Treatment Of Chronic Lymphocytic Leukemia Patients Research Articles

Reduced prognostic value of beta-2-microglobulin for time to first treatment in CLL patients with compromised kidney function

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults and characterized by a highly heterogeneous clinical course. The CLL-IPI and the OCLL-1 scores are among the best validated tools to predict time-to-first-treatment. In both models, elevated beta-2-microglobulin plasma level (B2M) is an independent prognostic factor. Yet, B2M is commonly increased in patients with chronic kidney disease (CKD) per se and both models were not adjusted for CKD. We analyzed the clinical outcomes of 297 treatment-naive CLL patients between 2000 and 2022. B2M was more frequently elevated in CKD patients and lost prognostic significance at the threshold > 2.5 mg/L. Both CLL-IPI and OCLL-1 failed to facilitate prognostic segregation in CKD patients. 22.2% of CKD patients were assigned to a higher CLL-IPI risk group due to elevated B2M. Our results suggest that both models overestimate the risk for disease progression and need to be interpreted with caution in CKD patients.

Диагностический потенциал регуляторных не кодирующих белок РНК при хроническом лимфоцитарном лейкозе

This paper reviews current knowledge about regulatory non-coding protein RNAs (ncRNAs) involved in the pathogenesis of chronic lymphocytic leukemia (CLL) and their potential capabilities as diagnostic markers. Diversity of clinical course as well as absence of detectable chromosomal aberrations and somatic mutations in 20 % of patients increase the interest to study the epigenetic aspects of pathogenesis. In this context, ncRNAs are believed to be promising diagnostic markers since their expression is commonly tissue-specific and they are quite stable in body fluids. Among the regulatory ncRNAs involved in the CLL pathogenesis, microRNAs and long (lncRNAs) have been most studied, whereas ring-like, or circulatory, ncRNAs (circRNAs) require further analysis. Aberrant expression of ncRNAs may account for the resistance to treatment in CLL patients without detected genomic abnormalities. Bioinformatics analysis of RNA sequencing databases allows to isolate novel candidate ncRNA molecules, including those associated with RNA-mediated suppression of the Piwi protein-interacting transposons. This paper proposes new independent predictive models based on the expression of 2 (LNC-KIA1755-4, LNC-IRF2-32-LNCRNA), 4 (miR-125b, miR-15b, miR-181c, miR-412), and 6 (PRKCQ, TRG.AS1, LNC00467, LNC01096, PCAT6, SBF2.AS1) simultaneously assessed different ncRNAs. Since risk- and stage classification of hematological malignancies is performed not only on the basis of clinical but also molecular genetic markers, the monitoring of regulatory ncRNA expression can provide an additional tool for more effective stratification of patients. The present review is concerned with the methodology issues in analytical procedures which impede widespread use of laboratory ncRNA tests.

Efficacy and safety outcomes of single-agent ibrutinib therapy in chronic lymphocytic leukemia and relapsed/refractory mantle-cell lymphoma

Objectives: We aimed to evaluate patients using ibrutinib for the treatment of chronic lymphocytic leukemia (CLL) and relapsed/refractory mantle-cell lymphoma (MCL), focusing on high-risk subgroups, predictors of efficacy, response levels, and safety profile. Methods: This retrospective cohort study included adult patients diagnosed with CLL and relapsed/refractory MCL who were started on ibrutinib as a single-agent between May 2015 and December 2021 in Bursa Uludag University, Department of Hematology. Results: Of the 45 patients (23 CLL, 22 MCL) started on ibrutinib, the median age was 65 (range: 48-86) years, and 66.7% were male. Del(17p) was present in 47.8% of CLL patients; there was no remarkable difference between del(17p) status and the rates of achieving CR. The median follow-up with ibrutinib treatment in CLL patients was 13.3 (range: 0.3-77.8) months. In univariate analysis, progression-free survival (PFS) and overall survival (OS) were associated with the advanced Eastern Cooperative Oncology Group (ECOG) score (p = 0.003 and p = 0.004, respectively), and > 2 lines treatment regimens before ibrutinib (p = 0.016 and p = 0.050, respectively). In multivariate analysis, the ECOG performance status remained significant for OS. The median use of ibrutinib for MCL patients was 6 (range: 1-48) months, and the proportion of patients who achieved CR was 27.3%. In the univariate analysis of MCL patients, the ECOG performance status for PFS and OS was statistically significant (p = 0.045 and p = 0.016, respectively). Patients' most common non-hematological adverse events were pneumonia and urinary tract infection. Conclusions: Our investigation of patient outcomes treated outside clinical trials confirms ibrutinib's sufficient efficacy and safety profile in CLL and relapsed/refractory MCL.

The Value of Neutrophil Gelatinase-Associated Lipocalin Receptor as a Novel Partner of CD38 in Chronic Lymphocytic Leukemia: From an Adverse Prognostic Factor to a Potential Pharmacological Target?

