Integration of Hi-C and Nanopore Sequencing for Structural Variant Analysis in AML with a Complex Karyotype: (Chromothripsis)²

Background. Acute myeloid leukemia (AML) with a complex karyotype (CK-AML) is an AML subtype with a still dismal outcome despite recent therapeutic advances. The prognosis is even worse when the underlying structural variants (SVs) lead to an extremely complex pattern of rearrangements, called chromothripsis, with a median overall survival of only 120 days. Except for the presence of inactivating TP53 aberrations in about 70% of all AML-CK cases, the pathogenesis is poorly understood. To gain novel insights into the molecular mechanisms underlying CK-AML reliable high precision SV delineation is needed, which so far has been a major limitation in cancer research. Aim. We developed a SV detection pipeline by integrating Oxford Nanopore Technology (ONT) based whole genome sequencing (WGS) and Hi-C sequencing. This pipeline generated precise characterization of SVs for which the impact on gene expression and the emergence of novel fusion genes was studied by RNA-seq and ONT transcriptome sequencing. Patients and Methods. We applied our WGS and Hi-C SV detection pipeline to a cohort of 11 AML-CK cases. Nanopore DNA Sequencing was performed until a genomic coverage >10x per patient was reached. The samples of 9 patients were also subjected to Nanopore cDNA sequencing for fusion gene analysis and Illumina based RNA-seq for transcript quantification. As controls for Hi-C and Illumina RNA sequencing, CD34+ hematopoietic stem cell enriched samples from five healthy donors were used. Results. Our SV detection pipeline enabled us to fully reconstruct the derivate chromosome structure even of very complex, chromothriptic rearrangements in CK-AML. This enabled us to identify features of chromothripsis, that could previously not be detected using conventual technologies. We found local clustering of breakpoints in three of the patients with up to 31 Inversions and Translocations located in a genomic region of just 2.7 kb. These breakpoints were present in the Hi-C as well as in our Nanopore SV dataset. Our SV pipeline also showed that in these highly clustered regions, the very small rejoined fragments (in many cases less than 1 kb in size) often showed an elevated copy number (CN) state, i.e. small amplifications. We termed this newly discovered phenomenon chromothripsis-in-chromothripsis or (chromothripsis)². The precise knowledge about these breakpoints, which were validated by two different technologies, enabled us to study the pathogenesis of CK-AML at a so far unprecedented resolution. Fusion transcripts could be very precisely mapped and the impact of the breakpoints and CN changes on gene expression levels could be validated, thereby indicating functional relevance of the respective aberrations. Conclusions. The combination of Hi-C and long-read sequencing for SV detection proved to be a powerful tool for precise SV detection. Our SV pipeline allowed us to discover a new level of complexity in chromothripsis. Application of this pipeline to leukemias as well as other types of cancer can improve the precision of SV detection, thereby raising new opportunities for functional interpretation of complex genomic aberrations of pathogenic relevance. Disclosures Döhner: Sunesis Pharmaceuticals: Research Funding; Astex Pharmaceuticals: Consultancy; Pfizer: Research Funding; Bristol-Myers Squibb: Research Funding; Arog: Research Funding; Roche: Consultancy; Novartis: Honoraria, Research Funding; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Abbvie: Consultancy; Agios: Consultancy; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Research Funding; Astellas Pharma: Consultancy; Celgene: Consultancy, Honoraria. Schrezenmeier:Alexion Pharmaceuticals Inc.: Honoraria, Research Funding. Bullinger:Amgen: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Hexal: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Menarini: Membership on an entity's Board of Directors or advisory committees.

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Dysregulation of Splicing in Multiple Myeloma: The Splicing Factor SRSF1 Supports MM Cell Proliferation Via Splicing Control

Blood ◽

10.1182/blood-2018-99-118845 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4500-4500

Author(s):

Mariateresa Fulciniti ◽

Michael A Lopez ◽

Anil Aktas Samur ◽

Eugenio Morelli ◽

Hervé Avet-Loiseau ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Alternative Splicing ◽

Board Of Directors ◽

Research Funding ◽

Splicing Factor ◽

Splicing Factors ◽

Rna Seq ◽

Advisory Committees ◽

Disease Biology

Abstract Gene expression profile has provided interesting insights into the disease biology, helped develop new risk stratification, and identify novel druggable targets in multiple myeloma (MM). However, there is significant impact of alternative pre-mRNA splicing (AS) as one of the key transcriptome modifier. These spliced variants increases the transcriptomic complexity and its misregulation affect disease behavior impacting therapeutic consideration in various disease processes including cancer. Our large well annotated deep RNA sequencing data from purified MM cells data from 420 newly-diagnosed patients treated homogeneously have identified 1534 genes with one or more splicing events observed in at least 10% or more patients. Median alternative splicing event per patient was 595 (range 223 - 2735). These observed global alternative splicing events in MM involves aberrant splicing of critical growth and survival genes affects the disease biology as well as overall survival. Moreover, the decrease of cell viability observed in a large panel of MM cell lines after inhibition of splicing at the pre-mRNA complex and stalling at the A complex confirmed that MM cells are exquisitely sensitive to pharmacological inhibition of splicing. Based on these data, we further focused on understanding the molecular mechanisms driving aberrant alternative splicing in MM. An increasing body of evidence indicates that altered expression of regulatory splicing factors (SF) can have oncogenic properties by impacting AS of cancer-associated genes. We used our large RNA-seq dataset to create a genome wide global alterations map of SF and identified several splicing factors significantly dysregulated in MM compared to normal plasma cells with impact on clinical outcome. The splicing factor Serine and Arginine Rich Splicing Factor 1 (SRSF1), regulating initiation of spliceosome assembly, was selected for further evaluation, as its impact on clinical outcome was confirmed in two additional independent myeloma datasets. In gain-of (GOF) studies enforced expression of SRSF1 in MM cells significantly increased proliferation, especially in the presence of bone marrow stromal cells; and conversely, in loss-of function (LOF) studies, downregulation of SRSF1, using stable or doxy-inducible shRNA systems significantly inhibited MM cell proliferation and survival over time. We utilized SRSF1 mutants to dissect the mechanisms involved in the SRSF1-mediated MM growth induction, and observed that the growth promoting effect of SRSF1 in MM cells was mainly due to its splicing activity. We next investigated the impact of SRSF1 on allelic isoforms of specific gene targets by RNA-seq in LOF and confirmed in GOF studies. Splicing profiles showed widespread changes in AS induced by SRSF1 knock down. The most recurrent splicing events were skipped exon (SE) and alternative first (AF) exon splicing as compared to control cells. SE splice events were primarily upregulated and AF splice events were evenly upregulated and downregulated. Genes in which splicing events in these categories occurred mostly did not show significant difference in overall gene expression level when compared to control, following SRSF1 depletion. When analyzing cellular functions of SRSF1-regulated splicing events, we found that SRSF1 knock down affects genes in the RNA processing pathway as well as genes involved in cancer-related functions such as mTOR and MYC-related pathways. Splicing analysis was corroborated with immunoprecipitation (IP) followed by mass spectrometry (MS) analysis of T7-tagged SRSF1 MM cells. We have observed increased levels of SRSF phosphorylation, which regulates it's subcellular localization and activity, in MM cell lines and primary patient MM cells compared to normal donor PBMCs. Moreover, we evaluated the chemical compound TG003, an inhibitor of Cdc2-like kinase (CLK) 1 and 4 that regulate splicing by fine-tuning the phosphorylation of SR proteins. Treatment with TG003 decreased SRSF1 phosphorylation preventing the spliceosome assembly and inducing a dose dependent inhibition of MM cell viability. In conclusions, here we provide mechanistic insights into myeloma-related splicing dysregulation and establish SRSF1 as a tumor promoting gene with therapeutic potential. Disclosures Avet-Loiseau: Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Munshi:OncoPep: Other: Board of director.

