HLA Is a Determinant Of The Ethnic Predisposition Of Chronic Lymphocytic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1620-1620
Author(s):  
LOREN GRAGERT ◽  
Stephanie DiPrima ◽  
Mark Albrecht ◽  
Martin Maiers ◽  
Matt Kalaycio ◽  
...  
Keyword(s):  
African Americans ◽  
Chronic Lymphocytic Leukemia ◽  
Allele Frequency ◽  
Odds Ratio ◽  
Lymphocytic Leukemia ◽  
Hla Typing ◽  
P Values ◽  
Hla Alleles ◽  
Asian Populations ◽  
Allele Family

Abstract Introduction Chronic Lymphocytic Leukemia (CLL) displays remarkable ethnic predisposition for Caucasians with a lower incidence of disease in African Americans and relative sparing of Asian Populations. In addition, CLL displays one of the highest familial predispositions of all hematologic malignancies, yet the genetic basis for these differences is not fully defined. Large scale genome wide association studies (GWAS) of CLL have identified the Human Leukocyte Antigen (HLA) locus and other loci associated with the development of CLL but the contribution of individual HLA alleles has not been extensively examined. The National Marrow Donor Program (NMDP) registry contains a large number of disease cases and donor controls across multiple populations as well as high quality classical HLA typing, making it a powerful source of data for inferring HLA associations for hematologic diseases. Using the NMDP dataset, we report the largest study of the HLA associations for chronic lymphocytic leukemia (CLL) to date. Methods Cases consisted of 2,689 US Caucasian and 298 African-American patients with CLL who underwent HLA typing performed for allogeneic donor search reported to the National Marrow Donor Program (NMDP). 50,000 controls were randomly selected for each population from US individuals who voluntarily enrolled in the NMDP unrelated donor registry. To address ambiguous haplotype phasing inherent to HLA typing, we used population haplotype frequencies to calculate probabilities for genotypes for each possible pair of HLA haplotypes. We then incorporated this typing ambiguity into a statistical model using multiple imputation, allowing us to report high-resolution allele, haplotype, and genotype associations. Patients and controls were compared using multivariate logistic regression using a generalized linear model for the covariates of age, birthdate, gender, and geographical location. To control for multiple testing, we adjusted p-values using False Discovery Rate with a cutoff of 0.05. Results We identified several HLA alleles that are associated with an increased susceptibility to the development of CLL as well as several alleles that are protective in the Caucasian as well as the African American (AA) US population. In Caucasians, A*02:01 (OR = 1.203, p = 0.00053), B*15:01 (OR = 1.264, p = 0.0105), B*38:01 (OR = 1.396, p = 0.0198), DRB1*04:02 (OR = 1.675, p = 0.0006) and DRB1*07:01 (OR = 1.223, p = 0.0006) were predisposing and A*01:01 (OR = 0.849, p = 0.01077), B*08:01 (OR = 0.853, p = 0.0437), B*27:05 (OR = 0.743, p = 0.0356) were protective. For African Americans, we find a different collection of high resolution HLA alleles: B*37:01, B*15:17 and DRB1*09:01 were predisposing to the development of CLL (OR 3.017, 3.089 and 2.144, respectively) with highly statistically significant p-values (0.0344, 0.0344, 0.00057, respectively). At the allele family level, B*44 was found to be protective in both Caucasian (OR = 0.843, p = 0.0115) and AA populations (OR = 0.485, p = 0.0309). The allele family C*07 (OR 0.848, p = 0.0046) was protective in Caucasians while C*04 was protective in AA populations (OR = 0.645, p = 0.0422). Association results also revealed additive effects of individual predisposing alleles within the same haplotype, such as the A*02:01∼C*06:02∼B*13:02∼DRB1*07:01 haplotype, which had a higher odds ratio (OR = 2.2, p = 0.000086) than either A*02:01 or DRB1*07:01. We performed a population risk analysis by applying the currently identified allelic disease associations for Caucasians to the known allele frequency seen in other populations, and determined that HLA allele frequency differences contribute to the lower risk of CLL development for both the AA and Asian populations. Conclusions We have identified several HLA associations that confer increased risk of development of CLL in both U.S. Caucasians and African Americans as well as several protective alleles. These results confirm the previously identified association with HLA*02:01 (OR 1.32, Di Bernardo, et al) with highly similar odds ratio. In addition, we identify many HLA allele associations that have not been previously observed due to low statistical power. Differences in HLA predispositions and HLA allele frequency between Caucasian, AA and Asian populations contribute to the difference in incidence of CLL in these populations and may confer inherent biologic differences due to their impact on immune surveillance. Disclosures: Hill: Celgene: Honoraria, Research Funding.

scholarly journals HLA allele repertoire in Russian chronic lymphocytic leukemia patients with an unfavorable prognosis

