Progressive Chronic Lymphocytic Leukemia Patients Research Articles

MER Signaling Axis: A Novel Therapeutic Target in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD19+/CD5+/CD23+ monoclonal B-cells in peripheral blood, bone marrow and lymphoid tissues with a highly variable disease course. B-cell receptor (BCR)-signal inhibitors, specifically Ibrutinib which targets Bruton's tyrosine kinase (BTK) and Idelalisib which targets PI3Kδ, are effective in relapsed/refractory CLL patients. Despite BCR-signal-inhibitors are changing the management of CLL, these agents are not curative and resistance can develop, often leading to the transformation of CLL into more aggressive B-cell lymphomas limiting their therapeutics options. Thus, improved understanding of the nature and extent of other active receptor tyrosine kinase (RTK) signals in CLL cells is critical for development of additional therapies to treat these high-risk patients. Our recent discovery of MER, a RTK of the TAM (Tyro3, AXL, MER) family, in CLL cells may offer such an option. In order to analyze MER expression status in CLL cells from previously untreated CLL patients, we employed western blot, flow cytometry and qRT-PCR analyses. We detected low to high level expression of MER in CLL cells from ~50% of CLL patients as compared to normal, purified B cells. We also found that MER in CLL cells remained constitutively phosphorylated (active). Furthermore, our preliminary analysis suggested that MER expression in CLL cells may be positively associated with high-risk, unmutated IGHV CLL patients. In vitro ligation of Gas6 (growth arrest-specific gene 6), a specific ligand for all the TAM receptors, robustly activated MER as compared to AXL or Tyro3. Interestingly, activation of MER preferentially activated Erk1/2, NF-κB, and BTK of the BCR pathway in CLL cells. Partial depletion of MER in CLL cells using a specific-siRNA resulted in inhibition of P-Erk1/2, P-NF-κB and P-BTK, but not P-AKT, suggesting that MER may crosstalk with the BCR signaling pathway in CLL cells likely via interaction with CD79a, LYN or BTK. To address this hypothesis, we immunoprecipitated MER from CLL cell lysates and found a direct complex formation between MER and LYN. We also analyzed leukemic B cells from the Eµ-TCL1 transgenic mice which recapitulate highly aggressive human CLL for the expression of MER by western blot/flow cytometry. Results showed robust expression of MER in TCL1 leukemic B cells purified from mice with advanced CLL. Finally, treatment of CLL mice with a high-affinity MER inhibitor (UNC-2025) via oral gavage significantly reduced leukemic tumor burden. In total, our findings suggest that expression/activation of MER may potentiate CLL cell survival via a crosstalk with the BCR signal thus, could be an attractive therapeutic target for the progressive CLL patients.

Genomic mutation profile in progressive chronic lymphocytic leukemia patients prior to first-line chemoimmunotherapy with FCR and rituximab maintenance (REM).

Chronic Lymphocytic Leukemia (CLL) is the most prevalent leukemia in Western countries and is notable for its variable clinical course. This variability is partly reflected by the mutational status of IGHV genes. Many CLL samples have been studied in recent years by next-generation sequencing. These studies have identified recurrent somatic mutations in NOTCH1, SF3B1, ATM, TP53, BIRC3 and others genes that play roles in cell cycle, DNA repair, RNA metabolism and splicing. In this study, we have taken a deep-targeted massive sequencing approach to analyze the impact of mutations in the most frequently mutated genes in patients with CLL enrolled in the REM (rituximab en mantenimiento) clinical trial. The mutational status of our patients with CLL, except for the TP53 gene, does not seem to affect the good results obtained with maintenance therapy with rituximab after front-line FCR treatment.

PD-1 Overexpression in Richter's Transformation (RT) and Aggressive Chronic Lymphocytic Leukemia (CLL) after Progression on Ibrutinib Increases Bcl-2 Expression Via Akt/mTOR Pathway

