Time To First Treatment Research Articles

XPO1 mutations identify early-stage CLL characterized by shorter time to first treatment and enhanced BCR signalling.

Here we evaluated the epigenomic and transcriptomic profile of XPO1 mutant chronic lymphocytic leukaemia (CLL) and their clinical phenotype. By ATAC-seq, chromatin regions that were more accessible in XPO1 mutated CLL were enriched of binding sites for transcription factors regulated by pathways emanating from the B-cell receptor (BCR), including NF-κB signalling, p38-JNK and RAS-RAF-MEK-ERK. XPO1 mutant CLL, consistent with the chromatin accessibility changes, were enriched with transcriptomic features associated with BCR and cytokine signalling. By combining epigenomic and transcriptomic data, MIR155HG, the host gene of miR-155, and MYB, the transcription factor that positively regulates MIR155HG, were upregulated by RNA-seq and their promoters were more accessible by ATAC-seq. To evaluate the clinical impact of XPO1 mutations, we investigated a total of 957 early-stage CLL subdivided into 3 independent cohorts (N = 276, N = 286 and N = 395). Next-generation sequencing analysis identified XPO1 mutations as a novel predictor of shorter time to first treatment (TTFT) in all cohorts. Notably, XPO1 mutations maintained their prognostic value independent of the immunoglobulin heavy chain variable status and early-stage prognostic models. These data suggest that XPO1 mutations, conceivably through increased miR-155 levels, may enhance BCR signalling leading to higher proliferation and shorter TTFT in early-stage CLL.

Prognostic Value of Serum CD200 Concentrations in Chronic Lymphocytic Leukemia.

little is known regarding the prognostic value of soluble CD200 (sCD200)in chronic lymphocytic leukemia patients. Therefore, the objective of our study is to address the prognostic value of sCD200 antigen concentration on CLL patients outcome. Determination of serum sCD200 was done using ELISA kit in 158 CLL patients at diagnosis before start of therapy beside 21 healthy controls. sCD200 concentration levels was significantly higher in CLL patients as compared to healthy controls. High sCD200 was associated with poor prognostic markers (high expression of CD38+% and ZAP70+, high LDH, high risk Rai stages, unfavorable cytogenetic finding, time to first treatment (TTT) as well as patients outcome (P<0.001 for All). sCD200 at cut-off value ( 752.5 pg/ml) could predicts TTT with specificity 83.4%. Determination of sCD200 concentration levels at diagnosis could be used as a prognostic biomarker in CLL patients.

CLINICAL AND BIOLOGICAL FEATURES AND OUTCOMES OF PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA: A REAL‐WORLD STUDY OF 2005 PATIENTS IN CHINA

Introduction: Chronic lymphocytic leukemia (CLL) is less common in Han Chinese compared with persons of Europeans. The data about the clinical and biological features and outcomes of Chinese with CLL are limited, thus promoting us to perform this analysis on a large cohort of 2005 patients with CLL in China. Methods: Clinical data of 2005 patients with CLL were analyzed in Blood Diseases Hospital, Chinese Academy of Medical Sciences from February 2000 to December 2020. Results: In this cohort, the median age of patients at diagnosis was 61(20-92) years, which was younger than that for Western CLL patients (65-70 years). Patients showed similar male predominance as Europeans with CLL with an M:F ratio of about 1.8. 69.3% of patients had Binet stage B or C at diagnosis, higher than those of predominately European descent (20%-40%). A higher proportion of mutated immunoglobulin heavy chain variable region (IGHV) (64.0%) was shown in patients with CLL in this cohort than Europeans. IGHV4-34 rather than IGHV1-69 was the most commonly used fragment. Compared with Europeans, patients in this cohort had a higher proportion of subset eight stereotype and a lower proportion of subset two. The most frequent abnormality detected by FISH was RB1 deletion (21.1%), followed by trisomy 12 (20.4%), IGH translocation (16.8%), del(17p) (11.4%) and del(11q) (10.9%), respectively. It was shown that the frequencies of the chromosomal abnormalities in this study population were similar to the frequencies in Western countries. Of the 1476 patients for whose treatment data were available, 1191 (80.7%) received treatment. Median time to first treatment (TTFT) for CLL patients was 7 months. Among patients treated, chemotherapy, immunochemotherapy and targeted therapy accounted for 69.6%, 14.1% and 16.3% of first line, yielding 68.4%, 90.0% and 94.2% of overall response rate (ORR), respectively. After median 75-month follow up, median progression free survival (PFS) was 42 months, 5-year and 10-year PFS rate was 35.5% and 12.5%, respectively. Median overall survival (OS) was 130 months, 5-year and 10-year OS rate was 74.9% and 52.3%, respectively. The outcomes were similar to those reported in previous Chinese and Western studies. As expected, PFS and OS were better in CLL patients receiving immunochemotherapy and targeted therapy than those receiving chemotherapy. In addition, patients who achieved partial remission (PR) or complete remission (CR) after first-line treatment had a longer PFS and OS. Interestingly, females with CLL had a longer OS than males. Conclusions: This is the largest real-world study involving Chinese patients with CLL so far, which might lay a foundation for clinical investigation of Chinese CLL in future. Although the patients were younger, had a later stage at diagnosis and had different genetic backgrounds compared with Europeans, the outcomes were not different from those reported in Western studies. Keyword: chronic lymphocytic leukemia (CLL) No conflicts of interests pertinent to the abstract.

