Chronic Lymphocytic Leukemia Cases Research Articles

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 <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 <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 <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 >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 <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.

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.

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 < 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<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>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<0.001) or still on treatment (1.8%, p<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<0.001) or still on treatment (1.8%; p<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

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

The Use of CD200 in the Differential Diagnosis of B-Cell Lymphoproliferative Disorders.

Background: B-Cell Lymphoproliferative Disorders (B-LPDs) are a group of heterogenous disorders characterised by the accumulation of B-cells in peripheral blood, bone marrow, lymph nodes and spleen. They have a variable disease course and outcome and many share similar features making differential diagnosis challenging. Therefore, accurate diagnosis is fundamental in particular for determining treatment options. Immunophenotyping by flow cytometry plays a crucial role in the diagnosis of B-LPDs. However, overlapping immunophenotyping patterns exist and the use of novel monoclonal antibodies has become increasingly important in immunophenotyping analysis. More recently differential expression of CD200 has been reported in various B-LPDs and that CD200 may improve the differentiation between chronic lymphocytic leukaemia (CLL) and mantle cell lymphoma (MCL). In this study CD200 expression is evaluated in different B-LPDs. Methods: A total of 100 samples were collected and analysed by immunophenotyping flow cytometry over a period of 1year (2017-2018), by a panel of monoclonal antibodies including CD200. The percentage of CD200 and its expression intensity was evaluated and compared between different groups of B-LPDs. Results: All of the 50 cases of CLL expressed CD200 with moderate to bright intensity, 6 MCL cases lacked the expression of CD200. Furthermore, all 5 cases of hairy cell leukaemia (HCL) expressed CD200. Out of all B-LPDs evaluated, CD200 expression in HCL cases was noted to be the brightest. The other 39 cases were not found to be B-LPDs. Conclusion: CD200 has an important role in differentiating CLL from MCL, HCL has a consistent bright expression of CD200. By adding CD200 to the combinations of markers in routine testing panel, Immunophenotyping by flow cytometry can be an effective tool in the diagnosis of B-LPDs especially in cases with atypical immunophenotyping pattern. Our result support that CD200 can be added to routine testing panel as it is useful in differentiating them.

Lifetime exposure to brominated trihalomethanes in drinking water and swimming pool attendance are associated with chronic lymphocytic leukemia: a Multicase-Control Study in Spain (MCC-Spain)

BackgroundChronic lymphocytic leukemia (CLL) etiology is poorly understood, and carcinogenic chemicals in drinking and recreational water are candidates.ObjectiveTo evaluate the association between drinking-water exposure to trihalomethanes (THMs) and nitrate as well as lifetime swimming pool attendance and CLL.MethodsDuring 2010–2013, hospital-based CLL cases and population-based controls were recruited in Spain, providing information on residential histories, type of water consumed and swimming pool attendance. Average THMs and nitrate levels in drinking water were linked to lifetime water consumption. Odds ratios (OR) and 95% confidence intervals (CI) were estimated using mixed models.ResultsFinal samples for residential tap water analyses and swimming pool attendance analyses were 144 cases/1230 controls and 157 cases/1240 controls, respectively. Mean (SD) values for average lifetime residential brominated THMs and chloroform in tap water (μg/L), and ingested nitrate (mg/day) were 48.1 (35.6), 18.5 (6.7) and 13.7 (9.6) respectively in controls; and 72.9 (40.7), 17.9 (5.4), and 14.1 (8.8) in CLL cases. For each 10 μg/L increase of brominated THMs and chloroform lifetime-average levels, the ORs (95% CI) were 1.22 (1.14, 1.31) and 0.54 (0.34, 0.87), respectively. For each 5 mg/day increase of ingested nitrate, the OR of CLL was 0.91 (0.80, 1.04). The OR of lifetime pool users (vs. non-users) was 2.38 (1.61, 3.52). Upon performing annual frequency of attending pools analysis through categorization, the second and third categories showed an ORs of 2.36 (1.49, 3.72) and 2.40 (1.51, 3.83), respectively, and P-trend of 0.001.Impact statementThis study identifies an association of long-term exposure to THMs in drinking water, at concentrations below the regulatory thresholds and WHO guidelines, and swimming pool attendance, with chronic lymphocytic leukemia (CLL). These unprecedented findings are highly relevant since CLL is an incurable cancer with still unknown etiology and because the widespread exposure to chlorination by-products that remain in drinking and recreational water worldwide. Despite the demonstrated carcinogenicity in animals of several chlorination by-products, little is known about their potential risks on human health. This study makes a significant contribution to the search for environmental factors involved in the etiology of CLL and to the evidence of the health impact of these high prevalent water contaminants.

