Characteristics Of Tumor Immunoglobulin Gene Rearrangements In Malignant B Lymphocytes And Their Impact On Clinical Prognosis

Part ? Immunoglobulin gene rearrangements in Chinese patients with chronic lymphocytic leukemiaObjectives:Chronic lymphocytic leukemia(CLL)is the most common type of leukemia in the Western countries,whereas in Asia this incidence is about 10 times lower.The reasons for this epidemiologic heterogeneity remains unknown.The B cell receptor(BCR)is composed of immunoglobulin(IG)which is responsible for the recognition and selection of specific antigens during B cell maturation and differentiation.CLL patients can be divided into 2 different prognostic subgroups based on the presence or absence of somatic hypermutations on the IG heavy variable(IGHV)genes.Earlier studies have reported differential usage of IGHV gene segments between Asian and Caucasian CLL patients,however the analyzed series were rather small.The aim of this study was to characterize IGHV gene segment usage and BCR stereotype in a large cohort of Chinese CLL,in order to offer potential explanations for the different clinical features between Chinese and Caucasian CLL patients.Method:A total of 623 Chinese CLL patients and 789 Italian CLL patients were included in this study.PCR amplification and sequence analysis of IGHVDJ rearrangements were performed.Productive rearrangements were analyzed by IMGT database(www.IMGT.org).BCR stereotype was identified using multiple sequence alignment ClustalW2 software.Multiple parameters between Chinese and Italian CLL patients were compared,including IGHV mutational status,IGHV gene segment usage and the frequency of each BCR stereotype.Results:Chinese patients showed a higher proportion of mutated IGHV and a more frequent usage of IGHV3-7,IGHV3-74,IGHV4-39 and IGHV4-59 genes than did Italian patients.A significantly lower usage of IGHV1-69 and IGHV1-2 was documented,with comparable IGHV3-21 frequency(3%Chinese vs 4%Italian CLL,both lower than that of northern European patients).The proportion of known stereotyped receptors was significantly lower in Chinese(20%)than in Italian(26%)CLL.A significantly higher frequency of subset#8(Chinese 27%vs.Italian 4%;P<0.001)and lower frequency of subset#2(Chinese 2%vs.Italian 10%;P=0.02)was also noticed.Moreover,we identified 16 novel subsets in Chinese patients,suggesting the different immunogenetic features between Chinese and Caucasian CLL.However,on the other hand,CLL patients using the same IGHV gene had similar clinical outcome regardless of the ethnic groups.We confirmed previously reported associations between usage of unmutated IGHV and poor prognosis and between subset#8 and Richter transformation in Chinese patients.Therefore,we conclude that despite of different usage of each IGHV gene between Chinese and Caucasian CLL patients,CLL cells displaying the same IGHV were biologically conservative.Conclusions:Our results offer the most extensive catalogue of the BCR features of Chinese CLL,being based on the largest series of Chinese CLL so far reported.In comparison to Caucasian CLL,most of the Chinese IGHV genes show different frequencies,but maintain the same propensity towards mutations and stereotype,with the same clinical impact.Part II Frequencies of SF3B1,NOTCH1,MYD88,BIRC3 mutations in Chinese with chronic lymphocytic leukemia:associations with IGHV gene usage and disparities with EuropeansBackground:Chronic lymphocytic leukemia(CLL)is a clinically heterogeneous disease with different molecular and cytogenetic abnormities.Several recurrent gene alterations in CLL detected by next-generation sequencing have shown promising prognostic value including mutations within SF3B1,NOTCH1,MYD88 and BIRC3.Interestingly,the presence of these gene mutations was associated with preferential usage of certain IGHV gene,suggesting that these genetic lesions might facilitate the prognostic impact of IGHV gene in CLL.However,the precise prognostic value of these novel gene mutations is still under dispute and there is no integrated study of these mutations in Chinese CLL cohort.The aim of this study is to assess the incidence of SF3B1,NOTCH1,MYD88 and BIRC3 mutation and prognostic impact in a large cohort of Chinese CLL patients and offer clues for the establishment of Chinese specific CLL risk stratification model.Method:A total of 307 patients were included in this study.The median age was 60 years old with a male/female ratio of 1.98.The median follow-up was 35 months.Sanger sequencing was performed to detect the mutations of exon 14-16 of SF3B1,exon 3-5 of MYD88,PEST domain of NOTCH1,exon 6-9 of BIRC3 and exon 4-9 of TP53.The correlations between above-mentioned gene mutations and diverse clinical parameters including IGHV gene usage and cytogenetic aberrations were studied.The prognostic impact of these gene mutations were also