Paired Bone Marrow Research Articles

Molecular Features and Clinical Outcomes of Extramedullary Plasmacytomas

The treatment of multiple myeloma (MM) continues to evolve with new drugs, resulting in dramatically improved outcomes. Despite these advances, MM remains an incurable disease, with Extraosseous/Extramedullary disease (EMD) playing a major role in the development of relapsed refractory disease. EMD is defined by the development of plasma cell neoplasms that arise in tissues other than bones. EMD can present at diagnosis or develop during the disease course of MM. Sometimes EMD can be solitary without bone marrow (BM) involvement. Determining the molecular underpinnings of EMD are critical to advance the care of such patients. A few reports suggest a possible role of Ras mutations in the intramedullary to extramedullary transition in a limited number of patients. Therefore, the molecular biology of developing EMD has not yet been clearly defined.We sought to identify molecular features of EMD and associated these with clinical outcomes.We analyzed samples from 443 MM patients who presented to Massachusetts General Hospital between 2013 and 2021. All patients voluntarily signed informed consent approved by the institutional review board for SNaPshot (molecular) testing. BM aspirate, biopsy and EMD tumor specimens underwent pathological analysis as well as FISH testing. The diagnosis of EMD was determined by either CT, PET-CT or MRI done as part of their clinical course. In some cases, EMD was confirmed by biopsy. Although there were some cases of EMD adjacent to bones, we strictly defined EMD as non-adjacent to bones based on imaging.Nucleic acids were extracted from BM and EMD samples obtained from MM patients. Multiplexed mutational analysis was done with primers designed to cover 111 genes including genes known to be oncogenic. SNaPshot was done and the threshold for allele frequencies was determined as 8% based on our laboratory cut-offs.The median follow-up of the entire MM population was 63.7 months (range 1 to 408). Overall, 96 of 443 patients (21.6%) developed EMD as previously defined. Sixty-five out of 96 patients had biopsy confirmed EMD, while 31 patients were diagnosed by imaging only. SNaPshot molecular testing was performed on 30 EMD samples from the 65 patients who had biopsies. Interestingly, all EMD samples except for 1 had either NRAS, KRAS, or BRAF mutations. There were 14 EMD samples with NRAS mutations, 6 with KRAS mutations, 4 with BRAF mutations, and 1 with both KRAS and BRAF mutations. All BRAF mutations coexisted with other mutations, such as TP53, ATM, and ARID1A whereas some of the NRAS and KRAS mutations were observed alone.Next SNaPshot was analyzed for 8 paired BM and EMD samples. Three BM samples were negative for NRAS mutations while all EMD samples were positive.Patients with KRAS/NRAS/BRAF mutations who developed EMD had poorer prognosis than non-EMD patients with KRAS/NRAS/BRAF mutations. In patients with KRAS mutations, the median overall survival (OS) was 36.9 months (EMD) vs not reached (non-EMD) (p<0.01); and BRAF mutations, the median OS was 58.5 months (EMD) vs 112.4 months(non-EMD) (p=0.194). In patients with NRAS mutations, the median OS was 94.5 months(EMD) vs 124.5 months (non-EMD) (p=0.11). Average time from diagnosis of MM to developing EMD in 68 patients with longitudinal follow up was 41.6 months and average time from developing EMD to death in 54 patients with longitudinal follow-up was 7.6 months. There were significant differences in BM FISH analysis in KRAS/NRAS/BRAF mutation harboring EMD vs non-EMD; Chromosome 1 abnormalities (79.2% vs 34.2%), TP53 deletion (41.7% vs 22.4%) and hyperdiploidy (37.5% vs 67.1%).Our data suggest that KRAS/NRAS/BRAF mutations may play an important role in the development of EMD. These and other associated molecular abnormalities portend a poorer prognosis and may provide novel therapeutic insights. DisclosuresNardi: Loxo Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees. Yee: Amgen: Consultancy; Bristol Myer Squibb: Consultancy; Adaptive: Consultancy; GSK: Consultancy; Janssen: Consultancy; Oncopeptides: Consultancy; Sanofi: Consultancy; Takeda: Consultancy; Karyopharm: Consultancy. Branagan: Adaptive Biotechnologies: Consultancy; BeiGene: Consultancy; CSL Behring: Consultancy; Karyopharm: Consultancy; Pharmacyclics: Consultancy; Sanofi Genzyme: Consultancy. O'Donnell: Onocopeptide: Consultancy; Karyopharm: Consultancy; Janssen: Consultancy; Bristol Myer Squibb: Consultancy; Adaptive: Consultancy; Takeda: Consultancy. Raje: Celgene, Amgen, Bluebird Bio, Janssen, Caribou, and BMS: Other.

Circulating Tumor Cells (CTCs) in Smoldering and Active Multiple Myeloma (MM): Mechanism of Egression, Clinical Significance and Therapeutic Endpoints

Circulating Tumor Cells (CTCs) in Smoldering and Active Multiple Myeloma (MM): Mechanism of Egression, Clinical Significance and Therapeutic Endpoints

Survival Genie: A Web Portal for Single-Cell Data, Gene-Ratio, and Cell Composition-Based Survival Analyses

The genomics data-driven identification of gene signatures and pathways has been routinely explored for predicting cancer survival and making decisions related to targeted treatments. Many packages and tools have been developed to correlate single-gene features to clinical outcomes, but lack in performing such analysis based on multiple-genes, gene sets, and genes ratio. Furthermore, cluster marker genes associated with cell types, states and function from cancer single-cell transcriptomics studies remain an underutilized prognostic option. Additionally, no bioinformatics online tool evaluates associations between the enrichment of known cell types and survival outcome across cancers.We have developed Survival Genie (https://bbisr.shinyapps.winship.emory.edu/SurvivalGenie/, a web tool to perform survival analysis on single-cell RNA-seq data and a variety of other molecular inputs such as gene sets, genes ratio, tumor-infiltrating immune cells proportion, gene expression profile scores, and tumor mutation burden (Fig. 1). For comprehensive survival evaluation, the Survival Genie contains 53 datasets of 27 distinct malignancies from 11 different cancer programs for both adult and pediatric cancers including different types of leukemia. Users can upload single-cell data or gene sets and select partitioning methods (i.e., mean, median, quartile, cutp) to determine the effect of their levels on patient survival outcomes. The tool provides comprehensive results including box plots of low and high-risk groups, Kaplan-Meier plots with univariate Cox proportional hazards model, and correlation of immune cell enrichment and molecular profile (Fig. 1). The Survival Genie source code is written in the R programming language and the interactive web application with the R Shiny framework.We demonstrate the application of the Survival Genie tool by exploring the prognostic utility of blast cell and immune cell markers generated from single cell RNA-seq analysis of paired pediatric AML bone marrow samples taken at the time of diagnosis and end of induction (Thomas, Perumalla et al. 2020) . We identified AML blast specific signature consisting of 7 genes (CLEC11A, PRAME, AZU1, NREP, ARMH1, C1QBP, TRH) that depicted significant association with poor survival (HR=2.3 and Log Rank P-value=.007). Further analysis of AML relapse-associated single cell clusters showed increased levels of individual markers, including CRIP1, FLNA, and RFLNB/FAM101B and their significant association with poor survival in TARGET AML dataset. Additionally, expression of combined RFLNB/FAM101B and WDFY4 genes was associated with poor overall survival (HR=1.8 Log Rank P-value=0.01) and shorter event-free survival (HR=1.9, Log Rank P-value<0.0001). This clearly shows the usefulness of Survival Genie tool in exploring the prognostic association of genes as well as gene sets.Survival Genie is a one-stop web-portal for single-cell phenotype clusters, list of genes, and cell composition-based survival analyses across multiple cancer datasets including hematological malignancies. The analytical options and harmonized collection of multiple cancer types makes Survival Genie a comprehensive resource to correlate gene sets, pathways, cellular enrichment, and single cell clusters to clinical outcome to assist in developing next generation prognostic and therapeutic biomarkers. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

