Marker In Chronic Lymphocytic Leukemia Research Articles

An Integrative Computational Approach to Evaluate Genetic Markers for Chronic Lymphocytic Leukemia.

Recent studies reported hundreds of genes linked to chronic lymphocytic leukemia (CLL). However, many of these candidate genes were lack of replication and results were not always consistent. Here, we proposed a computational workflow to curate and evaluate CLL-related genes. The method integrates large-scale literature knowledge data, gene expression data, and related pathways/network information for quantitative marker evaluation. Pathway Enrichment, Sub-Network Enrichment, and Gene-Gene Interaction analysis were conducted to study the pathogenic profile of the candidate genes, with four metrics proposed and validated for each gene. By using our approach, a scalable CLL genetic database was developed including CLL-related genes, pathways, diseases and information of supporting references. The CLL case/control classification supported the effectiveness of the four proposed metrics, which successfully identified nine well-studied CLL genes (i.e., TNF, BCL2, TP53, VEGFA, P2RX7, AKT1, SYK, IL4, and MDM2) and highlighted two newly reported CLL genes (i.e., PDGFRA and CSF1R). The computational biology approach and the CLL database developed in this study provide a valuable resource that may facilitate the understanding of the genetic profile of CLL.

Genome-wide DNA methylation profiling integrated with gene expression profiling identifies PAX9 as a novel prognostic marker in chronic lymphocytic leukemia

BackgroundIn chronic lymphocytic leukemia (CLL), epigenomic and genomic studies have expanded the existing knowledge about the disease biology and led to the identification of potential biomarkers relevant for implementation of personalized medicine. In this study, an attempt has been made to examine and integrate the global DNA methylation changes with gene expression profile and their impact on clinical outcome in early stage CLL patients.ResultsThe integration of DNA methylation profile (n = 14) with the gene expression profile (n = 21) revealed 142 genes as hypermethylated-downregulated and; 62 genes as hypomethylated-upregulated in early stage CLL patients compared to CD19+ B-cells from healthy individuals. The mRNA expression levels of 17 genes identified to be differentially methylated and/or differentially expressed was further examined in early stage CLL patients (n = 93) by quantitative real time PCR (RQ-PCR). Significant differences were observed in the mRNA expression of MEIS1, PMEPA1, SOX7, SPRY1, CDK6, TBX2, and SPRY2 genes in CLL cells as compared to B-cells from healthy individuals. The analysis in the IGHV mutation based categories (Unmutated = 39, Mutated = 54) revealed significantly higher mRNA expression of CRY1 and PAX9 genes in the IGHV unmutated subgroup (p < 0.001). The relative risk of treatment initiation was significantly higher among patients with high expression of CRY1 (RR = 1.91, p = 0.005) or PAX9 (RR = 1.87, p = 0.001). High expression of CRY1 (HR: 3.53, p < 0.001) or PAX9 (HR: 3.14, p < 0.001) gene was significantly associated with shorter time to first treatment. The high expression of PAX9 gene (HR: 3.29, 95% CI 1.172–9.272, p = 0.016) was also predictive of shorter overall survival in CLL.ConclusionsThe DNA methylation changes associated with mRNA expression of CRY1 and PAX9 genes allow risk stratification of early stage CLL patients. This comprehensive analysis supports the concept that the epigenetic changes along with the altered expression of genes have the potential to predict clinical outcome in early stage CLL patients.

Cytogenetic and fluorescence in situ hybridization testing in veterans with chronic lymphocytic leukemia.

7526 Background: The presence of deletion 17p (del17), determined by chromosome analysis and/or fluorescence in situ hybridization (FISH), is a strong negative prognostic marker in chronic lymphocytic leukemia (CLL). Prior to the introduction of novel agents (ibrutinib, venetoclax), the clinical utility of cytogenetics/FISH was limited by the absence of chemoimmunotherapy regimens that were proven effective in patients with del17. Testing practices for chromosomal aberrations since the introduction of novel agents have not been reported. We report cytogenetic/FISH trends in a nationwide cohort of veterans diagnosed with CLL. Methods: CLL patients diagnosed 2008-2015 and receiving care at VA were identified through the VA Clinical Cancer Registry. Electronic medical records were used to determine cytogenetic/FISH testing (lab records), treatment histories (pharmacy dispensation records), and evidence of system use (heme-onc notes). Cytogenetic/FISH testing was identified by presence of specific keywords in the test name or Logical Observation Identifiers Names and Codes (LOINC) descriptions, then validated by human annotation. The testing rates are reported for the entire cohort, at time of diagnosis, time of regimen initiation (including the 12 months preceding initiation), during the novel era (2014 – 2015) and prior (2008–2013). Results: From 2008 to 2015, 3,638 CLL patients were diagnosed and received care at VA. Documented records of treatment regimens were available for 1,562 patients who received a total of 2,929 treatment regimens. Only 24% (998) of patients were tested at any point in time during their care at the VA, 17% (622) were tested at time of diagnosis, and 19% (542) of treatment courses were preceded by cytogenetic/FISH testing. No testing differences existed following the introduction of the novel agents at diagnosis (both ~ 17%), or prior to regimen initiation (20% vs 16%). Conclusions: Our study suggests CLL patients diagnosed and receiving care at the VA are not routinely undergoing cytogenetics/FISH testing at diagnosis or prior to treatment. Changing this practice pattern will personalize treatments so that del17 CLL patients receive less toxic and more effective therapies.

