Factor In Chronic Lymphocytic Leukemia Research Articles

Serum Vitamin D3 Levels in Chronic Lymphocytic Leukemia and Its Relevance with Clinical Prognostic Factors

Background: Studies conducted in recent years have proposed a protective effect of vitamin D3 (VitD3) against various types of cancers. Thus, in this research, we investigated the serum levels of VitD3and its relation with prognostic clinical factors in chronic lymphocytic leukemia (CLL). Method: In this cross-sectional study, 86 CLL patients were investigated for a one-year period and evaluated for the relationship between Vit D3serum levels, Rai stages, age, gender, and pathological factors. A control group was added to the study in order to compare the VitD3 levels of CLL patients with that of the normal population. Results: The mean serum VitD3level in CLL patients was 29.76 ± 18.75 ng/ml, and most of the patients were in stage I and II. There was no significant association between age, sex, and Rai stages of CLL with VitD3 level. Except for splenomegaly (P < 0.001), the leukemia-related phenotypes, complications, and prognostic factors had no meaningful association with VitD3 (P > 0.05) level. VitD3 serum level was meaningfully different between case and control groups (P < 0.001). Conclusion: In the current research, there was no significant relationship between VitD3serum levels and investigated variables, except splenomegaly; these findings are in concordance with other studies.

Smudge cells due to infectious mononucleosis.

The smudge cells are well studied as a prognostic factor for chronic lymphocytic leukemia, but it is also found in viral infections. We experienced a case of abnormal hyperleukocytosis (WBC 41,600/mm3) associated with infectious mononucleosis (IM). The smudge cells were observed in peripheral blood smears, and were counted by mistake for the normal lymphocytes by the automatic blood count meter.

A case of chronic lymphocytic leukemia with unmutated variable region of the immunoglobulin heavy chain gene with markedly high lactate dehydrogenase responding to ibrutinib

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. Several prognostic factors exist that help stratify patients into different risk categories. Unmutated status of the immunoglobulin heavy chain (IGHV) gene has been shown to be among the most important high-risk prognostic factors in CLL. Elevated serum level of lactate dehydrogenase has also been shown to be associated with worst outcomes. Recently, the management of CLL has progressed favorably and improved outcomes were seen with the use of the small-molecule Bruton's tyrosine kinase inhibitor, ibrutinib, when compared with traditional chemotherapy. Here, we present a CLL case with an unmutated IGHV gene status presenting with markedly elevated LDH and responding dramatically to ibrutinib.

Regulation and function of angiogenic factors in chronic lymphocytic leukemia

Regulation and function of angiogenic factors in chronic lymphocytic leukemia

Mean Platelet Volume Has Prognostic Value in Chronic Lymphocytic Leukemia.

PurposeMean platelet volume (MPV) is a readily accessible and commonly tested hematological indicator. Recent studies revealed a significant impact of MPV on the course and prognosis of many diseases, including some types of cancer, as well as on the incidence of atrial fibrillation and bleeding. The study aimed to perform a retrospective analysis of MPV in terms of time to first treatment (TTFT) and to determine its prognostic value in the group of patients with chronic lymphocytic leukemia (CLL). Moreover, the study includes a retrospective analysis of platelet parameters in patients treated with ibrutinib concerning bleeding and atrial fibrillation.Patients and MethodsThe study included 523 patients with CLL, for 344 the most important cytogenetic aberrations were reported. The Mann–Whitney, Kruskal–Wallis, Kaplan–Meier, chi-squared, log‑rank tests and multivariate Cox proportional hazard regression model were used to analyze collected data.ResultsThe receiver operating characteristic curve analysis was performed to identify optimal cut-off value for MPV. The analysis of survival curves showed that in the group of patients with higher values of MPV TTFT was significantly longer than in the group with lower MPV (17.9 vs 36 months, p=0.0015, cut-off value for MPV= 10.4 fl). In multivariate Cox proportional hazard regression model low MPV, the presence of del11q and del13q provided independent prognostic value for TTFT (HR=0.69, 95%-CI, 0.5293 to 0.9081; p=0.0078; HR=1.76, 95%-CI, 1.3000 to 2.3882, p=0.0003, HR=0.74, 95%-Cl, 0.5674 to 0.9588, p=0.0229, respectively). In the group treated with ibrutinib, 59 patients had no significant correlation between MPV level and the incidence of therapy complications, although in the group of patients with low MPV there was a tendency for more frequent occurrence of atrial fibrillation (p=0.259).ConclusionLow MPV values are associated with unfavorable prognosis and might represent a novel, independent prognostic factor in CLL.

