scholarly journals DNA Methylation and Genetic Profiles in 320 Patients with Myelodysplastic Syndromes

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1799-1799
Author(s):  
Suguru Morimoto ◽  
Hideki Makishima ◽  
Yasunobu Nagata ◽  
Niroshan Nadarajah ◽  
Constance Baer ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Gene Mutations ◽  
Epigenetic Alterations ◽  
Employment Equity ◽  
Group 3 ◽  
Equity Ownership ◽  
Epigenetic Regulators ◽  
Group 2 ◽  
Genetic Profiles ◽  
Group 1

Abstract MDS is a heterogeneous group of myeloid neoplasms caused by genetic and epigenetic alterations. During the past decade, the major driver mutations in MDS have been fully investigated. However, the role of epigenetic alterations, particularly those of DNA methylation, has less intensively been studied, even though abnormal DNA methylation has long been implicated in the pathogenesis of MDS. In this study, we analyzed DNA methylation status of bone marrow mononuclear cells from 320 cases with MDS-SLD (n = 7), MDS-RS (n = 63), MDS-MLD (n = 51), MDS-EB (n = 186), MDS-U (n = 1), and MDS with isolated del(5q) (n = 12), using Illumina 450K methylation array. Mutations in major driver genes (51 genes) and abnormal genomic copy numbers were also interrogated using targeted-capture sequencing. Using unsupervised consensus clustering, we identified 3 subgroups showing unique DNA methylation profiles. Subsequently, we assessed differentially methylated positions (DMPs) associated with each subgroup. Differentially hypermethylated positions (hyper-DMPs) were significantly more enriched in Group 3 (n = 82) (P < 0.001), while differentially hypomethylated positions (hypo-DMPs) were more prominent in Group 1 (n = 125). Group 1 was significantly enriched for SF3B1 (46%) mutations (q < 0.01), while Group 2 (n = 131) was characterized by the enrichment of ASXL1 (38%), RUNX1 (30%), TP53 (26%), STAG2 (15%), and SETBP1 (6.7%) mutations (q < 0.01). In contrast, Group 3 (n = 64) was significantly enriched for TET2 (67%) and IDH1/2 (12% and 15%, respectively) mutations (q < 0.01), suggesting a strong association between DNA methylation and gene mutations. To further elucidate mutation-specific DNA methylation patterns, supervised analysis was performed for each mutation. As expected from their enrichment in Group 3 (q < 0.01), TET2 and IDH1/2 mutations were significantly associated with hyper-DMPs (P < 0.001) involving 1891 and 8330 promotor sites, respectively. Conspicuously, among these hypermethylated promoter sites, >1616 were commonly hypermethylated, strongly supporting the common impact of TET2 and IDH1/2 mutations on deregulated DNA methylation. To clarify prognostic impact of abnormal DNA methylation, we first interrogated the correlation between unique methylation subgroups and revised IPSS. Patients with very low or low risk were significantly dominant (74%) in Group 1 (q < 0.01), and very high or high risk cases were significantly enriched (68%) in Group 2 (q < 0.01). In accordance with this finding, patients in Group 3 showed significantly shorter overall survival (OS) compared to Group 1 (HR: 1.94, 95%CI: 1.11-3.4, P < 0.05) and OS was even worse in Group 2 patients (vs. Group 1: HR: 5.18, 95%CI: 3.21-8.36, P < 0.001). Strong correlations between epigenetic and genetic profiles were further interrogated using a Bayesian statistical model; on the basis of DNA methylation and gene mutations, the original 3 clusters were re-classified into 5 discrete clusters, clusters A, B, C, D, and E (n = 124, 17, 74, 46, and 59, respectively); patients in Group 1 and 3 largely clustered into Cluster A and E, respectively, while Group 2 was further subclassified into clusters B, C, and D. Clusters B and D were characterized by a conspicuos enrichment of DNMT3A (88%) and TP53 (69%) mutations (q < 0.001), while Cluster C was characterized by higher frequency of ASXL1 (71%), RUNX1 (54%), STAG2 (27%), and EZH2 (21%) mutations (q < 0.001). In contrast to significant associations between epigenetic regulators and unique methylation clusters, splice factor mutations tended to be clustered into multiple clusters, depending on type of co-occurring mutations. For example, combined SF3B1 and TET2 mutations (n = 20) were enriched in Cluster A, where highly associated with MDS-RS, while patients with SF3B1 and RUNX1 mutations (n = 9) were more grouped in Cluster C, mostly showing MDS-EB phenotype (89%). Similarly SRSF2 mutations with RUNX1 and/or ASXL1 mutations (n = 36) were enriched in Cluster C, largely associated with MDS-EB phenotype (80%), while those with TET2 or IDH1/2 (n = 39) were mainly grouped into Cluster C, many of which showed MDS-EB phenotype (74%). These findings highlight differential roles of mutated epigenetic regulators and splicing factors in abnormal DNA methylation. In conclusion, we elucidated the collaborative impact of DNA methylation profiles and mutation status on heterogeneous pathogenesis and prognosis in MDS. Figure. Figure. Disclosures Nadarajah: MLL Munich Leukemia Laboratory: Employment. Baer:MLL Munich Leukemia Laboratory: Employment. Nakagawa:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Inagaki:Sumitomo Dainippon Pharma Co., Ltd.: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

A Comparison of Minimal Residual Disease Detection Among ASO-PCR, Dd-PCR and Deep-Sequencing in Patients with Multiple Myeloma Who Underwent Autologous Stem Cell Transplantation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1782-1782 ◽  
Author(s):  
Rachel K Wee ◽  
Hiroyuki Takamatsu ◽  
Ryoichi Murata ◽  
Jianbiao Zheng ◽  
Martin Moorhead ◽  
...  
Keyword(s):  
Prognostic Value ◽  
Residual Disease ◽  
Digital Pcr ◽  
Employment Equity ◽  
Group 4 ◽  
Group 3 ◽  
Equity Ownership ◽  
Group 5 ◽  
Group 2 ◽  
Group 1