Prognosis biomarkers evaluation in chronic lymphocytic leukemia

Objective/BackgroundFrom clinical and biological points of view, chronic lymphocytic leukemia (CLL) is a heterogeneous disease characterized by a progressive accumulation of lymphocytes in the peripheral blood, bone marrow, and lymphoid organs. New prognostic markers in CLL may be useful to clinicians for predicting outcome and in clinical decision-making. The aim of this study was to evaluate the potential prognostic value of the apoptotic/survival-controlling proteins and protein tyrosine kinase ZAP-70 gene expression in CLL patients and control individuals, correlating such findings with patients’ clinical data. MethodsFifty-three patients diagnosed with CLL attending the hematology service of a clinical hospital, and 24 healthy individuals with no history of leukemia (Control group) were enrolled in this study. Analyses of apoptotic/survival-controlling proteins were performed by western blot and ZAP-70 gene expression was evaluated by real-time polymerase chain reaction. ResultsSignificant differences were observed for the p-p38, Mcl-1 long, and Mcl-1 short proteins when patients were compared with CLL and controls. A positive correlation between the results for Mcl-1 short and Mcl-1 long and lymphocyte count was observed, corroborating the hypothesis of an imbalance between proteins of cell survival pathways/apoptosis in CLL. ConclusionZAP-70 gene expression was not detected as a discriminant biomarker in these CLL patients. An imbalance between apoptosis-related proteins was observed in the present study, corroborating the hypothesis of increased survival of lymphocytes in CLL patients.

Determination of β2–microglobulin level as a prognostic marker of chronic lymphocytic leukemia

Disease prediction is rather important for the modern medicine. In the age of new and complex prognostic markers, β2–microglobulin is still simple, but important survival predictor in chronic lymphocytic leukemia (CLL) patients. The use of new biological markers is very expensive and not available for routine analysis in all regions. In contrast, β2–MG is significantly cheaper and simpler in its realization, as well as it is affordable for many non-specialized laboratories.Aim. To analyze β2–MG level prognostic significance and its correlation depending on the disease stage, tumor volume and response to treatment in CLL patients.Methods. 98 CLL patients, 37 women aged 42-75 years (average age 62.5 years) and 61 men aged 38-82 years (average age 62.5 years) were examined by us.All the patients had general clinical examination, lymphocytes immunophenotyping in peripheral blood or bone marrow – phenotype СD 5+, СD 19+, СD 20+, СD23+, β2–MG using ECLIA method (Electro-chemiluminescence immunoassay). Cobas 6000, Roche Diagnostics (Switzerland) analyzer and test system were used. Reference values were 0,8–2,2 mg/l.Results. In result of the study of β2–MG level in chronic lymphocytic leukemia (CLL) patients on the different stages of the disease, it was found, that β2–MG level correlates with CLL stage, directly depends on tumor volume, and is a reliable prognostic marker of response to treatment in CLL patients. β2–MG level >3,5 mg/l in blood serum is a bad prognostic predictor, which points to the high risk of disease.Conclusion. β2–MG level correlates with the disease stage, tumor volume, and is a reliable prognostic marker of response to treatment in CLL patients

CD73, a potential diagnostic marker in Egyptian patients with chronic lymphocytic leukemia

Objective This work aimed to study CD73 expression in chronic lymphocytic leukemia (CLL) patients and its correlation with other disease characteristics. Background CLL is a common B-lymphoproliferative disorder, which can be diagnosed in most cases by morphology and flowcytometry. It displays heterogeneity in clinical presentation, course, and response to treatment, which is likely due to the diversity of the biological nature of the disease. Diagnostic difficulty might arise in some patients due to atypical morphology and/or immunophenotype. CD73 is an extracellular enzyme that hydrolyses adenosine monophosphate to adenosine and is expressed on a subset of T and B lymphocytes. A number of studies have shown CD73 as a contributing factor in the multistep process of carcinogenesis by inducing favorable environment for tumor growth. However, it was shown in some studies to induce apoptosis. Patients and methods CD73 expression on both B and T lymphocytes was measured in 25 cases of newly diagnosed CLL and 15 healthy controls. The levels of expression were correlated to other disease characteristics. Results CD73 expression on CD19 + B lymphocytes was significantly lower in CLL patients compared with controls (mean 3.74 vs. 29.35%, respectively, P Conclusion CD73 expression is a potential diagnostic marker in CLL. This can be particularly relevant in cases with atypical immunophenotype using the current CLL scoring system. CD73 may have a role in CLL pathogenesis as low expression can lead to failure of termination of immune response with subsequent proliferation and clonal expansion of the involved clone.

