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As part of the Multiple Myeloma Research Foundation (MMRF) immune atlas pilot project, we compared immune cells of multiple myeloma bone marrow samples from 18 patients assessed by single-cell RNA sequencing (scRNA-seq), mass cytometry (CyTOF), and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to understand the concordance of measurements among single-cell techniques. Cell type abundances are relatively consistent across the three approaches, while variations are observed in T cells, macrophages, and monocytes. Concordance and correlation analysis of cell type marker gene expression across different modalities highlighted the importance of choosing cell type marker genes best suited to particular modalities. By integrating data from these three assays, we found International Staging System stage 3 patients exhibited decreased CD4+ T/CD8+ T cells ratio. Moreover, we observed upregulation of RAC2 and PSMB9, in natural killer cells of fast progressors compared with those of nonprogressors, as revealed by both scRNA-seq and CITE-seq RNA measurement. This detailed examination of the immune microenvironment in multiple myeloma using multiple single-cell technologies revealed markers associated with multiple myeloma rapid progression which will be further characterized by the full-scale immune atlas project. Significance: scRNA-seq, CyTOF, and CITE-seq are increasingly used for evaluating cellular heterogeneity. Understanding their concordances is of great interest. To date, this study is the most comprehensive examination of the measurement of the immune microenvironment in multiple myeloma using the three techniques. Moreover, we identified markers predicted to be significantly associated with multiple myeloma rapid progression.

Expression of cell type markers in CITE-seq and comparison of cell subset abundance between technical replicates and across different centers

supplementary table 1 shows cell type annotation markers, clinical information of patients, cross-technique comparison of cell subset abundance and expression of cell type marker genes in CITE-seq and CyTOF

As part of the Multiple Myeloma Research Foundation (MMRF) immune atlas pilot project, we compared immune cells of multiple myeloma bone marrow samples from 18 patients assessed by single-cell RNA sequencing (scRNA-seq), mass cytometry (CyTOF), and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to understand the concordance of measurements among single-cell techniques. Cell type abundances are relatively consistent across the three approaches, while variations are observed in T cells, macrophages, and monocytes. Concordance and correlation analysis of cell type marker gene expression across different modalities highlighted the importance of choosing cell type marker genes best suited to particular modalities. By integrating data from these three assays, we found International Staging System stage 3 patients exhibited decreased CD4+ T/CD8+ T cells ratio. Moreover, we observed upregulation of RAC2 and PSMB9, in natural killer cells of fast progressors compared with those of nonprogressors, as revealed by both scRNA-seq and CITE-seq RNA measurement. This detailed examination of the immune microenvironment in multiple myeloma using multiple single-cell technologies revealed markers associated with multiple myeloma rapid progression which will be further characterized by the full-scale immune atlas project.Significance:scRNA-seq, CyTOF, and CITE-seq are increasingly used for evaluating cellular heterogeneity. Understanding their concordances is of great interest. To date, this study is the most comprehensive examination of the measurement of the immune microenvironment in multiple myeloma using the three techniques. Moreover, we identified markers predicted to be significantly associated with multiple myeloma rapid progression.

A major cause of cancer recurrence following chemotherapy is cancer dormancy escape. Taxane-based chemotherapy is standard of care in breast cancer treatment aimed at killing proliferating cancer cells. Here, we demonstrate that docetaxel injures stromal cells, which release protumor cytokines, IL-6 and G-CSF, that in turn invoke dormant cancer outgrowth both in vitro and in vivo. Single-cell transcriptomics shows a reprogramming of awakened cancer cells including several survival cues such as stemness, chemoresistance, as well as an altered tumor microenvironment with augmented pro-tumor immune signaling. IL-6 plays a role in cancer cell proliferation, whereas G-CSF mediates tumor immunosuppression. Pathways and differential expression analyses confirmed MEK as the key regulatory molecule in cancer cell outgrowth and survival. Antibody targeting of protumor cytokines (IL-6, G-CSF) or inhibition of cytokine signaling via MEK/ERK pathway using selumetinib prior to docetaxel treatment prevented cancer dormancy outgrowth suggesting a novel therapeutic strategy to prevent cancer recurrence.

Increasing evidence suggests that many of the effector CD8+ T cells reactivated (‘rejuvenated”) by immunotherapy come from outside the tumor, derived from a circulating pool of “stem-like” memory or “precursor-exhausted” (TPEX) cells. These cells have been characterized in mice, but, despite their importance, circulating counterparts in humans have not yet been identified for study. We hypothesized that immunotherapy designed to enhance immunogenic antigen-presentation during chemotherapy might produce extensive reactivation of these precursor T cells. While the antigen-presentation step occurs in tissues, homing of the rejuvenated T cells to the tumor is via the circulation; thus, we hypothesized that they would be visible in blood. Informative patients were selected from two ongoing clinical trials of children with brain tumors treated with the IDO-inhibitor drug indoximod: a Phase 2 trial (NCT04049669) of indoximod plus chemotherapy; and a Phase 1 trial (NCT05106296) of indoximod, chemotherapy and the BTK-inhibitor ibrutinib, which synergistically destabilizes IDO (Immunity 54:2354-2371, 2021). Patient selection criteria included either (i) massive clonal expansion of activated CD8+ T cells on therapy (expanded clones reaching 16-25% of total CD8+ T cells); or (ii) complete radiographic tumor response (CR) on treatment; or (iii) both. Longitudinal blood samples (4-10 samples per patient) were obtained over a period of 6-24 months and analyzed by single-cell RNA-sequencing (scRNA-seq) with paired single-cell T cell receptor sequencing (scTCR-seq). TCR clonotypes of interest were identified based on robust clonal expansion on-treatment; then each clonotype was traced back through earlier samples to the pre-treatment baseline, or the earliest sample in which that clone could be detected. Clones were pooled, subjected to UMAP clustering, and differential gene-expression and trajectory analysis performed. At earliest appearance, each clonotype was enriched for a phenotype dominated by early transcription factors TCF7 and IKZF2 (Helios). These cells expressed little PDCD1 (PD-1) but showed a “hybrid” combination of genes associated with immaturity/arrest (BACH2, DUSP2, LTB, IL7R, CD160) and effector/memory (NKG7, GZMK, GZMA). Within each responding clone, this “precursor” phenotype could be observed to progressively transition into a mature cytotoxic/effector phenotype (PRF1, GZMB, GZMH, FGFBP2, KLRB1, IFNG). Trajectory analysis of this maturation sequence allowed us to analyze key gene-regulatory networks and transcription factor profiles at each stage. To our knowledge, this is the first study in humans to identify this key stem-like precursor population in circulation, allowing us to sequentially follow the molecular changes in these cells during rejuvenation. Citation Format: Theodore S. Johnson, Rafal Pacholczyk, Zuzana Berrong, Chenbin Huang, Eugene P. Kennedy, Eric Ring, Ramses F. Sadek, Sarthak Satpathy, Beena E. Thomas, Tobey J. MacDonald, Manoj Bhasin, David H. Munn. Indoximod or ibrutinib/indoximod based clinical chemo-immunotherapy drives conversion of extra-tumoral circulating stem-like precursor CD8+ T cells into clonally expanded, rejuvenated effector cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB200.

Men who have sex with men (MSM) in India are extremely marginalised and stigmatised, and therefore experience immense psychosocial stress. As current HIV prevention interventions in India do not address mental health or resilience to these stressors, we aimed to evaluate a resilience-based psychosocial intervention in the context of HIV and sexually transmitted infection (STI) prevention.We did a multicity, randomised, clinical efficacy trial in Chennai (governmental tuberculosis research institute) and Mumbai (non-governmental organisation for MSM), India. Inclusion criteria were MSM, aged 18 years or older, who were at risk of HIV acquisition or transmission, defined as having any of the following in the 4 months before screening: anal sex with four or more male partners (protected or unprotected), diagnosis of an STI, history of transactional sex activity, or condomless anal sex with a man who was of unknown HIV status or serodiscordant. Participants were required to speak English, Tamil (in Chennai), or Hindi (in Mumbai) fluently. Eligible individuals were randomly assigned (1:1) to either a resilience-based psychosocial HIV prevention intervention, consisting of group (four sessions) and individual (six sessions) counselling alongside HIV and STI voluntary counselling and testing, or a standard-of-care control comprising voluntary counselling and testing alone. The primary outcomes were number of condomless anal sex acts with male partners during the past month (at baseline and 4 months, 8 months, and 12 months after randomisation), and incident bacterial STIs (at 12 months after randomisation). Resilience-related mediators included self-esteem, self-acceptance, and depression. Recruitment is now closed. This trial is registered with ClinicalTrials.gov, NCT02556294.Between Sept 4, 2015, and June 28, 2018, we enrolled 608 participants; 305 (50%) were assigned to the psychosocial intervention condition and 303 (50%) were assigned to the control condition. 510 (84%) of 608 men completed an assessment at 4 months after randomisation, 483 (79%) at 8 months, and 515 (85%) at 12 months. 512 (99%) of 515 men had STI data from the 12-month assessment. The intervention condition had a 56% larger reduction in condomless anal sex acts (95% CI 35-71; p0·0001) from baseline to 4-month follow-up, 72% larger reduction (56-82; p0·0001) from baseline to 8-month follow-up, and 72% larger reduction (53-83; p0·0001) from baseline to 12-month follow-up, compared with the standard-of-care control condition (condition by time interaction; χA resilience-based psychosocial intervention for MSM at risk of HIV acquisition or transmission in India was efficacious in reducing condomless anal sex acts, with evidence for mediation effects in two key target resilience variables. HIV prevention programmes for MSM in India should address mental health resilience to augment reductions in the risk of sexually transmitted HIV.National Institute of Mental Health.

