Chronic Lymphocytic Leukemia Subgroups Research Articles

Chronic Lymphocytic Leukemia (CLL) with Borderline Immunoglobulin Heavy Chain Mutational Status, a Rare Subgroup of CLL with Variable Disease Course.

Chronic lymphocytic leukemia (CLL) exhibits substantial variability in disease course. The mutational status of the B-cell receptor immunoglobulin heavy variable (IGHV) chain is a critical prognostic factor, categorizing patients into mutated (M-IGHV) and unmutated (U-IGHV) groups. Recently, a third subgroup with borderline mutational status (BL-IGHV) has been identified, comprising approximately 5% of CLL cases. This study retrospectively analyzes the outcomes of 30 BL-IGHV mutated patients among a cohort of 653 CLL patients, focusing on time to first treatment (TTFT) and overall survival (OS). BL-IGHV patients had a short TTFT similar to U-IGHV patients (median 30.2 vs. 34 months; p = 0.9). Conversely, the OS of BL-IGHV patients resembled M-IGHV patients (median NR vs. 258 months; p = 1). Despite a similar incidence in unfavorable prognostic factors, the TTFT was shorter compared to other published cohorts. However, striking similarities with other experiences suggest that BL-IGHV mutated patients share common biological characteristics, biased IGHV gene usage and BCR subset frequency. These findings also underscore the need for multicentric efforts aggregating data on BL-IGHV CLL in order to elucidate its disease course and optimize therapeutic approaches for this rare subgroup. Until then, predicting outcomes and optimal management of BL-IGHV CLL will remain challenging.

Beta-2 Microglobulin As a Seric Marker for Diagnosis of Hematologic Neoplasms in First Contact Consultation with the Hematologist

Introduction Beta-2 microglobulin is a protein component of the Major histocompatibility complex I. Its concentration in blood has been long used as prognostic marker in some hematologic neoplasms, and even used as a tool for surveillance of relapse in other neoplasms. It is frequently elevated in non-Hodgkin lymphoma (NHL), Myelodysplastic syndrome (MDS), myeloma multiple (MM), chronic lymphocytic leukemia (CLL) and other Lymphoproliferative disorders. Although its utility as a diagnostic tool has been poorly described. Aim Describe the blood levels of Beta-2 Microglobulin in patients who are sent without established diagnosis to be evaluated by hematology department and its association with the different definitive diagnosis. Methods We captured information from outpatients who were evaluated in the first consultation at hematology department because of any citopenia or clinical suspicion of any hematologic neoplasm from September 2022 to July 2023. If the patient consented, we recorded it in database and measured blood levels of beta-2 microglobulin on first consultation, and then followed until definitive diagnosis was established. Analysis of data was made with IBM SPSS Statistics for Windows, Version 27.0. Armonk, NY: IBM Corp; we used Mann Whitney U Test for quantitative variable comparison, and Fisher exact test to determine differences between groups. Statistical significances were calculates using p<0.05. Results We captured a total of 87 patients, but 11 had to be excluded because of lack of information. Of the 76 patients included for analysis, 34 were male (44.7%) and 42 were female (55.3%). The medium age was 45.1 years (Range: 15-95 years). After the first consultation, 30 patients (39.5%) were suspected to have a neoplasm, and in follow-up, 35 (46.05%) ended up with a definitive diagnosis of hematologic neoplasm. Only one patient with initial suspected neoplasm ended up with benign disease, but 6 patients in which a bening condition was suspected ended up with a neoplasm diagnosis. There was a total of 35 (46.05%) patients with definitive neoplastic diagnostic, being the most frequent: NHL (n=15, 19.7%), Myeloproliferative neoplasm (MPNs) (n=6, 7.8%), and MM (4, 5.2%). In the no neoplastic pathology, the most frequent diagnosis was iron-deficit anemia (n=17; 22.36%), followed by megaloblastic anemia (n=4, 5.26%) and infectious disease (n=4, 26%). Beta-2 microglobulin was higher in the group with definitive neoplastic diagnosis (5.4mg/L vs 2.4mg/L, x 2=0.053), and was also, significatively higher in the NHL (4.94mg/L, x 2=0.005), MM (13.43mg/L, x 2=0.008), CLL (6.3mg/L, x 2=0.02) and MDS (8.05mg/L, x 2=0.034) subgroups. The sensibility for any hematologic neoplasm was 48.5% and specificity was 82.93%, with a positive LR of 2.84 and a Negative LR of 0.62. The subgroups with higher sensibility were MM (75%), CLL (66.6%), MPNs (66.6%) and MDS (100%), being the Positive LR of 4.3 for MM, 3.9 for CLL, 3.9 for MPNs and 5.86 for MDS. Groups with the lowest correlation were Hodgkin lymphoma (HL) with sensibility of 33.3%, and isolated plasmacytoma (PC) with sensibility of 0%. In the group with benign diagnosis, only 6 (7.89%) presented levels above upper normal limit of blood Beta-2 microglobulin, 4 (5.26%) with iron-deficit anemia, 1 (1.31%) with liver disease, 1 (1.31%) with Pulmonary tuberculosis and 1 (1.31%) with systemic lupus erythematosus; being the highest levels in pulmonary tuberculosis (14.9mg/L). Conclusions The blood levels of beta-2 microglobulin in our work were in concordance with other previous described papers, in which is frequently elevated in NHL, MM, and CCL. But in our case, it showed a marked elevation in MPNs, and MDS (higher correlation). It should be noted, no tumor burden was recorded in our study, which could directly affect beta-2 microglobulin levels. In conclusion, although not a strong correlation as an expert hematologist clinical evaluation, beta-2microglobulin could be of use to strengthen clinical suspicion of those hematologic neoplasms or reassess cases in which MDS/MPNs are a possible differential diagnosis.

Targeting conditioned media dependencies and FLT-3 in chronic lymphocytic leukemia

AbstractThe importance of the stromal microenvironment in chronic lymphocytic leukemia (CLL) pathogenesis and drug resistance is well established. Despite recent advances in CLL therapy, identifying novel ways to disrupt interactions between CLL and its microenvironment may identify new combination partners for the drugs currently in use. To understand the role of microenvironmental factors on primary CLL cells, we took advantage of an observation that conditioned media (CM) collected from stroma was protective of CLL cells from spontaneous cell death ex vivo. The cytokine in the CM-dependent cells that most supports CLL survival in short-term ex vivo culture was CCL2. Pretreatment of CLL cells with anti-CCL2 antibody enhanced venetoclax-mediated killing. Surprisingly, we found a group of CLL samples (9/23 cases) that are less likely to undergo cell death in the absence of CM support. Functional studies revealed that CM-independent (CMI) CLL cells are less sensitive to apoptosis than conventional stroma-dependent CLL. In addition, a majority of the CMI CLL samples (80%) harbored unmutated immunoglobulin heavy-chain variable (IGHV) region. Bulk-RNA sequence analysis revealed upregulation of the focal adhesion and RAS signaling pathways in this group, along with expression of fms-like tyrosine kinase 3 (FLT3) and CD135. Treatment with FLT3 inhibitors caused a significant reduction in cell viability among CMI samples. In summary, we were able to discriminate and target 2 biologically distinct subgroups of CLL based on CM dependence with distinct microenvironmental vulnerabilities.