Impact of type 2 diabetes on mortality, cause of death, and treatment in chronic lymphocytic leukemia.

Age-related comorbid conditions are exceedingly common in patients with chronic lymphocytic leukemia (CLL). As the prevalence of type 2 diabetes (T2D) is predicted to double during the next two decades, a better understanding of the interplay between CLL and T2D is of increasing importance. In this study, analyses were performed in parallel in two separate cohorts, based on Danish national registers and the Mayo Clinic CLL Resource. The primary outcomes were overall survival (OS) from time of CLL diagnosis, OS from time of treatment, and time to first treatment (TTFT), studied using Cox proportional hazard regression analysis and Fine-Gray regression analysis. In the Danish CLL cohort, the prevalence of T2D was 11%, in the Mayo CLL cohort, it was 12%. Patients with CLL and T2D had shorter OS both from time of diagnosis and from first-line treatment for were less likely to receive treatment for CLL compared with patients with CLL and without T2D. The increased mortality was largely driven by an increased risk of death due to infections, especially in the Danish cohort. The findings of this study emphasize a substantial subgroup of CLL patients with co-occurring T2D with an inferior prognosis and a possible unmet treatment need requiring additional interventions and further research.

Do GWAS-Identified Risk Variants for Chronic Lymphocytic Leukemia Influence Overall Patient Survival and Disease Progression?

Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults worldwide. Although genome-wide association studies (GWAS) have uncovered the germline genetic component underlying CLL susceptibility, the potential use of GWAS-identified risk variants to predict disease progression and patient survival remains unexplored. Here, we evaluated whether 41 GWAS-identified risk variants for CLL could influence overall survival (OS) and disease progression, defined as time to first treatment (TTFT) in a cohort of 1039 CLL cases ascertained through the CRuCIAL consortium. Although this is the largest study assessing the effect of GWAS-identified susceptibility variants for CLL on OS, we only found a weak association of ten single nucleotide polymorphisms (SNPs) with OS (p < 0.05) that did not remain significant after correction for multiple testing. In line with these results, polygenic risk scores (PRSs) built with these SNPs in the CRuCIAL cohort showed a modest association with OS and a low capacity to predict patient survival, with an area under the receiver operating characteristic curve (AUROC) of 0.57. Similarly, seven SNPs were associated with TTFT (p < 0.05); however, these did not reach the multiple testing significance threshold, and the meta-analysis with previous published data did not confirm any of the associations. As expected, PRSs built with these SNPs showed reduced accuracy in prediction of disease progression (AUROC = 0.62). These results suggest that susceptibility variants for CLL do not impact overall survival and disease progression in CLL patients.

Low Serum Cholesterol Level Is a Significant Prognostic Factor That Improves CLL-IPI in Chronic Lymphocytic Leukaemia.

Hypocholesterolaemia is associated with elevated cancer risk and mortality, yet the relation between chronic lymphocytic leukaemia (CLL) and serum lipid profile remains unclear. Our study aims to evaluate the prognostic value of cholesterol levels in CLL and develop a prognostic nomogram that incorporates lipid metabolism. We enrolled 761 newly diagnosed CLL patients and separated them into either derivation (n = 507) or validation (n = 254) cohorts. The prognostic nomogram was constructed through multivariate Cox regression analyses, with performance evaluated using C-index, the area under the curve, calibration, and decision curve analyses. Decreased total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) at diagnosis were significantly associated with worse time to first treatment (TTFT) and cancer-specific survival (CSS), and simultaneously, low HDL-C with low LDL-C was identified as an independent prognostic indicator for both TTFT and CSS. CLL patients achieving complete or partial remission post-chemotherapy had significantly increased TC, HDL-C, and LDL-C levels compared with the baseline, and post-therapeutic HDL-C and LDL-C elevation correlated with favourable survival. The prognostic nomogram augmenting the CLL international prognostic index with low cholesterol levels yielded higher predictive accuracy and discrimination capacity for both 3-year and 5-year CSS. In conclusion, cholesterol profiles can be used as a cheap and readily accessible tool for predicting prognosis in CLL practice.