Abstract Background: Most patients with multiple myeloma (MM) are considered to be incurable, and relapse, due to minimal residual disease (MRD), is the main cause of death among these patients. Even though allele-specific oligonucleotide real-time quantitative PCR (ASO-qPCR) of immunoglobulin heavy chain gene rearrangement has been used to assess the MRD in MM due to its excellent sensitivity and specificity, ASO-qPCR has a major limitation in its relative quantification method because it needs a reference standard curve, which is usually made with dilutions of diagnostic myeloma DNA or from plasmids containing the target IgH rearrangement gene. Recently, droplet digital PCR (ddPCR) was developed to perform reliable absolute quantification of target genes. Based on the principle of single target gene detection, the sensitivity can be increased when the larger amount of DNA is analyzed. Here we assessed the prognostic value of MRD assessment in autografts from MM patients in the autologous stem cell transplantation (ASCT) setting using ASO-qPCR, ddPCR and next-generation sequencing (NGS) approaches. Methods: Twenty-three Japanese patients with newly diagnosed MM who received various induction regimens prior to ASCT without any post-ASCT therapy were retrospectively analyzed. Median age 57 (range 39-67); males 11, females 12; ISS 1 (n=7), 2 (n=12), 3 (n=3), not assessed (n=1). 11 patients were analyzed by G-banding and FISH (t(4;14), del17p, t(14;16)) and 4 patients showed high-risk chromosomal abnormalities (t(4;14) (n=2), t(14;16) (n=1), -13 by G-banding (n=1)). All patients had achieved a very good partial response (VGPR) or better after ASCT. Analyzed samples included: (1) BM slides from 20 MM patients at diagnosis, (2) fresh/frozen BM cells from 3 MM patients at diagnosis, and (3) obtained autografts. IGH-based ASO-qPCR was performed as described previously (Methods Mol Biol 2009). ddPCR was performed by the QX200 Droplet Digital PCR system (Bio-RAD Inc.) with a total 6000 ng of genomic DNA combined with the same ASO-primers and TaqMan-probes used in the ASO-qPCR. Droplets were generated by the QX200 droplet generator. End-point PCR (40 cycles) was performed on a C1000 Touch Thermal cycler (Bio-RAD Inc). The PCR product was loaded in the QX200 droplet reader and analyzed by QuantaSoft 1.7.4 (Bio-Rad Inc). NGS-based MRD assessment was performed using the immunosequencing platform (Adaptive Biotechnologies, South San Francisco, CA) (Martinez-Lopez et al Blood 2014). Results: Nineteen patients could be analyzed by ASO-qPCR and ddPCR, while all 23 patients could be analyzed by NGS. We compared MRD results in autografts between ddPCR and NGS. We observed a high correlation between ddPCR and NGS results of MRD (r=0.82, P<0.0001). Although 19 samples were MRD negative by ASO-qPCR (MRDASO (-)), 12 samples were MRD positive by NGS (0.6-46 x 10-6, median 3.5 x 10-6) (MRDNGS (+)) and 7 samples were MRD positive by ddPCR (1.2-102 x 10-6, median 7.5 x 10-6) (MRDddPCR (+)), demonstrating the higher sensitivity of NGS (10-6 or higher) and ddPCR compared to ASO-qPCR (10-4 -10-5). Two high-risk chromosomal abnormality cases (t(4;14) (n=1), -13 by G-banding (n=1)) could achieve MRDNGS (-) in autograft. We evaluated the association of clinical outcome with MRD in autografts using ddPCR and NGS. To investigate the value of sensitive MRD detection by ddPCR and NGS, we compared PFS in 7 MRDddPCR (+) cases (Group 1) with 12 MRDddPCR (-) cases (Group 2) and 12 MRDNGS (+) cases (Group 3) with 11 MRDNGS (-) (Group 4). Group 2 and Group 4 showed significantly better PFS than Group 1 (P = 0.028) and Group 3 (P=0.033), respectively (Figure 1A and 1B). We also compared 5 MRDddPCR (-) & MRDNGS (+) cases (Group 5) with 11 MRDNGS (-) cases (Group 4). Group 4 tended to show better PFS than Group 5 (P=0.063, Figure 1C). Conclusions: In this study, we showed the prognostic value of ddPCR and NGS-based MRD assessment in autografts of patients with MM. Although the ddPCR had improved sensitivity in detecting MRD in autografts and demonstrated higher prognostic value compared with ASO-qPCR, the NGS platform showed the highest sensitivity and prognostic value among these methods. Disclosures Zheng: Adaptive Biotechnologies Corp: Employment, Equity Ownership. Moorhead:Adaptive Biotechnologies Corp: Employment, Equity Ownership. Faham:Adaptive Biotechnologies Corp.: Employment, Other: Stockholder.

scholarly journals Analysis of the Genetic Background in Patients with Myeloid Malignancies and Divergent Findings Regarding Blasts As Determined By Cytomorphology and Flow Cytometry

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2095-2095
Author(s):  
Wolfgang Kern ◽  
Manja Meggendorfer ◽  
Constance Baer ◽  
Claudia Haferlach ◽  
Torsten Haferlach
Keyword(s):  
Flow Cytometry ◽  
Genetic Background ◽  
Myeloid Leukemia ◽  
Employment Equity ◽  
Low Frequencies ◽  
Myeloid Malignancies ◽  
Group 3 ◽  
Equity Ownership ◽  
Group 2 ◽  
Group 1

Background: Diagnostics of hematologic malignancies is based on an integrated application of various methods. Different methods targeting the same structures, e.g. cytomorphology and flow cytometry both applied to identify and quantify pathologic cell populations, are aiming at revealing consistent results. While in the majority of cases achievement of such consistency is straight forward and in other cases both methods complement each other the alignment between cytomorphologically defined blasts and flow cytometrically defined myeloid progenitor cells (MPC) sometimes is challenging, in particular in myelodysplastic syndromes (MDS), acute myeloid leukemia (AML) and related diseases (thresholds defined for cytomorphology in WHO classification). We therefore identified such cases with higher percentages of cytomorphologically defined blasts than flow cytometrically defined MPCs and analyzed their genetic background in comparison to MDS cases with consistently low percentages by both methods and to acute myeloid leukemia (AML) cases with consistently high percentages by both methods. Aim: To clarify divergent findings between cytomorphology and flow cytometry on blasts and MPCs in patients with MDS and related diseases by analyzing the genetic background. Patients and methods: We identified 49 cases analyzed for myeloid malignancies, which were found to have higher percentages of cytomorphologically defined blasts than flow cytometrically defined MPCs (group 1). For comparison 83 patients with AML (group 2) and 53 cases with MDS (group 3) were selected in which percentages of cytomorphologically defined blasts (%B) were matching flow cytometrically defined percentages of MPCs (%MPC). All samples analyzed were bone marrow aspirates. Patients´ ages ranged from 40 to 60 (median 74), 23 to 89 (69) and 46 to 93 (76) years, respectively, in groups 1, 2 and 3 and sex distribution was 26/23 (f/m), 35/48 and 20/33, respectively. The median %B were 24% (range 11 to 62), 46% (20 to 98) and 5% (0 to 18), respectively, for groups 1, 2 and 3. The respective figures for %MPC were 9% (0.1 to 18), 52% (20 to 94) and 3% (0.3 to 16), respectively. The three groups were compared regarding cytogenetics based on chromosome banding analysis as well as regarding NGS data on mutations in 34 genes associated with myeloid malignancies. Results: Normal karyotypes were present in 37/49 (76%), 42/83 (51%) and 34/53 (64%) cases, respectively in groups 1, 2 and 3 (p=0.02). The respective figure for complex karyotypes was 5/49 (10%), 15/83 (18%) and 5/53 (9%, n.s.). Other chromosomal aberrations occurred less frequently and did not differ significantly between groups. Thus, karyotypes observed in group 1 were more similar to MDS than to AML. Regarding mutational profiling group 1 was characterized by a mutation spectrum similar to MDS developing into secondary AML while AML cases displayed a different spectrum as anticipated. Thus, in group 1, as compared to groups 2 and 3, the following gene were found more frequently mutated: ASXL1 (45% vs. 16% vs. 30%, p=0.001), SRSF2 (43% vs. 24% vs. 23%, p=0.042), TET2 (36% vs. 13% vs. 29%, p=0.014), RUNX1 (27% vs. 15% vs. 17%, n.s.). Interestingly, these genes were mutated at the lowest frequencies in group 2 most probably reflecting the low proportion of AML evolved as secondary disease after MDS. In contrast, SF3B1, which confers a favorable prognosis in MDS, was present at low frequencies in both groups 1 and 2 and at higher frequency in group 3 reflecting the low rate of AML progression in MDS with SF3B1 mutations (8% vs. 4% vs. 21%, p=0.005). Matching these findings mutations in genes typically associated with de novo AML were found most frequently in group 2 and only at low frequencies in groups 1 and 3: NPM1 (6% vs. 22% vs. 0%, p<0.001), FLT3-ITD (5% vs. 16% vs. 0%, p=0.001). No significant differences between the three groups were observed for mutations in the remaining genes. Conclusions: The genetic findings in the present series point to a similarity between cases with MDS and cases which display a lower level of %MPC and a higher level of %B at the same time. Therefore, while the latter cases based on WHO classification are defined as MDS-EB2 and AML, respectively, the genetic profile is matching better to the lower %PMC values. This may be used as a basis to more extensively integrate mutation data into the classification of these myeloid neoplasms. Disclosures Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Baer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