2020 ◽  
Vol 65 (3) ◽  
pp. 312-320
Author(s):  
B. V. Biderman ◽  
E. B. Likold ◽  
A. R. Abdrakhimova ◽  
E. A. Leonov ◽  
E. G. Khamaganova ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Cell Clone ◽  
Lymphocytic Leukemia ◽  
Hla Typing ◽  
Control Group ◽  
Antigen Receptors ◽  
Hla Alleles ◽  
Healthy Donors ◽  
Allele Group ◽  
Selection Of

Introduction. An unfavorable prognosis in chronic lymphocytic leukemia (CLL) is associated with unmutated status of rearranged IGHV genes. CLL is also characterized by a narrowing of the repertoire of IGHV genes and the formation of quasiidentical (stereotyped) receptors, which is probably associated with antigenic selection of the tumor B-cell clone in the pathogenesis of the disease. The HLA phenotype plays an important role in antigenic selection of B cells. On the other hand, the association of specifi c HLA alleles with various diseases has been described. Aim. To assess the frequencies of HLA alleles in CLL patients with unmutated IGHV genes and the most common stereotyped receptors (SARs). Materials and methods. The study included 100 CLL patients with unmutated IGHV genes - 50 with the most common stereotyped antigen receptors (SARs) and 50 with non-stereotyped antigenic receptors. Control group of healthy donors was also included. Results. Signifi cant differences in HLA-allele repertoire between this two groups of patients and groups of donors were found. B*18 allele group was found much more common in patients with SARs than in donors and in patients without SARs. HLA-B*39 was more frequent for patients with SARs compared to donors; in patients without SARs these alleles were not found. For all patients, the frequency of HLA-B*52 alleles was higher than for donors. HLA-C*12 allelic group was found more frequent in CLL patients than in donors. HLA-DRB1*15 in CLL patients with SARs was found twice as often as in healthy donors or patients without SARs, while HLA-DRB1*13, oppositely, was found twice as rare. HLA-DRB1*16 was signifi cantly more frequent in patients without SARs, compared with donors and the patients with SARs. No signifi cant differences were found in the HLA-A and HLA-DQB1 loci. Conclusion. The association of two HLA alleles with “unmutated” CLL and two others with CLL bearing prognostically unfavorable SARs was found. HLA typing of expanded samples of CLL patients with different prognosis and course of the disease will provide more information on the mechanisms of antigen selection in the pathogenesis of CLL and improve diagnostic and therapeutic approaches.

Clinical Impact of Clonal and Subclonal TP53, SF3B1, BIRC3, and ATM Mutations in Chronic Lymphocytic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4138-4138
Author(s):  
Ferran Nadeu ◽  
Julio Delgado ◽  
Cristina Royo ◽  
Tycho Bauman ◽  
Tatjana Stankovic ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Allele Frequency ◽  
Lymphocytic Leukemia ◽  
Molecular Characteristics ◽  
Prognostic Impact ◽  
Driver Genes ◽  
Germline Variants ◽  
Tp53 Mutations ◽  
Mutational Status ◽  
The Impact

