbvie: Honoraria, Other: Scientific advisory board; Janseen: Honoraria, Other: Scientific advisory board; Roche: Honoraria, Other: Scientific advisory board; Astra Zeneca: Honoraria, Other: Scientific advisory board; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Gaidano:AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astra-Zeneca: Consultancy, Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sunesys: Consultancy, Honoraria.

Multicentre Genome Wide Association Study Identifies Risk Alleles for Progressive Chronic Lymphocytic Leukaemia

The increased incidence of chronic lymphocytic leukemia (CLL) in first-degree relatives of affected patients indicates an element of genetic susceptibility to this malignancy, borne out in large scale genome-wide association studies (GWAS), which have identified over 40 constitutional risk alleles. Given the important genetic contribution to CLL susceptibility we hypothesized that constitutional genetic variants also affect disease progression. We employed GWAS methods in a large United Kingdom multi-center cohort study of well-characterized predominantly early-stage CLL cases to identify risk alleles for progressive CLL. We conducted six GWAS for single nucleotide polymorphisms (SNPs) associating with progressive CLL incorporating a total of 774 cases of European ancestry recruited to 6 clinical centers across the United Kingdom. CLL cases were genotyped on the Illumina OmniExpress platform and genotypes were determined using Illumina GenomeStudio software. After imputation, we combined the association test statistic for 5,199,911 autosomal SNPs common to all 6 GWAS after exclusion of those with an imputation quality score of &lt;0.9 and a minor allele frequency (MAF) of &lt; 2.5%, and conducted a meta-analysis under a fixed-effect model. The primary outcome assessed was time to first treatment (TTFT), defined as the interval between CLL diagnosis and first treatment or last follow-up. Pooling data from the 6 GWAS identified 5 SNPs at two genomic locations that surpassed genome-wide significance (P ≤ 5 x 10-8) for association with TTFT. The strongest statistical evidence for an association with progressive disease was for rs736456 (hazard ratio (HR) = 1.76, 95% confidence interval (CI) = 1.45-2.14; P = 1.26 x 10-8), which maps to chromosome 10q26.13. The second strongest association with progressive disease was for rs3778076 (HR = 2.03, 95% CI = 1.58-2.62; P = 3.89 x 10-8) which maps to chromosome 6p. Both markers showed consistent direction and magnitude of effect sizes across all six GWAS with no evidence of heterogeneity, and retained prognostic significance in multivariate models for disease progression, particularly in models restricted to Binet A patients. Whilst not as powerful as IGVH status, rs736456 and rs3778076 had prognostic utility equivalent to CD38 status, and are particularly powerful when considered together, identifying 5% of CLL patients carrying 2 or more risk alleles at high risk of progressive disease (Figure). To identify cis-regulated genes at each locus associated with progressive disease we interrogated gene expression data derived from a meta-analysis of 31,624 blood samples collated by the eQTLGen consortium. Of the thirteen genes annotated to within 1Mb of the chromosome 10 association signal rs736456 is eQTL for the PLEKHA1 gene (TAPP-1)(Benjamini-Hochberg corrected P-value [PBH] = 1.29 x 10-15). Of the 27 genes annotated to within 1Mb of the chromosome 6 signal, rs3778076 is eQTL for 5 genes including UHRF1BP1 (PBH = 6.73 x 10-139) and C6ORF106 (PBH = 3.54 x 10-64). Annotated genes at both loci have been implicated in modulating B-cell receptor or innate immune responses, key pathways in CLL pathogenesis. Data on post-treatment survival were available on 390 CLL cases, with 231 deaths and 159 censored at last follow-up, and neither the lead SNP at chromosome 10 or chromosome 6 were significantly associated with post-treatment survival in patients primarily treated with regimens that included chlorambucil, fludarabine or cyclophosphamide. It will be important to determine whether these markers predict overall survival in patients treated with novel targeted therapies. Taken together, these data identify rs736456 and rs3778076 as prognostic in early stage CLL patients demonstrating that progression of CLL from asymptomatic to symptomatic disease is determined by constitutional genetic variation as well as the more established somatic drivers. Constitutional genetic markers have the advantage of being easy to perform, highly reproducible and inexpensive making them ideal for incorporation into multivariate prognostication models for early stage CLL. Figure Disclosures Fegan: Roche: Honoraria; Janssen: Honoraria; Gilead: Honoraria; Abbvie: Consultancy, Other: Conference attendance sponsorship. Forconi:Gilead Sciences: Research Funding; Janssen-Cilag: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Novartis: Honoraria; Menarini: Consultancy; Abbvie: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Roche: Honoraria. Schuh:AbbVie: Consultancy, Speakers Bureau; Genentech: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy, Speakers Bureau; Verastem: Speakers Bureau; Gilead: Speakers Bureau; Seattle Genetics: Speakers Bureau; Jazz Pharmaceuticals: Speakers Bureau; Bristol-Myers Squibb: Research Funding; Janssen: Speakers Bureau; Kite: Speakers Bureau. Hillmen:Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses, Research Funding; Roche: Research Funding; Gilead: Research Funding; Apellis: Research Funding; Acerta: Membership on an entity's Board of Directors or advisory committees. Pratt:Binding Site, Amgen, Takeda, Janssen, Gilead: Consultancy, Honoraria, Other: Travel support.

