Taxane chemotherapy induces stromal injury that leads to breast cancer dormancy escape.

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 granulocyte colony stimulating factor (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 in a tumor stromal organoid (TSO) model, as well as an altered tumor microenvironment (TME) with augmented protumor immune signaling in a syngeneic mouse breast cancer model. 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.

Early Injury Landscape in Vein Harvest by Single-Cell and Spatial Transcriptomics.

BACKGROUND: Vein graft failure following cardiovascular bypass surgery results in significant patient morbidity and cost to the healthcare system. Vein graft injury can occur during autogenous vein harvest and preparation, as well as after implantation into the arterial system, leading to the development of intimal hyperplasia, vein graft stenosis, and, ultimately, bypass graft failure. Although previous studies have identified maladaptive pathways that occur shortly after implantation, the specific signaling pathways that occur during vein graft preparation are not well defined and may result in a cumulative impact on vein graft failure. We, therefore, aimed to elucidate the response of the vein conduit wall during harvest and following implantation, probing the key maladaptive pathways driving graft failure with the overarching goal of identifying therapeutic targets for biologic intervention to minimize these natural responses to surgical vein graft injury. METHODS: Employing a novel approach to investigating vascular pathologies, we harnessed both single-nuclei RNA-sequencing and spatial transcriptomics analyses to profile the genomic effects of vein grafts after harvest and distension, then compared these findings to vein grafts obtained 24 hours after carotid-carotid vein bypass implantation in a canine model (n=4). RESULTS: Spatial transcriptomic analysis of canine cephalic vein after initial conduit harvest and distention revealed significant enrichment of pathways (P<0.05) involved in the activation of endothelial cells (ECs), fibroblasts, and vascular smooth muscle cells, namely pathways responsible for cellular proliferation and migration and platelet activation across the intimal and medial layers, cytokine signaling within the adventitial layer, and ECM (extracellular matrix) remodeling throughout the vein wall. Subsequent single-nuclei RNA-sequencing analysis supported these findings and further unveiled distinct EC and fibroblast subpopulations with significant upregulation (P<0.05) of markers related to endothelial injury response and cellular activation of ECs, fibroblasts, and vascular smooth muscle cells. Similarly, in vein grafts obtained 24 hours after arterial bypass, there was an increase in myeloid cell, protomyofibroblast, injury response EC, and mesenchymal-transitioning EC subpopulations with a concomitant decrease in homeostatic ECs and fibroblasts. Among these markers were genes previously implicated in vein graft injury, including VCAN, FBN1, and VEGFC, in addition to novel genes of interest, such as GLIS3 and EPHA3. These genes were further noted to be driving the expression of genes implicated in vascular remodeling and graft failure, such as IL-6, TGFBR1, SMAD4, and ADAMTS9. By integrating the spatial transcriptomics and single-nuclei RNA-sequencing data sets, we highlighted the spatial architecture of the vein graft following distension, wherein activated and mesenchymal-transitioning ECs, myeloid cells, and fibroblasts were notably enriched in the intima and media of distended veins. Finally, intercellular communication network analysis unveiled the critical roles of activated ECs, mesenchymal-transitioning ECs, protomyofibroblasts, and vascular smooth muscle cells in upregulating signaling pathways associated with cellular proliferation (MDK [midkine], PDGF [platelet-derived growth factor], VEGF [vascular endothelial growth factor]), transdifferentiation (Notch), migration (ephrin, semaphorin), ECM remodeling (collagen, laminin, fibronectin), and inflammation (thrombospondin), following distension. CONCLUSIONS: Vein conduit harvest and distension elicit a prompt genomic response facilitated by distinct cellular subpopulations heterogeneously distributed throughout the vein wall. This response was found to be further exacerbated following vein graft implantation, resulting in a cascade of maladaptive gene regulatory networks. Together, these results suggest that distension initiates the upregulation of pathological pathways that may ultimately contribute to bypass graft failure and presents potential early targets warranting investigation for targeted therapies. This work highlights the first applications of single-nuclei and spatial transcriptomic analyses to investigate venous pathologies, underscoring the utility of these methodologies and providing a foundation for future investigations.

Psychosocial impact of COVID-19 pandemic on healthcare workers in India & their perceptions on the way forward - A qualitative study.

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.

Acceptability of the Medication Event Reminder Monitor for Promoting Adherence to Multidrug-Resistant Tuberculosis Therapy in Two Indian Cities: Qualitative Study of Patients and Health Care Providers.

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.

Mycobacterium tuberculosis strain lineage in mixed tribal population across India and Andaman Nicobar Island.

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.