Abstract Genomic studies have provided a complete profile of somatic mutations in chronic lymphocytic leukemia (CLL). These comprehensive approaches have revealed a relatively large number of mutated genes, the adverse prognostic value of some of which has been demonstrated in a number of reports. Recent studies have shown the clinical relevance of TP53 mutations at very low allele frequency. The presence and prognostic impact of minor mutated clones of other CLL driver genes and their clonal dynamics in the evolution of the disease is not well known. The goal of this study was to explore the presence of clonal and subclonal mutations of TP53, SF3B1, BIRC3, and ATM using an ultra-deep next-generation sequencing (NGS) strategy, to define the evolution of these subclones in different time-points of the disease, and to determine their influence in the outcome of the patients. Samples from 363 untreated CLL cases were included in this study. Copy number alterations were investigated by high density SNP-arrays or by quantitative PCR in 341 and 16 cases, respectively. Targeted ultra-deep NGS of TP53 (exons 4-10), ATM (exons 2-63), BIRC3 (exons 2-9), and SF3B1 (exons 14-16 and 18), including splicing sites, was performed using the Access-Array system (Fluidigm) and sequenced in a MiSeq equipment (Illumina). This methodology combined with a robust bioinformatic analysis based on well-known available tools allowed the identification of mutations down to 0.3% of variant allele frequency (VAF). Results obtained were fully verified by orthogonal techniques. Twelve per cent of VAF was used as threshold for the classification of clonal or subclonal mutations since 12% was the cut-off for detection of mutations by Sanger sequencing. Deletions of 11q comprising ATM or BIRC3 were found in 7% of the cases and were associated with mutations of the other ATM allele in 19/26 (73%) cases and BIRC3 in 3/23 (13%). Deletions of 17p were found in 19 (5%) cases and co-existed with TP53 mutations in 15 (79%) of them. Regarding the mutational status of the studied genes, TP53 mutations were present in 11.6% of patients (7.2% clonal, 4.4% subclonal), ATM mutations in 10% (7% clonal, 1% subclonal, 2% germline mutations considered pathogenic), SF3B1 mutations in 12% (7% clonal, 5% subclonal), and BIRC3 mutations in 4% (2% clonal, 2% subclonal). These subclonal mutations had similar molecular characteristics to their respective high-allele frequency mutations supporting a comparable pathogenic effect. In this regard, clonal and subclonal SF3B1 mutations were associated with shorter time to first treatment (TTT) independently of IGHV mutations. Clonal and subclonal TP53 mutations predicted for shorter overall survival (OS) together with the IGHV mutational status, although the impact of isolated TP53 mutations (i.e. without 17p deletion) on OS was not so evident, as has been the case in other studies. In addition, the outcome of patients with clonal and subclonal BIRC3 mutations showed a similar significant shorter OS. Regarding ATM, the effect of isolated subclonal ATM mutations could not be evaluated because of their low number, but ATM mutations as a whole had a significant impact on TTT even in the absence of 11q deletions. This study also reinforces the need to study the germline of the patients to fully characterize the ATM mutations observed in the tumors. Of note, germline variants previously described as pathogenic were associated with 11q deletions, confirming the hypothesis already suggested that these germline variants may influence disease progression through loss of the otherallele. Clonal dynamics was examined in longitudinal samples of 45 CLL patients. We confirmed the expansion of most TP53 mutated clones after therapy. However, both TP53 and SF3B1 mutations expanded also before any therapy in some patients, indicating that progressive dynamics of these clones is not only dependent on therapy selection. On the contrary, small ATM mutated clones seemed to be more stable. Although the number of cases is limited, we observed that clonal evolution in longitudinal samples had an unfavorable impact on OS. In conclusion, this study shows the presence of a high number of subclonal mutations of different driver genes in CLL and provides insights on the impact of these mutations on the outcome of the patients. These findings suggest that the characterization of the subclonal architecture may be relevant for a better management of CLL patients. Disclosures No relevant conflicts of interest to declare.

High-Throughput Sequencing For The Identification Of NOTCH1 mutations In Early Stage Chronic Lymphocytic Leukemia: Biological and Clinical Implications

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1622-1622
Author(s):  
Marta Lionetti ◽  
Sonia Fabris ◽  
Giovanna Cutrona ◽  
Luca Agnelli ◽  
Carmela Ciardullo ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Allele Frequency ◽  
Deep Sequencing ◽  
Mutant Allele ◽  
Sanger Sequencing ◽  
Early Stage ◽  
Lymphocytic Leukemia ◽  
Mutant Allele Frequency ◽  
Mutational Activation ◽  
Notch1 Mutations

Abstract NOTCH1 mutations have recently emerged as new genetic lesions significantly correlated with survival in chronic lymphocytic leukemia (CLL). NOTCH1 c.7541_7542delCT is by far the most frequently observed NOTCH1 mutation in the disease. To estimate the prevalence and clonal evolution of NOTCH1 c.7541_7542delCT mutation, and prospectively investigate its clinical significance in early stage CLL and clinical monoclonal B cell lymphocytosis (cMBL), we analyzed by next generation sequencing (NGS) 384 cases at diagnosis enrolled in the GISL O-CLL1 multicenter trial. The patient cohort included 100 cMBL and 284 Binet stage A CLL cases, 48 of whom were also longitudinally investigated at progression or during follow-up (32 and 16, respectively) in absence of treatment. Deep sequencing of the NOTCH1 mutation hotspot was performed by Roche 454 pyrosequencing on the Genome Sequencer Junior instrument. NOTCH1 mutation was validated by an extremely sensitive PCR-based approach and Sanger sequencing. The association between NOTCH1c.7541_7542delCT and clinical, molecular and biological variables, as well as its impact on progression free survival (PFS), were tested. Deep sequencing analysis of NOTCH1 mutation hotspot in our cohort (median depth of coverage 1510x, ranging from 605 to 2842) revealed a mutant allele frequency ranging from 0.02% to 75% of total reads in 145 cases. The occurrence of the mutation was subsequently assessed by an extremely sensitive ARMS (amplification refractory mutation system)-PCR, which allowed to confirm the presence of delCT in the 49 cases with frequency of mutated sequencing reads greater than 0.7%, specifically in 11% of cMBL (11/100) and 13.4% of CLL patients (38/284). Furthermore, mutated samples were subjected to DNA Sanger sequencing: in line with the expected sensitivity of the method, the mutation was identified only in samples with higher mutation loads according to NGS (mutant allele frequency ≥ 7%, n=25). Our data revealed that often NOTCH1 mutational activation affected a neoplastic sub-clone, especially in cMBL patients. NOTCH1 mutated patients utilized unmutated IGHV genes more frequently, and had higher expression of CD38 and ZAP-70 (P=3.2e-11, P=2.6e-08, P=3.4e-05, respectively). Trisomy 12 was more frequent in this patient group (P=5.4e-04), whereas 13q14 deletion was less represented than in the NOTCH1 wild-type patients (P=2.8e-03). NOTCH1 mutation was associated with the occurrence of stereotyped HCDR3 (P=5.6e-03); in addition, compared with other major BCR subsets, CLL subset #10 was significantly enriched in NOTCH1 mutations (P=0.032). The prevalence of the analyzed dinucleotide deletion was not significantly different between cMBL and CLL patients, even if only Rai 0 cases (28/197 cases, 14.2% mutation frequency) were considered. The percentages of variant sequencing reads in NOTCH1-mutated cases were slightly higher in CLL (median 19.6%) than in cMBL (median 4.2%), a finding confirmed by a regression analysis that highlighted the association of the CLL presentation with higher percentages of NOTCH1 delCT reads (P=0.033). NOTCH1-mutated cases, both at sub-clonal and clonal levels, displayed a significant reduction in median PFS (P=0.0018), although NOTCH1 mutation prognostic value, in multivariate analysis, was not independent if 11q and/or 17p deletion, IGHV mutational status, and cMBL or CLL status were considered. Finally, sequential analyses in a representative fraction of cases of our dataset indicated that (i) NOTCH1 mutation did not occur during the course of the disease and that (ii) the mutational load in positive cases was stable over time. These findings highlight the importance of using high sensitive methods for an accurate detection of NOTCH1 mutation in cMBL/early stage CLL. This is required for a better prognostic stratification and also to obtain useful information for potential therapeutic approaches, since sub-clonal mutations in untreated CLL can possibly anticipate the dominant genetic composition of the relapsing tumor. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Fine-mapping of HLA associations with chronic lymphocytic leukemia in US populations