High M-MDSC Percentage as a Negative Prognostic Factor in Chronic Lymphocytic Leukaemia.

Simple SummaryChronic lymphocytic leukaemia (CLL) is a malignancy of mature B cells. Tumour microenvironment is important for survival and proliferation of malignant cells. In the current study, we investigated the potential role of circulating monocytic myeloid-derived suppressor cells (M-MDSC) in CLL. We have observed an increased percentage of M-MDSC cells in CLL patients. Moreover, we have observed a close association with unfavourable prognostic markers, which suggests a potential role of M-MDSC as a prognostic factor in CLL. We have established an association between a high M-MDSC percentage on the one side and shorter time-to-treatment and overall survival on the other. Therefore, we strongly suggest to use M-MDSC percentage as another prognostic factor.In the current study, we analysed the role and prognostic value of myeloid-derived suppressor cells (MDSC) in chronic lymphocytic leukaemia (CLL). The frequency of circulating monocytic MDSC (M-MDSC; defined as CD14+CD11b+CD15-HLA-DR-/low cells) was assessed in correlation with clinical and laboratory parameters characterising the disease activity and patient immune status. Samples of peripheral blood from untreated CLL patients and healthy volunteers were stained with monoclonal antibodies for flow cytometry analysis. CLL patients with M-MDSC percentages above 9.35% (according to the receiver operating characteristic (ROC) analysis) had a shorter time-to-treatment and shorter survival time than the group with a lower percentage of M-MDSC. The M-MDSC percentage was higher in patients with adverse prognostic factors (i.e., 17p and 11q deletion and CD38 and ZAP-70 expression). A high M-MDSC percentage was linked to significantly lower expression of the CD3ζ in T cells. Furthermore, an analysis of immune regulatory molecules (arginase 1 (ARG1), nitric oxide synthase (NOS2), indoleamine 2,3-dioxygenase (IDO), transforming growth factor beta (TGF-β), and interleukin (IL)-10) was performed. By the means of flow cytometry and RT-qPCR, we showed an overexpression of three of them in M-MDSC of CLL patients. M-MDSC cells seem to be an important factor in the immunosuppressive microenvironment of CLL and seem to be a good and novel prognostic factor

Abstract 937: Quantitative assessment of CD200 and CD200R expression in lung cancer

Abstract Background: CD200 is a membrane-bound glycoprotein expressed on normal tissue, tumor and immune cells. The interaction of CD200 with its receptor leads to attenuation of the inflammatory process, resulting in tumor cell mediated immune suppression. CD200 has been characterized in hematologic malignancies and consists of an independent negative prognostic factor for chronic lymphocytic leukemia, acute myeloid leukemia and multiple myeloma patients. Here we assess both CD200 and CD200R expression in lung cancer and evaluate its association with clinicopathologic characteristics, mutation status, outcome and PD-L1 expression. Methods: We used quantitative multiplexed immunofluorescence (QIF) to measure the expression of CD200 and CD200R in 287 non-small cell lung cancer (NSCLC) patients and 30 patients with large cell neuroendocrine carcinomas (LCNEC) of the lung. We performed target measurement with tyramide-based QIF panels and analyzed the data using the PM2000 microscope and AQUA software. Targets were measured in cytokeratin-positive (CK+) tumor compartment and cytokeratin-negative (CK-) stromal compartment. Results: CD200 tumor positivity was found in 29.7% of NSCLC patients and 33.3% of LCNEC patients. CD200 demonstrated notable intratumoral heterogeneity (R2=0.09-0.31 between different TMA blocks versus 0.46-0.60 for CD200R). CD200R was expressed in immune cells in 25% of NSCLC and 41.3% of LCNEC patients. While CD200R is predominantly expressed in immune cells, rare tumor cell staining was seen in a highly heterogeneous pattern. CD200R expression in the stromal compartment was significantly higher in patients with squamous differentiation (p<0.0001). Neither CD200 nor CD200R were associated with other clinicopathologic characteristics or mutation status. Both CD200 and CD200R were not prognostic for disease-free or overall survival in NSCLC. Since these biomarkers are associated with immune modulation, both markers were assessed for correlation with PD-L1. CD200 showed moderate correlation with PD-L1 (R2=0.28 in the CK+ compartment and 0.40 in the CK- compartment); CD200R was less correlated with PD-L1 (R2=0.07 in the CK+ compartment and 0.10 in the CK- compartment). Conclusions: CD200 and CD200R pathway represents a novel immune checkpoint, frequently expressed in lung cancer patients. Co-expression patterns of CD200 and CD200R with PD-L1 suggest a potential role for targeting this pathway alone or in combination with PD-1/PD-L1 axis inhibitors. Citation Format: Ioannis A. Vathiotis, Tyler MacNeil, Jon Zugazagoitia, Sandra Martinez-Morilla, Fahad Ahmed, Konstantinos N. Syrigos, Aaron M. Gruver, Kyla Driscoll, David L. Rimm. Quantitative assessment of CD200 and CD200R expression in lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 937.