We have developed a personalized vaccine in which patientderived leukemia cells are fused to autologous dendritic cells, evoking a polyclonal T-cell response against shared antigens and neoantigens. We postulated that the dendritic cell/acute myeloid leukemia fusion vaccine would demonstrate synergy with checkpoint blockade by expanding tumor antigen-specific lymphocytes that would provide a critical substrate for checkpoint blockade-mediated activation. Using an immunocompetent murine leukemia model, we examined the immunological response to and therapeutic efficacy of vaccination in conjunction with checkpoint blockade with respect to leukemia engraftment, disease burden, survival and the induction of tumor-specific immunity. Mice treated with checkpoint blockade alone had rapid progression of leukemia and only a modest extension of survival. Vaccination with dendritic cell/acute myeloid leukemia fusions resulted in the expansion of tumor-specific lymphocytes and disease eradication in a subset of animals, while the combination of vaccination and checkpoint blockade induced a fully protective tumor-specific immune response in all treated animals. Vaccination followed by checkpoint blockade resulted in upregulation of genes regulating activation and proliferation in memory and effector T cells. Long-term survivors exhibited increased T-cell clonal diversity and were resistant to subsequent tumor challenge. The combination of dendritic cell/acute myeloid leukemia fusion vaccine and checkpoint blockade treatment offers unique synergy, inducing durable activation of leukemia-specific immunity, protection from lethal tumor challenge and the selective expansion of tumor-reactive clones.

India has one of the largest numbers of men who have sex with men (MSM) globally; however, geographic data on sexually transmitted infection (STI) prevalence and associations with sexual behavior are limited. Six-hundred and eight MSM in Chennai and Mumbai underwent screening for a behavioral trial and were assessed for bacterial STIs (syphilis, chlamydia, gonorrhea), HIV, and past-month self-reported condomless anal sex (CAS). Mumbai (37.8%) had a greater prevalence of any STI than Chennai (27.6%) (prevalence ratio [PR] = 1.37, 95% CI: 1.09, 1.73). This pattern also emerged for gonorrhea and chlamydia separately but not syphilis. Conversely, Mumbai MSM reported lower rates of CAS (mean = 2.2) compared to Chennai MSM (mean = 14.0) (mean difference = −11.8, 95% CI: −14.6, −9.1). The interaction of city by CAS on any STI prevalence (PR = 2.09, 95% CI: 1.45, 3.01, p

99DOTS is a cellphone-based strategy for monitoring tuberculosis medication adherence. In a sample of 597 Indian patients with tuberculosis, we compared 99DOTS’ adherence assessments against results of urine isoniazid tests collected during unannounced home visits. 99DOTS had suboptimal accuracy for measuring adherence, partly due to poor patient engagement with 99DOTS.

Background & objectives: The healthcare system across the world has been overburdened due to the COVID-19 pandemic impacting healthcare workers (HCWs) in different ways. The present study provides an insight into the psychosocial challenges faced by the HCWs related to their work, family and personal well-being and the associated stigmas. Additionally, the coping mechanisms adopted by them and their perceptions on the interventions to address these challenges were also explored. Methods: A qualitative study was conducted between September and December 2020 through in-depth telephonic interviews using an interview guide among 111 HCWs who were involved in COVID-19 management across 10 States in India. Results: HCWs report major changes in work-life environment that included excessive workload with erratic timings accentuated with the extended duration of inconvenient personal protection equipment usage, periods of quarantine and long durations of separation from family. Family-related issues were manifold; the main challenge being separated from family, the challenge of caregiving, especially for females with infants and children, and fears around infecting family. Stigma from the community and peers fuelled by the fear of infection was manifested through avoidance and rejection. Coping strategies included peer, family support and the positive experiences manifested as appreciation and recognition for their contribution during the pandemic. Interpretation & conclusions: The study demonstrates the psychological burden of HCWs engaged with COVID-19 care services. The study findings point to need-based psychosocial interventions at the organizational, societal and individual levels. This includes a conducive working environment involving periodic evaluation of the HCW problems, rotation of workforce by engaging more staff, debunking of false information, community and HCW involvement in COVID sensitization to allay fears and prevent stigma associated with COVID-19 infection/transmission and finally need-based psychological support for them and their families.

The TAM family receptor tyrosine kinases TYRO3, AXL and MERTK are potential therapeutic targets in a variety of cancers. In our previous studies, MERTK inhibition in the leukemia microenvironment significantly prolonged survival in a syngeneic B-cell acute leukemia (B-ALL) model, implicating MERTK as a promising immuno-oncology target. Strikingly, Mertk-/- mice were almost completely protected against leukemia. Here, we probed the mechanisms of anti-leukemia immunity mediated by MERTK inhibition and evaluated roles for TYRO3 and AXL in the leukemia microenvironment. Single cell RNA sequencing and flow cytometry studies revealed an increase in CD8α+ dendritic cells (DCs) with enhanced antigen-presentation capacity in naïve and leukemia-bearing Mertk-/- mice. These cells were also increased in leukemic wild type (WT) mice treated with the MERTK inhibitor MRX-2843 (currently in phase I/Ib clinical trials). Additionally, CD8+ T cells expressing high levels of the T cell exhaustion marker Tox were decreased in mice treated with MRX-2843, implicating a CD8α+ DC – T cell axis in the anti-leukemia immune response stimulated by MERTK inhibition. Indeed, combined depletion of CD8+ T cells and CD8α+ DCs completely abrogated the anti-leukemia response in Mertk-/- mice, while immunity remained partially intact in mice with selective depletion of CD8+ T cells. Similarly, protection from leukemia was abrogated in Mertk-/- scid mice, which lack functional B and T cells and have defects in DC function. These data demonstrate a critical immunosuppressive role for MERTK in DCs of the leukemia microenvironment. Similar to Mertk-/- mice, B-ALL growth was almost completely prevented in Tyro3-/- mice, while Axl-/- did not impact leukemogenesis, implicating TYRO3, but not AXL, as an additional immunotherapeutic target. However, in contrast to Mertk-/- mice, CD8α+ DCs with enhanced antigen-presentation capacity were not significantly increased in Tyro3-/- mice compared to WT, indicating differences in the underlying mechanisms by which MERTK and TYRO3 contribute to the immunosurveillance of leukemia cells. Indeed, in vivo depletion experiments confirmed differential roles for MERTK and TYRO3 in the leukemia microenvironment. In contrast to Mertk-/- mice, selective depletion of CD8+ T cells completely abrogated protection from leukemia in Tyro3-/- mice, indicating a mechanism less dependent on DCs. Together, these findings reveal novel and distinct mechanistic insights into the immunosuppressive roles for MERTK and TYRO3 in the leukemia microenvironment, demonstrate a critical role for MERTK in DC activity, and validate a similar mechanism mediated by MRX-2843. Thus, these studies provide strong rationale for development of MERTK and/or TYRO3-targeted immunotherapies for treatment of acute leukemia. Citation Format: Justus M Huelse, Swati S Bhasin, Kristen M Jacobsen, Beena E Thomas, Madison L Chimenti, Travon A Baxter, Xiaodong Wang, Stephen V Frye, Curtis J Henry, H. Shelton Earp, Manoj Bhasin, Deborah DeRyckere, Douglas K Graham. MERTK inhibition induces an anti-leukemia dendritic cell – T cell axis while TYRO3 inhibition protects by a separate mechanism [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A35.

Relapse- and continuous complete remission (CCR)-associated pediatric acute myeloid leukemia (AML) patient bone marrows collected at the time of diagnosis (Dx), end of induction (EOI) and relapse were analyzed by single cell RNA sequencing. A novel AML blasts-associated 7-genes signature (CLEC11A, PRAME, AZU1, NREP, ARMH1, C1QBP, TRH) displayed a strong correlation with blast percentages and overall survival in the TARGET AML dataset (HR = 2.3; P-value = .007). Distinct clusters of AML-blasts at Dx were observed for relapse- and CCR-associated samples with differential expression of genes associated with survival. Relapse-associated samples demonstrated enrichment of exhausted T cells and M2 macrophages as opposed to inflammatory M1 macrophages in CCR-associated samples at Dx. EOI treatment resistant blast cells overexpressed fatty acid oxidation, tumor growth and stemness genes. Also, a relapse-associated EOI samples T cells subset showed downregulation of MHC Class I and regulatory genes. Altogether, this study deeply characterizes pediatric AML relapse-/CCR-associated tumor microenvironment transcriptome landscape.

