Chinese Chronic Lymphocytic Leukemia Patients Research Articles

IGH Translocations in Chinese Patients With Chronic Lymphocytic Leukemia: Clinicopathologic Characteristics and Genetic Profile.

Immunoglobulin heavy chain translocations (IGH-t) have occasionally been reported in Chinese patients with chronic lymphocytic leukemia (CLL). The objective of the present study was to identify the clinicopathologic features of patients with IGH-t CLL and compare them with those of patients with non-IGH-t CLL. We performed fluorescence in situ hybridization (FISH) based on a routine CLL prognostic FISH panel using IGH, IGH-BCL2, BCL3, IGH-CMYC, and BCL6 FISH probes. Furthermore, we retrospectively evaluated the clinical features of 138 newly diagnosed CLL patients via chromosome banding analysis (CBA), FISH, and targeted next-generation sequencing. IGH-t was identified in 25 patients (18.1%). Patients with IGH-t CLL had lower flow scores than those with non-IGH-t CLL. The most frequent translocation was t(14;18) (10 patients), followed by t(14;19) (3 patients), and t(2;14)(p13;q32), t(7;14)(q21.2;q12), t(9;14)(p13;q32) (3 patients). The remaining nine patients included three with abnormal karyotypes without translocation involving 14q32, four with a normal karyotype, and two who failed CBA. The most frequently concomitant FISH-detected aberrations were 13q deletion, followed by +12 and TP53 deletion, while one case involved ATM deletion. Complex karyotypes were detected in five patients with IGH-t CLL, in whom all partner genes were non-BCL2. Available mutational information indicated that KMT2D mutation was the most frequent mutation among tested 70 patients, while TP53 mutation was the most frequent mutation in the IGH-t group. Moreover, the IGH-t group had higher FBXW7 (P=0.014) and ATM (P=0.004) mutations than the non-IGH-t group, and this difference was statistically significant. Our study demonstrates that IGH-t is not uncommon among Chinese CLL patients, and that its partner genes are multiple. The gene mutational profile of the IGH-t group was distinct from that of the non-IGH-t group, and the concomitant chromosomal abnormalities within the IGH-t CLL group differed. Thus, identification of IGH-t and its partner genes in CLL patients may help further refine risk stratification and strengthen the accurate management in CLL patients.

Distinct Age-Related Clinical Features and Risk Assessment in Chinese With Chronic Lymphocytic Leukemia.

The biological and clinical features of chronic lymphocytic leukemia (CLL) exhibited profound heterogeneity across Chinese and patients of predominately European descent. However, the age-related peculiarities and risk assessment of Chinese CLL patients remained ill-defined. The present study demonstrated that CLL patients were characterized by the earlier age at onset in China (median age at diagnosis: 63 years old) than in the United States (median age at diagnosis: 69 years old). Young patients from Shandong Provincial Hospital CLL database displayed prolonged overall survival than the Surveillance, Epidemiology, and End Results cohort. Furthermore, among Chinese CLL patients, young patients showed an increased relapse rate compared with elderly patients. To optimize the risk assessment of CLL patients, novel risk score models named PR-Score and HBG-Score were developed for predicting the outcomes of young and elderly CLL patients respectively. The neonatal survival prediction systems were superior to international prognostic index for CLL (CLL-IPI) and Binet stage in assessing the overall survival and progression free survival of CLL patients. The analyses highlighted refinement of risk evaluation for CLL patients in different age groups, providing insights into individualized diagnosis and treatment of CLL.

Comprehensive Profile of Young and Elderly Chronic Lymphocytic Leukemia Patients in China: The Younger Population Compared with Western Countries

IntroductionThe epidemiological features of chronic lymphocytic leukemia (CLL) are different in the United States (US) and China. In addition to variant incidence rates, the median age at diagnosis of CLL patients in the Shandong Provincial Hospital CLL (SPHCLL) database is younger than the Surveillance, Epidemiology, and End Results (SEER) database. However, investigations on the association between age at onset and other clinical characteristics of Chinese patients with CLL remain unclear. Hence, the aim of this study was to explore the clinical-pathological parameters and prognosis of Chinese patients with CLL which were divided into the young (<60 years) group and the elderly (≥60 years) group.MethodsThe clinical data of 510 Chinese CLL patients diagnosed between October 2010 and June 2020 were obtained from the SPHCLL database. Meanwhile, the survival data of 2580 CLL patients diagnosed between 2010 and 2018 were obtained with SEER*Stat 8.1.5 from the SEER database. Patients <60 years were divided into the young group and patients ≥60 years were divided into the elderly group. Clinical-pathological parameters were accessed from the hospital-based laboratory service within 24 hours after the first admission. SPSS 23 and R 4.0.5 were used in analyzing the data. Moreover, Kaplan-Meier survival analysis, univariate Cox regression analysis and multivariate Cox regression analysis were performed to evaluate the prognosis of CLL patients. Comparisons of clinical characteristics between young and elderly CLL patients were made using t test and Chi-square test. p < 0.05 was defined as the borderline of statistical significance.ResultsThe median age at diagnosis of Chinese patients, Asian American patients and patients of predominately European descent were 62, 66 and 69 years respectively (Figure 1A-C). After adjustments according to the population age distribution of China and the US, patients in the Chinese SPHCLL database presented earlier age at onset than the SEER database (Figure 1D).To further explore the clinical characteristics of young and elderly Chinese CLL patients, the general clinical data and laboratory parameters were analyzed among young and elderly CLL patients. Compared with elderly CLL patients, young CLL patients showed a higher percentage of cytogenetic aberrations detected by fluorescence in situ hybridization (p<0.05). The proportion of patients with combined diseases was higher in elderly patients than young patients, especially coronary heart disease and hypertension (p<0.05). The levels of prealbumin, albumin, Apolipoprotein A and Lp (a) were higher in young CLL patients, while the levels of β2-macroglobulin were lower in young CLL patients (p<0.05). Moreover, the proportion of young CLL patients who accepted CHOP/RCHOP treatment was significantly higher.The overall survival (OS) of young CLL patients was better than elderly patients in the SPHCLL cohort and the SEER cohort (Figure 1E-F). Intriguingly, young Chinese patients experienced improved OS compared with young patients of predominately European descent (Figure 1G-H). Furthermore, univariate Cox regression analysis presented that apolipoprotein A, hemoglobin, thrombocyte and total cholesterol were positive prognostic factors among young patients, while β2-microglobulin and ADA were negative prognostic factors. In elderly CLL patients, univariate Cox regression analysis showed that albumin, apolipoprotein A, hemoglobin and superoxide dismutasewere positive prognostic factors, while β2-microglobulin, ADA and cystatin were negative prognostic factors. Multivariate Cox regression analysis showed that cystatin was an independent prognostic factor in elderly CLL patients.ConclusionIn conclusion, Chinese CLL patients are characterized by earlier age at onset than patients of predominately European descent. The investigation presented a comprehensive profile of young and elderly CLL patients from China, contributing to the optimal management strategies of CLL patients in variant age groups. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Comparison of four prognostic scoring system in patients with early asymptomatic chronic lymphocytic leukemia patients

