Chronic Lymphocytic Leukemia Cases Research Articles

An overview of prognostic markers in patients with CLL

Chronic lymphocytic leukemia (CLL) is a low-grade B-cell lymphoproliferative disorder. It is the most prevalent type of leukemia in the western countries, with a median age at diagnosis of 70 years. In 2023, it is estimated that there will be 18,740 new cases of CLL, and an estimated 4,490 people will die of this disease. It represents 1.0% of all new cancer cases in the U.S. The rate of new cases was 4.6 per 100,000 men and women per year based on 2016–2020 cases, age-adjusted. Death rates from CLL are higher among older adults, or those 75 and older. The death rate was 1.1 per 100,000 men and women per year based on 2016–2020 deaths, age-adjusted. A common question that patients with CLL ask during their first clinic visit is: “How long will it be before I would need treatment?” Although this might seem like a simple question, the answer is not straight forward. CLL is a heterogenous disease, with a variable clinical course. Some patients may present with an aggressive disease requiring early initiation of treatment, while others have an indolent course and some, having so called smoldering CLL, may never need treatment. The variability in disease course can make predicting disease prognosis a complicated process. This brings forth the importance of establishing prognostic models that can predict disease course, time to treatment, and survival outcomes in such a heterogenous disease. The Rai and Binet staging systems were developed in the late 1970s to early 1980s. They separated patients into different stages based on clinical characteristics and laboratory findings. These simple staging systems are still in use; however, several prognostic markers need to be added for an individualized assessment and, with the recent development of genomic techniques leading to better understanding of CLL at the molecular level, newer prognostic markers have emerged.

Two recurrent types of IGH::5′ BCL2 breakpoints representing cytogenetic ins(14;18)(q32;q21q21) and t(14;18)(q32;q21), mediated by the VDJ and class switch recombination processes, respectively

We describe two types of IGH::BCL2 breakpoints involving the 5′ region of BCL2 (5′ BCL2). One was ins(14;18)(q32;q21q21) observed in 2 follicular lymphoma (FL) cases, in which IGH was cleaved at 3’ of IGHD and 5’ of IGHJ and BCL2 was cleaved at 5’ BCL2 and downstream regions, and a 281- or 201-kilobase pair fragment containing the BCL2 protein-coding sequences was invertedly inserted into IGH. In another type observed in 2 FL and 2 chronic lymphocytic leukemia (CLL) cases, breakage and reunion occurred within the switch region associated with IGHM (Sµ) and 5′ BCL2, creating IGH Sµ::5′ BCL2 fusion sequences on der(18)t(14;18)(q32;q21). The former is considered to be mediated by VDJ-recombination, while the latter by the class switch recombination process. There were no particular features in FL or CLL cases with IGH::5′ BCL2 breakpoints compared with those with t(14;18)(q32;q21)/IGH::BCL2 involving the 3′ breakpoint cluster regions.

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.

Abstract P01: Impact of Germline and Somatic ATM Variants in Chronic Lymphocytic Leukemia (CLL): Clinical Implications and Response to PARP Inhibition

Abstract Background: Next-generation sequencing has revealed numerous Variants of Unknown Significance (VUS) in the ATM gene, important in DNA repair and chronic lymphocytic leukemia (CLL) pathogenesis. This study evaluates the effect of rare ATM germline variants, including VUS, on features of CLL, ATM kinase activity, and response to PARP inhibition. Methods:ATM variants were detected in 885 CLL patients (327 DFCI; 276 German CLL Study Group; 282 ICGC) using DeepVariant, and compared to 5310 ancestry-matched controls. Concurrently, 278 CLL patients with ATM aberrations from clinical sequencing were retrospectively analyzed, annotated by population allele frequency (JCO 2023, Lampson et al.). ATM kinase activity was measured by ATM and KAP1 phosphorylation following gamma-irradiation in primary CLL samples. The impact of talazoparib, a PARP inhibitor, on ATM aberrant and wild-type CLL cell proliferation was also assessed. Results: Among the 885 CLL cases, 130 ATM germline variants were identified by DeepVariant, including 87 missense variants. The top 5 most frequent rare germline missense variants with high odds ratio for association with CLL were ATM p.L2332P (AF cases vs controls, 0.328% vs 0.0188%, OR 17.46), p.L2307F (OR 16.88), p.F763L (OR 18.89), p.Y1442H (OR 35.39) and p.S99G (OR 16.28). In our retrospective clinical cohort, we identified 294 ATM variants with 146 unique variants. Our previously proposed algorithm for classifying germline vs somatic identified 74 germline, with 70 missense, and 72 somatic. Phosphorylation of ATM and KAP1 (pATM, pKAP1) in response to IR was reduced in 13 patients assessed with germline ATM variants (9 rare and 4 non-rare; ATM variant alone group) compared to those without any somatic or germline ATM aberrancy (WT group) (P<0.01). Phosphorylation levels were also significantly lower in 17 patients with rare germline ATM variants with concurrent del(11q) (del11q + ATM germline variant group) compared to 15 with del(11q) alone (del 11q group) (P<0.01). We obtained similar results from Western blot analysis. To assess the sensitivity of ATM deficient cells to talazoparib, we compared proliferation inhibition across groups cocultured with talazoparib for 14 days. The del11q with ATM-somatic group exhibited significantly greater sensitivity to talazoparib at lower dosages compared to ATM WT (talazoparib 0.1 μM, P<0.05; 0.5μM, P=0.098). The monoallelic ATM deficient groups (del11q alone or ATM-germline alone) tended to have lower proliferation, but not significantly. Higher concentrations of talazoparib (2.5μM) strongly inhibited proliferation of all CLLs including WT. Conclusion: This study highlights the prevalence of rare ATM germline variants in CLL and their impact on ATM function in CLL. Our findings also suggest that these variants affect the DNA damage response in primary CLL cells and that ATM biallelic deficiency influences sensitivity to PARP inhibitors. Our evidence could lead to more personalized therapeutic strategies for CLL patients with distinct ATM aberrancies. Citation Format: Kiyomi Mashima, Nicholas Moore, Mariia Mikhaleva, Anna Petráčková, Samantha Shupe, Stacey M Fernandes, Amaro Taylor-Weiner, Riaz Gillani, Hoyin Chu, Seunghun Han, Sabrina Camp, Eric Kofman, Gad Getz, Catherine J. Wu, Svitlana Tyekucheva, Matthew S Davids, Eliezer Mendel Van Allen, Saud AlDubayan, Jennifer R Brown. Impact of Germline and Somatic ATM Variants in Chronic Lymphocytic Leukemia (CLL): Clinical Implications and Response to PARP Inhibition [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P01.