Telomere Length Is Associated with Epigenetic Programming in CLL and Is a Superior Predictor of Clinical Outcome with the Ability to Bifurcate Patients with the Same CLL-IPI Score

The characteristic clinical heterogeneity observed in chronic lymphocytic leukemia (CLL) reflects its biological diversity and as a consequence, better understanding of the biology of this disease has led to the identification of a number of useful prognostic tools. Advances in massively parallel sequencing technologies have unearthed genetic and epigenetic heterogeneity and reinforced the concept that specific genetic lesions and the development of genomic complexity can modulate the pathology of CLL. In addition, DNA methylation profiling has shown that CLL patients can be grouped into at least three clinically relevant epigenetic subgroups with distinct outcomes. However, despite this explosion in knowledge, defining the prognosis of individual CLL patients remains a significant clinical challenge. Consequently, there is a need to identify prognostic tests that can provide reliable personalized risk assessments.We have recently shown that a subset of CLL patients manifest telomere dysfunction, which drives genomic instability and clonal evolution. We went on to show an association between short, dysfunctional telomeres and ‘high-risk’ genetic lesions1,2. Importantly, longitudinal analysis confirmed that tumor cell telomere length remained remarkably stable throughout the course of the disease suggesting that the telomere length is fixed at an early point in disease pathogenesis making it a reliable predictor of both clinical progression and overall survival (OS) regardless of when the measurement is made. In this study, we generated telomere length profiles in 224 newly diagnosed, early stage CLL patients seen at the Mayo clinic in Rochester using high-throughput single telomere length analysis (HT-STELA). We then tested the ability of telomere length to predict time to treatment (TTT) and OS and compared its predictive power with DNA methylation profiling and CLL-IPI scores.Patients with a mean telomere length inside the fusogenic range (IFR) had a significantly shorter time to treatment (P<0.0001; HR = 3.2) and OS (P = 0.0027; HR = 2.3) when compared to patients with a mean telomere length outside the fusogenic range (OFR) (Figure 1A and B). When considering epigenetic subgroups, we found a strong association between telomere length and ‘epitype’; high programmed (HP) CLL cells had significantly longer telomeres than intermediate programmed (IP), and low programmed (LP) CLL cells (P = 0.0002 and P<0.0001 respectively). Furthermore, bifurcation of the HP and IP groups into telomere length OFR and IFR subsets identified patients with distinctly different TTT (P = 0.0008; HR = 2.74) and OS (P = 0.004; HR = 2.4). 79/81 (97.5%) of the LP group had short telomere length IFR, but when we divided the LP group above and below the mean telomere length of the fusogenic range (≤/>2.17kb), patients with the shortest telomeres had a significantly worse OS (P = 0.034; HR = 2.83). Telomere length was also strongly associated with CLL-IPI score; the CLL-IPI low-risk group (CLL-IPI 1) had the longest telomeres and the CLL-IPI high-risk group (CLL-IPI 4) had the shortest telomeres (P<0.0001). More heterogeneity in telomere length was observed in the CLL-IPI 1 and 2 groups and patients with telomere length IFR in these ‘low-risk’ groups showed significantly shorter TTT (P<0.0001; HR = 4.24) and overall survival (P<0.0001; HR = 10.1) (Figure 1C and 1D). Finally, multivariate modelling, using Cox proportional hazards with forward selection, revealed that telomere length retained independent prognostic significance when CLL-IPI score was entered in the model for time to treatment (HR = 1.42; P = 0.023) and OS (HR = 1.68; P = 0.008). In conclusion, telomere length analysis provides more accurate prognostic information about the progression and outcome of CLL patients and can augment the prognostic power of the CLL-IPI for early stage CLL.ReferencesLin TT et al. Blood. 2010;116(11):1899-907.Britt-Compton et al. Leukemia. 2012; 26(4):826-30. [Display omitted] DisclosuresNorris:Cardiff University: Patents & Royalties: Telomere measurement patent. Shanafelt:GlaxoSmithKline: Research Funding; Genentech: Research Funding; Pharmacyclics: Research Funding; Jansen: Research Funding. Kay:Acerta: Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees. Fegan:Napp: Honoraria; Gilead Sciences, Inc.: Honoraria; Roche: Honoraria; Abbvie: Honoraria; Janssen: Honoraria. Baird:Cardiff University: Patents & Royalties: Telomere measurement patents. Pepper:Cardiff University: Patents & Royalties: Telomere measurement patents.