Prognostic factors for the progression of chronic lymphocytic leukemia

Objective: to determine a complex of the most significant prognostic factors in chronic lymphocytic leukemia (CLL) for the purpose of evaluation of the probability of the disease progression. Material and methods. The study included 127 CLL patients whose clinical and laboratory parameters (sizes of the lymph nodes, liver and spleen, general and biochemical blood tests, β2-microglobulin, thymidine kinase, tissue polypeptide antigen (TPA), immunophenotypic markers) had been evaluated at the setting of the diagnosis. As a result of a dynamic followup, the patients were divided into 2 groups: 71 patients who had been observed in the outpatient setting and had had no signs of CLL progression within 3 years of the followup (group 1), and 56 patients with clinical signs of the disease progression that had manifested themselves after 3 years of the followup (group 2). Results. The study has revealed statistically considerable differences (Mann-Whitney test) between the groups in the size of the lymph nodes, liver and spleen, in the Binet stages, leukocyte count, absolute values of lymphocytes, the counts of red blood cells, hemoglobin and platelets, as well as the levels of β2-microglobulin, thymidine kinase, TPA, and immunophenotypic markers of CD24, ZAP-70, and CD38 in the blood, which indicates the interconnection of the levels of these parameters at the setting of the diagnosis with subsequent rapid CLL progression. According to the results of the use of logistic regression and Cox regression analysis, 10 parameters showing the strongest interconnection between the progression and 3-year progression free survival (PFS) have been selected. The use of the step-by-step selection of variables has made it possible to formulate a qualitative Cox regression equation including 5 main parameters: β2-microglobulin, thymidine kinase, TPA, ZAP-70, and CD38, which is indicative of the possibility to use this combination of the markers to predict CLL progression. Conclusion: The prognostic value of the complex of the laboratory parameters (β2-microglobulin, thymidine kinase, TPA, ZAP-70, and CD38) has been determined to assess the probability of CLL progression at the setting of the diagnosis, which can be used for the determination of the management tactics and selection of the treatment scheme for these patients.

Prognostic and predictive impact of genetic markers in patients with CLL treated with obinutuzumab and venetoclax

AbstractGenetic parameters are established prognostic factors in chronic lymphocytic leukemia (CLL) treated with chemoimmunotherapy, but are less well studied with novel compounds. We assessed immunoglobulin heavy variable chain (IGHV) mutation status, common genomic aberrations, and gene mutations in 421 untreated patients within the CLL14 trial (NCT02242942), comparing obinutuzumab+chlorambucil (GClb) vs obinutuzumab+venetoclax (VenG). The incidences of genomic aberrations considering the hierarchical model were del(17p) 7%, del(11q) 18%, +12 18%, and del(13q) 35%, whereas IGHV was unmutated in 60% of patients. NOTCH1 mutations were most common (23%), followed by SF3B1 (16%), ATM (13%), and TP53 (10%). Although the overall response rate (ORR) for GClb was lower in patients with del(17p), del(11q), mutated TP53, ATM, and BIRC3, none of these parameters reduced complete remission (CR) rate and ORR with VenG. At a median follow-up of 28 months, del(17p) and mutated TP53 were the only abnormalities with an effect on progression-free survival (PFS) for both treatment groups: GClb (hazard ratio [HR], 4.6 [P < .01]; HR, 2.7 [P < .01], respectively) and VenG (HR, 4.4 [P < .01]; HR, 3.1 [P < .01], respectively). No other factors affected outcome with VenG, whereas for GClb del(11q), BIRC3, NOTCH1, and unmutated IGHV were associated with shorter PFS. Multivariable analysis identified del(17p), del(11q), unmutated IGHV, and mutated TP53, BIRC3, and SF3B1 as independent prognostic factors for PFS with GClb, whereas for VenG, only del(17p) was significant. VenG was superior to GClb across most genetic subgroups. Patients with adverse genetic markers had the strongest benefit from VenG, particularly subjects with unmutated IGHV, which was identified as a predictive factor in a multivariable treatment-interaction analysis.