The Role of Methylation in Chronic Lymphocytic Leukemia and Its Prognostic and Therapeutic Impacts in the Disease: A Systematic Review.

Epigenetic regulation has been thoroughly investigated in recent years and has emerged as an important aspect of chronic lymphocytic leukemia (CLL) biology. Characteristic aberrant features such as methylation patterns and global DNA hypomethylation were the early findings of the research during the last decades. The investigation in this field led to the identification of a large number of genes where methylation features correlated with important clinical and laboratory parameters. Gene-specific analyses investigated methylation in the gene body enhancer regions as well as promoter regions. The findings included genes and proteins involved in key pathways that play central roles in the pathophysiology of the disease. Τhe application of these findings beyond the theoretical understanding can not only lead to the creation of prognostic and predictive models and scores but also to the design of novel therapeutic agents. The following is a review focusing on the present knowledge about single gene/gene promoter methylation or mRNA expression in CLL cases as well as records of older data that have been published in past papers.

SOLUBLE CD150 ISOFORM LEVEL IN PLASMA OF CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS.

SLAMF1/CD150 is an active player in B cell signaling networks in chronic lymphocytic leukemia (CLL). CD150-mediated signaling initiates through a homophilic CD150 binding, which spans the adjacent cells, or the interaction with the soluble CD150 isoform (sCD150). The expression of sCD150 isoform at the mRNA and protein levels ex vivo was confirmed. However, it is unclear whether sCD150 isoform present in the blood plasma of CLL patients is a factor in the constitutive activation of CD150+ cells. The aim of this study was to develop an ELISA assay for the specific sCD150 evaluation and assess the sCD150 levels in the blood plasma of CLL patients with different CD150 expression on B cells. Blood plasma samples and peripheral blood mononuclear cells from 40 previously untreated CLL patients were analyzed. An ELISA method, ex vivo drug sensitivity assay, and a cell viability assay were used. The sCD150 isoform was found in all studied plasma samples of CLL patients at different levels regardless of the cell surface CD150 expression status of B cells and sCD150 mRNA expression. CLL cases with low levels of the cell surface CD150 expression in B cells are characterized by high levels of sCD150 in blood plasma in contrast to the CLL cases with high cell surface CD150 expression on B cells. The elevated levels of sCD150 in blood plasma are associated with a better sensitivity of malignant B cells to cyclophosphamide and bendamustine. The sCD150 isoform is actively secreted by CLL B cells with its accumulation in blood plasma, which may be regarded as an additional factor in the CLL clinicopathologic variability.

IMMUNOPHENOTYPE OF LEUKEMIC CELLS IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS WITH NOTCH1 AND SF3B1 GENE MUTATIONS.

The typical chronic lymphocytic leukemia (CLL) immunophenotype is vital for diagnosis, but the expression of some antigens varies and has prognostic value. There are data that reduced CD20 expression is associated with NOTCH1 and SF3B1 gene mutations. To determine a high-risk group of CLL patients for prediction of unfavorable NOTCH1 and SF3B1 gene mutations based on immunophenotyping of leukemic cells. Flow cytometric and molecular-genetic analysis (mutations of NOTCH1, SF3B1, and TP53 genes using the polymerase chain reaction followed by direct sequencing) was performed in a group of 86 previously untreated CLL patients. The immunophenotype of leukemic cells of all examined patients met the criteria of CLL diagnosis. NOTCH1 gene mutations were found in 21 patients (24.4%), and SF3B1 gene mutations - in 7 patients (8.1%). There were no TP53 gene mutations among the examined patients. A decreased number of CD20+CD5+ cells and a downward trend in the relative index of mean fluorescence intensity (iMFI) of CD20+ cells were found in patients with NOTCH1 and SF3B1 gene mutations. Based on the iMFI level (higher and/or lower than 3.0) and the number of CD20+CD5+ cells among all B-cells (higher and/or lower than 50%), we distinguished CLL cases with low and relatively high levels of CD20 antigen expression. Using ROC analysis and the parameter of low CD20 antigen expression, we could predict the presence of NOTCH1 and SF3B1 gene mutations in 73.3 ± 0.06% of patients (p = 0.001). The risk of NOTCH1 and SF3B1 gene mutations in cases with low CD20 antigen expression was 6.96 (95% CI = 2.53-19.18; p = 0.0001). The revealed regularities were statistically significant for patients in whom the diagnosis was established in all Binet - Rai stages except A0-AI. Our data confirmed a reduced CD20 expression in CLL patients with NOTCH1 and SF3B1 mutations. In addition, an approach was proposed to identify high-risk CLL patients for prediction of such mutations: previously untreated CLL patients at advanced Binet - Rai stages (BII, CIII, CIV) with a reduced number of double-positive CD20+CD5+ cells in peripheral blood and/or low iMFI of CD20+ cells.

