Custom Amplicon Panel Research Articles

Lysosomal genes contribute to Parkinson’s disease near agriculture with high intensity pesticide use

Parkinson’s disease (PD), the second most common neurodegenerative disorder, develops sporadically, likely through a combination of polygenic and environmental factors. Previous studies associate pesticide exposure and genes involved in lysosomal function with PD risk. We evaluated the frequency of variants in lysosomal function genes among patients from the Parkinson’s, Environment, and Genes (PEG) study with ambient pesticide exposure from agricultural sources. 757 PD patients, primarily of White European/non-Hispanic ancestry (75%), were screened for variants in 85 genes using a custom amplicon panel. Variant enrichment was calculated against the Genome Aggregation Database (gnomAD). Enriched exonic variants were prioritized by exposure to a cluster of pesticides used on cotton and severity of disease progression in a subset of 386 patients subdivided by race/ethnicity. Gene enrichment analysis identified 36 variants in 26 genes in PEG PD patients. Twelve of the identified genes (12/26, 46%) had multiple enriched variants and/or a single enriched variant present in multiple individuals, representing 61% (22/36) of the observed variation in the cohort. The majority of enriched variants (26/36, 72%) were found in genes contributing to lysosomal function, particularly autophagy, and were bioinformatically deemed functionally deleterious (31/36, 86%). We conclude that, in this study, variants in genes associated with lysosomal function, notably autophagy, were enriched in PD patients exposed to agricultural pesticides suggesting that altered lysosomal function may generate an underlying susceptibility for developing PD with pesticide exposure. Further study of gene-environment interactions targeting lysosomal function may improve understanding of PD risk in individuals exposed to pesticides.

Abstract 6939: Residual disease after HER2 inhibition is driven by a primary tumor subpopulation expressing an ERBB2-low associated transcriptional program

Abstract Background: In HER2+ breast cancer (BC), residual disease (RD) after neoadjuvant HER2 inhibition is associated with increased risk of recurrence. However, the genetic and non-genetic resistance mechanisms underlying RD remain incompletely characterized. Here we used single nuclei (sn) RNA and DNA sequencing to profile genetic and transcriptional changes in growing, residual, and recurrent tumors from an inducible mouse tumor model of HER2+ breast cancer. Methods: We used mammary tumors/tumor bed (n=2-3/time point) from female MMTV-rtTA; TetO-ERBB2 mice at 9 weeks DoxOn (HER2on), 7 days DoxOff (HER2off) and 12-21 weeks Doxoff (recurrent). Samples were snap frozen and single nuclei isolation was performed. snRNAseq was performed using 10 × 5’ v2 kits for library preparation and sequencing. snDNAseq was performed using a custom amplicon panel targeting the mouse genome and human ERBB2 locus via the MissionBio Tapestri platform. Results: For snRNAseq analysis, tumor cell clusters were identified by Epcam and ERBB2 gene expression. Tumor cells were further subclustered into 18 unique clusters that separated by ERBB2 expression. Seven subclusters of HER2on tumor cells were characterized by low ERRB2 expression: 1.38 vs 2.94 (median normalized log counts) in HER2on-ERBB2high cells. Top 5 differentially expressed genes (DEG) per condition (log2 fold change >1.5, adjusted p value < 0.05) were: HER2on: Erbb2, Csn3, Lcn2, Csn2, Wfdc18; HER2on-ERBB2high: Csn3, Igfbp5, Csn2, Lcn2, Trf; HER2on-ERBB2low: Lars2, Gm47283, Cmss1, Gm42418, Neat1; HER2off: Lars2, Hexb, Cmss1, Gphn, Gm42418; Recurrent: Capza2, Vim, Lgals1, Crabp1, S100a6. HER2on-ERBB2low cells were more closely related in transcriptional space to HER2off residual tumor cells than HER2on-ERBB2high tumor cells. Trajectory analysis suggested that HER2off RD evolved from HER2on-ERRB2low cells and formed the basis of recurrent disease. To determine clonal evolution over time, we used common top 10 DEGs between HER2on vs HER2off vs HER2low snRNAseq data and intersected that set with significantly enriched single nucleotide variants (SNVs) from our snDNAseq data. We identified 16 clones containing at least 1 SNV at >1% frequency across all samples. We observed clonal persistence, relative enrichment and extinguishment between HER2on, HER2off and recurrent samples but without clonal dominance. Conclusion: Time-series analysis of genetic and transcriptional evolution in a mouse model of HER2+ BC RD suggests that a tumor cell subpopulation characterized by an ERBB2low-associated transcriptional program drives RD after HER2 inhibition. Citation Format: Sheheryar K. Kabraji, Weihua Wang, Yali Zhang, Johann Bergholz, Tao Jiang, Vittal Kurisetty, Maia Homsi, Jianmin Wang, Jean Zhao. Residual disease after HER2 inhibition is driven by a primary tumor subpopulation expressing an ERBB2-low associated transcriptional program [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6939.

Clonal Heterogeneity and Evolution during Treatment in High Hyperdiploid B-Cell Acute Lymphoblastic Leukemia

Introduction: Acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, is caused by the accumulation of multiple genomic aberrations in developing lymphocytes. In children, high hyperdiploidy (HeH) is the most prevalent subtype of B-cell ALL (B-ALL), which is associated with a superior outcome. However, because of the high incidence of HeH B-ALL in children, this subtype is responsible for about 25% of all relapses. The prediction of relapse remains difficult, especially in these good risk subtypes. Recent studies indicate that ALL is a heterogeneous disease, with multiple leukemia clones at diagnosis, each having a distinct set of mutations. We hypothesize that these different subclones have a different sensitivity to treatment and that samples with more heterogeneity are more prone to relapse. We used single-cell sequencing to determine the clonal heterogeneity of HeH B-ALL at diagnosis and followed the clonal evolution during chemotherapy treatment. Methods: Bone marrow samples of 11 patients with HeH B-ALL were collected at diagnosis, during treatment (for 9 cases) and at relapse (for 1 case) (Table 1). Samples during treatment were enriched for leukemia cells using magnetic antibodies targeting cell membrane markers (CD10, CD19, CD34) with the MARS® gentle sorter. We performed targeted single-cell DNA sequencing with the Mission Bio Tapestri® Platform, using our custom amplicon panel of 414 amplicons across 39 commonly mutated genes. This method detected single nucleotide variants (SNVs) and small insertions/deletions (indels) based on targeted PCR amplification in about 4000 cells per sample. Results: We sequenced diagnostic samples from 11 HeH B-ALL patients. For 9 patients we also tested samples during treatment, either at day 15 of treatment (1 case) or at the end of induction (EOI) (8 cases), and for 3 cases an additional sample at the end of consolidation (EOC). Furthermore, for one patient, we analyzed samples of 3 subsequent relapses. In total, 117,148 single cells were processed, with an average of 4500 cells per sample. We detected multiple subclones in almost all diagnostic samples (9/11), ranging from 2 to 10 subclones per case. Most frequently mutated genes were KRAS, NRAS, PTPN11 and FLT3, as well as NSD2. Four cases had 2 or more different KRAS and/or NRAS subclones. Three cases had a subclone with a KRAS/ NRAS mutation co-occurring with a NSD2 mutation. For the patients with multiple subclones at diagnosis, we subsequently performed single-cell analysis on one or more follow-up samples, either during treatment or at relapse. In the samples taken at EOI, we detected residual leukemia cells in all 8 cases: multiple subclones were detected in 5 cases and only one subclone persisted in 3 cases. Residual leukemia cells were also detected in all samples collected at EOC, though in a lesser amount as compared to the previous timepoint sequenced. Interestingly, in the matched diagnosis-relapse case, we found that a minor subclone with a NSD2, PTPN11and KRAS mutation became the dominant clone at first relapse (Figure 1). Conclusions: Single-cell DNA sequencing reveals a strong genetic heterogeneity in HeH B-ALL based on a variety of mutations in KRAS, NRAS, PTPN11, FLT3 and NSD2. RAS mutations and FLT3 mutations are mutually exclusive in subclones, whereas NSD2 mutations and RAS mutations are frequently co-occurring in the same subclone. This cooperation is highlighted in our relapse patient, having a minor subclone with a KRAS and NSD2 mutation at diagnosis that became the largest subclone at relapse. Even though few leukemia cells persisted during treatment, we were able to genetically identify the residual subclones with a potentially higher therapy resistance.