Clinical Staging and Flowcytomteric CD38 and Zap 70 Prognostic Indicators in Sudanese Patients with Chronic Lymphocytic Leukemia

Background: The clinical course of chronic lymphocytic leukemia is highly variable. The determination of ZAP70 and CD38 is increasingly utilized as prognostic factor for chronic lymphocytic leukemia. The aim of conducting this study was to investigate the frequency of CD38 and ZAP70 expression among Sudanese Chronic lymphocytic leukemia (CLL) patients and to relate them to the Binet and Rai clinical staging systems.&#x0D; Method: A total of 93 patients (mean age; 62.29 ± 11.68, sd) were enrolled in this cross-sectional study. CD38 and ZAP70 expression levels were measured with four color flowcytometry using the cut-off values of 20% for ZAP70 and 30% for CD38 expression. Staging was assessed by using clinical examination and CBC for all patients. Data were analyzed using the Statistical Package for Social science for Windows (SPSS), version 22.&#x0D; Results: There were 93 CLL patients and the median age of the group was 63 years (36–95 years). About 71% of the patients presented with lymphadenopathy, 53.8% with splenomegaly, 73.1% with anemia, and 45.2% with thrombocytopenia. There was higher frequency of Binet stage C and Rai stage IV (62 [66.6%] patients and 34 [36.5%] patients, respectively). In addition, CD38 and ZAP70 showed higher frequency among Binet and Rai advance stages. ZAP70 and CD38 positivity were detected in 21 patients (22.6%) and 31 patients (33.3%), respectively. There was no statistically significant association between ZAP70 and CD38 and clinical staging systems (P-value &gt; 0.05).&#x0D; Conclusion: No significant association was observed between Flowcytometric (CD38 and Zap70) Prognostic Indicators and clinical staging systems.&#x0D; Keywords: chronic lymphocytic Leukemia, Flowcytometry, ZAP70, CD38, clinical staging systems

Klinički značaj aberacija TP53 gena i IGHV mutacionog statusa u hroničnoj limfocitnoj leukemiji

Chronic lymphocytic leukemia (CLL) is a very heterogeneous disease with a variable clinical course. On the one side of the spectrum, there are patients with aggressive and resistant disease, of which they die only a few months after diagnosis while, on the other side, there are patients with an indolent, slowly progressive disease that does not require treatment for decades. The reasons for this are only partially known, but they have been the subject of numerous scientific studies during the last several decades. Consequently, the concept of prognostic and predictive factors in CLL have emerged, which aims to predict the clinical course and its therapeutic outcome. With the improvement of understanding the pathophysiology of this disease, the lists of prognostic and predictive factors are getting longer every year, but they also overlap. In this review, we singled out the aberrations of the TP53 gene and the IGHV (immunoglobulin heavy variable) gene mutational status as the two most important and most studied factors that have both prognostic and predictive significance.