Study of immunophenotypic markers and clinico-hematological findings in chronic lymphocytic leukemia

Background and objective: Chronic lymphocytic leukemia (CLL) is a clonal, mature B-cell neoplasm, its clinical features, cell morphology, and immunophenotyping are used to establish the diagnosis. Immunophenotyping by flow cytometry is the most accurate procedure for diagnosing CLL, there is no single marker exclusively expressed in CLL, however, a complex of immunophenotypic markers that incorporates several B-cell markers and assists in differentiating CLL from other mature B-cell neoplasms, the expression of these immunophenotypic markers is considered in the specific CLL scoring system (Moreue scoring system).this study aimed to assess the clinico – hematological features and immunophenotypic characteristics of CLL patients and to assess the role of immunophenotyping in the differential diagnosis of CLL. Methods: an observational prospective and retrospective study was carried out at Nanakaly hospital for blood diseases and oncology in Erbil city, Iraq. That was conducted on 100 patients newly diagnosed with mature B cell neoplasm which included 68 cases of CLL and 32 cases with other mature B cell neoplasm (MBN), usinga convenience sampling method. The study period was from the1st of September 2021 to the end of April 2022. An interview questionnaire was used to collect the study data from the patients. Results: The mean of age ± standard deviation of the CLL patients (58.85 ± 10.69) years and (66.2%) was male, The most frequent clinical presentations for CLL cases were fatigue (50%), lymphadenopathy (41.2%), and splenomegaly (41.2%), (50%) of CLL patients had anemia, thrombocytopenia was seen in (32.4%) and leukocytosis was seen (98.5%), all patients had lymphocytosis and (97%) had an absolute B lymphocyte count of more than 5 x109 /L. all the studied CLL patients expressed both CD45 and CD19 markers, the expression of the immunophenotypic markers was as following: CD5 (98.5%), CD23 (97.1%) and CD200 (97.1%), Half of the patients (50%) showed expression of monoclonal lambda light chain, sIgM (4.4%), CD20 (88.2%) and CD43 (54.4%) and CD38 (26.5%). The expression of the following marker had a significant role in the differentiation between CLL and other MBN: CD5, CD23, CD79b, FMC7, sIgM, CD200, and CD43. Conclusion: Our results have shown that the clinical presentations and hematological profile of Iraqi CLL patients were not significantly different from that of previous local and global studies, the distinct immunophenotyping, as well as it has been found that immunophenotyping is a promising method for supporting the clinical and morphological characterization of CLL.

Quality and Quantity of Nucleic Acids Extracted from Formalin-Fixed Paraffin-Embedded Lymphoma Biopsies from Nigerian Archived Biopsy.

Integrity of nucleic acids derived from archived formalin-fixed paraffin-embedded (FFPE) cancer specimens affects diagnosis, prognosis, and therapy. Several factors affect the quality and quantity of extracted nucleic acids and one of such factors is storage period. We investigated the impact of storage duration on the quality and quantity of nucleic acids extracted from archived FFPE lymphoma biopsies in Nigeria. A total of 53 FFPE biopsies diagnosed as lymphoma stored over several years (2008-2019) were analyzed. They were 22 chronic lymphocytic leukemia (CLL) cases, 17 Hodgkin lymphoma (HL) cases, and 14 diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS). DNA was extracted from all the lymphoma samples which were analyzed for integrity and amplifiability using the four pairs of control genes polymerase chain reaction (PCR) primers of BIOMED-2 protocol, whereas RNA extraction was from 6 CLL cases used for qPCR analysis of RNU43. For CLL, the mean DNA yield was 193.6 ng/µl (range: 3.0-533.0 ng/µl), whereas the mean A260/A280 ratio was 1.7 (1.2-1.9). For DLBCL, NOS, and HL, 255.5 ng/µl (range: 32.9-605.4 ng/µl), 1.8 (1.5-2.0) and 242.7 ng/µl (range: 1.3-886.0 ng/µl), and 1.7 (0.9-1.8), respectively. The extracted DNA gave amplifiable products of at least 200bp, whereas the RNA analysis showed CT values of <38 in all the samples. The mean RNA yield was 462.2 ng/µl (range: 74.7-1082.1), whereas the mean A260/A280 was 1.7 (1.5-1.8). Quantity and quality of nucleic acids from FFPE tissues stored for different time periods showed no significant difference in yield and quality.

Chronic Lymphocytic Leukemia (CLL) in Adolescents and Young Adults (AYA): Clinical Characteristics, Prognostic Factors and Survival for 498 Patients

Background Chronic lymphocytic leukemia (CLL) is primarily a disease of the elderly; however, it can also present in Adolescents and Young Adults (AYA) aged 15 to 39 years. Recent information of case series no larger than 227 patients each described clinical outcomes such as a 5-year Overall Survival (OS) of 90% and a 10-year OS of 78% for AYA 1-4. Data on the effect of age on OS in CLL is conflictive among studies 2,3. A higher proportion of males with CLL have been reported in the younger ages compared with olders in some series 2,4 but not in others 3. Sociodemographic characteristics and distribution between younger and older CLL patients have not been extensively reported 2-4. The objective of our study is to describe the clinical and sociodemographic characteristics, prognostic factors and survival in AYA diagnosed with CLL. Materials and Methods Cases of CLL were obtained from the Surveillance, Epidemiology, and End Results (SEER) Database from 2000 to 2020. Statistical analyses were conducted with SPSS® version 25. Patient characteristics were reported in frequencies and compared with the Chi-squared test. The Kaplan-Meier method was used to estimate the median Overall Survival (OS). Cox Regression was done to identify independent prognostic factors of survival. Results A total of 51,992 cases of CLL were identified. The median age of diagnosis was 69 years (interquartile range: 18 years). 498 AYA cases (1% of total population) were found (Table 1). For them, 66.1% were males, 70.3% were non-Hispanic whites and 90.2% lived in metropolitan areas. A median income of ≥$75,000 was noted in 45.6% of the patients. Chemotherapy was received by 32.7% and radiation therapy was given to 1.6%. The median OS for AYA population was not reached (Figure 1), with a 5-year and 10-year OS of 91% and 84%, respectively. OS was different between AYA vs older population, and age was found to be an independent prognostic factor for survival in the entire population of CLL (p&amp;lt;0.001, Figure 1). The age group comparison also found more proportion of males in AYA vs olders (66.1% vs 58.5%, p&amp;lt;0.001), more proportion of non-white patients in AYA group (29.6% vs 15.3%, p&amp;lt;0.001) and that AYA patients were more prone to receive chemotherapy (32.7% vs 14.8%, p&amp;lt;0.001). The multivariate analysis specific for AYA population found that American Indian/Alaska Native Non-Hispanic patients had poor prognosis compared with other races (HR=19.1, CI 95%: 2.2-166.5, p=0.008). Also, receiving radiotherapy (Beam radiation) was associated with poor prognosis (HR=5.5, CI 95%: 1.6-19.6, p=0.008). Compared to people with income &amp;lt;$35,000, all patients with higher income had better prognosis (for patients with ≥$75,000 median income the HR was 0.01, 95% IC: 0.001-0.11, p&amp;lt;0.001). Chemotherapy was not found to be an independent prognostic factor in this population (p=0.8). Conclusions To the best of our knowledge, this is the largest epidemiological study available for CLL in AYA to this date. Sociodemographic differences and epidemiologic prognostic factors described in this research contribute to understand this disease. Interventional studies are required to identify possible targeted therapies that could improve survival outcomes in this population. We believe that the differences in distribution and prognosis for the described variables in our study can contribute towards optimizing the randomization in interventional trials in the future.