Tailored approaches grounded on immunogenetic features for refined prognostication in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) patients with differential somatic hypermutation status of the immunoglobulin heavy variable genes, namely mutated or unmutated, display fundamental clinico-biological differences. Considering this, we assessed prognosis separately within mutated (M-CLL) and unmutated (U-CLL) CLL in 3015 patients, hypothesizing that the relative significance of relevant indicators may differ between these two categories. Within Binet A M-CLL patients, besides TP53 abnormalities, trisomy 12 and stereotyped subset #2 membership were equivalently associated with the shortest time-to-first-treatment and a treatment probability at five and ten years after diagnosis of 40% and 55%, respectively; the remaining cases exhibited 5-year and 10-year treatment probability of 12% and 25%, respectively. Within Binet A U-CLL patients, besides TP53 abnormalities, del(11q) and/or SF3B1 mutations were associated with the shortest time-to-first-treatment (5- and 10-year treatment probability: 78% and 98%, respectively); in the remaining cases, males had a significantly worse prognosis than females. In conclusion, the relative weight of indicators that can accurately risk stratify early-stage CLL patients differs depending on the somatic hypermutation status of the immunoglobulin heavy variable genes of each patient. This finding highlights the fact that compartmentalized approaches based on immunogenetic features are necessary to refine and tailor prognostication in CLL.

Microenvironmental regulation of the IL-23R/IL-23 axis overrides chronic lymphocytic leukemia indolence.

Although the progression of chronic lymphocytic leukemia (CLL) requires the cooperation of the microenvironment, the exact cellular and molecular mechanisms involved are still unclear. We investigated the interleukin (IL)-23 receptor (IL-23R)/IL-23 axis and found that circulating cells from early-stage CLL patients with shorter time-to-treatment, but not of those with a more benign course, expressed a defective form of the IL-23R complex lacking the IL-12Rβ1 chain. However, cells from both patient groups expressed the complete IL-23R complex in tissue infiltrates and could be induced to express the IL-12Rβ1 chain when cocultured with activated T cells or CD40L+ cells. CLL cells activated in vitro in this context produced IL-23, a finding that, together with the presence of IL-23 in CLL lymphoid tissues, suggests the existence of an autocrine/paracrine loop inducing CLL cell proliferation. Interference with the IL-23R/IL-23 axis using an anti-IL-23p19 antibody proved effective in controlling disease onset and expansion in xenografted mice, suggesting potential therapeutic strategies.