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of neoplastic B lymphocytes that escape death, and correlates with the expression of negative prognostic markers such as the CD38 antigen. Although certain new drugs approved by the US Food and Drug Administration improve the clinical outcome of CLL patients, drug resistance and disease relapse still occur. Like CD38, neutrophil gelatinase-associated lipocalin receptor (NGAL-R) is frequently overexpressed in CLL cells. Here, we evaluated the concomitant surface expression of NGAL-R and CD38 in leukemic blood cells from 52 CLL patients (37 untreated, 8 in clinical remission, and 7 relapsed). We provide evidence of a positive correlation between NGAL-R and CD38 levels both in the interpatient cohorts (p < 0.0001) and in individual patients, indicating a constitutive association of NGAL-R and CD38 at the cell level. Patients with progressing CLL showed a time-dependent increase in NGAL-R/CD38 levels. In treated CLL patients who achieved clinical remission, NGAL-R/CD38 levels were decreased, and were significantly lower than in the untreated and relapsed groups (p < 0.02). As NGAL-R and CD38 participate in CLL cell survival, envisioning their simultaneous inhibition with bispecific NGAL-R/CD38 antibodies capable of inducing leukemic cell death might provide therapeutic benefit for CLL patients.

Immunogenicity of the 13-Valent Pneumococcal Conjugated Vaccine Followed by the 23-Valent Polysaccharide Vaccine in Chronic Lymphocytic Leukemia.

Patients with Chronic Lymphocytic Leukemia (CLL) have a 29- to 36-fold increased risk of invasive pneumococcal disease (IPD) compared to healthy adults. Therefore, most guidelines recommend vaccination with the 13-valent pneumococcal conjugated vaccine (PCV13) followed 2 months later by the 23-valent polysaccharide vaccine (PPSV23). Because both CLL as well as immunosuppressive treatment have been identified as major determinants of immunogenicity, we aimed to assess the vaccination schedule in untreated and treated CLL patients. We quantified pneumococcal IgG concentrations against five serotypes shared across both vaccines, and against four serotypes unique to PPSV23, before and eight weeks after vaccination. In this retrospective cohort study, we included 143 CLL patients, either treated (n = 38) or naive to treatment (n = 105). While antibody concentrations increased significantly after vaccination, the overall serologic response was low (10.5%), defined as a ≥4-fold antibody increase against ≥70% of the measured serotypes, and significantly influenced by treatment status and prior lymphocyte number. The serologic protection rate, defined as an antibody concentration of ≥1.3 µg/mL for ≥70% of serotypes, was 13% in untreated and 3% in treated CLL patients. Future research should focus on vaccine regimens with a higher immunogenic potential, such as multi-dose schedules with higher-valent T cell dependent conjugated vaccines.

Efficacy and safety of add-on anti-CD20 monoclonal antibody to Bruton tyrosine kinase inhibitor treatment for chronic lymphocytic leukemia: a meta-analysis

The efficacy of Bruton tyrosine kinase inhibitors (BTKi) remains suboptimal in chronic lymphocytic leukemia (CLL) treatment. A systematic review and meta-analysis were conducted to compare the outcomes of combining anti-CD20 monoclonal antibodies (mAb) with BTKi therapy versus BTKi monotherapy for patients with CLL. We searched for relevant studies in the Pubmed, Medline, Embase, and Cochrane databases until December 2022. We estimated the effective results using a hazard ratio (HR) for survival outcomes and relative risk (RR) for response outcomes and safety. Four randomized controlled trials (including 1056 patients) were found until November 2022 and fulfilled the inclusion criteria. Progression-free survival was significantly improved with the addition of anti-CD20 mAb to BTKi over BTKi (HR 0.70, 95% confidence interval (CI) 0.51–0.97), whereas pooled analysis of overall survival did not favor combination therapy compared to BTKi monotherapy (HR 0.72, 95% CI 0.50–1.04). Combination therapy was related to a statistically better complete response (RR, 2.03; 95% CI 1.01 to 4.06) and an undetectable minimal residual disease rate (RR, 6.43; 95% CI 3.54 to 11.67). The risk of grade ≥ 3 adverse events was comparable between the two groups (RR, 1.08; (95% CI 0.80 to 1.45). Overall, adding anti-CD20 mAb to BTKi revealed superior efficacy than BTKi alone in untreated or previously treated CLL patients without affecting the safety of single-agent BTKi. Conducting further randomized studies to confirm our results and determine the optimal therapy for managing patients with CLL is essential.

Ofatumumab regimens in chronic lymphocytic leukemia: a meta-analysis.

Ofatumumab's therapeutic impact on patients with chronic lymphocytic leukemia (CLL) has been the subject of increasing clinical research. However, in recent years, no studies have yet provided a pooled assessment of the treatment effect of ofatumumab vs. non-ofatumumab regimens. Therefore, we conducted a progression meta-analysis to evaluate the efficacy of ofatumumab-based treatment in CLL patients using data from clinical studies. Relevant publications from PubMed, Web of Science and ClinicalTrials.gov were searched. The efficacy outcomes were progression-free survival (PFS) and overall survival (OS). The articles reviewed in the mentioned databases and matching the specified keywords were searched until January 2023. The pooled efficacy analysis showed that there was a significant difference in PFS [hazard ratios (HR) = 0.62, 95% confidence interval (CI) = 0.52-0.74] and no significant difference in OS (HR = 0.86, 95% CI = 0.71-1.03) between ofatumumab-based therapy and non-ofatumumab therapy. Our analysis showed the pooled efficacy for PFS was statistically significantly improved with ofatumumab-based treatments for CLL compared with other groups. Also, ofatumumab had no statistically significant improvement in the OS of patients with CLL. Thus, ofatumumab-based therapies for CLL patients could be improved by other combinational-based regimens.