Chronic Lymphocytic Leukemia and Fatal Tissue Infiltration

Chronic lymphocytic leukemia (CLL) is typically characterized as an indolent process that is most often a comorbid condition rather than an immediate cause of death. While it is well-known that CLL can infiltrate tissues and manifest as small lymphocytic lymphoma, diffuse parenchymal infiltration with perforation and hemorrhage is most often associated with more aggressive lymphomas, such as diffuse large B-cell lymphoma. Here we present a series of three cases of CLL in which fatal hemorrhage (two spontaneous and one procedure-related) resulted from tissue compromise due to infiltration of organ parenchyma by the patient's known CLL. In the first case, the patient died of spontaneous duodenal perforation and hemorrhage associated with CLL. This patient also had heart failure related to myocardial infiltration and diffuse hepatic infiltration. A second patient died of spontaneous pulmonary hemorrhage associated with CLL. The final case involved fatal pulmonary hemorrhage during bronchoscopy in a patient with unsuspected bronchial involvement by CLL. At autopsy, all three cases showed histologic and immunohistochemical features of typical CLL. None of the cases showed evidence of Richter transformation, and none of the patients had clinically evident coagulopathy. The literature contains few reports of parenchymal infiltration by CLL with fatal perforation or hemorrhage. This pattern may represent an under-appreciated mechanism of CLL contribution to morbidity and mortality. Further characterization of how CLL invades tissue and ultimately leads to compromise in the integrity of that tissue may be of clinical significance. In addition, continued research into the prevalence of CLL-related organ compromise, risk assessment, and disease progression monitoring is warranted to improve patient outcomes and quality of life.

Pregnancy in Patents with CLL Systematic Review

Introduction: Chronic Lymphocytic Leukemia (CLL) is a hematologic malignancy characterized by the clonal expansion of non-functional, mature-appearing lymphocytes in the bone marrow, peripheral blood, and lymphoid tissues. While cases of CLL during pregnancy are rare,it is associated with many complications. It was reported that women with CLL are more susceptible to infections due to a weakened immune system; possibly leading to more complications. There is also increased risk of thrombocytopenia, which can increase the risk of bleeding during pregnancy and childbirth. This review aimed at identifying literature available on the complications in CLL and pregnancy as well as the data available on the potential therapeutic strategies. Methods: A scoping review of case reports that discuss pregnant patients who were diagnosed with CLL were isolated. The effects of the disease on pregnancy outcomes regarding both mother and fetus were identified. Animal studies, reviews or non-original articles, and non-English articles were excluded from our study. FDA labels of medications in CLL were reviewed for data on pregnancy and animal outcomes. Information on those medications in pregnant patients were also identified where available. Results: The search strategy across four databases yielded 448 articles. After eligibility assessment, 14 articles were included in the review. Cases were subdivided according to timing of pregnancy relative to CLL diagnosis (diagnosed before pregnancy or during pregnancy at early or late gestational periods). The pre-pregnancy reports were further subclassified based on the presence or absence of infection during pregnancy. None of the cases reported required immediate initiation of CLL therapy and only one case reported mortality post-delivery. This occurred in a patient with Richter transformation and no prior CLL treatments. There were no reported fetal malformations or severe adverse outcomes related to the infants in any of the reported pregnancies ( Figure 2). The included studies provided insights into various aspects of CLL during pregnancy, including patient demographics, diagnosis timing and methods, acquired infections, changes in white blood cell count, treatment approaches, pregnancy outcomes, and complications for both mother and fetus. From the marketed pharmacological alternatives for CLL, most were not studied in pregnant humans with CLL ( Figure 1). However, venetoclax and rituximab have some data in pregnant patients with non-CLL indications. A case reported a pregnant refractory AML patient who received venetoclax with high dose cytarabine and mitoxantrone at 24 weeks of gestation. Bone marrow at 27 weeks showed complete remission and labor was induced at 28 weeks + 5 days after corticosteroid-induced lung maturation. The infant had hyperbilirubinemia, transient B-cell depletion and required ventilation. The baby recovered from all complications, although possible long-term effects are still unclear. The mother underwent allogeneic hematopoietic stem cell transplant. In a 30-year-old female (pregnant at 28 weeks) with primary CNS lymphoma, weekly rituximab was given for 4 weeks with dexamethasone. After delivery through C-section at 31 weeks, the patient started definitive treatment with chemotherapy and autologous transplant. The infant required brief mechanical ventilation and had transient B-cell depletion at delivery until 4 months post-delivery. The baby required a course of post-exposure acyclovir despite not developing an infection. Both mother and baby survived with good outcomes at last follow-up (4 years post-delivery) Conclusion: Despite the rarity of CLL occurrence during pregnancy, there is evident need of further studies and management guidelines in this population. This review provides insights into the possible pregnancy-related concerns in women with CLL at different disease stages. While watchful waiting is generally recommended due to CLL's slow progression, individual evaluation is crucial. If treatment is necessary, delaying therapy until the second or third trimesters may help mitigate the risk of fetal malformations. The increased susceptibility to infections resulting from the combined immunosuppressive effects of CLL and pregnancy should be carefully considered

Clinical and Economic Burden Among Patients with Chronic Lymphocytic Leukemia Using a Novel Real-World Patient-Centered Database