Blood ◽  
2014 ◽  
Vol 124 (17) ◽  
pp. 2657-2665 ◽  
Author(s):  
Loren Gragert ◽  
Stephanie Fingerson ◽  
Mark Albrecht ◽  
Martin Maiers ◽  
Matt Kalaycio ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
Chronic Lymphocytic Leukemia ◽  
Fine Mapping ◽  
Lymphocytic Leukemia ◽  
Malignant Cells ◽  
Hla Alleles ◽  
Hla Genes ◽  
Key Points

Key Points Polymorphisms in HLA genes may impact the ability of the immune system to detect malignant cells and direct T cells to eliminate them. Several HLA alleles and haplotypes are associated with development of chronic lymphocytic leukemia across different US populations.

Polymorphisms of Mir-34b/c, Mir-146a and Mir-196a-2 and Predisposition to Chronic Lymphocytic Leukemia and Monoclonal B-Cell Lymphocytosis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4585-4585 ◽  
Author(s):  
Krzysztof Jamroziak ◽  
Janusz Szemraj ◽  
Andy Rawstron ◽  
Zofia Szemraj-Rogucka ◽  
Olga Grzybowska-Izydorczyk ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Tumor Suppressor ◽  
B Cell ◽  
Allele Frequency ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Cell Leukaemia ◽  
Nucleotide Polymorphisms ◽  
Liver Cancers ◽  
The Impact

Abstract Abstract 4585 Since microRNAs control expression of protein-coding oncogenes and tumor suppressor genes, functional microRNAs single nucleotide polymorphisms (SNPs) may modulate the risk of tumorigenesis including susceptibility to chronic lymphocytic leukemia (CLL). In this case-control study we investigated whether pri-miR-34b/c rs4938723 (T-to-C), miR-196a2 rs11614913 (C-to-T) and miR-146a rs2910164 (G-to-C) SNPs influence predisposition to CLL or monoclonal B-cell lymphocytosis (MBL). miR-34 family members are direct transcriptional targets of tumor suppressor p53, and miR-34b/miR-34c have been recently proposed as regulators of TCL1 (T-cell leukaemia/lymphoma 1) expression in CLL. A rs4938723 SNP in the promoter region of pri-miR-34b/c might affect transcription factor GATA binding and pri-miR-34b/c expression. Alterations in miR-196a2 and miR-196a2 may represent common cancer predisposition pathways as miR-196a2 rs11614913 and miR-146a rs2910164 have been recently associated with altered risk of different solid tumors including breast, lung, prostate and liver cancers. Additionally, we verified the impact of these microRNA SNPs on prognostic factors and clinical course of CLL. Genotyping was performed using PCR-based assays in a total of 561 Caucasians including 195 patients with CLL, 166 patients with MBL and 200 healthy control individuals. The assessed minor allele frequencies (MAFs) of the investigated SNPs were as follows: for rs4938723 SNP C allele frequency was 0.37 in CLL, 0.36 in MBL and 0.30 in controls, for rs11614913 SNP T allele frequency reached 0.43 in CLL, 0.42 in MBL and 0.39 in controls, and for rs2910164 SNP C allele frequency was 0.29 in CLL, 0.30 in MBL and 0.26 in controls. Logistic regression analysis did not detect significant associations of CLL or MBL with studied genotypes or alleles (p>0.05). Moreover, none of the tested genetic variants was found to influence CLL patients’ progression-free survival (PFS) or overall survival (OS) with median follow-up time from diagnosis of 3.0 (0–13.9) years. In conclusion, our data suggest that investigated SNPs in pri-miR-34b/c, miR-146a and miR-196a-2 genes are not likely to play a major role in the susceptibility to CLL and MBL or in clinical outcome of CLL. Disclosures: No relevant conflicts of interest to declare.

