Marker In Chronic Lymphocytic Leukemia Research Articles

Significance of miRNA-192 as a Prognostic Marker in Chronic Lymphocytic Leukemia

Abstract Background Chronic lymphocytic leukemia (CLL) is the most common human leukemia in western world. This disease can arise in two forms, aggressive and indolent, both characterized by the accumulation of incompetent CD5+ B lymphocytes. MicroRNAs (miRNAs): represent a class of small non-coding RNAs (ncRNAs) of 19–25 nucleotide (nt) length, which post-transcriptionally regulate protein expression, impacting on key cellular functions. miRNAs control numerous cellular/molecular processes such as apoptosis, cell proliferation and differentiation. Deregulated expression of miRNAs contributes to the initiation or development of numerous diseases such as cancer. One of these microRNA is miR-192 which has been suggested to be a positive regulator of p53 (a tumor suppressor gene) and its expression is deregulated in biological samples of cancer patients. Objective To evaluate miRNA-192 as a prognostic biomarker in CLL patients, correlate miRNA-192 expression levels to p53 gene in CLL and find out the association between miRNA- 192 and p53 genes expressions and prognostic criteria of CLL. Methods The present study was conducted on 40 newly diagnosed CLL patients who attended the Hematology outpatient clinic-Ain Shams University hospital during the years from 2018 to 2019 and were included in the study in addition to ten healthy subjects. Quantitation of plasma miR-192 and P53 expression levels were done by real-time quantitative polymerase chain reaction. Results A significant statistical association between poor patients’ outcome and advanced clinical Rai staging together with unfavorable cytogenetic profile was found. Moreover, significant statistical association was also observed in patients with poor outcome expressing low levels of miRNA-192 with p value (P = 0.00) and increased levels of P53. miRNA-192 expression levels in our work were significantly lower in patients with lymphadenopathy and in patients with unfavorable cytogenetic profile. However, it was significantly higher among patients with good prognosis in comparison to patients with worse one. Conclusion miRNA-192 expression levels was significantly correlated with some standard prognostic factors as TLC, Hb &insignificantly with platelets, P53 expression level and favorable cytogenetic profile indicated that circulating miR-192 can serve as non-invasive biomarker that can be used for predicting prognosis and monitoring of therapy in CLL patients.

Current ideas on the pathogenesis of osteoporosis in chronic lymphatic leukemia (literature review)

Background. Chronic lymphocytic leukemia (CLL) is the second most common hematological malignancy without a trend towards a decrease in its incidence. 66 % of patients with CLL experience bone fractures as a result of osteoporosis in all age groups, and the detection frequency is no more than 15 %. Insufficient understanding of the osteoporosis pathogenesis in CLL leads to problems in diagnosis, prevention and therapy.The aim of the study. To analyze modern data on the features of the osteoporosis pathogenesis in chronic lymphocytic leukemia.Results and discussion. Osteoporosis is formed when osteoresorption prevails over osteosynthesis due to intercellular interactions of bone tissue and the immune system, dysregulation of intracellular signaling pathways RANKL/RANK/OPG, Wnt, FoxO, RUNX2, initiated by cytokines, growth factors, prostaglandins, and hormones. The degree of osteoresorption in CLL is associated with the severity of the clinical course, chemotherapy and hormonal deprivation. The osteoporosis pathogenesis in CLL is considered as part of a complex set of events, including, firstly, the interaction between leukemic cells (overexpression of PTHrP, RANKL) and bone cells (synthesis of growth factors), which forms a vicious circle of osteoresorption and tumor growth. Secondly, pro-inflammatory markers in CLL (tumor necrosis factor α, interleukin (IL) 1β, IL-6, IL-8, IL-11, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, transforming growth factor β, prostaglandin E2) limit osteoblast-induced osteosynthesis and stimulate the expansion of osteoclasts from monocytic suppressor cells of myeloid origin with or without the participation of the RANKL/RANK system. Thirdly, oxidative stress in CLL and impaired efficiency of antioxidant protection with the participation of fibroblast growth factor 23, transcription factor Nrf-2 with activation of JNK, ERK1/2, NF-κB, and also an increase in the RANKL/OPG ratio lead to inhibition of osteoblastogenesis.Conclusion. Analyzing and systematizing data on the osteoporosis pathogenesis in CLL are instrumental for the development of diagnostic criteria for osteoporosis in chronic lymphocytic leukemia that are much-needed in clinical practice and for the improvement of therapeutic tactics.