Detailed Clinical, Immunological and Molecular Analysis of NOTCH1, SF3B1 and MYD88 mutations in Chronic Lymphocytic Leukemia Patients Reveals Accumulation of Negative Prognostic Features in NOTCH1 and SF3B1 mutated Individuals

The clinical course of chronic lymphocytic leukemia (CLL) is highly heterogeneous, from stable to a rapidly progressive. The variety of prognostic factors has been already described, nevertheless they are not fully efficient in predicting the course of CLL, especially when the disease is diagnosed at an early stage. Recently, beside well established cytogenetic prognostic factors, novel molecular mutations of predictive value have been identified. Many of them have been thoroughly characterized, including TP53 mutation, which is commonly considered as a strong, negative prognostic factor. The use of next-generation sequencing technology has also revealed previously unknown genomic alterations, such as neurogenic locus notch homolog protein1 (NOTCH1), splicing factor 3B subunit 1 (SF3B1) or myeloid differentiation primary response 88 (MYD88). These new mutations could partly explain the CLL heterogeneity and help in identifying clinically relevant groups of patients.The aim of the study was to characterize CLL patients with NOTCH1, MYD88 and SF3B1 mutations with regard to molecular and immunological prognostic markers in CLL. Peripheral blood mononuclear cells (PBMCs) were obtained from 369 CLL patients at the moment of diagnosis and the median age reached 65 years. Sixty percent of the patients were male. Distribution of disease stages according to the Rai classification was the following: 0 stage=93, I stage=52, II stage=69, III stage=14, IV stage=26. Clinically, the prognostic significance in terms of time to first treatment (TTFT) was assessed for 202 CLL patients.DNA samples were extracted from PBMCs obtained after Ficoll density gradient centrifugation. NOTCH1 c.7544_7545delCT (n=316) in PEST domain (exon 34) and MYD88 L265P (n=323) mutations were investigated by ARMS PCR. Screening for SF3B1 (n=364) mutations K700, E622/R625 and H662/K666 (exons 14 and 15) were performed using HRM analysis and the results were confirmed by Sanger sequencing. The IGHV gene mutations were investigated by Sanger sequencing.NOTCH1 mutations were found in 19/316 (6.0%) patients. Patients harbouring NOTCH1 mutations prevalently have unfavourable prognostic factors including unmutated IGHV gene status, expression of CD38 (>30%) and expression of ZAP-70 (>20%). The complete analysis of correlations between NOTCH1, MYD88 and SF3B1 mutations and prognostic markers in CLL are presented in Table 1. Analysis of IGHV subsets in patients with NOTCH1 mutation revealed frequent presence of subset #1 in n=2/19 (10.5%), which is associated with particularly poor prognosis in CLL. Patients belonging to subsets #5, #6, #201 and #202 were also present, each in single NOTCH1 mutated CLL case (5.2%). MYD88 mutation occurred in 12/323 (3.7%), of whom one patient was characterized as subset #2 and another as subset #4. MYD88 mutations were nearly equally distributed in patients with mutated/unmutated IGVH status (5 vs. 7). SF3B1 mutations occurred in 17/364 (4.7%) patients, furthermore two of them carrying negative prognostic features of subsets #2 and one subset #1. Patients belonging to subsets #3 and #6 were also present.Certain negative prognostic factors accompany poor clinical outcome. The assessment of median TTFT revealed the significant differences between patients from various prognostic groups (Fig. 1). Patients with unmutated IGHV gene status were characterized by significantly shorter TTFT than patients harbouring the mutation (p<0.0001). The similar correlation occurs in patients with ZAP-70 positive (p=0.04) and CD38 positive (p=0.0003). There were no significant differences in patients with mutated and unmutated NOTCH1 and MYD88, while in patients harbouring SF3B1 mutation the tendency to lower median TFTT was revealed (p=0.08). Interestingly, the significant difference in median TTFT was observed in groups of men and women, showing the better outcome in female patients (median 10 vs 28 months, p=0.01).NOTCH1 and SF3B1 mutations accompany certain biological markers of unfavourable prognosis. Undeniably, the mutations may contribute to the identification of poor-risk CLL patients and in combination with conventional lesions of CLL may be the key to accurate estimation of the disease prognosis.Acknowledgments: This work was supported by Polish Ministry of Science and Higher Education Scientific Grant "The best from the best" ("Najlepsi z Najlepszych") No. 506-0000-39-0000. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

How to Improve the Definition of Chronic Lymphocytic Leukemia Outcome Using a Simple Flow Cytometric Score Based on CD49d and Homing Marker Expression