Functional humoral and cellular response of monovalent COVID-19-vaccines against Omicron BA.2 variant of SARS-CoV-2 in patients with chronic lymphocytic leukemia

We designed a prospective study to evaluate the humoral (using a surrogate virus neutralization test) and cellular (using an IFN-γ ELISpot) immune response among patients with chronic lymphocytic leukemia (CLL) against Wuhan-Hu-1 and Omicron BA.2 strains of SARS-CoV-2, after mRNA-based vaccination. The proportion of patients with a functional humoral response was higher among untreated CLL patients compared to treated CLL patients against both Wuhan-Hu-1 and Omicron BA.2 after the second and the third dose of vaccination, and at 12 months after the first dose. The proportion of positive cellular response against the peptide pool covering the full-length Wuhan-Hu-1 spike protein was similar between untreated and treated CLL patients at all three timepoints. The cellular response to the mutated regions of BA.2 spike protein was lower than the response to the corresponding regions in the ancestral spike after the second dose, but this difference was eliminated after the third dose.

Differences in Humoral and Cellular Response to COVID-19 Vaccine Against Different Variants over Time in Chronic Lymphocytic Leukemia

Background Patients with chronic lymphocytic leukemia (CLL) are at an increased risk of adverse clinical outcomes following COVID-19 infection, due to impaired humoral and cellular response to infection and vaccination. Most of the data available focus on humoral responses to SARS-CoV-2 vaccination or T cell responses following 2-dose vaccine series. However, the effect of emerging variants of concern (VoC) on T cell recognition in this population has not been previously reported. Aim of the study To evaluate functional humoral response and functional SARS-CoV-2 spike-specific T cell response in CLL patients after the second, the third dose, and 12-months since the first dose. To determine the effect of treatment on immune outcomes. To investigate T cell response to Omicron (BA.2) and ancestral strains, and the impact of Omicron-defining mutations on total spike-specific functional response. Methods CLL patients from Mayo Clinic, Rochester who submitted a blood sample soon after the second and the third dose, and 12-months after the first dose of an mRNA-based vaccination were included. The samples collected following a documented SARS-CoV-2 infection were excluded. Spike-specific T cell responses were quantified using IFN-γ ELISpot assay (R&D Systems). PBMCs were stimulated with a peptide pool spanning the complete sequence of ancestral SARS-CoV-2 spike and two 68-peptide pools: the BA.2 mutation pool selectively covering the mutated spike regions, and reference pool containing the wild-type sequences for the same regions (Peptivator pools, Miltenyi Biotec). The results were considered positive if at least two of the following three were met: average spot count > 5 SFUs/2x10^5; mean spot count >3-fold higher than in negative control; and statistically significant difference (paired t-test<0.5) between Ag-specific and negative control wells.For evaluation of humoral response, we utilized an assay measuring antibody capable of blocking spike receptor binding domain (RBD)-ACE2 (cPASS GenScript). Statistical analysis was performed using t-test and Mann-Whitney test for continuous variables; Chi-square (or Fisher's Exact test) was used for comparing categorical variables. The Mayo Clinic IRB approved the study. Results Thirty patients with CLL were included in the study. Eight patients received a fourth vaccine dose before the 12-months blood draw. The rate of positivity for receptor blocking activity was significantly higher in the untreated group after the 2ndand 3rd dose (0% vs. 75%, 38.5% vs. 93.3%, p>0.05). This difference was not observed for the cellular response to spike-specific ancestral in all the three timepoints. At 12 months 54.16 % of the patients had positive IFN-γ ELISpot response, with no significant difference between treated and untreated (58.3% vs. 50%, p=1.0), and patients with and without a fourth dose (62.5% vs. 50%, p=0.67). Among treated CLL patients, seven (70%) and five (41.6%) patients without neutralizing antibodies had detectable cellular response after the 2nd and 3rd dose, respectively. On average, less than 25% of the total spike-specific T cell reactivity was directed against the epitopes in the regions mutated in BA.2 subvariant. Moreover, we did not detect statistically significant differences in IFN-γspot counts between the mutational and reference 68-peptide pools at any timepoint (median spots T1: 1.62 vs. 2.33, p=0.4; T2: 5 vs. 4.33, p=0.31; T3: 5 vs. 6, p=0.49). Conclusion Treatment status impacts humoral, but no cellular response to SARS-CoV-2 vaccination CLL patients. However, the overall rate of positive cellular response one year after the first dose is low. This, combined with no boosting of cellular response upon the 4th dose, reflects an impaired response even after additional doses. Suboptimal cellular immunity may put CLL patients at higher risk of severe COVID-infection. We did not observe significant difference in the cellular response between ancestral and Omicron BA.2 strains, and only the smaller fraction of T cell response was directed against regions harboring BA.2-specific mutations. These findings confirm the greater observed cross-reactivity to VoC with cellular compared to humoral immune responses. They also suggest significant cellular immunity may remain in the face of Omicron circulation, at least among patients who have response to the vaccine. Given the small sample size, our findings need to be confirmed. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Immunogenicity of the Currently Recommended Pneumococcal Vaccination Schedule in Patients with Chronic Lymphocytic Leukaemia