Targeting sphingolipid metabolism in chronic lymphocytic leukemia

Elevated levels of circulating C16:0 glucosylceramides (GluCer) and increased mRNA expression of UDP-glucose ceramide glycosyltransferase (UGCG), the enzyme responsible for converting ceramides (Cer) to GluCer, represent unfavorable prognostic markers in chronic lymphocytic leukemia (CLL) patients. To evaluate the therapeutic potential of inhibiting GluCer synthesis, we genetically repressed the UGCG pathway using in vitro models of leukemic B cells, in addition to UGCG pharmacological inhibition with approved drugs such as eliglustat and ibiglustat, both individually and in combination with ibrutinib, assessed in cell models and primary CLL patient cells. Cell viability, apoptosis, and proliferation were evaluated in vitro, and survival and apoptosis were examined ex vivo. UGCG inhibition efficacy was confirmed by quantifying intracellular sphingolipid levels through targeted lipidomics using mass spectrometry. Other inhibitors of sphingolipid biosynthesis pathways were similarly assessed. Blocking UGCG significantly decreased cell viability and proliferation, highlighting the oncogenic role of UGCG in CLL. The efficient inhibition of UGCG was confirmed by a significant reduction in GluCer intracellular levels. The combination of UGCG inhibitors with ibrutinib demonstrated synergistic effect. Inhibitors that target alternative pathways within sphingolipid metabolism, like sphingosine kinases inhibitor SKI-II, also demonstrated promising therapeutic effects both alone and when used in combination with ibrutinib, reinforcing the oncogenic impact of sphingolipids in CLL cells. Targeting sphingolipid metabolism, especially the UGCG pathway, represents a promising therapeutic strategy and as a combination therapy for potential treatment of CLL patients, warranting further investigation.

Laboratories Can Reliably Detect Clinically Relevant Variants in the TP53 Gene below 10 % Allelic Frequency: A Multicenter Study of ERIC, the European Research Initiative on CLL

The presence of mutations in the TP53 gene is a powerful prognostic and predictive marker in chronic lymphocytic leukemia (CLL). Widespread use of NGS has enabled the detection of variants ≤10 % variant allelic frequency (low-VAF variants); however, the overall reliability and reproducibility of NGS techniques to identify such variants have been questioned repeatedly. Individual studies using sensitive, custom NGS-based assays have mostly demonstrated the shortened overall survival (OS) and event-free survival in patients with low-VAF TP53 variants treated with chemoimmunotherapy (CIT) regimens with median survival ranging between that of TP53 variants >10 % VAF (high-VAF) and wild-type TP53 (wt- TP53). Within an ERIC multicenter study, we tested the ability of NGS methods used in diagnostic and research laboratories to detect low-VAF TP53 variants and analyzed the impact of the identified low-VAF variants on patients' survival. In the first phase of the study (Fig. 1), seven sample mixes containing 23 pathogenic TP53 variants (range, 0.7-6.3% VAF) were analyzed in 41 ERIC centers using 44 NGS-based assays. All variants were validated with droplet digital PCR (ddPCR); obtained values were used as a reference for the assessment of each NGS method's performance. NGS results were categorized as true positive (TP), false positive (FP; not present in original samples and reported by one center each), and not reported/false negative (FN). In total, laboratories reported 77.8% of all variants (784 out of 1008), reaching a sensitivity [TP/ (TP + FN)] of 85.6%, 94.5%, and 94.8% at 1%, 2%, and 3% VAF cut-off, respectively. While the VAFs of individual variants reported by laboratories varied, median values strongly correlated with ddPCR (R 2=0.9841). Thirty-eight FP variants were reported by 10 laboratories, mainly <2% VAF (23 FP of VAF ≤1%, 14 FP of VAF >1 and ≤2%, 1 FP > 2%). Individual feedback was provided to improve the methods' performance and to help set an appropriate detection limit. In the second phase of the study, 12 centers provided results of TP53 NGS-based analysis of 1092 CLL clinical samples taken before first-line treatment (median time from sample to treatment 40 days). The impact of low-VAF variants (1-10% VAF; N=59) on time to second treatment (TTST; event: second treatment, death) and OS calculated from 1 st treatment initiation was compared to that of high-VAF variants (N=123) and wt- TP53 using logrank test with Benjamini-Hochberg correction of p-values. TTST (Fig. 2) of the low-VAF group was significantly shorter compared to wt- TP53 (P=0.013; median TTST wt- TP53 3.6 y, low-VAF 2.8 y, high-VAF 1 y) in patients not treated with targeted agents (N=999). If del(17p) status was considered, median TTST was the shortest in patients with a combination of del(17p) and either high (0.8 y) or low-VAF (1 y) TP53 mutations, followed by high-VAF (1.5 y) and low-VAF (2.8 y) mutations in the absence of del(17p) (P<0.001, P=0.032, P<0.001, P=0.026, respectively, compared to wt- TP53/no del(17p) (3.6 y)). In patients receiving frontline targeted agents (N=73; enriched for TP53 mutations), the results suggested shorter TTST for the high-VAF group only, but the difference was not significant (Fig. 2; P=0.06; median wt- TP53 n.r., low-VAF 4.8 y and high-VAF 3.6 y). OS of patients with low-VAF variants was significantly shorter compared to the wt- TP53 group in patients never treated with targeted treatment (P=0.033; median OS wt- TP53 6.6 y, low-VAF 3.2 y and high-VAF 2.1 y). Targeted therapy in 2 nd or later therapy lines diminished the difference and only OS of the high-VAF group differed significantly from wt- TP53 (P<0.001; median OS wt- TP53 10.6 y, low-VAF 8.6 y, and high-VAF 5.1 y). Altogether, we show that the cumulative reliability (no FN and FP) of methods tested increased continuously with VAF (Fig. 1), reaching 30% and 64% for variants ≥1.1% and 2% VAF, respectively. The reliability was affected by the type of NGS method and bioinformatic pipeline settings. We conclude that no strict threshold can be suggested from a technical standpoint. However, our results emphasize a strong need to validate/verify the NGS method, describe its limits, and report only reliable results. From a clinical standpoint, while low-VAF variants impact clinical outcomes for patients receiving CIT in the frontline setting, their clinical impact for patients treated with novel therapies remains to be evaluated in larger cohorts.