Background Nonadherence to tuberculosis medications is associated with poor outcomes. However, measuring adherence in practice is challenging. In this study, we evaluated the accuracy of multiple tuberculosis adherence measures. Methods We enrolled adult Indians with drug-susceptible tuberculosis who were monitored using 99DOTS, a cellphone-based technology. During an unannounced home visit with each participant, we assessed adherence using a pill estimate, four-day dose recall, a last missed dose question, and urine isoniazid metabolite testing. We estimated the area under the receiver operating characteristic curve (AUC) for each alternate measure in comparison to urine testing. 99DOTS data were analyzed using patient-reported doses alone and patient- and provider-reported doses, the latter reflecting how 99DOTS is implemented in practice. We assessed each measure’s operating characteristics, with particular interest in specificity—i.e., the percentage of participants detected as being nonadherent by each alternate measure, among those who were nonadherent by urine testing. Results Compared to urine testing, alternate measures had the following characteristics: 99DOTS patient-reported doses alone (AUC 0.65, specificity 70%, 95%CI:58—81%), 99DOTS patient- and provider-reported doses (AUC 0.61, specificity 33%, 95%CI: 22—45%), pill estimate (AUC 0.55, specificity 21%, 95%CI:12—32%), four-day recall (AUC 0.60, specificity 23%, 95%CI: 14—34%), and last missed dose question (AUC 0.65, specificity 52%, 95%CI:40—63%). Conclusions Alternate measures missed detecting at least 30% of people who were nonadherent by urine testing. The last missed dose question performed similarly to 99DOTS using patient-reported doses alone. Tuberculosis programs should evaluate the feasibility of integrating more accurate, objective measures, such as urine testing, into routine care.

In India, the tribal population constitutes almost 8.6% of the nation’s total population. Despite their large presence, there are only a few reports available on Mycobacterium tuberculosis (M. tb) strain prevalence in Indian tribal communities considering the mobile nature of this population and also the influence of the mainstream populations they coexist within many areas for their livelihood. This study attempts to provide critical information pertaining to the TB strain diversity, its public health implications, and distribution among the tribal population in eleven Indian states and Andaman & Nicobar (A&N) Island. The study employed a population-based molecular approach. Clinical isolates were received from 66 villages (10 states and Island) and these villages were selected by implying situation analysis. A total of 78 M. tb clinical isolates were received from 10 different states and A&N Island. Among these, 16 different strains were observed by spoligotyping technique. The major M. tb strains spoligotype belong to the Beijing, CAS1_DELHI, and EAI5 family of M. tb strains followed by EAI1_SOM, EAI6_BGD1, LAM3, LAM6, LAM9, T1, T2, U strains. Drug-susceptibility testing (DST) results showed almost 15.4% of clinical isolates found to be resistant to isoniazid (INH) or rifampicin (RMP) + INH. Predominant multidrug-resistant (MDR-TB) isolates seem to be Beijing strain. Beijing, CAS1_DELHI, EAI3_IND, and EAI5 were the principal strains infecting mixed tribal populations across India. Despite the small sample size, this study has demonstrated higher diversity among the TB strains with significant MDR-TB findings. Prevalence of Beijing MDR-TB strains in Central, Southern, Eastern India and A&N Island indicates the transmission of the TB strains.

Introduction: Multiple myeloma (MM) is a genetically complex and clinically heterogeneous disease. Disease biology and phenotype is heavily influenced by the tumor microenvironment and the interaction between the immune milieu and malignant plasma cell population. Understanding the molecular profile of tumor along with the immune ecosystem can provide insights into key pathways that are important in disease pathobiology. Therefore, in this study, we have used single-cell RNA-Seq (scRNA-Seq) to compare the detailed maps of the bone marrow microenvironment of patients with rapid progressing disease (PFS < 18 months) with those whose disease had not progressed at the time of analysis (PFS < 4 years) Methods: MM patients (n=18) with rapid and no progression were identified from the Multiple Myeloma Research Foundation (MMRF) CoMMpass study, a longitudinal genomic study of patients with newly diagnosed, active multiple myeloma (NCT01454297). To generate a robust scRNA-Seq profile with minimal false positive, we profiled multiple technical replicates/aliquots of viably frozen CD138-negative bone marrow cells from each patient at three medical centers/universities (Beth Israel Deaconess Medical Center, Boston, Washington University in St. Louis and Mount Sinai School of Medicine, NYC using droplet-based single-cell barcoding technique. After batch correction and normalization, the cellular clusters were identified using principal component analysis and Uniform Manifold Approximation and Projection (UMAP) approach (Becht et al, 2018). Differential expression, pathways and systems biology analysis between rapid and non-progressors revealed differences for specific cell clusters (Panigrahy, Gartung et al. 2019). To determine association of plasma cell overexpressed genes with survival in CoMMpass study, survival analysis was performed using Kaplan-Meier (K-M) approach. Results: In this study, comparative analysis was performed of the bone marrow microenvironment of patients with aggressive and indolent disease by generating single-cell profiles of ~102,207 cells from 48 samples of 18 patients with MM. The UMAP approach identified multiple transcriptionally diverse clusters of plasma (CD138+), immune (PTPRC+) and erythroid (GYPA1/2+) cells (Fig 1a). Interestingly, the analysis identified CD138+ plasma/tumors cells clusters in a subset of samples from patients with rapid -progression and these clusters depicted a high degree of inter-patient heterogeneity (Fig 1a). Further characterization of plasma tumor cells depicted significant activation (Z score >2 and P-value In summary, this multi-site study provides insights into potentially significant differences in the transcriptomic landscape of multiple myeloma patients with rapid and non-progression of disease. The non-progressive patients depict significant enrichment of activated T cells and myeloid lineage populations, suggesting their role toward better outcomes. These findings will be further expanded by ongoing single cell analyses of the CoMMpass tissue bank under the MMRF Immune Atlas initiative. Figure 1 Disclosures Bhasin: Canomiiks Inc: Current equity holder in private company, Other: Co-Founder. Dhodapkar:Roche/Genentech: Membership on an entity's Board of Directors or advisory committees, Other; Amgen: Membership on an entity's Board of Directors or advisory committees, Other; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Other; Janssen: Membership on an entity's Board of Directors or advisory committees, Other; Kite: Membership on an entity's Board of Directors or advisory committees, Other; Lava Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other. Kumar:Merck: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy; Genecentrix: Consultancy; Tenebio: Other, Research Funding; Celgene/BMS: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Genentech/Roche: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Oncopeptides: Consultancy, Other: Independent Review Committee; IRC member; Kite Pharma: Consultancy, Research Funding; Novartis: Research Funding; Sanofi: Research Funding; MedImmune: Research Funding; Karyopharm: Consultancy; BMS: Consultancy, Research Funding; Cellectar: Other; Carsgen: Other, Research Funding; Dr. Reddy's Laboratories: Honoraria; Janssen Oncology: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Takeda: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; AbbVie: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Amgen: Consultancy, Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments, Research Funding.

India has the highest-burden of tuberculosis (TB) patients globally and TB control has been the focus of the Government of India’s Revised National TB Control Programme (RNTCP) [1–3]. The National ...

BackgroundAt present, there are no validated quantitative scales available to measure patient-centred quality of care in health facilities providing services for tuberculosis (TB) patients in India and low-income and middle-income countries.MethodsInitial themes and items reflective of TB patient’s perceived quality of care were developed using qualitative interviews. Content adequacy of the items were ascertained through Content validity Index (CVI) and content validity ratio (CVR). Pilot testing of the questionnaire for assessing validity and reliability was undertaken among 714 patients with TB. Sampling adequacy and sphericity were tested by Kaiser-Meyer-Olkin and Bartlett’s test, respectively. Exploratory and confirmatory factor analysis was undertaken to test validity. Cronbach’s α and test–retest scores were used to test reliability.ResultsA 32-item tool measuring patient-perceived quality of TB distributed across five domains was developed initially based on a CVI and CVR cut-off score of 0.78 and cognitive interviews with patients with TB. Bartlett’s test results showed a strong significance f (χ2=3756 and p1 which accounted for 60.9% of the total variance of items. Correlation (z-value >1.96) between items and factors was highly significant and Cronbach’s α was acceptable for the global scale (0.76) for the four factors. Intraclass correlation coefficient and the test retest scores for four factors were (ConclusionWe validated a measurement tool for patient-perceived quality of care for TB (PPQCTB) which measured the patient’s satisfaction with healthcare provider and services. PPQCTB tool could enrich quality of care evaluation frameworks for TB health services in India.

Background The relationships between first-line drug concentrations and clinically important outcomes among patients with tuberculosis (TB) remain poorly understood. Methods We enrolled a prospective cohort of patients with new pulmonary TB receiving thrice-weekly treatment in India. The maximum plasma concentration of each drug was determined at months 1 and 5 using blood samples drawn 2 hours postdose. Subtherapeutic cutoffs were: rifampicin Results Among 404 participants, rifampicin, isoniazid, and pyrazinamide concentrations were subtherapeutic in 85%, 29%, and 13%, respectively, at month 1 (with similar results for rifampicin and isoniazid at month 5). Rifampicin concentrations were lower with human immunodeficiency virus coinfection (median, 1.6 vs 4.6 µg/mL; P = .015). Unfavorable outcome was observed in 19%; a 1-μg/mL decrease in rifampicin concentration was independently associated with unfavorable outcome (adjusted incidence rate ratio [aIRR], 1.21 [95% confidence interval {CI}, 1.01–1.47]) and treatment failure (aIRR, 1.16 [95% CI, 1.05–1.28]). A 1-μg/mL decrease in pyrazinamide concentration was associated with recurrence (aIRR, 1.05 [95% CI, 1.01–1.11]). Conclusions Rifampicin concentrations were subtherapeutic in most Indian patients taking a thrice-weekly TB regimen, and low rifampicin and pyrazinamide concentrations were associated with poor outcomes. Higher or more frequent dosing is needed to improve TB treatment outcomes in India.