First Prospective Data on Minimal Residual Disease (MRD) Outcomes after Fixed-Duration Ibrutinib Plus Venetoclax (Ibr+Ven) Versus Chlorambucil Plus Obinutuzumab (Clb+O) for First-Line Treatment of CLL in Elderly or Unfit Patients: The Glow Study

First Prospective Data on Minimal Residual Disease (MRD) Outcomes after Fixed-Duration Ibrutinib Plus Venetoclax (Ibr+Ven) Versus Chlorambucil Plus Obinutuzumab (Clb+O) for First-Line Treatment of CLL in Elderly or Unfit Patients: The Glow Study

The Spatial Heterogeneity in Newly Diagnosed Multiple Myeloma Patients - from Sub-Clonal Architecture to the Immune Microenvironment

The Spatial Heterogeneity in Newly Diagnosed Multiple Myeloma Patients - from Sub-Clonal Architecture to the Immune Microenvironment

Pathogenesis of Extramedullary Multiple Myeloma: From Resistance to Identification of Novel Therapeutic Targets

IntroductionExtramedullary disease (EMD) is a less frequent manifestation of multiple myeloma (MM), where MM plasma cells become independent of the bone marrow (BM) microenvironment and infiltrate other tissues and organs. The incidence of EMD is increasing and is associated with worse prognosis and drug resistance. The specific and efficient treatment is lacking. Therefore, a better understanding of EMD pathogenesis is desperately needed.AimsTo identify biological pathways leading to EMD development and to evaluate therapeutic targets in EMD plasma cells with further focus on EMD tumor microenvironment to reveal presence of effector immune cells that are crucial for immunotherapy.MethodsTo identify EMD specific genes, FACS/MACS sorted aberrant plasma cells were collected from: i) fresh 11 EMD relapse tumors for which we had ii) 7 corresponding cryopreserved paired BM samples from the time of MM diagnosis (NDMM), iii) 9 unpaired fresh NDMM without EMD confirmed by PET-CT and iv) 6 unpaired fresh relapsed MM (RRMM). For library preparation, we used total RNA with rRNA depletion protocol and Illumina sequencing. Residual rRNA was filtered out by SortMeRNA. Differential expression analysis was performed using Salmon for read mapping and quantification and Deseq2 package. For single-cell RNAseq we used 10x Genomics technology for sequencing and CellRanger and Seurat for data processing and analysis.ResultsTo better understand the aggressive nature of EMD, we have analyzed bulk RNA samples (7 EMD samples plus 7 corresponding cryopreserved paired BM samples from the time of MM diagnosis). Our preliminary analysis revealed a unique EMD profile (Fig 1A) with 423 up-regulated and 421 down-regulated genes in EMD samples (adjusted p-value < 0.1; absolute fold change > 1.5), with G2M checkpoint proteins being the most enriched hallmark pathways pointing to higher proliferation of EMD cells. EMD down-regulated genes mainly belong to genes of the adaptive immune response which together with lower immunoglobulin production suggest loss of mature plasma cell function.Among the top genes uniquely overexpressed in EMD (versus RRMM or NDMM) were SCD and ELOVL6 that regulate crucial steps in unsaturated fatty acids synthesis. Also their transcription factor SREBF1 was significantly up-regulated. The importance of these genes in EMD pathogenesis can be supported by the involvement of SREBP1 in stem cell differentiation and mediation of bortezomib resistance by ELOVL6 (Yi et al. 2018, Lipchick et al. 2021). Our dataset also revealed several deregulated lncRNA in EMD compared to NDMM. MALAT1 was highly expressed, however, we did not confirm results by Handa et al. 2017 showing lncRNA MALAT1 as upregulated in EMD.Furthermore, we aimed to evaluate expression of known immunotherapy MM targets being currently in use or under investigation. We compared the information about expression level in EMD vs paired NDMM, with unpaired NDMM without EMD lesion confirmed by PET/CT, and with RRMM. The analysis revealed a decrease in the expression of several antigens commonly used in anti-MM immunotherapy (e.g. CD38, SLAMF7, BCMA or PDL1) on EMD PCs (Fig 1B). Intriguingly, our data show EMD specific elevated expression of EZH2 gene being promising target in preclinical MM investigation which can prove efficient especially for the aggressive MM stage - EMD.Effective immunotherapy depends on the presence of effector immune cells. Therefore, we have evaluated immune cell types and their proportion in EMD tumors. Using flow cytometry we identified T and NK cells as the only immune cell subsets present in EMD tumors (median 0.9% and 0.5%, respectively). Single-cell RNAseq analysis of two EMD samples supported these findings.ConclusionsHere, we present up to our knowledge the worldwide largest cohort of 11 EMD samples (including 7 longitudinal pre-EMD/EMD samples) analysed using RNAseq with focus on biological pathways and dysregulation of particular genes leading to EMD development. Drop of expression of several known drug targets may suggest limited efficacy of the modern treatment in EMD as already presented by Jelinek et al., 2021. Importantly, we are also providing the initial insight into the microenvironment (including single-cell RNA analysis) of EMD tumors, where we detected presence of T cell and NK cells in very limited numbers. [Display omitted] DisclosuresHajek: Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Novartis: Consultancy, Research Funding; Pharma MAR: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Intrapatient Complex Transcriptional Responses to Conventional Chemotherapy in Relapsed Acute Myeloid Leukemia Revealed By Single Cell RNA-Seq Analysis