Comparison of HLA Alleles in Follicular Lymphoma and Chronic Lymphocytic Leukemia Patients Referred for Allogeneic Stem Cell Transplantation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4218-4218
Author(s):  
Fleur M. Aung ◽  
Benjamin Litchtiger ◽  
Issa Khouri
Keyword(s):  
Stem Cell ◽  
Chronic Lymphocytic Leukemia ◽  
Stem Cell Transplantation ◽  
Follicular Lymphoma ◽  
Cell Transplantation ◽  
Normal Population ◽  
Lymphocytic Leukemia ◽  
Control Group ◽  
P Value ◽  
Hla Alleles

Abstract Abstract 4218 Introduction: Data recently published showed that besides the several well-known parameters, long term outcome after allogeneic stem cell transplantation (SCT) in chronic lymphocytic leukemia (CLL) may be influenced by the presence or absence of certain HLA class I alleles (HLA-A1+/A2-/B44-) (Khouri et. Al. Cancer 2011). We have also recently published an 11-year progression free survival (PFS) rate of 72% in relapsed follicular lymphoma (FL) after SCT (Khouri et al. Blood 2012). A higher relapse rate has been observed in CLL patients when compared to FL (50% vs. 4%). Since HLA subtypes played an important role in CLL, our goal in this study was to assess and compare over expressed HLA alleles in FL and CLL patients who received a SCT at our center. Methods: Two cohorts of patients who received SCT were retrospectively studied. Group I consisted of 59 Caucasian patients (23 [39%] F: 36 [61%] M) with FL and Group II consisted of 119 Caucasian patients (27 [23%] F: 112 [77%] M) with CLL. The HLA alleles at HLA-A, -B,-C and -DRB1 loci of both groups were analyzed and the HLA typing was performed by polymerase chain reaction (PCR) amplification and oligonucleotide hybridization using commercial kits from Invitrogen (Carlsbad, Ca) or One Lambda (Canoga Park, Ca) that resulted in intermediate resolution. Patients were also typed for these loci using high-resolution methods with PCR amplification and nucleotide sequencing (Abbott, Abbott Park, Ill). The antigen frequencies of all of the alleles of HLA-A, -B,-C AND DRB1 were calculated. Antigen frequency was defined as the percentage of the population possessing the antigen. Antigen frequency comparisons were only done for North American whites due to sample size and control group constraints. The control group was based on a sample analysis of 643 normal North American Whites. The Pearson x2goodness-of-fit test was used to validate the Hardy-Weinberg genetic equilibrium for phenotypic data. The association of various alleles with the control group was determined by using a chi-square test with Yates correction in a 2 × 2 table with 1 degree of freedom (SAS software, version 6.12, SAS Institute Inc, Cary NC). P values < 0.05 at the 95% confidence interval (95% CI) were considered significant. Results: A male predominance was noted in both patient groups. A total of 17 HLA-A, 29 HLA-B, 13 HLA-C and 13 HLA-DRB1 distinct alleles for FL patients and 16 HLA-A, 24 HLA-B, 13 HLA-C and 11 HLA-DRB1 distinct alleles were identified for the CLL patients. Since the predominant ethnic type in both groups were North American Whites, statistically valid comparisons of HLA antigen frequencies were only possible in this population. The observed heterozygosity for FL/CLL patients was 0.93220./0.831932 for HLA-A, 0.915254/0.949579 for HLA-B, 0.779661/0.882352 for HLA-C and 0.847457/0.941176 for HLA-DRB1. There were no untyped patients and all of the patients underwent hematopoietic stem cell transplantation. Our analysis reveals an over expression of HLA-A*03, HLA-C*04, HLA-DRB1*01, HLA-DRB1*07 and HLA-DRB1*15 with frequencies of 25.4%, 24.1%, 22.9%, 28.8% and 8.5 % in FL patients which was significantly higher than the frequencies of 15.1% for HLA-A*03 (p value 0.005), 1.1% for HLA-C*04 (p value < .00001), 10.3% for HLA-DRB1*01 (p value <.0001), 14.4% for HLA-DRB1*07 (p value < .0001) and 15.7% for HLA-DRB1*15 (p value < .0495) in the normal population showing for the first time an over representation of these alleles in patients with FL. In the CLL group our analysis revealed a 24.8% (p value 0.0014) frequency for HLA-A*01 which was significantly higher than the frequency of 16% (p value 0.0014) in the normal population showing an overrepresentation of this allele. The underrepresented allele was HLA-B*38 with a frequency of 3.4% compared to 12.4% (p value 0.0335) in the normal population. When the two groups FL and CLL patients were analyzed, the significant alleles were HLA-A*01, HLA-A*03, HLA-C*04, HLA-DRB1*01 and HLA-DRB1*07. Conclusion: Our results demonstrate a significant difference in HLA expression in Follicular Lymphoma and Chronic Lymphocytic Leukemia patients with the over representation of HLA-A*03, HLA-C*04, HLA-DRB1*01, HLA-DRB1*07 and HLA-DRB1*15 alleles in FL and HLA-A*01 and HLA-B*38 alleles in CLL. We do not know whether these variances account for a different graft-versus-malignancy susceptibility to donor cells between the two groups and this remains to be studied. Disclosures: No relevant conflicts of interest to declare.