Genome-wide and panel-based cell-free DNA characterization of patients with resectable esophageal adenocarcinoma.

Circulating tumor DNA (ctDNA) holds promise in resectable esophageal adenocarcinoma (EAC) to predict patient outcome but is not yet sensitive enough to be clinically applicable. Our aim was to combine ctDNA mutation data with shallow whole-genome sequencing (sWGS)-derived copy number tumor fraction estimates (ichorCNA) to improve pathological response and survival prediction in EAC. In total, 111 stage II/III EAC patients with baseline (n = 111), post-neoadjuvant chemoradiotherapy (nCRT) (n = 68), and pre-surgery (n = 92) plasma samples were used for ctDNA characterization. sWGS (<5× coverage) was performed on all time-point samples, and copy number aberrations were estimated using ichorCNA. Baseline and pre-surgery samples were sequenced using a custom amplicon panel for mutation detection. Detection of baseline ctDNA was successful in 44.3% of patients by amplicon sequencing and 10.5% by ichorCNA. Combining both, ctDNA could be detected in 50.5% of patients. Baseline ctDNA positivity was related to higher T stage (cT3, 4) (p = 0.017). There was no relationship between pathological response and baseline ctDNA positivity. However, baseline ctDNA metrics (variant allele frequency > 1% or ichorCNA > 3%) were associated with a high risk of disease progression [HR = 2.23 (95% CI 1.22-4.07), p = 0.007]. The non-clearance of a baseline variant or ichorCNA > 3% in pre-surgery samples was related to early progression [HR = 4.58 (95% CI 2.22-9.46), p < 0.001]. Multi-signal analysis improves detection of ctDNA and can be used for prognostication of resectable EAC patients. Future studies should explore the potential of multi-modality sequencing for risk stratification and treatment adaptation based on ctDNA results. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Assessing sequence variation and genetic diversity of currently untapped Y-STR loci

With the advent of massively parallel sequencing (MPS) the full genetic variation of short tandem repeat (STR) markers can be assessed. Most MPS efforts of forensic interest to date have focused on autosomal STRs, with far less attention to the potential genetic variation contained within Y-STR loci. Many of the non-commercialized Y-STR loci described previously have not been reassessed with MPS to determine the full level of genetic diversity of these markers at the sequence level. In this study, genomic data were mined from the Phase 3 1000 Genomes Project to identify forensically relevant STRs using lobSTR, HipSTR, and an in-house pipeline. These loci then were typed in a dual-phase approach with an Illumina Custom Amplicon (281 markers; ≤ 425 bp) panel followed by an AmpliSeq Custom Amplicon (83 markers; ≤ 275 bp) panel using four populations (African American, US Caucasian, US East Asian, and US Southwest Hispanic). STRs also were assayed with the ForenSeq Signature Prep Kit. The genetic diversity of the loci was assessed and ranked for a final set of 81 loci. When ranking all loci with respect to genetic diversity highest-to-lowest, seven of the top ten loci are not utilized in a current commercial assay.

SNPs Identification in production related genes in strains of white leghorn chicken

This is the first comprehensive study aimed to determine Single Nucleotide Polymorphisms (SNPs) in candidate genes (GHR, IGF1, OCX-32 and GDF9) reported to associated with egg production at the age of 64 weeks (EP64) in strains of White Leghorn chicken (Anand Synthetic White Leghorn and Anand Bantamised White Leghorn by sequencing the coding region of respective genes. Both the strains of White Leghorn chicken were categorized into four different groups viz., Anand Synthetic White Leghorn High Production (ASHP), Anand Synthetic White Leghorn Low Production (ASLP), Anand Bantamised White Leghorn High Production (ABHP) and Anand Bantamised White Leghorn Low Production (ABLP) to find out SNPs in coding region of candidate genes and its association with EP64. The genomic DNA was extracted for 96 chickens and then further sequenced with the designed custom amplicon panel on the Illumina MiSeq platform. Raw data collected after sequencing was used for the bio in formatic analysis to confirm its association with EP64. Consequently, over 99% target regions were amplifed and 87 SNPs were available after quality filtering. Only thirty-three variants were novel while majority of the identified variants were catalogued in dbSNP database as known variants. The T21912084G, C21912423A and rs318030570 of OCX-32 and C13415980T of GHR gene was found to have a significant association (p

Cell-Type and Allele Specific Distribution of Multiple TET2 Mutations in Two Patients with Chronic Myelomonocytic Leukemia (CMML)

Background: Chronic myelomonocytic leukemia (CMML) is an aging disease that arises on the background of clonal hematopoiesis, commonly involving multiple TET2 mutations or TET2/SRSF2 co-mutations. In a prior study of 1084 CMML patients (Leukemia 2020; 34:1407-1421), we demonstrated a high prevalence of TET2 mutations (56%) in CMML, with approximately 30% of patients having >1 TET2 mutation. The cell-type and allele specific distribution of these mutations remains of great interest and are thus far poorly defined. To visualize the genotypic and immunophenotypic correlates, we performed single-cell DNA analysis and simultaneous protein profiling on 2 CMML patients with multiple TET2 mutations.Methods: After Mayo Clinic IRB approval, two CMML patients with dual TET2 mutations and available peripheral blood mononuclear cells were included in the study. Patient samples were labeled with TotalSeq TM -D Heme Oncology Cocktail (BioLegend, San Diego, CA) consisting of 45 unique oligo-conjugated cell surface markers. The samples were then washed and loaded into a Tapestri microfluidics cartridge, where single cells (3,500 cells/µL) were encapsulated, lysed and barcoded using the MissionBio Myeloid custom amplicon panel consisting of 312 amplicons in 45 genes relevant to myeloid disorders (MissionBio, San Francisco, CA). The individual barcoded DNA and protein for each cell was purified, recovered, and labeled with Illumina i5/i7 indices, quanitifed by Agilent Bioanalyzer and pooled for sequencing. The libraries were sequenced via Illumina NovaSeq by the Mayo Genomics Core, and data was recovered and analyzed using MissionBio Tapestri Insights v3.0.2.Results: Two dual TET2 mutated CMML patients were included in the study, both male, ages 79 and 83, and both with CMML-0. Case 1 had TET2 p.G1172Efs*53 (variant allele fraction/VAF-6%) and p.H1732Tfs*13 (VAF-13%) mutations, while case 2 had TET2 p.Y620Ffs* (VAF-85%), p.G898* (VAF-82%), and SRSF2 p.95H (VAF-45%) mutations, respectively. In both patients, TET2 mutations were found to be preferentially distributed in monocytes (CD11b+, CD14+, CD64+, CD13+, CD33+, HLA-DR+), with very few classically defined granulocyte (CD16+,CD13+.CD33+, HLA-DR neg), B (CD19+,CD22+)/T (CD2+,CD4+,CD5+,CD7+) lymphocytes, NK cells (CD16+/CD56+/HLA-DR neg) and plasma cells (CD38+/CD138+), demonstrating mutant TET2. Among monocytes, TET2 mutations were enriched in classical/M01 (CD11b+, CD14+,CD16-) and intermediate/M02 monocytes and were infrequent in the MO3 fraction, commensurate with monocyte repartitioning seen in CMML. Interestingly, a large number of single cells with TET2 mutations also demonstrated an aberrant surface phenotype, with universal expression of CD14+ and CD11b+, along with a mixture of other markers (CD163+, CD11c, CD4+, CD2+, and/or CD56+). With regards to allelic distribution, while several permutations and combination were seen involving heterozygosity and homozygosity for the TET2 mutations identified on bulk sequencing, data was corroborative for both TET2 mutations being located on trans alleles.Conclusions: In this study, we demonstrate that TET2 mutations in CMML are highly enriched in classical circulating monocytes and infrequently occur in classical granulocytes, B/T and NK cells; with multiple TET2 mutations most commonly co-existing on trans alleles. Analyses on additional samples along with copy number variation assessments are ongoing.Figure 1: Lollipop plot of TET2 exons with dual TET2 mutations labeled above and oxygenase domain labeled in green; below are number of cells identified with each genotype for Case 1 (A) and Case 2 (B). Mutations are labeled proximal (yellow) and distal (red) and alleles are in grey. The directionality of the arrows to different genotypes is based on decreasing cell number and genotype. Uniform manifold approximation and projection (UMAP) plot of Case 1 (C) and Case 2 (D) with cells clustered by immunophenotype and labeled by genotype (non TET-2 mutated purple, heterozygous TET2 in green, homozygous TET2 in yellow). Relative protein expression of T-cell phenotype (CD14-, CD3+, CD4+/-,CD8+/-, CD5+, CD7+, CD2+), or classical monocytes (CD14+/CD16-) for Case 1 (E & F) and Case 2 (G & H) overlayed on UMAP. Royal blue represents non TET2-mutated cells, and mutated cells are labeled in pastel blue, green and light orange by mutation type. [Display omitted] DisclosuresDurruthy-Durruthy: Mission Bio Inc.: Current Employment. Arribas-Layton: Mission Bio, Inc.: Current Employment. Patnaik: StemLine: Research Funding; Kura Oncology: Research Funding.