目的比较四种预后评分系统预测Binet A期中国慢性淋巴细胞白血病（CLL）患者诊断到治疗时间（TTFT）的预后评估价值。方法回顾性分析南京医科大学第一附属医院（浦口慢淋中心）2009年6月至2020年1月诊断的110例Binet A期CLL患者的基线临床资料，采用无症状早期CLL国际预后评分（IPS-E）、CLL国际预后指数评分（CLL-IPI）、CLL1预后评分模型（CLL1-PM）与巴塞罗那预后评分（Barcelona-Brno）模型对患者进行危险度分层和预后评估。结果110例Binet A期CLL患者中位年龄58（25～84）岁，中位随访时间35（4～189）个月，其中57例（51.8％）患者因病情进展达到治疗指征启动治疗。对患者年龄、Rai分期、淋巴细胞绝对计数（ALC）、淋巴结大小、淋巴细胞倍增时间（LDT）、β2-微球蛋白、IGHV突变状态、TP53缺失和（或）突变、11q缺失等9个因素进行Log-rank检验，其中RaiⅠ～Ⅲ期、ALC>15×109/L、淋巴结≥1 cm、β2-微球蛋白>3.5 mg/L、IGHV无突变、TP53缺失和（或）突变、11q缺失是影响TTFT的独立危险因素。使用一致性指数（C-index）和赤池信息准则（AIC）对上述模型进行预后评估效能评价，其中CLL1-PM C-index＝0.736，AIC＝777；CLL-IPI C-index＝0.722，AIC＝933；IPS-E C-index＝0.683，AIC＝1004；Barcelona-Brno C-index＝0.663，AIC＝986。结论四种预后评分模型均具有预测TTFT的效能。IPS-E因纳入指标的临床可及性高，价格较低，可作为指导临床检测的理想工具。对于完善FISH及二代测序检查的患者，使用CLL-IPI或CLL1-PM可进行更为全面的预后评价。

Low prevalence and independent prognostic role of del(11q) in Chinese patients with chronic lymphocytic leukemia

The 11q deletion (del(11q)) is a conventional cytogenetic aberration observed in chronic lymphocytic leukemia (CLL) patients. However, the prevalence and the prognostic value of del(11q) are still controversial. In this research, we retrospectively explored the prevalence, association, and prognostic significance of del(11q) in 352 untreated and 99 relapsed/refractory Chinese CLL patients. Totally 11.4% of untreated and 19.2% of relapsed/refractory patients harbored del(11q). Del(11q) was more common in patients with β2-microglobulin > 3.5 mg/L, positive CD38, positive zeta-chain associated protein kinase 70, unmutated immunoglobulin heavy variable-region gene and ataxia telangiectasia mutated mutation. Kaplan-Meier method and univariate Cox regression indicated that del(11q) was an independent prognostic factor for overall survival (OS). Based on the results of univariate Cox regression analysis, two nomograms that included del(11q) were established to predict survival. Desirable area under curve of receiver operating characteristic curves was obtained in the training and validation cohorts. In addition, the calibration curves for the probability of survival showed good agreement between the prediction by nomogram and actual observation. In summary, the prevalence of del(11q) is relatively low in our cohort and del(11q) is an unfavorable prognostic factor for untreated CLL patients. Besides, these two nomograms could be used to accurately predict the prognosis of untreated CLL patients.