HLA Haplotypes Are Associated with Multiple Myeloma Risk in the African American Multiple Myeloma Study (AAMMS)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3250-3250
Author(s):  
Loren Gragert ◽  
Amie Hwang ◽  
Leon Bernal-Mizrachi ◽  
Sikander Ailawadhi ◽  
Seema Singhal ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
African American ◽  
Hla Class I ◽  
Hla Typing ◽  
Class I ◽  
European Ancestry ◽  
Sample Sizes ◽  
P Values ◽  
Hla Alleles ◽  
Large Sample

Abstract Background: Persons of African ancestry (AA) have a 2-3-fold higher risk of multiple myeloma (MM) than persons of European ancestry (EA). Like other B-cell malignancies, genome-wide association scans (GWAS) have identified MM risk variants in the HLA region in persons of EA. We conducted a case-control analysis with data from the National Marrow Donor Program (NMDP)1comprising MM patients typed for bone marrow transplant to donor controls matched by race-ethnicity, and found associations between specific HLA alleles/haplotypes and MM risk that varied by race and ethnicity. To confirm our results and identify additional novel signals, we have now investigated associations between HLA alleles and haplotypes and MM risk in the African American Multiple Myeloma Study (AAMMS) Cohort. Methods: The source of subjects was the AAMMS, in which AA MM patients were identified from 10 cancer centers and 4 Surveillance, Epidemiology and End-Results (SEER) Program cancer registries in order to identify genetic risk factors for MM among AAs. A GWAS was conducted using the Illumina Human Core BeadChip array on DNA samples from 1,305 AA MM patients in the AAMMS comparing results to those from 7,078 AA controls with GWAS data generated from the Illumina 1MDuo2. The major histocompatibility complex (MHC) region single nucleotide polymorphisms (SNPs) were imputed to classical HLA variants using HIBAG. Unconditional logistic regression was used to estimate HLA associations, adjusting for sex, age and the first 2 principal components. P-values were adjusted for false discovery rate (FDR) for each locus group. Results: We did not identify any single HLA alleles associated with MM risk among AAs. However, several B*07:02-containing haplotypes were associated with MM risk (odds ratios [OR] ranging from 2.38 to 2.64 and FDR P-values ranging from 1.43 x 10-6 to 3.57 x 10-8). We found associations between MM risk and genotypes containing DRB3*02:02, including DRB3*02:02~DRB1*11:01+ DRB3*02:02~DRB1*11:01 (OR=1.93, PFDR= 9.36 x 10-5) similar to those observed in the NMDP study1. Novel findings included associations between MM risk and HLA Class I haplotypes B*53:01+ B*57:01 (OR=1.94, PFDR= 0.003) and C04:01~B*53:01+C*06:02~B*57:01 (OR=1.96, PFDR= 0.0050). Results from an ongoing meta-analysis between the two data sets (one based on an imputed GWAS and one based on NMDP HLA typing) will be presented. Conclusions: This study is the second to examine HLA alleles and risk of MM among AA's and is by far the largest. We confirmed a previously observed association between an HLA Class II DRB3 variant and MM risk and confirmed an association with B*07 haplotypes previously observed among EAs1. We also identified novel associations between other HLA Class I haplotypes and MM risk in AA's. Because HLA is highly polymorphic, many HLA alleles are rare variants for which genetic associations are difficult to detect without very large sample sizes. Further investigation with large sample sizes will be necessary to refine these associations in order to better identify the underlying causal alleles and determine the functional significance of these HLA associations. 1Beksac M, Gragert L, Fingerson S, et al.: HLA polymorphism and risk of multiple myeloma.Leukemia. 2016 Jul 27. doi: 10.1038/leu.2016.199. 2Rand KA, Song C, Hwang AE, et al. Genetic susceptibility markers of multiple myeloma in African-Americans. Abstract # 2030, 56th Annual American Society of Hematology Meeting, San Francisco, California, 2014. Disclosures Ailawadhi: Pharmacyclics: Consultancy; Novartis: Consultancy; Amgen Inc: Consultancy; Takeda Oncology: Consultancy. Nooka:Spectrum, Novartis, Onyx pharmaceuticals: Consultancy. Zonder:Pharmacyclics: Other: DSMC membership; Prothena: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Lonial:BMS: Consultancy; Novartis: Consultancy; Millenium: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Merck: Consultancy; Celgene: Consultancy; BMS: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Janssen: Consultancy; Onyx: Consultancy.