Background Patients with chronic lymphocytic leukemia (CLL) are at increased risk of both invasive pneumococcal disease (IPD) and community acquired pneumonia (CAP) caused by Streptococcus pneumoniae. Therefore, international guidelines recommend pneumococcal vaccination for all CLL patients, preferably before initiation of treatment. The vaccination schedule recommended in the European Conference on Infections in Leukaemia (ECIL) 7 guideline consists of Prevnar13®, a 13-valent pneumococcal conjugated vaccine (PCV13) followed 2 months later by Pneumovax23®, a 23-valent polysaccharide vaccine (PPSV23). However, the immunogenicity of this sequential pneumococcal vaccination schedule in CLL patients is unknown. The aim of this study is to assess the immunogenicity of PCV13 followed by PPSV23 in untreated and treated CLL patients, as CLL treatment has been shown to be a major determinant of COVID-19 vaccine immunogenicity. Methods We quantified pneumococcal immunoglobulin G (IgG) concentrations to 5 pneumococcal serotypes shared across both vaccines (6B, 9V, 14, 19F, 23F) and 4 serotypes unique to PPSV23 (8, 15B, 20, 33F) before and 4-8 weeks after vaccination using a validated multiplex immunoassay, Luminex®. The primary outcome was the serologic response rate (SRR), defined as a ≥4-fold increase in ≥70% of the serotype-specific anti-pneumococcal IgGs. Secondary outcome was serologic protection, defined as an antibody concentration of ≥1.3 mcg/ml for ≥70% of all measured serotypes, in accordance with the definition by the American Academy of Allergy, Asthma & Immunology. Additional secondary outcomes were serotype-specific serologic protection and IgG level dynamics. Results We included 113 CLL patients (mean age 66 years; 39/113 female) in one academic and six teaching hospitals in the Netherlands. All completed the vaccination and measurement schedule. Median time since CLL diagnosis was 3.6 years. Of the 113 patients 33 had previously received CLL treatment, including rituximab (22 patients), cytotoxic chemotherapy (11), ibrutinib (10) and/or venetoclax (7). Seven patients received intravenous immunoglobulin prior to the vaccinations, while 6 received this during the vaccination scheme. The SRR was 11.5% (15% in untreated and 3% in treated patients) (Table 1). A statistically significant increase in all serotype-specific antibody concentrations was observed after vaccination (p <0.001) (Figure 1). The immunogenicity varied per serotype: the percentage of patients mounting a 4-fold increase was lowest for serotype 20 (16%) and highest for serotype 9V (40%). The serotype-specific anti-pneumococcal IgGs after vaccination were all significantly lower in treated than in previously untreated patients. Nevertheless, treatment status was not statistically significant associated with the SRR. Total IgG concentration prior to vaccination was the only evaluated factor associated with the SRR (OR 5.69; p = 0.008). Serologic protection increased from 1.8% before vaccination to 10.6% after both vaccinations (13.8% in untreated and 3% in treated patients). Serotype-specific protection was highest for serotype 14 and 33F (43.4% of patients obtained a concentration of ≥1.3 mcg/ml) and lowest for serotype 6B (16.8%). Conclusion The serologic response to combined PCV13 and PPSV23 vaccination is impaired in previously treated CLL patients as well as in treatment-naive patients, a contrast with COVID-19 vaccination. These findings emphasize the importance of further investigation into the pneumococcal vaccination response in CLL patients. Since patients with CLL are insufficiently protected after a single dose of PCV13, research should focus on multi-dose PCV schedules with the currently available higher valent T cell dependent conjugate vaccines. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Changes in the T Helper Cell Compartment during Treatment with the BTK Inhibitor Acalabrutinib