Introduction: Given the established role of PD-1 in mediating immune suppression in chronic lymphocytic leukemia (CLL), we tested and reported the efficacy of PD-1 blocking antibody pembrolizumab in relapsed and transformed CLL patients. A selective response of pembrolizumab (~40%) in patients with RT, particularly after prior ibrutinib, was observed (Ding, Blood, 129:3419). Correlative analysis showed PD-1 expression in tumor B-cells of patients with RT and aggressive CLL after progression on ibrutinib. PD-1, an inhibitory receptor expressed on CLL T-cells, inhibits the immune synapse and cytotoxic T cell functions via the interactions with its ligands. However, the expression pattern and role of PD-1 in tumor B-cells is not well defined. In this study we investigated the functional implication of the PD-1 signaling axis in B-cell pathobiology in CLL and RT patients.Methods: 26 CLL-involved lymph node (LN) and 20 RT-involved LN were tested for PD-1 expression by immunohistochemistry (mouse clone NAT105, Abcam). For in vitro study, we checked PD-1 expression in 11 lymphoma cell lines and 1 CLL cell line by both flow cytometry and Western blot (WB) analysis. Effect of PD-1 knockdown using CRISPR/Cas9 system (Addgene) and over-expression of PD-1 using pLEX-lentiviral (Thermoscientific) or pRetro-retroviral (Clonetech) system were evaluated on pro-survival and apoptotic signaling pathways by Western blot analysis. Gene expression signatures in CLL and RT patients were also evaluated by Illumina-based RNA sequencing using FFPE-nodal tissue obtained by clinical biopsy (Tempus Labs; Chicago, IL).Results: The expression of PD-1 was significantly increased in RT-LN compared to CLL-LN. (mean ± SEM in RT vs. CLL, 30.6 ± 4.7 vs. 11.5 ± 2.8, p < 0.001). PD-1 expression was highest in patients with RT where the immediate prior CLL therapy was ibrutinib (Figure 1A). Among all cell lines tested for PD-1 expression, the expression of PD-1 by WB and flow was highest in Mino (mantle cell lymphoma line), followed by moderate expression in Jvm2 (B-PLL line) and Mec1 (CLL line), and very low-level expression in both Jeko-1 (B-NHL line) and lymphoma line ‘Karpas299‘. CRISPR/Cas9 mediated depletion of PD-1 in Mino cells inhibited constitutively active Akt, p70S6K and mTOR pathway, accompanied by significant downregulation of the anti-apoptotic proteins, Bcl-2, Mcl-1 and XIAP, but P-ERK1/2 was not affected. Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in Jeko-1 and doxycycline regulated inducible retroviral (pRetro-PD-1) mediated overexpression of PD-1 in Karpas299 activated Akt, mTOR and p70S6K pathway. Overexpression of PD-1 in Jeko-1 significantly increased Bcl-2 and Mcl-1 and in Karpas299 increased Bcl-2, Mcl-1 and XIAP expression (Figure 1B). A parallel genetic analysis using RNA sequencing was performed on 5 nodal tissues involved by either RT or progressive CLL after these patients developed clinical progression after prior ibrutinib therapy. In all 5 patients, overexpression of PD-1 was associated with increased expression of Bcl-2 and mTOR regardless of the genetic mutations detected (including TP53, ATM, BTK, NOTCH1, XPO1, SF3B1, TET2 etc). However, in 2 patients who received prior chemoimmunotherapy, similar overexpression of gene signature was not observed by RNA sequencing analysis, alternative pathways including Met or NFkB overexpression was detected. Given these clinical and laboratory findings, we have treated 2 RT patients with a combination of BTK and Bcl-2 inhibitors (ibrutinib and venetoclax, respectively) whose CLL transformed after prior ibrutinib. Significant reduction of tumor burden was observed in both cases with one complete response and one mixed response.Conclusion: An increased expression of PD-1, Akt/mTOR and Bcl-2 gene signature was first observed in RT patients after prior ibrutinib therapy. PD-1 overexpression in the tumor B-cells of RT and progressive CLL patients likely regulate AKT/mtOR to upregulate Bcl-2. Targeting both BTK and Bcl-2 pathways in addition to PD-1 blockade appear to be a promising strategy to treat these aggressive diseases. DisclosuresParikh:Gilead: Honoraria; Janssen: Research Funding; Abbvie: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding. Kenderian:Novartis: Patents & Royalties; Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding. Ansell:Takeda: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Celldex: Research Funding; Merck & Co: Research Funding; Regeneron: Research Funding; LAM Therapeutics: Research Funding; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Research Funding; Pfizer: Research Funding. Kay:Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees. Ding:Merck: Research Funding.