LncRNA CRNDE is downregulated and associated with poor prognostic markers in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is characterized by a very heterogeneous clinical outcome. Thus, a plethora of prognostic factors and systems has been identified to place patients into different risk categories and to guide therapy decisions. The classic clinical staging models by Rai and Binet have been the cornerstone of patient management for several years. The greater insight into the molecular biology of CLL facilitated the advent of prognostic genetic biomarkers that are expected to impact clinical practice soon in the future. Therefore, we aimed to investigate the expression of long non-coding RNA (lncRNA) CRNDE in patients with CLL, and to analyze its relationship with the clinicopathological parameters of CLL. In this study, 40 untreated CLL patients and 30 age- and gender-matched controls were enrolled. The analysis of lncRNA CRNDE expression was determined using reverse transcription-quantitative polymerase chain reaction technique. Our result confirmed the downregulated expression of LncRNA CRNDE in CLL patients compared to controls (p < 0.001). The low expression of CRNDE was significantly associated with poor prognostic markers including advanced stage of CLL, high levels of serum beta-2 microglobulin and lactic dehydrogenase, and the presence of del17p (p = 0.029, p = 0.013, p = 0.003, p = 0.028; respectively). Our study demonstrated that LncRNA CRNDE is significantly downregulated and associated with poor prognostic markers in CLL. It provides a rationale to assess its biological and prognostic impact in CLL.

Clinical Outcomes in a Large Canadian Centralized CLL Clinic Based on Treatment and Molecular Factors over a Decade.

FISH cytogenetics, TP53 sequencing, and IGHV mutational status are increasingly used as prognostic and predictive markers in chronic lymphocytic leukemia (CLL), particularly as components of the CLL International Prognostic Index (CLL-IPI) and in directing therapy with novel agents. However, testing outside of clinical trials is not routinely available in Canada. As a centralized CLL clinic at CancerCare Manitoba, we are the first Canadian province to evaluate clinical outcomes and survivorship over a long period of time, incorporating the impact of molecular testing and the CLL-IPI score. We performed a retrospective analysis on 1315 patients diagnosed between 1960 and 2018, followed over a 12-year period, where 411 patients had molecular testing and 233 patients had a known CLL-IPI score at the time of treatment. Overall, 40.3% (n = 530) of patients received treatment, and 47.5% (n = 252) of patients received multiple lines of therapy. High-risk FISH and CLL-IPI (4-10) were associated with higher mortality (HR 2.03, p = 0.001; HR 2.64, p = 0.002), consistent with other studies. Over time, there was an increase in the use of targeted agents in treated patients. The use of Bruton's tyrosine kinase inhibitors improved survival in patients with unmutated IGHV and/or TP53 aberrations (HR 2.20, p = 0.001). The major cause of death in patients who received treatment was treatment/disease-related (32%, n = 42) and secondary malignancies (57%, n = 53) in those who were treatment-naïve. Our data demonstrate the importance of molecular testing in determining survivorship in CLL and underpinning the likely immune differences in outcomes for those treated for CLL.

Differential prognosis of single and multiple TP53 abnormalities inhigh-count MBL and untreated CLL.

TP53 aberrations, including mutations and deletion of 17p13, are important adverse prognostic markers in chronic lymphocytic leukemia (CLL) but are less studied in high count monoclonal B-cell lymphocytosis (HCMBL), an asymptomatic pre-malignant stage of CLL. Here we estimated the prevalence and impact of TP53 aberrations in 1,230 newly diagnosed treatment-naïve individuals (849 CLL, 381 HCMBL). We defined TP53 state as: wild-type (no TP53 mutations and normal 17p), single-hit (del(17p) or one TP53 mutation), or multi-hit (TP53 mutation and del(17p), TP53 mutation and loss of heterozygosity, or multiple TP53 mutations). Cox regression was used to estimate hazard ratios (HR) and 95% confidence intervals (CI) for time to first treatment and overall survival by TP53 state. We found 64 (7.5%) CLL patients and 17 (4.5%) HCMBL individuals had TP53 mutations with variant allele fraction >10%. Del(17p) was present in 58 (6.8%) of CLL and 11 (2.9%) of HCMBL cases. Most individuals had wild-type (N=1,128, 91.7%) TP53 state, followed by multi-hit (N=55, 4.5%) and then single-hit (N=47, 3.8%) TP53 state. The risk of shorter time to therapy and death increased with the number of TP53 abnormalities. Compared to wild-type patients, multi-hit patients had 3-fold and single-hit patients had 1.5-fold increased risk of requiring therapy. Multi-hit patients also had 2.9-fold increased risk of death compared to wild-type. These results remained stable after accounting for other known poor prognostic factors. Both TP53 mutations and del(17p) may provide important prognostic information for HCMBL and CLL that would be missed if only one were measured.

Cell Surface Lipoprotein Lipase Enzyme as A Prognostic Indicator in B-Cell Chronic Lymphocytic Leukemia

Lipoprotein lipase (LPL) has emerged as a distinct prognostic marker for chronic lymphocytic leukemia (CLL), a heterogeneous disease with a similar heterogeneity in life expectancy. While many prior studies have focused on tumor expression of LPL mRNA, LPL surface protein expression has been less robustly studied as a prognostic marker. A novel antibody developed at Geisel School of Medicine at Dartmouth has been previously utilized in immunohistochemistry assays of breast and prostate cancer. With this study we aimed to use this antibody as a flow cytometry marker of surface LPL expression correlated with overall survival and time to first treatment. Of the 19 patients studied, our data show that LPL surface protein expression as measured by flow cytometry trended toward a protective effect on overall survival and overall better prognosis.