Use of a hybrid intelligence decision tree to identify mature B-cell neoplasms.

Mature B-cell neoplasms are challenging to diagnose due to their heterogeneity and overlapping clinical and biological features. In this study, we present a new workflow strategy that leverages a large amount of flow cytometry data and an artificial intelligence approach to classify these neoplasms. By combining mathematical tools, such as classification algorithms and regression tree (CART) models, with biological expertise, we have developed a decision tree that accurately identifies mature B-cell neoplasms. This includes chronic lymphocytic leukemia (CLL), for which cytometry has been extensively used, as well as other non-CLL subtypes. The decision tree is easy to use and proposes a diagnosis and classification of mature B-cell neoplasms to the users. It can identify the majority of CLL cases using just three markers: CD5, CD43, and CD200. This approach has the potential to improve the accuracy and efficiency of mature B-cell neoplasm diagnosis.

IGH 3'RR recombination uncovers a non-germinal center imprint and c-MYC-dependent IGH rearrangement in unmutated chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is an incurable indolent non-Hodgkin lymphoma characterized by tumor B cells that weakly express a B-cell receptor. The mutational status of the variable region (IGHV) within the immunoglobulin heavy chain (IGH) locus is an important prognosis indicator and raises the question of the CLL cell of origin. Mutated IGHV gene CLL are genetically imprinted by activation-induced cytidine deaminase (AID). AID is also required for IGH rearrangements: class switch recombination and recombination between switch Mu (Sμ) and the 3' regulatory region (3'RR) (Sμ-3'RRrec). The great majority of CLL B cells being unswitched led us to examine IGH rearrangement blockade in CLL. Our results separated CLL into two groups on the basis of Sμ-3'RRrec counts per sample: Sμ-3'RRrecHigh cases (mostly unmutated CLL) and Sμ-3'RRrecLow cases (mostly mutated CLL), but not based on the class switch recombination junction counts. Sμ-3'RRrec appeared to be ongoing in Sμ-3'RRrecHigh CLL cells and comparison of Sμ-3'RRrec junction structural features pointed to different B-cell origins for both groups. In accordance with IGHV mutational status and PIM1 mutation rate, Sμ-3'RRrecHigh CLL harbor a non-germinal center experienced B-cell imprint while Sμ-3'RRrecLow CLL are from AID-experienced B cells from a secondary lymphoid organ. In addition to the proposals already made concerning the CLL cell of origin, our study highlights that analysis of IGH recombinatory activity can identify CLL cases from different origins. Finally, on-going Sμ-3'RRrec in Sμ-3'RRrecHigh cells appeared to presumably be the consequence of high c-MYC expression, as c-MYC overexpression potentiated IGH rearrangements and Sμ-3'RRrec, even in the absence of AID for the latter.

Consumption of aspartame and other artificial sweeteners and risk of cancer in the Spanish multicase-control study (MCC-Spain).

Use of artificial sweeteners (AS) such as aspartame, cyclamate, saccharin and sucralose is widespread. We evaluated the association of use of aspartame and other AS with cancer. In total 1881 colorectal, 1510 breast, 972 prostate and 351 stomach cancer and 109 chronic lymphocytic leukaemia (CLL) cases and 3629 population controls from the Spanish Multicase-Control (MCC-Spain) study were recruited (2008-2013). The consumption of AS, from table-top sweeteners and artificially sweetened beverages, was assessed through a self-administered and validated food frequency questionnaire (FFQ). Sex-specific quartiles among controls were determined to compare moderate consumers (<third quartile) and high consumers (≥ third quartile) vs non consumers (reference category), distinguishing aspartame-containing products and other AS. Unconditional logistic regression models were used to estimate adjusted OR and 95%CI, and results were stratified by diabetes status. Overall, we found no associations between the consumption of aspartame or other AS and cancer. Among participants with diabetes, high consumption of other AS was associated with colorectal cancer (OR = 1.58, 95% CI 1.05-2.41, P trend = .03) and stomach cancer (OR = 2.27 [0.99-5.44], P trend = .06). High consumption of aspartame, was associated with stomach cancer (OR = 2.04 [0.7-5.4], P trend = .05), while a lower risk was observed for breast cancer (OR = 0.28 [0.08-0.83], P trend = .03). In some cancers, the number of cases in participants with diabetes were small and results should be interpreted cautiously. We did not find associations between use of AS and cancer, but found associations between high consumption of aspartame and other AS and different cancer types among participants with diabetes.