Genome-wide DNA methylation profiling integrated with gene expression profiling identifies PAX9 as a novel prognostic marker in chronic lymphocytic leukemia

BackgroundIn chronic lymphocytic leukemia (CLL), epigenomic and genomic studies have expanded the existing knowledge about the disease biology and led to the identification of potential biomarkers relevant for implementation of personalized medicine. In this study, an attempt has been made to examine and integrate the global DNA methylation changes with gene expression profile and their impact on clinical outcome in early stage CLL patients.ResultsThe integration of DNA methylation profile (n = 14) with the gene expression profile (n = 21) revealed 142 genes as hypermethylated-downregulated and; 62 genes as hypomethylated-upregulated in early stage CLL patients compared to CD19+ B-cells from healthy individuals. The mRNA expression levels of 17 genes identified to be differentially methylated and/or differentially expressed was further examined in early stage CLL patients (n = 93) by quantitative real time PCR (RQ-PCR). Significant differences were observed in the mRNA expression of MEIS1, PMEPA1, SOX7, SPRY1, CDK6, TBX2, and SPRY2 genes in CLL cells as compared to B-cells from healthy individuals. The analysis in the IGHV mutation based categories (Unmutated = 39, Mutated = 54) revealed significantly higher mRNA expression of CRY1 and PAX9 genes in the IGHV unmutated subgroup (p < 0.001). The relative risk of treatment initiation was significantly higher among patients with high expression of CRY1 (RR = 1.91, p = 0.005) or PAX9 (RR = 1.87, p = 0.001). High expression of CRY1 (HR: 3.53, p < 0.001) or PAX9 (HR: 3.14, p < 0.001) gene was significantly associated with shorter time to first treatment. The high expression of PAX9 gene (HR: 3.29, 95% CI 1.172–9.272, p = 0.016) was also predictive of shorter overall survival in CLL.ConclusionsThe DNA methylation changes associated with mRNA expression of CRY1 and PAX9 genes allow risk stratification of early stage CLL patients. This comprehensive analysis supports the concept that the epigenetic changes along with the altered expression of genes have the potential to predict clinical outcome in early stage CLL patients.

Distinct patterns of global promoter methylation in early stage chronic lymphocytic leukemia

Genomic and epigenomic studies of chronic lymphocytic leukemia (CLL) are reshaping our understanding of the disease and have provided new perspectives for a more individualized diagnosis and new potential therapeutic targets. In this study, the global promoter methylation profile was determined in highly purified B-cells from 37 (Binet stage A) CLL patients, using high-resolution methylation microarrays (27,578 CpG). Overall, the methylation pattern correlated with the major biological (ZAP-70 and CD38), and molecular (IGHV mutation) markers, distinguishing CLL cases according to IGHV mutational status. Cell adhesion molecules were enriched in the signature of unmutated (UM) versus mutated (M-) CLL. Moreover, in M-CLL CpG hyper-methylation in three genes, including SPG20, was significantly anti-correlated with the corresponding gene expression level. Finally, the correlation between the methylation pattern and clinical parameters was investigated. Notably, out of 42 methyl-probes that were significantly associated with progression free survival (PFS), hyper-methylation of SPG20 was also positively associated with PFS. These data support the notion that epigenetic changes have clinical impact in CLL and may contribute to the identification of novel candidate disease-associated genes potentially useful to predict the clinical outcome of early stage CLL patients.

Early Versus Deferred Treatment With Combined Fludarabine, Cyclophosphamide and Rituximab (FCR) Improves Event-Free Survival In Patients With High-Risk Binet Stage A Chronic Lymphocytic Leukemia – First Results Of a Randomized German-French Cooperative Phase III Trial