PRIMA-1MET cytotoxic effect correlates with p53 protein reduction in TP53-mutated chronic lymphocytic leukemia cells

TP53 gene defects represent the most unfavorable prognostic factor in chronic lymphocytic leukemia (CLL). Although recently introduced small-molecule B-cell receptor signalling inhibitors have revolutionized CLL treatment, data for ibrutinib still point to impaired prognosis for TP53-affected patients. Among cancer-associated TP53 mutations, missense substitutions predominate and typically result in a high mutated-p53 protein level. Therefore, rescuing the p53 tumor suppressor function through specific small molecules restoring p53 wild-type (wt) conformation represents an attractive therapeutic strategy for cancer patients with TP53 missense mutations. We tested the effect of mutated-p53 reactivating molecule PRIMA-1MET in 62 clinical CLL samples characterized for TP53 mutations and p53 protein level. At the subtle PRIMA-1MET concentrations (1–4 μM), most samples manifested concentration-dependent viability decrease and, conversely, apoptosis induction, with the response being similar in both the TP53-mutated and TP53-wt groups, as well as in the TP53-mutated samples with p53 protein stabilization and without it. PRIMA-1MET was able to reduce mutated p53 protein in a proportion of TP53-mutated CLL samples, and this reduction correlated with a significantly stronger viability decrease and apoptosis induction than samples with stable p53 levels. CLL cells are mostly sensitive to PRIMA-1MET apart from those with stable mutated p53.

Adherence to the 2018 WCRF/AICR cancer prevention guidelines and chronic lymphocytic leukemia in the MCC-Spain study

IntroductionPreventable risk factors for chronic lymphocytic leukemia (CLL) remain largely unknown. The aim of this study was to evaluate the association between adherence to nutrition-based guidelines for cancer prevention and CLL, in the MCC-Spain case–control study. MethodsA total of 318 CLL cases and 1293 population-based controls were included in the present study. The World Cancer Research Fund/American Institute for Cancer Research (WCRC/AICR) score based on the 2018 recommendations for cancer prevention (on body fatness, physical activity, and diet) was constructed. We used logistic regression analysis adjusting for potential confounders. ResultsIndividuals in the highest tertile of the WCRF/AICR score had an odds ratio for CLL of 1.25 (95 % CI 0.91; 1.73) compared with individuals with low adherence (p-trend = 0.172). Each point increment in the score was associated with an OR for CLL of 1.06 (95 % CI 0.91; 1.23). Analyses by severity of disease did not show significant heterogeneity of effects. ConclusionOverall, our results do not support an association between the WCRF/AICR score and CLL, yet we might have been limited by statistical power and study design to detect modest associations. Further research, ideally with a prospective design, long follow-up, and including additional lymphoma subtypes, is warranted to confirm the impact of composite healthy lifestyle behaviors on lymphoma risk.

Detection of Immunoglobulin Heavy Chain Gene Clonality By High-Throughput Sequencing for Minimal Residual Disease Monitoring in Chronic Lymphocytic Leukaemia