investigated.Results:Mutation frequencies were SF3B1,5%,NOTCH1,8%,MYD88,8%,BIRC3,2%,TP53,15%and IGHV,60%in the studied cohort.Compared to Caucasian CLL,higher incidence of MYD88 mutation and lower incidence of SF3B1 mutation was observed in Chinese CLL patients.The incidence of mutations within SF3B1,NOTCH1,and TP53 were closely related to the usage of unmutated IGHV,whereas MYD88 mutations were more common in patients with mutated IGHV.Moreover,NOTCH1 mutation were associated with biased usage of IGHV4-39 and IGHV1-69,while SF3B1 mutation correlated with IGHV4-59 usage.Cytogenetically,NOTCH1 mutation correlated with trisomy 12,so did TP53 mutation and del(17p13).Unmutated IGHV,TP53 aberration and NOTCH1 mutation were selected as independent unfavorable prognostic factors for treatment free survival in the multivariate analyses,but the prognostic value of SF3B1 mutation was minimal in Chinese CLL patients.Conclusion:The incidence of novel gene mutations were associated with the usage of specific IGHV gene segments.We show differences in incidence and prognostic impact of these gene mutations in Chinese CLL compared to Caucasian CLL patients.These data may give insights into the etiology and biology of CLL and suggests different risk stratification models may be needed for different CLL populations.Part ? Molecular characterization of immunoglobulin genetic repertoires in diffuse large B cell lymphoma provides insight for cell-of-origin and clinical outcomeObjective:Clinical heterogeneity is a major challenge for the treatment of diffuse large B cell lymphoma(DLBCL).Different cell-of-origin may contribute to the distinct biology of DLBCL as suggested by the classic germinal center-like and activated B cell(ABC)-like DLBCL classification system.Characterization of immunoglobulin(IG)gene helps to identify cell-of-origin of mature B cell malignancies such as chronic lymphocytic leukemia,whereas its role in the pathogenesis of DLBCL is poorly understood.The aim of this study was to characterize IG gene usage and intraclonal variations in a large cohort of de novo DLBCL patients with different cell-of-origin subgroups,and to identify the role of IG gene analysis in tracing the cell-of-origin of DLBCL.Method:A total of 378 biopsy specimens obtained from patients with de novo DLBCL were included in this study.Genomic DNA was extracted using QIAamp DNA FFPE Tissue Kit.The amplification and sequencing of IG heavy-and light-chain were performed using the Adaptive Biotechnologies ImmunoSEQTM platform.After correcting sequencing errors via a clustering algorithm,CDR3 segments were annotated according to the International ImMunoGeneTics collaboration,identifying which V,D,and J genes contributed to each rearrangement.Ongoing somatic hypermutation(SHM)or intraclonal variation is identified as subclone sequences which(1).Have identical SHM locations as the dominant or clonal sequence;(2).derive from the same germline sequence as the dominant sequence;(3).the frequency of point mutation within the alignment length is greater than 2%.The total number of ongoing sequences was used to determine the level of intraclonal verification.Gene expression profiling was applied in order to identify differentially expressed genes among diverse DLBCL subgroups.Results:Analysis of IGH and IGK/IGL was performed on the DNA samples of 378 and 269 patients with DLBCL,respectively.Due to the heavily mutated IG variable(IGV)region,productive complete V(D)J rearrangements were only detected in 37%(heavy chain)and 61%(light chain)patients.IGHV4-34 was the most common IGHV gene segment accounting for 17%of the whole cohort,and the biased usage of IGHV4-34 was closely related to the ABC phenotype.Also,we found 5 pairs of common CDR3 sequences,suggesting potential antigen selective pressure during lymphomagenesis of DLBCL.Most patients with DLBCL were IGV-mutated and showed evidence of ongoing SHM on their IG sequences indicating intraclonal variation.Importantly,our data supports the novel B-cell-associated gene signatures(BAGS)classification system based on the normal B cell hierarchy by demonstrating that the SHM rate was higher in germinal center-B-cell-like centroblast(CB)and centrocyte(CC)subgroups than others,and that CB-like DLBCL had higher level of intraclonal variation than the CC-like DLBCL.Moreover,we found high level of intraclonal variation had a poor prognostic impact in patients with CC-like DLBCL.Gene expression profiling suggested this group of patients only acquired partial centrocyte phenotype but lacked typical gene changes compared to CB-like DLBCL.Conclusion:Our data showed distinct immunoglobulin gene features in patients with DLBCL and demonstrated that immunoglobulin gene analysis is a useful tool for the cell-of-origin identification of DLBCL.Sustaining ongoing SHM might contribute to the progression of DLBCL.