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Inotuzumab Ozogamicin (Ino) May Overcome the Impact of Philadelphia Chromosome (Ph)-like Phenotype in Adult Patients (pts) with Relapsed/Refractory (R/R) Acute Lymphoblastic Leukemia (ALL)

Blood ◽

10.1182/blood-2019-126940 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1641-1641 ◽

Cited By ~ 1

Author(s):

Elias Jabbour ◽

Kathryn G. Roberts ◽

Koji Sasaki ◽

Yaqi Zhao ◽

Chunxu Qu ◽

...

Keyword(s):

Gene Expression ◽

Overall Survival ◽

Stem Cell ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Advisory Committees ◽

The Impact

Background: Ino showed significant activity in phase II trials in pts with R/R ALL, that was subsequently confirmed in Phase III trial where Ino demonstrated higher response rates and superior overall survival vs standard of care chemotherapy (SOC) in adults with relapsed/refractory B-cell precursor acute lymphoblastic leukemia (R/R ALL).Ph-like or BCR-ABL1-like ALL possesses a gene expression profile similar to that of BCR-ABL1 ALL but lacks the BCR-ABL1 fusion protein. It is characterized by increased expression of hematopoietic stem-cell genes, deletion of B-cell lineage genes and kinase-activating alterations. Ph-like ALL is associated with refractoriness to standard induction/consolidation chemotherapy and poor prognosis. Aim: To evaluate the outcomes of pts with R/R Ph-like ALL treated in phase II trial with Ino monotherapy. Methods: We performed an integrated analysis of whole genome sequencing (to identify sequence mutations, structural variations and DNA copy number alterations), and transcriptome sequencing (RNAseq; to quantify gene expression, determine Ph-like gene expression profile and identify fusions) on 53 patients' samples treated with Ino between June 2010 and September 2012. Results: Fifty-three evaluable pts with R/R ALL with stored baseline samples were analyzed. Pts characteristics are summarized in Table 1. Median age was 50 years. Ino was given as Salvage 1, Salvage 2, and Salvage 3 and beyond in 20 (38%), 18 (34%), and 15 (28%) pts, respectively. Figure 1 reflects the different genomic subgroups identified among 53 evaluable pts. Ph-like gene signature was found in 12 pts (22.6%). Among these 12 pts, 6 had IGH-CRLF2, 2 IGH-EPOR, 1 SNX2-ABL1, and 3 had no fusions identified. The overall response rates (ORR) were 54% [complete remission (CR) 20%, CR with partial hematologic recovery (CRh) 32%, and marrow CR (CRi) 2%]. Among pts with morphologic remission, 46% and 82% achieved minimal residual disease (MRD) negativity at CR and at any time, respectively. The ORR for pts with Ph-like ALL, Ph-positive ALL, ALL with KMT2A, and others were 58% (CR=25%; CRh=33%), 42% (CR=8%; CRh=33%), 57% (CR=14%; CRh=29%; CRi=14%), and 56% (CR=26%; CRh=30%), respectively. The respective overall MRD negativity rates were 71%, 100%, 75%, and 83% (Table 1). The median follow-up was 60 months. The median event-free (EFS) and overall survival (OS) were 3.3 and 5.4 months, respectively. There was no difference in EFS and OS between the subgroups analyzed (P=0.464; P=0.824). The median EFS and OS were 4.5 and 4.5 months for pts with Ph-like, 3.1 and 7.2 months for those with Ph-positive ALL, 2.8 and 4.4 months for those with KMT2A, and 2.2 and 4.6 months for others (Table 1). 21 (40%) pts had subsequent allogeneic stem cell transplant; 6 (50%), 3 (25%), 4 (57%), and 8 (36%) in each subgroup, respectively. The rate of VOD was 3 (6%) with no difference among different subgroups. Conclusion: The current analysis suggest that Ino therapy may overcome the impact of Ph-like phenotype in pts with ALL. Confirmation of these findings in a larger cohort and in frontline ALL patients is needed. Disclosures Jabbour: Takeda: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Adaptive: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Cyclacel LTD: Research Funding. Sasaki:Pfizer: Consultancy; Otsuka: Honoraria. Jain:Precision Biosciences: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics, an AbbVie company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Adaptive Biotechnologies: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ravandi:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Xencor: Consultancy, Research Funding; Macrogenix: Consultancy, Research Funding; Menarini Ricerche: Research Funding; Selvita: Research Funding; Cyclacel LTD: Research Funding. Short:AstraZeneca: Consultancy; Takeda Oncology: Consultancy, Research Funding; Amgen: Honoraria. Garcia-Manero:Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding. Konopleva:Cellectis: Research Funding; Agios: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Ascentage: Research Funding; Eli Lilly: Research Funding; Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Kisoji: Consultancy, Honoraria; Ablynx: Research Funding; Genentech: Honoraria, Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Astra Zeneca: Research Funding. Mullighan:Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding; AbbVie: Research Funding; Loxo Oncology: Research Funding; Amgen: Honoraria, Other: speaker, sponsored travel. Kantarjian:Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria, Research Funding; Ariad: Research Funding; Novartis: Research Funding; Amgen: Honoraria, Research Funding; Immunogen: Research Funding; AbbVie: Honoraria, Research Funding; Astex: Research Funding; BMS: Research Funding; Cyclacel: Research Funding; Daiichi-Sankyo: Research Funding; Pfizer: Honoraria, Research Funding; Jazz Pharma: Research Funding; Takeda: Honoraria.