The Development and Expansion of Resistant Subclones Precedes Relapse during Ibrutinib Therapy in Patients with CLL

▪The Bruton's Tyrosine Kinase (BTK) inhibitor ibrutinib is very effective in chronic lymphocytic leukemia (CLL). Resistance in CLL is at least in part mediated by acquired mutations in BTK or down-stream PLCG2. Here we describe the largest institutional cohort of CLL patients treated with ibrutinib, focusing on risk factors for relapse, prevalence of known resistance mutations, and development of a monitoring strategy to identify patients at risk for relapse.All 308 patients from The Ohio State University Comprehensive Cancer Center participating in 4 clinical trials of ibrutinib were included. Deep sequencing for BTK and PLCG2 was performed using Ion Torrent Personal Genome Machine and covered coding regions of both genes. Preclinical experiments used XLA cell lines (Coriell Institute) infected with lentiviral constructs containing empty vector, wild type BTK, or C481S BTK.With a median follow-up of 40.5 months (range 4-71 months), 136 patients remain on study, 14 have received transplant or therapy elsewhere, and 158 have discontinued. 83 patients experienced disease progression, classified as Richter’s or PLL transformation (n=28) and progressive CLL (n=55). Using multivariable models, baseline risk factors for ibrutinib discontinuation due to transformation include complex karyotype (p<0.01) and MYC abnormalities on FISH (p=0.051). Age<65, del(17p) by FISH, and complex karyotype all associated with discontinuation due to CLL progression (p<0.05). Relapse generally has a poor prognosis, with a median survival from ibrutinib discontinuation for patients with transformation and progressive CLL of 3.9 (95% CI 2.0 to 10.1) and 22.7 (95% CI 13.5 to not reached) months, respectively.46 patients with progressive CLL had samples at relapse available for deep sequencing. Of these, 39 had mutations in BTK and/or PLCG2 (84.8%; 95% CI 71.1-93.7). Distribution of mutation included patients with BTK C481 mutation only (n=30), mutation in PLCG2 only (n=3) and both BTK/PLCG2 genes (n=6).Given the poor prognosis at relapse and presence of acquired mutations in the majority of progressive CLL patients, we were interested to evaluate whether the emergence of small clones of mutated cells could be used as a biomarker to predict relapse. For 15 patients with BTK or PLCG2 mutations, serial samples were available prior to relapse, and a clone of resistant cells could first be detected a median of 9.3 months prior to clinical relapse (95% CI: 7.6-11.7). Based on the finding that patients who relapsed on ibrutinib often had rapid progression and poor outcomes, we initiated a clinical-grade monitoring strategy in our institutional CLIA-certified molecular lab starting in November 2014. Mutational analysis of the entire coding regions of BTK and PLCG2 was performed on a cohort of 112 patients every 3 months. To date, 8 patients have clinically relapsed and all 8 had BTK C481S mutations with expansion of the clone prior to relapse. BTK C481S mutations of over 1% allelic frequency were detected in an additional 8 patients. All patients have had expansion of the resistant clones and all but one, who discontinued therapy without clinical relapse, have had increasing circulating CLL cells in the peripheral blood. Four of these 7 patients have also had increasing lymph node size on CT scan.In 11 patients who relapsed with BTK C481S, samples were available following discontinuation. In 5 patients treated subsequently with venetoclax, allelic fractions of C481S decreased dramatically or were eliminated, but in 6 patients treated with other agents, the mutant clone persisted. To investigate whether this was due to differences in biology between wild type and C481S BTK, we created XLA cell lines without BTK protein expression stably expressing BTK, or C481S BTK. C481S BTK showed enhanced BCR signaling as evidenced by pERK and cMYC expression, and enhanced migration compared to wild type BTK (p=0.04). This demonstrates for the first time that this acquired resistance mutation fundamentally alters the tumor cell biology.These data confirm our initial findings that relapse on ibrutinib is primarily mediated through mutations in BTK and PLCG2 and suggest that the presence of these mutations may have utility as an early biomarker to predict clinical relapse. Early identification of relapsing patients could improve outcomes by facilitating initiation of adjunct therapies such as venetoclax to eliminate the small resistant clone. DisclosuresWoyach:Karyopharm: Research Funding; Morphosys: Research Funding; Acerta: Research Funding. Jones:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding. Andritsos:Hairy Cell Leukemia Foundation: Research Funding. Awan:Innate Pharma: Research Funding; Pharmacyclics: Consultancy; Novartis Oncology: Consultancy. Lozanski:Beckman Coulter: Research Funding; Stemline Therapeutics Inc.: Research Funding; Boehringer Ingelheim: Research Funding; Genentech: Research Funding.