NOTCH PATHWAY MUTATION CONTRIBUTES TO INFERIOR PROGNOSIS IN HBV‐INFECTED CHRONIC LYMPHOCYTIC LEUKEMIA

Chronic lymphocytic leukemia (CLL) patients with hepatitis B virus (HBV) infection have a poor prognosis, underlying mechanism remains unclear. NOTCH mutations are frequent in CLL and associated with disease progression and drug resistance. It is also reported to be associated with hepatitis infection in lymphoid malignancies. In order to investigate the relation between NOTCH pathway and HBV-associated CLL, we studied 98 previously untreated HBV positive CLL patients and 244 HBV-negative CLL. A total of 342 treatment naïve patients diagnosed with CLL at the First Affiliated Hospital of Nanjing Medical University from 1 January 2010 to 31 October 2021 were enrolled in our study. The mutation hotspots of NOTCH pathway genes were analyzed by Next Generation Sequencing (NGS) of DNA that used PCR for the identification of genomic mutation. Fisher exact and χ2 tests were used to assess the correlation between HBV infection and clinical, demographic factors in CLL patients. Survival curves were generated by Graphpad 9.5. NOTCH mutations were more frequent in HBV positive CLL subgroup (17.3% vs. 7.4%, p = 0.033). By survival analysis, HBV infection was associated with disease progression and poor survival (p = 0.0099 for overall survival [OS] and p = 0.0446 for time-to-treatment [TTT]). Any lesions of the NOTCH pathway (NOTCH1, NOTCH2 and SPEN) aggravated prognosis. In multivariate analysis, NOTCH mutation retained an independent significance for HBV-infected patients (p = 0.016 for OS and p = 0.023 for TTT). However, HBV positive with NOTCH unmutated had no statistical difference in prognosis compared with HBV negative patients (p = 0.1706 for OS and p = 0.2387 for TTT), which indicated that NOTCH pathway mutation contributed to inferior prognosis in HBV-infected CLL. In conclusion, a cohort of CLL patients with HBV positive displayed a worse clinical outcome and the status of NOTCH signaling pathway might play a crucial role. Keywords: chronic lymphocytic leukemia (CLL), diagnostic and prognostic biomarkers No conflicts of interests pertinent to the abstract.

Tumor Heterogeneity-Based Sscr-Score Predicts Prognosis and Reveals Mechanism of SORL1 in Restraining CLL Progression

Introduction Complex heterogeneity is a hallmark of chronic lymphocytic leukemia (CLL) that challenges oncological research and optimal treatment regimens. Although genetic markers with prognostic values have been developed for risk assessment in CLL, the existing prognostic models are far from satisfaction. Single-cell RNA sequencing (scRNA-seq) technologies have made it possible to illustrate the genetic heterogeneity and cell differentiation status at the single-cell level. A prognostic model associated with CLL cell differentiation status was established to refine the risk assessment of CLL patients. Methods Primary CLL cells from 90 patients and CD19+ B cells from 20 healthy donors were collected with informed consents according to the Declaration of Helsinki. Expression levels of SORL1 mRNA and protein in CLL cells were determined by quantitative RT-PCR and western blotting. Cell viability was measured by the CCK-8 assay. Cell cycle and apoptosis were analyzed by PI and Annexin V-PE/7AAD staining. Fatty acid oxidation rate was assessed with Fatty Acid Oxidation Complete Assay Kit. Red oil O staining was performed to evaluate the lipid deposition in CLL cells. Single cell sequencing data were analyzed by the Seurat package and Monocle package in R4.0.5. Results The scRNA-seq profile revealed remarkable intra- and inter-tumoral heterogeneity of CLL. According to the differentiation status, this study reconstructed the evolutionary history of CLL cells, which reflected the molecular classification and risk subgroups of CLL. At the key point of cell differentiation, CLL cells were divided into two subsets with progressive and indolent genetic signatures respectively. Genes differentially expressed between aggressive and indolent subsets were identified. Through univariate and multivariate analysis, four genes (SORL1, SGK1, CYBB, RPL22L1) with significant prognostic values were selected to construct a risk model named SSCR-Score. The risk score was calculated and validated in two independent validation cohorts (Fig 1A). SSCR-Score has revealed the positive prognostic value of SORL1 in CLL. Increased expression of SORL1 was associated with prolonged overall survival and improved time to first treatment in CLL. Among patients with ZAP70 (-) or IGHV mutated status, the expression levels of SORL1 were up-regulated. Although increased expression of SORL1 was associated with favorable prognosis, the biological mechanism of SORL1 still remained ill-defined. This study demonstrated that overexpression of SORL1 restrained cell proliferation, induced cell apoptosis and G2/M cell cycle arrest in CLL cells. In accordance with the RNA sequencing profile, overexpression of SORL1 suppressed abnormal lipid reposition and fatty acid oxidation in CLL cells. On a molecular level, SORL1 down-regulated the expression of lipid-metabolism related genes, including lipoprotein lipase (LPL), carnitine palmitoyltransferase 1A (CPT1A) and carnitine palmitoyltransferase 2 (CPT2). To illustrate the SORL1-mediated regulation of LPL, immunofluorescence staining was performed. Results showed that SORL1 accelerated the degradation of LPL. After the inhibition of lysosome function, accelerated degradation of LPL was not observed in CLL cells overexpressing SORL1. Rescue experiments substantiated that SORL1 impeded progression of CLL through downregulating the activity of LPL which promoted lipid metabolism. Immuno-coprecipitation experiment showed that anti-SORL1 antibody precipitated LPL protein. Additionally, 6-Shohaol induced overexpression of SORL1 in CLL cells. It exhibited potent anti-CLL efficacy by suppressing cell proliferation, promoting cell apoptosis and G2/M cell cycle arrest in CLL cells (Fig 1B). Conclusion CLL presented remarkable heterogeneity which reflected different biological subclasses and risk groups. Through identifying genes differentially expressed between aggressive and indolent cell subsets, a novel prognostic model named SSCR-Score was developed to optimize risk stratification of CLL patients. Moreover, SORL1 restrained progression of CLL by inhibiting lipid metabolism regulated by LPL in CLL cells. SORL1 may serve as a potential strategy for the development of novel therapeutic target in CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