Resistance to anthracycline and cytarabine based conventional chemotherapy often occurs and results in extremely poor prognosis in patients with acute myeloid leukemia (AML). Although chemotherapy resistance is the most critical clinical problem, the mechanisms by which AML confers resistance to conventional chemotherapy are not yet fully understood. In this study, we investigated the key mechanisms of chemotherapy resistance through single cell RNA-sequencing analysis using paired bone marrow AML cells longitudinally collected from two AML-MRC patients at diagnosis and relapse after anthracycline-based chemotherapy. AML blasts were sorted by CD45/SSC gating and subjected to single cell RNA-seq analysis. Single cell RNA-seq was performed using 10x Genomics' Chromium System. Mean estimated number of cells per sample was 3.403 (2,731-4,200) and median detected genes per cell ranged 3,030 to 3,918 among four samples. Data collected from paired samples were combined in following analysis. Transcriptome based clustering following UMAP dimensionality reduction distinguished 5 and 9 cluster groups in each paired sample. Chemotherapy sensitive cluster groups dominant at diagnosis and chemotherapy resistant cluster groups dominant at relapse were clearly divided. In each paired sample, a few AML cells at diagnosis were allocated to chemotherapy resistant cluster groups. This suggested that transcriptionally identifiable less frequent cells resistant to chemotherapy existed at diagnosis and may expand during and/or after chemotherapy maintaining its transcriptional features. Next, to determine whether these transcriptional features are correlated with DNA mutation profiles, we labeled DNA mutation status to each cell and compared frequencies of mutation. As far as we detected, AML recurrent mutations such as DNMT3A R882C and TP53 missense mutation were not related to chemotherapy resistant cluster groups, although this method was relatively limited by the nature of RNA-seq-based mutation detection. Then we sought to determine transcriptional features of resistant clones. Gene set enrichment analysis identified some gene groups such as E2F signaling pathway, MYC signaling pathway, hedgehog signaling pathway and TNFA signaling pathway as transcriptional signatures related to emergence after chemotherapy. Analysis of known hematopoietic differentiation gene signatures showed distinct differentiation profiles in each cluster groups, whereas resistant cluster groups were not necessarily related to hematopoietic stem cell signatures. Intrapatient variations of transcriptional signatures among the resistant cluster groups were detected, which indicated that accurate detection of transcriptional features related to chemotherapy resistance may be difficult by using bulk RNA-seq method. As for other cluster groups which were not dominant both at diagnosis and relapse, these cluster groups hardly changed its frequencies between at diagnosis and relapse, which suggested less proliferative leukemia cells persisted during chemotherapy and have various transcriptional features although whether these persisting cells contribute to relapse was unclear. Since enriched transcriptional signatures in resistant cluster groups were not consistent between the two patients, further analysis using samples collected from more patients would be needed to determine common critical chemotherapy resistant transcriptional signature. In conclusion, our analysis suggested that a transcriptionally identifiable small fraction of cells showing gene signatures related to chemotherapy resistance at diagnosis may expand during chemotherapy and revealed intrapatient transcriptional complexity of response to chemotherapy, which cannot be uncovered by bulk RNA-sequencing. DisclosuresHonda: Takeda Pharmaceutical: Other: Lecture fee; Otsuka Pharmaceutical: Other: Lecture fee; Chugai Pharmaceutical: Other: Lecture fee; Ono Pharmaceutical: Other: Lecture fee; Jansen Pharmaceutical: Other: Lecture fee; Nippon Shinyaku: Other: Lecture fee. Kurokawa: MSD K.K.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; Daiichi Sankyo Company.: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Teijin Limited: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; AbbVie GK: Research Funding, Speakers Bureau.

The Spliceosome As a New Therapeutic Target in Cytarabine-Resistant Acute Myeloid Leukemia

Introduction. Despite the recent approval of several drugs for the treatment of AML, the 3 + 7 regimens remain as the standard of care for many patients. Its lack of efficacy represents the main cause of death, since only 10% of patients who show refractoriness/relapse overcome the disease. Therefore, there is still an urgent need for seeking more effective treatments. Aberrant RNA splicing has been described in AML, but its relevance as mechanism of resistance is unclear. In this study, we deepen the mechanism of resistance to cytarabine and the role of splicing factors SR proteins, involved in the spliceosome functionality, to seek more effective therapies for AML.Methods. First, the expression levels of genes encoding SR proteins were analyzed with the GEPIA2 platform, comparing the data from the TCGA-LAML (AML patients) and GTEx (healthy) projects. Then, the gene expression of one of the most overexpressed genes, SRRM2, was validated by qPCR in samples of AML patients compared to controls and other myeloid disorders, as MDS and MPN (n=54). The resistance-associated phospho-proteomic profile was analyzed by LC-MS / MS after IMAC enrichment in paired samples from 3 AML patients. The expression of SR proteins and their phosphorylated forms was studied by immunohistochemistry (IHC) before and after resistance in paired bone marrow samples from 3 AML patients. We also analyzed by IHC the prognostic value of phospho-SR proteins at the moment of diagnosis in 64 patients with different responses to cytarabine (non-responders and responders). In order to validate an altered function of SR proteins, the analysis of the differential use of exons of paired samples from 25 AML patients was performed using RNAseq. Then, we evaluated in vitro the efficacy of some splicing modulators, and its combination with other approved drugs, in cytarabine-sensitive and resistant cells. The combination of H3B-8800, a spliceosome inhibitor, with venetoclax was tested in ex vivo samples from AML patients and healthy donors.Results. We found that the gene expression levels of SRSF9, SRSF12 and SRRM2 were altered in AML (Fig 1A-B). Immunohistochemical studies revealed that, although at the protein level no differences were found in SR proteins expression between the diagnosis and relapse moment, an increase in the levels of phosphorylated SR proteins was associated at the time of relapse (Fig 1C). Indeed, the phosphorylation levels of SRRM2, among other SR proteins, were found to be increased during cytarabine resistance by phospho-proteomics (Fig 1D). Moreover, the phosphorylation levels of SR proteins predicted the response to cytarabine treatment, as AML patients that were non-responders presented significantly higher levels compared to responders ones (Fig 1E). The observed alterations in the phosphorylation of these proteins were correlated with a differential use of exons in some of their known targets, when comparing the diagnostic condition and drug resistance moment. Based on this evidence, the efficacy of combining different therapeutic options was evaluated in vitro using sensitive or cytarabine-resistant cell models (Fig 1F). The combination of H3B-8800 together with venetoclax was the most effective in vitro and also presented synergic effects ex vivo in AML patients samples (Fig 1G). Furthermore, this combination did not show toxicity over healthy hematopoietic progenitors, since the same doses that were effective in AML did not show toxicity in a healthy context (Fig 1H).Conclusions. The results of this work shed light on the role of the RNA splicing process in cytarabine resistance in AML. Interestingly, the high levels of phosphorylated splicing factors SR proteins at diagnosis in refractory patients, would allow us to use them as a predictive biomarker of response to cytarabine treatment. Otherwise, due to the need to search effective and safe treatments in this disease, we have found that the combination of splicing inhibitors with venetoclax should be a good strategy for the treatment of AML.Acknowledgment. This work has been possible thanks to the granting of the project PI19/01518 from the Carlos III Health Institute and the CRIS Against Cancer Foundation. ML.M. enjoys a research grant from the Spanish Society of Hematology and Hemotherapy and R.GV. a FPU grant from the Ministry of Science, Innovation and Universities. [Display omitted] DisclosuresSanchez: Altum sequencing: Current Employment. Ayala: Incyte Corporation: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Celgene: Honoraria.