Interactions with T helper cells are part of the supportive lymph node microenvironment promoting chronic lymphocytic leukemia (CLL) cell growth and survival. Treatment of CLL patients with ibrutinib, the first-in-class BTK inhibitor, induces profound changes in the T cell compartment, with normalization of elevated T cell numbers and plasma cytokine levels, and diversification of the T cell receptor (TCR) repertoire (Yin Q et al., J Immunol 2017). Acalabrutinib, a second generation BTK inhibitor, is more selective for BTK, with less off-target effects towards other kinases, including IL-2-inducible T cell kinase (ITK), an important kinase for T cell signaling. To characterize acalabrutinib effects on T cell numbers and function in patients with CLL, we analyzed serial samples from 12 acalabrutinib-treated CLL patients at baseline, after 1 week, and after 1, 3, 6, and 12 months of continuous acalabrutinib treatment (with monthly Obinutuzumab added during months 3-9) in an ongoing Phase 2 trial (NCT04505254). Six age-matched healthy donors (HD) were analyzed for comparison. T cell subsets were characterized by flow cytometry. Functional T cell response were assessed after stimulation with phorbol myristate acetate and ionomycin, and proliferation by Ki-67 expression. Prior to treatment, untreated CLL patients had elevated numbers of CD4+ (1864±32/ml) and CD8+ (2533±452/ml, mean T cells which normalized after 12 months of therapy (CD4+ 514±65/ml, p<0.01; and CD8+ 747±271/ml, p<0.05). Two key subsets, T follicular helper cells (Tfh) and T regulatory cells (Tregs), also decreased significantly after 12 months of treatment (Tfh from 2.9±0.4% to 0.2±0.1%, p<0.05; and Tregs from 2.4±0.4% to 0.9±0.2%, p<0.05). Next, we analyzed expression of Tfh and Treg surface markers and found significant down-modulation of CD69 (on Tfh from 20±3.7% to 3.3±0.6%, p<0.001; on Tregs from 10±1.8% to 1.7±0.5%, p<0.01), ICOS (on Tfh from 33±2.6% to 13±1.6%, p<0.01), PD-1 (on Tregs from 7.8±1.3% to 1.2±0.3%, p<0.01), and CTLA-4 (on Tfh from 16±3.2% to 3.8±1.0%, p<0.05; on Tregs from 14±1.5% to 3.1±0.7%, p<0.001) after 12 months of treatment. Additionally, this therapy resulted in reduced cytokine production by Tfh and Tregs when compared to baseline samples, as quantified by flow cytometry after cytoplasmatic staining. For example, induced IL-21 expression decreased from 23±1.3% in baseline samples to 12±0.5% after 12 months, p<0.05 (for comparison, 5.7%±1.3% in HD), IL-10 decreased from 7.3±1.1% to 0.9±0.2% at 12 months, p<0.05 (0.9%±0.1% in HD), and TGFb (from 6.2±0.9% to 0.8±0.2% at 12 months, p<0.01 (1.6%±0.5% in HD). Moreover, circulating Tfh cells from pre-treatment samples expressed higher levels of the co-stimulatory molecule CD40L upon restimulation at baseline compared to after 12 months of treatment and the levels in HD (from 23.0±1.6% to 11.0±1.1% at 12 months, p<0.05; versus 3.8%±1.0% in HD, p<0.001). Looking at Ki-67 expression, we noted, as expected, a significant decrease in CLL cell proliferation from 3.9±0.8% down to 0.8±0.6% after 1 month of therapy (p<0.001), similar data were seen at later time points. Interestingly, Tregs also had reduced Ki-67 expression, down from 16±0.6% to 7.3±0.9% after 6 months (p<0.01) and to 7.9%±1.1% after 12 months of therapy (p<0.05). Collectively, the reduction in T cells and T helper cell subset numbers, along with changes in surface activation markers, and reduced cytokine and Ki-67 expression suggest that an activated pre-treatment T helper cell compartment normalizes during continuous acalabrutinib therapy. These findings are reminiscent of the T cell data from patients receiving ibrutinib therapy, suggesting that off-target effects of the BTK inhibitor may not dictate T helper cell changes, and these may rather be due to activation by the CLL clone, which diminishes as the underlying disease responds to treatment.

CLL-035 Long-Term Survival After Fludarabine, Cyclophosphamide, and Rituximab Treatment in Previously Untreated Chronic Lymphocytic Leukemia Patients

Chronic lymphocytic leukemia (CLL) is characterized by a lower incidence rate in Iraq and Kurdistan as compared to Western countries. However, a good prognosis of CLL is dependable on diagnosis, risk stratification, and a better choice of an appropriate treatment regimen. To evaluate the effectiveness of fludarabine, cyclophosphamide, and rituximab (FCR) regimen in comparison to other chemotherapy regimens in the management of patients with CLL in Kurdistan, Iraq. A retrospective review study carried out in three cancer centers in the Kurdistan region of Iraq for a duration of 10 years through the period from January 1, 2010 to December 31, 2019, on 152 CLL patients. CLL was diagnosed according to the International Workshop on CLL. The treatment of CLL patients was either by FCR chemoimmunotherapy regimen or other chemotherapies. The FCR chemoimmunotherapy was the treatment of 38.8% of CLL patients, while 61.2% of CLL patients were treated by other chemotherapies. There was a significant association between younger age patients and the use FCR chemoimmunotherapy (P = 0.001). There was a significant association between a complete response and treatment by FCR chemoimmunotherapy (P = 0.02). The mean overall survival duration and progression free survival of CLL patients treated by FCR chemoimmunotherapy were significantly longer than the mean survival time of CLL patients treated by other chemotherapies (P = 0.01). Complete response and survival of CLL patients treated by FCR chemoimmunotherapy were better than the complete response and survival of CLL patients treated by other chemotherapies.

CLL-093 Long-Term Survival After Fludarabine, Cyclophosphamide, and Rituximab Treatment in Previously Untreated Chronic Lymphocytic Leukemia Patients

Chronic lymphocytic leukemia (CLL) is characterized by a lower incidence rate in Iraq and Kurdistan as compared to Western countries. However, a good prognosis of CLL is dependent on diagnosis, risk stratification, and a better choice of an appropriate treatment regimen. To evaluate the effectiveness of fludarabine, cyclophosphamide, and rituximab (FCR) regimen in comparison to other chemotherapy regimens in the management of patients with CLL in the Kurdistan region of Iraq. A retrospective review study was carried out in three cancer centers in the Kurdistan region of Iraq for a duration of 10 years from January 1, 2010, to December 31, 2019, on 152 CLL patients. CLL was diagnosed according to the International Workshop on CLL. The treatment of CLL patients was either by FCR chemoimmunotherapy regimen or other chemotherapies. The FCR chemoimmunotherapy was the treatment of 38.8% of CLL patients, while 61.2% of CLL patients were treated by other chemotherapies. There was a significant association between younger age patients and the use of FCR chemoimmunotherapy (P=0.001). There was a significant association between a complete response and treatment by FCR chemoimmunotherapy (P=0.02). The mean overall survival duration and progression-free survival of CLL patients treated by FCR chemoimmunotherapy were significantly longer than the mean survival time of CLL patients treated by other chemotherapies (P=0.01). The complete response and survival of CLL patients treated by FCR chemoimmunotherapy were better than the complete response and survival of CLL patients treated by other chemotherapies.