The TAM family receptor tyrosine kinases TYRO3, AXL and MERTK are potential therapeutic targets in a variety of cancers. In previous studies, inhibition of MERTK decreased PD-1 checkpoint proteins in the leukemia microenvironment and prolonged survival in a syngeneic BCR-ABL+/Arf-/- B-cell acute leukemia model, implicating MERTK as a promising immune-oncology target in leukemia. Strikingly, Mertk-/- mice were largely protected from leukemia. In our current studies, Tyro3-/- almost completely prevented development of leukemia, comparable to Mertk-/-, while Axl-/- mice died with similar timing to wild type (WT) mice (20-40 days). These data demonstrate differential roles for TAM kinases in the anti-leukemia immune response. Depletion studies were conducted to evaluate potential roles for T cells and dendritic cells (DCs) in anti-leukemia immunity in Mertk-/- mice. Selective depletion of CD8+ T cells abrogated protection from leukemia in Mertk-/- mice, but survival was still prolonged relative to WT. Thus, while CD8+ T cells were required for complete protection from leukemia, the anti-leukemia response remained partially intact even in the absence of CD8+ T cells, implicating an innate immune mechanism. Indeed, combined depletion of CD8+ T cell and CD8α+ DC subsets completely abrogated the anti-leukemic effects in Mertk-/- mice, revealing a critical immunosuppressive role for MERTK in DCs in the leukemia microenvironment. In contrast to Mertk-/- mice, selective depletion of CD8+ T cells completely abrogated protection from leukemia in Tyro3-/- mice, indicating a mechanism less dependent on DCs. Similarly, single cell RNA sequencing revealed CD8+ DCs with a more mature and antigen-presenting phenotype in Mertk-/- mice compared to WT, while antigen-presenting DCs were not increased in Tyro3-/- mice. Single cell sequencing data also suggest induction of an anti-leukemic DC - T cell axis in WT leukemic mice treated with the MERTK-selective inhibitor MRX-2843. DCs were nearly absent in leukemic bone marrow from saline-treated mice and were dramatically increased in response to treatment with MRX-2843. Treatment with MRX-2843 also decreased the incidence of CD8+ T cells expressing high levels of Tox, which has been associated with T cell exhaustion. These changes coincided with decreased leukemic blasts, even in the context of established disease.Together, our findings support a model whereby MERTK inhibition promotes DC function and CD8+ T cell activity, leading to anti-leukemia immunity. In contrast, anti-leukemia immunity in response to TYRO3 inhibition is less dependent on DCs. Differential roles for the TAM kinases in the leukemia microenvironment provide rationale for development of MERTK and/or TYRO3 targeted immunotherapies to treat acute leukemia. Citation Format: Justus M. Huelse, Swati S. Bhasin, Beena E. Thomas, Madison L. Chimenti, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Manoj Bhasin, Deborah DeRyckere, Douglas K. Graham. MERTK inhibition induces an anti-leukemia dendritic cell - T cell axis while TYRO3 inhibition protects through a separate mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 240.

To understand the diabetic wound healing microenvironment, we profiled 174,962 single cells from foot, forearm, and PBMCs using single-cell RNA sequencing (scRNASeq) approach. Our analysis shows enrichment of a unique population of fibroblasts overexpressing MMP1, MMP3, MMP11, HIF1A, CHI3L1, and TNFAIP6 genes and M1 macrophage polarization in the DFU patients with healing wounds. Further, scRNASeq of spatially separated samples from same patient and spatial transcriptomics (ST) revealed preferential localization of these healing associated fibroblasts toward deep wound/ulcer bed as compared to wound edge or non-wounded skin. ST also validated our findings of higher enrichment of M1 macrophages in healers and M2 macrophages in non-healers. Our analysis provides deep insights into the wound healing microenvironment, identifying cell types that could be critical in promoting DFU healing, and may inform novel therapeutic approaches for DFU treatment.

BACKGROUND: Approximately 10% of incident TB cases worldwide are attributable to alcohol. However, evidence associating alcohol with unfavorable TB treatment outcomes is weak.METHODS: We prospectively evaluated men (≥18 years) with pulmonary TB in India for up to 24 months to investigate the association between alcohol use and treatment outcomes. Unhealthy alcohol use was defined as a score of ≥4 on the Alcohol Use Disorders Identification Test-Concise (AUDIT-C) scale at entry. Unfavorable TB treatment outcomes included failure, recurrence, and all-cause mortality, analyzed as composite and independent endpoints.RESULTS: Among 751 men, we identified unhealthy alcohol use in 302 (40%). Median age was 39 years (IQR 28–50); 415 (55%) were underweight (defined as a body mass index [BMI] 2); and 198 (26%) experienced an unfavorable outcome. Unhealthy alcohol use was an independent risk factor for the composite unfavorable outcome (adjusted incidence rate ratio [aIRR] 1.47, 95% CI 1.05–2.06; P = 0.03) and death (aIRR 1.90, 95% CI 1.08–3.34; P = 0.03), specifically. We found significant interaction between AUDIT-C and BMI; underweight men with unhealthy alcohol use had increased risk of unfavorable outcomes (aIRR 2.22, 95% CI 1.44–3.44; P < 0.001) compared to men with BMI ≥18.5 kg/m2 and AUDIT-C CONCLUSION: Unhealthy alcohol use was independently associated with unfavorable TB treatment outcomes, highlighting the need for integrating effective alcohol interventions into TB care.

Background Poor adherence to tuberculosis (TB) treatment is associated with disease recurrence and death. Little research has been conducted in India to understand TB medication nonadherence. Methods We enrolled adult drug-susceptible TB patients, approximately half of whom were people with human immunodeficiency virus (PWH), in Chennai, Vellore, and Mumbai. We conducted a single unannounced home visit to administer a survey assessing reasons for nonadherence and collect a urine sample that was tested for isoniazid content. We described patient-reported reasons for nonadherence and identified factors associated with nonadherence (ie, negative urine test) using multivariable logistic regression. We also assessed the association between nonadherence and treatment outcomes. Results Of 650 participants in the cohort, 77 (11.8%) had a negative urine test. Nonadherence was independently associated with daily wage labor (adjusted odds ratio [aOR], 2.7; confidence interval [CI], 1.1–6.5; P = .03), the late continuation treatment phase (aOR, 2.0; CI, 1.1–3.9; P = .03), smear-positive pulmonary disease (aOR, 2.1; CI, 1.1–3.9; P = .03), alcohol use (aOR, 2.5; CI, 1.2–5.2; P = .01), and spending ≥30 minutes collecting medication refills (aOR, 6.6; CI, 1.5–29.5; P = .01). People with HIV reported greater barriers to collecting medications than non-PWH. Among 167 patients reporting missing doses, reported reasons included traveling from home, forgetting, feeling depressed, and running out of pills. The odds of unfavorable treatment outcomes were 4.0 (CI, 2.1–7.6) times higher among patients with nonadherence (P < .0001). Conclusion Addressing structural and psychosocial barriers will be critical to improve TB treatment adherence in India. Urine isoniazid testing may help identify nonadherent patients to facilitate early intervention during treatment., We evaluated adherence to tuberculosis medications in 650 Indian patients by conducting urine isoniazid tests collected during unannounced home visits. Structural barriers to collecting medication refills and psychosocial barriers emerged as the most important factors contributing to medication nonadherence.

Background and objectives Understanding the drivers for care-seeking among those who present with symptoms of TB is crucial for early diagnosis of TB and prompt treatment, which will in turn halt further TB transmission. While TB is a challenge among the tribal population, little is known about the care-seeking behaviour and the factors influencing care-seeking behaviour among the tribal population across India. Methodology This community-based descriptive study was carried out in 17 states of India across 6 zones, covering 88 villages from tribal districts with over 70% tribal population. The sample population included individuals ≥15 years old who were screened through an interview for symptoms suggestive of pulmonary TB (PTB), currently and/or previously on anti-TB treatment. Those with symptoms were then assessed on their health-seeking behavior using a semi-structured interview schedule. Results Among 74532 eligible participants screened for symptoms suggestive of TB, 2675 (3.6%) were found to be presumptive TB cases. Of them, 659 (24.6%) sought care for their symptoms. While 48.2% sought care after a week, 19.3% sought care after one month or more, with no significant difference in the first point of care; 46.9% approaching a private and 46.7% a public facility. The significant factors influencing care-seeking behaviour were knowledge on TB (OR: 4.64 (3.70–5.83), p < 0.001), age Conclusion Delayed healthcare-seeking behaviour among those with symptoms presumptive of TB in the tribal population is a major concern. Findings point to differences across gender about symptoms that prompt care-seeking in this population. Gender-sensitive interventions with health system strengthening are urgently needed to facilitate early diagnosis and treatment among this population.