scholarly journals Landscape of DNA Methylation and Genetic Profiles in 291 Patients with Myelodysplastic Syndromes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5205-5205
Author(s):  
Yasunobu Nagata ◽  
Hiromichi Suzuki ◽  
Vera Grossmann ◽  
Genta Nagae ◽  
Yusuke Okuno ◽  
...  
Keyword(s):  
Dna Methylation ◽  
High Frequency ◽  
Research Funding ◽  
Gene Mutations ◽  
Consensus Clustering ◽  
Epigenetic Alterations ◽  
Employment Equity ◽  
Demethylating Agents ◽  
Equity Ownership ◽  
Causative Link

Abstract DNA hypermethylation has long been implicated in the pathogenesis of myelodysplastic syndromes (MDS) and also highlighted by the frequent efficacy of demethylating agents to this disease. Meanwhile, recent genetic studies in MDS have revealed high frequency of somatic mutations involving epigenetic regulators, suggesting a causative link between gene mutations and epigenetic alterations in MDS. The accumulation of genetic and epigenetic alterations promotes tumorigenesis, hypomethylating agents such as Azacitidine exert their therapeutic effect through inhibition of DNA methylation. However, the relationship between patterns of epigenetic phenotypes and mutations, as well as their impact on therapy, has not been clarified. To address this issue, we performed genome-wide DNA methylation profiling (Infinium 450K) in combination with targeted-deep sequencing of 104 genes for somatic mutations in 291 patients with MDS. Beta-mixture quantile normalization was performed for correcting probe design bias in Illumina Infinium 450k DNA methylation data. Of the >480,000 probes on the methylation chip, we selected probes using the following steps: (i) probes annotated with "Promotor_Associated" or "Promoter_Associated_Cell_type_specific; (ii) probes designed in "Island", "N_Shore" or "S_Shore"; (iii) removing probes designed on the X and Y chormosomes; (iv) removing probes with >10% of missing value. Consensus clustering was performed utilizing the hierarchical clustering based on Ward and Pearson correlation algorithms with 1000 iterations on the top 0.5% (2,000) of probes showing high variation by median absolute deviation across the dataset using Bioconductor package Consensus cluster plus. The number of cluster was determined by relative change in area under cumulative distribution function curve by consensus clustering. Unsupervised clustering analysis of DNA methylation revealed 3 subtypes of MDS, M1-M3, showing discrete methylation profiles with characteristic gene mutations and cytogenetics. The M1 subtype (n=121) showed a high frequency of SF3B1 mutations, exhibiting the best clinical outcome, whereas the M2 subtype (n=106), characterized by frequent ASXL1, TP53 mutations and high-risk cytogenetics, showed the shortest overall survival with the hazard ratios of 3.4 (95% CI:1.9-6.0) and 2.2 (95% CI:1.2-4.0) compared to M1 and M3, respectively. Finally, the M3 subtype (n=64) was highly enriched (70% of cases) for biallelic alterations of TET2 and showed the highest level of CpG island methylation and showed an intermediate survival. In the current cohort, we had 47 patients who were treated with demethylating agents, including 11 responders and 36 non-responders. When DNA methylation status at diagnosis was evaluated in terms of response to demethylating agents, we identified 54 differentiated methylated genes showing >20% difference in mean methylation levels between responders and non-responders (q < 0.1). Twenty-five genes more methylated in responders were enriched in functional pathways such as chemokine receptor and genes with EGF-like domain, whereas 29 less methylated gene in responders were in the gene set related to regulation of cell proliferation. Genetic alterations were also assessed how they affected treatment responses. In responders, TET2 mutated patients tended to more frequently respond (45% vs 34%), whereas patients with IDH1/2 and DNMT3A mutations were less frequently altered (0% vs 14%, 9% vs 14%) in responders, compared in non-responders. In conclusion, our combined genetic and methylation analysis unmasked previously unrecognized associations between gene mutations and DNA methylation, suggesting a causative link in between. We identified correlations between genetic/epigenetic profiles and the response to demethylating agents, which however, needs further investigation to clarify the mechanism of and predict response to demethylation agents in MDS. Disclosures Alpermann: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kiyoi:Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Novartis Pharma K.k.: Research Funding; Pfizer Inc.: Research Funding; Takeda Pharmaceutical Co.,Ltd.: Research Funding; MSD K.K.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Alexion Pharmaceuticals.: Research Funding; Teijin Ltd.: Research Funding; Zenyaku Kogyo Company,Ltd.: Research Funding; FUJIFILM RI Pharma Co.,Ltd.: Patents & Royalties, Research Funding; Nippon Shinyaku Co.,Ltd.: Research Funding; Japan Blood Products Organization.: Research Funding; Eisai Co.,Ltd.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Kyowa-Hakko Kirin Co.,Ltd.: Consultancy, Research Funding; Fujifilm Corporation.: Patents & Royalties, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Bristol-Myers Squibb.: Research Funding; Chugai Pharmaceutical Co.,LTD.: Research Funding; Mochida Pharmaceutical Co.,Ltd.: Research Funding. Kobayashi:Gilead Sciences: Research Funding. Naoe:Toyama Chemical CO., LTD.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Patents & Royalties, Research Funding; Pfizer Inc.: Research Funding; Astellas Pharma Inc.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Celgene K.K.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Miyazaki:Chugai: Honoraria, Research Funding; Shin-bio: Honoraria; Sumitomo Dainippon: Honoraria; Celgene Japan: Honoraria; Kyowa-Kirin: Honoraria, Research Funding.