Evidence of NF-ΚB Pathway Activation in Patients with Advanced, High Molecular Risk Myelofibrosis

Evidence of NF-ΚB Pathway Activation in Patients with Advanced, High Molecular Risk Myelofibrosis

Peripheral Blood Single-Cell Genotyping and Phenotyping in Multiple Myeloma Reveals Shared Mutations across Multiple Hematopoietic Cell Lineages

Background:Multiple myeloma (MM) is a hematologic malignancy of terminally differentiated plasma cells. While most genomic studies of the disease have focused on characterizing isolated bone marrow plasma cells, few have addressed somatic mutations within the immune microenvironment. We previously demonstrated that somatic mutations are significantly more numerous in unsorted blood compared to bone marrow and that the number of mutations correlated with elevated markers of disease. We also reported that genes implicated in clonal hematopoiesis of indeterminate potential (CHIP) are commonly mutated in the blood from patients with MM.Aim:To integrate single-cell DNA mutation analysis and protein expression to map the clonal relationship between circulating plasma cells and non-plasma cells within the peripheral blood of patients with MM.Methods:Although bulk DNA sequencing can estimate clonal heterogeneity using analytic deconvolution techniques, it is unable to distinguish which mutations occur in the same clone(s) nor can it correlate genotype with immunophenotype from the same cell. To accurately describe the clonal architecture of the blood, we combined targeted single-cell DNA sequencing (scDNA-seq) coupled with protein expression to define mutations specific to cell phenotypes using the Tapestri Platform (Mission Bio). For this analysis, we developed a custom amplicon panel covering 22 frequently mutated genes in MM and/or CHIP to perform scDNA-seq and 4 antibodies targeting CD138, CD19, CD3, and CD33 for immunophenotyping or plasma cells, B cells, T cells, and myeloid cells respectively.Results:Peripheral blood mononuclear cells were isolated from 17 blood samples collected from 12 patients with MM. Three samples were collected at diagnosis before treatment, 8 were collected post-treatment during remission, and 6 were collected at relapse. Serial samples were obtained before and after autologous stem cell transplant from 4 patients. A total of 85,262 cells were sequenced uncovering 154 unique variants in 22 genes after quality filtering. There were 47 somatic variants, defined as those present in less than 40% of cells per sample. There were 52,136 total clones, defined as cells with identical genotypes, within this heterogenous population of cells. The most common non-synonymous somatic mutations were identified in genes SETD2, KMT2C, PPM1D. An immunophenotype was assigned to 82% of cells according to the protein with the most abundant number of reads. Dimensionality reduction of single-cell genotype using t-distributed stochastic neighbor embedding (t-SNE) effectively clustered cells according to sample and by patient in cases where there were multiple samples from the same individual. Patients with active myeloma and known circulating plasma cells detected by clinical flow cytometry were found to have the greatest proportion of CD138+ cells as well as the highest mutational burden. It was rare to detect the same clone in two separate samples collected from the same patient at different time points (median time be collection was 248 days) which may be due to under-sampling or clonal evolution. Surprisingly, all somatic variants detected in plasma cells, were also present in other cell types suggesting they were acquired from a common progenitor cell rather than after differentiation.Conclusions:scDNA-seq coupled with immunophenotyping of the peripheral blood of patients with MM reveals somatic variants are shared across multiple hematopoietic cell lineages including plasma cells indicating these mutational are acquired from a common progenitor. DisclosuresDurruthy-Durruthy: Mission Bio Inc.: Current Employment. Arribas-Layton: Mission Bio, Inc.: Current Employment. Wang: Mission Bio, Inc: Current Employment.

Preexisting Mutations in Hematopoietic Stem Cells in Patients with Therapy-Related Myeloid Neoplasms

Introduction: Recently, it has been suggested that some hematopoietic stem cell clones (HSC) harbor preexisting genomic mutations which could be selected for antineoplastic treatments received against a first neoplasm (1st-neo) that might, eventually, lead the development of therapy-related myeloid neoplasms (TRMN).Aim: To analyze genetic mutational profile in a cohort of patients with TRMN from different national centers. To determine the presence of preexisting mutations in HSC by deep massive sequencing: genetic composition and evolutive behavior.Methods: We studied 103 patients with TRMN from 6 national hospitals. The average age was 62 years old (range: 31-89); 45% were men; as 1st-neo: 36% were breast cancer, 34% lymphoma, 5% multiple myeloma and 25% other type of neoplasm. In 18 patients, a paired sample of bone marrow at the moment of the 1st-neo was available [previous disease: lymphoma (n=13), multiple myeloma (n=4), breast cancer neo (n=1)]. We sequenced the complete coding region of the genes: BCOR, BRAF, CDKN2A, CEBPA, DNMT3A, ETV6, EZH2, GNAS, LUC7L2, NF1, PHF6, PTPN11, RAD21, RPS14, SF1, SF3A1, SMC3, SPARC, SRSF2, STAG2 and ZRSR2 and the hotspot regions of genes: ASXL1, MPL, NPM1, JAK2, KRAS, NRAS, TET2, U2AF1, KIT, IDH1, RUNX1, IDH2, SETBP1, TP53, WT1, CBL, SF3B1 and FLT3 with a custom amplicon panel (Ampliseq). Variants were selected according to VAF≥1%, MAF<0,01 and its effects upon the protein. Depth coverage in 1st-neo samples was 10,000x and 2,000x for TRMN.Results: At least one variation was detected in the studied genes in 98% of patients with TRMN (n=101) [321 mutations (263 missense and 58 indels); 3,2 mut/pa (0-12)]. Most frequently mutated genes were TP53 (35%), TET2 (32%), DNMT3A (17%), RAD21 (14%), SETBP1 (12%), IDH2 (8%), SF3B1 (8%) and SRSF2 (7%). Half of the patients with paired sample harbored mutations in the 1st-neo sample (14/18). In total, 27 pathologic variations were detected in these samples (24 SNV and 3 indels). In 4 cases, mutations prevailed after evolution (22%), increasing their variant allelic frequency (VAF) and suggesting the selection of a preexisting clone after antineoplastic treatment (Table 1).Conclusions: At least one variation in the studied genes was detected in 98% of the patients with TRMN, being TP53 mutations the most frequent (35%). In 22% of the cases with paired sample was confirmed the expansion of a clone present in the apparently normal bone marrow at the moment of the first neoplasm, suggesting a possible mechanism of selective pressure in the context of the received antineoplastic therapy.Research funded by CIBERONC CB16/12/00284, PI12/01047, RD12/0036/0014, PIE13/00046, PI13/01640, PI13/02837, PT13/0010/0026, PI14/01649, AC15/00068, PI16/011113 and PI16/00665. [Display omitted] DisclosuresMontesinos:Celgene Corporation: Honoraria, Research Funding. Sanz:Gamida Cell: Research Funding.