Global and MYC-Specific Translation Is Enhanced in Activated Chronic Lymphocytic Leukemia Cells Carrying NOTCH1 C.7541_7542delct Mutations

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 970-970 ◽  
Author(s):  
Annalisa D'Avola ◽  
Alison Yeomans ◽  
Samantha Drennan ◽  
Matthew Rose-Zerilli ◽  
Jonathan C. Strefford ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Allele Frequency ◽  
Mrna Translation ◽  
Lymphocytic Leukemia ◽  
Chromosome 9 ◽  
Variant Allele ◽  
Variant Allele Frequency ◽  
Genetic Lesion ◽  
13Q Deletion

Abstract Introduction: mRNA translation is increased in activated tumor cells of the aggressive form of Chronic Lymphocytic Leukemia (CLL), typically unmutated (U) immunoglobulin gene heavy-chain variable region (IGHV) with a strong sIgM signaling capacity (Yeomans et al, Blood 2016). C-MYC protein is a master regulator of cell performance and its expression is controlled at both transcriptional and translational levels. C-MYC protein is over-expressed in the proliferation centers of CLL and high c-MYC mRNA expression is associated with poor prognosis. In leukemic cell lines, c-MYC is an essential mediator and direct target of NOTCH1. Pro-activating c.7541_7542delCTmutations in NOTCH1 PEST domain of chromosome 9 exon 34 (NOTCH1ΔCT) are enriched in U-CLL with high sIgM levels/signaling capacity and associate with poorer prognosis in CLL (D'Avola et al, Blood, 2016), likely due to accumulation of more stable NOTCH1 protein and enhanced signaling in tissue activated CLL cells (Arruga et al, Leukemia, 2014). Aims and Methods: We investigated the consequences of NOTCH1ΔCT on global mRNA and c-MYC translation using a novel flow cytometry-based O-propargyl-puromycin (OPP) incorporation assay ('Click-iT' assay) and by c-MYC-specific immunoblotting in U-CLL. Since prolonged culture of CLL cells in vitro in the absence of stimuli led to spontaneous inactivation of NOTCH1 pathway, CpG-mediated TLR9 induction was used as a tool for activation of CLL cells in vitro. Cycloheximide (CHX) was used as a negative control for mRNA translation. For this study, 2 cohorts were investigated: i) a test "CLLΔCT cohort" of U-CLL with NOTCH1ΔCT (variant allele frequency [VAF] by droplet digital PCR, range 42.6-48.9%, median 47% of the CD19+CD5+ CLL cell population), but no additional genetic lesion other than 13q deletion, and ii) a control "CLLWT cohort" of U-CLL with no NOTCH1ΔCT (VAF<1% in all cases) or additional genetic lesion other than 13q deletion. CLL cells were incubated with 7.5 μg/ml CpG-ODN 2006 for 24 hours and assays were performed at baseline, 3 and 24 hours. NOTCH1 pathway γ-secretase inhibition was performed with DAPT GSi. Results: The CLLΔCT cohort had higher sIgM levels (range 31-372 MFI, median 81 MFI) and signaling capacity (Fab'2 anti-IgM induced intracellular calcium mobilization sIgM [iCa2+] range 47-54%, median 51) than the CLLWT cohort (sIgM levels range 19-288 MFI, median 47 MFI; IgM iCa2+ range 2-78%, median 25%). Following TLR9-mediated cell activation, the CLLΔCT cohort had sustained NICD (NOTCH1-intracellular cleaved domain) protein accumulation for up to 24 hours and expressed higher NOTCH1 target gene HES1 (hairy enhancer of split) transcript levels than in the CLLWT cohort. These data indicated NOTCH1 canonical pathway sustainment in the CLLΔCT upon activation. Global mRNA translation after 24 hours in the presence of CpG was 11.5 fold higher than that without CpG in the CLLΔCT cohort and only 4 fold higher in the CLLWT cohort, revealing significantly higher levels of translation in CLLΔCT than in CLLWT (p=0.03). CpG-induced global mRNA translation in the CLLWT cohort was similar to that in the CLLΔCT cohort treated with CHX. By using CpG-induced global mRNA translation in the presence of CHX inhibitor as background levels for each group, DAPT GSiat 2.5 to 10 μM showed from 47% to 63% inhibition of the residual CpG-induced global translation in CLLΔCT (p<0.05), but no effect in CLLWT. Remarkably, c-MYC mRNA translation after 3 hour culture with CpG was higher in CLLΔCT than in CLLWT (p= 0.02), and a similar trend was maintained in the cases investigated at 24 hour. Treatment of CLLΔCT cells with DAPT GSi decreased expression of c-MYC in a dose-dependent manner. Conclusion: NOTCH1ΔCT mutations associate with a very aggressive clinical behavior in CLL. These results now indicate that pro-activating mutations of NOTCH1 pathway associate with increased global mRNA translation and c-MYC expression. They highlight a mechanism by which NOTCH1 pathway may induce c-MYC overexpression in CLL, likely leading to increased proliferation and survival. The association of increased NOTCH1 variant allele frequency with sIgM levels and signaling capacity indicate that these mechanisms are predominant in the less anergic subgroup of U-CLL and make NOTCH1 mediated c-MYC translation an attractive target for therapeutic inhibition. Disclosures Steele: Portola Pharmaceuticals: Honoraria. Packham:Karus Therapeutics: Other: Share Holder & Founder; Aquinox Pharmaceuticals: Research Funding.