Treatment of Myelofibrosis Patients with the TGF-β 1/3 Inhibitor AVID200 (MPN-RC 118) Induces a Profound Effect on Platelet Production

Treatment of Myelofibrosis Patients with the TGF-β 1/3 Inhibitor AVID200 (MPN-RC 118) Induces a Profound Effect on Platelet Production

Single-Cell Characterization of the Immune and Leukemic Cells Following Anti-TIM3 and Hypomethylating Agent Combination Therapy in Patients with AML or MDS

BackgroundThe success of allogeneic stem cell transplantation supports the notion that immunotherapy can have curative potential in AML, but immune checkpoint therapies (e.g., anti-PD1) have shown only modest clinical efficacy. TIM3 is an immune-checkpoint molecule expressed both on immune and leukemic cells but not on healthy hematopoietic stem cells (HSCs), making it a particularly interesting target in AML. In myeloid malignancies, the combination of anti-TIM3 therapy with hypomethylating agents (HMA), which may prime the tumor microenvironment for immune therapies, has shown promising initial response rates up to 50%-60% of patients, but their mechanism of action is not fully understood.MethodsWe analyzed the effects of anti-TIM3 (sabatolimab, MBG453) in combination with decitabine in 11 refractory/relapsed AML patients and 1 MDS patient recruited in a phase Ib trial (NCT03066648), with 5/12 responders (3 CR, 2 CRi). We studied paired bone-marrow (BM) and peripheral blood samples with scRNA+TCRαβ-seq enriched for CD45+ immune cells (90% of input) and blast cells (10%) and flow cytometry.Additionally, to explore the expression of TIM3 and other immune checkpoints in different cell populations, we combined scRNAseq data from 160 BM aspirate samples across 10 different hematological malignancies and healthy controls.ResultsOur pan-heme scRNA-seq data analysis of over 500'000 cells revealed that unlike PD1 and CTLA4, HAVCR2 (TIM3) was primarily expressed in NK and myeloid cells (including dendritic cells [DCs], macrophages, and monocytes). In healthy controls, the expression of HAVCR2 was low in T-cells, but in patients with heme-malignancies, expression was seen on activated T-cells. In HSC populations, AML patients had generally upregulated HAVCR2 expression compared with healthy subjects.ScRNAseq data of 20 samples (n=7 patients) treated with anti-TIM3+HMA revealed that at baseline, DCs were more highly represented in samples from the responding (n=4) than from the non-responding patients (n=3). Following anti-TIM3+HMA treatment, DCs expanded significantly, and upregulated pathways related to interleukin production (IL-1b, IL-18) in responders, suggestive of an activated inflammasome response.At baseline, the most expanded NK-phenotype expressed the highest amounts of HAVCR2, which varied between patients from CD56 bright to adaptive NK cells. Anti-TIM3+HMA therapy modulated NK cells especially in responders, in which NK cells downregulated HAVCR2 and upregulated the NF-κB pathway. Importantly, the NF-κB pathway was upregulated in other cell types in responding patients, but not in non-responding patients. In contrast, the IFN-γ response was downregulated in both responding and non-responding patients in multiple different cell types.The highest expression of HAVCR2 in T cells was seen in cells co-expressing NK-receptors and with the highest cytotoxicity. Analysis of the scTCRαβ-seq revealed that the combination treatment did not have a marked effect on T-cell clonality, but one patient with CR had a significantly expanded large granular lymphocyte (LGL) clone covering 4%-25% of the repertoire. In responding patients, HAVCR2+ regulatory T-cells were more numerous at baseline, contracted following therapy, and lost response to IFN-γ, a pattern not seen in non-responding patients.The analysis of predicted cell-cell interactions between leukemic and immune cells did not show significant interactions between inhibitory PD1 or CTLA4, and their ligands, but ubiquitous LGALS9 - HAVCR2 interactions were predicted in leukemic bone marrows. Responding patients had more these interactions, which decreased following therapy. Non-responding patients had multiple interactions between T/NK cells and blasts via PVR and its ligands which were not seen in responding patients, which represent a putative resistance mechanism for anti-TIM3+HMA therapy.ConclusionsUnlike PD1 and CTLA4, TIM3 is expressed on leukemic, DC, myeloid, and NK cells, and consistent with this finding, the effects of TIM3 blockade in vivo were mainly observed in these cell types. In responding patients, NFκB pathway was activated in T/NK cells following anti-TIM3 and HMA treatment concomitant with a decrease in inhibitory interactions. Our results pinpoint the differential effects of TIM3-blockade on immune cells and may aid in developing predictive biomarkers for treatment outcome. [Display omitted] DisclosuresKreutzman: Novartis: Current Employment. Kontro: Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding. Orlando: Novartis: Current Employment. Cremasco: Novartis: Current Employment. Wagner: Novartis: Current Employment, Current holder of individual stocks in a privately-held company. Pelletier: Novartis: Current Employment. Sabatos-Peyton: Novartis: Current Employment. Rinne: Novartis: Current Employment; Qiagen: Consultancy. Mustjoki: Janpix: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; BMS: Research Funding.

The expression of PHOX2B in bone marrow and peripheral blood predicts adverse clinical outcome in non-high-risk neuroblastoma

Paired-like homeobox 2B (PHOX2B) is a highly sensitive and specific biomarker for diagnosing neuroblastoma, as well as detecting minimal residual disease in neuroblastoma. The clinical significance of PHOX2B expression in bone marrow (BM) and peripheral blood (PB) samples of newly diagnosed patients with very low-, low- and intermediate-risk neuroblastoma remains unknown, to the best of our knowledge. The expression level of PHOX2B in paired BM and PB samples of patients with newly diagnosed neuroblastoma was validated using reverse transcription-quantitative polymerase chain reaction (RTqPCR). Among the 132 patients, 26 exhibited a positive PHOX2B expression BM (19.7%) and 11 in PB (8.3%) samples. PHOX2B was highly expressed in BM and PB samples from patients aged <18 months, with International Neuroblastoma Risk Group Staging System stages M and MS, 1p loss of heterozygosity, and high levels of lactate dehydrogenase, serum ferritin and neuron-specific enolase (p < 0.05). In all eligible patients, the 2-year event-free survival (EFS) and overall survival (OS) rates were 94.7 ± 2.0% and 97.7 ± 1.3%, respectively. However, the 2-year EFS rates were significantly decreased to 76.9 ± 8.3% and 63.6 ± 14.5% in patients with a positive PHOX2B expression in BM and PB samples, respectively (p < 0.05). Similarly, the 2-year OS rates were also decreased to 88.5 ± 6.3% and 81.8 ± 11.6% in patients with a positive PHOX2B expression in BM and PB samples, respectively (p < 0.05). In conclusion, a positive PHOX2B expression in BM and PB samples at diagnosis had a strong adverse prognostic effect on patients with non-high-risk neuroblastoma.