Association between germline DNA damage repair-related genes mutation and tumor mutational burden in lung cancer patients.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e21163-e21163
Author(s):  
Wei Nie ◽  
Hua Zhong ◽  
Ding Zhang ◽  
Shiqing Chen ◽  
Baohui Han
Keyword(s):  
Dna Damage ◽  
Dna Damage Repair ◽  
Gene Mutations ◽  
Related Gene ◽  
Mutational Burden ◽  
Damage Repair ◽  
Tumor Mutational Burden ◽  
Group 3 ◽  
Group 2 ◽  
Group 1

e21163 Background: Deleterious somatic DNA damage repair (DDR) gene mutations are frequent in non-small cell lung cancer (NSCLC) and are associated with improved clinical outcomes of immunotherapy. DDR gene mutations are associated with higher tumor mutational burden (TMB) in cancer. However, the effect of germline DDR-related genes mutation with different functional annotations on TMB in NSCLC patients is still unclear. Methods: 1671 Chinese patients with NSCLC were enrolled in this study. Genomic profiling was performed on formalin-fixed paraffin-embedded tumor samples or peripheral blood by next generation sequencing (NGS) with 733 cancer-related genes panel. The germline mutation data were obtained. All annotations in clinical significance were according to the 2015 American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) guidelines. Results: 1076 patients (64.39%) had germline DDR-related gene mutations and 595 (35.61%) had no germline DDR-related gene mutations. Among patients with DDR-related gene mutations, 78 (7.25%) patients had the pathogenic (P) mutations or likely pathogenic (LP) mutations and 1056 (98.14%) had variants of unknown significance (VOUS) mutations. In total, the median TMB was 3.91 mutations/MB (range, 0-68.16) and 4.47 mutations/MB (range, 0-51.40) in patients with P, LP or VOUS mutations and no germline DDR-related gene mutations, respectively. To the further analysis, we divided patients with germline DDR-related gene mutations into three groups: only P or LP mutations (Group 1), only VOUS mutations (Group 2) and concurrence with P/LP/VOUS mutations (Group 3). Compare to the DDR-negative group, TMB was significantly lower in Group 2 (P < 0.001). No significant differences in Group 1 and Group 3 were observed. In addition, we found that mutations in different DDR pathway could not affect TMB value significantly. Conclusions: Germline DNA damage repair-related genes mutation may be not associated with TMB.

Effect of Nilotinib (NIL) on Molecular Response in Chronic Myelogenous Leukemia - Chronic Phase (CML-CP) Patients (pts) with a Suboptimal Molecular Response to Imatinib (IM)–ENABL Study Update

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2771-2771 ◽  
Author(s):  
Sikander Ailawadhi ◽  
Carole B. Miller ◽  
Anand P. Jillella ◽  
Nebu Koshy ◽  
Brian Tudor ◽  
...  
Keyword(s):  
Research Funding ◽  
Molecular Response ◽  
Employment Equity ◽  
Transcript Levels ◽  
Log Reduction ◽  
End Point ◽  
Day Range ◽  
Equity Ownership ◽  
Group 2 ◽  
Group 1

Abstract Abstract 2771 Background: NIL is a potent, highly selective Bcr-Abl kinase inhibitor approved for newly diagnosed adult pts with Philadelphia-chromosome positive (Ph+) CML-CP and in Ph+ CML-CP and accelerated-phase pts who are resistant or intolerant to IM. Achieving complete cytogenetic response (CCyR) and major molecular response (MMR, 3-log reduction of Bcr-Abl transcript level from the baseline mean) are favorable prognostic factors for CML. This multicenter, open-label study (ENABL) was designed to explore nilotinib Bcr-Abl effects in pts with CCyR but who have suboptimal molecular response to IM. Methods: This study evaluates change in Bcr-Abl trends in 2 groups of CML-CP pts (total n = 18) who achieved CCyR but have suboptimal molecular response to IM defined as: (Group 1) treated ≥ 1 year with IM, but Bcr-Abl transcript levels did not reach ≤ 0.1% on the international scale (IS) (MMR); or (Group 2) > 1-log increase in Bcr-Abl transcript levels from best response regardless of IM treatment duration. Pts are treated with NIL 300 mg twice daily for ≥ 1 year. RQ-PCR analysis is performed by a central lab at screening, then every 3 months (mos) for Group 1. Group 2 pts are monitored by RQ-PCR monthly for the first 3 mos, then every 3 mos. The 1° end point is change in Bcr-Abl transcript levels from a standardized baseline value by RQ-PCR at 12 mos. The data cutoff date for this analysis was June 30, 2011. Results: Eighteen pts (Group 1, n = 17; Group 2, n = 1) have been treated with NIL for a median of 17 mos on study (range 3–34 mos). Thirteen pts have been treated for ≥ 6 mos and 10 for ≥ 12 mos. One pt was deemed ineligible due to lack of evidence of CCyR at baseline but is included in the analysis because there was at least 1 post-baseline evaluation performed. The remaining 17 pts had CCyR at baseline. Before enrollment, pts were treated with at least 400 mg once-daily IM; the mean dose of prior IM treatment was 487 mg/day (range 342–786 mg/day). Median duration of prior IM treatment was 3.4 yrs (range 1.3–10.2 yrs). Three pts had prior interferon treatment. All 18 pts were treated for ≥ 3 mos and had ≥ 1 post-baseline RQ-PCR result. Overall, 15 of 18 evaluable pts (83%) achieved MMR during treatment; 10 pts by 3 mos, 1 pt by 4.5 mos (measured at end of study), 1 pt by 6 mos, 2 pts by 9 mos, and 1 pt by 30 mos (Figure 1). The 3 pts who did not reach MMR at any point were only followed for up to 3 mos before discontinuing from the trial but showed a decreasing Bcr-Abl trend. Overall, pts achieved a median log reduction of PCR transcript levels of 3.1 (0.08% IS) at 3 mos; median 3.3-log reduction (0.05% IS) at 6 mos, and median 3.5-log reduction (0.035% IS) at 9 mos. Four pts had > 4-log (≤ 0.01% IS) reduction in Bcr-Abl; of these, 2 pts reached > 4.5-log (≤ 0.0032% IS) reduction in Bcr-Abl at least once during the study. Median Bcr-Abl transcript log reduction at 12 mos was 3.6 (0.025% IS, 1° end point) for 10 evaluable pts. All these pts reached MMR during NIL treatment; 9 pts by 12 mos, 1 pt after 30 mos. NIL was well tolerated and brief dose interruptions were sufficient to manage most adverse events (AEs). Seven of 18 pts were dose reduced for NIL-related AEs and re-escalated if the patient recovered from the AEs. Patients were permitted to dose escalate to 400 mg b.i.d. per physician's discretion if MMR was not achieved after 6 mos (n = 1). The Grade 3 AEs reported include 2 cases of rash and 1 case each of pneumonia, squamous cell carcinoma, bladder prolapse, uterine prolapse, bradycardia, hypertension, hyperbilirubinemia and hypophosphatemia. The rashes and bradycardia were suspected to be related to NIL. No Grade 4 AEs were reported. The median dose intensity was 600 mg/day (range 300–683 mg/day). Five pts were discontinued from the study (3 due to abnormal laboratory values, 1 due to an AE, and 1 due to protocol violation). No pts who experienced QTcF changes had differences > 33 msec from baseline. No QTcF prolongation > 500 msec was observed. Conclusions: NIL treatment results in high molecular response rates in CML-CP pts with suboptimal molecular responses to IM. Overall 83% of pts who switched to NIL achieved MMR, and the median Bcr-Abl log reduction for pts who reached 12 mos on study was 3.6 (0.025% IS). The IRIS study has shown that MMR rates increase with time in pts treated with IM (Hughes Blood 2010); however, this study appears to demonstrate that MMR is achieved relatively quickly in suboptimal molecular IM-treated pts when switched to NIL. Disclosures: Ailawadhi: Novartis Pharmaceuticals: Consultancy, Speakers Bureau. Miller:Incyte: Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau. Akard:Eisai: Speakers Bureau; Bristol Myers-Squibb: Speakers Bureau; Novartis: Speakers Bureau; Millenium: Speakers Bureau; Chemgenex: Consultancy. Ericson:Novartis Pharmaceuticals Corporation: Employment, Equity Ownership. Lin:Novartis: Employment, Equity Ownership. Radich:Novartis: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau. DeAngelo:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy.