Validation of the Chinese EORTC chronic lymphocytic leukaemia module \u2013 application of classical test theory and item response theory

PurposeThe association of chronic lymphocytic leukemia (CLL) with health-related quality of life (HRQoL) is rarely studied globally. This study evaluated the psychometric properties of the EORTC-Chronic Lymphocytic Leukaemia (CLL17 [phase III]) module, a newly developed assessment on CLL patients’ HRQoL, among Chinese CLL patients.MethodsThe Chinese CLL17, comprised of three subscales (symptom burden [SB], physical condition [PC] and worries/fears [WF]), was provided by the developer team through EORTC. A cross-sectional online survey was conducted to collect data. The classical traditional theory (CTT) and the item response theory (IRT) were used to evaluate the psychometric properties of CLL17. Internal consistency reliability was determined by the Cronbach’s alpha and item-total correlation. Dimensionality was verified through confirmatory factor analysis (CFA). Convergent validity was also assessed. The generalized partial credit model was used for the IRT. The difficulty, discrimination, item fit, and differential item functioning (DIF) were calculated to assess the instrument’s psychometric properties.ResultsIn all, 318 patients, aged between 26 and 82 years, completed the questionnaire. A good level of internal reliability was achieved (Cronbach’s alpha = 0.92). The item-total correlation coefficient ranged from 0.46 to 0.72. There was a mid-to-high correlation between CLL17 and domains of EQ-5D and QLQ-C30. The IRT model showed a satisfactory homogeneity, item fit and good discrimination of items, except for item 4, 6 and 16 (< 1.0). low information provided by item 16 and 17. SB and PC provided more information with theta > 0, whereas WF provided more information with theta < 0. Item 17 perform inconsistently for respondents from different age groups (DIF).ConclusionThe EORTC-CLL17 Chinese version shows acceptable reliability and validity, making it a valuable instrument to evaluate the impact on the HRQoL of Chinese CLL patients.

Genomic Landscape of Chinese Patients with Chronic Lymphocytic Leukemia By Whole-Exome Sequencing

Introduction: Through previous studies using Sanger sequencing and targeted gene sequencing, we found that the frequencies of certain genes in Chinese patients with chronic lymphocytic leukemia (CLL) were different from those in CLL patients from the western countries. However, until now, there are no published whole-exome sequencing (WES) studies in Chinese CLL patients. To better define the genomic landscape of CLL cases in China, we conducted WES studies of CLL cases in our center. Methods: This study include 43 CLL patients, of whom 40 were untreated at the time of sampling. The median age of the 43 patients were 61 years, the male-to-female ratio was 2:1. We isolated peripheral blood mononuclear cells, then CD19 positive cells were purified by using magnetic microbeads in some cases. The buccal cells or the leukocytes that were depleted of CD19 positive cells were used as germline controls. We purified genomic DNA and used the purified DNA for library construction, after which we sequenced the library using the Illumina HiSeq2500 platform. After sequencing, software including Burrows-Wheeler Aligner (BWA) and GATK were used for the bioinformatic analysis. Results: In the 43 Chinese patients with CLL, the median number of somatic mutations was 67 (range:10-128) and the median number of nonsynonymous mutations was 26 (range: 5-55). We found that the number of mutations in patients older than 60 was significantly higher than that in the younger patients. Somatic mutations were detected across 991 different genes, of which 24 genes were identified as drivers in the study from the Dana-Faber Cancer Institute (DFCI). The 24 genes included NOTCH1, SF3B1, MYD88, POT1, TP53 and so on. Seventy-nine genes were recurrently mutated and 13 genes were found to be mutated in 3 or more cases. The most frequently mutated gene was NOCTH1 (6 cases, 14.0%). FBXW7, another gene that is involved in the regulation of the NOTCH1 pathway, was also mutated in 3 cases. IGLL5, MUC4 and MYD88 were also among the most frequently mutated genes, with each of them being mutated in 5 cases (11.6%). We found that the frequencies of mutations in IGLL5 (11.6% vs. 2.2%, P=0.0056), MYD88 (11.6% vs. 3.0%, P=0.0147), BCOR (9.3% vs. 2.2%, P=0.0246), and SF3B1 (4.7% vs. 21.0%, P=0.0084) were significantly different between Chinese CLL patients and patients from western countries. BCOR-mutated patients had more frequent aberrations involving NOTCH1 pathway (NOTCH1 and/or FBXW7 mutation) than BCOR-unmutated patients (3/4 [75.0%] vs. 6/39 [15.4%], P=0.0242) (Figure 1A). Analysis of 10967 tumor samples from the Cancer Genome Atlas (TCGA) program revealed that tumors with BCOR mutations had a higher frequency of NOTCH1 aberrations (NOTCH1 and/or FBXW7 mutation) than those without BCOR mutations (116/349 [33.2%] vs. 722/10618 [6.8%]; odds ratio: 6.824, 95%CI: 5.393-8.634, P&lt;0.0001) (Figure 1B). These data suggest BCOR mutations and NOTCH1 aberration may cooperate in the pathogenesis of CLL and other types of tumors. NFKBIA mutations were identified in 2 cases (Figure 1C) and we found that the mRNA level of NFKBIA was significantly lower in the NFKBIA mutated CLL cells than in the normal B cells (Figure 1D-E); in the MEC-1 cell line, significant activation of NF-кB pathway was observed after knockdown of NFKBIA using siRNA (Figure 1F). Conclusion: Patients with CLL in China and patients with CLL in the western countries have different mutational landscapes. BCOR gene mutations may cooperate with aberrations involving the NOTCH1 pathway in the pathogenesis of CLL. NFKBIA could be a potential tumor suppressor in the pathogenesis of CLL. Figure 1 Disclosures No relevant conflicts of interest to declare.