P-058: The dynamics of nucleotide variants in the progression from myeloma precursor conditions to multiple myeloma using targeted sequencing of serial bone marrow samples

<h3>Background</h3> Multiple myeloma (MM) is known to evolve from the premalignant precursor conditions Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM). The exact mechanisms driving this progression are still not completely understood. The continuum between MGUS-SMM-MM provides the opportunity to investigate its evolution. However, collecting paired/serial samples from low and intermediate risk precursors progressing to MM are significantly hindered by the (i) only incidental diagnosis of asymptomatic precursors, (ii) invasive bone marrow (BM) sampling, (iii) very low number of aberrant BM plasma cells (PCs), and (iv) rather irregular disease follow-up. <h3>Methods</h3> In this study, a unique retrospective collection of paired BM samples was available because of an effective biobanking effort. We had access to 68 archival diagnostic May-Grünwald–Giemsa (MGG)-stained BM smears from 21 progressing low to intermediate risk myeloma precursor patients, 19 MGUS and 2 SMM, with a median time to progression of 6 years. DNA was extracted from these BM smears and Next Generation Sequencing (NGS) was performed using a custom targeted capture-based sequencing panel (Illumina) including coding exons or hotspots of 81 selected myeloma-related genes with the aim to study the evolution of single nucleotide variants (SNVs) and short insertions and deletions (indels). The pooled libraries were paired-end sequenced on a MiSeq instrument (Illumina). <h3>Results</h3> Data was analyzed with Local Run Manager and annotated with VariantStudio. After filtering, only exonic nonsynonymous and loss-of-function variants with a coverage >30 and a variant allele frequency (AF) >1% were retrieved. A variant detected in the MM phase but not in the prestage of that patient was manually inspected in the data visualization tool of Integrated Genome Viewer (IGV) to assess its presence. With a mean coverage of 636x of the targeted captured region the sequencing depth was sufficiently high to detect low burden variants down to an AF of 1%. A median of 2 variants per patient (range 0 to 5 with a total of 38 variants) was detected. While in 19 patients at least one variant was detected in the MM phase, in 2 patients not a single variant was detected in any of the 81 genes. Interestingly, the majority of variants in MM could already be detected at low AFs in the BM smears sampled in the precursor phase, even many years before progression. The reason why some variants were not detected in a prestage is likely due to sensitivity issues (BM PCs ≤2.5%). The median time of detection of a variant in the precursor stage BM was 49 months (>4 years; range 10 to 105 months) prior to MM progression. <h3>Conclusion</h3> In conclusion, targeted sequencing of unsorted BM smears from myeloma precursor conditions can already provide relevant insights into the behavior of mutant clones. Paired sample analysis can reveal the genetic architecture of somatic variation in the prestage even with low proliferative PCs in the BM.

OAB-014: Newly diagnosed Multiple Myeloma patients with high levels of circulating tumor cells are distinguished by increased bone marrow plasma cell proliferation

<h3>Background</h3> Circulating tumor cells (CTCs) are present in the blood of all patients with (precursor)stages of Multiple Myeloma (MM). The levels of CTCs vary widely between patients, yet the biology behind this variability is unknown. Importantly, MM patients with a higher percentage of CTCs have an inferior prognosis independent of high-risk cytogenetics, suggesting that CTC numbers are a relevant reflection of tumor cell biology. Here, we set out to identify differences in CTCs and paired bone marrow plasma cells (BM PCs). Additionally, we compared BM PCs from patients with high and low levels of CTCs with the goal of identifying mechanistic drivers of high-risk disease. <h3>Methods</h3> We isolated PCs from viably frozen mononuclear cells of peripheral blood and bone marrow aspirates of newly diagnosed MM patients. Common high-risk mutations were present in both groups. We performed single cell RNA sequencing of paired CTCs and BM PCs from five patients with a high percentage of CTCs (0.5%-8%). In addition, we generated single cell transcriptomes of BM PCs of eight patients with a high percentage of CTCs (2-22%) and 13 patients with low percentage of CTCs (0.004%-0.08%) <h3>Results</h3> Single cell transcriptomes were generated from 44,779 CTCs and 35,697 bone marrow PCs. When paired CTCs and BM PCs were integrated, we identified 9 common clusters, no cell specific cluster for either source was detected. Moreover, only 25 genes were significantly differentially expressed between CTCs and BM PCs. The absence of unique clusters in either CTCs or BM PCs, and the transcriptional overlap between these two sources indicate that CTC levels are not driven by the emergence of a transcriptionally different migratory clone, but are likely a reflection of altered BM PC biology. To identify such alterations, we compared bone marrow PCs from patients with high and low percentages of CTCs. Single cell transcriptomes were generated from 74,830 bone marrow PCs. Integration of all patients lead to the identification of 8 distinct PC clusters, one of which was characterized by active proliferation as defined by transcription of STMN1 and MKI67. Interestingly, this proliferative cluster was larger in patients with a high percentage of CTCs. Furthermore, cell cycle analyses based on canonical G2M and S phase markers revealed that actively cycling PCs were more frequent in the BM of patients with a high percentage of CTCs (64% versus 30%, p<0.001), irrespective of the cluster in which these cells were contained. <h3>Conclusions</h3> Through single cell transcriptomic analyses we reveal that CTCs and MM cells from BM are transcriptionally similar. Importantly, we identify increased BM PC proliferation as a significant difference between patients with high and low levels of CTCs, implicating an increased entry into the cell cycle as one of the mechanisms driving CTC levels and MM disease pathobiology.

Metabolic Profiling during Acute Myeloid Leukemia Progression Using Paired Clinical Bone Marrow Serum Samples.

Cellular metabolic changes reflect the characteristics of patients with acute myeloid leukemia (AML) caused by genetic variations, which are important in establishing AML treatment. However, little is known about the metabolic profile of patients with genetic variation-induced AML. Furthermore, the metabolites differ with disease progression. Here, metabolites in the bone marrow serum of ten patients with AML and healthy individuals were analyzed using gas chromatography–mass spectrometry. Compared with that in healthy individuals, expression of most metabolites decreased in patients with AML; hydroxylamine, 2-hydroxybutyric acid, monomethylphosphate, and ethylphosphate expression was unusually increased in the patients. We further examined serial metabolite changes across the initial diagnosis, postremission, and relapse phases. Patients with relapse showed increased metabolite expression compared with those in the diagnostic phase, confirming that patients with AML had aggressively modified leukemic cells. However, a clear difference in metabolite distribution was not observed between the diagnosis and complete remission phases, suggesting that the metabolic microenvironment did not change significantly despite complete remission. Interestingly, metabolite profiles differed with genetic variations in leukemic cells. Our results, which were obtained using paired samples collected during AML progression, provide valuable insights for identifying vulnerable targets in the AML metabolome and developing new treatment strategies.