The identification of new molecular markers in Chronic Lymphocytic Leukemia (CLL) allowed to better define prognosis and clinical outcome. The actual staging systems could estimate the prognosis, but not the rapidity of disease evolution. Neither the identification of new molecular markers did allow to foresee the evolution and clinical response, because discordant findings were mostly reported. The aim of the present study was (1) to confirm the independent prognostic role of CD49d as a single marker in CLL patients, (2) to investigate the relationship between CD49d and other well-established CLL-membrane predictor markers (CD5, CD11c, CD20 and CD38) or clinical staging systems and (3) to evaluate the role of an immunophenotypic score based on the flow-cytometric detection of CD5, CD11c, CD20, CD38 and CD49d in the work up of CLL staging.Heparinized whole blood was collected from 68 CLL patients for immunophenotyping using the following antibodies: anti kappa, anti-lambda, CD5, CD11c, CD19, CD20, CD23, CD38, CD45, CD49d. A scoring system was elaborated combining 5 membrane markers: CD5, CD11c, CD20, CD38 and CD49d. Antigens were divided in two groups, favorable (CD5 and CD20) and unfavorable (CD11c, CD38 and CD49d) prognostic markers, and the cut-off of positivity was chosen according to the literature (30% for CD5, CD11c, CD20 and CD38, and 45% for CD49d). A value of "0" or "1" ("2" only for CD49d positivity) was assigned according to antigen expression. Finally, we defined a favorable phenotype when the sum of all the cytometric features was equal or less than 2, conversely the unfavorable phenotype was defined for a sum equal or greater than 3 (between 3 and 6).Flow cytometric analysis showed high CD49d expression in CD19+ cells in 47% of patients (n=32), and high CD38 expression in 44% of subjects (n=30), simultaneously expressed in 28% of patients (n=19). The 19% (n=13) of all CLL patients were CD5-, and interestingly the 85% of them showed higher expression of CD49d. Linear correlation was found between CD49d and CD38 (r2=0.08772, p=0.0142), and between CD49d and CD20 expression (r2=0.2490, p<0.0001). For CD5, the opposite tendency was registered (r2=0.1944, p=0.0002). Strong negative correlation between higher CD49d expression and total lymphocyte count was found (Pearson r = -0.3577, r2=0.1279, p=0.0068), but not for hemoglobin level and platelet count. The statistical power of each parameter of the score was also calculated by Chi-square test and all markers displayed a statistically significant weight (p<0.0001). After assessed the prognostic power of each marker, we applied the score to staged patients. Forty patients (59%) had a Favorable Phenotype and 28 of them (70%) an early stage disease; in this group of patients, only 7.5% (n=3) showed CD49d high expression. The overall response rate (ORR) was of 58%. The other 28 patients (41%) showed an Unfavorable Phenotype and 21 of them (75%) had an early stage disease. In this arm, all subjects carried>45% of CD49d positive cells. Four patients with Unfavorable Phenotype received chemotherapy with an ORR of 25%. Furthermore, a small population (n=16) of our CLL cohort was also studied for genetic abnormalities using FISH technique. According to FISH analysis, 25% of studied patients were classifies as very low-risk and, interestingly, no one of them showed an Unfavorable Phenotype (only one patient carried CD49d as unique negative marker). In our cohort, 50% of patients were low-risk with no genetic abnormalities or +12, but 63% of them showed an Unfavorable Phenotype with high CD49d and CD38 expression in 100% and 60% of cases, respectively.Our data confirm the independent negative prognostic role of CD49d and suggest a stronger prognostic power compared to CD38 in the definition of CLL outcome, because of its complex activity as homing marker, signaling receptor and anti-apoptotic molecule. Thus, the prognostic significance of CD49d may be enhanced when considered in comparison with other established markers, as CD11c and CD38. In conclusion, our results propose the use of the CD49d marker in combination with other B-cell membrane antigens as an additional tool for routine diagnosis and risk-stratification of CLL patients. Identification of high-risk phenotype with a simple scoring method could improve the treatment of these patients, who could take advantage of the most recent molecular targeting therapies. DisclosuresNo relevant conflicts of interest to declare.

Abstract B131: Quantitative modeling of Syk regulation in B cell chronic lymphocytic leukemia

Abstract Spleen tyrosine kinases (Syk) are initiators of B cell receptor-mediated (BCR) signaling events, enabling cells to drive chronic lymphocytic leukemia (CLL) progression. However, the mechanistic principles of biochemical signaling pathways that contribute to CLL pathogenesis are poorly understood. This study investigates Syk-mediated molecular pathways of the BCR signaling network using computational modeling. An expression of ζ chain-associated protein kinase of 70 kD (ZAP-70, Syk-family protein kinase) in the BCR signaling pathway is an adverse prognostic marker in CLL. Therefore, we need a better understanding of molecular mechanisms of Syk family protein kinases and effects of their inhibitors in the BCR signaling pathway. In this work, we dissected several computational models of ZAP-70 and SYK regulation to design an integrated mechanistic description of the BCR signaling pathway, and to explain the observed differences in the clinical behaviors of ZAP-70+ / ZAP-70- phenotypes of CLL patients. Specifically, we characterized the effects of different ZAP-70 and SYK expression and phosphorylation levels on the BCR activation threshold, using stochastic simulations of the BCR signaling network. Preliminary findings suggest that ZAP-70 serves as an adaptor protein that facilitates the recruitment of SYK to the BCR complex. We find that the expression of ZAP-70 enhances the BCR signaling, and therefore correlates with a more aggressive disease course. Our results will help in the development of novel therapies targeting Syk functions to regulate B-CLL responses. Citation Format: Maria P. Frushicheva. Quantitative modeling of Syk regulation in B cell chronic lymphocytic leukemia [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B131.