A comprehensive assessment of lymphocyte subsets, their prognostic significance, and changes after first-line therapy administration in patients with chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), changes in the peripheral blood lymphocyte subsets play an important role in disease progression and infectious complications. The impact of chemoimmunotherapy (CIT) on these changes has not been extensively studied METHODS: We used multi-color flow cytometry, to prospectively measure absolute and relative numbers of CD4+ and CD8+ T-cells and their subsets in 45 patients with indolent untreated CLL, 86 patients indicated for first-line treatment, and 34 healthy controls. In 55 patients, we analyzed the impact of CIT RESULTS: CLL patients had a significant increase in most cell populations in comparison to controls. Progression of CLL was characterized by significantly elevated counts with the exception of a lower percentage of naïve T-cells. After treatment, the percentage of naïve T-cells further decreased at the expense of effector memory T-cells (TEM). In patients with indolent CLL, higher percentages of naïve CD4+ (p= 0.0026) and naïve CD8+ (p= 0.023) T-cells were associated with a longer time to first treatment (TTFT). The elevation of CD4+ central memory T-cells (TCM) (p= 0.27) and TEM (p= 0.003) counts and a higher percentage of CD4+ TEM (p= 0.0047), were linked with shorter TTFT. In treated patients, increased regulatory T-cells count was associated with shorter time to next treatment (TTNT) (p= 0.042), while higher CD4+ TCM count with shorter TTNT (p= 0.035) and shorter overall survival (p= 0.041). Our results indicate that naïve cell depletion and CD4+ TCM and TEM increases are detrimental to CLL patients' prognosis.

Anti-PD-L1/PD-L2 therapeutic vaccination in untreated chronic lymphocytic leukemia patients with unmutated IgHV.

Chronic lymphocytic leukemia (CLL) patients with unmutated immunoglobulin heavy chain (IgHV) are at risk of early disease progression compared to patients with mutated IgHV. As a preventive strategy, we treated 19 previously untreated CLL patients with unmutated IgHV in a phase 1/2 trial (clinicaltrials.gov, NCT03939234) exploring the efficacy and toxicity of a therapeutic cancer vaccine containing peptides derived from programmed death ligand 1 (PD-L1) and ligand 2 (PD-L2), hoping to restore immunological control of the disease. According to the International Workshop on Chronic lymphocytic Leukemia (iwCLL) response criteria, no patients obtained a response; however, during follow-up, one patient had complete normalization of the peripheral lymphocyte count and remained in biochemical remission after a follow-up time of 15 months. At the end of treatment, one patient had progressed, and 17 patients had stable disease. During follow-up with a median time of 23.5 months since inclusion, seven patients had progressed, and eight patients had stable disease. The median time to first treatment (TTFT) from diagnosis was 90.3 months with a median follow-up time of 50.1 months. This apparent favorable outcome in TTFT needs to be investigated in a randomized setting, as our population may have been biased. More than 80% of patients obtained vaccine-specific immune responses, confirming the immunogenicity of the vaccine. The vaccine was generally well tolerated with only grade I-II adverse events. Although there were some signs of clinical effects, the vaccine seems to be insufficient as monotherapy in CLL, possibly due to a high tumor burden. The efficacy of the vaccine should preferably be tested in combination with novel targeted therapies or as a consolidating treatment.

Association of Clonal Hematopoiesis of Indeterminate Potential with Time to First Treatment, Incidence of Richter's Syndrome, and Overall Survival in Patients with Chronic Lymphocytic Leukemia