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GFI1 Is a Downstream Target of EVI1 in Normal Hematopoiesis

Blood ◽

10.1182/blood-2020-134071 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 33-33

Author(s):

Akira Chiba ◽

Yosuke Masamoto ◽

Hideaki Mizuno ◽

Mineo Kurokawa

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Board Of Directors ◽

Research Funding ◽

Hematopoietic Progenitor Cells ◽

Rna Seq ◽

Hematopoietic Progenitor ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Knock Out

Acute myeloid leukemia (AML) with high expression of a transcriptional factor, Ecotropic viral integration site 1 (EVI1), is associated with extremely poor prognosis. EVI1 is, however, also essential for maintaining normal hematopoietic stem cells (HSCs), rendering it potentially difficult to target this molecule. To overcome this therapeutic difficulty, it is important to comprehensively elucidate differentially regulated downstream targets between normal and leukemia cells. In this study, we searched downstream targets of EVI1 in normal hematopoiesis by combining a chromatin immunoprecipitation sequence (ChIP-seq) and RNA-sequence (RNA-seq) analysis using a mouse hematopoietic cell line 32D-cl3 with high EVI1 expression. We deleted Evi1 using CRISPR/Cas9 in 32D-cl3 cells. Evi1 knock-out (KO) 32D-cl3 cells showed comparable cell growth with parental cells in the presence of IL-3, which enables them to proliferate permanently without differentiation. When they are allowed to differentiate by adding G-CSF, the number of KO cells decreased sharply at day 5-6, compared with parental 32D-cl3 cells. Along with the decreased cell number, KO cells also demonstrated higher positive rate of Gr-1 at day 7, a typical marker of differentiation into granulocytes, indicating accelerated differentiation of KO cells. These results indicated that EVI1 is required to maintain undifferentiated status of 32D-cl3 cells in a differentiation-permissive conditions, which can model normal hematopoiesis. We knocked in 3×FLAG tag at the 3' end of the Evi1 gene to perform ChIP-seq using anti-FLAG antibody. By using these knock-in cells, ChIP-seq was performed on day 0 and day 3 of G-CSF treatment, when they had started to differentiate with still maintained EVI1 expression. The peaks observed in undifferentiated day 0 sample were considered to contain a group of genes involved in undifferentiated hematopoietic cells in cooperation with EVI1. Genes associated HDAC class I, RAC1 signaling were enriched in these genes. To investigate the functional implications of the result of ChIP-seq, RNA-seq data using two clones of KO cells and parental cells were combined. We found that 152 genes were significantly up-regulated, and 155 genes were down-regulated in the KO cells, with false discovery rate less than 0.05. Twenty-four genes were identified by extracting common genes between ChIP-seq and RNA-seq; namely, genes which had day 0-specific peaks in ChIP-seq, and whose expression were decreased in the KO cells. In order to further examine the physiological implications of 24 genes in vivo, we referred to the results of RNA-seq using murine bone marrow transplantation model, where murine hematopoietic progenitor cells retrovirally transduced with Evi1 were transplanted into irradiated syngeneic mice, finally leading to AML after a long latency. Samples obtained early after post transplantation and those after AML onset were compared to those of normal hematopoietic progenitor cells. Among the above 24 genes, the expression of 5 genes was increased early after transplantation and decreased after the onset of AML, that is, these genes were up-regulated by EVI1 but don't seem to be involved in AML maintenance. We functionally validated the role of these genes in 32D-cl3 cells. Of the above, CRISPR/Cas9-mediated knock-out of Gfi1(Growth Factor Independent 1 Transcriptional Repressor) and Mfsd2b (Major facilitator superfamily domain containing 2B) in 32D-cl3 cells led to high Gr-1 positivity at day 7 like Evi1-KO cells, suggesting that these genes are involved in the functions of EVI1 in the normal hematopoiesis. The mRNA expression of these genes was compared in LSK (Lineage- Sca1+ c-kit+) cells from the bone marrow of Evi1 conditional knockout (cKO) mice and control mice. The expression of Gfi1 and Mfsd2b was decreased in LSK cells from Evi1 cKO mice. Furthermore, retroviral expression of Gfi1 in LSK cells restored the reduced colony-forming ability of Evi1 cKO cells. These results collectively suggest that GFI1 is regulated by EVI1 and is involved in the function of EVI1 regulating the stemness of hematopoietic stem and progenitor cells in normal hematopoiesis. These findings provide us with the novel insights on EVI1-mediated HSC maintenance as well as on the therapeutic strategy that specifically targets leukemia-specific EVI1 effectors while preserving normal hematopoiesis. Disclosures Kurokawa: Shire Plc: Speakers Bureau; Jansen Pharmaceutical: Speakers Bureau; Ono: Research Funding, Speakers Bureau; Boehringer Ingelheim: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Eisai: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Teijin: Research Funding; Takeda: Research Funding, Speakers Bureau; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Otsuka: Research Funding, Speakers Bureau; Pfizer: Research Funding; Sanwa-Kagaku: Consultancy; MSD: Consultancy, Research Funding, Speakers Bureau; Chugai: Consultancy, Research Funding, Speakers Bureau; Bioverativ Japan: Consultancy; Celgene: Consultancy, Speakers Bureau; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Nippon Shinyaku: Research Funding, Speakers Bureau.

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Impact of KIR/HLA Incompatibilities after Posttransplant Cyclophosphamide Based T Cell-Replete Haploidentical Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood-2019-125243 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3340-3340

Author(s):

Sarah Lindner ◽

Tobias Berg ◽

Christian Seidel ◽

Franziska Kalensee ◽

Michael A. Rieger ◽

...

Keyword(s):

T Cell ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Chronic Gvhd ◽

Relapse Free Survival ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Free Survival ◽