scholarly journals Effect of Ara-C on T-Cell Function and Flotetuzumab Activity in Pediatric Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1387-1387
Author(s):  
Anilkumar Gopalakrishnapillai ◽  
Anne Kisielewski ◽  
Ezio Bonvini ◽  
John Muth ◽  
Jan K Davidson-Moncada ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Median Survival ◽  
Cell Function ◽  
Concurrent Administration ◽  
Employment Equity ◽  
Group 3 ◽  
Equity Ownership ◽  
Group 2 ◽  
Pediatric Aml

Acute myeloid leukemia (AML) in children still has a poor prognosis despite the use of maximally intensive chemotherapy associated with severe short-term and long-term side effects. Therefore, development of targeted therapeutics is necessary to improve outcomes in pediatric AML. CD123 (IL3RA) is overexpressed in most of pediatric AML patients (Bras et al., Cytometry B Clin Cytom, 963:134, 2019) and has been pursued as a target for immunotherapy. The efficacy of a dual affinity retargeting agent (CD123xCD3; MGD006 or flotetuzumab), was evaluated in two patient-derived xenograft models of pediatric AML. In addition, concurrent administration of cytarabine with MGD006 was performed to determine the effect of cytarabine on T-cell function and flotetuzumab efficacy. NSG-SGM3 mice were transplanted with 2.5 x 106 cells AML PDX cells. After 18 days post transplant, when human cells were detectable in mouse blood, mice were randomly assigned to one of 8 treatment groups - 1) untreated, 2) T-cells, 3) T-cells with MGD006 (0.5 mg/Kg, Q5d), 4) T-cells with Ara-C (50 mg/Kg, Q5d), 5) T-cells with concurrent administration of Ara-C and MGD006, 6) MDG006 and 7) Ara-C. Mice belonging to groups 2-5 were intravenously injected with 2.0 x 106 human pan T-cells (StemCell Technologies, Cat No. 70024.1), prior to i.p. administration of MGD006 and/or Ara-C. Mice were monitored daily and peripheral blood was collected periodically to evaluate leukemia progression (CD45+CD3-) and T-cell expansion (CD3+CD45+) by flow cytometry. Mice were euthanized when they showed systemic signs of leukemia based on weight and body condition score. The growth of human cell percentage in mouse blood over time was plotted and Kaplan-Meier survival plots were generated. On the day after treatment was terminated, AML cell percentage was greatly reduced, in mice treated with T-cells + MGD006 (Fig. 1, group 3) or T-cells + MGD006 + Ara-C (group 5), compared to the other groups. In addition, exposure to MGD006 (groups 3 and 5) enhanced expansion of adoptively transferred T-cells compared to AML PDX mice receiving T-cells alone (group 2). The ability of MDG006 to enhance the expansion of T-cells in vivo was not attenuated by treatment with Ara-C. Similar results were obtained in a second PDX model (Fig. 2). Taken together, MGD006 enhanced T-cell engraftment with or without Ara-C accompanied by marked reduction in the burden of AML blats in the peripheral blood. As expected, MGD006 in the absence of the effector T-cells (group 6) had minimal effect on reducing leukemic burden or survival (Fig. 3A, B). Mice injected with T-cells alone (group 2) showed 40-day improvement in survival, likely due to the allogeneic effect of T-cells. Regardless, the addition of MGD006 with T-cells (group 3) amplified the effect as mice did not reach experimental endpoints upon study termination at 210 days (Fig. 3B, brown line). Ara-C treatment (group 7) delayed leukemia progression and prolonged median survival by 22.5 days compared to untreated mice (Fig. 4A, B). Consistent with the T-cell expansion induced by Ara-C (Fig. 1), mice treated with T-cells + Ara-C (group 4) survived longer (median survival 180 days) than those treated with Ara-C (group 7) or T-cells alone (group 2) (median survival 116 and 135 days respectively). Mice administered with MGD006 concurrently with Ara-C following T-cell injection (group 5) also did not reach experimental endpoints upon study termination (Fig. 3B, purple line). These mice had 0.1% residual AML cells when the study was terminated (Fig. 3A, solid purple line), which was significantly lower than mice receiving T-cells + MGD006 (group 3, 2% AML cells, P=0.0047). These data demonstrate the activity of MGD006 in the presence of T-cells in prolonging survival in pediatric AML PDX models. Inclusion of Ara-C to this regimen was more efficient in reducing AML burden. Disclosures Bonvini: MacroGenics, Inc.: Employment, Equity Ownership. Muth:MacroGenics, Inc.: Employment, Equity Ownership. Davidson-Moncada:MacroGenics, Inc.: Employment, Equity Ownership.

scholarly journals Genome Data Allow in silico Pharmacogenetic Studies Using the Genetic Makeup of Both the Individual Patient As Well As the Disease