Abstract A39: Targeting tumor-microenvironmental dependencies as a therapeutic approach in Chronic Lymphocytic Leukemia (CLL)

Abstract Interaction between CLL and its microenvironment is a major factor in drug resistance. Bone marrow derived stroma cells provide a protective and supportive niche to allow CLL cells to survive drug-induced apoptosis. Therefore, a current challenge is to develop new strategies to target the microenvironment. To understand the role of tumor microenvironment in CLL survival, we studied conditioned media (CM) derived from NKTert cells. Human NKTert stroma cells provide a survival advantage to primary CLL cells by protecting them from spontaneous apoptosis in ex-vivo culture. We identified three cytokines (IL6, IL8 and CCL2) from CM as candidates for mediating survival. We exposed CLL cells to neutralizing antibodies against each cytokine and found significant decrease in CLL viability in presence of anti-CCL2 suggesting its role in CLL survival. BH3 profiling revealed an increase in apoptotic priming with anti-CCL2 treatment as well. Using dynamic BH3 profiling (DBP), we found that anti-CCL2 increased dependence on BCL-2 as well as BCL-xL in primary CLL cells suggesting a potential role of CCL2 on CLL survival. To our surprise, we also identified a CLL group (CM independent, CMI) that were resilient to cell death in the absence of stroma support in ex-vivo culture. In addition to this, anti-CCL2 antibody showed no effect on their survival. Next, we exposed CMI CLL cells to venetoclax and found that these CLL cells were less sensitive to apoptosis compared to the CLL samples that are stroma dependent (CM dependent, CMD). BH3 profiling showed that CMI CLL cells are less primed for apoptosis. Consistent with our findings, DBP with anti-CCL2, venetoclax or the combination didn’t increase any drug induced apoptotic priming in CMI CLL samples. In our study, 80% of CMI harbor unmutated IgHV. To provide additional insight to the molecular mechanism, we performed western blot analyses for BCL-2 family proteins and found decreased BAX ad BCL-2 expression in CMI samples. This result suggests altered BCL-2 family proteins expression might be a mechanism for differential priming. Bulk RNA seq analysis revealed upregulation of focal adhesion genes and Ras signaling pathway in CMI samples. In summary, our study suggest that in the CMD subset of CLL, anti-CCL2 therapy enhances apoptotic priming and venetoclax efficacy. Furthermore, we have also functionally defined two biologically distinct CLL subgroups based on stroma dependence. Citation Format: Salma Parvin, Aditi Aryal, Shanye Yin, Matthew S. Davids, Geoffrey G. Fell, Catherine J. Wu, Anthony Letai. Targeting tumor-microenvironmental dependencies as a therapeutic approach in Chronic Lymphocytic Leukemia (CLL) [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A39.

Identification of Clinically Relevant Subgroups of Chronic Lymphocytic Leukemia Through Discovery of Abnormal Molecular Pathways.

Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in the Western world with a highly variable clinical course. Its striking genetic heterogeneity is not yet fully understood. Although the CLL genetic landscape has been well-described, patient stratification based on mutation profiles remains elusive mainly due to the heterogeneity of data. Here we attempted to decrease the heterogeneity of somatic mutation data by mapping mutated genes in the respective biological processes. From the sequencing data gathered by the International Cancer Genome Consortium for 506 CLL patients, we generated pathway mutation scores, applied ensemble clustering on them, and extracted abnormal molecular pathways with a machine learning approach. We identified four clusters differing in pathway mutational profiles and time to first treatment. Interestingly, common CLL drivers such as ATM or TP53 were associated with particular subtypes, while others like NOTCH1 or SF3B1 were not. This study provides an important step in understanding mutational patterns in CLL.

Different Time-Dependent Risk for Disease Evolution in Chronic Lymphocytic Leukemia with Distinct IGHV Gene Somatic Hypermutation Status

A great challenge for early stage patients with chronic lymphocytic leukemia (CLL) is to determine if and, consequently, when they will require treatment, highlighting the need for robust prognostic assessment. Today, immunogenetic and genomic features are considered essential for accurate risk stratification in CLL. However, when utilizing such biomarkers, the prognosis is assessed assuming stable predictability over the disease course. Here, we assessed the risk for CLL evolution overtime by evaluating time-to-first-treatment (TTFT) for 2366 CLL patients not requiring treatment at the time of diagnosis (Binet A: n=1900, 80.3%/Binet B: n=287, 12.1%), hypothesizing that risk evolution might depend on both time and the biological characteristics of the clone. Based on the somatic hypermutation status (SHM) of the immunoglobulin heavy variable genes, 1364 (58%) and 1002 (42%) patients were classified as mutated (M-CLL) and unmutated CLL (U-CLL), respectively. Regarding genomic subgroups of CLL, we focused on high risk patients carrying aberrations within the TP53 gene (TP53 abn, deletion of chromosome 17p and/or TP53 mutations) and low risk patients exhibiting isolated deletion of chromosome 13q or normal FISH according to the Döhner hierarchical model (del(13q)/normal FISH).First, we estimated the risk overtime by the hazard curve. This enables to better visualize changes in risk overtime compared to a Kaplan-Meier curve by showing the estimated proportion of cases receiving treatment in a defined interval, given that a person is still treatment-free at the start of this interval. An interpolation method was then used to estimate the values of the hazard curve at each distinct year from the time of diagnosis, and the corresponding differences in hazard values between M-CLL and U-CLL. These differences constituted the basis for the overtime analysis. In particular, the overall time frame was divided in 5-year intervals, and the distributions of differences in hazard were statistically compared between consecutive 5-year intervals. P-values less than 0.05 would indicate different evolution for the hazard curve overtime for the M-CLL and U-CLL.First, we assessed all Binet A patients, regardless of their genomic aberrations background, and found significant differences between M-CLL vs U-CLL with p[0,5]Vs(5,10]=0.006, p(5,10]Vs(10,15]=0.009, p(10,15]Vs(15,20]=0.009, p(15,20]Vs(20,25]=0.014, and p(overall)<0.001, reflecting the constant decrease of the distance between the M-CLL and U-CLL hazard curves (Fig 1a), deemed as statistically significant between consecutive 5-year intervals. Next, we focused on different genomic risk subgroups and found that del(13q)/normal FISH patients followed the M-CLL/U-CLL rule with p[0,5]Vs(5,10]=0.006, p(5,10]Vs(10,15]=0.009, p(10,15]Vs(15,20]=0.009, and p(overall)<0.001, indicating little additional impact on risk evolution beyond that dictated by the SHM status (Fig 1b). In sharp contrast, within TP53 abn patients we found p[0,5]Vs(5,10]=0.006, which reflects the statistically significant increase of the distance between the two hazard curves for M-CLL and U-CLL after the 5th year (Fig 1c), hence indicating decreasing risk for TP53 abn M-CLL and, vice versa, constantly increasing risk for TP53 abn U-CLL after the 5th year. For Binet B patients, we did not observe any significant differences with p[0,5]Vs(5,10]=0.2, highlighting a similar hazard evolution for M-CLL and U-CLL (Fig 1d). Low numbers precluded assessing the impact of genomic aberrations within this subgroup of patients.In conclusion, within all early stage patients as well as in subgroup analysis of high or low genomic risk Binet A patients, M-CLL was found to display a rather stable overtime risk for TTFT, independently of the presence of TP53 abn. This sharply contrasted U-CLL patients, where the overtime risk for TTFT was clearly influenced by the genomic background since del(13q)/normal FISH patients showed a similar pattern with M-CLL whereas TP53 abn patients exhibited a continuously increasing risk for disease progression. From a different standpoint, these findings corroborate the concept that the precise prognostic value of TP53 abn depends on the biological background of the CLL clone that is altogether different in patients with distinct SHM status. [Display omitted] DisclosuresStrefford:Roche: Research Funding. Delgado:Abbvie, Jansen, Gilead, Roche: Consultancy, Speakers Bureau. Stavroyianni:Janssen Pharmaceuticals: Honoraria; Gilead Sciences: Honoraria; Takeda: Honoraria. Gaidano:Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Ghia:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding; Roche: Honoraria; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Stamatopoulos:Abbvie: Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Novartis SA: Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding.