B cells and macrophages pursue a common path toward the development and progression of chronic lymphocytic leukemia.

The development and progression of chronic B-cell tumors depend on a complex microenvironmental network of cells that include monocyte-derived macrophages. In chronic lymphocytic leukemia (CLL) the survival of malignant cells is supported in vitro by nurse-like cells, which differentiate from CD14+ monocytes and have been identified as tumor-associated macrophages (TAMs). The role of the monocyte/macrophage lineage in CLL has been extensively studied in vitro, but only recently has been investigated in in vivo models. We here discuss how the cellular and molecular interactions that physiologically occur between B cells and macrophages can be subverted in chronic B lymphoid malignancies. Clinical approaches for the therapeutic targeting of TAMs are under evaluation. Promising strategies, along with a direct impact on the malignant cells, affect crucial pathways involved in the interaction of leukemic cells with TAMs. As an example, ibrutinib reduces CLL cell chemoattraction by inhibiting macrophage secretion of CXCL13. Lenalidomide and trabectedin prevent TAM recruitment mainly through CCL2 blockade. Most advanced strategies aim at depleting macrophages by targeting the CSF1/CSF1R pathway, which is fundamental for TAM survival. Of note, CSF1 transcripts are significantly more abundant in progressive CLL patients when compared with stable CLL and the frequency of CSF1R+ TAMs correlates with poor survival in hematological malignancies. The successful combination of CSF1R inhibition with currently available agents targeting malignant cells might represent the next therapeutic frontier in CLL. Conceivably these approaches may become applicable to numerous chronic B lymphoid malignancies.

Chronic Lymphocytic Leukemia-Associated Chromosomal Abnormalities and Risk of Secondary Malignancies

Introduction: Incidence of secondary malignant neoplasm in patients with chronic lymphocytic leukemia (CLL) is already established. Genetic predisposition has been considered to be a possible contributory factor for the preponderance of second cancers in this population. In the present study, we examined the frequency, characteristics, and clinical outcomes of secondary malignancies in patients with CLL based on their Interphase fluorescence in situ hybridization (FISH) panel.Methods: We reviewed the medical records of consecutive patients with CLL observed or treated at Ellis Fischel Cancer Center during the period from 2007-2014. We collected demographic data, CLL Rai stage, treatment history, Interphase fluorescence in situ hybridization (FISH) panel results and the presence of a secondary malignancy (skin cancers, solid tumors and hematologic malignancy excluding CLL transformation).Our aim was to investigate the association between secondary malignancy and chromosomal abnormalities and other risk factors using Chi2test for categorical variables and Wilcoxon rank-sum test for continuous variables. Cox proportional hazard models and logistic regression models were used to evaluate risk factors of developing secondary malignancy and overall survival.Results: We identified 142 CLL patients who were either observed (n=62) or had a history of treatment (n=80) for CLL. 67% were male and 33% were female. 85% of patients were Caucasians, 10% African Americans, and 5% other. Familial CLL was present in 6% of cases. 51% of patients were non-smokers. 5% of patients had cancers preceding the diagnosis of CLL (non-melanoma skin cancers=3, melanoma=1, Hodgkin’s lymphoma=1, Prostate=2). 28% of patients developed secondary malignancies after the diagnosis of CLL; among them non-melanoma skin cancers were the most common (78%), followed by cutaneous melanoma (9.5%) and papillary thyroid cancer (4.7%). Other malignancies included lung (2.3%), kidney (2.3%), prostate (2.3%) and ocular melanoma (2.3%). 12% patients had aggressive non-melanoma recurrent skin cancers that require multiple treatments along-with systemic therapy for progressive CLL.CLL-FISH panel at diagnosis was available in 98 patients. Among these patients,13q deletion was present in 45%. Within the 13q deletion group, secondary malignancy was noted in 50% during a median follow up of 7 years. All cases of papillary thyroid cancers were also present in 13q deletion. In contrast, 11q deletion was detected in 15% and trisomy 12 in 18% of patients with incidence of secondary malignancy 40% and 10%, respectively. No solid malignancy other than skin was identified in the 11q deletion and trisomy 12 groups. 17p deletion was present in 5% of cases and 60% of 17p deletion cases had secondary cancers (skin 80%). Normal FISH panel was present in 17% cases and 20% of normal FISH had secondary malignancy (solid 10%, skin 10%).The median overall survival time is 6 years for the entire cohort (IRQ: 3-9 years). Secondary malignancy was associated with worse overall survival (OS) (HR=0.57, 95% CI:0.33-0.98, p=0.04) compare to patients who did not have secondary malignancy. The risk of secondary malignancy is increased in patients with advanced age (OR=1.05, 95% CI:1.00-1.10, p=0.04) and in those who were treated with alkylating agent for CLL OR=6.62, 95% CI:2.08-21.03, p=0.001. However, there were no significant difference on risk of developing secondary malignancy based on specific chromosomal FISH result, Rai stage, smoking, family history and gender.Conclusion: Secondary malignancies are frequent in patients with detectable chromosome abnormality on FISH panel. However, the increased risk of secondary malignancy does not correlate with specific cytogenetic abnormality. Non melanoma skin cancers are exceedingly common in CLL patients and carries aggressive couse in progressive CLL patients. Larger studies are required to identify subtype of CLL based on integrated mutational and cytogenetic subgroup that are at increased risk of specific secondary malignancies. DisclosuresNo relevant conflicts of interest to declare.