EVALUATION OF CD38 ANTIGEN EXPRESSION IN CHRONIC LYMPHOCYTIC LEUKEMIA: THE EXPERIENCE OF CLINICAL IMMUNOLOGY DEPARTMENT, NATIONAL RESEARCH CENTER FOR RADIATION MEDICINE, NATIONAL ACADEMY OF MEDICAL SCIENCES OF UKRAINE

Summary. CD38 antigen is a negative prognostic marker of chronic lymphocytic leukemia (CLL). The question of what cut-off level of CD38 antigen expression should be considered as prognostically significant, remains debatable. Aim: to evaluate the optimal cut-off level for CD38 antigen in primary untreated CLL patients for the prognosis of disease and its association with the mutational status of immunoglobulin heavy chain genes (IGHV genes). Objects and methods: flow cytometric and molecular-genetic analysis (mutational status of IGHV genes using polymerase chain reaction followed by direct sequencing) were performed in the groups of 106 primary previously untreated CLL patients. Time-to-treatment, progression-free survival and overall survival were evaluated using the Kaplan-Meier method and Cox regression. Results: The prognostic value of cut-off level CD38 antigen expression ≥ 20% or ≥ 30% does not differ significantly. Lowering of cut-off level to 7% or 5% was unsignificant. The specificity of the evaluation of the mutational status of IGHV genes based on CD38 antigen expression was low, since CD38 expression in almost half of CLL cases with unmutated IGHV genes not differ from CLL cases with mutated IGHV genes. Conclusion: the obtained data confirmed the prognostic value of CD38 antigen expression in patients at the initial stages of disease (A0-AI), primarily for assessing the duration of overall survival.

A novel next-generation sequencing capture-based strategy to report somatic hypermutation status using genomic regions downstream to immunoglobulin rearrangements.

The somatic hypermutation (SHM) status of the clonotypic, rearranged immunoglobulin heavy variable (IGHV) gene is an established prognostic and predictive marker in chronic lymphocytic leukemia (CLL). Analysis of SHM is generally performed by polymerase chain reaction (PCR)-amplification of clonal IGHV-IGHD-IGHJ gene rearrangements followed by sequencing to identify IGHV gene sequences and germline identity. Targeted-hybridization next-generation sequencing (NGS) can simultaneously assess clonality and other genetic aberrations. However, it has limitations for SHM analysis due to sequence similarity between different IGHV genes and mutations introduced by SHM, which can affect alignment efficiency and accuracy. We developed a novel SHM assessment strategy using a targeted-hybridization NGS approach (EuroClonality- NDC assay) and applied it to 331 samples of lymphoproliferative disorder (LPD). Our strategy focuses on analyzing the sequence downstream to the clonotypic, rearranged IGHJ gene up to the IGHM enhancer (IGHJ-E) which provides more accurate alignment. Overall, 84/95 (88.4%) CLL cases with conventional SHM data showed concordant SHM status, increasing to 91.6% when excluding borderline cases. Additionally, IGHJ-E mutation analysis in a wide range of pre- and post-germinal center LPD showed significant correlation with differentiation and lineage status, suggesting that IGHJ-E analysis is a promising surrogate marker enabling SHM to be reported using NGS-capture strategies and whole genome sequencing.

Patterns of use of biological and genetic markers for chronic lymphocytic leukemia and acute myeloid leukemia in Puerto Rico.

The use of markers has stimulated the development of more appropriate targeted therapies for chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). We assessed the use and prevalence of biological and genetic markers of CLL and AML in the homogeneous Hispanic population of Puerto Rico. We used the Puerto Rico CLL/AML Population-Based Registry, which combines information from linked databases. Logistic regression models were used to examine factors associated with biological and genetic testing. A total of 926 patients 18 years or older diagnosed with CLL (n=518) and AML (n=408) during 2011-2015 were included in this analysis. Cytogenetic testing (FISH) was reported for 441 (85.1%) of the CLL patients; of those, 24.0% had the presence of trisomy 12, 9.5% carried deletion 11q, 50.3% carried deletion 13q, and 6.3% carried deletion 17p. Regarding AML, patients with cytogenetics and molecular tests were considered to determine the risk category (254 patients), of which 39.8% showed poor or adverse risk. Older age and having more comorbidities among patients with CLL were associated with a lower likelihood of receiving a FISH test. Although prognostic genetic testing is required for treatment decisions, the amount of testing in this Hispanic cohort is far from ideal. Furthermore, some tests were not homogeneously distributed in the population, which requires further exploration and monitoring. This study contributes to the field by informing the medical community about the use and prevalence of biological and genetic markers of CLL and AML. Similarly, it has the potential to improve the management of CLL and AML through benchmarking.