Lipoprotein Lipase as a Prognostic Marker in Chronic Lymphocytic Leukemia.

The marked clinical heterogeneity of CLL makes early prognosis assessment important. Lipoprotein lipase (LPL) has been shown to confer adverse prognosis in CLL, recent data indicating it might also contribute to CLL cell survival and metabolism. We determined LPL mRNA expression in unselected peripheral blood of 84 CLL patients by RT PCR. Results were correlated with other prognostic markers and outcome. 30/84 (40%) of cases were LPL positive based on the cutoff established by ROC analysis. In LPL positive patients significantly shorter median survival (136 vs 258months, p<0.0001) and time to first treatment intervals (36 vs 144months, p<0.002) were documented. LPL values correlated with male gender, higher stages, more treatment requirement, CD38 positivity and unmutated IgVH genes. Among cases with 13q deletion, LPL positivity identified a subcohort with poor outcome (median survival 108months vs NR, p<0.0001). In multivariate analysis, cytogenetic aberrations and LPL had significant impact on survival. Our results confirm that LPL is a strong predictor of outcome in CLL, able to improve prognostic accuracy in good risk cytogenetic subgroups. The relationship between its prognostic and functional role in CLL needs to be explored further.

Major prognostic value of complex karyotype in addition to TP53 and IGHV mutational status in first‐line chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder of remarkable heterogeneity as demonstrated by cytogenetics and molecular analyses. Complex karyotype (CK), TP53 deletions and/or mutations (TP53 disruption), IGVH mutational status, and, more recently, recurrent somatic mutations have been identified as prognostic markers in CLL. On a cohort of 110 patients with CLL treated with first-line fludarabin, cyclophosphamide, and rituximab treatment compared with 33 untreated (watch and wait) patients with CLL, we report more frequent complex karyotypes (34 vs 15%; P=.05), unmutated IGHV (70 vs 21%; P<.0001), ATM deletion (25 vs 6%, P=.02), and NOTCH mutation (3 vs 17%, P=.04). Among treated patients, 39 relapsed during the follow-up period. These patients were characterized before treatment by a higher incidence of trisomy 12 (38 vs 11%, P<.001) and TP53 disruption (31 vs 4%, P=.0002). A significantly shorter 5-year overall survival was found for treated patients with CK (72.4 vs 85.8%; P=.007), unmutated IGHV (70 vs 100%; P=.04), or TP53 disruption (55.7 vs 82.7%; P<.0001). Three risk groups were defined based on the status of TP53 disruption or unmutated IGVH, which differed significantly in terms of 5-year overall survival. Moreover, the presence of CK impacted pejoratively 5-year overall survival and progression-free survival in all these 3 groups. Conventional karyotyping therefore appears to be of value, CK being an additional factor, undetectable in classical FISH, in patients with CLL at the stage when therapy becomes required.

Assessment of red blood cell distribution width as a prognostic marker in chronic lymphocytic leukemia.

Red blood cell distribution width (RDW) is a quantitative measure of the variability in size of circulating erythrocytes. It was recently reported that RDW is a prognostic factor for infection diseases, cardiovascular and pulmonary diseases, as well as some neoplasms. Moreover, RDW is remarkably strong predictor of longevity, including all causes of death, for adults aged 45 years and older. To explain this occurrence it was proposed that persistent IGFs/mTOR signaling is one of the factors that play a role in affecting the RDW and mortality.The above observations induced us to analyze the prognostic role of RDW in chronic lymphocytic leukemia (CLL) being the most frequent type of adult leukemia in Western countries. The obtained results have shown that RDW may be considered as a potential CLL prognostic marker. Elevated RDW level at the moment of diagnosis was associated with advanced disease and presence of other poor prognostic factors. It is also connected with overall survival indicating shorter time in patients with elevated RDW. It is possible that the presently observed correlation between mortality and RDW of the CLL patients is affected by their metabolic (IGF-1/mTOR driven)- rather than chronological- aging. The patients with high level of RDW are expected to have an increased persistent level of IGF-1/mTOR signaling. Within the framework of personalized therapy, these CLL patients therefore would be expected to be more sensitive to the treatment with mTOR inhibitors.

Association of Bax Expression and Bcl2/Bax Ratio with Clinical and Molecular Prognostic Markers in Chronic Lymphocytic Leukemia.