Mutations in DNMT3A, U2AF1, and EZH2 Identify Intermediate-Risk Acute Myeloid Leukemia Patients with Poor Outcome after CR1

Introduction: Intermediate-risk acute myeloid leukemia (IR-AML) is a clinically heterogeneous disease, for which optimal post-remission therapy has been debated. Several studies using next-generation sequencing have identified important prognostic information and led to modifications in risk stratification. However, the utility of this genetic information in decision making for IR-AML has yet to be elucidated. We investigated the clinical relevance of recurrent driver gene mutations in a well-characterized cohort of IR-AML patients in first complete remission (CR1) after induction chemotherapy.Methods: A total of 1,589 AML patients treated at the Cleveland Clinic (CC) between 2002 and 2016 were screened for eligibility, of whom 825 were in European Leukemia Net (ELN) IR category. Of them, 100 IR-AML patients, who had mutational information at diagnosis and achieved CR after ≥ 1 round of intensive induction were included. CR was defined as <5% bone marrow blasts, absolute neutrophil counts ≥ 1x109/L, and platelet counts ≥ 100x109/L. CR with inadequate count recovery (CRi) was same as CR, except for residual neutropenia or thrombocytopenia. The induction regimen was 7+3, and patients with persistent disease at day 14 marrow were re-induced with 7+3 or 5+2. Patients who did not receive intensive therapy or achieve CR/CRi were excluded. A TruSeq Custom Amplicon panel targeting exons of 62 genes was used for deep sequencing at diagnosis. We used The Cancer Genome Atlas (TCGA) data for validation of our findings.Results: In the CC cohort, median age was 58.5 (24-75) years, 64% had normal cytogenetics, and 31% required >1 induction cycles to achieve CR1. DNMT3A and FLT3-ITD mutations were the most common (19%), followed by RUNX1 (15%), TET2 (12%), IDH2 (12%), NPM1 (12%), ASXL1 (11%), and U2AF1 (11%). The frequency of persistent disease after initial induction was higher in patients with mutations in DNMT3A (47% vs 27%, p= .03) and U2AF1 (55% vs 28%, p= .01), while CRi was more common in patients with BCOR mutation (50% vs 8.5%, p= .02). In univariable analysis, patients with mutations in DNMT3A, U2AF1, and EZH2, and patients who achieved CRi after induction had worse overall (OS) and relapse-free survival (RFS). Patients who had hematopoietic cell transplant (HCT) in CR1 achieved better OS (HR, 0.4; 95% CI, 0.2-0.7; p=0.003) and RFS (HR, 0.3; 95% CI, 0.1-0.6; p< 0.001) than those who did not. Other clinical factors analyzed, such as age, sex, cytogenetics, and the number of induction courses to achieve CR1 were not significant predictors for survival after CR1. To evaluate the impact of these three mutations on risk stratification, we compared patients whose AML harbored any of the mutations with those who did not. The median OS and RFS for patients with mutations were 13 and 9 months, respectively, compared to 41 and 16 months in patients without (p< 0.0001 and p= .003). Similarly, TCGA patients with mutations had significantly poorer OS (median, 9.8 vs 46.8 months, p= .0002) and RFS (median, 9.3 vs 17 months, p= .02). In multivariable analysis, mutations maintained an independent effect on survival (Table 1).Since mutations and HCT were the major predictors of outcome after CR1, we analyzed the outcomes of patients harboring mutations in relation to HCT (Figure 1). In both cohorts, patients who did not have the mutations and underwent HCT had the best outcomes. In the CC cohort, median OS and RFS for patients who had mutations and HCT were 14 and 11 months, respectively, compared to 7.5 and 5 months in patients who did not undergo HCT (p= .04 for OS and p= .002 for RFS). In the TCGA cohort, patients who harbored mutations and did not undergo HCT had significantly worse OS (median, 6.3 vs 24.6 months, p= .001) and RFS (median, 7.8 vs 12.5 months, p= .003). In view of the recently proposed ELN 2017 criteria that place IR patients with RUNX1 or ASXL1 mutations into the adverse risk, we explored outcomes of these patients at the time of CR1. Presence of these mutations did not predict OS and RFS in our cohort, and performance of HCT did not confer a significant survival benefit in patients with these mutations.Conclusions: In two independent cohorts, DNMT3A, U2AF1, and EZH2 mutations were predictors of relapse and OS in IR-AML at CR1, and performance of HCT translated into survival benefit. Our results provide evidence of clinical utility in considering mutation screening to stratify IR-AML patients to guide therapeutic decisions. [Display omitted] DisclosuresSekeres:Celgene: Membership on an entity's Board of Directors or advisory committees. Gerds:CTI BioPharma: Consultancy; Incyte: Consultancy. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Speaker Fees; Ra Pharma: Consultancy; Apellis Pharmaceuticals: Consultancy. Advani:Pfizer: Consultancy; Takeda/ Millenium: Research Funding.

Molecular Characterisation of Participants in the Phazar Trial Reveals Prognostic Impact of Mutations in Advanced-Phase-MPN

Molecular Characterisation of Participants in the Phazar Trial Reveals Prognostic Impact of Mutations in Advanced-Phase-MPN

Prognostic Prediction Clinical-Molecular Model for Allogeneic Transplant Outcomes in Patients with Myelodysplastic Syndrome