High Frequency Mutations of MYD88 and KMT2D in Chinese Chronic Lymphocytic Leukemia

Background: Chronic lymphocytic leukemia (CLL) is one of the most common forms of adult leukemia in the Western population but rare in Chinese descent. As Chinese CLL patients have distinct clinical features, higher frequency of mutated IGHV as well as unique BCR stereotype, we hypothesize that genetic alterations may contribute to the disease phenotype differences. We profiled the known recurrent mutated genes associated with Western CLLs in 338 Chinese CLL samples, with the goal to define the somatic mutation pattern in the Chinese CLL population. Methods: We first assessed 96 CLL mutated genes in a discovery cohort that includes 126 CLL samples using Illumina MiSeq platform. We then validated 20 recurrent mutated genes in a validation cohort that comprises of 212 CLL samples with Ion Torrent platform. The presence of mutation in DNA and mRNA were further validated by Sanger sequence. Results: A total of 303 untreated and 35 relapsed samples were investigated. The average sequencing depth was 1957X (range: 112 to 4254). In the discovery cohort, we identified recurrently mutated genes in TP53 (17.5%), SF3B1 (14.3%), ATM (13.7%), KMT2D (12.7%), MYD88 (10.3%), NOTCH1 (8.7%), FBXW7 (8.7%), BIRC3 (7.9%), POT1 (4.8%), KRAS (4.0%). This was further confirmed in the validation cohort. In comparison to the frequency of these mutations in the western studies, we observed an unexpected significantly higher frequency of mutations in MYD88 and KMT2D (Chi-squared test, p&lt;0.01). Mutations of KMT2D occurred with a similar frequency in treatment-naïve (8%) and relapsed samples (9%). 19/30 (63%) mutations were loss-of-function as they are predicted to generate truncated proteins due to lacking part or all of the C-terminal domains (Figure 1A). We observed a significant co-occurrence between KMT2D mutations and trisomy 12 (Chi-squared test, p&lt;0.01, Figure 1E), suggesting there could be intrinsic linkage between these lesions. Further integration with IGHV mutation status revealed that CLLs with KMT2D mutations were characterized by an overrepresentation of VH4-34 and VH4-39 (p&lt;0.01, p=0.03, respectively) and enriched in stereotyped subset #8 (4/12, 33.3%) (Figure 1B). Consistent with previous reports that VH4-34 and VH4-39 are commonly used in Asians CLLs, we confirmed the high frequency of subset #8 in Chinese patients [1], indicating KMT2D as a uniquely mutated gene in Chinese CLLs. MYD88 mutations were found in 38/303 (13%) untreated CLLs. 17/38 (45%) of the mutations were at the L265P site (Figure 1C). Additional hotspot mutations were also detected (V217F 29%; S219C 13%). These mutations are associated with a significant male predominance (Chi-squared test, p&lt;0.01), increased monoclonal gammopathy (15%, p=0.01), and a higher rate of Hepatitis B virus (HBV) infection (HBV core antibody positivity 56%, p=0.02). Most of these mutations (87%) were clonal and IGHV mutated (97%). It is worth to note that the usage of VH3-7 and VH4-34 were more dominant in patients with MYD88 V217F/S219C (6/16, 38%) and with L265P (6/17, 35%), respectively. Mutations of MYD88 are mutually exclusive with TP53 mutation and del(17p) (Chi-squared test, p&lt;0.01). Moreover, we found a superior time to first treatment in MYD88-mutated patients compared with wide-type patients (Log-rank test, p=0.03). To further understand the linkage between mutations and clinical outcome, we divided 303 treatment-naive patients into two groups: Non-progressor (NPRO, 39%) and Progressor (PRO, 61%). NPRO and PRO were classified as patients who developed symptoms that need treatment either more than or less than three years after sampling. Epigenetic modifiers pathway mutations (KMT2D, CREBBP, EP300, TET2, ARID1A and IKZF1) were more frequently in PRO group (17% vs. 8%, p=0.02). Inflammatory pathway mutations (MYD88, BIRC3, CARD11 and TRAF3) was more involved in NPRO group, compared to PRO or Relapsed samples (21%, 15% and 0%, p&lt;0.01). Conversely, DNA damage pathway mutations (TP53, ATM and POT1) was more frequently found in Relapsed and PRO groups compared to NPRO group (49%, 30% and 7%, p&lt;0.01). Conclusion: In summary, we observed high frequency mutations in KMT2D and MYD88 that correlated with specific IGHV repertoire in Chinese CLL population. This suggests that pathogenesis of CLL between the Chinese and Western population may be different and potentially have therapeutic value. [1] Marinelli M, et al. Oncotarget. 2016 Apr 12;7(15):20520-31. Figure 1 Disclosures No relevant conflicts of interest to declare.

SF3B1 mutation predicts unfavorable treatment-free survival in Chinese chronic lymphocytic leukemia patients.

Splicing factor 3b subunit 1 (SF3B1), a splicing factor modulating RNA alternative splicing, is frequently mutated in multiple hematological malignancies including myelodysplastic syndromes and chronic lymphocytic leukemia (CLL). The clinical impact of SF3B1 mutation on CLL remains controversial especially for patients of Asian descent. We retrospectively analyzed the frequency of SF3B1 mutation by Sanger sequencing in 399 newly diagnosed Chinese CLL patients. SF3B1 mutation was detected in 5.5% (22/399) of the studied cohort with 59.1% of them being c.A2098G (p.K700E). SF3B1 mutation was common in patients with unmutated immunoglobulin heavy chain variable region gene, positive CD38 and positive ZAP-70. Survival analysis showed that SF3B1 mutation was associated with short treatment-free survival (TFS), but not overall survival (OS). We then developed 2 new risk models, named CLL-IPI-S and CLL-PI, according to the SF3B1 mutation status and CLL-international prognostic index (CLL-IPI); CLL-PI showed greater power to predict TFS than CLL-IPI in Chinese CLL patients. Our data suggest a low incidence and adverse clinical significance of SF3B1 mutation in newly diagnosed Chinese CLL patients.

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.

