ALBERT

Description: Background: The relapse rate is around 20-40% in adult B-cell acute lymphoblastic leukemia (B-ALL). The genetic defects are the major reasons for the relapse and poor outcome. We screened the genomic variants with Pan-cancer panel in B-ALL patients and whole exome-seq (WES) in the paired de novo-relapsed B-ALL samples. Methods: The xGen Pan-Cancer Panel (IDT), which has been designed with the probes targeting 127 significantly mutated genes from the TCGA database across 12 tumor tissue types (Nature, 502:333-339) was used. Agilent SureSelect Human All Exon V4+UTRs (Agilent) was used to target coding exons plus UTRs for the WES analysis. The genomic DNA from 81 Philadelphia chromosome positive (Ph+) B-ALL patient samples (71 de novo and 10 relapsed, 14 to 77 years old) collected between June 2008 and June 2016 at Zhongda Hospital Southeast University were used for the Pan-cancer panel screening. All DNA samples were sheared and generated approximately 260bp DNA fragments. The fragmented DNA was processed into Illumina compatible sequencing libraries using the Kapa Hyper Prep Kit. Each library was uniquely barcoded and captured by either the Pan-cancer panel or WES probes, followed by PCR amplification and sequencing on a HiSeq 2500 (Illumina) with 2x100 bp reads. The sequencing reads were aligned to the human genome (hg19) by following Broad Institute's GATK best practice pipeline to call germline short variants (SNPs and Indels). Called variants were annotated using ANNOVAR (version 2.3). Variants with exonic, nonsynonymous, stopgain, or stoploss, novel SNPs (not covered by dbSNP138), and with predicted deleterious/damaging functions were manually surveyed by IGV to confirm. Two paired de novo-relapse samples from Ph(+) B-ALL patients were performed the WES analysis. Results: We identified a total of 3945 single nucleotide variants (SNVs), 2222 insertions and deletions (INDELs) in the Pan-cancel panel analysis in 81 Ph(+) B-ALL patients. Among these, 101 genomic variants are with amino acid change, 8 are with stopgain, and 5944 have not been previously reported. We evaluated the frequency and distribution of likely pathogenic variants (PVs) detected in the cohort. Likely PVs were defined by SIFT algorithm which predicts whether an amino acid substitution is likely to affect protein function. Defined by the SIFT's qualitative score 'deleterious', we detected 46 PVs. Among these, PVs were commonly detected in KMT2C, APC, CDKN1A, NSD, BRCA1, EPHA3, and PIK3CG. The PVs were also validated with the Sanger DNA sequencing in the patients. The patients with the likely PVs have significantly higher WBC count (61*10^9/L vs. 24.45*10^9/L, P=0.004). Survival analysis showed that the patients with likely PVs had a worse event-free survival (EFS) and overall survival (OS), the difference was statistically significant (8 months vs. 15 months, P=0.017 and 14 months vs. 25 months, P=0.021). In order to gain an insight to the gene mutations contributing to the disease relapse, we compared the mutants spectrum between the de novo and relapsed samples. We found the genomic variants in NF1, CDK12, mTOR, and USP9X genes appeared mostly in relapsed samples, indicating their roles in the relapse. Using the WES, we further analyzed the genomic variants in two paired de novo and relapsed samples. We detected totally 40354 (de novo) and 16822 (relapsed) genomic variants, and among these 10415 (de novo) and 3082 (relapsed) are with amino acid change in patient 1; likewise, 30130 (de novo) and 14003 (relapse), and among these 7200 (de novo) 2534 (relapsed) with amino acid changes in patient 2. Totally 216 genomic variants with amino acid changes in 162 genes appeared only in the two relapsed samples, among which 10 genomic variants in ADAMTS8, CDK11B, EFCAB4B, FBXL21, HYDIN, IRF2BPL, MIR205HG, PRIM2, ZNF717, ZNF880 appeared in both relapsed samples, revealing their driver roles in relapse. Also, 110 of the 216 genomic variants are not previously reported. Conclusion: Genomic variants in common human cancer driver genes were also detected in B-ALL patients. The new genomic variants detected in the relapse samples may be involved in the oncogenesis of the relapse, which will be further verified with functional analysis. Our data also suggested the significance of developing more efficient new therapies to prevent the relapse in hematological malignancies. Disclosures No relevant conflicts of interest to declare.