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.

Artificial Intelligence Enhances Diagnostic Flow Cytometry Workflow in the Detection of Minimal Residual Disease of Chronic Lymphocytic Leukemia.

Simple SummaryFlow cytometric immunophenotyping is critical in detecting minimal residual disease (MRD) in patients with chronic lymphocytic leukemia (CLL). However, flow cytometric analysis is complicated and time-consuming. Herein, we evaluated the performance of a deep neural network (DNN) in detecting CLL MRD and whether it could improve the diagnostic workflow in a clinical laboratory setting. Our findings demonstrated that a hybrid DNN approach had high accuracy in detecting CLL MRD; it standardized the gating strategy and dramatically reduced gating time, and it could be fully integrated into the existing clinical laboratory.Flow cytometric (FC) immunophenotyping is critical but time-consuming in diagnosing minimal residual disease (MRD). We evaluated whether human-in-the-loop artificial intelligence (AI) could improve the efficiency of clinical laboratories in detecting MRD in chronic lymphocytic leukemia (CLL). We developed deep neural networks (DNN) that were trained on a 10-color CLL MRD panel from treated CLL patients, including DNN trained on the full cohort of 202 patients (F-DNN) and DNN trained on 138 patients with low-event cases (MRD < 1000 events) (L-DNN). A hybrid DNN approach was utilized, with F-DNN and L-DNN applied sequentially to cases. “Ground truth” classification of CLL MRD was confirmed by expert analysis. The hybrid DNN approach demonstrated an overall accuracy of 97.1% (95% CI: 84.7–99.9%) in an independent cohort of 34 unknown samples. When CLL cells were reported as a percentage of total white blood cells, there was excellent correlation between the DNN and expert analysis [r > 0.999; Passing–Bablok slope = 0.997 (95% CI: 0.988–0.999) and intercept = 0.001 (95% CI: 0.000–0.001)]. Gating time was dramatically reduced to 12 s/case by DNN from 15 min/case by the manual process. The proposed DNN demonstrated high accuracy in CLL MRD detection and significantly improved workflow efficiency. Additional clinical validation is needed before it can be fully integrated into the existing clinical laboratory practice.

Long-Term Efficacy and Safety of Ibrutinib in the Treatment of CLL Patients: A Real Life Experience.

Ibrutinib has demonstrated a significant clinical impact in patients with de novo and relapsed/refractory chronic lymphocytic leukemia (CLL), even in cases with unfavorable cytogenetics and molecular markers. All CLL patients’ data treated at our Institute with ibrutinib have been retrospectively reviewed. Forty-six patients received ibrutinib either as frontline (10) or second or more advanced treatment (36). Five patients presented with TP53 mutations; 11 had the deletion of chromosome 17p; 17 displayed an unmutated immunoglobulin variable heavy chain status. The median number of cycles administered was 26. Among patients treated frontline, the best overall response rate (ORR) was 90.0%. In patients receiving ibrutinib as a second or later line ORR was 97.2%. Median progression-free survival was 28.8 and 21.1 months for patients treated frontline and as second/later line, respectively. Median overall survival was not reached for those treated frontline and resulted in 4.9 years for patients treated as second/later line. Grade 3–4 hematological toxicities were neutropenia, thrombocytopenia, and anemia. Grade 3–4 extrahematological toxicities included diarrhea, cutaneous rash, utero-vesical prolapse, vasculitis, and sepsis. Ibrutinib is effective and well tolerated in CLL. Responses obtained in a real-life setting are durable and the safety profile of the drug is favorable.

Humoral and cellular immune responses to recombinant herpes zoster vaccine in patients with chronic lymphocytic leukemia and monoclonal B cell lymphocytosis.