Transcriptome Sequencing Reveals Alternative Splicing Patterns and an Increased Sensitivity to Spliceosome Inhibition Associated MYD88 L265P Mutation in Chronic Lymphocytic Leukemia

Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in the Western countries but is relatively rare in East Asia. Mutations in myeloid differentiation primary response gene 88 (MYD88) occur in 2.0-4.4% of Caucasian patients with CLL. However, Asian subjects showed a higher MYD88 mutation frequency of 8% and MYD88 mutations were associated with unfavorable prognosis in Asian CLL patients with mutated IGHV gene. To further explore the molecular and pathological mechanisms of the MYD88 L265P mutation in CLL, we performed a pilot study to investigate the transcriptome-wide differential expression between CLL patients with and without MYD88 L265P mutation.Methods: mRNA sequencing was performed on a cohort of 15 Chinese CLL patients with MYD88 L265P mutation (n=5) or wild-type MYD88 gene (n=10). Differential expression and pathway enrichment analyses were performed based on FPKM values after processing the raw sequencing reads. The alternative splicing events were analyzed using JuncBASE and the differences in splicing patterns were compared between the two groups. The MYD88 L265P and wild-type genes were overexpressed in the MEC-1 cell line using lentiviral vectors, and stable cell lines were established after puromycin selection. Real time quantitative PCR was used to determine the mRNA expression of spliceosome pathway genes. The protein expression of TLR pathway was assessed with Western Blot. The PrestoBlue™ assay was used for determining IC-50 of spliceosome inhibitor-treated CLL cell lines, and flow cytometry was utilized for apoptosis and cell death analyses.Results: mRNA sequencing analysis revealed that 4231 genes showed significant differences in mRNA expression between MYD88 mutant and wild-type CLL patients. Of these genes, 3033 were up regulated and 1198 were down regulated in patients with MYD88 L265P mutation. Pathway analysis demonstrated that tumor necrosis factor (TNF) signaling, cell cycle, spliceosome, ribosome, p53 signaling, and NF-κB signaling pathways were significantly enriched among differentially expressed genes. Interestingly, about two thirds (19 out of 30) of differentially expressed genes in the spliceosome pathway were down regulated in CLL samples with MYD88 mutation. Quantitative RT-PCR confirmed the down regulation of spliceosome genes including SF3A1, SF3A2, SF3B4, SF3B5 and PUF60 in MYD88 mutated samples in an independent CLL patient cohort. Analysis of alternative splicing events revealed a total of 180 genes with inter-group splicing differences, among which differential splicing events were most common in the genes involved in immune system regulation, i. e. CD22, CD79B, CD200 and IKZF1 etc. The MEC1 cells stably overexpressing MYD88 L265P demonstrated decreases in RelA and TRAF6 expression as compared to wild type MYD88-overexpressing cells, which was in agreement with RNA-seq analysis results in CLL patient samples. In addition, we found that overexpression of MYD88 L265P mutant resulted in decreased mRNA expression of several spliceosome genes such as SF3A1, SF3A2, SF3B4, SF3B5 and PUF60 in the stable cell line. Furthermore, MYD88 L265P increased the sensitivity of MEC1 cells to spliceosome inhibitor sudemycin D6 (SD6) by 3 fold, while reducing their sensitivity to Ibrutinib by two fold.Conclusion: We discovered the differential mRNA expression landscapes related to MYD88 mutation status in CLL and their contributions to the progression of CLL through multiple pathways. Of particular interest was the down regulation of spliceosome pathway genes. In a CLL cell line model, we found that, compared with the MYD88 wild-type CLL cell line, CLL cells overexpressing the mutant MYD88 were more sensitive to spliceosome inhibitor SD6, suggesting that inhibiting spliceosome function may be a potential therapeutic target for CLL patients with mutant MYD88 gene. In summary, our findings provide novel insights for further understanding of the molecular and pathological mechanisms of MYD88 mutation in CLL. DisclosuresLagisetti:St. Jude Children's Research Hospital: Patents & Royalties: Chandraiah Lagisetti is listed as inventors on patents assigned to St. Jude Children's Research Hospital covering sudemycin D6 and was employed by SRI Biosciences at the time these experiments were carried out. Webb:St. Jude Children's Research Hospital: Patents & Royalties: Thomas R. Webb is listed as inventors on patents assigned to St. Jude Children's Research Hospital covering sudemycin D6 and was employed by SRI Biosciences at the time these experiments were carried out..

Chinese External Validation of the Cochrane Haematological Malignancies Group Prognostic Index for Chronic Lymphocytic Leukemia Patients