Introduction: The negative minimal residual disease (MRD) after treatment has been recently accepted as endpoint for Chronic Lymphocytic Leukaemia (CLL) clinical trials. Conventionally, MRD can be detected by using multi-color Flow Cytometry (FC) with high sensitivity. Determination of the clonal immunoglobulin gene rearrangement can be a useful monitoring marker in a broad range of B-cell lymphoproliferative neoplasms. Moreover, the mutational status of immunoglobulin heavy chain variable (IgHV) rearrangement is considered one of the most important prognostic factors in CLL. Therefore, the identification of the IgHV rearrangement can be a useful marker both at diagnostic and as monitoring marker for MRD. Nowadays, high-throughput sequencing (HTS) technologies has enabled highly sensitive cancer genomic testing in clinical laboratories. There are same initiatives based on HTS to use IgHV rearrangement as marker for MRD monitoring in Acute lymphoblastic leukemia or multiple myeloma, but it remains unharmonized for application on CLL in the clinical laboratory. Objective: We evaluated the performance and clinical applicability of HTS assay for IgHV rearrangement in CLL MRD monitoring in 69 samples from 19 CLL patients treated. Methods: The libraries including IGH locus were performed using the Sequencing Multiplex Kit on IGH consensus primers. To simplify and make automatic the analysis of the data obtained, we developed a specific bioinformatic pipeline that covers from preprocessing to final data summarization and interpretation. The backbone of the analysis includes read preprocessing, mapping against IMGT reference sequences, consensus IgHV reads pairwise alignment to determine mutational status and read classification into rearrangements. Assessment of IgHV mutational status by Sseq, genomic DNA (gDNA; 50-100 ng), were used for IgHV analysis. gDNA was amplified using locus-specific primer sets for IgHV designed to allow for the amplification of all known alleles of the germline IgH sequence, as described previously. Inmunophenotypic studies were performed on erythrocyte-lysed whole PB samples according to Euroflow procedures. PB white blood cells (WBC) was systematically stained with the eight color combination panel recently proposed by the ERIC group for MRD detection (Rawstron AC et al. 2016). Data acquisition was performed on a FACSCanto II flow cytometer Becton-Dickinson Biociences using the FACSDiva software (V8.0; BD). For data analysis, the Infinicyt softwareTM (Cytognos SL, Salamanca, Spain) was used. The MRD levels were reported as fraction of CLL cells of all nucleated cells. MRD negativity was define as a fraction &lt;10 -4 However, for treatments that preferentially clear the blood (for example monoclonal antibodies) the MRD was confirm in the bone marrow as has been recommended. Results: Patient demographics and the results of IGH clonality tests are summarized in Table 1. Interpretable results were obtained with higher sensitivity compared with Sseq at diagnosis stage (19/19 samples), and we are able to determine the same clone at subsequent samples. Among 50 follow-up samples, 44 MRD were positive in both techniques (HTS and FC); 3 follow-up samples, were negative in both determinations (HTS and FC); in contrast, 3 follow-up samples, were negative by FC but positive by HTS (8.3, 17.2, 18.2 samples). This is the case of Patient 8, in which the last follow-up sample, the detection of the primary clone is positive by HTS, while by FC is undetected (Figure 1). In addition, it was possible to detect IgHV clone in all Patient 7 samples. It was under long-term monitoring by HTS and FC, and 17 months after initial diagnosis and first line of treatment (7.5 sample), it was detected a loss of response (Figure 2.). Conclusions: We evaluated an HTS IgHV assay using initial and follow-up samples of 19 CLL patients. Using our pre-designed primer set in library preparation, and developed our specific bioinformatic pipeline that covers from preprocessing to final data summarization and interpretation, we were able to demonstrate that our method was more sensitive than FC in detecting positive follow-up samples and could be able to propose it for MRD monitoring in CLL. However, the prognostic impact of these low-level MRD detected by HTS should be validated with further investigations. Samples were provided by the INCLIVA Biobank. Funded by Gilead Felowship 257/17 Disclosures Ortiz: GILEAD SCIENCES: Research Funding. Terol:Abbvie: Consultancy; Gilead: Research Funding; Janssen: Consultancy, Research Funding; Astra Zeneca: Consultancy; Roche: Consultancy.

Examination of clonal evolution in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is one of the most frequent lymphoproliferative diseases. CLL is characterized by unusual heterogeneity, which probably reflects its biological and genetic lack of homogeneity. Clonal chromosome aberrations belong to the most important prognostic and predictive factors in CLL. This research was aimed at observing clonal evolution in CLL at the chromosomal level, and assessing its clinical significance in relation to selected prognostic factors. The study involved 72 untreated patients with CLL. The preliminary investigations using cytogenetic banding analysis (CBA) and FISH were performed at the time of diagnosis, and again after about 24 months to observe clonal changes (clonal evolution). In addition, other parameters were evaluated, i.e., the expression of ZAP-70 kinase, CD38 antigen, and the mutation statuses of IGVH and NOTCH1 genes. Classic prognostic factors, i.e., categorized ZAP70 and CD38 expressions as well as mutations in IGVH and NOTCH1 genes did not influence the course of clonal evolution in the examined group of patients. Clonal evolution was detected in 45.8% of patients by means of CBA, and in 19.4% patients with FISH. Analysis of chromosomal aberrations in the examined group of patients showed that the incidence of 17p deletions and translocations in karyotypes has a negative impact on overall survival. CE was found to be a risk factor for the occurrence of disease progression (OR = 2.22). Our observations indicate that combined CBA and FISH are the most optimal techniques for monitoring clonal evolution in the course of CLL.