Background: Prior work has shown that clonal hematopoeisis of indeterminate potential (CHIP) is present in 12% of 285 patients with chronic lymphocytic leukemia (CLL) (median age 71 years) (Br J Haem. 2022;198:103). Additionally, CHIP was common (19%) among 89 patients with relapsed/refractory CLL exposed to venetoclax (Blood 2022;139:1198). Beyond these data, CHIP in patients with CLL has not been extensively characterized. The objective of this study is to describe the prevalence of myeloid CHIP (M-CHIP) in patients with CLL, and to determine the association of M-CHIP with overall survival (OS), time to first treatment (TTFT), and incidence of Richter's syndrome (RS). Methods: We retrospectively analysed data from 975 patients from a prospective CLL tissue bank and database at Dana Farber Cancer Institute and who had a targeted 95-gene next-generation sequencing (NGS) panel performed as part of standard of care (J Mol Diagn. 2016;18:507). We utilized published classification criteria for M-CHIP to create a list of 56 candidate genes (Nat Med. 2021;27:1921). Given the overlap between M-CHIP and CLL driver genes, we excluded overlap genes that were more common in CLL than M-CHIP (SF3B1, TP53, BRAF, KRAS, BCOR, NRAS, CREBBP, SETD2; Knisbacher. Nat Genetics. In press). We analysed CHIP as a binary (present/absent) and categorical (>2 vs. 1 vs. 0 mutations) predictor. We controlled for the covariates of age at time of NGS panel and prior treatment; other covariates considered included IGHV and 17pdel/TP53 mutation status, but these did not differ between groups. We performed landmark analyses from time of NGS panel to assess the association of M-CHIP with OS, TTFT, and RS. We computed unadjusted and adjusted hazard ratios with 95% confidence intervals for the outcomes of interest. Results: Of the 975 patients assessed, 749 (77%) had NGS performed prior to CLL treatment (Group 1), while 226 (23%) had it performed after receiving treatment (Group 2). NGS was primarily performed on peripheral blood (96%; 4% on bone marrow). Median follow-up time was 700 days (interquartile range (IQR) 200-1246). Median age at time of NGS was 65 years (IQR 58-72) and 40% of patients were female (Table 1A for patient characteristics). The prevalence of M-CHIP was 12% (N=93) in Group 1, and 24% (N=54) in Group 2. The prevalence of M-CHIP appeared to increase by age in Group 1, but appeared consistent across age groups in Group 2 (by decile of age from <40 to >80; Group 1: 0%, 5%, 9%, 13%, 16%, 47%, p<0.0001; Group 2: 33%, 29%, 17%, 22%, 32%, 26%, p=0.6). Patients with M-CHIP in Group 1 were older than patients without M-CHIP in Group 1, while patients with M-CHIP in Group 2 were more likely to have previous chemotherapy/chemo-immunotherapy exposure (p=0.04), but there were no other significant differences between groups (Table 1A). In Group 1, CHIP as a categorical predictor (>2 mutations), but not a binary predictor, was associated with OS in both univariable and multivariable analyses (Table 1B, p=0.0099) but not associated with TTFT as either a binary or categorical predictor. In the entire cohort, CHIP as a categorical predictor (>2 mutations) was associated with OS in univariable and multivariable analyses (Table 1B, Figure 1, p=0.03). Among both Group 1 and the entire cohort, CHIP as a binary predictor was not associated with Richter's syndrome (N=28 events). In the entire cohort, CHIP as a categorical predictor (>2 mutations) was associated with Richter's syndrome in univariable analysis, but this association did not hold in multivariable cause-specific hazard analysis considering death as a competing event. Conclusions: In our cohort, the prevalence of M-CHIP in untreated (12%) and treated (24%) patients with CLL was similar to previous literature. M-CHIP prevalence appeared to increase with age in untreated patients, but appeared consistent across age groups in treated patients, suggesting that treatment (85% had prior chemotherapy) may have impact on M-CHIP emergence even in younger patients. The presence of two or more M-CHIP mutations in all patients was associated with OS, even accounting for prior treatment and age, but was driven by a small subset of patients (N=28). M-CHIP did not appear to be associated with TTFT or RS, but the latter analysis is limited in power due to few RS events. Further analyses are ongoing to assess whether increased deaths in patients with multiple M-CHIP mutations were related to non-CLL causes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Development of a Novel Chromatin Activation Score As a Powerful Independent Prognostic Factor in Chronic Lymphocytic Leukemia