Patients with Chronic Lymphocytic Leukemia are more Likely to Have TP53 Gene Mutations

Objective: The purpose was to quantify the prevalence of Tp53 gene mutation in chronic lymphocytic leukemia patients.. Study Design: Cross-sectional/ Descriptive study Place and Duration: This study was conducted in multiple centres including Mayo Hospital Lahore and Liaquat University of Medical and Health Sciences in the duration from August, 2022 to January, 2023. Methods: Total 112 cases of chronic lymphocytic leukaemia were included in this study. All the patients were received at laboratory for TP53 mutation analysis. To identify the TP53 mutation using the FISH method, five ml of whole blood or one ml of bone marrow aspirate sample were taken. Data input and analysis were done with the help of the statistical package for social sciences 22. Results: The mean age of the patients was 52.8±6.52 years and had mean BMI 23.7±14.52 kg/m2. Seventy-three (65.2%) patients were males and 39 (34.8%) cases were females. There were 52 (46.4%) patients educated and 60 (53.6%) cases were non educated. We found frequency of Tp53 gene mutation in 17 (15.2%) cases in which 11 cases were males and 6 cases were females. Among 17 cases of Tp53 gene mutation, 8 cases had age &gt;50 years, 6 cases were aged between 30-50 years and 3 cases were between 18-30 years. Conclusion: We concluded in this study that frequency of Tp53 gene mutation in patients of chronic lymphocytic leukaemia was 15.2% in which majority were males and had age &gt;50 years. Keywords: CLL, Tumour suppressor gene, Chronic lymphocytic leukaemia, FISH, TP53, Fluorescence in Situ hybridization

Shelter in place: Live CLL cells inside the bone marrow fibroblasts and its implication in drug resistance

Introduction: Cell-in-cell (CIC) describes a century old observation where an intact cell can be seen inside another whole cell. It has been associated with human cancers, especially hematopoietic malignancies. However, most observations have been published as case reports. In the absence of systematic studies of case cohorts, understanding of the pathological significance of live CIC remains challenging. Methods: In this investigation, we studied primary CLL samples using an ex vivo co-culture model system which mimics the CLL tumor microenvironment (TME). In these TME models, CLL cells were cocultured with bone marrow fibroblasts (BMF) in the presence of B-cell growth factors. The tumor cells survive for weeks, become larger, downregulate surface CXCR4 and upregulate Ki67 expression. Phenotypically, tumor cells proliferate and divide into daughter cells. Collectively, these features recapitulate the behavior of lymph node-resident CLL cells. In this study, cells were observed and analyzed with confocal microscopy and 3D reconstruction. Results: We observed that CLL cells were actively internalized by BMF in 41 CLL cases studied. The internalized CLL cells were alive and mobile within the BMF’s cytoplasm. Moreover, the tumor cells were seen leaving their BMF host and re-entering into other BMFs. The number of internalized CLL cells were quantified and correlated with patients’ clinical and pathological parameters. No correlations were found between cell internalization and widely used CLL prognostic indicators, including cytogenetic features and IGHV mutational status. However, we have found that treated patients had higher level of CIC than those not treated. We hypothesized that live CIC may contribute to drug resistance and internalization may be an important mechanism for tumor cells to evade harmful drug insult and survive as residual disease. To test the hypothesis, we exposed CLL cells in the TME models to BTK inhibitors including ibrutinib and pirtobrutinib. Our results showed that the drug exposure drove CLL cells into BMF. Additionally, we observed that increased CIC was associated with higher cellular proliferation capacity. Importantly, chemokine CXCL12-CXCR4 axis played a critical role in this process. Specifically, CXCL12 ligand promoted cell internalization, while a CXCR4 antagonist partially prevented CIC from occurring. The research was funded by: NCI Keywords: Chronic Lymphocytic Leukemia (CLL), Microenvironment, Molecular Targeted Therapies No conflicts of interests pertinent to the abstract.