Monoclonal B-cell lymphocytosis (MBL) and chronic lymphocytic leukemia (CLL) are clonal B-cell disorders associated with an increased risk of infections and impaired vaccination responses. We investigated the immunogenicity of recombinant zoster vaccine (RZV) in these patients. Individuals with MBL/untreated CLL and Bruton tyrosine kinase inhibitor (BTKi)-treated CLL patients were given two doses of RZV separated by 2 months. Responses assessed at 3 and 12 months from the first dose of RZV by an anti-glycoprotein E ELISA antibody assay and by dual-color Interferon-γ and Interleukin-2FLUOROSPOT assays were compared to historic controls matched by age and sex. About 62 patients (37 MBL/untreated CLL and 25 BTKi-treated CLL) were enrolled with a median age of 68 years at vaccination. An antibody response at 3 months was seen in 45% of participants, which was significantly lower compared to historic controls (63%, p= .03). The antibody response did not significantly differ between MBL/untreated CLL and BTKi-treated CLL (51% vs. 36%, respectively, p= .23). The CD4+ T-cell response to vaccination was significantly lower in study participants compared to controls (54% vs. 96%, p< .001), mainly due to lower responses among BTKi-treated patients compared to untreated MBL/CLL (32% vs. 73%, p= .008). Overall, only 29% of participants achieved combined antibody and cellular responses to RZV. Among participants with response assessment at 12 months (n= 47), 24% had antibody titers below the response threshold. Hypogammaglobulinemia and BTKi therapy were associated with reduced T-cell responses in a univariate analysis. Strategies to improve vaccine response to RZV among MBL/CLL patients are needed.

Efficacy and Safety of Chimeric Antigen Receptor T Cell Therapy in Chronic Lymphocytic Leukemia: A Systematic Review

Background:Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in adults. Despite the advancements in drug therapies, CLL is largely incurable, and relapsed/refractory (R/R) patients have a very poor prognosis. Chimeric antigen receptor T (CART) cell therapy has shown promising results in B-cell malignancies. We conducted a systematic review to assess the efficacy and safety of CART cell therapy in patients with CLL.Methods:PRISMA guidelines were followed to perform the literature search and selection of articles for this systematic review. A search was performed using databases including PubMed, Cochrane, Web of Science, Embase, and clinicaltrials.gov. We used the following Mesh and Emtree terms, “Chronic lymphocytic leukemia” AND “Adoptive immunotherapy” from the inception of literature till 06/11/2021. Out of 1319 articles, we screened and included nine clinical studies (N=208) measuring the efficacy (i.e., complete response, partial response, etc.) and safety (adverse events ≥grade 3) in clinical terms. We excluded case reports, pre-clinical studies, review articles, and meta-analyses.Results:In 9 clinical studies, 158 patients with CLL were treated with anti-CD19 CART cell therapy. The range of age of the patients was 38-75 years. A high dose was used in 38 patients, and ibrutinib was added in 19 patients with CLL. The therapy was well tolerated with ≥grade 3 cytokine release syndrome (CRS) and neurological toxicity reported in 23/151 (15%) and 20/151 (13%) of the patients, respectively. Table 1. Complete response (CR), partial response (PR), and overall response (OR) were seen in 40% (54/134), 17% (22/126), and 56% (103/183). In the clinical study by Frey et al. (N=51)., progression-free survival (PFS) and ORR were significantly higher in the high dose (5x10 8 cells/kg) group as compared to the low dose (5x10 7 cells/kg) group (1.8 months vs. one month and 53% vs. 29%, respectively) without significantly increasing treatment-related adverse events (TRAE). In the clinical study by Gauthier et al. (N=38)., ORR was significantly high in the anti-CD19 CART cell therapy + ibrutinib group compared to the no ibrutinib group (83% vs. 56%). Neutropenia, infection, thrombocytopenia, leukemia, hypocalcemia, elevated ALT, and tumor lysis syndrome were common ≥grade 3 TRAEs reported in these patients (Table 1). More clinical trials are targeting CD-20, CD-137, CD-7, CD-28, ROR1, etc (Table 2).Conclusion:Anti-CD19 CART cell therapy was safe and effective in the treatment of CLL patients. A high dose of 5x10 8/kg CART cell therapy was well tolerated and had superior efficacy. Adding ibrutinib to CART cell therapy was safe and more effective than anti-CD19 CART cell therapy alone. More placebo-controlled randomized multicenter studies are needed to confirm these results. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Targeting PAK1 Overcomes Resistance to Ibrutinib in Chronic Lymphocytic Leukemia