SummaryBackgroundIn chronic lymphocytic leukemia (CLL), in vivo apoptotic resistance of malignant B lymphocytes results, in part, from the intrinsic defects of their apoptotic machinery. These include genetic alterations and aberrant expression of many apoptosis regulators, among which the Bcl2 family members play a central role.AimThe aim of this study was to investigate the association of pro-apoptotic Bax gene expression and Bcl2/Bax ratio with the clinical features of CLL patients as well as with molecular prognostic markers, namely the mutational status of rearranged immunoglobulin heavy variable (IGHV) genes and lipoprotein lipase (LPL) gene expression.MethodsWe analyzed the expression of Bax mRNA and Bcl2/Bax mRNA ratio in the peripheral blood mononuclear cells of 58 unselected CLL patients and 10 healthy controls by the quantitative reverse-transcriptase polymerase chain reaction.ResultsWe detected significant Bax gene overexpression in CLL samples compared to non-leukemic samples (p=0.003), as well as an elevated Bcl2/Bax ratio (p=<0.001). Regarding the association with prognostic markers, the Bcl2/Bax ratio showed a negative correlation to lymphocyte doubling time (r=-0.307; p=0.0451), while high-level Bax expression was associated with LPL-positive status (p=0.035). Both the expression of Bax and Bcl2/Bax ratio were higher in patients with unmutated vs. mutated IGHV rearrangements, but this difference did not reach statistical significance.ConclusionsOur results suggest that dysregulated expression of Bcl2 and Bax, which leads to a high Bcl2/Bax ratio in leukemic cells, contributes to the pathogenesis and clinical course of CLL.

Using the geometric mean fluorescence intensity index method to measure ZAP-70 expression in patients with chronic lymphocytic leukemia.

Expression of ζ-chain-associated protein kinase 70 kDa (ZAP-70) in chronic lymphocytic leukemia (CLL) is associated with more aggressive disease and can help differentiate CLL from cases expressing mutated or unmutated immunoglobulin heavy chain variable region (IgHV) genes. However, standardizing ZAP-70 expression by flow cytometric analysis has proved unsatisfactory. The key point is that ZAP-70 is weakly expressed with a continuous expression pattern rather than a clear discrimination between positive and negative CLL cells, which means that the resulting judgment is subjective. Thus, in this study, we aimed at assessing the reliability and repeatability of ZAP-70 expression using the geometric mean fluorescence intensity (geo MFI) index method based on flow cytometry with 256-channel resolution in a series of 402 CLL patients and to compare ZAP-70 with other biological and clinical prognosticators. According to IgHV mutational status, we were able to confirm that the optimal cut-off point for the geo MFI index was 3.5 in the test set. In multivariate analyses that included the major clinical and biological prognostic markers for CLL, the prognostic impact of ZAP-70 expression appeared to have stronger discriminatory power when the geo MFI index method was applied. In addition, we found that ZAP-70-positive patients according to the geo MFI index method had shorter time to first treatment or overall survival (P=0.0002, P=0.0491). This is the first report showing that ZAP-70 expression can be evaluated by a new approach, the geo MFI index, which could be a useful prognostic method as it is more reliable, less subjective, and therefore better associated with improvement of CLL prognostication and prediction of clinical course.

Innate immune signaling in CLL

In this issue of Blood, Wagner et al describe a complex signaling model that explains the mechanism of action of a long-known prognostic marker in chronic lymphocytic leukemia (CLL) and integrates its function with the innate immune system and B-cell receptor signaling.

Abstract 4964: Nonsense MED12 mutation in a patient with T-cell acute lymphoblastic leukemia

Abstract Mediator complex subunit 12 (MED12) is part of a Mediator, which is a large, highly conserved protein complex that participates in the regulation of general as well as gene-specific transcription. First somatic MED12 mutations were identified when exome sequencing revealed highly specific mutations in exon 2 in 70% of uterine leiomyomas studied. Additional mutations have subsequently been observed in exon 1, which further highlight the role of MED12 in these tumors. Functional analyses have indicated that exon 1 and exon 2 mutations lead to a diminished interaction between MED12 and other components of the Mediator kinase module, reduced Mediator-associated CDK kinase activity, and a unique global gene expression pattern. Recently, we observed similar mutations in chronic lymphocytic leukemia (CLL). Mutations were observed in 5% of the patients and the positive mutation status was associated with well-known poor prognosis markers in CLL. All the observed mutations both in uterine leiomyomas and CLL have been missense mutations or small insertions or deletions leading to an in-frame transcript. Interestingly, the first MED12 mutation in a patient with T-cell acute lymphoblastic leukemia (T-ALL) was recently identified through exome sequencing. This is a nonsense mutation affecting the mutation hotspot observed in CLL. It is also the first nonsense mutation identified in the MED12 N-terminal mutation hotspot area in any tumor type. The patient has received bone marrow transplantation and is now in complete remission, but small amounts of cancer cells were collected before the treatment. RNA was extracted from the cells, and cDNA sequencing revealed that only the mutant allele is expressed. We have now generated a construct with the MED12 specific mutation and transfected it into HEK293 Flp-In cells. Wild-type MED12 is being used as a control. Western blotting with N- and C-terminal MED12 antibodies indicates that the mutant protein is being produced but that the N-terminal part is lacking. We now aim to further characterize the function of this highly unusual mutation. Mutant and wild type protein will be produced for proteome-wide mass spectrometry analyses to detect possible effects on protein-protein interactions and also to gain more information on the translation start site. Also transcriptome-wide expression data will be produced to study the effect of the mutation on global gene expression profile. Overall, a mutation affecting the 5′ terminus of MED12 has now been identified in yet another cancer type, T-ALL. Our preliminary analyses indicate that the observed nonsense mutation escapes nonsense-mediated RNA decay and leads to a truncated protein product. Besides providing additional knowledge on the role of MED12 mutations in cancer, this exceptional mutation has potential to produce novel information on the normal MED12 function. Citation Format: Tuomas Heikkinen, Kati Kämpjärvi, Salla Keskitalo, Mikko Turunen, Heikki Kuusanmäki, Netta Mäkinen, Satu Mustjoki, Caroline Heckman, Markku Varjosalo, Pia Vahteristo. Nonsense MED12 mutation in a patient with T-cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4964. doi:10.1158/1538-7445.AM2015-4964