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3360-3360
Author(s):  
Manja Meggendorfer ◽  
Wencke Walter ◽  
Stephan Hutter ◽  
Constance Baer ◽  
Claudia Haferlach ◽  
...  
Keyword(s):  
In Silico ◽  
Drug Target ◽  
Target Genes ◽  
Response To Treatment ◽  
Employment Equity ◽  
Equity Ownership ◽  
Genetic Makeup ◽  
Group 2 ◽  
The Individual ◽  
Group 1

Pharmacogenomics is becoming more and more important due to personalized medicine. To understand the association between the genetic background of a patient and the response to a tailored treatment would strongly influence the choice of therapy and reduce trial-and-error strategies. Various screening assays have been established, e.g. to test the impact of acquired gene mutations in cancer on the response to different agents and combinations in cell culture models. In the era of genome data, in silico analyses are becoming an interesting tool and the hope is growing to predict a patient's response to therapy by association studies using the genetic makeup of both, the individual patient as well as the disease. The aim of this study was to get insights into the genetic background of AML patients refractory to treatment and to investigate associations of polymorphisms (SNPs) in cancer treatment target genes and disease associated mutations contributing to an altered response to treatment. The cohort consisted of 247 AML patients diagnosed by cytomorphology following the WHO classification. All patients were treated intensively with a standard chemotherapy protocol such as 7+3 and response to treatment was assessed after first and second induction. Following ELN guidelines patients were grouped into responder (group 1), showing cytomorphological complete remission after first induction (n=186), and treatment failure (group 2) with only partial remission or progressive disease after second induction (n=61). Whole genome sequencing (WGS) was performed with 90x coverage for all samples at diagnosis to assess their mutational profiles as well as their germline background. In depth analyses were restricted to exonic SNPs of 217 drug target genes (DrugBank 5; Schärfe et al., 2017) associated with cancer treatments and 20 recurrently mutated genes in AML. Morphologic subtypes as well as mutation frequencies in AML genes were similar in the responder and treatment failure groups. In total 9,742 unique SNPs were found with a median of 1,603 per patient. 37% of these SNPs were found in single patients only and another 27% were found in less than 5% of the cases. SNPs and somatic variants for the addressed genes were combined into a single matrix and co-occurrences of two variants were assessed by matrix multiplication for each group. First, we were interested in group-specific co-occurrences of SNPs and somatic variants. Here we found considerably less co-occurrences in group 1 compared to group 2. All identified co-occurrences in group 1 were unique whereas 772 co-occurrences could be found in more than one patient in group 2. For group 2 a co-occurrence network was created based on recurrent combinations of somatic variants and missense germline variants (figure A). 51% of the patients from group 2 were involved in these interactions, while group 1 did not show any recurrent co-occurrences. It was interesting to note that IDH2, KMT2A-PTD, SF3B1 and TP53 recurrently co-occurred with multiple missense SNPs. Second, we addressed the association of two SNPs in the investigated drug target genes. Looking at SNPs co-occurring in at least 10% of the patients again reflected the heterogeneity of group 1, showing no recurrently co-occurring SNPs, while group 2 showed 6 SNP pairs associated with MAPK family signaling cascades (figure B). Last, it was noteworthy that multiple non-coding SNPs were found in the 3'UTR of the drug target genes. These variants showed a higher frequency in group 2 compared to group 1. SNPs in the 3'UTR region might modify mRNA - miRNA interactions by creating, abolishing, or changing the miRNA-binding sites (figure C). Recently multiple miR-SNPs have been described that were prognostic for treatment outcome, suggesting a potential as predictive biomarkers. In conclusion, WGS data allow comprehensive genome wide but also targeted association analyses. We found three different mechanisms potentially altering treatment sensitivity based on the genetic makeup of individual patients. 1) Association of somatic mutations and SNPs located in drug target genes, 2) association of two SNPs, and 3) SNPs in the 3'UTR modulating miRNA interaction. All three mechanisms occurred only in the refractory AML cases and not in the responder group. This study shows the power of genomic data and in silico analyses that are the basis for future tools to predict response to treatment for individual patients. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Walter:MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Baer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

scholarly journals Impact of Gene Mutations on Overall Survival in Older Patients with Acute Myeloid Leukemia (AML) Treated with Azacitidine (AZA) or Conventional Care Regimens (CCR)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2859-2859 ◽  
Author(s):  
Lin Tang ◽  
Anna Dolnik ◽  
Kyle J. MacBeth ◽  
Hervé Dombret ◽  
John F. Seymour ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Overall Survival ◽  
Board Of Directors ◽  
Older Patients ◽  
Research Funding ◽  
Gene Mutations ◽  
Molecular Heterogeneity ◽  
Employment Equity ◽  
Advisory Committees ◽  
Equity Ownership