Archival bone marrow smears are useful in targeted next-generation sequencing for diagnosing myeloid neoplasms.

Gene abnormalities, including mutations and fusions, are important determinants in the molecular diagnosis of myeloid neoplasms. The use of bone marrow (BM) smears as a source of DNA and RNA for next-generation sequencing (NGS) enables molecular diagnosis to be done with small amounts of bone marrow and is especially useful for patients without stocked cells, DNA or RNA. The present study aimed to analyze the quality of DNA and RNA derived from smear samples and the utility of NGS for diagnosing myeloid neoplasms. Targeted DNA sequencing using paired BM cells and smears yielded sequencing data of adequate quality for variant calling. The detected variants were analyzed using the bioinformatics approach to detect mutations reliably and increase sensitivity. Noise deriving from variants with extremely low variant allele frequency (VAF) was detected in smear sample data and removed by filtering. Consequently, various driver gene mutations were detected across a wide range of allele frequencies in patients with myeloid neoplasms. Moreover, targeted RNA sequencing successfully detected fusion genes using smear-derived, very low-quality RNA, even in a patient with a normal karyotype. These findings demonstrated that smear samples can be used for clinical molecular diagnosis with adequate noise-reduction methods even if the DNA and RNA quality is inferior.

Exploring the Tumor Clonal Dynamics between Diffuse Large B-Cell Lymphomas and Bone Marrow Involvement By Deep Immunoglobulin Heavy Chain VDJ Sequencing

INTRODUCTION : The BM of patients with diffuse large B-cell lymphomas (DLBCL) can be involved as DLBCL (morphologically concordant), small B-cell lymphoma (SBCL) (morphologically discordant) or a monotypic B-lymphocyte population (MBCL) detected by flow cytometry (FC). The tumor clonal dynamics in the bone marrow (BM) of patients with DLBCL has not been studied and is poorly understood. We examined DLBCL and their paired BM, and comparatively analyzed their clonal evolution patterns and dynamics based on V(D)J usage and IGH somatic hypermutation profiles by deep sequencing.METHODS : DLBCL and paired BM from 39 patients with DLBCL were included. In 14 of 39 cases, there was morphologic, immunophenotypic and/or cytogenetic (CG) evidence of BM involvement: 7 with DLBCL, 3 with SBCL, 3 with MBCL by FC only, and 1 with abnormal CG only. The IGH VDJ regions were PCR amplified using framework 1 (FR1) and/or FR2 primers and the amplicons generated from diagnostic DLBCL and BM were subjected to paired-end sequencing in separate flow cells, resulting in an average of ~0.75 million reads per sample. The V(D)J region with the highest count of read-pairs was defined as the major rearrangement combination. V(D)J usage and somatic hypermutations of all identifiable clones were analyzed. Clonal and subclonal comparisons were visualized by phylogenetic trees of the matched tumor and BM samples.RESULTS : In all 14 cases in which there was prior morphologic, immunophenotypic and/or CG evidence of BM involvement by B-cell lymphoma, VDJ reads of various abundance related to the dominant clone in the original extra-medullary tumors (DoT) could be identified in the BM. They represented either tumor clones identical (DoBM) or divergent/ancestral (DiBM) to the DoT. All of these 14 cases showed tumor clones of the DiBM type in the BM. In 8 of these 14 cases, DoBM-type clones could also be detected, but DiBM-type clones dominated over the DoBM-type in 5 of these 8 cases. In 4 of the 10 cases which were involved by DLBCL or SBCL, the most abundant VDJ clones in the BM had distinctly different VDJ rearrangements compared to the DoT, implying that tumor clones unrelated to DoT represented the most abundant clones in the BM (UnrBM). Overall, DiBM- or UnrBM-type clones were the dominant clones in 90% (9/10) of cases with morphologic evidence of lymphoma involvement in BM. In 5 of 7 (71%) cases in which the BM showed involvement by DLBCL, i.e. morphologically concordant, only UnrBM- and/or DiBM-type clones were identified; in the remaining 2 cases, both DiBM and DoBM-type clones were present, with DiBM-type clone as dominant in 1 of the 2 cases. Minor clones similar to the DiBM- or UnrBM-type clones could be identified in a subset of the diagnostic extramedullary tumors.Among the negative BM cases, deep sequencing detected lymphoma clones at a relatively low abundance in 16 of 25 cases (64%). In 14 of these 16 cases, DoBM-type clones were identified; 6 of these 14 also harbored DiBM-type clones. Two cases (2/16) had DiBM-type clones only. Notably, in two cases, the negative staging BM harbored minute DiBM- or UnrBM-type clones which persisted post-chemotherapy and resembled the dominant clone of the relapsed DLBCL, suggesting that this minute clone was chemo-resistant and may have served as a potential precursor for relapse.CONCLUSIONS : Our studies indicate a complex tumor clonal dynamics in the BM of patients with DLBCL. Lymphoma involvement of the BM in patients with DLBCL, in most cases, represents expansion of tumor cells clonally divergent or unrelated from the dominant tumor clone in the primary tumor. In some instances, such tumor clones in the marrow can be traced back to the original diagnostic tumor. These findings underscore the extent of tumor heterogeneity between the primary DLBCL and the lymphoma in the BM that has not been previously recognized by routine pathologic examination. The frequent detection of low-abundance DoBM or DiBM-type clones in pathologically negative BM suggests that expansion of these clones depends on the appropriate microenvironment, which may be provided by the neoplastic lymphoid tissue but not the BM. In addition, minute clones of the DiBM- or UnrBM-type detected in the BM by deep VDJ sequencing but not by conventional diagnostic methods may be a harbinger of frank lymphoma development in BM or extramedullary relapse; thus, their detection may be of clinical significance. DisclosuresNo relevant conflicts of interest to declare.