Objection:Chronic lymphocytic leukemia (CLL) is a chronic lymphoproliferative disease characterized by highly clinical and biological heterogeneity. A number of biomarkers have been identified in predicting the overall survival (OS) over the last decades besides the traditional clinical staging. Recently, an international prognostic index (IPI) combing clinical staging and biomarkers was developed by the investigators of the Cochrane Haematological Malignancies Group. Due to genetic differences between Caucasic and Chinese CLL patients, our study was to validate the guiding function of IPI on Chinese CLL cases.Method:We performed a validation of the IPI proposed by the Cochrane Haematological Malignancies Group to stratify Chinese CLL patients prognostically in 225 CLL cases registered at our center. The five parameters (age, TP53 abnormalities, IGHV mutation status, b2-microglobulin and Binet stage) involved in the IPI were collected by clinical data, serum test, PCR and fluorescence in situ hybridization (FISH). Chi-square test, survival analysis, log-rank test and cox hazard regression analysis were utilized in the validation.Result:In the 225 Chinese CLL cases analysed in the validation, all five parameters involved in the IPI were associated with overall survival (OS) independently. The multivariate analysis demonstrated that age above 65 years old (HR 2.22; [1.15-4.30]; P=0.018), b2-microglobulin over 3.5 mg/L (HR 2.46; [1.22-4.94]; P=0.001), Binet staging B/C (HR 3.40; [1.02-11.33]; P=0.046), TP53 abnormalities (HR 2.72; [1.50-4.94]; P=0.012) and IGHV unmutation (HR 5.19; [2.51-10.77]; P<0.001) were OS related risk factors respectively. Then a total point score was calculated for each patient according to the grading system proposed by the Cochrane Haematological Malignancies Group investigators. There were 60 (26.7%) patients at low-risk (scoring 0-1), 57 (25.3%) patients at intermediate-risk (scoring 2-3), 65(28.9%) patients at high-risk (scoring 4-6) and 43 (19.1%) patients at very high-risk (scoring 7-10). The IPI allowed different prediction of time to treatment (TTT) in all groups (Fig. 1). The estimated median TTT were: 102 months for low-risk, 12 months for intermediate-risk and 1 month for high-risk group. However, the low-risk and intermediate-risk groups showed similar overall survival (P=0.424). Beyond that, significant difference was found between the intermediate, high and very high-risk groups. We combined the low-risk and the intermediate-risk groups into one to accommodate to the Chinese CLL cases. 117 (52%) patients were at low & intermediate-risk (scoring 0-3), thus leading to the significantly different prognostic value between groups (Fig. 1) . The estimated median survival times were: not reached for low&intermediate-risk, 63 months for high-risk and 128 months for very high-risk group.Conclusion:Our results basically validated the IPI proposed by the Cochrane Haematological Malignancies Group to prognostically stratify CLL patients in China, which confirmed the value of the novel prognostic index externally. However, a slight adaption was made to accommodate the Chinese cases better via the combination of the low-risk and intermediate-risk groups. We considered that a universally recognized prognostic model would be utilized to predict the disease progression and guide the treatment when initially diagnosed. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Expression and regulation of CacyBP/SIP in chronic lymphocytic leukemia cell balances of cell proliferation with apoptosis.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western countries, with incidence in Chinese populations also increasing. CLL involves an accumulation of abnormal B cells which result in dysregulation of cell proliferation and apoptosis rates. The calcyclin-binding protein/Siah-1-interacting protein (CacyBP/SIP) plays a pivotal role in tumorigenicity and cell apoptosis. Here, we investigated the function of CacyBP/SIP in CLL cell proliferation and apoptosis. CacyBP/SIP expression levels were measured in peripheral blood mononuclear cells from 23 Chinese CLL patients and three healthy donors by western blotting. Correlation analysis was performed to assess associations between CacyBP/SIP expression and clinical stage, chromosome abnormalities and zeta-chain-associated protein kinase 70 (ZAP-70) expression. We silenced CacyBP/SIP expression in MEC-1 cells using a lentivirus system and analyzed cell vitality, cell cycle and tumorigenicity. Apoptosis was also analyzed following the upregulation of CacyBP/SIP expression in MEC-1 cells. Downregulation of CacyBP/SIP expression in CLL patients was negatively correlated with CLL clinical stage, but not with patient sex, age, del(13q14) or del(17q-) presence, or ZAP-70 expression. CacyBP/SIP silencing significantly enhanced cell proliferation and tumorigenicity. CacyBP/SIP silencing promoted accumulation of cells in S phase by upregulation of β-catenin, cyclin D1 and cyclin E, and downregulation of p21. Moreover, CacyBP/SIP overexpression facilitated CLL apoptosis through the activation of pro-caspase-3. CacyBP/SIP is a useful indicator of CLL disease processes and plays an important role in sustaining the balance of cell proliferation and apoptosis.

Distinct IGHV Repertoire Features In Chinese Chronic Lymphocytic Leukemia Patients

Mounting evidence indicates that immunoglobulin variable heavy-chain (IGHV) repertoire and mutational status in chronic lymphocytic leukemia (CLL) patients are prognostically relevant. However, rare data is available in Chinese CLL population. Our group investigated 270 Chinese CLL patients for their IGHV sequences and discovered significant differences between Chinese and European CLL patients. First of all, 169 (62.6%) patients in our group got mutated IGHV and 101 (37.4%) were unmutated, rendering a considerable higher percentage of mutated subgroup compared with European patients (55%) (Figure 1). While IGVH3 is still the most frequently used IGVH gene in Chinese CLL patients (135/270, 50%), discrepancy occurs in the usage of IGVH1 gene, which only presents in 13.7% (37/274) patients in our cohort whereas 23.79% for European (Figure 2). Regarding IGHV subgroups, IGHV3-23 and IGHV4-34 are more often used in Chinese CLL patients (10.7% and 10.4%, respectively). Remarkably, IGHV1-69, the most prevalent IGHV subgroup in European CLL patients (12.81%), only accounts for 5.2% (14/270) Chinese cases. [Display omitted] [Display omitted] [Display omitted] We further studied the distribution of stereotyped BCR in our cohort. Thirty-eight patients (14.07%) with stereotyped BCR that belonged to 21 subsets were identified, with 1 to 7 sequences contained each. Among them, subset 1 and subset 8 are the most common types with 6 and 7 cases respectively. Three new subsets were discovered (Table 1). Notably, only 1 case belonged to subset 2, the subset with largest group size in western world. Hence, we conclude that Chinese CLL patients show unique IGHV repertoire features compared to patients from western countries. While the mechanism within remains unknown, the discrepancy might due to antigenic difference in geographically remote areas.Table 1Three new subsets of BCR stereotypy in Chinese CLL patientsNO.IGHVIGHDIGHJM/UMIdentityHCDR3 AA sequenceLengthNovel 1NJ-15IGHV4-59*083-22*016*03UM100,00%ARGNYYDSSGYYYVGYYYYYMDV23NJ-31IGHV4-59*013-22*016*03UM99,65%ARGDYYDSSGYYYVGYYYYYMDV23Novel 2NJ-186IGHV3-23*013-22*014*02M96.60%AKGYRDNYDGDQSSVFDS18NJ-23IGHV3-23*012-21*014*02M96,53%AKGYRDNYDGDQSSVFDS18Novel 3NJ-36IGHV4-34*016-6*015*02M93,33%AKLMAGRPNWFDP13NJ-123IGHV4-34*016-6*015*02M91,67%AKLMAGRPNWFDP13 Disclosures:No relevant conflicts of interest to declare.