Abstract Background: AML is characterized by molecular heterogeneity and specific mutations are prognostically important (Papaemmanuil, Gerstung et al, NEJM,2016). Mutational analysis of NPM1, CEBPA, and FLT3 is included in the 2010 European LeukemiaNet recommendations for AML (Döhner et al, Blood, 2010). Additional recurrently mutated genes have since been identified with potential value for prognosis and prediction of treatment (Tx) response. The phase 3 AZA-AML-001 study showed AZA prolonged median overall survival (OS) vs CCR (10.4 vs 6.5 months [mos]; P=0.101) and improved 1-year survival (46.5% vs 34.2%) in older patients (pts) with AML (Dombret et al, Blood, 2015). Aim: To investigate relationships between gene mutations and OS in the subpopulation of AZA-AML-001 pts with available baseline bone marrow (BM) for molecular analyses ("biomarker" cohort). Methods: Eligible pts were age ≥65 years with newly diagnosed AML (>30% BM blasts), ECOG performance status (PS) score 0-2, WBC count ≤15x109/L, and NCCN-defined intermediate- or poor-risk cytogenetics. Pts received AZA (75 mg/m2/day [d] x 7d/28d cycle) or a preselected CCR: intensive chemotherapy (7 + 3 regimen), low-dose ara-C, or best supportive care only. DNA was isolated from BM mononuclear cells and targeted sequencing of 39 genes was performed with Haloplex target enrichment (Agilent) on Illumina HiSeq 2500 using 2x100bp read lengths. FLT3 tyrosine kinase domain (TKD) mutations were determined by next-generation sequencing (NGS) and internal tandem duplications (ITD) were determined by capillary electrophoresis sizing of PCR amplicons. Target regions varied by gene from all exons to hot-spots. Log-rank test, stratified by ECOG PS score (0-1 vs 2) and cytogenetic risk (intermediate vs poor) at baseline, was used to assess OS of pts with mutations (mut) in genes detected in ≥5 pts vs OS in pts with wild-type (wt) genes within the AZA and CCR arms. Median OS was estimated using Kaplan-Meier methods. Results: The biomarker cohort comprised 156 of all 488 pts in AZA-AML-001 (32%; AZA n=83, CCR n=73). Baseline characteristics and hematologic response rates were well-matched between biomarker and non-biomarker pts. Mutations were detected in 33 of 39 sequenced genes. The most frequently mutated genes were DNMT3A (27%), TET2 (25%), IDH2 (23% [R140 15%, R172 8%]), TP53 (21%), RUNX1 (18%), NPM1 (16%), NRAS (12%), FLT3 (12% [-ITD 10%, -TKD 5%]), ASXL1 (11%), and STAG2 (10%). Stratified log-rank tests showed that median OS was significantly reduced for CCR pts with TP53mut (2.4 vs 12.5 mos with TP53wt; P=0.026) and with NRASmut (4.3 vs 10.3 mos with NRASwt; P=0.020). In the AZA arm, median OS was not significantly different between pts with TP53mutor TP53wt (7.2 vs 12 mos; P=0.40) or between pts with NRASmut or NRASwt (11.8 vs 8.9 mos; P=0.95), but was reduced in pts with FLT3mut (5.4 vs 12.0 mos with FLT3wt; P=0.017). Compared with similar pts treated with CCR, pts with TP53mut and/or NRASmut treated with AZA had nominally better median OS (7.2 vs 2.4 mos for TP53mut; 11.8 vs 4.3 mos for NRASmut), and pts with FLT3mut had nominally worse OS (5.4 vs 6.4 mos) (Table). Median OS was similar for pts with or without mutations in each of the genes known to influence DNA methylation (DNMT3A, IDH1, IDH2, and TET2); however, there was a statistical difference in OS within the AZA arm for pts with TET2mut (P=0.005) despite similar median OS for pts with TET2mut vs TET2wt (9.6 vs 9.5 mos) that was not observed within the CCR arm (P=0.45). Median OS for pts with a mutation in any 1 of the DNA methylation genes listed above was similar in the AZA and CCR arms (Table). Conclusion: These exploratory analyses suggest older AML pts with TP53 or NRAS mutations have a better prognosis when treated with AZA than with CCR. Mutations in genes that regulate DNA methylation did not impact median OS with AZA Tx, although the potential negative effects of TET2mut and FLT3mut warrant further evaluation. Prognostic implications of isolated gene mutations can vary due to co-mutations; larger pt cohorts are needed to establish the influence of recurring co-mutational patterns in AZA-treated pts. Disclosures Tang: Celgene: Employment, Equity Ownership. MacBeth:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties, Research Funding. Dombret:Agios: Honoraria; Sunesis: Honoraria; Ambit (Daiichi Sankyo): Honoraria; Karyopharm: Honoraria; Kite Pharma.: Honoraria, Research Funding; Menarini: Honoraria; Menarini: Honoraria; Astellas: Honoraria; Janssen: Honoraria; Servier: Honoraria; Seattle Genetics: Honoraria; Roche/Genentech: Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Pfizer: Honoraria; Ariad: Honoraria, Research Funding; Novartis: Honoraria; Celgene: Consultancy, Honoraria; Jazz Pharma: Honoraria, Research Funding. Seymour:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding, Speakers Bureau; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Stone:Pfizer: Consultancy; Sunesis Pharmaceuticals: Consultancy; Karyopharm: Consultancy; ONO: Consultancy; Jansen: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celator: Consultancy; Roche: Consultancy; Agios: Consultancy; Amgen: Consultancy; Novartis: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Consultancy; Merck: Consultancy; Seattle Genetics: Consultancy; Xenetic Biosciences: Consultancy. Beach:Celgene Corporation: Employment, Equity Ownership.

scholarly journals Phase 1 Study of AMG 900, an Orally Administered Pan-Aurora Kinase Inhibitor, in Adult Patients (Pts) with Acute Myeloid Leukemia (AML)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4929-4929 ◽  
Author(s):  
Michael W. Schuster ◽  
Mikkael A. Sekeres ◽  
Erick Gamelin ◽  
Erik Rasmussen ◽  
Gloria Juan ◽  
...  
Keyword(s):  
Respiratory Failure ◽  
Board Of Directors ◽  
Dose Escalation ◽  
Single Agent ◽  
Objective Response ◽  
Employment Equity ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Group 2 ◽  
Group 1

Abstract Background: Aurora kinases form a family of highly conserved serine/threonine protein kinases that regulate key steps in mitosis. Aurora kinases A and B are amplified and/or overexpressed in many malignancies, including various types of leukemia, and are associated with high proliferation rates, poor prognosis, and therapeutic resistance. AMG 900 is an investigational, orally administered, highly potent, selective, small-molecule pan-aurora kinase inhibitor that has shown single-agent activity in heavily pretreated pts with chemotherapy-resistant/refractory solid tumors. This open-label, multicenter, sequential dose escalation study (NCT01380756) assessed AMG 900 in adult pts with AML. Methods: The primary objectives of this study were to evaluate the safety and tolerability of AMG 900, to evaluate pharmacokinetics after multiple oral administrations, to determine the optimal dose schedule, and to determine the maximum tolerated dose (MTD). A secondary objective was to evaluate antitumor activity in pts with AML. Key inclusion criteria included definitively diagnosed AML that had failed standard treatments or for which no standard therapy was available, Eastern Cooperative Oncology Group (ECOG) performance status ≤ 2, and life expectancy > 3 months. Dose escalation included parallel evaluation of 2 schedules in separate groups. In group 1, AMG 900 was administered daily 4 days on/10 days off at doses of 15, 25, 40, 60, 80, 100, 125, and 150 mg. In group 2, AMG 900 was administered daily 7 days on/7 days off at doses of 30, 40, 50, 60, and 75 mg. Dose escalation was conducted using a 3+3+3 design; intrapatient dose escalation was not allowed. The relationship between gene expression at baseline and clinical response was an exploratory objective. RNA levels for preselected genes were measured by microarray in mononuclear cells obtained from bone marrow (BM) aspirates. Results: A total of 35 pts were enrolled: 22 in group 1 and 13 in group 2. Twenty-three pts (65.7%) were male, 27 (77.1%) were white, and mean (SD) age was 66.1 (12.2) years. ECOG status was 0 in 7 pts (20.0%), 1 in 22 pts (62.9%), and 2 in 6 pts (17.1%). Pts received a median (min, max) of 14 (4, 49) doses of AMG 900. Mean maximum plasma concentration (Cmax) and area under the concentration-time curve (AUC) increased with increasing dose. Thirty pts (85.7%) had treatment-related adverse events (AEs). The most common AEs (in ≥ 10% of pts overall) were nausea (31.4%), diarrhea (28.6%), febrile neutropenia (28.6%), fatigue (22.9%), vomiting (17.1%), alopecia (14.3%), dyspnea (11.4%), epistaxis (11.4%), mucosal inflammation (11.4%), and rash (11.4%). Nine pts (25.7%) died during the study from lung infection and respiratory failure (2 pts each) and acute respiratory failure, cardiorespiratory arrest, respiratory distress, sepsis, and septic shock (1 pt each). Only respiratory failure and septic shock (1 pt each) were considered potentially related to AMG 900 by the investigators. All 35 pts discontinued treatment. Reasons for discontinuation were disease progression (65.7%), AEs (11.4%), death (8.6%), withdrawal of consent (5.7%), other reasons (5.7%), and requirement for alternative therapy (2.9%). Two pts (5.7%) experienced dose-limiting toxicities: 1 pt from group 1 (40 mg) had grade 3 pancytopenia, and 1 pt from group 2 (50 mg) had febrile neutropenia and grade 3 abdominal pain. The MTD of AMG 900 was not formally reached in either dose schedule. Three pts (8.6%) from group 1 (40 mg [2 pts] and 60 mg [1 pt]) had a best response of complete response with incomplete count recovery (CRi). Overall, the objective response rate for CRi was 8.6% (80% confidence interval: 3%, 18%). Higher gene expression of BIRC5, AURKB, AURKA, TTK, and CCNB1 was associated with objective response in univariate logistic regression models (P < .02), and was still significant after adjusting for average dose and percentage of blasts in the BM in a multivariate model (P < .01). Expression profiles of responders were clustered together in a principal component analysis. Conclusions: AMG 900 had an acceptable safety profile in this grievously ill population of adult pts with recurrent/refractory AML. Prolonged cytopenias hampered further dose escalation in this single-agent treatment setting. Combination of low doses of AMG 900 with other anticancer agents should be evaluated in future studies. Disclosures Schuster: Amgen Inc.: Equity Ownership, Honoraria, Speakers Bureau. Sekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Gamelin:Amgen Inc.: Employment, Equity Ownership. Rasmussen:Amgen Inc.: Employment, Equity Ownership. Juan:Amgen Inc.: Employment, Equity Ownership. Anderson:Amgen Inc.: Employment, Equity Ownership. Chow:Amgen Inc.: Employment, Equity Ownership. Friberg:Amgen Inc.: Employment, Equity Ownership. Vogl:Amgen Inc.: Employment, Equity Ownership.