Introduction Hematopoietic stem-cell transplantation (HSCT) has demonstrated only curative potential for patients with myelodysplastic syndrome (MDS), still prognosis after HSCT are highly different in MDS patients. Over the past decade, several studies have demonstrated that outcomes after HSCT are influenced by clinical and molecular factors, which suggests that a prognostic model combining clinical and molecular-related factors are needed to be identified. Method A total of 138 MDS patients who underwent allogeneic HSCT between 2010 and 2018 at the Institute of Hematology and Blood Disease Hospital were considered for inclusion in this study. Median follow-up duration was 33 months. A Custom Amplicon panel targeting exons of 185 genes was used for deep sequencing before transplant. We used a LASSO Cox regression model to achieve shrinkage and variable selection from prognostic factors (P&amp;lt;0.1 in log-rank tests) to build risk score for predicting overall survival. A nomogram was constructed to display the risk of death in individuals. The discrimination of the risk score was measured by the concordance index (C-index) and areas under time-dependent receiver-operating characteristics (ROC) curves (AUCs), and the calibration of the risk score was explored graphically by calibration plots. Patients from the whole cohort were randomly assigned into a training group comprising two-third of the patients, and validation cohort, comprising the remaining one-third of the patients. Results In our cohort, U2AF1 was the most recurrently mutated genes (31%), followed by ASXL1 (23.9%), RUNX1 (14.5%), TP53 (10%) and TET2 (10%). Univariate analyses in training cohort shows, HCT-CI≥3, Blast&amp;gt;0.065, IPSS-R score, TERT, ANKRD26 and PDGFRB mutations were significantly associated with poor OS (P &amp;lt; 0.05). Five variables were incorporated in our scoring model by LASSO, including IPSS-R score, HCT-CI, TERT, PDGFRB, ANKRD26 mutation. A risk scoring model was developed incorporating the weighted coefficients of these variables. The risk score grouped patients into two subgroups: low risk (LR, score ≤1.7, n= 78) and high risk (HR, score &amp;gt; 1.7, n= 13) groups. The 3-year OS for LR and HR groups were 80.5% and 23.1% (P &amp;lt; 0.0001), and 3-year DFS were 73.9% and 23.1% (P &amp;lt; 0.0001), respectively (Figure A-B). The similar results were also observed in validation cohort (3-year OS 88.9% vs. 50.0%, P= 0.0032; 3-year DFS were 83.3% and 50.0%, P =0.016, Figure C-D). Concordance index [train: 0.682, 95% CI (0.63, 0.734), validation: 0.747, 95% CI (0.655, 0.839)] demonstrated well discrimination power and calibration plots showed that the nomograms did well compared with an ideal model. Conclusions Here we create a new model combining clinical and molecular-related factors provides better survival stratification for patients with MDS receiving HSCT, provide a useful tool to predict transplant outcomes for MDS patients before transplant. This could have important implications as it can help better identify MDS patients who will benefit the most from transplantation, and may impact the choice of intensity of preparative regimens for transplantation as well as post-transplant maintenance therapy. Additional independent cohorts and prospective studies are needed to externally validate our model before implementation into clinics. Figure 1 Disclosures No relevant conflicts of interest to declare.

Evaluation of an amplicon-based custom gene panel for the diagnosis of hereditary tumors.

Genetic testing based on next-generation sequencing (NGS) analysis has recently been used to diagnose hereditary diseases. In this study, we explored the usefulness of our custom amplicon panel that targeted 23 genes related to hereditary tumors given in the American College of Medical Genetics and Genomics recommendations. We applied our custom NGS panel to samples from 12 patients previously diagnosed by Sanger sequencing as having the diseases or diagnosed clinically by meeting the diagnostic criteria in this study. Our gene panel not only successfully identified all variants detected by Sanger sequencing but also identified previously unrecognized variants that resulted in confirmation of the disease, or even in the revision of the diagnosis. For instance, a patient identified with an SDHD gene mutation actually had von Hippel-Lindau (VHL) syndrome, as determined by the presence of a pathogenic VHL gene variant. We also identified false-positive results that were generated by amplification of genome regions that are not intended to be investigated. In conclusion, NGS-based amplicon sequencing is a highly effective method to detect germline variants, as long as they are also carefully reviewed by manual inspection.

Spectrum of Genetic Defects and Phenotype-Genotype Correlation in Dyserythropoietic Anemias: Bench to Bedside Approach in the Indian Scenario

Introduction Congenital dyserythropoietic anemias (CDA) are rare inherited red cell disorders characterized by ineffective erythropoiesis and inappropriate reticulocytopenia. CDAs are usually difficult to diagnose due to variable phenotypes and overlapping bone marrow (BM) morphology with other disorders. Numerous implicated causal genes make Sanger sequencing a less likely approach and hence, the use of targeted resequencing can expedite molecular diagnosis. This study aimed at determining the genetic spectrum of CDAs and translating the results into patient care. Methods Twenty nine patients with clinical and laboratory evidence suggestive of CDA and 1 patient suggestive of CDA with thrombocytopenia by BM morphology were studied. Various biochemical and molecular tests were done to exclude common hemolytic anemias. Common SEC23B: p.Tyr462Cys variant in our patients with CDA was screened by Sanger sequencing. DNA libraries were prepared using TruSight One Sequencing Panel and TruSeq Custom Amplicon Panel and sequenced on Illumina platform. After data analysis variants were classified and the most likely disease-causing variants were validated by Sanger sequencing followed by pedigree analysis. Results Out of 27 patients of suspected CDA, SEC23B: p.Tyr462Cys variant was found in 10 patients. Rest of the remaining 17 patients were subjected to targeted resequencing. Data analysis revealed novel potentially pathogenic variants in compound heterozygosity in SEC23B in 4 patients and 1 patient had a heterozygous variant in SEC23B. There could be the possibility of intronic or large indel in her. The variants were distributed throughout the SEC23B gene. Notably, in 7 patients with suspected CDA, the final molecular diagnosis were hemolytic anemias. Of them, 4 showed likely pathogenic variants in PKLR gene and 1 each had probably causal variant in MTRR, SPTB and PIEZO1 genes. In the patient's with pyruvate kinase deficiency, screening by enzyme assays were normal. Except for the patient with MTRR gene defect all 6 had transfusion dependent anemia and BM showed dyserythropoiesis. One patient each of GATA1 gene variant (novel) and a known pathogenic variant p.Glu325Lys in KLF1 gene (CDA type IV) was detected. Of 17 cases subjected to targeted resequencing the diagnosis was achieved in ~76% (13/17) of cases. The phenotypes correlated with the genetic defects found in the SEC23B gene. The homozygous and compound heterozygous defects in this gene cause CDA type II. As anticipated GATA1 gene defect (p.Val205Leu) was found in a patient of X-linked thrombocytopenia with dyserythropoietic anemia. Patient with KLF1 had high levels of fetal hemoglobin along with features of dyserythropoiesis in BM compatible with the phenotype of variant p.Glu325Lys causing CDA type IV. Phenotype-genotype correlation was discrepant in 7 cases of CDA. In 4 cases pyruvate kinase deficiency (PKLR) was found and each case of hereditary xerocytosis (PIEZO1), membrane defect (SPTB) and MTRR defect was found. Conclusion(s) CDA showed a highly varied etiology. Our experience demonstrates a high diagnostic yield (~76%) of targeted resequencing for molecular diagnosis of suspected CDAs. Discrepancy was noted in 41% (7/17) cases with suspected CDA which were diagnosed as hemolytic anemia after molecular analysis. Establishing the correct diagnosis of pyruvate kinase deficiency led to an evidence-based decision of splenectomy that eliminated transfusion dependence. In the patient with MTRR defect change in therapy was suggested. Prenatal diagnosis was done for 2 families, where in 1 of the family both the SEC23B variants were novel and in compound heterozygosity. This study highlights the importance of genetic testing in patients under frequent blood transfusions and suspected CDAs, to provide accurate diagnosis and therapeutic interventions. Disclosures No relevant conflicts of interest to declare.