Clinical characteristics of 11 patients with chronic lymphocytic leukemia with t (14;19) (q32;q13)

Objective: To analyze the clinicopathological characteristics of 11 cases of chronic lymphocytic leukemia (CLL) with t (14;19) (q32;q13) . Methods: The case data of 11 patients with CLL with t (14;19) (q32;q13) in the chromosome karyotype analysis results of the Blood Diseases Hospital, Chinese Academy of Medical Sciences from January 1, 2018, to July 30, 2022, were retrospectively analyzed. Results: In all 11 patients, t (14;19) (q32;q13) involved IGH::BCL3 gene rearrangement, and most of them were accompanied by +12 or complex karyotype. An immunophenotypic score of 4-5 was found in 7 patients and 3 in 4 cases. We demonstrated that CLLs with t (14;19) (q32;q13) had a mutational pattern with recurrent mutations in NOTCH1 (3/7), FBXW7 (3/7), and KMT2D (2/7). The very-high-risk, high-risk, intermediate-risk, and low-risk groups consisted of 1, 1, 6, and 3 cases, respectively. Two patients died, 8 survived, and 2 were lost in follow-up. Four patients had disease progression or relapse during treatment. The median time to the first therapy was 1 month. Conclusion: t (14;19) (q32;q13), involving IGH::BCL3 gene rearrangement, is a rare recurrent cytogenetic abnormality in CLL, which is associated with a poor prognosis.

Network analysis reveals a major role for 14q32 cluster miRNAs in determining transcriptional differences between IGHV-mutated and unmutated CLL

Chronic lymphocytic leukaemia (CLL) cells can express unmutated (U-CLL) or mutated (M-CLL) immunoglobulin heavy chain (IGHV) genes with differing clinical behaviours, variable B cell receptor (BCR) signalling capacity and distinct transcriptional profiles. As it remains unclear how these differences reflect the tumour cells’ innate pre/post germinal centre origin or their BCR signalling competence, we applied mRNA/miRNA sequencing to 38 CLL cases categorised into three subsets by IGHV mutational status and BCR signalling capacity. We identified 492 mRNAs and 38 miRNAs differentially expressed between U-CLL and M-CLL, but only 9 mRNAs and 0 miRNAs associated with BCR competence within M-CLL. Of the IGHV-associated miRNAs, (14/38 (37%)) derived from chr14q32 clusters where all miRNAs were co-expressed with the MEG3 lncRNA from a cancer associated imprinted locus. Integrative analysis of miRNA/mRNA data revealed pronounced regulatory potential for the 14q32 miRNAs, potentially accounting for up to 25% of the IGHV-related transcriptome signature. GAB1, a positive regulator of BCR signalling, was potentially regulated by five 14q32 miRNAs and we confirmed that two of these (miR-409-3p and miR-411-3p) significantly repressed activity of the GAB1 3′UTR. Our analysis demonstrates a potential key role of the 14q32 miRNA locus in the regulation of CLL-related gene regulation.

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

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

Computational flow cytometry provides accurate assessment of measurable residual disease in chronic lymphocytic leukaemia.

Undetectable measurable residual disease (MRD) is associated with favourable clinical outcomes in chronic lymphocytic leukaemia (CLL). While assessment is commonly performed using multiparameter flow cytometry (MFC), this approach is associated with limitations including user bias and expertise that may not be widely available. Implementation of unsupervised clustering algorithms in the laboratory can address these limitations and have not been previously reported in a systematic quantitative manner. We developed a computational pipeline to assess CLL MRD using FlowSOM. In the training step, a self-organising map was generated with nodes representing the full breadth of normal immature and mature B cells along with disease immunophenotypes. This map was used to detect MRD in multiple validation cohorts containing a total of 456 samples. This included an evaluation of atypical CLL cases and samples collected from two different laboratories. Computational MRD showed high correlation with expert analysis (Pearson's r > 0.99 for typical CLL). Binary classification of typical CLL samples as either MRD positive or negative demonstrated high concordance (>98%). Interestingly, computational MRD detected disease in a small number of atypical CLL cases in which MRD was not detected by expert analysis. These results demonstrate the feasibility and value of automated MFC analysis in a diagnostic laboratory.