▪ IntroductionPatients with asymptomatic early Rai or Binet stage chronic lymphocytic leukemia (CLL) do not benefit from mono-chemotherapy. Therefore, clinical observation without treatment (watch&wait; W&W) has been the gold standard for the management of these patients. Chemoimmunotherapy with FCR improves the outcome of patients with advanced CLL, but its efficacy in early stage disease has not been investigated. Several clinical and biological variables identify those patients who have a high risk of an aggressive disease course and who might benefit from early interventions. Consequently, this trial was conducted to test the value of FCR treatment in patients with early stage, high-risk CLL. MethodsThis report represents the endpoint and safety analysis of a randomized German-French cooperative phase III trial comparing the efficacy of early versus deferred FCR therapy in treatment-naïve Binet stage A CLL patients with a high risk of disease progression. Risk assessment was performed using 4 prognostic markers: Lymphocyte doubling time <12 months, serum thymidine kinase >10 U/L, an unmutated immunoglobulin heavy chain variable region gene (IGHV) status, and presence of unfavorable cytogenetics (del11q, del17p, trisomy 12) by fluorescence-in-situ hybridization. Presence of at least 2 versus less than 2 of these factors defined “high-risk” versus “low-risk” CLL. High-risk CLL patients were further randomized to receive either 6 cycles FCR (HR-FCR) or to be followed by a W&W strategy (HR-W&W). Patients with low-risk CLL were observed only (LR-W&W). ResultsBetween 2005 and 2010, a total of 824 patients was enrolled, 423 patients in 69 centers of the German CLL Study Group and 401 patients in 25 centers of the French Cooperative Group on CLL. The diagnosis of CLL needed to be established no longer than 12 months prior to enrollment and patients were required to present with previously untreated stage Binet A CLL at the time of inclusion. Overall, 800 patients (97.1%) were stratified, 201 of them categorized as high-risk CLL (25.1%). There was no significant difference between high-risk patients from the two study groups regarding common baseline characteristics (e.g., age, sex, comorbidity, immunophenotype) and the distribution of risk factors used for stratification.100 out of 201 high-risk patients were randomized to receive FCR therapy (HR-FCR), while 101 patients were allocated to the HR-W&W arm. 18 out of 100 patients (18%) withdrew consent for FCR therapy before treatment was started. 71 (86.6%) of 82 treated patients completed ≥4 cycles. The most common of 228 CTC grade III/IV adverse events reported within 12 months after treatment initiation were hematotoxicity (73.2% of patients) and infections (19.5% of patients). Three patients (3.7%) developed fatal CTC grade V infections (2 septic bacteremias, 1 of them with pulmonary aspergillosis; 1 encephalitis). Out of 79 patients available for response assessment until month 12 after treatment start, 76 showed a complete or partial remission (ORR 96.2%), 2 patients had stable disease (2.5%) and 1 patient had progressed (1.3%). After a median follow up of 46 months (range 0-88 months), HR-FCR patients demonstrated a significantly improved event-free survival (EFS) compared to HR-W&W patients (median EFS not reached versus 24.5 months, respectively, P<0.0001, Fig. 1). Overall survival was not significantly different between HR-FCR and HR-W&W with 181 high-risk patients (90%) being alive at last follow up. Both, HR-FCR and HR-W&W patients exhibited a significant shorter event-free and overall survival than LR-W&W patients, demonstrating an efficient prognostic segregation of patients by the risk assessment used for this trial (analysis based on the German LR-W&W cohort only, complete German-French LR-W&W data will be presented at the meeting). [Display omitted] ConclusionThis is the first randomized phase III trial investigating the efficacy of FCR chemoimmunotherapy in early stage CLL. So far, the study has revealed two major results: 1. A combination of clinical and biological factors can be used to identify early stage CLL patients who experience a rapid disease progression with unfavorable outcome, 2. FCR chemoimmunotherapy substantially improves event-free survival in early stage high-risk CLL. Disclosures:Langerbeins:Roche: travel grants Other. Cazin:roche: meeting invitation Other, Membership on an entity's Board of Directors or advisory committees; GSK: meeting invitation, meeting invitation Other, Membership on an entity's Board of Directors or advisory committees. Fischer:Mundipharma: Travel grants, Travel grants Other; Roche: Travel grants Other. Stilgenbauer:Roche: Consultancy, Research Funding, Travel grants Other; Mundipharma: Consultancy, Research Funding. 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. Hallek:F. Hoffmann-La Roche: Consultancy, Honoraria, Research Funding.

Small nucleolar RNAs as new biomarkers in chronic lymphocytic leukemia.