Background & Aim: In a previous study, we identified a signature of 498 regulatory regions with de novo increased H3K27ac levels (a mark of chromatin activation) in patients with chronic lymphocytic leukemia (CLL), which seemed to be present in all cases studied (Beekman et al., Nat Med 2018). In this abstract, we show that such de novo chromatin activation is not stable among CLL patients but heterogeneous, and therefore, we aim at exploring whether such heterogeneity has a biological and clinical impact. Results: The signature of 498 de novo active regulatory regions was related to 434 specific H3K27ac peaks based on chromatin immunoprecipitation sequencing (ChIPseq) data from 104 CLLs. Analyzing the signal variability among patients, we observed a high degree of heterogeneity. An unsupervised clustering revealed two different sets of regions whose mean H3K27ac signal was associated with time to first treatment (TTFT) using a univariate Cox model. In detail, a higher mean H3K27ac intensity was associated with a shorter TTFT in one of the sets (P=0.001 and hazard ratio (HR)=3.5) while, intriguingly, showed a trend towards longer TTFT (P=0.09 HR=0.6) in the other set. These findings suggest that not only disease progression but also disease stability seem to be active processes in CLL involving de novo chromatin changes. Then, we argued that the clinical behavior of CLL patients could be better captured by the balance between the activation levels of these two sets of regions. Indeed, the ratio of both means was statistically more significant (P=1.4x10-9 HR=15.4). Next, we further refined these groups by selecting individual regions associated with shorter (HR>1, 77 peaks) or longer (HR<1, 144 peaks) TTFT (FDR 0.2). We subsequently defined the score SPP as the mean signal in the pro-progressive (PP) group (HR>1), the score SPS as the mean signal in the pro-stable (PS) disease group (HR<1), and SB as their ratio. Ultimately, this led to an improvement of the statistical significance of the SB with TTFT (P=1.4x10-14 HR=8.6). From a biological perspective, we explored the validity of the SB performing ChIPseq for H3K27ac in 10 primary CLLs before and after a co-culture system inducing strong proliferation (Mangolini et al., Nat Comms in press). In these experiments, we observed an increased SB (P<10-3) that was mostly associated with a decreased SPS while maintaining the SPP levels. To further validate the biological implications of our findings, we mined RNAseq data in 64 cases with concurrent ChIPseq experimentation. We identified target genes whose expression levels correlated with H3K27ac levels (43 genes associated with the PP regions and 62 target genes associated with the PS regions). Both lists included genes previously described in CLL (e.g. CTLA4, FMOD or KSR2). Analogously to H3K27ac, we defined three new scores based on expression levels: SPP-exp, SPS-exp and SB-exp. These scores highly correlated with the H3K27ac-based scores (Pearson r between 0.82 to 0.88). Interestingly, cases with higher SB-exp were enriched in TNF-α and NF-κB pathways and in RELB, FOS, JUN, JUNB and EGR1 transcription factors regulons. We also identified 21 genetic driver alterations related to a higher SB-exp including trisomy 12, 11q loss and mutations in IGLV3-21R110, ATM, SF3B1, NOTCH1 and U1. Finally, we evaluated the potential of SB and SB-exp as independent prognostic factors in multivariate models. In the ChIPseq series (n=104), SB was significant independently of the IGHV mutational status, Binet stage and number of genetic driver alterations. Similarly, using three independent CLL series of gene expression data (n=110, 174 and 140), we observed that the SB-exp was a highly significant prognostic factor independent from IGHV status, Binet stage and number of drivers. To favour potential clinical translation, we generated a reduced SB-exp with only 12 genes which maintained the clinical impact. Conclusions: This study revealed an unexpected heterogeneity within de novo active chromatin regions in CLL, and that such heterogeneity has a dual clinical impact on CLL. A balance score between regions related to the two tendencies (i.e. more progressive vs more stable disease) identified groups of patients with different biological features. Finally, using both chromatin and gene expression data as a surrogate, the balance score was a highly significant independent prognostic factor in independent CLL series.

The role of NPM1 alternative splicing in patients with chronic lymphocytic leukemia.

ObjectivesChronic lymphocytic leukemia (CLL) is a lymphoproliferative disease with heterogeneous clinical course. Recent studies revealed a link between NOTCH1 mutation and the overexpression of MYC and MYC-related genes involved in ribosome biogenesis and protein biosynthesis, such as nucleophosmin-1 (NPM1), in CLL cells. In the present study, we aim to evaluate the impact of the NOTCH1 mutation on the MYC and MYC induced NPM1 expression in CLL cells via quantification of their transcripts.MethodsUsing qRT-PCR, we analyzed the levels of MYC and three main NPM1 splice variants in 214 samples collected from CLL patients. We assessed the impact of each splice variant on CLL prognostic markers, including the IGHV, TP53, NOTCH1, SF3B1, and MYD88 mutational status, cytogenetic aberrations, and laboratory features.ResultsSignificantly higher levels of NPM1.R1 transcripts in patients with unmutated compared to mutated IGHV status were found. The median time to first treatment (TTFT) in patients with a high level of NPM1.R1 was significantly shorter compared to the group with low NPM1.R1 levels (1.5 vs 33 months, p = 0.0002). Moreover, in Multivariate Cox Proportional Hazard Regression Model NPM1.R1 splice variant provided an independent prognostic value for TTFT.ConclusionIn conclusion, our study indicates the prognostic significance of the level of NPM1.R1 expression and suggests the importance of splicing alterations in the pathogenesis of CLL.