PB1869 PERSISTENT COMPLEMENT ACTIVATION IS PROVOKED BY IGG‐AGGREGATES IN A SUB‐POPULATION OF CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS

Background:Therapeutic monoclonal antibodies used in Chronic lymphocytic leukemia (CLL) act through complement‐mediated cytotoxicity and other mechanisms, and thus depend on the complement (C) availability and activity. Recently we showed abnormal Western analysis pattern of the C5 component in some CLL patients, that was associated with high basal levels of C activation markers, and decreased activity of the classical pathway (CP). We hypothesized that the CP may be constantly activated, and thus exhausted, in a sub‐group of CLL patients, identified by the abnormal appearance of C5.Aims:The aim was to study the circulating abnormal C5 and its association with the observed decrease in CP activity, and to search for potential CP‐activating factors in CLL plasma.Methods:Blood samples were collected from 40 naïve CLL patients and 15 normal controls (NC). Activities of the CP and of the CP C5‐convertase were studied. C5, C5a and C5b were studied by Western blot analysis. The presence of high molecular weight (HMW) protein complexes, such as IgM or hexameric‐IgG, as potential CP activators was assessed by sera fractionation (gel‐filtration chromatography) followed by C activation with the obtained HMW, and low molecular weight (LMW) fractions. The levels of IgM were measured. Data from patients with abnormal C5 was compared to normal NC subjects and to patients with normal C5.Results:In patients with abnormal C5, activities of the CP and of the CP C5‐convertase were decreased, compared to NC subjects and to patients with normal C5. The abnormal C5 was identified as a complex of immunoglobulin with C5a (Ig‐C5a). In agreement, the high levels of C activation markers showed negative and significant correlation with activity of the CP, but not with the alternative pathway. The data on C activation by the HMW fraction indicated the presence of IgG‐aggregates, and not IgM, as the C activating factor in sera.Summary/Conclusion:In some CLL patients, factors such as IgG‐aggregates that are present in sera constantly trigger the activation of the CP. This constant activation results in the release of activation products, such as C5a, that form the complex Ig‐C5a. Another result is the exhaustion of the CP and the inability to exert CP activity that is comparable to normal subjects. The data provides a potential prognostic tool that may personalize therapy by identifying a sub‐group of CLL patients who display Ig‐C5a and impaired CP activity, and are likely to be less responsive to immunotherapy due to compromised CP activity.

Paraoxonase 1 (PON1) Q192R and L55M Polymorphisms as Potential Predisposition Factors for Chronic Lymphocytic Leukemia.

PON1 gene has an executive role in antioxidant defense, protecting cells from genotoxic factors. Q192R and L55M PON1 polymorphisms reduce catalytic activity of the encoded protein. These polymorphisms were studied in 300 chronic lymphocytic leukemia (CLL) patients and 106 healthy donors. They were also associated with patients' cytogenetic findings, to investigate their possible implication in CLL pathogenesis. SNP genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Karyotypic analysis was also performed by chromosome G-banding analysis and fluorescence in situ hybridization. Genotypic and allelic distribution of Q192R polymorphism showed a statistically significant higher frequency of mutant genotypes and mutant alleles in patients compared to controls. The same observation was noted in patients with abnormal karyotypes and those carrying abn14q32 and del(6q). A statistically increased frequency for the mutant allele was also revealed in patients with del(11q). On the contrary, L55M polymorphism showed a similar distribution between patients and controls. Q192R polymorphism plays a role in CLL predisposition and the formation of specific chromosomal aberrations.

Integrated epigenomic and transcriptomic analysis reveals TP63 as a novel player in clinically aggressive chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) stereotyped subsets #6 and #8 include cases expressing unmutated B cell receptor immunoglobulin (BcR IG) (U-CLL). Subset #6 (IGHV1-69/IGKV3-20) is less aggressive compared to subset #8 (IGHV4-39/IGKV1(D)-39) which has the highest risk for Richter's transformation among all CLL. The underlying reasons for this divergent clinical behavior are not fully elucidated. To gain insight into this issue, here we focused on epigenomic signatures and their links with gene expression, particularly investigating genome-wide DNA methylation profiles in subsets #6 and #8 as well as other U-CLL cases not expressing stereotyped BcR IG. We found that subset #8 showed a distinctive DNA methylation profile compared to all other U-CLL cases, including subset #6. Integrated analysis of DNA methylation and gene expression revealed significant correlation for several genes, particularly highlighting a relevant role for the TP63 gene which was hypomethylated and overexpressed in subset #8. This observation was validated by quantitative PCR, which also revealed TP63 mRNA overexpression in additional nonsubset U-CLL cases. BcR stimulation had distinct effects on p63 protein expression, particularly leading to induction in subset #8, accompanied by increased CLL cell survival. This pro-survival effect was also supported by siRNA-mediated downregulation of p63 expression resulting in increased apoptosis. In conclusion, we report that DNA methylation profiles may vary even among CLL patients with similar somatic hypermutation status, supporting a compartmentalized approach to dissecting CLL biology. Furthermore, we highlight p63 as a novel prosurvival factor in CLL, thus identifying another piece of the complex puzzle of clinical aggressiveness.