Notch1 mutation in B Chronic Lymphocytic Lymphocytes Induces Proliferation through Cell Cycle G1/S Phase-Specific Cyclin Overexpression

Background and Aim: Chronic lymphocytic leukemia (CLL) is an incurable neoplasm with considerable clinical heterogeneity that is underpinned by distinct genetic changes. This poses a great challenge to the development of effective treatment, as responses to novel therapies may vary depending upon the biological subtype of disease. NOTCH1 has emerged as the most commonly mutated gene in CLL at diagnosis, its frequency rises with progression, and the variant c.7541_7542delCT accounts for approximately 80% of cases. NOTCH1-mutated (NOTCH1mut) CLL subset represents a subtype that responds poorly to anti-CD20 drugs, has worse outcomes, and a proliferative profile. We aimed, among the proliferative signals, to study the D-type cyclins that govern cell cycle entry in a NOTCH1mut subset which lacks an accurate targeted therapy. Methods: We performed targeted sequencing on 289 CLL cases with a TruSeq Custom Amplicon panel from Illumina, containing 13 genes recurrently mutated in CLL. Paired-end sequencing reached a mean depth of 938 reads/base. Out of 30 NOTCH1mut patients, RNA from the CD19+ fraction was available in 25 cases. Cyclins D1, D2, D3 and cyclin dependent kinases CDK4 and CDK6 expression was measured by RT-qPCR in 25 NOTCH1mut and 57 NOTCH1wild type (NOTCH1wt). CD19+ B-CLL cells from 7 NOTCH wt and 2 NOTCH1mut patients were cultured in Expansion Human Media supplemented with CD40L, IL-4 antibiotics and inactivated BFS. On day ten 2,5x105 B cells were added to a combination of ON-TARGETplus SMART pool siRNA NOTCH1, pool siRNA CCND1, SMART pool siRNA CCND2, or ON-TARGETplus siCONTROL as negative control. Electroporation was performed with Neon Transfection System in 10 ul tips using settings of 1400 volts, 10ms width, and 3 pulses. Transfected cells were double stained with Annexin V-FITC conjugate and PI in isotonic solution, and then analyzed by FC. Viable cells were defined as double negative cells. Results: A total of 82 CLL patients, 25 NOTCH1mut and 57 NOTCH1wt were included in the RT-qPCR study. Figure 1a shows fold change expression and p value comparing NOTCH1mutvs.NOTCH1wt of CDK4 (FC= 11,1; p&amp;lt;0,001), CDK6 (FC= 11,2; p&amp;lt;0,001), CCND1 (FC= 8,9; p&amp;lt;0,001), CCND2 (FC= 8,4; p&amp;lt;0,001), and CCND3 (FC= 15; p&amp;lt;0,001). As cyclin D2 and CDK4 are located both on chromosome 12, we next checked whether that overexpression was due to copy number gains. However, we found no significant differences in the expression of Cyclin D2 in trisomy 12 NOTCH1mut cases (n=3) vs. non trisomy 12 NOTCH1mut cases (n=22) (0,28 vs. 0,37; p= 0,8) while CDK4 expression was higher in trisomy 12 NOTCH1mut vs. non trisomy 12 NOTCH1mut cases with a trend to reach statistical significant (0,74 vs. 0,29; p= 0,06). Cell culture from 7 CLL patients was stable until optimal day of culture and confluency for transfection and apoptosis assay. Viability was studied by the trypan blue dye exclusion test. Patients with NOTCH1mut kept a higher cell count and viability than NOTCH1 wt (70% cell survival in NOTCH1mut vs. 45% in NOTCH1wt on day +8). Next we transfected cultured primary cells from two c.7541_7542delCTNOTCH1mut and 5 NOTCH1wt patients. Figure 1b shows the cell viability assay after 48 h of the transfection, highlighting a marked reduction in viable cells in NOTCH1mut cases in each silencing experiment, (siRNA_Control: 77% NOTCH1sirna: 71 %, CCND2siRNA: 73% and CCND1: 72%) which was not paralleled in NOTCH1wt cases (siRNA_Control: 81% NOTCH1sirna: 80 %, CCND2siRNA: 84% and CCND1: 78 %). Figure 1C shows how the apoptosis index increased in every silencing experiment compared with NOTCH1mut siRNA pool control. However, for therapeutic purposes the accurate approach should increase apoptosis, leading the cells to a controlled death, and reduce necrosis, which is responsible of inflammation and collateral damage. The optimal apoptosis/necrosis ratio in our experiments, it is shown by the silencing of CCDN2 in NOTCH1mut (ratio A/N=5,15) where percentage of necrotic cells was markedly reduced (fig.1d). Conclusions: We found a marked upregulation of cyclins governing the transition from G1 to S phase of the cell cycle in patients with CLL and NOTCH1 mutation compared with those wild type cases for this lesion. In addition, silencing of cyclin D2 in the NOTCH1mut CLL subset was able to reach a similar degree of apoptosis than silencing NOTCH1, with a more favourable reduction of necrosis, emerging as a suitable candidate for a therapeutic target. Disclosures Jerez: Novartis: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Molecular Analysis in the Pacritinib Dose-Finding PAC203 Study in Patients with Myelofibrosis Refractory or Intolerant to Ruxolitinib

PAC203 is a global multicenter dose-finding study of pacritinib (PAC), an oral JAK2/IRAK1 inhibitor in patients with primary or secondary myelofibrosis refractory or intolerant to ruxolitinib, including patients with severe thrombocytopenia. Patients were randomized 1:1:1 (PAC 100mg QD, 100mg BID or 200mg BID) and stratified by baseline platelet count. The mutational landscape of this patient group is not well characterised, and the impact of mutation status on disease response and hematologic parameters is unknown. We carried out baseline mutational analysis on 105 (of total 164 recruited; 161 treated) patients using an ISO accredited Illumina TruSeq Custom Amplicon Panel, including 32 gene mutation hotspots and exons (~36,000 bp, 287 amplicons). CALR mutation screening was carried out independently. Accepted coverage was achievement of a depth of ≥100 reads per base in ≥95% of targeted bases. Median follow-up time for this cohort was 163 (28-476) days. 57% of patients had a diagnosis of primary myelofibrosis (PMF), whereas 27% and 15% had post-polycythemia vera MF (PPV-MF) and post-essential thrombocythemia MF (PET-MF), respectively. The median baseline platelet count was 64.5 x109/L, with &lt;50 x109/L in 38.3% of patients and baseline Hb &lt;10g/dL in 67.8% of patients. The median age was 67.5 (37-87) years. The majority of patients were JAK2 V617F-mutated (77%), followed by MPL-mutated (8.6%), with a notably low incidence of CALR-mutated (8.6%; type 1 CALR n=6, type 2 CALR n=3) and "triple-negative" (4.8%) cases. Non-myeloproliferative neoplasm driver mutations (NDM) were present in 77.1% (n=81) with ≥3 NDMs in 18.1% of patients. Similar to previous reports, the most prevalent NDMs were TET2 and ASXL1 (n=27 and n=25 respectively). Splicing factor (SF) mutations were mutually exclusive and detected in 32.3% of patients: SF3B1 [13], U2AF1 [14], SRSF2 [6], ZRSR2 [1]. RAS pathway mutations (KRAS/NRAS) were found at a higher frequency than previously in MF cohorts; 16.2% (n=17). SF mutations were present in more PMF (45.6%) than PPV-MF (3.7%, n=1/27) or PET-MF patients (33.3%, n=5/15), P=.002. SF3B1-mutated patients had higher trial entry platelet counts (66.7% platelets ≥100x109/L, P=.014). Baseline red cell transfusion dependency was more often associated with U2AF1 mutations 24.1% (n=7/29) as compared with red cell transfusion independence, 4.5% (n=2/44), P=.047 and U2AF1-mutated patients had lower baseline hemoglobin &lt;10g/dL (n=11/12), P=.015. Overall, 41 (39%) of patients had a high molecular risk mutation (HMR; IDH1/2, SRSF2, ASXL1, SRSF2, U2AF1Q157), and 8 patients had TP53 mutations. In those with molecular analysis available, the highest rates of ≥35% SVR were observed in the 200mg BID arm: 11.1% (n=4/36) followed by 3.2% (n=1/32) in 100mg BID arm and 0% (n=0/30) in 100mg QD arm). Rates of TSS reduction ≥50% were similar across arms. There were no significant correlations between mutations and SVR or TSS response. More grade 3/4 anemia occurred in TET2-mutated patients (OR 5.7, 95% CI 1.6-20.4, P=.007). RAS pathway mutations were associated with grade 3/4 thrombocytopenia (OR 4.4, 95% CI 1.3-14.8, P=0.016). Treatment discontinuation was not influenced by mutation status. In summary, the PAC203 cohort is molecularly high risk, with a high incidence of HMR and low incidence of CALR mutations. We identified novel associations between mutation profiles and hematologic events in this population with advanced MF. Disclosures Taylor: Baxalta: Research Funding; CTI BioPharma: Employment, Equity Ownership. Gerds:Roche: Research Funding; Incyte: Consultancy, Research Funding; Pfizer: Consultancy; CTI Biopharma: Consultancy, Research Funding; Imago Biosciences: Research Funding; Sierra Oncology: Research Funding; Celgene Corporation: Consultancy, Research Funding. Buckley:CTI BioPharma: Employment, Equity Ownership. Harrison:Incyte: Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Promedior: Honoraria; Celgene: Honoraria, Speakers Bureau; AOP: Honoraria; Sierra Oncology: Honoraria; Janssen: Speakers Bureau; Gilead: Speakers Bureau; Roche: Honoraria; CTI: Speakers Bureau; Shire: Speakers Bureau. Oh:Novartis: Consultancy; Incyte: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees. Mead:CTI: Honoraria, Research Funding; Bristol Myers-Squibb: Consultancy; Novartis: Consultancy, Honoraria, Other: Travel/accommodation expenses, Research Funding, Speakers Bureau; Celgene: Consultancy, Research Funding; Pfizer: Consultancy.