Expression of stereotyped B-cell receptor and its clinical value in chronic lymphocytic leukemia

To investigate the expression pattern of stereotyped B-cell receptor (BCR) and its prognostic significance in Chinese chronic lymphocytic leukemia (CLL) patients and evaluate the relationship to other prognostic markers. Multiplex polymerase chain reaction (PCR) was used to identify the immunoglobulin variable heavy-chain (IGHV) segment and its mutation status in 116 CLL patients from April 1992 to April 2010. For CDR3-driven clustering, all in-frame IGHV-D-J rearrangements were aligned by the multiple sequence alignment software ClustalW2. There were 102 of 116 samples from newly diagnosed CLL were successfully analyzed. IGHV CDR3 genes were identified in 73/102 cases. Fourteen patients (19.2%) carried stereotyped BCR. A high percentage of carried stereotyped BCR was observed in IGHV non-mutated group versus mutated group (40.9% vs 11.8%, P = 0.005). Patients with stereotyped BCR had a higher frequency of deletion (17q) (33.3% vs 10.7%, P = 0.045). The median progression-free survival (PFS) in patients with stereotyped BCR was much shorter than other patients (39 vs 84 months, P = 0.002). The patients with stereotyped BCR have a poor prognosis. It highlights the importance of immunoglobulin mediated stimulation in the development of CLL.

Comprehensive assessment of prognostic factors predicting outcome in Chinese patients with chronic lymphocytic leukemia treated with fludarabine and cyclophosphamide

To determine whether prognostic factors remain relevant to chronic lymphocytic leukemia (CLL) patients treated with fludarabine and cyclophosphamide (FC), we prospectively evaluated 86 Chinese CLL patients who received FC in first-line therapy. Twenty-four patients (27.9%) achieved complete remission (CR), and overall response rate was 75.6%. With a median follow-up of 41 months, the median progression-free survival (PFS) was 36.0 months and median overall survival (OS) has not been reached. The strong correlations of lower CR rate with advanced Binet stage, unmutated IGHV, cytogenetic abnormalities of del(17p13) or del(11q23), and p53 mutations were observed by univariable analyses. Stepwise logistic regression identified that unmutated IGHV and p53 abnormality (p53 deletion or mutation) were associated with a decreased odds of achieving CR. The less cycles of treatment, not achieving CR, advanced Binet stage, and p53 abnormality significantly correlated with a shortened PFS. Furthermore, in a multivariate analysis, p53 abnormality and advanced Binet stage were identified as being significant risk factors for early relapse. Not achieving CR, advanced Binet stage, ZAP-70-positive, and p53 abnormality were the adverse factors in determining OS. Only p53 aberration was independently associated with significantly shorter OS by a multivariate analysis. These results suggest that patients with p53 abnormality should be considered for alternative therapies.

Expression and prognostic significance of TOSO in CD19+ B cells from Chinese CLL patients

To investigate the expression and its prognostic significance of TOSO in CD19(+) B cells from Chinese chronic lymphocytic leukemia (CLL) patients. The expression of TOSO was detected by absolute quantitative reverse transcription-polymerase chain reaction (RT-PCR) in a cohort of 85 untreated CLL patients from March 2006 to September 2010. Their status of immunoglobulin heavy chain variable (IGVH) somatic mutation, ZAP70 and CD38 were analyzed. The expression of TOSO was significantly elevated in CLL patients versus the healthy population (8.30 ± 2.99 vs 6.63 ± 1.22, P = 0.036) and other B cell lymphoproliferative diseases (8.30 ± 2.99 vs 7.12 ± 1.13, P = 0.023). The expression of TOSO was elevated in the IGVH non-mutated group versus the mutated group (9.87 ± 1.08 vs 7.61 ± 3.03, P = 0.000). The expression of TOSO was significantly elevated in Binet stage C patients versus in Binet stage B and Binet stage A patients (9.91 ± 3.03 vs 8.73 ± 1.86 vs 7.27 ± 2.83, P = 0.000). The expression of TOSO rose to (9.37 ± 2.12) in the chemotherapy group. And it was significantly higher than that in the observation group (7.19 ± 3.23, P = 0.001). At the cut-off point of 55 years old, the younger patients had a higher expression of TOSO than the elders (9.10 ± 2.06 vs 7.95 ± 3.22, P = 0.049). The expression of TOSO in ZAP70 and CD38-positive groups had no difference with those in the negative groups. The over-expressed TOSO in CLL cells is associated with a progressive disease. It may be an important prognostic factor for CLL.