Evaluation of the Accuracy of 99DOTS, a Novel Cellphone-based Strategy for Monitoring Adherence to Tuberculosis Medications: Comparison of DigitalAdherence Data With Urine Isoniazid Testing.

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.

Pretreatment loss to follow-up of tuberculosis patients in Chennai, India: a cohort study with implications for health systems strengthening.

BACKGROUND: Pretreatment loss to follow-up (PTLFU) is a barrier to tuberculosis (TB) control in India's Revised National TB Control Programme (RNTCP). PTLFU studies have not been conducted in India's mega-cities, where patient mobility may complicate linkage to care. METHODS: We collected data from patient registries for May 2015 from 22 RNTCP designated microscopy centers (DMCs) in Chennai and audited addresses and phone numbers for patients evaluated for suspected TB to understand how missing contact information may contribute to PTLFU. From November 2015 to June 2016, we audited one month of records from each of these 22 DMCs and tracked newly diagnosed smear-positive patients using RNTCP records, phone calls, and home visits. We defined PTLFU cases as including: (1) patients who did not start TB therapy within 14 days and (2) patients who started TB therapy but were lost to follow-up or died before official RNTCP registration. We used multivariate logistic regression to identify factors associated with PTLFU. RESULTS: In the audit of May 2015 DMC registries, out of 3696 patients evaluated for TB, 1273 (34.4%) had addresses and phone numbers that were illegible or missing. Out of 344 smear-positive patients tracked from November 2015 to June 2016, 40 (11.6%) did not start TB therapy within 14 days and 36 (10.5%) started therapy but were lost to follow-up or died before official RNTCP registration, for an overall PTLFU rate of 22.1% (95%CI: 17.8%-26.4%). Of all PTLFU patients, 55 (72.4%) were lost to follow-up and 21 (27.6%) died before starting treatment or before RNTCP registration. In the regression analysis, age > 50 years (OR 2.9, 95%CI 1.4-6.5), history of prior TB (OR 3.9, 95%CI 2.2-7.1), evaluation at a high patient volume DMC (OR 3.2, 95% CI 1.7-6.3), and absence of legible patient contact information (OR 4.5, 95%CI 1.3-15.1) were significantly associated with PTLFU. CONCLUSIONS: In an Indian mega-city, we found a high PTLFU rate, especially in patients with a prior TB history, who are at greater risk for having drug-resistance. Enhancing quality of care and health system transparency is critical for improving linkage of newly diagnosed patients to TB care in urban India.

Transactional sex and the challenges to safer sexual behaviors: a study among male sex workers in Chennai, India.

Male sex workers (MSW) are a significant but invisible population in India who are at risk for HIV/sexually transmitted infections (STIs). Few studies from India have documented HIV risk factors and motivations for sex work in this population. Between 2013 and 2014, a community-based convenience sample of 100 MSW in Chennai (south India) completed a baseline risk assessment as part of a behavioral intervention. Participants were ≥18 years, and reported current sex work. We report medians and proportions, and Wilcoxon-Mann-Whitney and chi-square tests are used to examine differences between sex work and sexual behavior measures by income source. Participants were engaged in sex work for 5.0 years (IQR = 2.3-10.0), and earned 3000 (IQR = 2000-8000) Rupees (<50 USD) per month from sex work. Sixty-four percent reported ever testing for HIV and 20.2% for any STI. The most common reasons for starting sex work were money (83.0%) and pleasure (56.0%). Compared to participants with an additional source of income, those whose only source of income was sex work reported more male clients in the past month (10.0 vs. 6.0, p = .017), as well as more condomless anal sex acts with male clients (8.0 vs. 5.0, p = .008). Nearly 70.0% were offered more money not to use a condom during sex with a client, and 74.2% reported accepting more money not to use a condom. Three-quarters reported having experienced difficulty using condoms with clients. MSW in India engage in high levels of sexual risk for HIV/STIs. Money appears to be a driving factor for engaging in sex work and condomless sex with clients. HIV prevention interventions with MSW should focus on facilitating skills that will support their ability to negotiate sexual safety in the context of monetary disincentives.

The Tuberculosis Cascade of Care in India's Public Sector: A Systematic Review and Meta-analysis.