scholarly journals Identification of a Specific Immunophenotype Associated with a Consistent Pattern of Genetic Mutations Including Srsf2 and Gene Expression Profile in MDS

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4255-4255
Author(s):  
Wolfgang Kern ◽  
Wencke Walter ◽  
Manja Meggendorfer ◽  
Constance Baer ◽  
Claudia Haferlach ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profile ◽  
Genetic Background ◽  
Drug Targets ◽  
Employment Equity ◽  
Cd11b Expression ◽  
Cd16 Expression ◽  
Equity Ownership ◽  
Group 2 ◽  
Group 1

Background: Myelodysplastic syndromes (MDS) comprise a heterogeneous group of diseases classified by comprehensive diagnostics. Identification of homogeneous subgroups is desirable to understand differences in clinical course and to develop targeted treatment approaches. We identified a specific immunophenotype in MDS and analyzed its genetic background. Aim: To assess the genetic background of MDS with specific CD11b/CD16 expression pattern in granulocytes and reduced CD45 expression in myeloid progenitor cells (MPC) by whole genome (WGS) and whole transcriptome sequencing (WTS). Methods: During routine flow cytometric evaluation for suspected MDS a specific immunophenotype was identified (Figure) which was found consistently associated with SRSF2 mutations. Normal granulocytic maturation starts with steep increase in CD11b expression paralleled by only slight increase of CD16 expression. This is followed by steep increase in CD16 expression paralleled by only slight further increase in CD11b expression (giraffe pattern). The immunophenotype newly identified in MDS patients is characterized by an increase of CD11b expression without CD16 expression followed by an increase in CD16 expression without further CD11b expression (rectangle pattern) and only dim CD45 expression of MPC. 32 such cases, all SRSF2mut (group 1) were selected to evaluate the molecular differences to 51 MDS SRSF2mut cases without this specific immunophenotype (group 2) by WGS and WTS. Variants were called with the Illumina tumor/unmatched normal workflow. A mixture of genomic DNA from multiple anonymous donors served as control sample. Variants with a global population frequency > 1% were removed and the final analysis was performed on protein-altering and splice-site variants only. For gene expression analysis, estimated gene counts were normalized and the resulting log2 counts per million were used as a proxy of gene expression in each sample. Unsupervised hierarchical clustering (HC) with Ward's method as clustering method and Euclidean distance as distance measure were used to segregate the patients according to their whole transcriptional profile. Analysis of differentially expressed (DE) genes was performed considering an adjusted p<0.05 and an absolute logFC>2 as significant. Results: HC resulted in a dendrogram clearly showing two distinct clusters of samples consistent with groups 1 and 2 as defined based on the immunophenotype. 116 genes were DE, including 13 transcription factors (e.g. MYCN, ZNF462, HOXA10), 4 FDA approved drug targets (MMP2, PRLR, AR, MME) and 6 potential drug targets (AASS, PXDN, CYP27A1, SLC16A2, UCHL1, GUCY1A3) as indicated by the human protein atlas (Uhlén et al, 2015). The identified gene signature was significantly enriched for cellular developmental process, cell adhesion and extracellular matrix organization (adjusted p<0.05 for each pathway). Analysis of the mutational profiles strikingly revealed that STAG2 mutations were exclusively found in group 1 (17/32, 53% vs. 0%, p<0.001). Hence, >50% of cases with the specific immunophenotype were characterized by co-mutations in SRSF2 and STAG2. STAG2 is a key member of the cohesion complex and its regulatory functions include chromosome segregation and formation of DNA loops that influence gene expression. Recently it was shown that the deletion of STAG2 severely impacts chromatin accessibility and cell fate decisions in hematopoiesis (Viny et al. 2019). Furthermore, group 1 was characterized by higher frequencies of mutations in ASXL1 (25/32, 78% vs. 9/51, 18%, p<0.001), NRAS (6/32, 19% vs. 3/51, 6%, p=0.07) and RUNX1 (11/32, 34% vs. 5/51, 10%, p=0.007). Of the 15 STAG2 unmutated cases from group 1, 6 (40%) had mutations in at least two genes out of ASXL1, NRAS and RUNX1. The same constellation was present in only 2/51 (4%) cases from group 2. Taken together, the immunophenotype of group 1, besides predicting SRSF2 mutations, is significantly associated with a specific mutation profile which is found in 23/32 (72%) such cases and which occurs infrequently in other SRSF2mut MDS cases (2/51, 4%, p<0.001). Conclusions: We here for the first time describe a specific immunophenotype which defines MDS cases with SRSF2 mutations and a consistent and specific mutational and gene expression profile. This comprehensive data may qualify for the definition of a new sub-entity which should be addressed in future research. Disclosures Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Walter:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Baer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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