Genome-Wide DNA Methylation Profiling of Chronic Lymphocytic Leukemia Subsets Carrying Stereotyped B Cell Receptors

In recent years, subsets of chronic lymphocytic leukemia (CLL) patients carrying quasi-identical or stereotyped B cell receptors (BcRs) have been identified that share clinicobiological features and disease outcome. While these stereotyped subsets show distinct gene expression and genomic profiles, the DNA methylation landscape remains largely unexplored. By applying high-resolution 450K methylation arrays, we investigated 176 CLL subset cases belonging to: (i) the clinically aggressive, IGHV-unmutated (U-CLL) subsets #1 (clan I genes/IGKV(D)1-39, n=37) and #8 (IGHV4-39/IGKV1(D)-39, n=21); (ii) the IGHV1-69-expressing U-CLL subsets #3 (n=12), #5 (n=9), #6 (n=22), and #7 (n=12); and, (iii) the indolent, IGHV-mutated (M-CLL) subset #4 (IGHV4-34/IGKV2-30, n=28). In addition, we included subset #2 cases (IGHV3-21/IGLV3-21, mixed mutation status, n=35) that have a poor outcome independent of IGHV mutation status. For comparative purposes, we included a cohort of CLL cases that do not express stereotyped BcRs (‘non-subset’, n=325). These patients were subgrouped according to the recently proposed epigenetic classification of CLL, i.e. poor-prognostic, naïve-like CLL (n-CLL, n=102), favorable-prognostic, memory-like CLL (m-CLL; n=176), broadly corresponding to U-CLL and M-CLL, respectively, and a third intermediate CLL subgroup (i-CLL; n=47), which express borderline mutated IGHV genes and have an intermediate prognosis. Finally, a series of sorted normal subpopulations spanning different stages of B-cell differentiation [precursors (n=22), naïve B cells (n=19) and germinal center/memory B-cells (n=33)] were also included in the analysis. Overall, unsupervised analysis of subset vs. non-subset CLL revealed that all U-CLL subsets clustered with n-CLL, subset #4 clustered with m-CLL, while subset #2 clustered separately with i-CLL (Figure 1). Supervised analysis revealed a limited number of CpG sites that were differentially methylated when comparing each U-CLL or M-CLL subset with non-subset cases. In contrast, almost all subset #2 cases clustered separately from i-CLL in supervised analysis, indicating that this subset might represent a distinct subgroup of i-CLL. We recently demonstrated that the number of epigenetic changes that a tumor acquires, compared to its cellular origin (i.e. ‘epigenetic burden‘), may be a powerful predictor of clinical aggressiveness (Queiros et al, Cancer Cell 2016). When adopting this approach in CLL, comparison of specific subsets vs. their non-subset cases matched by epigenetic subgroup, revealed significant differences in the epigenetic burden amongst the various groupings; for instance, in subset #1 vs. n-CLL (72K vs. 67K, p<0.05) and in subset #2 vs. i-CLL (76K vs. 68K, p=0.001), while no difference was observed between subset #4 vs. m-CLL (83K vs. 82K, p=not significant). Subset #2 cases frequently carry del(11q) and harbor SF3B1 mutations, however, neither the IGHV mutation status nor the presence of del(11q) or SF3B1 mutations had any impact on the epigenetic burden within subset #2. In conclusion, U-CLL and M-CLL subsets generally clustered with n-CLL and m-CLL categories, respectively, implying common cellular origins. In contrast, subset #2 emerged as the first defined member of the i-CLL group, which in turn alludes to a distinct cellular origin and/or pathogenetic process for subset #2 and i-CLL patients. [Display omitted] DisclosuresPapakonstantinou:Janssen Pharmaceuticals: Research Funding; Gilead: Research Funding. Smedby:Janssen: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees. Gaidano:Roche: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Ghia:AbbVie: Consultancy; Adaptive: Consultancy; Gilead: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy; Roche: Consultancy; Novartis: Research Funding. Stamatopoulos:Novartis SA: Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding.