Role of microRNA in chronic lymphocytic leukemia onset and progression.

B-cell chronic lymphocytic leukemia (CLL) is the most common human leukemia occurring as indolent or aggressive form. CLL clinical features and genetic abnormalities are well documented, but molecular details are still under investigation. MicroRNAs are small non-coding RNAs involved in several cellular processes and expressed in a tissue-specific manner. MicroRNAs regulate gene expression, and their deregulation can alter expression levels of genes involved in development/progression of tumors. In CLL, microRNAs can function as oncogenes or tumor suppressors and can also serve as markers for CLL onset/progression. Here, we discuss the most recent findings about the role of microRNAs in CLL and how this knowledge can be used to identify new biomarkers and treatment approaches.

Somatic MED12 mutations are associated with poor prognosis markers in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. We performed systematic database search and identified highly specific MED12 mutations in CLL patients. To study this further, we collected three independent sample series comprising over 700 CLL samples and screened MED12 exons 1 and 2 by direct sequencing. Mutations were identified at significant frequency in all three series with a combined mutation frequency of 5.2% (37/709). Positive mutation status was found to be associated with unmutated IGHV and ZAP70 expression, which are well-known poor prognosis markers in CLL. Our results recognize CLL as the first extrauterine cancer type where 5'terminus of MED12 is mutated at significant frequency. Functional analyses have shown that these mutations lead to dissociation of Cyclin C-CDK8/19 from the core Mediator and to the loss of Mediator-associated CDK kinase activity. Additional studies on the role of MED12 mutation status as a putative prognostic factor as well as mutations' exact tumorigenic mechanism in CLL are warranted.

Clinical Outcomes for Chronic Lymphocytic Leukemia (CLL) Patients with 11q Deletion in British Columbia (BC), Canada: Results of a Population-Based Cohort