Abstract 5222: Genetic variants associated with progression from monoclonal B-cell lymphocytosis (MBL) to chronic lymphocytic leukemia (CLL)

Abstract Low count (LC) MBL is characterized by a circulating population of clonal B-cells and is a precursor state to CLL. Most individuals with LC MBL do not progress to CLL and never come to clinical attention. An active area of research is to identify factors that distinguish those individuals with MBL who progress from those who do not. Currently, 41 single nucleotide polymorphisms (SNPs) are known to be associated with CLL risk, and a portion of these SNPs have also been found to be associated with MBL risk. We hypothesize that these CLL-susceptibility SNPs associated with both MBL and CLL risk are SNPs associated with developing a B-cell clone; whereas the remaining SNPs found not to be associated with MBL risk are associated with progression of the B-cell clone (i.e., progression from MBL to CLL). To test this hypothesis, we evaluated the association of these 41 SNPs with risk of progression to CLL using a case-control study of 1,911 CLL cases and 1,579 individuals with MBL from Mayo Clinic. Individuals with MBL were identified through screening using 8-color flow cytometry assay. Individuals with LC MBL (defined by having a clonal B-cell population with the percent clonal B-cells out of total B-cell count &amp;lt;85%) who had an immunophenotype consistent with CLL were included as controls. CLL cases were obtained through the Mayo Clinic hematology practice. DNA was genotyped from whole blood. We analyzed the 41 CLL susceptibility SNPs with risk of progression to CLL using logistic regression and estimated odds ratio (OR) and 95% confidence interval (CI), adjusting for sex and age. We evaluated the CLL polygenic risk score (CLL-PRS), comprised of a weighted average of the 41 SNPs, with risk of CLL. Out of the 41 established CLL susceptibility SNPs, 15 were associated with CLL progression from MBL to CLL (all P&amp;lt;0.05). Nine of these 15 significant SNPs were not previously found to be associated with risk of MBL. The most significant SNPs were rs9880772 (OR=1.23, CI: 1.11-1.36, P=6.1 × 10−5), rs888096 (OR=1.25, CI: 1.12-1.38, P=3.0 × 10−5), and rs305065 (OR=1.28, CI: 1.14-1.43, P=1.5 × 10−5). One SNP (rs77551289) had a suggestive association (OR=1.21, P=0.069). The remaining 25 SNPs had no evidence of association with progression (ORs ranging between 0.9-1.1 and all P&amp;gt;0.1). The CLL-PRS was also associated with CLL risk (continuous OR = 1.25, CI: 1.15 - 1.35, P = 6.7 × 10−8). In our study accounting for the presence of B-cell clonal population, we identified 15 out of 41 CLL-susceptibility SNPs to be significantly associated with progression from LC MBL to CLL. The remaining 25 non-significant CLL-SNPs were not associated with progression and may instead be associated with development or initiation of the B-cell clone. These findings suggest that the established CLL susceptibility SNPs may play differing role (i.e., development or progression of the clone) in the etiology of CLL. Citation Format: Raphael Mwangi, Geffen Kleinstern, Sara J. Achenbach, Dennis Robinson, Aaron D. Norman, Kari G. Rabe, Janet E. Olson, Neil E. Kay, Rosalie G. Waller, Nicholas J. Boddicker, James R. Cerhan, Esteban Braggio, Sameer A. Parikh, Curtis A. Hanson, Celine M. Vachon, Tait Shanafelt, Susan L. Slager. Genetic variants associated with progression from monoclonal B-cell lymphocytosis (MBL) to chronic lymphocytic leukemia (CLL). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5222.