The Impact

Introduction: Posttransplantation cyclophosphamide (PTCy) based T cell-replete haploidentical (haplo) hematopoietic stem cell transplantation (HSCT) is a valid option for patients with indication for allogeneic HSCT without a human leucocyte antigen (HLA) matched donor. However, selection criteria to determine the optimal among several available haplo donors are still a matter of debate. Especially, the impact of killer cell immunoglobulin-like receptor (KIR)/human leukocyte antigen (HLA) incompatibilities (inc) in the setting of PTCy T cell-replete haplo HSCT is unclear. PTCy has been reported to eliminate most mature donor NK cells infused with the graft, including single KIR+ NK cells, thereby blunting NK cell alloreactivity in this setting (Russo et al., Blood 2018). Willem et al. (J Immunol 2019) reported (i) a significant loss of KIR2DL2/3+ NK cells at day +30 in patients with inhibitory KIR/HLA incompatibility (inc.) suggesting that PTCy might target responsive KIR NK cells and (ii) a correlation of genetic KIR2DL/HLA inc. with less relapse, but more graft-versus-host-disease (GvHD). Similarly, NK alloreactivity defined as KIR receptor-ligand mismatch or group B KIR haplotype with the presence of KIR2DS2 has been correlated with improved survival (Salomon et al., BBMT 2018). Aims of our study were to evaluate the impact of (i) HLA/KIR inc, (ii) donor KIR genotype and (iii) HLA-DP mismatch status on survival and incidence of relapse, acute and chronic GvHD in our homogeneously treated, independent patient cohort. Patients and methods: We retrospectively analyzed the outcome of 51 consecutively transplanted patients (AML/MDS (n=28/5), ALL (n=9), HD (n=2), NHL (n=5), CML (n=1), PMF (n=1)) receiving a PTCy based T cell-replete haplo HSCT between 01/2011-12/2018. All patients received a myeloablative conditioning regimen (fludarabine/total body irradiation (FTBI) or thiotepa/busulfan/fludarabine (TBF)) with unmanipulated bone marrow (98%) as the preferred graft (median CD34+ cells: 3.02 x 106/kg (range, 1.50-6.90) and median CD3+ T cells: 3.54 x 107/kg (range 1.52-43.74)). GvHD prophylaxis with ciclosporin A started on day 0, mycophenolate-mofetil on day +1, PTCy was applied on day +3 and +5. Results: Patient, donor and transplant characteristics as detailed in table 1 were well balanced between the inh. KIR/HLA inc. group (n=29) vs. no inh. KIR/HLA inc. group (n=22) with the exception of the median donor age (41.7 (range, 23.4-73.7) vs. 33.6 years (range, 19.0-56.2), resp. All patients engrafted. At day +28 (range, 20-29; n=26) CD3+ cells were 88.5/nL (range, 3-665), CD3+CD4+ cells 22.5/nL (range, 0-277.0), CD3+CD8+ cells 117.0/nL (range, 7-478), CD19+ cells 1.0/nL (range, 0-12), CD56bright cells 74.4/nL (range11.1-93.4), CD56dim cells 25.5/nL (range, 6.4-88.9) measured by flow cytometry and without differences between the inh. KIR/HLA inc. group vs. no inh. KIR/HLA inc. group. Cytomegalovirus (CMV) reactivation occurred in 73.3% of patients at risk and median time of occurrence was 32 days (range, 12-97) without difference between groups. Median follow-up for surviving patients was 26.1 months (range, 2.8-92.8) and we found no significant differences in 2-year overall survival (OS; 65.3±10.3 vs. 89.6±7.0, p=0.311), 2-year relapse-free survival (RFS; 66.0±9.4 vs 77.8±10.2, p=0.235), GvHD- and relapse-free survival (GRFS; 48.4±9.8 vs 60.5±12.0, p=0.182) as well as cumulative incidence (CI) of relapse (23.3% vs 16.2%, p= 0.283), acute GvHD grade 2-4 (27.6% vs 31.8, p=0.563), moderate-severe chronic GvHD (22.2% vs. 9.9%, p=0.227) and NRM (16.3% vs 5.3%, p=0.283) between the inh. KIR/HLA inc. group vs. no inh. KIR/HLA inc. group. This was also the case for donor KIR genotype AA vs AB (n=46; 2-y OS: 74.9±13.0% vs. 73.0±9.9%, p=0.844; 2-y RFS: 60.0±14.8% vs 74.5±8.4%, p=0.645) and HLA-DP-identical/permissive mismatch (MM) vs non permissive MM (n=45; 2-y OS: 70.7±10.0% vs 72.7±13.4%, p=0.945; 2-y RFS: 73.2±8.2% vs 63.6.0±14.5%, p=0.798) Conclusion: Our outcome data support the hypothesis of PTCy eliminating mature donor NK cells infused with the graft and thereby reducing the impact of alloreactivity in this setting. However, our patient number is quite small and the findings need to be validated in larger cohorts and preferably prospective studies. Disclosures Lindner: Celegene, Sanofi, Neovii: Honoraria, Research Funding. Berg:Riemser Pharma GmbH: Consultancy, Honoraria; Incyte, Abbvie, Astellas, Alexion and Celgene: Other: travel support. Bug:Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene Neovii: Other: travel grant; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grants; Hexal: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Other: Travel grants; Sanofi: Other: travel grants. Schwaeble:Uniqure BV: Research Funding. Ullrich:CellGenix: Honoraria, Research Funding; Novartis: Research Funding.

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Distinct Pathogenesis of Clonal Hematopoiesis Revealed By Single Cell RNA Sequencing Integrated with Highly Sensitive Genotyping Method

Blood ◽

10.1182/blood-2020-142157 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 34-34

Author(s):

Masahiro Marshall Nakagawa ◽

Ryosaku Inagaki ◽

Yutaka Kuroda ◽

Yasuhito Nannya ◽

Lanying Zhao ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Single Cell ◽

Board Of Directors ◽

Research Funding ◽

Simultaneous Detection ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Single Cell Sequencing ◽

Clonal Hematopoiesis

Background Recent evidence suggests that age-related clonal hematopoiesis (CH) might represent the earliest precursor of myeloid neoplasms. Although the exact mechanism of clonal selection that shapes CH is still to be elucidated, both cell intrinsic and non-cell intrinsic effects of mutations, including the interplay between mutated cells and the bone marrow environment, are thought to play important roles, which are best studied using single-cell sequencing analysis of both mutations and gene expression. Methods We performed single-cell sequencing of hematopoietic stem and progenitors (HSPCs) from BM of the 16 patients with CH along with 16 control patients without CH identified by screening otherwise healthy individuals who received hip joint replacement, using a novel platform that enables simultaneous detection of gene mutations and expression based on the Fluidigm C1-HT system. Sequence data were analyzed with Seurat (Stuart et al Cell 2019) with integration of genotyping information. Cells were clustered and each cluster was assigned by marker-gene expressions for major cell-types in HSPCs, including hematopoietic stem cell (HSC)-like and erythroid progenitors. Cells were grouped by their genotypes and pathway analysis were performed. Results In total, we identified 35 subjects who had CH-related mutations, including those affecting DNMT3A, TET2, ASXL1, SF3B1, PPM1D, IDH1, GNB1 and TP53, of which 11 had more than one CH-related mutation. Most of these mutations showed a low variant allele frequency (VAF) ≤ 0.05. However, clones having double mutations of DNMT3A/TET2 or those having biallelic TET2 mutations tended to show a higher VAF as high as 0.4, suggesting an enhanced clonal advantage for clones having multiple mutations. Using our novel single-cell platform, we analyzed 3,767 cells from control patients without CH and 1,474 mutated cells and 7,234 wild-type (WT) cells from patients with CH. By targeting both genomic DNA and RNA, we successfully obtained a sufficient number of single-cell reads for genes whose expression was too low to evaluate by only targeting RNA, such as TET2 and DNMT3A. Although some clones having a high-VAF mutation caused a skewed clustering to be detected as a CH clone, many clones with low-VAF mutations did not make distinct clusters, indicating the importance of genotyping at a single cell level to identify and characterize mutated cells. Simultaneous detection of genotype and expression allowed us to see the effect of CH-mutations on cell phenotype and differentiation. For example, cells having compound TET2/DNMT3A mutations were significantly enriched in the erythroid cluster, while another clone with double TET2 mutations were more enriched in the HSC-like cluster, compared to cells from individuals without CH (WTcont). These are in line with the previous findings of TET2/DNMT3A double knockout mice or TET2 knockout mice, respectively. In another case with an IDH1 mutation, IDH1-mutated (MUTIDH1) cells less contributed to the HSC-like fraction, showing an enhancement of cell proliferation-signature, compared to WT (WTIDH1) cells in the same patient. Strikingly, compared to WTcont cells, WTIDH1 cells were significantly enriched in the HSC-like fraction and showed an enhanced expression of cytokine-related pathway genes, which was in line with a finding seen in mouse cells treated with 2-hydroxy-glutalate, an mutant IDH-related oncometabolite. Similarly, when compared to WTcont cells, WT cells from patients with DNMT3A- (WTDNMT3A) or TET2- (WTTET2) mutated CH significantly showed an enhanced cell proliferation. HSC-like WTTET2 cells also showed aberrant IFN-response signatures compared to corresponding WTcont cells, which was confirmed in competitive transplantation of Tet2 heterozygous knockout (hKO) and WT cells in a mouse model; HSPCs of WT competitors transplanted with Tet2-hKO cells showed a significant enhancement of IFN-response signatures compared to those transplanted with WT cells. Intriguingly, monocytes of Tet2-hKO donors showed aberrant expression of S100a8/a9, which might contribute to the non-cell intrinsic effect of Tet2-hKO cells. Conclusions In CH, not only mutated cells but also surrounding WT cells show an aberrant gene expression phenotype, suggesting the presence of non-cell autonomous phenotype or an altered bone marrow environment that favors the positive selection of CH-clones. Disclosures Nakagawa: Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Inagaki:Sumitomo Dainippon Pharma Co., Ltd.: Current Employment. Ogawa:Eisai Co., Ltd.: Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Isocitrate Dehydrogenase (IDH) 1 and 2 Mutation Is an Independent Predictor of Better Outcome in Patients with Acute Myeloid Leukemia Undergoing Allogeneic Hematopoietic Stem Cell Transplantation: A Study of the ALWP of EBMT