BackgroundSmall nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs are non-coding RNAs involved in the maturation of other RNA molecules. Alterations of sno/scaRNA expression may play a role in cancerogenesis. This study elucidates the patterns of sno/scaRNA expression in 211 chronic lymphocytic leukemia (CLL) patients (Binet stage A) also in comparison with those of different normal B-cell subsets.MethodsThe patterns of sno/scaRNA expression in highly purified CD19+ B-cells of 211 CLL patients and in 18 normal B-cell samples - 6 from peripheral blood, and 12 from tonsils (4 germinal center, 2 marginal zone, 3 switched memory and 3 naïve B-cells) - were analyzed on the Affymetrix GeneChip® Human Gene 1.0 ST array.ResultsCLLs display a sno/scaRNAs expression profile similar to normal memory, naïve and marginal-zone B-cells, with the exception of a few down-regulated transcripts (SNORA31, -6, -62, and -71C). Our analyses also suggest some heterogeneity in the pattern of sno/scaRNAs expression which is apparently unrelated to the major biological (ZAP-70 and CD38), molecular (IGHV mutation) and cytogenetic markers. Moreover, we found that SNORA70F was significantly down-regulated in poor prognostic subgroups and this phenomenon was associated with the down-regulation of its host gene COBLL1. Finally, we generated an independent model based on SNORA74A and SNORD116-18 expression, which appears to distinguish two different prognostic CLL groups.ConclusionsThese data extend the view of sno/scaRNAs deregulation in cancer and may contribute to discover novel biomarkers associated with the disease and potentially useful to predict the clinical outcome of early stage CLL patients.

Total body computed tomography scan in the initial work‐up of Binet stage A chronic lymphocytic leukemia patients: Results of the prospective, multicenter O‐CLL1‐GISL study

Total body computed tomography (TB-CT) scan is not mandatory in the diagnostic/staging algorithm of chronic lymphocytic leukemia (CLL). The aim of this study was to determine the value and prognostic significance of TB-CT scan in early stage CLL patients. Baseline TB-CT scan was performed in 240 Binet stage A CLL patients (179 Rai low- and 61 Rai intermediate-risk) included in a prospective multicenter observational study (clinicaltrial.gov ID:NCT00917549). The cohort included 69 clinical monoclonal B lymphocytosis (cMBLs). Patients were restaged considering only radiological data. Following TB-CT scans, 20% of cases reclassified as radiologic Binet (r-Binet) stage B. r-Binet B patients showed a higher incidence of unfavorable cytogenetic abnormalities (P = 0.027), as well as a shorter PFS (P = 0.001). At multivariate analysis, r-Binet stage [HR = 2.48; P = 0.004] and IGHV mutational status [HR = 3.01; P = 0.002] retained an independent predictive value for PFS. Among 179 Rai low-risk cases, 100 were redefined as r-Rai intermediate-risk based upon TB-CT scan data, showing a higher rate of cases with higher ZAP-70 (P = 0.033) and CD38 expression (P = 0.029) and β2-microglobulin levels (P < 0.0001), as well as a shorter PFS than those with r-Rai low-risk (P = 0.008). r-Rai stage [HR = 2.78; P = 0.046] and IGHV mutational status [HR = 4.25; P = 0.009] retained a significant predictive value for PFS at multivariate analysis. Forty-two percent of cMBL patients were reclassified as r-small lymphocytic lymphomas (r-SLLs) by TB-CT scan. TB-CT scan appears to provide relevant information in early stage CLL related to the potential and the timing of patients to progress towards the more advanced disease stages.

Chromosome 2p gain in monoclonal B‐cell lymphocytosis and in early stage chronic lymphocytic leukemia