Dysregulation of PRMT5 in Chronic Lymphocytic Leukemia Supports Transformation to Diffuse Large B-Cell Lymphoma

Background: Richter's transformation (RT) is the progression of chronic lymphocytic leukemia (CLL) to a high-grade lymphoma, most commonly resembling a diffuse large B-cell-like lymphoma, that has a bleak prognosis of 6-12 months. Many of the recurrent somatic alterations identified in RT are also associated with the CLL-phase of disease, suggesting additional unidentified genetic, epigenetic, or molecular events underlie transformation. Aberrant PRMT5 expression is known to promote and maintain oncogenic signaling in non-Hodgkin's lymphomas but its role in CLL and RT remains unclear. Methods: Tissue microarrays and western blots were used to evaluate PRMT5 expression in CLL/SLL and RT lymph nodes (LN). Differentially expressed genes between patient-matched RT LNs and CLL PBMCs were identified with scRNA-seq & scV(D)J-seq. ScRNA-seq was utilized to identify transcriptional changes between Eμ-PRMT5/TCL1 & Eμ-TCL1 mice. Tumor allografts were generated by administering 5 x 10^6 CD19+CD5+ spleen cells from Eμ-PRMT5/TCL1 mice intravenously to C57/BL6J mice. One week post-engraftment mice were enrolled in treatment groups (PRT382 10mg/kg, n=10; vehicle, n=7) with all treatments administered for 4 contiguous days per week by oral gavage. Results: Tissue microarray analysis on 70 CLL and 15 RT LN cases showed robust PRMT5 staining in RT LNs and minimal staining in CLL tissues. Immunoblot analysis revealed that CLL cells from patients that eventually undergo transformation displayed significant increase in PRMT5 expression months prior to transformation, whereas CLL patients maintained variable to minimal PRMT5 expression. ScRNA-seq & scV(D)J-seq on RT LN biopsies and CLL PBMCs from two patients revealed that PRMT5 expression was restricted to distinct nodal B cell subpopulations co-expressing MYC, BIRC5, CD83, and CD69. One PRMT5+ cluster displayed overlapping expression of proliferative markers (Mki67, TOP2A, PCNA, CALM2/3, and HMGB1/2) with enriched EIF4 signaling. Anti-apoptotic and immune modulating genes (BCL21A, IL4I1, TCL1A, and CCL3/4) were enriched in the second PRMT5+ nodal cluster. Interestingly, transcriptional diversity was enhanced in clonally related RT B cell populations with an increased proportion of cells expressing PRMT5 when compared to the CLL-phase of disease. To further evaluate the role of PRMT5 in CLL disease progression, we conducted histopathologic analysis on the spleen and lymph nodes of 6 month old animals from Eµ-PRMT5/TCL1 & Eµ-TCL1 mouse models, revealing a RT-like histology with loss of germinal centers and effacement of normal architecture exclusively in Eµ-PRMT5/TCL1 animals. Transcriptional changes underlying phenotypic differences between Eµ-PRMT5/TCL1 & Eµ-TCL1 mice were also assessed by scRNA-seq, where splenic B cells of the Eµ-PRMT5/TCL1 exhibited increased expression of oncogenic and immune regulating genes (Myc, Mki67, Egr1, Cxcr5, Ccr7, Il-10, Ctla4, and Pd-L1). Interestingly, the dominant B-cell clusters enriched in Eµ-PRMT5/TCL1 spleen and LNs showed significant transcriptional overlap with human RT PRMT5 expressing LN clusters (e.g., Myc, Cd83, and Cd69). With the observation that PRMT5 may play a significant role in CLL-to-RT evolution, we then aimed to evaluate the use of targeted PRMT5 inhibition against murine RT-like tumors in vivo. Using the selective SAM-competitive PRMT5 inhibitor, PRT382, in Eµ-PRMT5/TCL1 allografts, we observed PRT382 treatment delayed leukemic expansion, increased median survival (p<0.0001), and reduced spleen mass compared to vehicle treated mice. Conclusion: We show that PRMT5 is upregulated in the lymph nodes of CLL patients prior to and after transformation, suggesting a role for PRMT5 in CLL transformation to RT. Likewise, the Eμ-PRMT5/TCL1 mouse model develops a RT-like histology and shows significant overlap in the transcriptional profile to human RT tumors. And further, inhibition of PRMT5 with PRT382 provides significant improvement in median overall survival and slows disease progression in Eμ-PRMT5/TCL1 adoptive transfer models. Our preclinical data identifies PRMT5 as a high-risk marker in CLL and provides rationale for the clinical evaluation of PRMT5 inhibition in the treatment of high-risk CLL/RT.

Functional Anti-Apoptotic Protein Dependence and Its Relation to Genomic Prognostic Markers and Response to Treatment in Chronic Lymphocytic Leukemia