IGLV3-21R110 identifies an aggressive biological subtype of chronic lymphocytic leukemia with intermediate epigenetics

AbstractB-cell receptor (BCR) signaling is crucial for chronic lymphocytic leukemia (CLL) biology. IGLV3-21–expressing B cells may acquire a single point mutation (R110) that triggers autonomous BCR signaling, conferring aggressive behavior. Epigenetic studies have defined 3 CLL subtypes based on methylation signatures reminiscent of naïve-like (n-CLL), intermediate (i-CLL), and memory-like (m-CLL) B cells with different biological features. i-CLL carries a borderline IGHV mutational load and significantly higher use of IGHV3-21/IGLV3-21. To determine the clinical and biological features of IGLV3-21R110 CLL and its relationship to these epigenetic subtypes, we characterized the immunoglobulin gene of 584 CLL cases using whole-genome/exome and RNA sequencing. IGLV3-21R110 was detected in 6.5% of cases: 30 (38%) of 79 i-CLLs, 5 (1.7%) of 291 m-CLLs, and 1 (0.5%) of 189 n-CLLs. All stereotype subset 2 cases carried IGLV3-21R110, whereas 62% of IGLV3-21R110 i-CLL cases had nonstereotyped BCR immunoglobulins. IGLV3-21R110 i-CLL had a significantly higher number of SF3B1 and ATM mutations and total number of driver alterations. However, the R110 mutation was the sole alteration in 1 i-CLL and was accompanied only by del(13q) in 3. Although IGHV mutational status varied, IGLV3-21R110 i-CLL transcriptomically resembled n-CLL/unmutated IGHV CLL with a specific signature including WNT5A/B overexpression. In contrast, i-CLL lacking IGLV3-21R110 mirrored m-CLL/mutated IGHV. Patients with IGLV3-21R110 i-CLL had a short time to first treatment and overall survival similar to those of n-CLL/unmutated IGHV patients, whereas patients with non-IGLV3-21R110 i-CLL had a good prognosis similar to that of patients with m-CLL/mutated IGHV. IGLV3-21R110 defines a CLL subgroup with specific biological features and an unfavorable prognosis independent of IGHV mutational status and epigenetic subtype.

Proteogenomic Subtyping of Chronic Lymphocytic Leukemia Identifies a Novel Poor Outcome Subgroup with a Distinct Drug Response Profile

Proteogenomic Subtyping of Chronic Lymphocytic Leukemia Identifies a Novel Poor Outcome Subgroup with a Distinct Drug Response Profile

Biological Impact of Monoallelic and Biallelic BIRC3 Loss in Del(11q) Chronic Lymphocytic Leukemia Progression

Chronic lymphocytic leukemia (CLL) patients harboring 11q22.3 deletion, del(11q), are characterized by a rapid disease progression. One of the suggested genes to be involved in the pathogenesis of this deletion is BIRC3, a negative regulator of NF-κB, which is monoallelically deleted in ~80% of del(11q) CLL cases. In addition, truncating mutations in the remaining allele of this gene can lead to BIRC3 biallelic inactivation, which accounts for marked reduced survival in CLL. Nevertheless, the biological mechanisms by which monoallelic or biallelic BIRC3 lesions could contribute to del(11q) CLL pathogenesis, progression and therapy response are partially unexplored. We used the CRISPR/Cas9 system to model monoallelic and biallelic BIRC3 loss in vitro. First, we generated an isogenic HG3 CLL cell line harboring monoallelic del(11q) - HG3-del(11q) - by the introduction of 2 guide RNAs targeting 11q22.1 and 11q23.3 (~17 Mb). Loss-of-function BIRC3 mutations (MUT) were introduced in the remaining allele, generating 3 HG3-del(11q) BIRC3MUT clones. In addition, single BIRC3MUT were introduced in HG3 and MEC1 CLL-derived cells for experimental validation (n = 3 clones/cell line). We first questioned whether monoallelic and biallelic BIRC3 loss had an impact in the DNA-binding activity of NF-κB transcription factors. Interestingly, HG3-del(11q) had higher p52 and RelB (non-canonical NF-κB signaling) activity than HG3WT cells (P = 0.005; P = 0.007), being this activity further increased in HG3-del(11q) BIRC3MUT cells (P &amp;lt; 0.001; P &amp;lt; 0.001). In depth analysis of the non-canonical signaling components by immunoblot revealed that HG3-del(11q) and, to a greater extent, HG3-del(11q) BIRC3MUT cells presented NF-κB-inducing kinase (NIK) cytoplasmic stabilization, high p-IKKα levels and p52-RelB nuclear translocation. Besides, HG3-del(11q) BIRC3MUT cells showed increased levels of the anti-apoptotic proteins BCL2 and BCL-xL. We next assessed this pathway ex vivo in stroma and CpG-stimulated primary CLL cells with or without BIRC3 deletion (n = 22; 11 each group). Remarkably, stimulated BIRC3-deleted primary cells showed higher p52 and RelB activity than BIRC3WT cases (P = 0.01; P = 0.07), and the percentage of BIRC3-deleted cells correlated with p52 activity in del(11q) cases (P = 0.04). We further performed western blot analyses in a homogenous cohort of del(11q) cases including (n = 4) or not including (n = 3) BIRC3 within the deleted region. Interestingly, del(11q)/BIRC3 deleted cases presented high levels of stabilized NIK, which correlated with higher p52 processing (P = 0.003). These patients also showed higher BCL2 levels than those del(11q)/BIRC3 undeleted, and we could further observe a correlation between p52 and BCL2 levels (P = 0.01). Given this p52-dependent BCL2 upregulation, we treated the CRISPR/Cas9 edited clones with venetoclax, demonstrating that HG3-del(11q) BIRC3MUT cells were more sensitive upon BCL2 inhibition than HG3WT clones (mean IC50 3.5 vs. 5.75 μM; P = 0.005). In vitro proliferation assays were performed to interrogate the impact of BIRC3 loss in CLL cell growth, revealing that HG3 BIRC3MUT cell lines had higher growth rates than BIRC3WT cells (P = 0.001). HG3-del(11q) BIRC3MUT cells also showed enhanced proliferation in comparison to HG3-del(11q) clones (P = 0.009). We further determined the clonal dynamics of del(11q) and/or BIRC3MUT cell lines in clonal competition experiments, showing that HG3 BIRC3MUT and HG3-del(11q) BIRC3MUT cells progressively outgrew HG3WT and HG3-del(11q) cells, respectively, overtime (P = 0.02; P = 0.006). Furthermore, we injected these edited cell lines into NSG mice (n = 20) in vivo, showing that mice xenografted with HG3 BIRC3MUT and HG3-del(11q) BIRC3MUT cells presented, by flow cytometry, an increase of human CD45+ cells in spleen 14 days after injection, compared to HG3WT and HG3-del(11q) cells (P = 0.02; P = 0.015). In summary, this work demonstrates that biallelic BIRC3 deletion through del(11q) and mutation triggers non-canonical NF-κB signaling, driving BCL2 overexpression and conferring clonal advantage, which could account for the negative predictive impact of BIRC3 biallelic inactivation in CLL. Taken together, our results suggest that del(11q) CLL patients harboring BIRC3 mutations should be considered as a CLL subgroup at a high risk of progression that might benefit from venetoclax-based therapies. Funding: PI18/01500 Disclosures No relevant conflicts of interest to declare.