Clonal Abundance Determines The Prognostic Relevance Of a NOTCH1 Mutation In Chronic Lymphocytic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2863-2863
Author(s):  
Stefanie Rost ◽  
Michael Möllmann ◽  
Lewin Eisele ◽  
Stefanie Weber ◽  
Ulrich Dührsen ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Real Time ◽  
Allele Frequency ◽  
Real Time Pcr ◽  
Lymphocytic Leukemia ◽  
Allele Frequencies ◽  
Operating Characteristics ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Over Time

Abstract Introduction The transmembrane receptor NOTCH1 operates as a ligand-activated transcription factor controlling developmental processes, proliferation and apoptosis. In the context of cancer, activating NOTCH1 mutations are the most frequent oncogenic events in T-cell acute lymphoblastic leukemia and have been implicated in chronic lymphocytic leukemia (CLL) as well. The most prevalent CLL NOTCH1 mutation (N1ΔCT) leads to a truncation of the protein (p.P2515Rfs*4) and has been associated with impaired overall survival (OS). Here, we applied three different methods to study the N1ΔCT prevalence and subclone size in a cohort of n=275 CLL patients. Methods Presence of the N1ΔCT mutation was analyzed using newly established restriction fragment length polymorphism (RFLP) and allele-specific PCR (AS-PCR) methodologies. A novel real-time PCR (qRT-PCR) assay was used to precisely quantify the N1ΔCT allele frequency. Presence of the N1ΔCT mutation was confirmed by conventional Sanger sequencing. Results Using RFLP analysis we detected the N1ΔCT mutation in n=17 CLL patients. In parallel, we used a more sensitive AS-PCR and identified n=12 additional N1ΔCT-mutated cases resulting in a total N1ΔCT mutation rate of 10.5% (n=29/275) in our cohort. The OS of RFLP-positive patients (RFLP+) was significantly shorter than the OS of N1ΔCT-unmutated patients (wt) (mean OS; RFLP+, 87 months vs. wt, 218 months; p=0.017). In contrast, OS of AS-PCR-positive cases (AS-PCR+) did not differ significantly from the OS of wt patients (mean OS; AS-PCR+, 175 months vs. wt, 218 months; p=0.42). These data prompted us to design a quantitative real-time PCR (qRT-PCR) assay, which is capable of precisely quantifying the size of the N1ΔCT-mutated subclones (allele frequency, %) in our CLL cohort. As expected, significantly different allele frequencies between RFLP+ (mean±SEM 27.1±3.4%), AS-PCR+ (3.7±0.6%) and wt patients (0.6±0.04%) were revealed by qRT-PCR (p<0.0001). In order to determine a methodology-independent cut-off which correlates with the clinical significance of the N1ΔCT mutation, we employed Receiver Operating Characteristics (ROC) analysis based on survival status and calculated a N1ΔCT allele frequency cut-off of 15.2% (AUC=0.71). Next, we determined N1ΔCT allele frequencies over time to investigate clone dynamics within individual patients (n=15 patients, mean observation period 87.4 months; range 5-186 months). Unexpectedly, the N1ΔCT allele frequencies remained relatively constant and none of the patients with N1ΔCT allele frequencies below 15.2% rose above this cut-off over time. Conclusions Our data demonstrate that high abundance of a N1ΔCT-mutated CLL clone correlates with an aggressive disease course. In our CLL cohort a N1ΔCT allele frequency below 15% was of negligible clinical relevance. Thus, mere qualitative detection of a N1ΔCT mutation by PCR is not inevitably associated with shortened survival. Surprisingly, we did not observe that a minor N1ΔCT clone became dominant over time. Disclosures: No relevant conflicts of interest to declare.

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