Introduction Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults, with an estimated 18,740 new cases of CLL in 2023. This study describes current treatment patterns, healthcare resource use (HCRU) and patient voice in patients with CLL in the US using a novel patient-centered database. Methods A retrospective study using data from the Inspire Integrated Analytical Database (IIAD), which links Inspire patient voice with electronic health records (EHR) and medical and pharmacy claims through HIPAA-compliant tokenization. Patients worldwide with various conditions join Inspire's online health community and contribute their experience through online posts and survey participation (see Figure 1). Medical and pharmacy claims in IIAD range from 2013 to present, with EHR data ranging from 2010 to present. Inspire members were included in the study if they resided in the US and had at least two diagnoses for CLL (ICD-10 codes C91.10, C91.11 or C91.12) on at least two distinct dates that were between 30 and 60 days of each other. The first observed CLL diagnosis date was considered the index date. Patients with an index date on or before 6/30/2022 were included in the analysis. Baseline patient characteristics and HCRU were evaluated. CLL treatment was identified from medical and pharmacy claims. Online posts which CLL patients have viewed at least once were evaluated. Post topics are defined as named entities recognized by Inspire's Natural Language Processing (NLP) engine from all of the discussion posts written by any Inspire members. Descriptive statistics were reported. Results A total of 331 CLL patients were identified in the online community with linked claims and EHR data. Median follow-up time was 59 months from index date. The median age at diagnosis was 73 and 49% of patients were female. Patients were geographically represented across the US. 63% had ≥100 claims from a median of 25 care sites across 33 providers. During follow-up a median of 3 hospitalizations per patient were observed. 5% of patients experienced a Richter's transformation and 22% developed a secondary malignancy (lung, gastrointestinal or skin cancer) after their index CLL date. 104 patients (31%) of the cohort had available data for CLL treatment. Median time to first CLL treatment was 11.7 months (range: 1 day - 86.8 months). Ibrutinib was the most common medication (n = 43), followed by venetoclax (n = 30) and rituximab (n = 20). The 5 most common topics viewed by patients on the Inspire community website included: skin, side-effect, muscle, biopsy, and infection. Prior to starting therapy, searches on the Inspire platform included terms such as “imbruvica,” “calquence,” and “living with CLL.” Searches after CLL treatment revealed that patients broadened search terms to include other cancer types such as “pelvic cancer,” “liver metastasis” and “melanoma.” Conclusions This report utilized a unique dataset that integrates patient voice with EHR and claims data to describe clinical characteristics, and treatment patterns of CLL patients drawn from the patient-centered Inspire digital health community. The online population had a median age of 73 at diagnosis, consistent with the average age of CLL diagnosis (70 yrs.). Patients in our cohort initiated treatment approximately 1 year after CLL diagnosis. Online activities on the Inspire platform demonstrated that CLL patients are engaged in searching and reading discussion posts concerning the impact of side effects and treatments on quality of life, and emphasized emerging concerns about the potential impact of secondary malignancies. Future real-world studies need to integrate patient voice to fully understand the consequences of a cancer diagnosis.

Evidence of Defective Cell-Autonomous Signalling By B Cell Receptors in CLL Stereotyped Subset #1

B cell receptor (BCR)-mediated signalling is central in the pathogenesis of chronic lymphocytic leukaemia (CLL), and represents a target for pharmacological intervention. Notably, so far characterized CLL-derived BCRs are capable of triggering cell-autonomous intracellular signalling through homotypic interactions without requirement for exogenous antigens. Approximately 41% of all CLL cases can be classified in subsets based on homologous, otherwise known as stereotyped, BCRs expressed by the malignant clones, sharing remarkably similar sequences in the VH CDR3 region and highly restricted immunoglobulin heavy and/or light variable genes. Since cases within a subset display clinicobiological consistency in contrast with the pronounced heterogeneity of CLL at large, analysis of the mechanism of BCR-mediated signalling can unveil correlations between molecular details of BCR activation and the course of the disease. Our present study focused on BCRs of stereotyped subset #1, the second largest stereotyped subset, known for a particularly aggressive clinical course. Cases belonging to subset #1 express unmutated IGHV genes of phylogenetic clan I (namely, IGHV1, IGHV5 and IGHV7 subgroup genes) partnered by light chains encoded by the IGKV1-39/1D-39 gene. First, we used X-ray crystallography to determine the high-resolution crystal structures of the BCR Fab fragments from three subset #1 cases utilizing the IGHV1-2, IGHV1-3, and IGHV5-10 genes partnered with IGKV1-39/1D-39 to obtain information on the combining sites' structures and possible modes of intermolecular interactions that may mediate cell-autonomous signalling. Analysis of the experimental structures revealed that, despite the high sequence homology of the three proteins, the heavy and light chain CDR loops in the three BCR Fabs are arranged differently to shape binding cavities with distinct dimensions and polarity, thus suggesting that distinct antigens may be the physiological target of each subgroup. Unlike BCRs from other subsets previously characterised, the intermolecular contacts between the subset #1 Fab molecules in the three crystals involve diverse residues that are not indicative of a shared homotypic interaction. In addition, the surfaces involved in the contacts are small and the computed energies for the protein-protein contacts observed are suggestive at best of very weak interactions. In accordance to this, when we used analytical ultracentrifugation along with chromatographic and calorimetric methods to detect BCR Fab oligomerization in solution, no evidence of self-association was obtained, thus indicating that the receptors in solution either do not interact, or are characterized by self-dissociation constants that exceed the sensitivity limit of the techniques employed. To assess whether the cellular environment could promote self-association and signalling activity of the full-length BCR, we expressed subset #1 BCRs in the model triple knockout (TKO) pre-B-cell line, designed to measure Ca 2+ ion influx using cytofluorimetry. All subset #1 BCRs tested were unable to mobilize autonomously the Ca 2+ ions, despite being fully expressed on the cell surface and capable of signalling upon stimulation with anti-BCR antibody as a proxy for exogenous ligand. This result is in sharp contrast with the behaviour of all other CLL-derived BCRs so far characterised in this system that could activate a cell-autonomous signal as evidenced from a persistent Ca 2+ ion influx in the transduced cells without requiring external stimuli. Hence, subset #1 BCRs differ from the so far characterized BCRs from CLL cases belonging to other stereotyped subsets, including clinically aggressive subset #2 and its satellite subset #169 as well as the clinically indolent stereotyped subset #4. In conclusion, our combined biochemical and cellular analysis demonstrates that a constitutively active cell-autonomous signalling may not be a unifying feature of all CLL clones as previously proposed, and that other mechanisms may be at the roots of the BCR-mediated signal required for leukemic cell proliferation and development of CLL.