Abstract 3482: METTL3-mediated m6A modification controls splicing factor abundance and contributes to CLL progression

Abstract RNA splicing dysregulation is a hallmark of cancers and underlies the onset and progression of diseases. Chronic lymphocytic leukemia (CLL) is one of the most common adult leukemia in western countries. Spliceosome mutations occur in ~20% of patients. However, the mechanism for splicing defects in spliceosome unmutated CLL cases remains elusive. Through an integrative transcriptomic and proteomic analysis of primary CLL samples, we discovered proteins involved in RNA splicing are post-transcriptionally upregulated. Coupled with clinical annotation, we found spliceosome protein abundance is an independent risk factor and associated with poor prognosis. Splice variants found in CLL are highly overlapped with those driven by high spliceosome abundance but not splicing factor mutations, indicating high spliceosome abundance contributes to genetic lesion-independent splicing defects. To identify potential regulators for spliceosome, we proteome-widely analyzed the proteins that highly correlated with splicing factors expression. Analysis of 113 CLL samples has consistently identified METTL3 upregulation with positive correlation with 77.6% of detected splicing factors. METTL3 is an RNA methyltransferase that modifies N6-methyladenosine (m6A) on mRNA and regulates the translation of m6A-installed transcripts. m6A level on mRNA is increased in CLL cells with differential m6A highly enriched on splicing related transcripts. Moreover, high METTL3 expression in CLL is also associated with poor clinical outcomes. These results suggested that METTL3 translationally controls splicing factors through m6A and plays a role in CLL progression. Toward this end, we demonstrated that METTL3 is essential for CLL growth in both in vitro and in vivo studies. Knock out (KO) and pharmaceutical inhibition of METTL3 decreased cell growth and splicing factor expression. Overexpression of wildtype but not catalytic mutant METTL3 restored both defects in METTL3 KO cells, indicating that the regulation of splicing factor is m6A-dependent. To dissect the underlying mechanism, we performed an integrated Ribo-seq, RNA-seq, and MeRIP-seq on CLL cells with or without METTL3. KO of METTL3 decreased overall translation efficiency (TE) with RNA splicing as the most significantly affected pathway. Splicing factors with reduced TE displayed either hypo-m6A at stop codon region or hyper-m6A at CDS regions upon METTL3 KO, as direct or indirect targets of METTL3. Moreover, we found that m6A at stop codon and CDS regions regulates splicing factor translation via ribosome recycling and ribosome pausing, respectively. Taken together, our results uncovered a novel regulatory axis for METTL3 that controls splicing factor translation and contributes to CLL progression. Our study highlights a post-transcriptional layer of m6A modification as a major contributor to genetic lesion-independent splicing defects in CLL. Citation Format: Yiming Wu, Meiling Jin, Mike Fernandez, Kevyn Hart, Aijun Liao, Stacey M. Fernandes, Tinisha McDonald, Zhenhua Chen, Daniel Röth, Lucy Ghoda, Guido Marcucci, Markus Kalkum, Raju K. Pillai, Alexey V. Danilov, Jianjun Chen, Jennifer R. Brown, Steven T. Rosen, Tanya Siddiqi, Lili Wang. METTL3-mediated m6A modification controls splicing factor abundance and contributes to CLL progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3482.

Hypophosphatemia related to the use of ibrutinib.

Tyrosine kinase inhibitors (TKis) and Bruton's TKi (BTKis) constitute broadly used antitumor drug groups with almost completely tolerable and manageable side-effect profiles. Mainly side effects are cardiovascular and gastrointestinal for the TKi group. Hypophosphatemia is documented frequently in many studies with TKis but rarely mentioned with ibrutinib use up to the present. A 61-year-old patient with the diagnosis of chronic lymphocytic leukemia had hypophosphatemia-related complaints and symptoms when ibrutinib use was preferred for his second relapse of the disease. After drug discontinuation, we started ibrutinib again with an alternating dose. We managed to control hypophosphatemia, and the patient has been following up for 2 years in remission status without any support or a second drug need. We have presented here a chronic lymphocytic leukemia case that developed mild-severe hypophosphatemia associated with ibrutinib use. By using an alternating dose of ibrutinib, we managed to control the disease and drug side effects. TKis and BTKis are widely in use for different indications. Hypophosphatemia is rare but it can cause drug discontinuation or change if it is not manageable. It is mentioned that hypophosphatemia can be seen due to a common group effect with the mechanism of causing secondary hyperparathyroidism and renal tubulopathy. In our case, we could explain the side effect of hypophosphatemia with secondary hyperparathyroidism and renal tubulopathy. Prospective, large-group studies are needed to explain the hypophosphatemia and other side effects of ibrutinib and new BTKis in detail.