Background Patients with multidrug-resistant tuberculosis (MDR-TB) face challenges adhering to medications, given that treatment is prolonged and has a high rate of adverse effects. The Medication Event Reminder Monitor (MERM) is a digital pillbox that provides pill-taking reminders and facilitates the remote monitoring of medication adherence. Objective This study aims to assess the MERM’s acceptability to patients and health care providers (HCPs) during pilot implementation in India’s public sector MDR-TB program. Methods From October 2017 to September 2018, we conducted qualitative interviews with patients who were undergoing MDR-TB therapy and were being monitored with the MERM and HCPs in the government program in Chennai and Mumbai. Interview transcripts were independently coded by 2 researchers and analyzed to identify the emergent themes. We organized findings by using the Unified Theory of Acceptance and Use of Technology (UTAUT), which outlines 4 constructs that predict technology acceptance—performance expectancy, effort expectancy, social influence, and facilitating conditions. Results We interviewed 65 patients with MDR-TB and 10 HCPs. In patient interviews, greater acceptance of the MERM was related to perceptions that the audible and visual reminders improved medication adherence and that remote monitoring reduced the frequency of clinic visits (performance expectancy), that the device’s organization and labeling of medications made it easier to take them correctly (effort expectancy), that the device facilitated positive family involvement in the patient’s care (social influences), and that remote monitoring made patients feel more cared for by the health system (facilitating conditions). Lower patient acceptance was related to problems with the durability of the MERM’s cardboard construction and difficulties with portability and storage because of its large size (effort expectancy), concerns regarding stigma and the disclosure of patients’ MDR-TB diagnoses (social influences), and the incorrect understanding of the MERM because of suboptimal counseling (facilitating conditions). In their interviews, HCPs reported that MERM implementation resulted in fewer in-person interactions with patients and thus allowed HCPs to dedicate more time to other tasks, which improved job satisfaction. Conclusions Several features of the MERM support its acceptability among patients with MDR-TB and HCPs, and some barriers to patient use could be addressed by improving the design of the device. However, some barriers, such as disease-related stigma, are more difficult to modify and may limit use of the MERM among some patients with MDR-TB. Further research is needed to assess the accuracy of MERM for measuring adherence, its effectiveness for improving treatment outcomes, and patients’ sustained use of the device in larger scale implementation.

BACKGROUND Patients with multidrug-resistant tuberculosis (MDR-TB) face challenges adhering to medications, given that treatment is prolonged and has a high rate of adverse effects. The Medication Event Reminder Monitor (MERM) is a digital pillbox that provides pill-taking reminders and facilitates the remote monitoring of medication adherence. OBJECTIVE This study aims to assess the MERM’s acceptability to patients and health care providers (HCPs) during pilot implementation in India’s public sector MDR-TB program. METHODS From October 2017 to September 2018, we conducted qualitative interviews with patients who were undergoing MDR-TB therapy and were being monitored with the MERM and HCPs in the government program in Chennai and Mumbai. Interview transcripts were independently coded by 2 researchers and analyzed to identify the emergent themes. We organized findings by using the Unified Theory of Acceptance and Use of Technology (UTAUT), which outlines 4 constructs that predict technology acceptance—performance expectancy, effort expectancy, social influence, and facilitating conditions. RESULTS We interviewed 65 patients with MDR-TB and 10 HCPs. In patient interviews, greater acceptance of the MERM was related to perceptions that the audible and visual reminders improved medication adherence and that remote monitoring reduced the frequency of clinic visits (performance expectancy), that the device’s organization and labeling of medications made it easier to take them correctly (effort expectancy), that the device facilitated positive family involvement in the patient’s care (social influences), and that remote monitoring made patients feel more cared for by the health system (facilitating conditions). Lower patient acceptance was related to problems with the durability of the MERM’s cardboard construction and difficulties with portability and storage because of its large size (effort expectancy), concerns regarding stigma and the disclosure of patients’ MDR-TB diagnoses (social influences), and the incorrect understanding of the MERM because of suboptimal counseling (facilitating conditions). In their interviews, HCPs reported that MERM implementation resulted in fewer in-person interactions with patients and thus allowed HCPs to dedicate more time to other tasks, which improved job satisfaction. CONCLUSIONS Several features of the MERM support its acceptability among patients with MDR-TB and HCPs, and some barriers to patient use could be addressed by improving the design of the device. However, some barriers, such as disease-related stigma, are more difficult to modify and may limit use of the MERM among some patients with MDR-TB. Further research is needed to assess the accuracy of MERM for measuring adherence, its effectiveness for improving treatment outcomes, and patients’ sustained use of the device in larger scale implementation.

IntroductionDelaying care-seeking for tuberculosis (TB) symptoms is a major contributor to mortality, leading to worse outcomes and spread. To reduce delays, it is essential to identify barriers to care-seeking and target populations most at risk of delaying. Previous work identifies barriers only in people within the health system, often long after initial care-seeking.MethodsWe conducted a community-based survey of 84 625 households in Chennai, India, to identify 1667 people with TB-indicative symptoms in 2018–2019. Cases were followed prospectively to observe care-seeking behaviour. We used a comprehensive survey to identify care-seeking drivers, then performed multivariate analyses to identify care-seeking predictors. To identify profiles of individuals most at risk to delay care-seeking, we segmented the sample using unsupervised clustering. We then estimated the per cent of the TB-diagnosed population in Chennai in each segment.ResultsDelayed care-seeking characteristics include smoking, drinking, being employed, preferring different facilities than the community, believing to be at lower risk of TB and believing TB is common. Respondents who reported fever or unintended weight loss were more likely to seek care. Clustering analysis revealed seven population segments differing in care-seeking, from a retired/unemployed/disabled cluster, where 70% promptly sought care, to a cluster of employed men who problem-drink and smoke, where only 42% did so. Modelling showed 54% of TB-diagnosed people who delay care-seeking might belong to the latter segment, which is most likely to acquire TB and least likely to promptly seek care.ConclusionInterventions to increase care-seeking should move from building general awareness to addressing treatment barriers such as lack of time and low-risk perception. Care-seeking interventions should address specific beliefs through a mix of educational, risk perception-targeting and social norms-based campaigns. Employed men who problem-drink and smoke are a prime target for interventions. Reducing delays in this group could dramatically reduce TB spread.

Heterocyclic aryl hydrocarbon receptor agonists modulate lymphocyte-mediated tissue repair in a murine model of colitis., Aryl hydrocarbon receptor (AHR) is an essential regulator of gut immunity and a promising therapeutic target for inflammatory bowel disease (IBD). Current AHR agonists are inadequate for clinical translation due to low activity, inadequate pharmacokinetics, or toxicity. We synthesized a structurally diverse library and used integrated computational and experimental studies to discover mechanisms governing ligand-receptor interaction and to design potent drug leads PY109 and PY108, which display physiochemical drug-likeness properties, desirable pharmacokinetic profiles, and low toxicity. In a murine model of dextran sulfate sodium–induced colitis, orally administered compounds increase interleukin-22 (IL-22) production and accelerate mucosal healing by modulating mucosal adaptive and innate lymphoid cells. AHR and IL-22 pathway induction was confirmed using RNA sequencing and characterization of the lymphocyte protein-protein interaction network. Significant induction of IL-22 was also observed using human T cells from patients with IBD. Our findings support rationally designed AHR agonists for IBD therapy.

AimTo determine the prevalence of pulmonary tuberculosis, socio-cultural practices and health seeking behaviour of tribal people in four districts of Odisha.MethodologyThis was an action research study with qualitative and quantitative design following a sequential approach implemented in a 4-phased manner. It was carried out in the 6 selected villages from July,2015 to June,2017. The screening for active TB among chest symptomatics is followed as per the guidelines of the (RNTCP) Revised National Tuberculosis Control Program in India.ResultsIn all, 1455 households were surveyed in the 6 tribal dominated villages of 4 districts, namely Balangir, Dhenkanal, Kandhmal and Mayurbhanj. Total population of the villages was 6681. Based on the eligibility, 5144 (97.7%) individuals were screened. About 139 (2.3%) could not be screened due to non-availability in their households during day time. Out of these, 126 chest symptomatic individuals were identified. Sputum samples were collected from these and sent to the National Reference Laboratory, RMRC, Bhubaneswar using public transport and maintaining cold chain. 35 patients were found to be having active TB disease. 18 were culture positive. The prevalence of pulmonary TB is 0.68%. The risk factors seemed to be ignorance about TB symptoms, addiction to alcoholic drinks, difficulty reaching the health facilities owing to the long distances, lack of communication and transport. In addition, other morbidities like Malaria, diabetes, hypertension, malnutrition, etc. were observed in the tribes of the study sites.ConclusionObserved prevalence suggests further strengthening of TB control program. This study is the first of its kind in this State.

99DOTS is a cellphone-based strategy for monitoring tuberculosis medication adherence. In a cohort of 650 Indian tuberculosis patients, we compared 99DOTS’ adherence record against results of urine isoniazid tests collected during unannounced home visits. 99DOTS had suboptimal accuracy for measuring adherence, partly due to poor patient engagement with 99DOTS.