NOTCH1 mutations identify a chronic lymphocytic leukemia patient subset with worse prognosis in the setting of a rituximab-based induction and consolidation treatment.

Induction therapy with fludarabine followed by rituximab and consolidation plus maintenance with rituximab improved response duration (RD) and overall survival (OS) in our patients with chronic lymphocytic leukemia (CLL). The aim of our study was to investigate the clinical impact of NOTCH1 mutations in this setting of patients. The study included 123 progressive CLL patients homogeneously assigned to first-line induction treatment with fludarabine followed by rituximab. Fifty-nine patients either in complete remission (CR) minimal residual disease positive (MRD+) after induction (n = 39) or in partial remission (PR, n = 20) underwent consolidation/maintenance therapy with rituximab. Sixteen patients in CR MRD + or PR underwent observation only. The presence of NOTCH1 mutations was investigated by amplification refractory mutation system (ARMS) PCR and by Sanger sequencing. NOTCH1 mutations occurred in 20 out of 123 (16.3 %) cases. Consolidated patients showed longer OS than unconsolidated patients (p = 0.030). Both NOTCH1 mutated and CR MRD+ or PR NOTCH1 mutated patients showed significantly shorter OS after treatment (p = 0.00014 and p = 0.0021, respectively). Moreover, NOTCH1 wild-type consolidated cases experienced significantly longer RD and OS than NOTCH1 mutated consolidated or not consolidated cases (p = 0.00001 and p = 0.018, respectively). Finally, the independent prognostic impact of NOTCH1 mutations for OS was confirmed in multivariate analysis (p < 0.001). The presence of NOTCH1 mutations identifies a CLL subset with worse prognosis in the setting of a rituximab-based induction and consolidation treatment.

The Tyrosine Kinase Receptor ROR1 Is Constitutively Phosphorylated in Chronic Lymphocytic Leukemia (CLL) Cells

Phosphorylation of receptor tyrosine kinases (RTKs) has a key role in cellular functions contributing to the malignant phenotype of tumor cells. We and others have previously demonstrated that RTK ROR1 is overexpressed in chronic lymphocytic leukemia (CLL). Silencing siRNA downregulated ROR1 and induced apoptosis of CLL cells. In the present study we analysed ROR1 isoforms and the phosphorylation pattern in CLL cells (n=38) applying western blot and flow-cytometry using anti-ROR1 antibodies and an anti-phospho-ROR1 antibody against the TK domain. Two major ROR1 bands with the size of 105 and 130 kDa respectively were identified, presumably representing unglycosylated (immature) and glycosylated (mature) ROR1 respectively as well as a 260 kDa band which may represent dimerized ROR1. A ROR1 band of 64 kDa that may correspond to a C-terminal fragment was also noted, present only in the nucleus. The 105 kDa ROR1 isoform was more frequently expressed in non-progressive as compared to progressive CLL patients (p=0.03). The 64, 105, 130 and 260 kDa bands were constitutively phosphorylated both at tyrosine and serine residues. Phosphorylation intensity of the mature (130 kDa) isoform was significantly higher in progressive than in non-progressive disease (p<0.001). Incubation of CLL cells with a mouse anti-ROR1 KNG or an anti-ROR1 CRD mAb respectively induced dephosphorylation of ROR1 before entering apoptosis. In conclusion CLL cells expressed different isoforms of ROR1 which were constitutively phosphorylated. The mature, phosphorylated ROR1 isoform was associated with a progressive disease stage. Targeting ROR1 by mAbs induced specific dephosphorylation and leukemic cell death. ROR1 might be an interesting therapeutic target.