The percentage of cells with 17p deletion and the size of 17p deletion subclones show prognostic significance in chronic lymphocytic leukemia.

TP53 disruption is considered to be the most important prognostic factor in chronic lymphocytic leukemia (CLL), but not all patients with TP53 disruption have similar dismal outcomes. We evaluated the prognostic value of TP53 disruption in CLL patients without treatment indications. Data of 305 CLL patients were analyzed. 41 of them (13%) had TP53 disruption. Patients with lower percentage of cells with del(17p) had significantly better survival. Patients with mutated IGHV, β2-microglobulin ≤3.5 mg/L, wild-type TP53, age ≤65 years or without complex karyotype (CK) had relatively favorable outcomes in the del(17p) group. Furthermore, patients with del(17p) as a minor clone showed survival advantage compared with those with del(17p) as a major clone. These data suggest that the percentage of cells with del(17p), the size of the del(17p) subclone, CLL International Prognostic Index, and CK should be considered to build refined prognostication models for patients with TP53 disruption.

98% IGHV Gene Identity Is Still the Optimal Cutoff to Predict the Prognosis of Chronic Lymphocytic Leukemia Patients in China

Background: Immunoglobulin heavy chain variable region (IGHV) has been an important prognostic factor for chronic lymphocytic leukemia (CLL) for decades. 98% being a cut-off value for IGHV is a mathematical choice and researches on the best cut-off value have never been stopped. Chinese CLL patients are known to differ from Caucasian CLL patients on both clinical and genetical features. However, the optimal cutoff for IGHV mutational status has not yet been studied in this particular ethnic group.Method: We carried out a study on 595 Chinese CLL patients in order to find out whether 98% is the best cut-off value for IGHV in Chinese CLL patients. Genomic DNA from peripheral blood or bone marrow was subjected to PCR amplification following the IGH Somatic Hypermutation Assay v2.0 protocol (InVivoScribe). Sequences were aligned to ImMunoGeneTics/V-QUEry and Standardization (IMGT/-VQUEST) database.Result: 600 sequences were received after IGHV rearrangement sequencing. IGHV3-23, IGHV4-34, IGHV3-7, IGHV4-39 and IGHV1-69 were the most frequently used IGHV genes. 352 (58.7%) cases were IGHV-mutated while 248 (41.3%) cases were IGHV-unmutated if the classical 98% classification by ERIC was used. In order to determine the optimal cut-off value, we used 1% as the interval to divide the entire cohort into 7 groups according to the mutational rate, which were <95%, 95%-95.99%, 96%-96.99%, 97%-97.99%, 98%-98.99%, 99%-99.99% and 100% respectively. Binet A patients had a relatively indolent course of disease and cases with different IGHV mutational rates had no significant differences in time to first treatment (TTFT) apart from truly unmutated (100%) cases. For the whole study cohort, significant difference appeared at 98% interval (P<0.001 and P=0.005 for TTFT and OS respectively) while intervals less than 98% had no significant difference compared with the <95% group. Similarly, there was no clear dissimilarities among 98%, 99% and 100% intervals (Table 1a and b). All the other prognostic factors including del(17p), del(11q), TP53 mutation, MYD88 mutation, NOTCH1 mutation, SF3B1 mutation, CD38, ZAP-70, Binet staging, gender, β2-microglobulin and EBV-DNA were differently distributed between group <98% and group ³98%, but not among subgroups in ³98%. In multivariate analysis, the 98% IGHV was also an independent prognostic factor for TTFT and OS.Conclusion: 98% is the optimal cutoff value for IGHV mutational status to predict the prognosis of CLL patients in China. DisclosuresNo relevant conflicts of interest to declare.