Prognostic significance of serum immunoglobulin paraprotein in patients with chronic lymphocytic leukemia

The aim of this study was to explore the clinical and other associated laboratory features of chronic lymphocytic leukemia (CLL) patients with immunoglobulin (Ig) paraproteinemia. Serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE) were performed to measure serum Ig paraprotein. The correlations between serum Ig paraprotein and other prognostic factors were analyzed. Univariate and multivariate Cox regression analyses were used to assess associations between survival time and potential risk factors. In 133 Chinese CLL patients, 27 (20.3%) patients occurred Ig paraproteinemia at diagnosis. According to the correlation analysis, advanced Binet stage (r=0.314, P<0.001), direct antiglobulin test (DAT)-positive (r=0.366, P<0.001), high level of serum β2-microglobulin (β2-MG) (r=0.296, P=0.001) and thymidine kinase (TK) 1 (r=0.227, P=0.037), unmutated immunoglobulin heavy chain variable gene (IGHV) status (r=0.284, P=0.002), ZAP-70-positive (r=0.305, P=0.001), CD38-positive (r=0.284, P=0.002), and cytogenetic abnormalities of del(17p13) or del(11q22.3) (r=0.208, P=0.032) emerged as factors significantly related to the occurrence of Ig paraproteinemia. Survival analysis showed that the patients with Ig paraproteinemia had significantly shorter survival times than the patients without serum Ig paraprotein (P=0.013). Binet stage (P=0.028), high levels of lactate dehydrogenase (LDH) (P=0.004), IgG paraproteinemia (P=0.048), IgM paraproteinemia (P=0.001), ZAP-70-positive (P=0.003), DAT-positive (P=0.013), unmutated IGHV status (P=0.009), and del(17p13) (P=0.001) were the adverse factors in determining overall survival (OS). Del(17p13) (P=0.006), ZAP-70 (P=0.030), and IgM paraproteinemia (P=0.040) were the variables strongly associated with OS by multivariate Cox regression analysis. It was showed that serum Ig paraprotein might be applied for the assessment of prognosis in patients with CLL.

Research on the Characteristics of IGHV Gene In Chronic Lymphocytic Leukemia of China.

AbstractAbstract 4609 ObjectiveThe usage, mutation status and prognostic impact of immunoglobulin heavy chain variable (IGHV) gene in Chinese patients with chronic lymphocytic leukemia (CLL) is unclear. We set out to define the characteristics of IGHV gene and its relevance to clinical and biological parameter in our patients. MethodsIGHV gene mutations were detected by multiplex PCR in 202 Chinese CLL patients and the purified PCR amplification products were sequenced. IGHV somatic hypermutation status and gene usage were analyzed by IMGT/V-QUEST software. The association analysis between IGHV somatic mutation status and the clinical and biological features, including Binet staging, immunophenotype, cytogenetic aberrant, were also emphasized in this study. ResultsThe results showed that 129 patients had mutated (M) IGHV, and the remaining 73 patients had unmutated (UM) IGHV according to the cutoff value of accordance rate 98%. The most frequent VH gene family was found to be VH3 (47.5%), followed by VH4 (34.7%), VH1 (11.4%), VH2 (2.5%), VH5(1.5%), VH7(1.5%) and VH6(0.9%) gene families, which was similar to other Asian populations. The overall survival (OS) time of UM IGHV group was significant shorter than M IGHV group (P=0.025). Significance was found in the expression of CD38 and ZAP-70 between patients with and without IGHV mutations (P<0.0001 and P=0.015, respectively). Binet staging was significantly different with IGHV mutation status (P<0.001). “Unmutated” sequences had significantly longer heavy chain complementarity-determining region 3 (HCDR3). Seven of these patients used VH1-69, which was similar to other Asia countries, but in striking contrast to those in Western countries, where VH1-69 was one of the most frequently used genes. FISH was performed in 117 cases, del(11q22) was considered as high risk factors, and 10(10/42, 23.8%) cases with UM IGHV gene. On the other hand, there were 7(7/75, 9.3%) cases with M IGHV gene (P=0.033). No significance was found in del(17p),del(13q),del(6q),add(12),IGH translocation between IGHV mutation and unmuatioan patients. A total of five stereotyped BCR were identified, IGHV3-21/IGHD3-9/IGHJ6, IGHV4-34/IGHD2-15/IGHJ6, IGHV1-3/IGHD6-19/IGHJ4, IGHV4-59/IGHD3-22/IGHJ6 and IGHV4-39/IGHD6-13/IGHJ5. ConclusionsThe usage of IGHV gene families indicates significant difference in Chinese CLL patients compared with Western patients, suggesting involvement of ethnic and/or environmental factors in CLL disease initiation. In the development course of CLL, BCR play an important role in the immunological recognition and selection. There are intimate relationships between mutation status of IGHV gene and prognosis. The usage of IGHV provides enlightment for the occurrence mechanism of CLL. Disclosures:No relevant conflicts of interest to declare.

Immunoglobulin variable heavy chain region genetic constitution and mutation status in Chinese patients with chronic lymphocytic leukemia

To evaluate the genetic constitution and mutation status of the immunoglobulin variable heavy chain region (IGVH) gene expression in Chinese patients with chronic lymphocytic leukemia (CLL). The IGVH mutation was detected by multiplex PCR and direct sequencing of the purified PCR products from 64 CLL patients. The segments of VH, DH and JH family and mutations were analyzed by IMGT/V-QUEST and IGBlast. In the 64 patients, the most common usage was VH3 (31/64, 48%), followed by VH4 (26/64, 41%), VH1 (4/64, 6%), VH2 (2/64, 3%) and VH7 (1/64, 2%). The results also showed that 44 patients (69%) had mutated VH, 6 cases (9%) had identical germline sequences. Among the 64 sequences of DH segments, DH3 gene family was used most frequently (25/64, 39%), among which 11 cases had unmutated VH. The most frequent usage of the JH segments was JH6. There is significant difference in the frequency of the IGVH gene family in Chinese CLL patients compared to Western patients, suggesting the involvement of antigen selection in different ethnic and/or environmental factors in CLL disease initiation, and its prognostic significance needs further investigation.