A STUDY ON THE ROLE OF THE RB1 GENE AS A PROGNOSTIC FACTOR IN UNTREATED PATIENTS WITH B-CHRONIC LYMPHOCYTIC LEUKEMIA

Chronic lymphocytic leukemia (CLL) is one of the most common leukemias in adults. After 2000, the FDA approved drugs for its treatment with mechanisms of action different from those of the then-known cytostatics. This new therapeutic approach, employing targeted therapy, is based on a better understanding of the biology and key pathogenetic mechanisms driving the development and progression of the disease. Deletion of the long arm of chromosome 13 is one of the most frequently described cytogenetic aberrations among CLL patients, the prognostic value of which is still debated, given that part of the tumor suppressor genes are located in this chromosome. Our study aimed to investigate the frequency of deletions affecting the retinoblastoma gene (RB1) and their impact on the time to first treatment (TTFT) in untreated B-CLL patients. The cohort included 40 patients with a confirmed diagnosis of CLL and deletion of the DLEU1 gene. Fluorescence in situ hybridization (FISH) was used to determine cytogenetic aberrations affecting the DLEU1 and Rb1 genes. The difference in time to first treatment in patients with/without deletion in the Rb1 gene was determined based on the log-rank test. In our study, we found a shorter time to initiation of first treatment in patients with a deletion in the Rb1 gene. Future clinical studies, including a more significant number of patients with an isolated deletion in the Rb1 gene, would confirm or reject the importance of the mentioned aberration as a prognostic factor and the necessity of its examination in routine practice.

Expression of BCL11A in chronic lymphocytic leukaemia.

The B-cell lymphoma/leukaemia 11A (BCL11A) gene encodes a Krüppel-like transcription factor involved in lymphocyte development during normal haematopoiesis. Aberrant expression of BCL11A has been observed in several haematological malignancies, including chronic lymphocytic leukaemia (CLL). However, its functions in the regulatory networks of malignant B lymphocytes are poorly understood, as are the relations to clinical course and outcome of B-cell malignancies, particularly CLL. The expression of BCL11A was analysed in peripheral blood mononuclear cells of 87 newly-diagnosed CLL patients by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), and association with clinical and molecular variables was assessed. BCL11A was significantly overexpressed in CLL samples compared to control samples (p < 0.001). BCL11A expression level exhibited no association with age, sex, leukocyte, lymphocyte and platelet counts, haemoglobin level, serum β2-microglobulin, CD38 status and cytogenetic abnormalities. On the other hand, high BCL11A expression was associated with low serum lactate dehydrogenase (p=0.031), Binet A stage (p=0.047) and mutated IGHV (p=0.028). In addition, a positive correlation with BCL2/BAX mRNA ratio was observed (r=0.36; p < 0.001). Regarding the association with the time to first treatment (TTFT), a trend towards longer median TTFT in BCL11A high- versus BCL11A low-expressing cases was detected (21 vs. 6months; p=0.164). The results of this study show that BCL11A is upregulated in CLL patients, and that high BCL11A expression at diagnosis may be associated with better prognosis. These data are consistent with the role of BCL11A expression in CLL biology, and imply its potential prognostic relevance.

5′ Rapid amplification of cDNA ends (5′RACE): A simpler method to analyze immunoglobulin genes and discover the value of the light chain in chronic lymphocytic leukemia

The mutational status of the variable region of the immunoglobulin heavy chain gene (IGHV) is a very important biomarker for chronic lymphocytic leukemia (CLL) patients. However, the routine detection of IGHV mutational status is time-consuming and costly. Therefore, we performed 5′ Rapid amplification of cDNA ends (5′ RACE) in 81 CLL patients who previously underwent detection using Biomed-2. The agreement rate of these two methods was 93.8 %. Regarding the discordant cases, 5′ RACE was more sensitive to identify unproductive and multiple rearrangements. Furthermore, 5′ RACE can also be used to simultaneously sequence light chains. In most CLL cases, the mutational statuses of heavy and light chains are concordant, except in IGLV3–21. Most IGLV3–21 (24/25) rearrangement shared a similar LCDR3 (QVWDSSSDHPWV) and harbored a single point mutation, namely, IGLV3–21R110. Compared to mutated-CLL non IGLV3–2R110, IGLV3–21R110-CLL exhibited a shorter overall survival (OS) and time to first treatment (TTFT) (p = 0.05, p < 0.0001, respectively) even though 75 % (18/24) of these patients expressed mutated heavy chains. Altogether, IGLV3–21R110 defines a CLL subgroup with specific biological features and an unfavorable prognosis independent of the IGHV mutational status and emphasizes the important value of the light chain. This study is the first to use 5′ RACE to detect the mutational status of IGH in CLL. Here, 5′ RACE was a reliable and effective method to test the mutational status of heavy and light chains. In addition, 5′ RACE can be combined with other assays in the NGS workflow to obtain more detailed insight into subclonal architecture and intraclonal diversity.