A Risk Score Combined Clinical and Molecular Profiles Identifies a High-Risk Subgroup within Intermediate-Risk Acute Myeloid Leukemia

Introduction Acute myeloid leukemia (AML) is categorized into favorable-, intermediate- and adverse-risk groups according to European LeukemiaNet (ELN) risk stratification. The intermediate-risk AML comprises a substantial proportion of AML, and even within the intermediate-risk AML, the biology and prognosis are highly different, which suggests that more prognostic factors are needed to be identified. Method A total of 265 newly diagnosed AML patients treated between January 2010 and January 2019 who had cryopreserved DNA for mutational analyses diagnosed and treated in Changhai Hospital were included. 121 patients were aged ≤ 65y, and classified as intermediate-risk (IR)-AML. A Custom Amplicon panel targeting exons of 210 genes was used for deep sequencing at diagnosis. We used a LASSO Cox regression model to achieve shrinkage and variable selection from prognostic factors (P&lt;0.1 in log-rank tests) to build risk score for predicting overall survival. A nomogram was constructed to display the risk of death in individuals. The discrimination of the risk score was measured by the concordance index (C-index) and areas under time-dependent receiver-operating characteristics (ROC) curves (AUCs), and the calibration of the risk score was explored graphically by calibration plots. Patients with IR-AML and aged ≤ 65y in The Cancer Genome Atlas (TCGA) data (n= 41) was used as a validation cohort. Results The median age was 44 (11-75) years, and 54% had normal karyotype. NRAS and CEBPA were the most recurrently mutated genes (both 26%), followed by KIT (24%), DNMT3A (23%), ARID1B (20%), FLT3-ITD (19%), PCLO (17%), and TET2 (17%). In univariate analyses, age ≥ 55 years, WBC ≥ 10×109/L , PLT &gt; 40×109/L, high LDH counts at diagnosis, DNMT3A and Signaling Pathway genes mutations and the number of mutated genes ≥ 8 were significantly associated with poor OS (P &lt; 0.05), and age ≥ 55 years, WBC≥ 10×109/L, DNMT3A and FLT3-ITD mutations were associated with worse relapse free survival(RFS, P &lt; 0.05). Three variables were incorporated in our scoring model by LASSO, including Signaling Pathway genes mutations (including NRAS, KIT, FLT3, KRAS etc), DNMT3A mutation and WBC≥ 10×109/L. A risk scoring model was developed incorporating the weighted coefficients of these variables:0.6749 × Signaling Pathway + 1.1147 × DNMT3A + 0.7829 × WBC. The risk score grouped IR-AML patients into two subgroups: intermediate-low risk (ILR, score &lt; 1, n= 48) and intermediate-high risk (IHR, score ≥ 1, n= 62) groups (Table1). Concordance index[OS: 0.703, 95% CI (0.643, 0.763); RFS: 0.681, 95%CI (0.620, 0.741)] demonstrated well discrimination power and calibration plots showed that the nomograms did well compared with an ideal model. The 3-year OS for ILR and IHR groups were 72.3% and 29.4% (P &lt; 0.0001), and 3-year RFS were 63.9% and 19.4% (P &lt; 0.0001), respectively (Figure 1A-B). The similar results were also observed in TCGA cohort (3-year OS 57.7% vs. 26.2%, P= 0.019; Figure 1C). The survival was equivalent for patients with ILR when treated with chemotherapy and allogeneic hematopoietic stem cell transplantation (alloHSCT, 3-year OS: 74.7% vs. 70.8%, P= 0.936, RFS: 76.4% vs. 55.0%, P= 0.225; Figure 2A-B). However, alloHSCT benefited patients with IHR. Patients who received alloHSCT had survival advantages over those who treated with chemotherapy only (3-year OS: 51.6% vs. 20.7%, P= 0.022; 3-year RFS: 38.1% vs. 7.8%, P= 0.008; Figure 2C-D). The prognosis of patients with IHR was as poor as those with adverse-risk AML (n=65 , 3-year OS: 29.5% vs. 33.3%, P= 0.794; 3-year RFS: 19.4% vs. 39.1, P= 0.294). Conclusions In this study, we developed and validated a novel scoring model that incorporated molecular and clinical profiles. According to our score model, IR-AML patients could be further stratified into two subgroups with distinct clinical outcomes. And the prognosis of patients with IHR was similar with patients with adverse-risk. Moreover, alloHSCT would override the poor outcome of patients with IHR. In summary, our scoring model might help identify patients with IR-AML who are most likely to benefit from alloHSCT. The results are needed to be validated in other independent cohorts and prospective studies before implementation into clinics. Disclosures No relevant conflicts of interest to declare.

The Necessary for Long-Term Follow-up of Mutated Genes and Clonal Evolutions in Multiple Myeloma Combined with cfDNA Assay