Introduction Due to its heterogenous biology, chronic lymphocytic leukemia (CLL) is a well-suited disease for identifying new relationships between genomic and functional biomarkers for drug response. Here, we combined a multi-omics approach and ex vivo drug-treatment data with functional characterization of apoptotic sensitivity to correlate genetic features and drug sensitivity with specific anti-apoptotic signaling patterns measured by BH3 profiling in primary CLL samples. Methods BH3 profiling is a functional assay to measure the proximity of cells to undergo apoptosis via overall priming as well as to identify specific anti-apoptotic proteins the cells are dependent on for survival. BH3 peptides and mimetic such as BIM and PUMA were used to identify overall priming or proximity to apoptosis, BAD and ABT199 were used to identify BCL-2 dependence, and FS1, MS1 and HRK were used to identify BFL-1, MCL-1 and BCL-xL dependences respectively (Ryan et al, Biol Chem, 2016). Results We analyzed primary CLL samples from 73 patients and using BH3 profiling identified the specific associations between anti-apoptotic protein dependence and genetic features (such as IGHV status, trisomy-12, del(17p) and TP53 mutation. Our univariate models demonstrated that BCL-2 dependence, as measured by ABT199 drug (p=0.001) and BAD (p=0.004) BH3 peptide, are highly correlated with mutated IGHV status, a favorable prognostic marker in CLL (Figure 1A). Using ex vivo treatment with multiple classes of small molecule inhibitors, we further identified several specific drug response or sensitivity patterns correlating with anti-apoptotic protein dependence as well as CLL prognostic factors (Figure 1B). In particular, our multivariate models (LASSO regularized) demonstrated that viability following ex vivo treatment with the MDM-2 inhibitor Nutlin-3 was positively correlated with TP53 mutation/deletion, but inversely correlated with BFL-1 (FS1 peptide) and BCL-xL (HRK peptide) dependence as well as trisomy-12 (Figure 1B), though the inverse correlation between Nutlin-3 and BFL-1 or BCL-xL dependence is relatively weak. CLL cell viability following ex vivo treatment with the dual BCL-2/BCL-xL inhibitor navitoclax was negatively correlated with BCL-2 (BAD peptide) and BCL-xL (HRK peptide) dependence, though primarily BCL-2 dependence, suggesting that sensitivity to navitoclax is highly dependent on BCL-2 (Figure 1B). At the transcriptomic level, CLL cell dependence on BCL-2 positively correlated with NOXA expression, NFκB signaling, and reduced intracellular oxidative level, various pathways that could potentially maintain BCL-2 dependence. Finally, we utilized BH3 profiling to retrospectively predict in vivo drug response, with high BCL-2 dependence (p=0.016) correlating with greater absolute lymphocyte count decrease. Conclusion Functional characterization of apoptotic signaling in CLL demonstrated that patients with mutated IGHV are more dependent on BCL-2 for survival, and that BH3 profiling could retrospectively predict in vivo sensitivity to BCL-2 inhibition. This functional precision medicine approach may help to stratify patients with CLL beyond genetic markers and should be further explored for correlation with clinical outcomes in prospective clinical trials. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CD20 Expression as a Possible Novel Prognostic Marker in CLL: Application of EuroFlow Standardization Technique and Normalization Procedures in Flow Cytometric Expression Analysis.

Simple SummaryUp to 50% of patients with chronic lymphocytic leukemia relapse within four years after anti-CD20-directed treatment with a need for a subsequent treatment, which can potentially include anti-CD20 agents again. To retrospectively study the influence of CD20 expression in CLL patients receiving anti-CD20 directed therapy on therapy response, we designed and evaluated a bead-based normalization approach for the analysis of CD20 expression levels to reduce variation of flow cytometric measurements due to technical issues. Normalization significantly reduced the variability of median fluorescence intensities of fluorochrome-conjugated beads without artificially rendering the instruments’ performances, and longitudinal biological variations of marker expression based on MFI values could be robustly assessed. In a cross-trial comparison, strong MRD response correlated with the CD20 expression level before therapy started in patients receiving Ofatumumab, but not in patients receiving Obinutuzumab.Background: CD20 expression is a controversial issue regarding response prediction to anti-CD20 therapy in chronic lymphocytic leukemia (CLL). Methods: Median fluorescence intensities (MFIs) of standard fluorescence beads from the daily calibration of flow cytometers according to EuroFlow protocols were used to establish a normalization approach to study CD20 expression on CLL cells. CD20 MFI was retrospectively assessed prior to and during treatment from flow cytometric measurements of peripheral blood in patients with different depths of molecular response in the four phase-II CLL2-BXX trials (BIG; BAG; BIO; BCG; N = 194) administering either Obinutuzumab or Ofatumumab in combination with targeted agents. Results: No significant difference was observed between the normalized and measured MFIs of CD19 and CD20 on CLL cells. During treatment, CD20 expression levels on CLL cells did not significantly differ between the four investigated different treatment schemes, but a strong molecular response to Ofatumumab seemed to correlate with higher CD20 expression prior to therapy. Conclusions: Standardized staining and instrument monitoring enable a robust assessment of longitudinal biological variations of marker expression based on MFI values. Obinutuzumab showed a higher proportion of patients with a strong MRD response independent from initial CD20 expression, whereas high pre-therapeutic CD20 expression levels seem to correlate with a profound response to Ofatumumab.

CLL-345 Retrospective Analysis of Real-World Outcomes of Chronic/Small Lymphocytic Leukemia (CLL/SLL) Patients Treated With First-Line Ibrutinib Versus Chemoimmunotherapy (CIT)