Recent studies have described chromosome 2p gain as a recurrent lesion in chronic lymphocytic leukemia (CLL). We investigated the 2p gain and its relationship with common prognostic biomarkers in a prospective series of 69 clinical monoclonal B-cell lymphocytosis (cMBL) and 218 early stage (Binet A) CLL patients. The 2p gain was detected by FISH in 17 patients (6%, 16 CLL, and 1 cMBL) and further characterized by single nucleotide polymorphism-array. Overall, unfavorable cytogenetic deletions, i.e., del(11)(q23) and del(17)(p13) (P = 0.002), were significantly more frequent in 2p gain cases, as well as unmutated status of IGHV (P < 1 × 10(-4) ) and CD38 (P < 1 × 10(-4) ) and ZAP-70 positive expression (P = 0.003). Furthermore, 2p gain patients had significantly higher utilization of stereotyped B-cell receptors compared with 2p negative patients (P = 0.009), and the incidence of stereotyped subset #1 in 2p gain patients was significantly higher than that found in the remaining CLLs (P = 0.031). Transcriptional profiling analysis identified several genes significantly upregulated in 2p gain CLLs, most of which mapped to 2p. Among these, NCOA1 and ROCK2 are known for their involvement in tumor progression in several human cancers, whereas among those located in different chromosomes, CAV1 at 7q31.1 has been recently identified to play a critical role in CLL progression. Thus, 2p gain can be present since the early stages of the disease, particularly in those cases characterized by other poor prognosis markers. The finding of genes upregulated in the cells with 2p gain provides new insights to define the pathogenic role of this lesion.

Expansion of a CD8+PD-1+ Replicative Senescence Phenotype in Early Stage CLL Patients Is Associated with Inverted CD4:CD8 Ratios and Disease Progression

Patients with chronic lymphocytic leukemia (CLL) display immune deficiency that is most obvious in advanced stage disease. Here we investigated whether this immune dysfunction plays a pathologic role in the progression of early stage disease patients. We carried out eight-color immunophenotyping analysis in a cohort of 110 untreated early stage CLL patients and 22 age-matched healthy donors and correlated our findings with clinical outcome data. We found a significant reduction in naive CD4(+) and CD8(+) T cells in CLL patients. Only the CD4(+) subset showed significantly increased effector memory cells (T(EM) and T(EMRA)) in the whole cohort (P = 0.004 and P = 0.04, respectively). However, patients with inverted CD4:CD8 ratios (52 of 110) showed preferential expansion of the CD8 compartment, with a skewing of CD8(+) T(EMRA) (P = 0.03) coupled with increased percentage of CD57(+)CD28(-)CD27(-) T cells (P = 0.008) and PD-1 positivity (P = 0.027), consistent with a replicative senescence phenotype. Furthermore, inverted CD4:CD8 ratios were associated with shorter lymphocyte doubling time (P = 0.03), shorter time to first treatment (P = 0.03), and reduced progression-free survival (P = 0.005). Our data show that the emergence of CD8(+)PD-1(+) replicative senescence phenotype in early stage CLL patients is associated with more aggressive clinical disease. Importantly, these findings were independent of tumor cell prognostic markers and could not be accounted for by patient age, changes in regulatory T-cell frequency, or cytomegalovirus serostatus (n = 217).

Overexpression of Fc mu receptor (FCMR, TOSO) gene in chronic lymphocytic leukemia patients

Rai and Binet staging systems that have been used as a standard method for evaluating the prognosis of chronic lymphocytic leukemia (CLL) have some restrictions in distinguishing the early stage CLL patients that will progress rapidly. To solve this shortcoming, prognostic parameters other than staging have become important in the recent years. Intracellular upregulation of Fc mu receptor (FCMR, FAIM3/TOSO) gene in the leukemic lymphocytes of the patients with CLL may be an important parameter in predicting the progression of the disease. In this study, FCMR mRNA expression levels were evaluated in 50 CLL patients and in 50 healthy controls. FCMR mRNA expression was found to be significantly higher in CLL patients than in healthy controls. We, then, evaluated FCMR mRNA levels according to the stages of CLL. Rai stage 0, I, II cases were compared with stage III and IV, and Binet A was compared with Binet B and C according to FCMR mRNA levels. In cases with higher risks, Rai stage III, IV and Binet stage B and C, FCMR mRNA levels were also significantly higher. In addition, overexpression of the FCMR seems to be promoting the chromosomal abnormalities. As a result, we found that the mRNA levels of FCMR in the CLL patients are 23-fold higher than that of the control group and this may suggest that it can be associated with the disease progression and survival. For this reason and because of the simplicity of analyzing with Q-PCR, it can be a useful clinical parameter, after its importance has been shown in larger and multi-variate studies.