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.

A gene expression assay based on chronic lymphocytic leukemia activation in the microenvironment to predict progression

AbstractSeveral gene expression profiles with a strong correlation with patient outcomes have been previously described in chronic lymphocytic leukemia (CLL), although their applicability as biomarkers in clinical practice has been particularly limited. Here we describe the training and validation of a gene expression signature for predicting early progression in patients with CLL based on the analysis of 200 genes related to microenvironment signaling on the NanoString platform. In the training cohort (n = 154), the CLL15 assay containing a 15-gene signature was associated with the time to first treatment (TtFT) (hazard ratio [HR], 2.83; 95% CI, 2.17-3.68; P < .001). The prognostic value of the CLL15 score (HR, 1.71; 95% CI, 1.15-2.52; P = .007) was further confirmed in an external independent validation cohort (n = 112). Notably, the CLL15 score improved the prognostic capacity over IGHV mutational status and the International Prognostic Score for asymptomatic early-stage (IPS-E) CLL. In multivariate analysis, the CLL15 score (HR, 1.83; 95% CI, 1.32-2.56; P < .001) and the IPS-E CLL (HR, 2.23; 95% CI, 1.59-3.12; P < .001) were independently associated with TtFT. The newly developed and validated CLL15 assay successfully translated previous gene signatures such as the microenvironment signaling into a new gene expression–based assay with prognostic implications in CLL.

TP53 Abnormalities Are Underlying the Poor Outcome Associated with Chromothripsis in Chronic Lymphocytic Leukemia Patients with Complex Karyotype.

Chromothripsis (cth) has been associated with a dismal outcome and poor prognosis factors in patients with chronic lymphocytic leukemia (CLL). Despite being correlated with high genome instability, previous studies have not assessed the role of cth in the context of genomic complexity. Herein, we analyzed a cohort of 33 CLL patients with cth and compared them against a cohort of 129 non-cth cases with complex karyotypes. Nine cth cases were analyzed using optical genome mapping (OGM). Patterns detected by genomic microarrays were compared and the prognostic value of cth was analyzed. Cth was distributed throughout the genome, with chromosomes 3, 6 and 13 being those most frequently affected. OGM detected 88.1% of the previously known copy number alterations and several additional cth-related rearrangements (median: 9, range: 3-26). Two patterns were identified: one with rearrangements clustered in the region with cth (3/9) and the other involving both chromothriptic and non-chromothriptic chromosomes (6/9). Cases with cth showed a shorter time to first treatment (TTFT) than non-cth patients (median TTFT: 2 m vs. 15 m; p = 0.013). However, when stratifying patients based on TP53 status, cth did not affect TTFT. Only TP53 maintained its significance in the multivariate analysis for TTFT, including cth and genome complexity defined by genomic microarrays (HR: 1.60; p = 0.029). Our findings suggest that TP53 abnormalities, rather than cth itself, underlie the poor prognosis observed in this subset.

Genetic Testing at Diagnosis Has Prognostic Value in Patients with Chronic Lymphocytic Leukemia including at Early Stages

Chronic lymphocytic leukemia (CLL) has a variable clinical evolution, with some patients living treatment-free for decades while others require therapy shortly after diagnosis. In a consecutive series of 217 CLL patients, molecular biomarkers with prognostic value (IGHV status, TP53 mutations, and cytogenetics), whose analysis is recommended prior to treatment start, were studied at diagnosis. Multivariate analyses identified prognostic variables for overall survival (OS) and time to first treatment (TTFT) and validated the CLL-IPI and IPS-E variables for all or early-stage patients (Rai 0–2/Binet A), respectively. Unmutated IGHV was associated with shorter OS and TTFT, even for early-stage patients. Lymphocyte count was not statistically significant for TTFT of early-stage patients in multivariate analysis. Our results validate the prognostic value of IGHV mutational status at diagnosis for OS and TTFT, including for early stages. Our findings suggest a role for molecular and mutational analysis at diagnosis in future prospective studies.