Objective:Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder that mainly affects the elderly and is characterized by the expansion of small mature B-cells. New targeted drugs, such as the BTK inhibitor ibrutinib, have greatly improved patient survival but have also posed the challenge of drug resistance. The three-dimensional (3D) spatial structure of chromatin is highly dynamic and varies greatly between cell types and developmental stages, with the maintenance of chromatin homeostasis being of major significance in disease prevention. Accumulating evidence has suggested that changes in 3D genomic structures play an important role in cell development and differentiation, disease progression, as well as drug resistance. Nevertheless, the characteristics and functional significance of chromatin conformation in the resistance of CLL to ibrutinib remain unclear. In this study, we aimed to investigate the mechanism underlying ibrutinib resistance through multi-omics profiling, including the study of chromatin conformation. Thus, we would be able to demonstrate the importance of chromatin spatial organization in CLL and highlight the oncogenic factors contributing to CLL development and mediating ibrutinib resistance.Methods:An ibrutinib-resistant cell line was established by exposing cells to increasing doses of ibrutinib. High-throughput chromosome conformation capture (Hi-C), assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), bulk RNA sequencing (RNA-seq), and Tandem Mass Tag (TMT) were performed to explore differences between ibrutinib-resistant and parental cells. Peripheral blood mononuclear cells (PBMCs) from 53 CLL patients were collected for RNA-seq. Mitochondrial respiration and glycolysis were assessed via Seahorse analysis. The growth-inhibitory effects of tested drugs were evaluated via a CCK8 assay, and the combination index (CI), indicating synergy, was calculated using CompuSyn software. Apoptosis was detected via annexin V staining.Results:Between ibrutinib-resistant and parental cells changes in some chromosomes, including chr11 were observed (Figure 1A). p21-activated kinase 1 (PAK1), which is located on chr11 and frequently overexpressed or excessively activated in almost all cancer types and involved in almost every stage of cancer progression, was first explored for its role in CLL progression and drug resistance. The oncogene PAK1 was observed locate in a region where B-to-A compartment switching occurred (Figure 1B). Consistent with the results of ATAC-seq, RNA-seq, and TMT, Hi-C analysis revealed a transcriptional upregulation of PAK1 in ibrutinib-resistant CLL cells (Figure 1C). Functional analysis demonstrated that PAK1 overexpression significantly promoted cell proliferation, while knockdown markedly suppressed cell viability (Figure 1D). Cell viability assays indicated that the depletion of PAK1 increased ibrutinib sensitivity (Figure 1E). In addition, PAK1 positively regulates glycolysis and oxidative phosphorylation in CLL cells (Figure 1F and G).To verify the results of sequencing and further explore the role of PAK1 in CLL, B-cells from healthy volunteers and PBMCs from CLL patients were collected. The level of PAK1 mRNA expression was significantly higher in CLL primary cells than in B-cells from healthy volunteers (Figure 1H). Kaplan-Meier survival analysis of qRT-PCR data confirmed that patients with high PAK1 expression had a significantly lower OS (Figure 1I).IPA-3, the small molecular inhibitor of PAK1 suppressed the proliferation of ibrutinib-resistant and parental CLL cells in a dose-dependent manner. The combination of IPA-3 and ibrutinib exerted potent cell growth inhibition (Figure 1J), and the combination index (CI) calculated using the CompuSyn software confirmed the synergistic effect (CI<1) of this combinatorial therapy (Figure 1K).Conclusions:In the current study, we have provided a genome-wide view of alterations in 3D chromatin organization between ibrutinib-resistant and parental CLL cells and confirmed the oncogenic role of PAK1 in CLL. Most importantly, our research provides promising therapeutic targets for overcoming ibrutinib resistance. In particular, the treatment of CLL patients with a combination of IPA-3 and ibrutinib may improve clinical outcomes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CLL-115: First Results of a Head-to-Head Trial of Acalabrutinib Versus Ibrutinib in Previously Treated Chronic Lymphocytic Leukemia

Context: Increased selectivity of the Bruton tyrosine kinase inhibitor (BTKi) acalabrutinib versus ibrutinib may improve tolerability. Objective: To compare acalabrutinib versus ibrutinib in patients with chronic lymphocytic leukemia (CLL). Design: Open-label, randomized, noninferiority, phase 3 trial. Patients: Previously treated CLL patients with del(17p) or del(11q) by central lab. Interventions: Oral acalabrutinib 100 mg twice daily (BID) or ibrutinib 420 mg once daily (QD) (stratified by del(17p) status, Eastern Cooperative Oncology Group performance status [2 vs ≤ 1], and number of prior therapies [1–3 vs ≥4]) until progression or unacceptable toxicity. Main Outcome Measures: Primary endpoint was progression-free survival (PFS), as assessed by independent review committee; secondary endpoints of all-grade atrial fibrillation (AF), grade ≥3 infection, Richter transformation, and overall survival (OS) were assessed in hierarchical order. Results: 533 patients (acalabrutinib, n=268; ibrutinib, n=265) were randomized (median age 66 years; median 2 prior therapies; del(17p) 45.2%; del(11q) 64.2%). At a median follow-up of 40.9 months (range 0.0–59.1), acalabrutinib was noninferior to ibrutinib with a median PFS of 38.4 months in both arms (HR: 1.00; 95% CI: 0.79–1.27). Acalabrutinib was statistically superior to ibrutinib in all-grade AF incidence (9.4% vs 16.0%; P=0.023). Among other secondary endpoints, incidences of grade ≥3 infection (acalabrutinib: 30.8%, ibrutinib: 30.0%) and Richter transformation (acalabrutinib: 3.8%, ibrutinib: 4.9%) were comparable between arms. Median OS was not reached in either arm (HR: 0.82; 95% CI: 0.59–1.15), with 63 (23.5%) deaths in the acalabrutinib arm and 73 (27.5%) in the ibrutinib arm. Among any-grade adverse events (AEs) in ≥20% of patients in either arm, acalabrutinib was associated with lower incidence of hypertension (9.4%, 23.2%), arthralgia (15.8%, 22.8%), and diarrhea (34.6%, 46.0%) but a higher incidence of headache (34.6%, 20.2%) and cough (28.9%, 21.3%). AEs led to treatment discontinuation in 14.7% of acalabrutinib- versus 21.3% of ibrutinib-treated patients. Any-grade AEs of clinical interest that were less frequent with acalabrutinib included cardiac (24.1% vs 30.0%), hypertension (above), and bleeding events (38.0% vs 51.3%). Conclusions: In this first head-to-head trial of BTKis in CLL, acalabrutinib demonstrated noninferior PFS with less cardiotoxicity and fewer discontinuations due to AEs versus ibrutinib.