Blood ◽

10.1182/blood-2021-149794 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2920-2920

Author(s):

Razan Mohty ◽

Abdul Hamid Bazarbachi ◽

Myriam Labopin ◽

Jordi Esteve ◽

Nicolaus Kröger ◽

...

Keyword(s):

Stem Cell ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Wild Type ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Free Survival ◽

Mutational Status ◽

The Impact

Abstract Isocitrate dehydrogenase (IDH) 1 and 2 mutations occur in 20% of acute myeloid leukemia (AML). Patients with AML carrying IDH1-2 mutations have a similar prognosis compared to patients without these mutations (DiNardo et al, AM J H, 2015). However, the impact of IDH1-2 mutations on patients with AML undergoing allogeneic hematopoietic stem cell transplantation (allo-HCT) is not well known. In this study, we investigate the prognostic impact of IDH1-2 mutational status on AML patients undergoing alloHCT in complete remission (CR). In this retrospective registry-based analysis, we identified 710 consecutive adult patients (46.2% female; median age: 58.5 years [range, 18-78]) with AML undergoing allo-HCT in CR between 2015 and 2019 at 85 EBMT participating centers. Cord blood, ex-vivo graft manipulated transplants, and patients with favorable cytogenetics were excluded. Median follow-up was 15 months [95% CI 13.4-16.6]. Patients were categorized based on IDH1-2 mutational status, with 300 (42%) mutated and 410 (58%) wild type. Six hundred and fifty-two (92%) and 58 (8%) patients had de novo and secondary AML, respectively, and 141 (20%) patients had poor-risk cytogenetics. IDH1-2 mutation was positively correlated with NPM1 mutation (40% in IDH1-2 mutated vs 27% in wild type, p=0.0001) and more frequently encountered in middle-aged patients (p=0.01). No correlation was noted between IDH1-2 and FLT3 mutation or other patient characteristics. Minimal residual disease (MRD) data was available for 344 patients, 53% of which were MRD-negative at transplant in both groups. Six hundred and twenty-three (88%) and 87 (12%) patients were in first and second CR at time of transplant, respectively. Patients received grafts from a matched sibling (24%), unrelated (62%), or haploidentical (14%) donor, and myeloablative conditioning (MAC) was used in 42%. Ninety-three percent of the patients received peripheral blood as the stem cell source. At day 180, the cumulative incidence of grade II-IV acute graft-versus-host disease (aGVHD) was significantly lower in IDH1-2 mutated compared to wild-type patients (22% vs 33%, p=0.002). No differences in chronic GVHD rates were noted between the 2 groups (39% vs 40%, p=0.87). The 2-year cumulative relapse incidence (RI) was significantly lower and the GVHD-free, relapse-free survival (GRFS) was also improved in IDH1-2 mutated compared to wild-type patients (14.4% vs 27%, p=0.001 and 47% vs 39%, p=0.006, respectively). This led to an improved leukemia-free survival (LFS) in IDH1-2 patients (69% vs 59%, p=0.01), however, it did not translate into an overall survival (OS) difference. No significant difference was noted in non-relapse mortality (NRM) between the 2 groups (17% vs 14.2%, p=0.26). These findings were confirmed in multivariate analysis. In fact, IDH1-2 mutation was associated with significant improvement in RI (hazard ratio [HR]=0.4 [95%CI 0.25-0.64], p=0.0001), LFS (HR=0.7 [95%CI 0.51-0.95], p=0.022), aGVHD II-IV (HR=0.63 [95%CI 0.45-0.87], p=0.005) and GRFS (HR=0.69 [95%CI 0.54-0.89], p=0.004). Conversely, the presence of adverse cytogenetics and undergoing allo-SCT in second CR increased the RI (HR=2.29 [95%CI 1.46-3.61], p=0.0003 and HR=2.84 [95%CI 1.64-4.91], p=0.0002, respectively) and were associated with a shorter LFS (HR=1.67 [95%CI 1.18-2.36], p=0.004 and HR=1.61 [95%CI 1.06-2.44], p=0.025) while reduced intensity (RIC) conditioning had a worse impact on OS compared to MAC (HR=1.56 [95%CI 1.07-2.29], p=0.022). Additionally, in the subgroup of patients with available MRD data, MRD positivity at transplant significantly increased RI (HR=2.15 [95%CI 1.09-4.23], p=0.027) with no impact on survival. In conclusion, our data suggest that the presence of IDH1-2 mutations acts as an independent prognostic factor and is associated with improved outcome in patients with AML in CR undergoing allo-HCT. Indeed, patients with IDH1-2 mutations had significantly lower rates of RI and aGVHD, which translated into improved LFS and GRFS. Nevertheless, patients with MRD positivity at time of transplant had significantly increased RI. Further studies investigating allo-HCT outcomes in IDH1-2 mutated patients with AML in the era of IDH inhibitors (both in the pre- and post-transplant setting) would help to further define the impact of these mutations in this setting and thus optimize an individualized treatment approach. Disclosures Labopin: Jazz Pharmaceuticals: Honoraria. Esteve: Abbvie: Consultancy; Pfizer: Consultancy; Astellas: Consultancy; Novartis: Research Funding; Novartis: Consultancy, Research Funding; Bristol Myers Squibb/Celgene: Consultancy; Jazz: Consultancy. Kröger: Novartis: Research Funding; Riemser: Honoraria, Research Funding; Sanofi: Honoraria; Neovii: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Gilead/Kite: Honoraria; Celgene: Honoraria, Research Funding; AOP Pharma: Honoraria. Blaise: Jazz Pharmaceuticals: Honoraria. Socie: Alexion: Research Funding. Ganser: Jazz Pharmaceuticals: Honoraria; Novartis: Honoraria; Celgene: Honoraria. Yakoub-Agha: Jazz Pharmaceuticals: Honoraria. Bazarbachi: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Hikma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees.