MiR-146b-5p regulates IL-23 receptor complex expression in chronic lymphocytic leukemia cells

AbstractChronic lymphocytic leukemia (CLL) cells express the interleukin-23 receptor (IL-23R) chain, but the expression of the complementary IL-12Rβ1 chain requires cell stimulation via surface CD40 molecules (and not via the B-cell receptor [BCR]). This stimulation induces the expression of a heterodimeric functional IL-23R complex and the secretion of IL-23, initiating an autocrine loop that drives leukemic cell expansion. Based on the observation in 224 untreated Binet stage A patients that the cases with the lowest miR-146b-5p concentrations had the shortest time to first treatment (TTFT), we hypothesized that miR-146b-5p could negatively regulate IL-12Rβ1 side chain expression and clonal expansion. Indeed, miR-146b-5p significantly bound to the 3′-UTR region of the IL-12Rβ1 mRNA in an in vitro luciferase assay. Downregulation of miR-146b-5p with specific miRNA inhibitors in vitro led to the upregulation of the IL-12Rβ1 side chain and expression of a functional IL-23R complex similar to that observed after stimulation of the CLL cell through the surface CD40 molecules. Expression of miR-146b-5p with miRNA mimics in vitro inhibited the expression of the IL-23R complex after stimulation with CD40L. Administration of a miR-146b-5p mimic to NSG mice, successfully engrafted with CLL cells, caused tumor shrinkage, with a reduction of leukemic nodules and of IL-12Rβ1–positive CLL cells in the spleen. Our findings indicate that IL-12Rβ1 expression, a crucial checkpoint for the functioning of the IL-23 and IL-23R complex loop, is under the control of miR-146b-5p, which may represent a potential target for therapy since it contributes to the CLL pathogenesis. This trial is registered at www.clinicaltrials.gov as NCT00917540.

Optical Genome Mapping: A Promising New Tool to Assess Genomic Complexity in Chronic Lymphocytic Leukemia (CLL).

Simple SummaryGenome complexity, detected by chromosome banding analysis or chromosomal microarray analysis, is a poor prognostic factor for chronic lymphocytic leukemia (CLL). Herein, we aimed to assess the performance of optical genome mapping (OGM) for the cytogenomic characterization of CLL patients, with a special focus on risk stratification based on genomic complexity. A cohort of 42 patients enriched in complex karyotypes was assessed by OGM, and the results were compared with those obtained from current methods. Moreover, clinical–biological characteristics and time to first treatment were analyzed according to the OGM-defined complexity. Globally, OGM identified 90% of the known alterations and provided novel structural information about these aberrations in 55% of patients. Regarding genomic complexity, OGM allowed us to identify a complex group (≥10 alterations) displaying enrichment of TP53 abnormalities and poorer evolution. Altogether, we confirmed that OGM is a valuable tool for the cytogenomic assessment and prognostic stratification of CLL patients.Novel treatments in chronic lymphocytic leukemia (CLL) have generated interest regarding the clinical impact of genomic complexity, currently assessed by chromosome banding analysis (CBA) and chromosomal microarray analysis (CMA). Optical genome mapping (OGM), a novel technique based on imaging of long DNA molecules labeled at specific sites, allows the identification of multiple cytogenetic abnormalities in a single test. We aimed to determine whether OGM is a suitable alternative to cytogenomic assessment in CLL, especially focused on genomic complexity. Cytogenomic OGM aberrations from 42 patients were compared with CBA, FISH, and CMA information. Clinical–biological characteristics and time to first treatment (TTFT) were analyzed according to the complexity detected by OGM. Globally, OGM identified 90.3% of the known alterations (279/309). Discordances were mainly found in (peri-)centromeric or telomeric regions or subclonal aberrations (<15–20%). OGM underscored additional abnormalities, providing novel structural information on known aberrations in 55% of patients. Regarding genomic complexity, the number of OGM abnormalities had better accuracy in predicting TTFT than current methods (C-index: 0.696, 0.602, 0.661 by OGM, CBA, and CMA, respectively). A cut-off of ≥10 alterations defined a complex OGM group (C-OGM, n = 12), which included 11/14 patients with ≥5 abnormalities by CBA/CMA and one patient with chromothripsis (Kappa index = 0.778; p < 0.001). Moreover, C-OGM displayed enrichment of TP53 abnormalities (58.3% vs. 3.3%, p < 0.001) and a significantly shorter TTFT (median: 2 vs. 43 months, p = 0.014). OGM is a robust technology for implementation in the routine management of CLL patients, although further studies are required to define standard genomic complexity criteria.