Circulating Tumor Cells As a Surrogate Marker for Bone Marrow Minimal Residual Disease and an Adverse Prognostic Factor for Patients with Multiple Myelom

Circulating Tumor Cells As a Surrogate Marker for Bone Marrow Minimal Residual Disease and an Adverse Prognostic Factor for Patients with Multiple Myelom

Improving Depth of Response with Continued Ibrutinib Therapy in CLL Patients

Introduction: Minimal residual disease negativity (MRD-) predicts long-term survival of CLL patients after chemoimmunotherapy. Data on depth of response and its clinical significance in patients receiving ibrutinib is limited. The fact that ibrutinib is a time-indefinite therapy, as opposed to the time-limited approach with classic chemoimmunotherapy, raises the question of temporal MRD changes during continued treatment. We sequentially quantified MRD in peripheral blood (PB) and bone marrow (BM) by flow cytometry in CLL patients on ibrutinib.Methods and Patients: 86 CLL patients received single-agent ibrutinib 420mg once daily under a phase II investigator-initiated trial (NCT01500733). 3-year safety and efficacy results from this trial were previously reported [Farooqui et al, 2015 ASH Abstract 2937]. Briefly, eligible patients had either TP53 aberration or were ≥65 years old. The study included both treatment-naïve and relapsed/refractory patients. The primary endpoint was response at 6 months according to 2008 International Workshop on Chronic Lymphocytic Leukemia (iwCLL) criteria incorporating 2012 clarifications. Response assessments at 6 and 12 months and annually thereafter included PB and BM flow cytometry and BM biopsy. MRD- was defined as <10-4 CLL cells per leukocytes based on 8-color flow cytometry. Spearman's analysis was used to assess the correlation between PB and BM MRD. Linear mixed models for repeated measures were used to estimate annual reduction of disease burden. Statistical analyses were performed using R (version 3.4.0).Result: Of 86 patients enrolled, 51 (59.3%) patients had TP53 aberration and 35 (40.7%) patients were ≥65 years old. More than half of the patients had previously untreated CLL (53 [62.6%]), advanced Rai stage (58 [67.4%]), and unmutated IGHV (U- IGHV) status (57 [66.3%]). At the median follow-up of 48 months, 52 (60.5%) remained on study. CLL disease burden significantly and continuously decreased with therapy in both PB and BM. Comparison of the paired PB and BM MRD levels revealed strong correlation between the two compartments (P <0.0001). Past the first year, residual CLL burden decreased by 36% in PB and 28% in BM with each additional year of treatment. Median MRD at 3 years was 5.3x10-2 in PB and 7.3x10-2 in BM. Although depth of response significantly improved over time, MRD- (<10-4) was rare even with long-term therapy. Three patients achieved MRD- in PB (each at 2, 3 and 4 years) and three patients achieved MRD- in BM (two at 3 years and one at 4 years, respectively). Low MRD (<10-2) was achieved by 16 (25.4%) of 63 patients at 3 years. We correlated MRD levels to iwCLL response. The CR rate of 38% in the low MRD group was not significantly different from 23% in the high MRD group (P=0.33). Likewise, both MRD groups showed comparable levels of residual disease in lymph nodes, spleen, and BM. Next, we performed subgroup analyses using baseline characteristics to identify factors associated with differential levels of MRD. MRD levels were similar between subgroups stratified by TP53 aberration and prior treatment status. Notably, patients with U- IGHV had significantly lower MRD in PB than patients with mutated IGHV (M- IGHV) starting from the first year on ibrutinib (P <0.05; Figure 1). However, BM MRD levels were similar between IGHV subgroups.Conclusion: Depth of response improved with continued ibrutinib, leading to low levels of MRD. PB and BM MRD levels were highly correlated, suggesting non-invasive testing could replace BM assessments on ibrutinib. Patients with M- IGHV had significantly higher levels of PB MRD than those with U- IGHV, consistent with known differences in redistribution lymphocytosis between two subgroups. Studies using MRD as a decision point for treatment duration with ibrutinib need to take into consideration the different levels of PB MRD achieved in the two IGHV subgroups. [Display omitted] DisclosuresWiestner:Pharmacyclics: Research Funding; Acerta Pharma: Research Funding.

Genomic Analysis and Pathway Characterization of Genes with Somatic Variants in Infant Acute Lymphoblastic Leukemia

IntroductionInfant acute lymphoblastic leukemia (ALL) is an aggressive cancer with poor relapse-free and overall survival. Other than MLL (KMT2A ) rearrangement (MLL -r), infant ALL contains remarkably few genomic lesions. We previously reported results of whole genome sequencing (WGS) on 14 matched diagnosis-remission-relapse (DX-RM-RL) MLL -r infant ALL trios (cohort A) treated on Children's Oncology Group (COG) trial AALL0631 (Kostadinov, et al. Blood 2016, 128(22):1735).Here, we examine the presence of somatic variants in both MLL -r (n=12) and non- MLL -r (n=15) infant ALL cases (COG AALL0631), which, to date, have not relapsed (cohorts B and C, respectively). For MLL -r cases, MLL partners were 6 AF4 (AFF1 ), 5 ENL (MLLT1 ), and 1 unspecified. For all cases, paired RM samples served as germline controls. In addition, we manually curated genes with somatic variants into functional pathway categories to determine whether any pathways were recurrently altered amongst all 3 cohorts.MethodsWe performed WGS on 27 paired DX-RM blood or bone marrow samples using an Illumina Hiseq 4000 or 2500 to a minimum depth of 90Gb. Alignment and variant calling were performed using BWA and GATK. Variants present in the DX sample and absent in the paired RM sample were considered somatic. Filters included an allelic depth threshold of 4 reads, minor allele frequency ≤0.1%, and a variant annotation of known pathogenic, likely pathogenic, or uncertain significance per RUNES (Saunders, et al. Sci Transl Med 2012, 4(154):154). Next, somatic variant genes (SVG) in these 2 cohorts, as well as cohort A (n=25 at DX and 156 at RL), were categorized into 1 or more of 41 functional pathways using publically available protein databases (UniProt, GenAtlas, BioGPS) and published literature. We then compared the prevalence of pathways affected across all 3 cohorts, specifically examining relapsed versus non-relapsed cases, MLL -r versus non- MLL -r, DX versus RL, and MLL - AF4 versus MLL - ENL , to identify any differences.ResultsWe found 23 non-synonymous somatic variants (NSSV) across 22 genes in cohort B; 4 were small (<40 bp) insertions/deletions (indels) and 19, single nucleotide variants (SNVs), of which 17 were missense, 1 nonsense, and 1 splicing. The mean variant allele frequency (VAF) was 0.42 ± 0.12 (range (r) 0.14-0.58). The mean number of NSSV per case at DX was 1.92 ± 1.73 (r 0-6). Mutations in KRAS (n=2), PIK3R1 (n=1), and NRAS (n=1) were observed. In cohort C, 29 NSSV across 28 genes were identified; 3 were small indels and 26, SNVs: 23 missense, 1 nonsense, and 2 splicing. The mean VAF was 0.37 ± 0.13 (r 0.13-0.67). The mean number of NSSV per case at DX was 2.0 ± 1.73 (r 0-6). Mutations in KRAS (n=2) and JAK2 (n=1) were observed. Using the same analysis filters for cohort A, we found that the mean number of NSSV per case was 2.1 at DX, and 11.5 at RL. The most frequently mutated genes included NRAS and FLT3 (n=3 cases each) at DX and KRAS , NRAS , and TP53 ,at RL (n=2 cases each).Pathway classification revealed that 2 categories-signal transduction and transcriptional regulation-contained the greatest number of SVG (n≥8) in each cohort at DX. Other categories with a high number of SVG (n≥4) across cohorts at DX included: cell cycle, metabolism, and developmental regulation. Within the signal transduction pathway group, the RAS/MAPK/ERK and PI3K/AKT/mTOR signaling pathways contained the greatest number of SVG; interestingly, these were more likely to be found in MLL-AF4 cases (n=9 and 10, respectively, of 14 total) than MLL - ENL (n=0 and 3, respectively, of 10 total). SVG involved in NF-kappa-B signaling were only found in MLL -r cases (n=2 of 26) versus non- MLL -r (n=0 of 15). SVG involved in WNT signaling and ion transport were more common at DX in cases that eventually relapsed (n=3 and 4, respectively, of 14 total) than those that did not (n=0 and 1, of 12 total). Of all the SVG gained at relapse (n=136), most fell into the signal transduction and transcriptional regulation categories.ConclusionsOverall, the mean number of NSSV per case at DX for each cohort was low. Functional pathways containing the highest numbers of SVG were also largely similar across cohorts, though we have highlighted some potentially important differences in subsets of cases. Further correlation with the type of variant found, as well as the expression of SVG across infant ALL cases, is ongoing. DisclosuresNo relevant conflicts of interest to declare.