BACKGROUND 99DOTS is a cell phone–based strategy for monitoring tuberculosis (TB) medication adherence that has been rolled out to more than 150,000 patients in India’s public health sector. A considerable proportion of patients stop using 99DOTS during therapy. OBJECTIVE This study aims to understand reasons for variability in the acceptance and use of 99DOTS by TB patients and health care providers (HCPs). METHODS We conducted qualitative interviews with individuals taking TB therapy in the government program in Chennai and Vellore (HIV-coinfected patients) and Mumbai (HIV-uninfected patients) across intensive and continuation treatment phases. We conducted interviews with HCPs who provide TB care, all of whom were involved in implementing 99DOTS. Interviews were transcribed, coded using a deductive approach, and analyzed with Dedoose 8.0.35 software (SocioCultural Research Consultants, LLC). The findings of the study were interpreted using the unified theory of acceptance and use of technology, which highlights 4 constructs associated with technology acceptance: performance expectancy, effort expectancy, social influences, and facilitating conditions. RESULTS We conducted 62 interviews with patients with TB, of whom 30 (48%) were HIV coinfected, and 31 interviews with HCPs. Acceptance of 99DOTS by patients was variable. Greater patient acceptance was related to perceptions of improved patient-HCP relationships from increased phone communication, TB pill-taking habit formation due to SMS text messaging reminders, and reduced need to visit health facilities (performance expectancy); improved family involvement in TB care (social influences); and from 99DOTS leading HCPs to engage positively in patients’ care through increased outreach (facilitating conditions). Lower patient acceptance was related to perceptions of reduced face-to-face contact with HCPs (performance expectancy); problems with cell phone access, literacy, cellular signal, or technology fatigue (effort expectancy); high TB- and HIV-related stigma within the family (social influences); and poor counseling in 99DOTS by HCPs or perceptions that HCPs were not acting upon adherence data (facilitating conditions). Acceptance of 99DOTS by HCPs was generally high and related to perceptions that the 99DOTS adherence dashboard and patient-related SMS text messaging alerts improve quality of care, the efficiency of care, and the patient-HCP relationship (performance expectancy); that the dashboard is easy to use (effort expectancy); and that 99DOTS leads to better coordination among HCPs (social influences). However, HCPs described suboptimal facilitating conditions, including inadequate training of HCPs in 99DOTS, unequal changes in workload, and shortages of 99DOTS medication envelopes. CONCLUSIONS In India’s government TB program, 99DOTS had high acceptance by HCPs but variable acceptance by patients. Although some factors contributing to suboptimal patient acceptance are modifiable, other factors such as TB- and HIV-related stigma and poor cell phone accessibility, cellular signal, and literacy are more difficult to address. Screening for these barriers may facilitate targeting of 99DOTS to patients more likely to use this technology.

Introduction Despite recent improvement in outcomes for de novo disease, pediatric T-cell acute lymphoblastic leukemia (T-ALL) remains challenging to treat at relapse. Investigation into genomic markers of treatment response and therapy resistance offers an opportunity to further enhance outcomes for these patients. We previously identified a T-ALL blast-associated gene signature at diagnosis (Dx) and characterized the immune microenvironment in Dx T-ALL marrow samples using single cell transcriptome analysis (Bhasin et al. Blood 2020(ASH)). This approach allowed us to generate a granular expression map of both the T-ALL landscape and the Dx bone marrow (BM) immune microenvironment. Here we expand this work by evaluating samples collected from the same patients Dx and End of Induction (EOI) BM samples from pediatric T-ALL patients. The use of paired samples provides insight into treatment-induced changes in the microenvironment. Further, the inclusion of both minimal residual disease (MRD) positive and MRD negative samples allowed us to compare differences between these groups. Methods Using the 10X genomics platform, we profiled the single cell transcriptome of ~18,000 BM and immune microenvironment cells from viably frozen samples collected from T-ALL patients at Dx or EOI. Five paired Dx and EOI samples and one EOI sample from a patient with relapsed T-ALL were evaluated, for a total of 11 samples. Three paired samples were MRD positive at EOI and two were MRD negative; the relapsed sample was MRD negative. Cell clustering was performed using the Seurat package and differential expression analysis was performed using R/Bioconductor packages (Hao et al. Cell 2021). Cell communication analysis was conducted using the CellChat R tool (v 1.0.0) to infer cell-cell communication within the EOI MRD positive and MRD negative subsets and compare their communication networks (Jin et al. Nature Comm 2021). Results Using our previously described blast-associated gene signature (Bhasin et al. ASH 2020) we were able to identify residual blast populations at EOI in MRD-positive samples. Comparative analysis of gene profiles at Dx and EOI showed significant changes in the microenvironment cell populations with highest increase in erythroid cell populations after induction therapy. The gene expression profiles were significantly different for immune cells at Dx and EOI and the relapsed sample had greater similarity to the Dx samples indicating a persistent immunosuppressive environment. Clustering analysis of the EOI samples (3 MRD positive and 2 MRD negative) demonstrated the presence of patient specific blast cells in MRD positive samples that retained patient-specific transcriptomeheterogeneity at EOI (Fig.1A). Analysis of communication networks between different cell types based on receptor and ligand expression levels between different cell types identified a CD34 + cluster of stem cells that had different interactions with other immune populations in the MRD positive and negative subsets. Differential expression analysis between the MRD positive and MRD negative cells in this CD34 + stem cell cluster identified higher expression of myeloid associated genes such as CEBPB, CEBPD, AZU1 in the MRD negative group relative to the MRD positive cells, which showed higher expression of B-cell related genes such as IGHM, VPREB1, CD79A/ B along with upregulation of P13K signaling in B-lymphocytes, B-cell receptor signaling and autophagy pathways. Analysis of upstream regulators based on the differential gene signature between the MRD positive and MRD negative group demonstrated upregulation of MYC and TCF3 activity and inhibition of TGFB1, CSF3 and CEBPA in MRD positive compared to MRD negative samples (Fig.1B). Conclusions: Leukemic blasts exhibit patient-specific gene expression signatures that are present at EOI in MRD positive samples. Exploration of the impact of minimal residual disease at EOI revealed differential gene expression patterns in stem cells from MRD positive samples, characterized by activation of B cell related signaling pathways and regulators such as MYC and TCF3. In contrast, a more myeloid-like expression signature was observed in stem cells from MRD negative samples. These findings open the avenues for exploration of therapeutic targets of T-ALL progression. Figure 1 Figure 1. Disclosures DeRyckere: Meryx: Other: Equity ownership. Graham: Meryx: Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership.

Introduction: Multiple myeloma (MM) is a complex hematological malignancy with the heterogenous immune bone marrow (BM) environment contributing to tumor growth, drug resistance, and immune escape. T-Cells play a critical role in the clearance of malignant plasma cells from the tumor environment. However, T-Cells in multiple myeloma demonstrate impaired cytotoxicity, proliferation, and cytokine production due to the activation of immune inhibitory receptors from ligands produced by the myeloma cells. In this study, we investigate the behavior of T-Cells in MM patients by using single-cell RNA-Seq (scRNA-Seq) to compare the transcriptomic profiles of BM T-Cells of patients with rapid progressing (FP; PFS < 18mo) and non-progressing (NP; PFS > 4yrs) disease. Methods: Newly diagnosed MM patients (n=18) from the Multiple Myeloma Research Foundation (MMRF) CoMMpass study (NCT01454297) were identified as either rapid progressors or non-progressors based on their progression free survival since diagnosis. To capture transcriptomic data, scRNA-Seq was performed on 48 aliquots of frozen CD138-negative BM cells at three medical centers/universities (Beth Israel Deaconess Medical Center, Boston, Washington University in St. Louis, and Mount Sinai School of Medicine, NYC). Samples were collected at diagnosis prior to treatment. Surface marker expression for 29 proteins was captured for at least one sample per patient using CITE-Seq. After integration and batch correction, clustering was performed to identify cells of T or NK lineage. Uniform Manifold Approximation and Projection (UMAP) and differential expression were used to identify T-Lymphoid subtypes, and differences in NP and FP samples. Results: In this study, single cell transcriptomic profiles were identified for ~102,207 cells from 48 samples of 18 MM patients. 40,328 T (CD3+) and NK (CD3-, NKG7+) cells were isolated, and subclustered for further analysis (Fig 1A). Using differentially expressed markers for each cluster, the T-Lymphoid subset was refined into seven subtypes, consisting of various CD4+ T-Cells, CD8+ T-Cells, and NK cells (Fig 1B). The CD8+ cells were divided into three distinct phenotypes, namely a GZMK-, GZMB- CD8+ T-Cell cluster, a GZMK+ CD8+ Exhausted T-Cell cluster enriched in TIGIT and multiple chemokines (CCL3, CCL4, XCL2), and a GZMB+ NkT cluster enriched in cytolytic markers (PRF1, GNLY, NKG7) (Fig 1C). Differential expression between NP and FP samples in this CD8+ subset showed enrichment of the NkT cytotoxic markers in NP samples, while FP samples were enriched in the CD8+ Exhausted chemokine markers (Fig 1D). Furthermore, the proportion of CD8+ Exhausted T-Cells was enriched in FP samples (p.val < 0.05) (Fig 1E). Exhaustion markers were measured through both RNA and surface marker levels. In RNA, TIGIT was uniquely associated with the FP-enriched CD8+ Exhausted T-Cell cluster, and CD160 was uniquely expressed in FP samples (Fig 1F). CITE-Seq surface marker expression confirms enrichment of both TIGIT and PD1 in the CD8+ Exhausted T-Cell cluster, and along with more exhaustion in FP samples (p.val < 0.01). Conclusion: In this study, we have identified significant differences in T-Cell activity in patients with non-progressing and rapid-progressing multiple myeloma. T-Cells in rapid progressing patients appear to be in a suppressed state, with low cytolytic activity and enriched exhaustion markers. This GZMK+ T-Cell population shows strong similarities with an aging-associated subtype of effector memory T-Cells found to be enriched in older populations (Mogilenko et al, Immunity 54, 2021). These findings will be further validated in an expanded study, consisting both of a larger number of samples, and multiple samples at different timepoints from the same patient. Figure 1 Figure 1. Disclosures Jayasinghe: MMRF: Consultancy; WUGEN: Consultancy. Vij: BMS: Research Funding; Takeda: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; BMS: Honoraria; GSK: Honoraria; Oncopeptides: Honoraria; Karyopharm: Honoraria; CareDx: Honoraria; Legend: Honoraria; Biegene: Honoraria; Adaptive: Honoraria; Harpoon: Honoraria. Kumar: Carsgen: Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Beigene: Consultancy; Bluebird Bio: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Tenebio: Research Funding; Oncopeptides: Consultancy; Antengene: Consultancy, Honoraria; Roche-Genentech: Consultancy, Research Funding; Merck: Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Amgen: Consultancy, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding. Avigan: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Kite Pharma: Consultancy, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partner Tx: Membership on an entity's Board of Directors or advisory committees; Karyopharm: 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; Aviv MedTech Ltd: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Legend Biotech: Membership on an entity's Board of Directors or advisory committees; Chugai: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Parexcel: Consultancy; Takeda: Consultancy; Sanofi: Consultancy.