Clinical and Biological Impact of TP53 Alterations in Del(11q) Chronic Lymphocytic Leukemia

Large-scale next-generation sequencing (NGS) studies have suggested common patterns of co-occurrence or mutual exclusivity between genetic alterations in chronic lymphocytic leukemia (CLL). However, little is known about how most of these alterations cooperate to drive CLL pathogenesis, as well as the impact of these concurrencies in clinical outcome. In this regard, we investigated the clinical and biological impact of the co-occurrence of high-risk lesions such as del(11q)/ATM mutation and del(17p)/TP53 mutation by integrating NGS and CRISPR/Cas9 approaches. To address these questions, we first analyzed the mutational profile of 271 CLLs (17.3% del(11q); 10.7% del(17p)). The most frequently mutated genes were NOTCH1 (20%), TP53 (14%), SF3B1 (11%) and ATM (10%). Within del(11q), 32% showed TP53 alterations (53% biallelic; 47% monoallelic). Interestingly, patients harboring combined del(11q) and TP53 alterations by either mutation or deletion (del(11q) TP53ALT) exhibited significantly shorter overall survival (OS) than del(11q) CLLs without TP53 alterations (del(11q) TP53WT) and those TP53 altered without del(11q) (no del(11q) TP53ALT) (median 17 vs. 88, 36 months; P=0.0004, P=0.02). Conversely, we observed a significant lack of ATM mutations in CLLs with biallelic TP53 alterations (P=0.002) and a mutual exclusivity between biallelic TP53 and biallelic ATM losses (P=0.03)(Fig 1A). Based on the NGS results, we next used the CRISPR/Cas9 system to model monoallelic and biallelic ATM and TP53 loss in vitro. We generated isogenic HG3-Cas9 CLL-derived cell lines harboring monoallelic del(11q) (targeting 11q22.1/11q23.3 regions) and further loss-of-function mutations in ATM and/or TP53 to mimic all the possible combinations observed in our CLL cohort. By proliferation assays, we noted that the introduction of TP53 mutations increased the proliferation rates in both HG3WT and HG3-del(11q) cells. In contrast, the introduction of an ATM truncating mutation on the remaining allele of the HG3-del(11q) TP53MUT clone, suppressed this proliferative advantage, with growth rates comparable to those of HG3-del(11q). Accordingly, DNA content analysis by propidium iodide revealed that cells harboring biallelic ATM and TP53 loss also showed mitotic and cell cycle defects. To further evaluate the implications of these alterations in the clonal dynamics of CLL in vivo, we performed fluorescence-based clonal competition experiments by injecting these edited cell lines intravenously into NGS mice. First, we observed that HG3-TP53MUT cells outgrew HG3WT cells in spleen of xenotransplanted mice 14 days after injection (P&amp;lt;0.001). In a second experiment, HG3-del(11q), HG3-del(11q) TP53MUT and HG3-del(11q) ATMMUTTP53MUT cells were injected. Strikingly, HG3-del(11q) TP53MUT cells were able to outcompete HG3-del(11q) cells in spleen and bone marrow (P&amp;lt;0.001; P&amp;lt;0.001). By contrast, HG3-del(11q) ATMMUTTP53MUT cells failed to engraft neither in spleen nor bone marrow, being outcompeted by the other injected cells (P&amp;lt;0.001), providing biological insights on the mutual exclusivity of these genetic events in CLL (Fig 1B). We next assessed whether these cell models could predict responses of these combined abnormalities to BTK and PI3K inhibitors. We observed that HG3-del(11q) TP53MUT and HG3-TP53MUT cells showed partial response to ibrutinib and idelalisib, although the IC50 values were still higher than the ones observed in HG3WT clones, especially with idelalisib (27.4 and 20.6 vs. 1.8 uM, respectively). Nonetheless, we found that HG3-del(11q) TP53MUT cells were highly sensitive to novel preclinical drugs that have been shown to be effective in TP53 deficient cells such ATR inhibitors, with an IC50 value comparable to HG3WT cells (mean IC50 0.55 vs. 0.67 uM) (Fig 1C). In summary, we show that mutations in TP53 can appear in a subset of monoallelic del(11q) CLL cases, conferring a synergistic clonal advantage in vivo, and therefore a dismal clinical impact on the OS of this CLL subgroup. In addition, the biological basis of mutual exclusivity of biallelic ATM and TP53 alterations in CLL was assessed, underscoring the importance of the number of alleles affected by these alterations, and establishing novel pre-clinical models for the study of the biology and therapeutic response of concurrent genetic abnormalities in the disease. Funding: PI18/01500 FI19/00191 CD19/00222 *MQA CPC equal contr Disclosures No relevant conflicts of interest to declare.

Clonal lineage analysis of CLL research samples by IGH chain sequencing to reveal novel CLL subgroups defined by ongoing class-switch recombination and somatic hypermutation.

e20001 Background: B cell somatic hypermutation (SHM) and class switch recombination (CSR) are mechanistically related but distinct processes requiring precisely targeted generation and repair of single and double strand DNA breaks. Within the context of chronic lymphocytic leukemia (CLL), the presence of ongoing SHM or CSR may reveal the functionality of DNA damage repair pathways, with potential relevance to therapeutic strategies involving the generation of DNA breaks or inhibition of DNA repair machinery. Here we apply clonal lineage analysis of IGH chain sequencing data to evaluate CSR and SHM in a cohort of CLL and splenic marginal zone lymphoma (SMZL) research samples. We present evidence of ongoing CSR and SHM in a significant subset of samples. Methods: Multiplex primers targeting the IGH framework 1 region and isotype region (Oncomine IGH-LR assay; detection of all nine isotypes) were used for IGH repertoire sequencing via the Ion Gene Studio S5 from 25ng peripheral blood total RNA derived from 63 individuals with CLL and 4 individuals with SMZL. Clonotyping and clonal lineage analysis was performed by Ion Reporter, whereby clonal lineages are defined as sets of unique rearrangements having a shared variable and joining gene, the same CDR3 length, and a minimum CDR3 nucleotide similarity of 85%. Ongoing CSR was defined as the presence of IgM/IgD and at least one switched isotype (IgG, IgA, or IgE), or a combination of switched isotypes, within the same lineage. Ongoing SHM was defined as the presence of subclones that differ within the VDJ region sequence compared to other clonal lineage members. Results: 11/68 cases showed evidence of ongoing CSR or SHM. Of the 57 cases showing no evidence of ongoing CSR or SHM, variable gene mutation analysis revealed the presence of three distinct subgroups having either no SHM, intermediate SHM (average 98% sequence identity) or high SHM &lt; 94% identity). 3 of 4 SMZL cases showed evidence of ongoing CSR or SHM. Conclusions: These results reveal previously underappreciated heterogeneity within CLL and suggest the subdivision of CLL based on a combination of IGHV mutation level and presence of ongoing SHM or CSR. The described heterogeneity may serve as a valuable criterion for stratifying CLL patients in the future.