T Cells From CLL Patients Recognize Spontaneously Peptides Derived of the Receptor Tyrosine Kinase Ror1

AbstractAbstract 3603 Background:The receptor tyrosine kinase Ror1 is a tumor-associated molecule over-expressed in chronic lymphocytic leukemia (CLL) and a variety of other malignancies with potential implication for targeted immunotherapy. Little is known about the functional role of Ror1 in the leucomogenesis of CLL. Aims: To assess spontaneous T cell response against Ror1 in CLL patients. Methods:Autologous T cell response against Ror1 was studied in 9 CLL patients and 6 healthy subjects expressing the HLA-A2 allele. Four synthetic 9-mer HLA-A2 restricted peptides selected from different parts of the Ror1 molecule were loaded on dendritic cells and co-cultured with enriched autologous T cells. T cell response was determined by enumeration of the frequency of IFN-γ, IL-5 and IL-17A secreting T cells by ELISPOT. Cell proliferation was determined by 3H-thymidine incorporation. Results:Our results demonstrated a significantly higher frequencies of IFN-γ producing T cells (p<0.05-0.0001) and to a lesser extent IL-17A secreting autologous T cells (p<0.05) in response to Ror1 peptides in CLL patients compared to healthy controls. No IL-5 secreting T cells were noted. The frequency of IFN-γ secreting T cells was significantly higher in non-progressive as compared to progressive CLL patients (P<0.05). No differences were observed between patients with mutated and unmutated immunoglobulin heavy chain variable region (IGHV) genes. Conclusion:Ror1 may spontaneously induce mainly a type 1 T cell response in CLL patients, particularly in those with non-progressive disease as compared to progressive disease. Ror1 may be an immunodominant antigen in CLL which, has also been suggested by Fakuda et al. PNAS, 105, 3047, 2008. Active immunotherapy using Ror1 as a target might be an interesting approach to test in the clinic. Disclosures:No relevant conflicts of interest to declare.

Immunoglobulin heavy chain variable region gene usage and mutational status of the leukemic B cells in Iranian patients with chronic lymphocytic leukemia

The mutational status of the immunoglobulin variable region heavy chain genes (IGHV) is an important prognostic marker in chronic lymphocytic leukemia (CLL). The data accumulated in the literature has largely been derived from studies conducted on Caucasian Western populations. Little is known about Asian CLL patients. In this study the IGHV genes usage and somatic hypermutation status have been investigated in 87 Iranian CLL patients. Based on a cut-off of 98% nucleotide sequence homology, 64.4% and 35.6% of the patients expressed mutated and unmutated IGHV genes, respectively, with most non-progressive patients being in the mutated group (35/44 vs 19/40; P = 0.009). Progression-free survival (PFS) and time to first treatment (TTFT) were significantly higher in our mutated and non-progressive patients compared to unmutated and progressive subtypes, respectively. The most frequently used IGHV gene was IGHV3-7 (12.6%) followed by IGHV3-30 (11.4%), IGHV3-48 (9.2%), IGHV4-39 (6.9%), and IGHV1-8 (6.9%) genes, which taken together comprised nearly half of the IGHV genes expressed in the Iranian CLL patients. Of the IGHV genes, IGHV3-7 was significantly over-represented in non-progressive compared to progressive CLL patients (P = 0.036), whereas IGHV1-69 and IGHV1-2 were expressed at a higher frequency in unmutated compared to mutated CLL patients (P < 0.03). Comparison of IGHV gene usage in our patients with that of Western CLL patients revealed significant differences in expression of IGHV1-69, IGHV3-7, IGHV3-21, and IGHV4-34 genes. Analysis of the IGHV third complementary determining region (HCDR3) sequences revealed a high frequency use of certain HCDR3 motifs, such as YYYGMDV, in our samples. These findings imply contribution of antigen selection and regional (ethnic/geographic) parameters in the leukomogenesis of CLL.