Chromosome Banding and SNP-Array Analysis Are Complementary Methods to Characterize Karyotype Complexity in High-Risk Chronic Lymphocytic Leukemia

Introduction: Karyotype complexity is of prognostic significance in chronic lymphocytic leukemia (CLL). It can either be assessed by chromosome banding analysis (CBA) or by chromosome microarray analysis (CMA). How to integrate both methods into CLL diagnostic procedures is not defined. Material and Methods: To compare both methods in their ability to detect chromosomal aberrations, samples from 110 patients enrolled on the CLL2O trial (NCT01392079) were analyzed in parallel by Affymetrix ® 6.0 single nucleotide polymorphism arrays (SNP-arrays, CMA) and CBA (CpG oligonucleotide and IL-2 stimulation). The trial included genetically complex high-risk CLL cases defined by TP53 loss (treatment-naïve or relapsed) or refractoriness to purine-analogue based treatment. Intraindividual reference DNA for paired CMA was available for 79 samples. CMA-defined gains and losses outside known structural variations were counted as aberration irrespective of their size. Discontinuous gains and losses were counted as one aberration whenever segments had a similar log2-ratio. Results: CBA identified 561 chromosomal aberrations with a median of 4 aberrations per case (range: 0-21). With 653 DNA copy number alterations, CMA identified more aberrations partly afforded by a high number of unbalanced translocations (median: 5 aberrations per case, range: 0-19). With regards to karyotype complexity (KC), CBA and CMA allocated 67/110 cases (61%) to comparable risk groups as defined by Baliakas et al. (2019) and Leeksma et al. (2021). CMA assigned more cases to higher complexity risk groups than CBA, which was not observed when excluding gains and losses &amp;lt;5Mb not previously deemed to be significant. However, this approach did not improve the congruency in KC risk group stratification between both methods (see Table). Only 14/110 cases (13%) had a complete overlap in the chromosomes found to be affected by CBA and CMA. Overall, CBA discovered more additionally affected chromosomes than CMA (p=0.0104). Considering all localizable breakpoints of the CBA-defined structural chromosomal changes (N=922), 532 coincided with a cytoband or a cytoband directly adjacent to one harbouring CMA-defined copy number alterations (N=299 and N=233, respectively), whereas 128 CBA-defined breakpoints located to other cytobands on the same chromosome arm (N=85) or on the other arm (N=43). For 50 of these 128 breakpoints the distance between the CBA-identified cytoband and the CMA-defined breakpoint was &amp;lt;20 Mb. About one quarter of CBA-defined breakpoints (N=237, 23%) affected chromosomes not found to be altered in CMA. Potential explanations for non-detection by CMA were small clone sizes (78/237, 33%; considering breakpoints found in &amp;lt;25% of metaphases), association with an ultra-complex karyotype (140/237, 59%; referring to cases with ≥10 affected chromosomes and/or a composite karyotype), and no or poor coverage of cytobands by SNP-array probes (72/237, 30%; referring to cytobands with stretches &amp;gt;1Mb covered by ≤5 probes). Besides, the vast majority of the 237 breakpoints belonged to alterations that can in principle be balanced: 209 translocations, 8 insertions and 2 inversions. Only 18/237 breakpoints defined deletions, 15 of which met at least one of the criteria mentioned above as potential explanations for non-detection by CMA. Overall, 134 chromosomal alterations were detected by CMA alone. Four of these alterations were copy neutral losses of heterozygosity. Most of the 130 DNA copy number alterations were &amp;lt;20 Mb in size (N=104) and 22/130 alterations have previously been associated with a shorter overall survival (affecting 18/110 cases; 16%). Regarding loss of CDKN2A/ B and gain of MYC as genetic features associated with an increased risk for Richter transformation, CMA identified three cases each lacking abnormalities on the respective chromosome in CBA. CBA identified three cases with a potential MYC rearrangement and one case with a potential CDKN2A/ B loss despite having normal results for 9p21 in CMA. Conclusions: In this cohort of high-risk CLL cases, CBA and CMA were complementary methods for karyotype characterization. CBA was more sensitive in uncovering chromosome involvement than CMA, whereas CMA was superior in determining specific DNA copy number alterations previously associated with an inferior prognosis. As to what extend these findings apply to standard-risk CLL cases needs to be investigated.

Reappearance of CXCR4 dim/CD5 bright Proliferative Fraction Associates with BTK Mutations and Anticipates Progression in Ibrutinib-Treated CLL