Optimized Xenograft Protocol for Chronic Lymphocytic Leukemia Results in High Engraftment Efficiency for All CLL Subgroups.

Preclinical drug development for human chronic lymphocytic leukemia (CLL) requires robust xenograft models recapitulating the entire spectrum of the disease, including all prognostic subgroups. Current CLL xenograft models are hampered by inefficient engraftment of good prognostic CLLs, overgrowth with co-transplanted T cells, and the need for allogeneic humanization or irradiation. Therefore, we aimed to establish an effective and reproducible xenograft protocol which allows engraftment of all CLL subtypes without the need of humanization or irradiation. Unmanipulated NOD.Cg-PrkdcscidIl2rgtm1Sug/JicTac (NOG) mice in contrast to C.Cg-Rag2tm1Fwa-/-Il2rgtm1Sug/JicTac (BRG) mice allowed engraftment of all tested CLL subgroups with 100% success rate, if CLL cells were fresh, injected simultaneously intra-peritoneally and intravenously, and co-transferred with low fractions of autologous T cells (2%–4%). CLL transplanted NOG mice (24 different patients) developed CLL pseudofollicles in the spleen, which increased over 4–6 weeks, and were then limited by the expanding autologous T cells. Ibrutinib treatment studies were performed to validate our model, and recapitulated treatment responses seen in patients. In conclusion, we developed an easy-to-use CLL xenograft protocol which allows reliable engraftment for all CLL subgroups without humanization or irradiation of mice. This protocol can be widely used to study CLL biology and to explore novel drug candidates.

Outcomes of Patients with Chronic Lymphocytic Leukemia Admitted with Sepsis: An Analysis of National Inpatient Sample Database

Background: Chronic lymphocytic leukemia (CLL) has a very high prevalence in the Western hemisphere, secondary to increased life expectancy and improved therapeutic options. Infections are known to be the prime contributor to morbidity and mortality in this patient population, accounting for 50-60% of all deaths. This occurs secondary to humoral depression by the disease and immune suppression by the treatment. There is paucity of data regarding outcomes of sepsis in CLL patients. We aim to study in-hospital outcomes of sepsis in patients with CLL. Methods: This is a retrospective cohort analysis of National inpatient sample (NIS) database (year 2016). Patients with CLL who were admitted with a principal diagnosis of sepsis were identified using the associated ICD-10 codes. Admissions were further categorized based on remission status of CLL: group 1 was in remission, group 2 had not achieved remission and group 3 had relapse. Primary outcome was inpatient mortality and secondary outcome was the use of mechanical ventilation (as a surrogate for utilization of intensive care). Comparisons were made between the subgroups of CLL as listed above and non-CLL patients. Associated factors were analyzed using multivariable regression model. We used STATA for statistical analysis. Results: Among patients with CLL who were admitted, the most common principal diagnosis (reason for admission) was sepsis, accounting for 12.9% of all CLL admissions. Other common reasons for admissions were pneumonia (5.4%), acute kidney injury (2.3%), NSTEMI (1.8%) and COPD exacerbation (1.8%). A total of 9,315 admissions with sepsis in CLL patients were identified (780 in remission, 8280 had not achieved remission and 255 had relapse). Compared to non-CLL patients, CLL patients were older (mean age 75.1 vs 64.7, p=0.0001), had more males (62.3% vs 49.3%, p=0.00001) and higher percentage of Caucasians (81.8% vs 67.5%, p=0.00001). CLL patients had higher inpatient mortality (14% vs 10%, p=0.00001), longer length of stay (7.8 vs 7.3 days, p=0.003) and higher hospital charges ($83,000 vs $75,000, p=0.006). On multivariable logistic regression model, after adjusting for age, gender, race and number of comorbidities, CLL patients were comparable with non-CLL patients, in regard to mortality and mechanical ventilation. On analysis of subgroups of CLL, those with CLL in remission had lower mortality (OR 0.51, CI 0.28-0.91, p=0.024) when compared to non-CLL patients. Group 2 (CLL, not yet achieved remission) were not statistically significant, whereas, group 3 (CLL in relapse) had higher mortality (OR 2.97, CI 1.64-5.35, p=0.000). Only group 3 CLL patients had higher risk of requiring mechanical ventilation (OR 2.92, CI 1.52-5.61, p=0.001). Conclusions: Infectious complications continue to remain a major cause of mortality and morbidity in CLL. Sepsis constituted the most common principal diagnosis in CLL patients admitted to acute-care hospitals. CLL patients with sepsis had higher mortality, compared to their non-CLL counterparts, even after adjusting for age, gender and other variables. Patients with relapsed disease had higher inpatient mortality when admitted to sepsis and interestingly, those with CLL in remission had lower mortality when compared to non-CLL patients. Disclosures No relevant conflicts of interest to declare.