Description: Background: SH2B3 (Src homology 2 B3), an adaptor protein can suppress activation of the JAK-STAT signaling pathway and the cytokine signaling pathway. Co-existence of SH2B3 mutations with IKZF1 deletion and JAK mutations are involved in oncogenesis in myeloproliferative neoplasms (MPN) and acute lymphoblastic leukemia (ALL).SH2B3 mutations and their clinical significance in adult ALL patients are still not well understood. Methods: 106 patients with newly-diagnosed ALL (79 B-cell and 27 T-cell ALL; 14-77 years old) between June 2008 and June 2016 were studied at Zhongda Hospital Southeast University. The 15 normal bone marrow subjects were enrolled as controls. The study was approved by the Ethics Committee of the institute. The 1-7 exons of SH2B3 were amplified from the genomic DNA extracted from fresh mononuclear cells of the patients by polymerase chain reaction (PCR) and nested PCR. The PCR products were extracted by gel purification and sequenced. Median differences between the cohorts were evaluated using a Mann-Whitney U-test. Frequency differences were analyzed using uni- and multivariate Cox model. Relapse-free survival (RFS) and overall survival (OS) were estimated by the Kaplan-Meier method and compared by log-rank test. Results: Two kinds of SH2B3 mutations (c.724C〉T, p.P242S; c.724C〉T, p.P242S ) were detected in 107 ALL patients. The two SH2B3 SNPs were detected in 20 patients with B-ALL, which represents a detection rate is 25.6% (20/79), and in 4 patients with T-ALL, equating to a detection rate of 14.8% (4/27). There was no statistical difference between the detection rate in B-ALL and T-ALL (P〉0.05). Both of the SH2B3 SNPs localize in the PH domain. The c.724C〉T, p.P242S (rs78894077) on exon2 (n=23, 18.69%), are only detected in the heterozygous genotype (CT). The % CD13 (+) was lower in patients with the SH2B3 P242S mutation than those without the mutation (20% vs. 47%, P=0.031). Deletion of IKZF1, the Ikaros gene is associated with poor prognosis. IK6, is one of the most common and important types of the gene deletion. In our study, IK6 was detected in 13 of 72 ALL samples with adequate DNA. In these 13 patients, 4 of them co-existed with a SH2B3 P242S mutation. No significant difference of prognosis was observed in the 4 patients compared to those lacking SH2B3P242S mutation. However, in the 59 patients without IK6, there were 13 patients harboring the SH2B3 P242S mutant. The EFS (18 months vs. 5 months, P=0.019, OR=0.471, 95%CI= [0.233-0.950]) and OS (32 months vs. 13 months, P=0.038, OR=0.382, 95%CI= [0.146-1.003]) in these patients were significantly longer than the other 46 patients without a SH2B3 mutation. This data indicates that the SH2B3 P242S mutation may be associated with a better prognosis in patients. The c.784C〉T, p.R262W on exon4 is detected in 1 Chinese patient (0.93%). There is also a rs3184504 (c.784T〉C) SNP recorded in the database. However, we detected both homozygous (CC) and heterozygous (CT) genotypes on the nucleotide 784 site in this cohort. The CC genotypes was observed in 100% (15/15) of the normal controls and 99.07% of the ALL patients. Only one patient (0.93%) harbored the CT genotype. This data indicates that in the Chinese Han population, the CC genotype is dominant, and it should be the wild type genotype. The wild type for the Chinese Han population for the SH2B3 amino acid residue 262 is arginine "R" not tryptophan "W". The "W" in this site is the SH2B3 SNP (R262W) in the population. The patient with this SNP was a 62 year old male. He had a high WBC count (123*10^9/L), a low PLT count (25*10^9/L), BCR-ABL1 fusion (FISH: BCR-ABL1: 220/300), and also had expression of myeloid markers (CD13: 53.9%)when diagnosed. In addition, this patient had co-existing IL7R mutations (c.197T〉C, p.I66T; c.254G〉A, p.V138I and c.337C〉T, p.H165H ) and an IKZF1 deletion, both of which are important members of the JAK-STAT signaling pathway. The patient relapsed 3 times within one year, and died with central nervous system leukemia. Certainly a bigger ALL cohort is needed to confirm if SH2B3 R262W SNP is related to relapse and poor prognosis. Conclusion: Two novel SH2B3 mutations were identified in a Chinese adult ALL cohort study. Our data showed that the different SH2B3 mutations have distinct clinical features, and the SH2B3 mutations may be used as new molecular markers for clinical risk stratification and even prognosis evaluation following a much bigger cohort validation. Disclosures No relevant conflicts of interest to declare.