Introduction: Pediatric mixed phenotype acute leukemia (MPAL), a rare subgroup of leukemia, contains features of both myeloid and lymphoid lineage blasts, which makes the disease more difficult to diagnose/treat. More information is needed to understand the origins of the major pediatric MPAL subtypes, B/Myeloid (B-MPAL) and T/Myeloid (T-MPAL), and how they relate to other leukemias. Single-cell RNA sequencing (scRNA-seq) analysis of bone marrow (BM) can provide in-depth information about the leukemia microenvironment and reveal differences/similarities between MPAL subtypes and other types of leukemia that could be exploited to develop novel diagnostics/therapies. Methods: We analyzed ~16,000 cells from five pediatric MPAL BM samples to generate a transcriptomic landscape of B-MPAL and T-MPAL blasts and associated microenvironment cells. Samples collected at the time of diagnosis (Dx) were used to generate scRNA-Seq data using a droplet-based barcoding technique (Panigrahy et al. JCI 2019, Tellechea et al. JID, 2020). After data normalization, cell clusters were identified using principal component analysis (PCA) and Uniform Manifold Approximation and Projection (UMAP) approach (Becht et al. Nat. Biotech 2018). Meta-analysis was performed using single cell samples from ongoing studies in the Bhasin lab (Bhasin, et al. Blood 2020 (ASH), Thomas et al. Blood 2020 (ASH)) and publicly available single cell data from GEO biorepository. Unsupervised analysis using UMAP and PCA was performed to determine the overall relationship among B-MPAL, T-MPAL and other leukemias (acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia (B-ALL), T-cell ALL (T-ALL)). Supervised differentially expressed gene (DEG) analysis was performed to identify B- and T- MPAL blast cell signatures (P value < 0.001 and log2 FC > 0.5). Transcriptomic profiles in MPAL samples and normal BM stem and immune cells were compared using data from the Human Cell Atlas Data Portal (humancellatlas.org). Gene set enrichment analysis (GSEA) was performed, and significantly enriched pathways were compared in MPAL subtypes (P value < 0.001). Results: PCA analysis showed transcriptome similarity between B-MPAL and both B-ALL and AML, while T-MPAL transcriptome correlated with T-ALL and AML (Fig. 1A). B- and T-MPAL subtype blasts clustered separately from each other in UMAP analyses, with T-MPAL blasts clustering with T-ALL blasts, and B-MPAL somewhat overlapping with B-ALL blasts. Subtype DEG analysis of leukemia blasts and healthy BM revealed distinct significantly upregulated gene signatures in B-MPAL (YBX3, SOCS2, BCL11A, and HIST1H1C) and T-MPAL (ITM2A, HPGD, PDLIM1, and TRDC) blasts (Fig. 1B). Pathway analysis showed upregulated gene activity related to TNFA signaling via NFKB, B-cell survival, and the AP1, FRA, and NGF transcription factors in B-MPAL blasts. In contrast, IL-17 and IL-12, T-cell apoptosis, and Stathmin pathways were upregulated in T-MPAL blasts (Fig. 1C). T-MPAL T-cells also expressed higher levels of T-cell exhaustion markers compared to T-cells in B-MPAL samples and healthy bone marrow. After filtering out genes that are significantly expressed in immune cells, we identified genes that are differentially expressed at diagnosis in MPAL blasts from patients that relapsed after treatment (Dx1) versus patients that achieved remission (Dx2). These genes are potential prognostic markers for B-MPAL and T-MPAL relapse/remission. These include MDM2 and NEIL1 from Dx1 and FOSL2 and CDKN1A in Dx2 B-MPAL blasts. In T-MPAL, expression of HES4 and SPINK2 is associated with Dx1 blasts and GNAQ and ITGA4 with Dx2 blasts. Pathway enrichment analysis on B-MPAL blasts revealed upregulation of interferon gamma and PD-1 signaling in Dx1 samples and increased HSP27 and Cell Cycle pathways in the Dx2 subset. T-MPAL Dx1 associated pathways included prostaglandin synthesis and IL-17, while cell-cell junction and extracellular matrix interactions were increased in T-MPAL Dx2 samples (Fig. 1D). Conclusion: Single-cell profiling was used to characterize the molecular landscapes of MPAL blasts and the bone marrow microenvironment and identified gene signatures and pathways that are specifically enriched in B- and T-MPAL subtypes. Figure 1 Figure 1. Disclosures DeRyckere: Meryx: Other: Equity ownership. Graham: Meryx: Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership.

Multiple Myeloma (MM) is the second most common hematologic malignancy, marked by uncontrolled clonal expansion of plasma cells. MM genome is complex and heterogeneous, with a high frequency of structural variants (SVs) and copy-number abnormalities (CNAs). Single-cell sequencing technologies offer advantages over traditional bulk methods in cancer genomics research for evaluating cellular heterogeneity and investigating evolution of cellular subpopulations within the tumor. Thus, there is an impetus to translate the growing understanding of the genomic landscape of MM into dissecting the tumor heterogeneity using scRNA-seq. So far, there are no reported studies systematically comparing tumor and immune populations differences between MM NON-progressors (NPs) and RAPID-progressor (RPs) and investigating how clonal plasma cells contribute to progression in a large cohort. In addition, the bone marrow microenvironment plays an important role in the evolution of premalignant MM and MM progression. A previous study of single-cell transcriptomics analysis of Monoclonal gammopathy of undetermined significance (MGUS) and MM tumor microenvironment (TME) revealed that natural killer (NK) cell abundance is frequently increased in the early stages and associated with altered chemokine receptor expression. This study shed the light on the role of immune cells on disease progression from asymptomatic MGUS to symptomatic MM. However to date, how immune cells influence disease progression within symptomatic MM is still unclear. Here, we subjected 344 CD138-negative Bone Marrow Mononuclear cells (BMMC) samples to scRNA-seq. To control for the first line therapy, 272 patients were treated with RVD and Autologous stem cell transplant (ASCT) and were stratified into 3 groups based on progression: 200 NON-progressors (NPs) (PFS> 5 years), 7 Intermediate Progressors (IPs, PFS 2-4 years) and 38 RAPID-progressors (RPs) (PFS Our preliminary analysis of scRNA-seq of 6 samples treated by the same first-line therapies (RVD but without ASCT) revealed that plasma cells (PCs) from two patients (MMRF2550 and MMRF2562) with chromosome 13q deletion clustered together, whereas PCs from a patient (MMRF2187) with t(8;14) Myc translocation formed a distinct cluster. Interestingly, PCs from a patient (MMRF2271) with both chromosome 13q deletion and t(8;14) formed another independent cluster. These observations highlight the important role of genetic drivers in transcriptome profiles of plasma cells. In addition, we further investigated how progression features correlate with the immune microenvironment and identified differentially expressed genes between NK cells from RPs versus those from NPs, which could potentially serve as MM progression markers. Interestingly, several cytotoxicity genes are significantly upregulated in FPs, such as GZMB and KLRB1 (log Fold Change=1.44, and 8.41 respectively). Overall, our preliminary results provide a small glimpse of the interconnected nature of driver genetic alterations and progression features in MM tumor and TME. This study will provide a sufficiently broad, deep, and diverse vast dataset for accurately characterizing MM at single-cell resolution to help interrogate how genetic alterations and disease progression interplay MM tumor and TME. We hope this study could identify novel candidate targets for therapeutic approaches, and ultimately stratify patients by risk of progression for early intervention in the clinic. Disclosures Kumar: Roche-Genentech: Consultancy, Research Funding; Merck: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding; Novartis: Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Beigene: Consultancy; Carsgen: Research Funding; Bluebird Bio: Consultancy; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Consultancy; Tenebio: Research Funding; BMS: Consultancy, Research Funding; Antengene: Consultancy, Honoraria; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding. Oh: Abbvie: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees; Celgene Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Constellation: Membership on an entity's Board of Directors or advisory committees; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees; Disc Medicine: Membership on an entity's Board of Directors or advisory committees; Geron: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Kartos Therapeutics: Membership on an entity's Board of Directors or advisory committees; PharamaEssentia: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Membership on an entity's Board of Directors or advisory committees. Vij: BMS: Research Funding; Takeda: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; BMS: Honoraria; GSK: Honoraria; Oncopeptides: Honoraria; Karyopharm: Honoraria; CareDx: Honoraria; Legend: Honoraria; Biegene: Honoraria; Adaptive: Honoraria; Harpoon: Honoraria. Avigan: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Kite Pharma: Consultancy, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partner Tx: Membership on an entity's Board of Directors or advisory committees; Karyopharm: 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; Aviv MedTech Ltd: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Legend Biotech: Membership on an entity's Board of Directors or advisory committees; Chugai: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Parexcel: Consultancy; Takeda: Consultancy; Sanofi: Consultancy.