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Chromatin Accessibility Identifies Regulatory Elements Predictive of Oncogene Expression in Multiple Myeloma

Blood ◽

10.1182/blood-2020-140717 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 31-32

Author(s):

Benjamin G Barwick ◽

Vikas A. Gupta ◽

Shannon M Matulis ◽

Jonathan C Patton ◽

Doris R Powell ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Board Of Directors ◽

Research Funding ◽

Chromatin Accessibility ◽

Rna Seq ◽

Advisory Committees ◽

Myeloma Cells ◽

Intergenic Regions ◽

Highly Correlated

Introduction Extensive genomic characterization of multiple myeloma has identified subtypes with prognostic and therapeutic implications. In contrast, less is known about the myeloma epigenome. One challenge that has hindered epigenetic studies are assays amenable to biobanked specimens. Here, we sought to determine whether ATAC-seq and RNA-seq of myeloma cells from cryopreserved bone marrow aspirates recapitulated those from fresh samples and used this approach to investigate enhancers of myeloma oncogenes. Methods Consent and collection of specimens followed approved Institutional Review Board protocols. Mononuclear cells were enrichment by Ficoll gradient centrifugation and were either cryopreserved in 10% DMSO and RPMI media with 10% FBS or used to isolate viable CD138+CD38+ myeloma cells. RNA-seq used the mRNA HyperPrep kit (Kapa Biosystems) with RNA from 50,000 cells. ATAC-seq used the Tn5 transposase (Illumina) on 20,000 cells. Sequencing was performed on an HiSeq 4000 (Illumina). Sequencing data were quality and adapter trimmed using Trim Galore! And mapped to the GRCh37 genome using STAR (RNA-seq) or bowtie2 (ATAC-seq). MACS2 was used to determine chromatin accessible regions and R was used for downstream analyses. H3K27ac ChIP-seq from Jin et al. (Blood, 2018) were downloaded from the European nucleotide archive (PRJEB25605). RNA-seq from CoMMpass (NCT01454297) were downloaded from dbGaP phs000748.v7.p4. Enhancer RNAs were interrogated in intergenic regions excluding 500 bp upstream of TSSs and 5 kb downstream of transcription termination sites to avoid contamination from exonic mRNAs or intronic pre-mRNAs. Results We compared RNA-seq and ATAC-seq data from myeloma cells isolated from fresh bone marrow aspirates to those cryopreserved for up to 6 months from the same aspirate. RNA-seq and ATAC-seq data from fresh and frozen samples from the same aspirate were highly correlated with each other but distinct from other samples as depicted by principal component analysis (Fig. A,B). Inspection of CCND1 showed high levels of RNA in two patients and this was consistent in both fresh and frozen specimens as well as with FISH results indicating a t(11;14) translocation in these samples (Fig. C). Similarly, fresh and frozen specimens from the same patient showed consistent expression for CCND2 and MYC and these data corresponded with chromatin accessibility found near these genes (Fig. D, see regions shaded in gray). Based on these results we expanded our analysis to include 8 biobanked specimens, which identified 91,632 regions of chromatin accessibility that were enriched around plasma cell lineage genes such as IRF4, CD38, SLAMF7, and IGH. Chromatin accessibility often predicted proximal gene expression and this was especially pronounced for regions enriched for histone 3 lysine 27 acetylation (H3K27ac) - a mark of enhancers. Active enhancers are sometimes demarcated by enhancer RNAs (eRNAs) observable in RNA-seq data, thus we queried intergenic regions marked by chromatin accessibility and H3K27ac for eRNAs using RNA-seq data on 768 myeloma specimens from the CoMMpass study. This identified transcription at 4,729 of 13,452 potential regions. eRNA expression was highly correlated with proximal gene expression. To illustrate this point, we performed t-SNE clustering based on mRNA and eRNA expression and color-coded each sample by myeloma gene expression subtype (Fig. E). Interestingly, this identified several regions highly correlated with oncogene expression between myeloma subtypes. For example, an enhancer ~154 kb upstream of CCND2 was uniquely transcribed in the MAF subtype (Fig. F) and this was highly correlated with CCND2 expression (Fig. G). Conclusions Cryopreservation of myeloma bone marrow aspirates allows isolation and analysis of biobanked samples that produce RNA-seq and ATAC-seq data that are highly congruent with those obtained from fresh samples and this represents a strategy for retrospective genomic and epigenomic studies. Chromatin accessibility analysis identified distinct enhancer elements regulating oncogenes in myeloma subtypes providing mechanistic insight into myeloma pathology. Figure 1 Disclosures Lin: Amgen: Current Employment, Current equity holder in publicly-traded company. Hofmeister:Bristol Myers Squibb: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Nektar: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; Oncopeptides: Honoraria; Oncolytics Biotech: Research Funding; Imbrium: Honoraria; Karyopharm: Honoraria, Research Funding. Nooka:Celgene: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria; Adaptive Technologies: Consultancy, Honoraria; Spectrum Pharmaceuticals: Consultancy; Takeda: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Other: Personal Fees: Travel/accomodations/expenses, Research Funding; Karyopharm Therapeutics, Adaptive technologies: Consultancy, Honoraria, Research Funding; Oncopeptides: Consultancy, Honoraria. Lonial:GSK: Consultancy, Honoraria, Other: Personal fees; BMS: Consultancy, Honoraria, Other: Personal fees, Research Funding; Takeda: Consultancy, Other: Personal fees, Research Funding; Novartis: Consultancy, Honoraria, Other: Personal fees; Janssen: Consultancy, Honoraria, Other: Personal fees, Research Funding; Merck: Consultancy, Honoraria, Other: Personal fees; JUNO Therapeutics: Consultancy; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Honoraria; Onyx: Honoraria; Genentech: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy, Honoraria, Other: Personal fees; Sanofi: Consultancy; Abbvie: Consultancy. Boise:AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genetech: Membership on an entity's Board of Directors or advisory committees.

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Impact of Race and Geographic Location on Outcomes in Allogeneic Transplant

Blood ◽

10.1182/blood-2020-140012 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 17-18

Author(s):

Audrey M. Sigmund ◽

Qiuhong Zhao ◽

Justin Jiang ◽

Patrick Elder ◽

Don M. Benson ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Ohio State University ◽

Cell Transplant ◽

State University ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Significant Difference ◽