Reciprocal expression of CXCR4 and CD5 on the surface of chronic lymphocytic leukemia (CLL) cells can discriminate the proliferative fraction (CD5high/CXCR4dim, PF), recently egressed from the lymph node, from the resting fraction (CXCR4high/CD5dim, RF) of older, quiescent cells. BTK inhibitors affect the proliferative capacity of CLL cells and cause the redistribution from nodal compartments to the blood stream. Nevertheless, little is known about the impact of long-term treatment of the BTK-inhibitor ibrutinib (IBR) on the PF/RF phenotype. Here, we monitored the PF dynamics in two cohorts of IBR-treated CLL cases with long-term follow-up, and correlated with the emergence of IBR resistance. Longitudinal analysis of the PF under IBR in 31 CLL cases, enrolled in the IOSI-EMA-001 with sequential blood samplings (156 samples, median 6 samples/case) showed a depletion of the PF over time, from 17.7% at pre-treatment to 4.75, 0.4, 1.5, 0.6, 1.5% at later timepoints (0.5, 6, 12, 18, 24 months; p &amp;lt; 0.001, U test), along with loss of proliferative potential by Ki67 expression. We then retrospectively analyzed the PF in 100 IBR-treated CLL cases from the real world, referred for routine immunophenotyping (296 samples, median 3 samples/case). Median PF of pre-IBR samples was 12.0% (range 0.7-50), with a significant drop to 2.7% within one year of treatment and to 1.0% in the second year (p&amp;lt;0.001, U test). 77 cases discontinued IBR due to: toxicity (n=13), progression (n=51), other/death (n=13). Mutations of BTK/ PLCG2 were detected in 30 cases, 28 of which discontinued IBR due to progression and 2 for other/death. To correlate reappearance of the PF with cause of discontinuation, we focused on 64 cases collected within 12 months before/after IBR discontinuation due to toxicity (n=10), progression (n=47) or other/death (n=7), and compared with 22 samples still on treatment for the next 12 months after testing (Panel A). ROC analysis of the PF suggested a criterion of PF&amp;gt;3% as the most discriminating to predict IBR discontinuation due to progression, with a sensitivity of 86.7% and a specificity of 71.1%. This cut-off could separate patients with a longer time-to-progression (median 56 vs. 45 months, p=0.031). 26/47 progressing cases bore BTK/ PLCG2 mutations, with a PF higher than wild-type cases discontinuing for toxicity (8.4% vs. 2.2%; p&amp;lt;0.001) or still on treatment (1.8%, p&amp;lt;0.001). Notably, the 21 BTK/ PLCG2- wild-type progressing cases also showed higher PF compared to cases discontinuing for toxicity (6.9% vs. 2.2%; p&amp;lt;0.001) or still on treatment (1.8%; p&amp;lt;0.001). Notably, PF levels showed no significant correlation with white blood cell counts. Sequential dosages of plasmatic B2M in progressing cases (n=5) revealed steady levels below the 3.5 ug/mL threshold, despite the PF increase (median PF 18%). Cell sorting of the PF/RF fractions from 10 cases showing PF reappearance after prolonged IBR treatment (median 50.7 months) revealed a mean of 1.7 BTK mutations per sample (27 total, range 1-4), a median VAF higher in the PF (23.0%, 0.3-92.3) on average 2.8 times larger than in the RF (5.2%, 0.1-84.8; p=0.003, paired rank test). A similar skewing was present for PLCG2 mutations (median PF VAF: 2.0%, 0.1-6.2; RF VAF: 1.35%, 0.4-2.8; p=0.014). Finally, mRNA-seq was performed in 20 cases, on matching PF/RF fractions both at pre-IBR and progression (9 cases with BTK mutations). Differential expression signature of pre-IBR PF vs. RF, concordant with published gene sets, drove co-clustering of post-IBR PF/RF with their respective counterparts, indicating that PF at progression functionally resembles its pre-treatment counterpart. A strong enrichment of multiple proliferation-related signatures, present in pre-treatment samples, was significantly up-regulated also at progression, independently from BTK mutations (Panel B). In summary, here we report that CLL progression under IBR associates with reappearance of the CD5bright/CXCR4dim PF which precedes clinical progression, increase of serum B2M and WBC counts. Transcriptomic/genetic profiling of the post-IBR PF suggests presence of clonal selection dynamics and active bypass signaling pathways, bona fide taking place within the lymph node microenvironment, including BTK/ PLCG2 mutations. Clinically, longitudinal monitoring of the CXCR4/CD5 fractions by flow cytometry may provide a simple tool helping to intercept CLL progression under IBR therapy.

Glycerolipid Metabolism Via the MGA-MYC-NME1-PGP Axis As a Key Regulator of Oxidative Phosphorylation in Richter's Transformation

Despite advances in targeted therapies for chronic lymphocytic leukemia (CLL), Richter's transformation (RT) remains a clinical challenge. RT is an aggressive shift from CLL to lymphoma, associated with metabolic changes and aberrations in the MYC transcriptional network. Genomic analysis of paired CLL and RT cases revealed recurrent mutations or deletions in the MYC suppressor Max-gene-associated (MGA) increase from 5% (CLL) to 30% (RT) cases, highlighting its critical yet unexplored role as a driver event. We recently developed a murine RT model using B cell-restricted in vitro editing of LSK progenitors from Cd19Cre-Cas9-del(13q)-Sf3b1 ( Sf3b1-K700E) with Mga-targeting sgRNAs. This murine model demonstrated upregulation of Mga targets Myc and Nme1 (nucleoside diphosphate kinase), a distinct OXPHOS-based transcription signature, increased oxygen consumption rate, elevated ROS levels, suggesting a significant link between the Mga-Myc-Nme1 axis and OXPHOS dysregulation in RT. Given the crucial role of metabolic reprogramming in cancer, we seek to understand the molecular mechanisms of metabolites driving OXPHOS and accelerating CLL transformation. Unraveling these processes could revolutionize therapeutic approaches for managing RT in clinical settings. To identify metabolites driving dysregulated OXPHOS in RT, we first conducted paired RNA-seq and unlabeled metabolomic profiling on normal and RT mouse splenic B cells (n=3/group). To translate our discovery from the murine RT model to human disease, we performed metabolomic profiling using MEC1 and Nalm6 cell lines with MGA deletion. Our metabolomic analysis of murine RT cells revealed 44 upregulated metabolites, including TCA cycle substrates (glutamine fold change (FC)=9.6, p=0.001;asparagine FC= 4.0, p=0.006), fatty-acid intermediates (glycerol-3P FC=11.7, p=0.003; carnitine FC=4.3, p=0.02; palmitate FC= 4.1, p= 0.08), and glycolysis intermediates (3-phosphoglycerate FC= 4.0, p=0.03; UDP-glucose FC=12.7, p=0.02; glycine FC=4.7, p=0.03). Moreover, integrated metabolite analysis (MetaboAnalyst v5.0) identified 63 common upregulated metabolites in MGA KO (knockout) human cell lines and murine RT, enriching glycerolipid, and fatty acid oxidation, associated pathways, indicating a potential role for MGA deletion in driving dysregulated fatty acid metabolism. Pathway enrichment analysis using integrated RNA-seq and metabolite analyses showed that the glycerolipid pathway was one of the most enriched pathways among mouse RT and B-cell lines (Nalm6 and MEC1) with MGA KO. Additionally, two key fatty-acid enzymes, fatty acid synthetase (FASN) and phosphoglycerate phosphatase (PGP), displayed significant mRNA and protein changes, as validated by qPCR and immunoblotting. Furthermore, elevated lipid accumulation and increased free glycerol were confirmed based on lipi-green flow cytometry and glycerol-bioluminescent assays, suggesting an important role of glycerolipid metabolism in MGA KO-associated OXPHOS. PGP, a glycerol-3-phosphatase, regulates glycerol-3-P levels, a key intermediate in glucose, lipid, and energy metabolism. Overexpression of MYC and NME1 increases PGP levels in B-cell lines (MEC1, Nalm6, and Jeko), confirming PGP as a direct target of the MGA-MYC-NME1 axis. To determine the role of PGP in MGA KO (knock-out) cells, we used CRISPR/Cas9 to KO PGP in MEC1, Nalm6, and JeKo cell lines. KO of PGP decreased cell growth and reduced OXPHOS compared to controls based on CCK8 absorbance and Seahorse Mitostress assays respectively. Moreover, PGP KO downregulated the mTOR (p4E-BP1) pathway, emphasizing the significance of dysregulated glycerol and fatty acid metabolism in CLL to RT-driven OXPHOS. Our study reveals key metabolites driving RT and highlights the crucial role of the MGA-MYC-NME1-PGP axis in regulating OXPHOS in Mga KO RT cells. Our results thus suggest targeting glycerolipid metabolism and related pathways may offer effective strategies to conquer RT