Compared with traditional bulk sequencing technologies, single-cell technologies have advantages to evaluate cellular heterogeneity and investigate the evolution of cellular subpopulations from the tumor and microenvironment. Application of single-cell sequencing in Multiple Myeloma (MM) is especially beneficial given MM is a highly heterogeneous disease with uncontrolled clonal expansion of plasma cells. Single-cell RNA sequencing (scRNA-seq) has been previously utilized to understand this hematopoietic malignancy in both tumor and immune populations in MM (Ledergor G. et al., 2018, Zavidij, O. et al., 2020). Mass cytometry (CyTOF) has also been used to identify the expansion of novel memory B cells in MM. (Hansmann, L. et al., 2015). Among the various single cell techniques, cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) is a multimodal approach with simultaneous quantification of single-cell transcriptomes and surface proteins. Since these three single-cell approaches enable the identification of cell types, cell states and characterization of cellular heterogeneity at transcriptomic and/or protein levels, understanding the concordance of the measurements among these three modalities is of great interest. We applied scRNA-seq, CyTOF and CITE-seq on four baseline samples of CD138-negative 'immune cell' fractions from patients enrolled in the MMRF CoMMpass study (NCT01454297). Two subjects were fast progressors (PFS < 18 months) and two subjects were non-progressors (PFS not reached). With multi-center collaboration coordinated by Multiple Myeloma Research Foundation (MMRF), each sample was subject to scRNA-seq and CyTOF by 3 independent centers. All sites received aliquots of the same sample. On average, 1,060 immune cells were detected in each sample using scRNA-seq and >64K CD45+ cells were detected using CyTOF. CITE-seq was performed in one center and 4,805 CD138-negative immune cells were identified on average. To compare cell type abundance between scRNA-seq, CyTOF and CITE-seq, we calculated the cell subset frequency of each immune population relative to the CD45+ populations. Overall, all three approaches were concordant while there is a stronger concordance between scRNA-seq and CyTOF. Cell type abundance is especially consistent for B cells, monocytes/macrophages, and plasmacytoid dendritic cells (pDC) among the 3 methods. For the same patient of interest, natural killer (NK) cell frequency was detected at the lowest level in CyTOF relative to scRNA and CITE-seq. The T cell population showed the highest discrepancy among techniques, with highest abundance in scRNA-seq followed by CyTOF and lowest abundance in CITE-seq. Interestingly, CITE-seq detected far less CD4 T cells compared to the other two techniques while CD8 T cell frequency did not show drastic differences. In addition to cell type abundance, we further examined the concordance of expression of cell type signature genes between scRNA-seq and CyTOF. Overall, expression between RNA-level and protein-level is positively correlated with typical cell type markers highly expressed in both techniques, especially in the following cell populations: CD8+ T cells (CD3, CD8), NK cells (CD56, GranzymeB/GZMB, NKG2A/KLRC1), B cells (CD19, CD38), Monocytes (CD14, CD11c/ITGAX, CD33). To note, although the concordance in CD4+ T cells is generally good, we found the expression of CD4 is higher in CyTOF compared to scRNA-seq whereas CD127/IL7R tends to be overexpressed in scRNA-seq. This could explain why IL7R is often identified as a differentially expressed gene (DEG) in CD4+ T cell population while CD4 is barely seen in DEG list in scRNA-seq analysis. It is also interesting to notice that the expression of most NK cell markers has strong concordance between scRNA-seq and CyTOF except NKG2D/KLRK1, which has much higher expression in CyTOF relative to scRNA-seq. Our preliminary results suggested good concordance of immune cell type abundance identified in CyTOF, scRNA-seq and CITE-seq as well as concordant expression of some canonical cell type markers between RNA level and protein level. This work provides the field with reference data sets and shows more detailed examination of NK and T cell subsets is needed when handling single cell sequencing with different modalities. Disclosures Dhodapkar: Roche/Genentech: Membership on an entity's Board of Directors or advisory committees, Other; Lava Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other; Kite: Membership on an entity's Board of Directors or advisory committees, Other; Janssen: Membership on an entity's Board of Directors or advisory committees, Other; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Other; Amgen: Membership on an entity's Board of Directors or advisory committees, Other. Kumar:MedImmune: Research Funding; Amgen: Consultancy, Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments, Research Funding; AbbVie: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Janssen Oncology: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Carsgen: Other, Research Funding; Merck: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy; Celgene/BMS: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Genentech/Roche: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Oncopeptides: Consultancy, Other: Independent Review Committee; IRC member; Kite Pharma: Consultancy, Research Funding; Novartis: Research Funding; Sanofi: Research Funding; Takeda: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Tenebio: Other, Research Funding; Karyopharm: Consultancy; BMS: Consultancy, Research Funding; Genecentrix: Consultancy; Cellectar: Other; Dr. Reddy's Laboratories: Honoraria. Gnjatic:Neon Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; OncoMed: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Genentech: Research Funding; Immune Design: Research Funding; Agenus: Research Funding; Janssen R&D: Research Funding; Pfizer: Research Funding; Takeda: Research Funding; Regeneron: Research Funding; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees. Bhasin:Canomiiks Inc: Current equity holder in private company, Other: Co-Founder.

Introduction: While advances in front-line conventional chemotherapy have increased the likelihood of attaining remission in pediatric AML, relapse rates remain high (25-35%), and novel therapies are needed (Zhang, Savage et al. 2019). The clinical and molecular heterogeneity of AML makes it complex to study and creates challenges for the development of novel therapies (Bolouri, Farrar et al. 2018). It is important to identify cells and pathways underlying relapse to facilitate development of novel therapies. Single-cell RNA Sequencing (scRNA-Seq) allows in-depth analysis of the heterogeneous AML landscape to provide a detailed view of the tumor microenvironment, revealing populations of blasts and immune cells which may be relevant to relapse or complete remission. Methods: We analyzed ~36,500 cells from 14 pediatric AML bone marrow samples in our institutional biorepository, spanning different AML subtypes and 3 healthy children to generate a comprehensive scRNA-Seq landscape of immature AML-associated blasts and microenvironment cells. Samples collected at the time of diagnosis (Dx), end of induction (EOI), and relapse (Rel) were used to generate scRNA-Seq data using a droplet-based barcoding technique (Panigrahy, Gartung et al. 2019). After normalization of scRNA-Seq data, the cell clusters were identified using principal component analysis and Uniform Manifold Approximation and Projection (UMAP) approach (Becht et al, 2018). Differential expression, pathways and systems biology analysis between relapsed and remission patients reveal differences for specific cell clusters (Panigrahy, Gartung et al. 2019). To determine the clinical outcome association of our AML blast specific markers, survival analysis was performed on AML TARGET data (https://ocg.cancer.gov/programs/target) using cox proportional hazard survival approach. To characterize AML blast cells with high accuracy, we used support vector machine (SVM), an Artificial Intelligence based feature extraction and model development approach (Bhasin, Ndebele et al. 2016). Results:ScRNA-Seq analysis of paired Dx and EOI samples using UMAP identified three blast cell clusters with significant gene expression differences among different patients, indicating heterogeneity of AML blast cells (Fig 1a, b). Comparative analysis of the three Dx enriched blast cell clusters with other cells identified a "core blast cell signature" with overexpression of genes like AZU1, CLEC11A, FLT3, and NREP (Fig 1c). These core AML-blast genes were linked to significant activation of the Wnt/Ca2+, Phospholipase C, and integrin signaling pathways (Z score >2 and P-value The scRNA-Seq of AML specific blast cells from relapsed and remission samples exhibited a different clustering pattern indicating different transcriptome landscapes. Relapse-associated AML cell clusters expressed high levels of AZU1, S100A4, LGALS1, and GRK2 genes (Fig 2a). Analysis of the non-AML tumor microenvironment demonstrated enrichment of T/NK in relapsed samples, with differential expression of T cell regulatory/activation genes (Fig 2b, c). ScRNA-Seq showed enrichment of monocyte/macrophage cell clusters in remission samples with distinct relapse- and remission-specific clusters. Remission associated macrophage/monocyte clusters showed overexpression of S100A10, FTH1, CST3 and IFITM2 genes (Fig 2d). Similarly, enrichment of T cell and monocyte/macrophage clusters was observed in relapse and remission samples respectively during EOI. Conclusions: Using single cell transcriptomics we developed a novel potential gene signature to characterize heterogenous AML blast populations with high sensitivity. These genes and the pathways they regulate implicate potential therapeutic targets in pediatric AML. Single cell transcriptome analysis also enabled identification of cell clusters with modulated gene expression at both Dx and EOI that may be useful in predicting relapse/remission. Disclosures Bhasin: Canomiiks Inc: Current equity holder in private company, Other: Co-Founder.