The Ohio State University ◽

The Impact

Introduction: Allogeneic hematopoietic stem cell transplant (allo-HCT) is a potential curative therapy for a variety of both malignant and nonmalignant hematologic disorders. However, allo-HCT is costly and requires highly specialized, technologically advanced care that is only available in select healthcare centers across the country. Due to its cost and limited availability, minority populations are at risk for healthcare disparities in access to and outcomes of allo-HCT. Prior studies have focused on the impact of health disparities, including race, and geographic residence at time of transplant, on allo-HCT outcomes with variable results. The aim of this study was to evaluate the impact of race and location of residence on outcomes of allo-HCT at one major referral institution. Methods: We performed a retrospective cohort study of patients that underwent allo-HCT at the Ohio State University from 1984 to 2018. The impact of demographic factors including race and place of primary residence were assessed. Patients were divided into race defined as Caucasian, African American (AA), and other. They were also grouped by zip code into rural, suburban, and urban groups. Rural was defined as less than 1000 people per square mile, suburban between 1000-3000 people per square mile, and urban greater than 3000 people per square mile. 2018 population estimates were used. Patients were then stratified into 7 groups based on year (yr) of transplant for analysis. Group (gp) 1 included 1984-1988, gp 2 1989-1993, gp 3 1994-1998, gp 4 1999-2003, gp 5 2004-2008, gp 6 2009-2013, and gp 7 2014-2018. Primary endpoints were progression free survival (PFS) and overall survival (OS). PFS and OS were calculated using Kaplan Meier Curves and compared using log-rank test between race and residence groups. Results: A total of 1,943 patients were included in the study. Of these patients, median age at time of transplant was 50 years old (range 18-76), and 59.6% were male. AML/MDS patients made up the majority of the cohort at 46.3%, with the other most common diagnoses being non-Hodgkin's lymphoma (14.2%), acute lymphocytic leukemia (11.8%), and chronic myeloid leukemia (10.1%). Most patients (94.3%) identified as Caucasian, while 4.6% identified as AA, and 1.1% other. The majority of patients lived in a rural area at the time of transplant with 63.4% rural, 22.9% suburban, and 13.8% urban. There was no significant difference in OS or PFS between Caucasian and AA patients (Figure 1A and B; p=0.15, 0.21). Median OS for AA was 1.9 yrs [95% confidence interval (CI): 0.8-3.6] as compared to 2.3 yrs (95% CI: 1.9-2.9) for Caucasians, with 5 -yr OS of 33 vs. 42% and 10-yr OS of 21 vs. 36% for AA and Caucasian, respectively. Median PFS was 0.9 (95% CI: 0.5-2.7) and 1.3 yrs (95% CI 1.1-1.6), with 5 -yr PFS of 30 vs. 37% and 10-yr PFS of 21 vs. 32% for AA and Caucasian, respectively. There also was no significant difference in OS or PFS between rural, urban, and suburban patients (Figure 2A and 2B; p=0.39, 0.17), with median OS in the three groups 2.2 (95%CI: 1.7-2.9), 2.9 (95%CI: 1.6-4.5), and 2.2 (95% CI: 1.6-3.6) yrs, and 5-yr OS of 40 vs. 43 vs. 43% and 10-yr OS of 33 vs. 39 vs. 39%, respectively. Median PFS were 2.2 (95%CI: 1.7-2.9), 2.9 (95%CI: 1.6-4.5), and 2.2 yrs [95% CI: 1.6-3.6], with 5-yr PFS of 36 vs. 40 vs. 38% and 10-yr PFS of 30 vs. 37 vs. 35%, respectively. Conclusion: Our study suggests that once patients undergo allo-HCT, there is no significant difference in outcomes between patients based on race or residence. This finding suggests that while these underserved populations may initially have less access to specialized care for HCT, if they ultimately undergo allo-HCT, outcomes are similar to their counterparts. Our study did show a significantly lower rates of allo-HCT performed in non-Caucasian races (94% Caucasians vs 4.6% AA and 1% other), which may reflect disparities in access to care in these groups as well as a lack of donors. Further research is needed to assess the barriers for these underserved patients to undergo transplant and to help ameliorate these barriers. Disclosures Chaudhry: Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees. Bumma:Amgen: Speakers Bureau; Sanofi: Speakers Bureau. Khan:Amgen: Consultancy; Janssen: Consultancy. Devarakonda:Janssen: Consultancy. Vasu:Janssen: Membership on an entity's Board of Directors or advisory committees; Omeros: Membership on an entity's Board of Directors or advisory committees; Kiadis Inc: Other: Kiadis has obtained exclusive licensing requirements from The OHio State University. Jaglowski:Kite, a Gilead Company: Consultancy, Research Funding; Juno: Consultancy; Novartis: Consultancy, Research Funding; CRISPR: Consultancy. William:Merck: Research Funding; Celgene: Consultancy, Honoraria; Dova: Research Funding; Seattle Genetics: Research Funding; Incyte: Research Funding; Guidepoint Global: Consultancy; Kyowa Kirin: Consultancy, Honoraria. Mims:Syndax Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Kura Oncology: Membership on an entity's Board of Directors or advisory committees; Leukemia and Lymphoma Society: Other: Senior Medical Director for Beat AML Study; Agios: Consultancy; Novartis: Speakers Bureau; Jazz Pharmaceuticals: Other: Data Safety Monitoring Board. Brammer:Seattle Genetics, Inc.: Speakers Bureau; Celgene Corporation: Research Funding. Efebera:Celgene: Research Funding; Pharmacyclics: Research Funding; Takeda: Honoraria, Speakers Bureau; Ohio State University: Current Employment.

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JMML Fetal Identity Results Either from Retention of a Physiologic Signature or Aberrant Activation of Master Oncofetal Regulators

Blood ◽

10.1182/blood-2020-136367 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 4-5

Author(s):

Marion Strullu ◽

Aurélie Caye-Eude ◽

Loïc Maillard ◽

Chloé Arfeuille ◽

Elodie Lainey ◽

...

Keyword(s):