Effect of Demographic and Environmental Factors on Chronic Lymphocytic Leukemia(CLL) in the State of Colorado

Various publications have examined the relationship between environmental risk factors and the development of CLL. Factors such as household income, race, altitude and air pollution have been explored. Results have been inconclusive. In this study, we examine these four factors and their possible relationship to the prevalence of CLL. We accessed the state of Colorado all payer claims database of about 3.6 million people with dates of service from Jan 2017 to June 2020. We calculated age and sex in CLL patients. We used propensity score matching based on age and sex to generate our comparator group on a 1:1 matching scheme. We accessed US EPA 2022 data to get air quality index (AQI) for all zip codes in Colorado and US census data for household income and racial distribution by zip code. We accessed US geological survey data to determine average elevation by zip code. We assess these four factors in relation to CLL and the comparator group. We found 3622 cases of CLL in the total population. The average age of CLL patients was 69 and the sex distribution was 74% male. After propensity matching, we identified an equal number of non CLL patients as the comparator. In analyzing CLL patients versus the comparators, we found no relationship between average elevation, racial makeup, average household income, or air quality by zip code (P=NS). In this large statewide population, there was no effect of race distribution, household income, elevation or air quality on the prevalence of CLL.

Live Lymphoma and CLL Cells inside the Bone Marrow Fibroblasts: Implication in Residual Disease Persistence

Bruton tyrosine kinase inhibitors (BTKi) have generated remarkable clinical responses in patients with chronic lymphocytic leukemia (CLL) and other mature B-cell malignancies. However, deep, and durable response to BTK inhibition is uncommon. A significant number of patients with measurable residual disease relapse with resistance to therapies. Although mutational mechanisms responsible for resistance have been well described, it is not known what mechanism led to persistence of the minimal residual disease (MRD) in the first place. The tumor microenvironment (TME) contributes to drug resistance. Various roles for stroma participation have been described, however, none of these include the physical intracellular sheltering of tumor cells. Cell-in-cell (CIC) describes the microscopic observations of the embodiment of an intact cell by another whole cell ( Fig. 1A). Pathologists have reported this phenomenon sporadically in human fixed tissues for over a century. However, its biological, pathological, and clinical significance remains obscure. In the absence of systematic studies, understanding the significance of live CIC remains challenging. In this investigation, we studied primary human CLL samples using an ex vivo co-culture model system which mimics the CLL TME. In the TME system, CLL cells were cocultured with a primary bone marrow fibroblast line (BMF) in the presence of B-cell growth factors. The tumor cells survive for weeks, become larger, downregulate surface CXCR4. Phenotypically, tumor cells proliferate and divide into daughter cells. Collectively, these features recapitulate the behavior of lymph node-resident CLL cells. Using the TME system, we have demonstrated that modeled drug response correlates well with patient clinical response to BTK inhibitor ibrutinib and BCL2 inhibitor venetoclax. Interestingly, with confocal microscopy and 3D reconstruction, we observed that CLL cells placed in the model system were actively internalized by BMF in all 41 CLL cases studied. CLL cells were live and mobile. Moreover, one CLL cell was captured halfway in ( Fig. 1B). The number of internalized CLL cells was quantified and correlated with patients' clinical and pathological features. No correlations were found between CIC and widely used CLL prognostic indicators, including cytogenetic features and IGHV mutational status. However, we have found that patients who were on treatment for CLL at the time of sample collection had higher levels of CIC than those not treated. We hypothesized that live CIC may be an important mechanism contributing to residual disease persistence and drug resistance. To test the hypothesis, we exposed CLL cells in the TME model system to covalent and noncovalent BTKi including ibrutinib, zanubrutinib, and pirtobrutinib at clinically achievable concentrations. Our results showed that drug exposure drove CLL cells into BMF. This happened specifically to the BTK inhibitors as BCL2 inhibitor venetoclax was not observed to cause such an increase in CIC. These laboratory findings are consistent with the clinical observations that the rate of MRD negativity is much higher with venetoclax treatment than with BTKi. Additionally, CIC was observed in both follicular lymphoma (FL) cell line and primary FL cells suggesting the phenomenon is not limited to CLL. Further, we found that CIC was stimulated by several B-cell growth factors. The chemokine CXCL12-CXCR4 axis played a critical role in this process. Specifically, conditioned medium containing CXCL12 ligand promoted cell internalization, while plerixafor, a CXCR4 antagonist, largely prevented CIC from occurring. Furthermore, plerixafor blocks ibrutinib-driven CIC in cell lines as well as in primary CLL cells. Our study represents the first report implicating the CIC phenomenon in residual disease persistence using a cohort of patient-derived CLL cells assessed with 3D imaging and quantitative approach. These findings suggest that live CIC may be an important mechanism tumor cells use to avoid harmful drug insults, survive, persist, and later proliferate, eventually leading to clinical relapse. Targeting the CXCL12-CXCR4 axis may become a potential therapeutic strategy to minimize residual disease and future relapse. Fig.1. A. Images are reconstructed in 3D using Imaris v10.0. BMF cells are visualized as transparent red surfaces and CLL cells as green surfaces. B. One CLL cell was captured halfway in.

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

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