Ibrutinib is an oral, once-daily Bruton's tyrosine kinase inhibitor and is the only targeted treatment that has demonstrated progression-free survival and overall survival in multiple phase 3 trials across age, fitness, and high-risk markers for chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). To describe the trends in genomic testing and compare real-world outcomes of CLL/SLL patients treated with first-line (1L) ibrutinib or chemoimmunotherapy (CIT). A nationwide, Electronic Health Record-derived de-identified database (03/04/2016-08/31/2021) was used to collect treatment and outcome data. Patient baseline characteristics before treatment were compared between the two groups. Propensity score-based inverse probability of treatment weighting was used to adjust for baseline differences between groups. Time to next treatment (TTNT) from 1L initiation was analyzed using the Kaplan-Meier (KM) method. Subgroup analyses were performed on patients with high-risk cytogenetic markers (deletion 17p, 11q, or unmutated IGHV) and those without IGHV testing. Testing for high-risk markers in CLL patients was suboptimal. Although cytogenetic testing for del17p or del11q increased from 52.3% to 74.3%, IGHV testing remained infrequent and low at 37.7%. A total of 1882 patients (61.4%) received 1L ibrutinib monotherapy (mean age 71 years, median follow-up 26 months), and 1182 (38.6%) received a CIT regimen (mean age 68 years, median follow-up 33 months). More patients treated with ibrutinib had a known high-risk cytogenetic marker than those treated with CIT (44% vs. 34%), especially those with del17p (15% vs. 4%). The median TTNT was not reached in patients treated with ibrutinib and was 39.6 months in those treated with CIT. The hazard ratios of starting second-line treatment were 0.49 (95% CI: 0.44-0.54, p<0.001) overall, 0.40 (0.34-0.47, p<0.001) among high-risk patients, and 0.51 (0.44-0.58, p<0.001) among patients without IGHV testing. Consistent with clinical trials, 1L treatment with ibrutinib for CLL/SLL significantly lowers the risk of initiating subsequent treatment compared with 1L CIT, including in patients with high-risk cytogenetic markers and patients without IGHV testing. Assessment of cytogenetic/molecular risks before initiating treatment is vital to optimizing clinical outcomes.

S2007 Chronic Lymphocytic Leukemia Presenting as Colonic Ulcers

Introduction: Chronic Lymphocytic Leukemia (CLL) accounts for 25% of leukemias. One can present with hepatomegaly, splenomegaly, and lymphadenopathy. CLL infiltration in the gastrointestinal (GI) mucosa is rare. Colonic CLL can develop late in the disease course with mild to no GI symptoms. Awareness of CLL and its various extra-nodal presentations allow for prompt diagnosis and treatment. Case Description/Methods: An 80-year-old female with untreated CLL presented with 2 months of rectal bleeding. She denied weight loss, anorexia, night sweats, blood thinners, and NSAID use. She had WBC 34.3, platelets 139, and a normal Hgb of 13.2. Colonoscopy showed ulcerations at the ileocecal valve and transverse colon, a 30 mm ascending colon polyp, and multiple 10-20 mm transverse and descending colon polyps. Pathology showed only chronic inflammation of the ulcerated regions, and a sessile serrated adenoma at the ascending colon. The remaining polyps were not removed due to concern for cancer and potential colectomy. A CT chest, abdomen, and pelvis was unremarkable. Tumor markers showed CEA 8 and normal Ca19-9 and Ca-125. Due to an unclear diagnosis, a second colonoscopy was performed with repeat biopsies and removal of all polyps in the descending and sigmoid colon. Pathology results were negative for inflammatory bowel disease and infection. The polyps were tubular adenomas and hyperplastic. However, immunohistochemical analysis was positive for CD5/CD43 and CD23, indicative of CLL in the ulcerated mucosa. Treatment options were discussed with surgery and oncology. The patient was offered right colon resection or endoscopic removal of the remaining polyps prior to chemotherapy (Figure). Discussion: Colorectal lymphomas are rare and account for 1% of all GI malignancies with CLL usually found proximal to the hepatic flexure. In this case, the presence of ulcerations and large polyps were concerning for colorectal cancer. However, the initial work-up was negative. After a repeat colonoscopy, testing for specific CLL markers was requested. In a review of literature, chemotherapy alone for colonic CLL had a perforation risk of 15%. While it is unclear if surgical treatment is beneficial, it is a potential option. Due to the high risk of perforation on chemotherapy, recommendations included right hemicolectomy or endoscopic removal of the remaining polyps prior to starting treatment. This demonstrates the importance of knowing the varying presentations of colonic CLL as its treatment may require multidisciplinary discussion.Figure 1.: Colonic ulcers located at the transverse colon (top left) and IC valve (top right). H&E stain of the colonic mucosa (bottom) that showed positive markers for CLL on IHC analysis.

Super-Enhancer-Associated nine-gene prognostic score model for prediction of survival in chronic lymphocytic leukemia patients.

Chronic lymphocytic leukemia (CLL) is a type of highly heterogeneous mature B-cell malignancy with various disease courses. Although a multitude of prognostic markers in CLL have been reported, insights into the role of super-enhancer (SE)–related risk indicators in the occurrence and development of CLL are still lacking. A super-enhancer (SE) is a cluster of enhancers involved in cell differentiation and tumorigenesis, and is one of the promising therapeutic targets for cancer therapy in recent years. In our study, the CLL-related super-enhancers in the training database were processed by LASSO-penalized Cox regression analysis to screen a nine-gene prognostic model including TCF7, VEGFA, MNT, GMIP, SLAMF1, TNFRSF25, GRWD1, SLC6AC, and LAG3. The SE-related risk score was further constructed and it was found that the predictive performance with overall survival and time-to-treatment (TTT) was satisfactory. Moreover, a high correlation was found between the risk score and already known prognostic markers of CLL. In the meantime, we noticed that the expressions of TCF7, GMIP, SLAMF1, TNFRSF25, and LAG3 in CLL were different from those of healthy donors (p < 0.01). Moreover, the risk score and LAG3 level of matched pairs before and after treatment samples varied significantly. Finally, an interactive nomogram consisting of the nine-gene risk group and four clinical traits was established. The inhibitors of mTOR and cyclin-dependent kinases (CDKs) were considered effective in patients in the high-risk group according to the pRRophetic algorithm. Collectively, the SE-associated nine-gene prognostic model developed here may be used to predict the prognosis and assist in the risk stratification and treatment of CLL patients in the future.