BACKGROUNDThe 11q22.3 deletion (11q-) encompassing the ATM locus detected by fluorescence in situ hybridization (FISH) is present in up to 20% of CLL patients (pts), and is associated with an aggressive disease course and poor response to treatment (tx). However, there are few clinical studies that have specifically focused on pts with 11q-. Prognostic factors that may explain the variable outcome in this group have not been investigated, nor have survival outcomes in a population-based setting. Furthermore, few clinical studies have described practice patterns within the 11q- group. This study aims to improve our understanding of the prognosis, management and clinical course of CLL pts with 11q-.METHODSThe BC Provincial CLL Database, which includes all pts who have undergone CLL FISH testing in a provincially validated laboratory since 2004, was used to identify all pts in BC (population 4.5 million) with a confirmed 11q- >10% found at any point during the disease course, with or without other detectable FISH abnormalities. Overall survival (OS), treatment-free survival (TFS) (time from diagnosis to first tx/death) and time to second-line treatment (tx) were the main outcomes of interest, and were investigated in relationship to age (<60 vs ≥60 years [yrs]), sex, Rai stage (3-4 vs 0-2), CD38 status, % 11q-, and presence of other recurrent FISH abnormalities (17p deletion [17p-], trisomy 12, 13q deletion, and IGH translocation). Multivariate analysis (MVA) was performed using Cox proportional hazard models with a backwards stepwise selection process to determine predictors of OS/TFS.RESULTS125 out of 1044 (12%) pts were identified with 11q- detected at any point. Median age at CLL diagnosis was 61 yrs (range, 35-80). At a median follow-up of all patients of 6.3 yrs (range 0.5 - 26.2), median OS and TFS for the entire cohort are 12.6 yrs (standard error [SE] = 1.5 yrs) and 2.5 yrs (SE = 0.5 yrs), respectively. In MVA, advanced Rai stage (HR 3.7, 95% CI 1.28-10.8, P=.015), age ≥ 60 yrs (HR 2.2, 95% CI 1.2-4.3, P=.012) and male gender (HR 2.4, 95 % CI 1.5-5.3, P=.024) were significant predictors of OS.Clonal evolution (CE) to 11q- and/or other FISH abnormalities was documented in 12 pts. In 9/12 cases, 11q- developed after one or more tx courses whereas, in 3/12 cases, 11q- developed prior to tx initiation at median 5 yrs (range, 3.8-6.5) from initial FISH test.Sixty-nine pts had a documented 11q- pre-tx. At median follow-up of 5.0 yrs (range, 0.5 to 18.7), this group had median OS and TFS of 14.7 (SE =1.8) and 2.5 yrs (SE= 0.5), respectively (Fig 1). Pts with presence of 11q- and 17p- had a markedly worse prognosis compared to those without, with median OS 4.9 vs. 14.7 yrs (P< .001) and TFS of 0.2 vs. 2.5 yrs (P=0.31). In MVA, co-presence of 17p- (HR = 11.3, 95% CI 2.4-52.5, P=.02) and age ≥ 60 yrs (HR 3.8, 95% CI 0.83-17.1, P=.05) were adverse prognostic markers for OS. Age ≥ 60 yrs (HR =2.0, 95% CI 1.0-3.9, P=.03), presence of > 75% 11q- (vs. < 75 % 11q-) (HR = 1.8, 95%CI 1.0-2.3, P=.03) and advanced Rai stage (HR = 7.1, 95%CI 2.4-21.0, P=.01) were associated with shorter TFS.Of this group of 69, 52 pts (75%) received at least one tx course during follow-up. Thirty-three pts received fludarabine +/- rituximab (FR) as first line, 14 pts received alkylator based tx (FC +/- R in 8; CVP +/- R in 5; CHOP + R in 1) and 5 pts received chlorambucil-based +/- R. Pts treated with FR alone had median OS 12.8 yrs (SE=1.0), which was not statistically different from those treated with alkylators , where median OS was 6.8 yrs (SE=2.1), P=.35 (Fig 2). In MVA, receipt of fludarabine vs alkylator-based treatment was not associated with any significant difference in OS (P=.52) or time to 2nd tx (P=.65).CONCLUSIONThis study is to our knowledge the largest population-based report of CLL pts with 11q- and further enhances our understanding of the clinical course of such pts in a real world setting. Although median TFS of 11q- pts is short at 2.5 yrs, OS remains long at 12.6 yrs. The importance of combined 11q- and 17p- as adverse prognostic markers in CLL is highlighted. Most pts in BC with 11q- received FR as first line therapy. There is retrospective trial data suggesting that first line tx containing alkylators (ie. cyclophosphamide) can overcome the adverse prognostic features of 11q-. However, most of this trial data is from the pre-R era. Though limited in numbers, our study does not suggest that first line tx without alkylators is associated with negative outcomes. [Display omitted] [Display omitted] DisclosuresToze:Roche: Consultancy, Honoraria, Research Funding. Gerrie:F Hoffmann-La Roche: Other.

Association between polymorphism of GLI1 gene SNP rs2228226 and chronic lymphocytic leukemia in Chinese population.

A non-synonymous single-nucleotide polymorphism (SNP) (rs2228226C>G), in exon 12 of glioma-associated oncogene homolog 1 (GLI1) (Q1100E), encodes a change from glutamine to glutamic acid (Q1100E). The variant GLI1 protein exhibited reduced transactivation function in vivo, decreasing the ability of activating hedgehog signal, which has been proposed as an unfavorable prognostic marker in chronic lymphocytic leukemia (CLL). The GLI1 Q1100E (NCBI SNP ID: rs2228226) genotypes in 155 CLL patients were detected by direct sequencing. The difference between frequencies of GLI1 Q1100E genotype among CLL patients and controls was statistically significant (p < 0.001). Logistic regression analysis revealed that in comparison with G/G, GLI1 SNP1100 C/C genotype was associated with a significantly increased risk of CLL (OR 3.787, 95% CI 1.814-7.907, p < 0.001). Compared with the SNP1100 C/G genotype, C/C genotype significantly increased the risk of CLL (OR 3.860, 95% CI 1.827-8.153, p < 0.001). In addition, combining C/G with G/G, C/C genotype also significantly increased the risk of CLL (OR 3.820, 95% CI 1.885-7.742, p < 0.001). The comparison between frequencies of C and G allele of GLI1 was also statistically significant (p = 0.004). In the entire cohort, SNP1100 genotypes were found in association with Binet stage (p = 0.045) and trisomy 12 (p = 0.036). By comparing C/C with G allele (C/G+G/G) genotype, there was a significant correlation with trisomy 12 (p = 0.013). This study demonstrated that GLI1 Q1100E polymorphism was closely associated with CLL. C/C genotype contributes to the risk of developing CLL and correlates with trisomy 12. Patients with trisomy 12 are susceptible group of CLL.