Introduction)Somatic mutations in multiple myeloma (MM) are strongly related to the clinical outcome and clonal evolution over the clinical course, and are a major problem. From a clinical viewpoint, although numerous novel drugs have been utilized, achieving long-lasting and complete remission remains difficult. Recent studies have elucidated the mutated genes using next-generation sequencing, and have examined how clonal change can be acquired in myeloma. In this study, we traced the transition of the somatic mutations of bone marrow tumor cells in patients with MM over a long-term follow-up. Furthermore, we compared the somatic mutations found in serum cell-free DNA (cfDNA) and mutated genes obtained from bone marrow myeloma cells. Material and Methods)Patients diagnosed with multiple myeloma who provided written informed consent to participate in the study were enrolled. Patients were treated by immuno-chemotherapy with or without radiation between 2000 and 2017 at our institute. Bone marrow aspiration and biopsy were performed at the time of diagnosis and upon disease progression. Around the time of bone marrow aspiration, serum was obtained from a peripheral blood sample for cfDNA analysis. Myeloma cells were separated from bone marrow samples with MicroBeads of CD138 antibody and genomic DNA was extracted. The peripheral blood samples derived from myeloma patients. The cfDNA was extracted from the serum using a Maxwell RSC cfDNA Plasma kit. Using genomic DNA derived from cfDNA and bone marrow, multiplex polymerase chain reaction (PCR) was performed, and a sequence library was then constructed with an Ion Custom Amplicon panel. The panel for the sequence library was designed using an Ion AmpliSeq DesignerTM. 126 targeted genes were selected. The genomes were sequenced using the Ion ProtonTM System. This protocol was approved by the institutional review board and the Genomic Review Board of the Japanese Foundation for Cancer Research. Result)We followed 7 patients' long term-clinical course and the transition of mutations (8.5 year average). The expression of myeloma driver genes, such as RAS, BRAF, and MYC, were not critical. We did, however, detect a relationship between an increase in the dominant mutated gene, such as TP53, DIS3, FAM46C, KDM6B, and EGR1 and poor prognosis in patients with myeloma. Next, we calculated the cfDNA concentrations from 34 cases. The cfDNA concentrations were significantly higher than 10 control cases (average 62.0 ng/mL (0-200 ng/mL) and 8.18 ng/mL (4.3-14.1 ng/mL), P=0.0046). The 2.5 year-progression free survival (PFS) during the first treatment of MM were tend to be poorer in the group with cfDNA&gt;50 ng/mL (72.9%) than the group with cfDNA&lt;50 ng/mL(25.9%), however there are no statistical significance (P = 0.15).We caluculated concordance rate of derived mutations from bone marrow MM cells and cfDNA in 7 cases. The somatic mutations found in serum cell-free DNA (cfDNA) and bone marrow MM cells were determined the correlation coefficients. However, there are few difference expression pattern in each source. In cfDNA assay, CREEP, EGR1, HDAC4, HDAC6, and JMJD1C were highly expressed as 57.1% (4/7) - 85.7% (6/7), and these results were almost the same as those for bone marrow MM cells. On the other hand, KDM1A (85.7%), PI3KCD (71.4%), and KDM3B (57.1%) were highly detected in cfDNA, although those were not frequently expressed in bone marrow. Discussion)Our data demonstrate the importance of the long-term follow-up of somatic mutations during the clinical course of myeloma. Serum cfDNA is a useful alternative source for detecting somatic mutations in MM patients during long-term follow-up. Disclosures Mishima: Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy. Yokoyama:Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy. Nishimura:Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy; Celgene K.K.: Honoraria. Hatake:Celgene K.K.: Research Funding; Janssen Pharmaceutical K.K.: Research Funding; Takeda Pharmaceutical Co.,Ltd.: Honoraria. Terui:Bristol-Myers Squibb K.K.: Research Funding; Bristol-Myers Squibb, Celgene, Janssen, Takeda, MSD, Eisai, Ono, and Chugai-Roche Pharmaceuticals Co.,Ltd.: Honoraria.

Cell-Free DNA Genotyping Analysis in Diffuse Large B-Cell Lymphoma; The Correlation between TP53 Gene Mutation and Cereblon Gene Mutation

Introduction: Recently cell-free DNA (cfDNA) genotyping analysis has been utilized as a non-invasive procedure for detecting tumor specific genes or clarifying gene mutations instead of tumor sample biopsies. In diffuse large B-cell lymphoma (DLBCL), cfDNA analysis has been proceeding and the relationships between somatic gene mutation and the disease status have been considered. We retrospectively analyzed somatic gene mutations in DLBCL and examined the correlation between mutant genes and clinical outcomes by using serum cfDNA samples. Patients and Methods: 50 patients newly diagnosed with DLBCL (including 4 patients mixed with follicular components) in our institute between March 2016 and March 2017 were enrolled in this study. All cases were sub-divided into germinal center B-cell (GCB) or non-germinal center B-cell (NGC) through immuno-staining, as CD10, bcl-6, and MUM-1 as Hans algorithm with biopsied tumor specimen. The stage at diagnosis and evaluation of treatment effects were assessed by PET-CT scan and bone marrow aspiration. All patients were treated by R-CHOP-like regimens with or without radiation. The serum samples from the patients were obtained before treatment and the cfDNA was extracted from the serum using a Maxwell RSC cfDNA Plasma kit. Using genomic DNA derived from cfDNA, multiplex polymerase chain reaction (PCR) was performed, and a sequence library was then constructed with an Ion Custom Amplicon panel. The panel for the sequence library was designed using an Ion AmpliSeq DesignerTM. 121 targeted genes were selected. The genomes were sequenced using the Ion ProtonTM System. We compared the validation of the sequence results dependent on the characteristics and prognoses of the patients. Connections between each gene mutation and clinical features were assessed using Fisher's exact test. Mann-Whitney U test was used for evaluating factors associated with the number of gene mutations. Survivals were estimated by the Kaplan-Meier method and differences were compared using the log-rank test. This protocol was approved by the institutional review board and the Genomic Review Board of the Japanese Foundation for Cancer Research. Results: Median age was 65.4 years old (range 33-83), 27 patients were male and 23 were female. The stage of Ⅲ or Ⅳ were 17 (34%), IPI high or high intermediate were 17 (34%), and 20 (40%) had LDH&gt;ULN. 27 patients (54%) were GCB and 23 (46%) were NGC. With a median follow up was 26.6 months (range 6-35). Factors affecting both shorter overall survival (OS) and progression-free survival (PFS) were LDH&gt;ULN (P=0.037 and P&lt;0.001), NGC (P=0.012 and P=0.002), IPI≧3 (P=0.023 and P&lt;0.001) in univariate analysis. According to the cfDNA gene mutation analysis, the mutations of HDAC4 (88%), KDM3B (86%), HDAC6 (86%), KDM1A (84%), CREBBP (80%), JMJD1C (78%), and EGR1 (62%) were frequently detected in all DLBCL patients. CREBBP, PCLO, and KDM5A tended to be relatively abundant in NGC (P=0.084, 0.087, and 0.085, respectively). No other mutations had deviation in either GCB or NGC and correlated with other prognostic factors. 7 patients had TP53 mutation, 6 were TP53 p.N178H and 1 was TP53 p.P58L. All cases with TP53 p.N178H mutation were accompanied by more than 27 types of other gene mutations. This was significantly abundant compared to patients without TP53 p.N178H (mean of 38 types vs 16 types, P&lt;0.001). Patients with 15 or more types of gene mutations tended toward poor PFS (2-year PFS of 89.5% vs 77.2%, P=0.092). Additionally, 7 patients had cereblon (CRBN) p.F101S mutation and 5 of 7 were concurrent with TP53 p.N178H mutation. Discussion and Conclusion: This study showed serum cfDNA could be used as an alternative resource for analysis by tumor specimen. Our results indicated that TP53 mutation occurred in latter period undergoing multistep genetic variation and it showed the tendency that multistep mutations were related to poor prognosis. Interestingly, relative rate in TP53 mutation accompanied by CRBN mutation was high. In some reports, it was described that deletion p53 caused drug resistance including immunomodulator which targets CRBN in multiple myeloma cases. Zijun Y et al mentioned CRBN expression correlated with favorable prognosis in DLBCL with wild type TP53. Further studies are warranted to confirm the relationships between TP53 and CRBN mutations and its prognosis of DLBCL. Disclosures Mishima: Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy. Yokoyama:Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy. Nishimura:Celgene K.K.: Honoraria; Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy. Hatake:Takeda Pharmaceutical Co.,Ltd.: Honoraria; Celgene K.K.: Research Funding; Janssen Pharmaceutical K.K.: Research Funding. Terui:Bristol-Myers Squibb, Celgene, Janssen, Takeda, MSD, Eisai, Ono, and Chugai-Roche Pharmaceuticals Co.,Ltd.: Honoraria; Bristol-Myers Squibb K.K.: Research Funding.