Recurrent Genetic Variants of Long Non-Coding RNAs (lncRNAs) Independently Associate with Clinical Outcome of Younger Adults with Cytogenetically Normal Acute Myeloid Leukemia

BACKGROUNDAberrant expression of lncRNAs has been shown to independently associate with clinical outcome of younger adult patients (pts) [aged <60 years (y); Papaioannou et al. Haematologica 2017;102:1391-1400] and older pts (aged ≥60 y; Garzon et al. PNAS 2014;111:18679-84) with cytogenetically normal acute myeloid leukemia (CN-AML). However, the prognostic significance of genetic variants (single nucleotide polymorphisms or somatic mutations) in lncRNAs and their association with established prognostic gene mutations have not been studied. METHODSWe performed whole transcriptome profiling (RNA-seq) in 377 younger adults with de novo CN-AML and investigated the associations of recurrent genetic variants located within expressed lncRNAs with clinical outcome and gene mutations. All pts were treated on frontline Cancer and Leukemia Group B (CALGB)/Alliance for Clinical Trials in Oncology protocols. Analysis of 11 gene mutations, reported to be prognostic in AML, was performed by targeted amplicon sequencing (ASXL1, DNMT3A, FLT3 -TKD, IDH2, IDH1, NPM1, RUNX1, TET2, WT1), Sanger sequencing (double CEBPA) and fragment analysis (FLT3 -ITD). RESULTSWe first generated a comprehensive list of the genetic variants that are located in lncRNAs in transcriptomes of younger CN-AML pts. To analyze unequivocally non-coding genetic variants and to avoid ambiguity in their genomic location, all variants that overlapped exons of protein coding genes and those that mapped to segmental duplications or repeat masked regions of the genome were excluded from further analyses. We focused on 207 variants whose frequency differed between our AML cohort and the healthy population, and on 88 variants that were novel. Of the variants tested, 8 showed significant association with at least 1 outcome endpoint [disease-free (DFS), overall (OS) or event-free (EFS) survival], and 3 of them significantly correlated with DFS, OS and EFS. There was no association between complete remission rates and lncRNA variants. The 3 variants which associated with all survival endpoints were: a thymidine (T)-to-cytosine (C) variant in lncRNA RP5-1074L1.4, an adenosine (A)-to-C variant in lncRNA HCG4, and a C-to-T variant in lncRNA HLA-V . The C variant of RP5-1074L1.4 was detected in 182 (64%) of the 284 pts who expressed the lncRNA and associated with longer DFS (P <.001; 5-y DFS, 41% v 27%), OS (P=.04; 5-y OS, 40% v 35%) and EFS (P <.001; 5-y EFS, 36% v 22%). The C variant of HCG4 was found in 41 (18%) of the 234 pts with detectable expression of the lncRNA. Pts harboring the C variant had longer DFS (P=.01; 5-y DFS, 61% v 35%), OS (P=.02; 5-y OS, 58% v 39%) and EFS (P=.04; 5-year EFS, 49% v 29%). Finally, the T variant of HLA-V, which was detected in 58 (26%) of the 227 pts who expressed the transcript, associated with longer DFS (P=.005; 5-y DFS, 53% v 30%), OS (P=.01; 5-y OS, 55% v 33%) and EFS (P=.007; 5-y EFS, 45% v 24%).With regard to associations with prognostic gene mutations, pts with the C variant of RP5-1074L1.4 were more likely to harbor FLT3 -TKD than pts with the T variant (P=.02). Pts with the T variant in HLA-V were less likely to harbor RUNX1 mutations than pts with the C variant (P=.02). The C variant of HCG4 did not associate with differences in the frequencies of prognostic mutations.In multivariable analyses, the variables considered in the models (other than the prognostic lncRNA variants) were: white blood cell and platelet counts, hemoglobin, extramedullary involvement, the presence of FLT3 -TKD, FLT3 -ITD and CEBPA, DNMT3A, NPM1, RUNX1 and WT1 mutations, and expression levels of MN1 and miR-155. The C variant of RP5-1074L1.4 was an independent marker for longer DFS (HR: 0.53, P <.001), OS (HR: 0.64, P=.007) and EFS (HR: 0.55, P <.001). The C variant of HCG4 independently associated with longer DFS (HR: 0.52, P=.03) and OS (HR: 0.61, P=.05), whereas the T variant of HLA-V independently associated with longer DFS (HR: 0.46, P=.003), OS (HR: 0.38, P <.001) and EFS (HR: 0.48, P=.001).PCR assays and targeted Sanger sequencing experiments in leukemic blasts and paired remission bone marrow samples are currently being conducted to examine whether the prognostic variants are acquired genetic events or germline variants. CONCLUSIONSWe conclude that a small subset of genetic variants that are located within lncRNAs are clinically relevant and provide independent prognostic information in younger adults with CN-AML. DisclosuresBlum:Astellas: Consultancy; Pfizer: Consultancy; Boerhinger Ingelheim: Research Funding. Uy:Boehringer Ingelheim: Consultancy; Novartis: Consultancy, Other: Travel Suppport; GlycoMimetics: Consultancy. Stone:Celgene: Consultancy; Novartis: Consultancy; Fuji Film: Consultancy; Ono: Consultancy; Agios: Consultancy; Astellas: Consultancy; Abbvie: Consultancy; Amgen: Consultancy; Jazz: Consultancy; Sumitomo: Consultancy; Pfizer: Consultancy; Arog: Consultancy. Byrd:Acerta Pharma: Research Funding; Janssen: Research Funding; The Ohio State University: Patents & Royalties: OSU-2S; Genentech: Research Funding; Pharmacyclics: Research Funding.