Immunoglobulin Gene Sequence as an Inherited and Acquired Risk Factor for Chronic Lymphocytic Leukemia.

Simple SummaryChronic lymphocytic leukemia (CLL) is the most prevalent among adult leukemias. Over the years, several research efforts discovered a lot of intricate details about the cause of the disease, its mechanism, and the prognostic factors that help to understand the progression and outcome of the disease. Mutations in the immunoglobulin gene sequences in B cells are the most important prognostic factor for CLL. The cells having no to very less mutations show aggressive disease, while those having more mutations are either fairly indolent or non-aggressive. In this review, we discussed the current gain of knowledge about these mutations and their effects in the overall disease pathology.Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disease characterized by the accumulation of CD5+ CD19+ malignant B cells. Autonomous ligand-independent B-cell signaling is a key process involved in the development of CLL pathogenesis. Together with other cytogenetic alterations, mutations in the immunoglobulin heavy chain variable (IGHV) gene act as a prognostic marker for CLL, with mutated CLL (M-CLL) being far more indolent than unmutated CLL (U-CLL). Recent studies highlight the role of a specific light chain mutation, namely, IGLV3-21R110G, in the development and prognosis of CLL. Such a mutation increases the propensity of homotypic BCR–BCR interaction, leading to cell autonomous signaling. In this article, we review the current findings on immunoglobulin gene sequence mutations as a potential risk factor for developing CLL.

Flow Cytometric Expression of CD49d in Newly Diagnosed Chronic Lymphocytic Leukemia and Its Correlation with Established Prognostic Markers.

Introduction Chronic lymphocytic leukemia (CLL) is the commonest hematological malignancy in the West but is relatively uncommon in India. The prognosis of CLL is determined by well-established prognostic markers. CD49d has been emerging as a promising prognostic marker in CLL. CD49d expression in CLL has been found to have an aggressive clinical course, shorter time to first treatment, and poorer prognosis. The aim of this study was to analyze the flow cytometric expression of CD49d in newly diagnosed CLL and to correlate its expression with clinico-hematological parameters. Materials and Methods Twenty-five consecutive patients of CLL, diagnosed on flow cytometry, were included in the study. Patients on treatment or those with relapse were excluded. The panel for flow cytometry included the routine markers used for CLL diagnosis along with CD49d. The expression of CD49d was correlated with clinico-hematological parameters in all patients. "R" software was used for the statistical analysis. Fisher's exact test and Wilcox test were used to assess the correlation of CD49d to categorical and continuous data, respectively. Results The mean age of the patients was 62.6 ± 12.5 years, and 80% were symptomatic at diagnosis. CD49d expression was found in 44% cases, with a higher proportion being male patients. CD49d and prolymphocyte percentage showed a statistically significant correlation ( p = 0.0007). We found a statistically significant correlation between CD49d expression and lymphadenopathy and splenomegaly with p -values of 0.033 and 0.0472, respectively. CD49d positivity correlated significantly with a higher Rai stage ( p = 0.0196) and intermediate and high-risk cases according to Binet staging ( p = 0.033). Conclusion CD49d expression in the present study correlated with a higher prolymphocyte percentage, lymphadenopathy, splenomegaly, and higher Rai and Binet stages. CD49d expression on flow cytometry was reproducible and easy to interpret.

Molecular Basis and Role of Siglec-7 Ligand Expression on Chronic Lymphocytic Leukemia B Cells.

Siglec-7 (sialic acid–binding immunoglobulin-like lectin 7) is an immune checkpoint-like glycan recognition protein on natural killer (NK) cells. Cancer cells often upregulate Siglec ligands to subvert immunosurveillance, but the molecular basis of Siglec ligands has been elusive. In this study, we investigated Siglec-7 ligands on chronic lymphocytic leukemia (CLL) B cells. CLL B cells express higher levels of Siglec-7 ligands compared with healthy donor B cells, and enzymatic removal of sialic acids or sialomucins makes them more sensitive to NK cell cytotoxicity. Gene knockout experiments have revealed that the sialyltransferase ST6GalNAc-IV is responsible for the biosynthesis of disialyl-T (Neu5Acα2–3Galβ1–3[Neu5Acα2–6]GalNAcα1–), which is the glycotope recognized by Siglec-7, and that CD162 and CD45 are the major carriers of this glycotope on CLL B cells. Analysis of public transcriptomic datasets indicated that the low expression of GCNT1 (encoding core 2 GlcNAc transferase, an enzyme that competes against ST6GalNAc-IV) and high expression of ST6GALNAC4 (encoding ST6GalNAc-IV) in CLL B cells, together enhancing the expression of the disialyl-T glycotope, are associated with poor patient prognosis. Taken together, our results determined the molecular basis of Siglec-7 ligand overexpression that protects CLL B cells from NK cell cytotoxicity and identified disialyl-T as a potential prognostic marker of CLL.