PF395 COMPLEX EVALUATION OF CELLULAR AND HUMORAL IMMUNITY IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA

Background:Chronic lymphocytic leukemia (CLL) is typically associated with a significant immune deficiency resulting in elevated risk of infections ‐ the leading cause of death in CLL patients (pts). However, many specific mechanisms contributing to suppression of immunity in CLL are not yet well understood.Aims:Comprehensive assessment of immunological parameters in patients with CLL.Methods:We prospectively enrolled 126 CLL pts: 42 with clinically stable disease not requiring treatment (median age, 65 years; males, 50%; low/medium/high modified Rai risk, 59/36/5%) and 84 with CLL progression indicated for therapy (median age, 68 years; males, 64%; low/medium/high modified Rai risk, 0/43/57%). In addition, parameters of immunity were compared before and after first‐line chemoimmunotherapy in 39 pts (FCR, n = 13; BR, n = 15; G‐CLB, n = 5; R‐CLB, n = 6). Control group consisted of 46 healthy blood donors (median age, 40 years; males, 59%). Cellular subpopulations (CLL cells, T cells, Th and Tc cells, NK cells and polyclonal B cells) were quantified by flow cytometry (cytometer Navios, Beckman Coulter). Humoral immunity was evaluated by quantification of serum immunoglobulin concentrations (total IgG, IgG subclasses 1–4, IgA 1–2 and IgM) using Immunochemistry Image System (Beckman Coulter). Activation of complement was assessed by Wieslab Complement System Screen (Euro Diagnostica). Serum concentrations of immunity‐regulating cytokines (IL‐4, IL‐5, IL‐6, IL‐10, IL‐17A, IL‐17F, IL‐33 and IFN‐γ) were determined by xMAP multiplex technology (Luminex).Results:Serum IL‐4, IL‐6 and IL‐10 were elevated in CLL compared to controls (all p &lt;0.0001). Polyclonal B cells were decreased (p &lt; 0.0001) whereas Tc cells were increased (p = 0.019 and p &lt; 0.0001) in stable and progressive CLL in comparison to controls. Both CLL subgroups had significantly lower IgG, A and M when compared to controls (all p &lt; 0.0001). Progressive CLL pts had significantly higher numbers of NK cells (p = 0.0003) and stable CLL pts significantly higher IFN‐γ (p = 0.01) vs controls. Pts with progressive CLL had significantly lower concentrations of IgG, A, and M (p = 0.0012, p &lt; 0.0001 and p = 0.027) than stable pts. Chemoimmunotherapy resulted in significant decline of polyclonal B cells, NK cells, Th cells, Tc cells and T regs (all p &lt; 0.0001) but serum IgA and complement activation (both classical and alternative pathway) increased after therapy (p = 0.048, p = 0.002 and p = 0.0017).Summary/Conclusion:Conclusions: The present study identified multiple defects in cellular as well as humoral immunity in CLL pts. Clinical progression of CLL was associated with further decline of immune parameters. Cytoreductive therapy resulted in significant decrease in all physiological lymphocyte subpopulations; however, several markers of humoral immunity improved after treatment. The enrollment into the study continues and further analyses are underway including assessment of association of immune defects with infections and changes in immunological parameters over longer follow‐up. Supported by project PROGRES Q40/08, by DRO Ministry of Health (UHHK, 00179906) and by League Against Cancer Prague.image

PF363 UNDETECTABLE EXPRESSION OF WNT5A, A GENE ENCODING A ROR1 LIGAND, DEFINES MEMORY B-CELL LIKE CHRONIC LYMPHOCYTIC LEUKEMIA

Background: Chronic lymphocytic leukemia (CLL) is a malignancy with highly variable disease course among patients. Although many approaches distinguishing patients with worse and better prognosis have been published, IGHV somatic hypermutation status remains one of the strongest prognostic predictors. However, even within prognostically favorable IGHV-mutated (M-CLL) group, there is a subset of “grey-zone” patients with worse prognosis. We have shown recently that expression of WNT5A, a gene encoding a ligand activating WNT/PCP signaling pathway via ROR1 receptor, distinguishes the cases with worse prognosis within M-CLL (Janovska, Clin Cancer Res 2016). Still, over 80% of M-CLL patients had undetectable WNT5A expression, suggesting possible silencing of the gene expression through its methylation. Aims: To analyze methylation status of WNT5A promoter in the context of WNT5A mRNA expression, IGHV somatic hypermutations and methylation-based CLL classification (Querios, Leukemia 2015). Methods: Using bisulphite sequencing we analyzed methylation status of 3 CpG islands (CGI) located in WNT5A promoter and 5 marker CpG sites distinguishing methylation-based prognostic subgroups of CLL (located in B3GNTL1, CTBP2, TNF, SCARF1 and intergenic region on chromosome 14). mRNA expression was analyzed with quantitative real-time PCR using TaqMan probes (ThermoFisher Scientific): WNT5A, and HPRT1, TBP were used as controls. Results: Thirty-nine previously untreated CLL patients were chosen for the analysis. They were equally distributed among 4 groups based on IGHV status and WNT5A expression [WNT5A- M-CLL (10), WNT5A+ M-CLL (9), WNT5A- IGHV-unmutated CLL (U-CLL; 10), and WNT5A+ U-CLL (10), respectively]. Within the first of the three CpG islands of the WNT5A promoter (CGI1), we identified three CpG sites whose methylation status significantly correlated with the WNT5A expression in M-CLL (P = 0,004; P = 0,004; P = 0,0268; Mann-Whitney test). Furthermore, complete demethylation of the three CpG sites was associated with high WNT5A expression in M-CLL. In fact, complete demethylation of the whole tested CGI1 region was noted in these patients. Methylation analysis of 5 marker CpGs (Querios, Leukemia 2015) enabled us to classify patients into three methylation-based prognostic groups: 9/10 WNT5A- M-CLL patients belonged to the memory B-cell like CLL (m-CLL) group, and 7/9 WNT5A+ M-CLL patients to the intermediate CLL (i-CLL). U-CLL patients irrespective of WNT5A expression were assigned as naive B-cell like CLL (n-CLL) in 19/20 cases.Summary/Conclusion: Within M-CLL, methylation of the 3 CpGs located in the CGI1 of WNT5A promoter correlated significantly with WNT5A expression. Higher WNT5A expression was associated with complete demethylation of CGI1 locus. Our classification based on WNT5A expression and IGHV status overlapped with the methylation-profile-based CLL classification (m-CLL, i-CLL, n-CLL). Furthermore, memory B-cell like CLL group was associated with undetectable WNT5A level in our cohort. Supported by grants: MUNI/A/1105/2018, CEITEC2020 LQ1601, MZCR-RVO 65269705. Spanish Ministry of Health, Plan Estatal de I + D + I, ISCIII, FEDER, FIS PI15/01763ISIII PI15/01763 and PI18/01484.

Study of gene expressions' correlation structures in subgroups of Chronic Lymphocytic Leukemia Patients.

In chronic lymphocytic leukemia (CLL) the interaction of leukemic cells with the microenvironment ultimately affects patient outcome. CLL cases can be divided in two subgroups with different clinical course based on the mutational status of the immunoglobulin heavy variable (IGHV) genes: mutated CLL (M-CLL) and unmutated CLL (U-CLL). Since in CLL, the differentiated relation of genes between the two subgroups is of greater importance than the individual gene behavior, this paper investigates the differences between the groups' gene interactions, by comparing their correlation structures. Fisher's test and Zou's confidence intervals are employed to detect differences of correlation coefficients. Afterwards, networks created by the genes participating in most differences are estimated with the use of structural equation models (SEM). The analysis is enhanced with graph modeling in order to visualize the between group differences in the gene structures of the two subgroups. The applied methodology revealed stronger correlations between genes in U-CLL patients, a finding in line with related biomedical literature. Using SEM for multigroup analysis, different gene structures between the two groups of patients were confirmed. The study of correlation structures can facilitate the detection of differential gene expression profiles in CLL subgroups, with potential applications in other diseases. Comparison of correlations can be very useful in understanding the complex internal structural differences which signify the variations of a disease.