Gene Expression ◽

Stem Cell ◽

Board Of Directors ◽

Hematopoietic Stem Cell ◽

Hierarchical Clustering ◽

Research Funding ◽

Gene Set Enrichment Analysis ◽

Hematopoietic Progenitors ◽

Advisory Committees ◽

Hematopoietic Stem

Objectives: Juvenile myelomonocytic leukemia (JMML) is a rare but aggressive myeloproliferative/myelodysplastic neoplasm affecting infants and young children. The narrow age-window of onset suggests that a prenatal environment is needed for JMML oncogenesis. In search of a transcriptional reminiscence of embryo-fetal characteristics that would confirm this hypothesis, we investigated how the gene expression profile of JMML hematopoietic progenitors compared to their healthy counterpart isolated at different stages of ontogeny. Methods: Hematopoietic stem cell and progenitor cell (HSPC) fractions of JMML (n=16), bone marrow (BM) of healthy children (n=7), fetal liver (FL; n=3) and fetal BM (FBM; n=2) were phenotyped and sorted using signatures validated in the fetal and adult BM (Notta et al, Science 2011). RNAseq was performed using the TruSeq® Stranded Total RNASample preparation kit. Unsupervised hierarchical clustering analysis was done with the Bioconductor edgeR package. Differentially expressed genes were identified with the Bioconductor limma package. Results: To eliminate the impact of variations in the HSPC distribution, the JMML transcriptome was assessed on FACS-sorted common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP) and megakaryocyte-erythroid progenitor (MEP) cell fractions from 16 JMML and compared to healthy counterparts at different stages of ontogeny (FL, FBM, age-matched children BM). Unsupervised hierarchical clustering separated the samples into 4 groups (C1-4), primarily according to ontogeny, with 14/15 embryo-fetal fractions in C1 and all healthy post-natal progenitors in C2 (CMP, MEP) or C3 (GMP). Most JMML fractions clustered either with the prenatal fractions (C1; 17/47 fractions from 8/16 patients) or in a separate group containing no healthy sample (C4; 23/47 samples from 10/16 patients). Two groups were defined accordingly: one with JMML resembling embryo-fetal samples ('Fetal-JMML'; n=6/16), and a JMML-specific group ('Onco-JMML'; 8/16). Patients with Onco-JMML tended to be older, with a more severe presentation and elevated fetal hemoglobin levels. All PTPN11-mutated JMML were in this group whereas 5/6 Fetal-JMML had NRAS or KRAS mutations. Analysis of differential gene expression between Fetal and Onco-JMML highlighted 344 up-regulated genes versus 19 up-regulated genes in Onco-JMML. Surprisingly, LIN28B and WT1, both known to activate fetal pathways, were the most up-regulated genes in Onco-JMML. These key transcription factors were deregulated as early as the hematopoietic stem cell (HSC) compartment. Gene Set Enrichment Analysis (GSEA) confirmed enrichment in LIN28B and WT1-related signatures and showed enrichment in an AML signature in Onco-JMML. On the other hand, Fetal-JMML showed striking overexpression of monocytic /dendritic cell markers and inflammasome and innate immunity components. GSEA confirmed the strong monocyte identity of Fetal-JMML progenitors compared to onco-JMML or healthy postnatal progenitors. Part of the monocytic markers 'aberrantly' expressed in JMML progenitors was expressed in healthy fetal progenitors. Analysis of the HSC and multipotent progenitor (MPP) fractions showed that up regulation of monocytic markers was limited to the JMML progeny compartments. As we were able to confirm the transcriptional and functional identity of the sorted progenitors, these data suggest an early monocytic priming in these JMML progenitors, reminiscent of the monocyte-biased myelopoiesis characterizing physiologic fetal hematopoiesis. Conclusion: Our findings give a striking example of how ontogeny-related features are involved in childhood oncogenesis. They highlight a strong but complex link beween JMML and development, with a fetal identity resulting either from retention of a physiologic fetal monocytic signature or from aberrant (re)activation of master oncofetal regulators. Intriguingly, although LIN28B is thought to reprogram hematopoietic progenitors into a fetal-like state, its activation does not lead to an overall fetal profile in JMML, suggesting a regulatory mechanism distinct from that of physiological development. These two ontogeny-based signatures are likely to uncover the biology underlying previous classifiers based on AML-like profile or DNA methylation and suggest that a subset of JMML patient may benefit from immunomodulating therapies. Disclosures Dalle: Bellicum: Consultancy, Honoraria; bluebird bio: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi-Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Medac: Consultancy, Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; AbbVie Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Orchard: Consultancy, Honoraria; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees. Baruchel:Jazz Pharmaceuticals: Consultancy, Honoraria; Celgene Corporation: Consultancy, Honoraria; Astra Zeneca: Consultancy; Servier: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Shire: Research Funding; Bellicum: Consultancy.

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A New Gene Expression Profile Signature CRLF2 Overexpression Based Identifies Novel Adult "Triple Negative" Acute Lymphoblastic Leukemia Subgroups

Blood ◽

10.1182/blood-2018-99-118062 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5284-5284

Author(s):

Anna Ferrari ◽

Silvia Vitali ◽

Valentina Robustelli ◽

Andrea Ghelli Luserna Di Rora ◽

Simona Righi ◽

...

Keyword(s):

Gene Expression ◽

Board Of Directors ◽

Copy Number ◽

Research Funding ◽

Triple Negative ◽

Lymphoblastic Leukemia ◽

Gene List ◽

Advisory Committees ◽

The Impact ◽

Stat Pathway

Abstract Background: The heterogeneous and poor survival group of Philadelphia negative (Ph-) B-ALL patients (pts) that doesn't have the most recurrent adult rearrangements (BCR-ABL1 t(9;22); TCF3-PBX1 t(1;19); MLL-AF4 t(4;11)) are collectively referred to as "triple negative" (Ph-/-/-) ALL. CRLF2 is frequently altered in adult B-ALL, especially in Ph-like pts (50-75% of cases). Alterations that lead, in the majority of cases, to a CRLF2 overexpression. Adult pts with CRLF2 upregulated have poor outcome and novel strategies are needed to improve it. Aims: Clustering and biological characterization of Ph-/-/- ALL (that represents 61% of adult B-ALL; Roberts KG, J Clin Oncol. 2016), considering CRLF2 overexpression event, in order to define and assess biomarkers in this subgroup to test new drugs. Patients and Methods: Gene Expression Profiling (GEP; HTA 2.0 Affymetrix) were performed on 55 Ph-/-/- ALL, 29 B-ALL Ph+ at different time point of the disease and on 7 mononuclear cell of healthy donors. Data were normalized with the Expression Console Software. Successively we cluster triple negative GEP data with our validated pipeline, based on CRLF2 upregulation and in the top ten-gene list. Ph-/-/- ALL samples were then characterized for the presence of gene fusions, Copy Number Alterations (CNAs) and mutations using different approaches (TruSight Pancancer-Illumina; MLPA and/or dMLPA-MRC-Holland; SNP Array-Affymetrix; 454 Junior-Roche and PCR). Results: Clustering our Ph-/-/- gene expression data using the impact of the 10 single genes in our cohort, we could identify a defined 2-clusters-subdivision (Gr1 and Gr2; Fig 1A). The Gr2 is characterized by CTGF, CRLF2 and CD200 (Gr2=3C-up; Fig 1B) overexpression and it represents 14.1% of all B-ALL. The Gr2 GEP is similar to Ph+ one. Fusion copy number alteration and mutational screening done, detected that 3C-Up group has a higher frequency of Ph-like associated lesions (primarily CRLF2, JAK2, IL7R mutations or deletion), that mainly affect JAK-STAT pathway. Also IKZF1 and EBF1 deletions are significantly associated to Gr2 (p=0.003; p=0.016). RAS pathway genes are highly affected in Gr1. Molecular characterization shed light on a very heterogeneous scenario especially in the group 1, suggesting the need of a more discerning clustering for this group. In spite of the small number of cases is required, preliminary Gr1 subclustering discerns MLLr and ZNF384 gene expression subgroups. Notably p53 pathway is enriched in both groups but with different deregulated genes: CHEK2 is upregulated in the group1 and CDK6 in the Gr2. CRLF2 and CD200 immunoblotting and CD200 immunohistochemistry preliminary analyses suggest that protein expression of CRFL2 and CD200 are higher in Gr2 in comparison to Gr1. Conclusions: we identified a new signature, related to CRLF2 high expression, to classify Ph-/-/- ALL B-based on 10 genes. 3C-up represents 14.1% of all B-ALL and it is characterized by a) high co-expression of three main genes: CRLF2, CTGF and CD200; b) IKZF1 deletion; c) JAK-STAT pathway mutations/fusions/deletions. Gr1 represents 46.9% of all B-ALL. Gr2 GEP similarity to Ph+ one, suggests that this Gr2 could contain Ph-like pts. This new Ph-/-/- subclassification identify new potential therapeutic targets with available drug (α-CTGF, α-CD200, CDK2, CHK2 and CDK6 inhibitors; tyrosine kinase inhibitors already effective on Ph+ and Ph-like) to test. Supported by: ELN, AIL, AIRC, project Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project, HARMONY project, Fondazione del Monte BO e RA project. Figure. Figure. Disclosures Cavo: Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Martinelli:Novartis: Speakers Bureau; Abbvie: Consultancy; Jazz Pharmaceuticals: Consultancy; Janssen: Consultancy; Pfizer: Consultancy, Speakers Bureau; Roche: Consultancy; Celgene: Consultancy, Speakers Bureau; Ariad/Incyte: Consultancy; Amgen: Consultancy.

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