BACKGROUND: India has 23% of the global burden of active tuberculosis (TB) patients and 27% of the world's "missing" patients, which includes those who may not have received effective TB care and could potentially spread TB to others. The "cascade of care" is a useful model for visualizing deficiencies in case detection and retention in care, in order to prioritize interventions. METHODS AND FINDINGS: The care cascade constructed in this paper focuses on the Revised National TB Control Programme (RNTCP), which treats about half of India's TB patients. We define the TB cascade as including the following patient populations: total prevalent active TB patients in India, TB patients who reach and undergo evaluation at RNTCP diagnostic facilities, patients successfully diagnosed with TB, patients who start treatment, patients retained to treatment completion, and patients who achieve 1-y recurrence-free survival. We estimate each step of the cascade for 2013 using data from two World Health Organization (WHO) reports (2014-2015), one WHO dataset (2015), and three RNTCP reports (2014-2016). In addition, we conduct three targeted systematic reviews of the scientific literature to identify 39 unique articles published from 2000-2015 that provide additional data on five indicators that help estimate different steps of the TB cascade. We construct separate care cascades for the overall population of patients with active TB and for patients with specific forms of TB-including new smear-positive, new smear-negative, retreatment smear-positive, and multidrug-resistant (MDR) TB. The WHO estimated that there were 2,700,000 (95%CI: 1,800,000-3,800,000) prevalent TB patients in India in 2013. Of these patients, we estimate that 1,938,027 (72%) TB patients were evaluated at RNTCP facilities; 1,629,906 (60%) were successfully diagnosed; 1,417,838 (53%) got registered for treatment; 1,221,764 (45%) completed treatment; and 1,049,237 (95%CI: 1,008,775-1,083,243), or 39%, of 2,700,000 TB patients achieved the optimal outcome of 1-y recurrence-free survival. The separate cascades for different forms of TB highlight different patterns of patient attrition. Pretreatment loss to follow-up of diagnosed patients and post-treatment TB recurrence were major points of attrition in the new smear-positive TB cascade. In the new smear-negative and MDR TB cascades, a substantial proportion of patients who were evaluated at RNTCP diagnostic facilities were not successfully diagnosed. Retreatment smear-positive and MDR TB patients had poorer treatment outcomes than the general TB population. Limitations of our analysis include the lack of available data on the cascade of care in the private sector and substantial uncertainty regarding the 1-y period prevalence of TB in India. CONCLUSIONS: Increasing case detection is critical to improving outcomes in India's TB cascade of care, especially for smear-negative and MDR TB patients. For new smear-positive patients, pretreatment loss to follow-up and post-treatment TB recurrence are considerable points of attrition that may contribute to ongoing TB transmission. Future multisite studies providing more accurate information on key steps in the public sector TB cascade and extension of this analysis to private sector patients may help to better target interventions and resources for TB control in India.

Pulmonary tuberculosis among tribals in India: A systematic review & meta-analysis.

BACKGROUND & OBJECTIVES: There has been limited investigation on the prevalence of tuberculosis (TB) in tribal communities in India, a vulnerable section of Indian society. The lack of a population-based estimate prompted us to conduct a meta-analysis of existing studies to provide a single, population-based estimate of the TB prevalence for tribals. METHODS: Literature search was conducted in PubMed using the keywords - "tuberculosis", "tribals", "India", "prevalence", and "survey". References cited in the articles retrieved were also reviewed, and those found relevant were selected. TB prevalence rates estimated by the studies were used for our calculation of a pooled-estimate. RESULTS: The pooled estimate, based on the random effects model, was 703 per 100,000 population with a 95 % CI of 386-1011. The associated heterogeneity measures in terms of Cochran's Q was significant ( p=0 0.08 <0.1) and I [2] was moderate at 48 per cent. INTERPRETATION & CONCLUSIONS: The meta-analysis demonstrated a large variability in pulmonary TB prevalence estimates among the different studies with poor representation of the various tribal groups. The moderate level of heterogeneity found across the studies suggests that the pooled-estimate needs to be treated with caution. Our findings also highlight the need to assess the pulmonary TB burden in India.

Prevalence of chest symptoms amongst brick kiln migrant workers and care seeking behaviour: a study from South India.

BACKGROUND: Early detection and treatment of tuberculosis (TB) have been key principles of TB control. However, this can be a challenge with 'hard to reach' populations such as migrants. Brick kiln workers are one such group of migrants who are exposed to smoke, heat and dust from brick kilns which are one of the major causes of respiratory illnesses. METHODOLOGY: A cross-sectional community based study was carried out in Thiruvallur, Tamil Nadu, South India, from August 2011 to June 2012. A total of 4002 individuals from 55 brick kiln chambers were interviewed to determine the prevalence of chest symptoms and care seeking behaviour patterns. RESULTS: Three hundred and seventy-seven (9.4%) chest symptomatics were identified. The most significant variables associated with chest symptoms were illiteracy, alcohol abuse and heavy smoking. Of the chest symptomatics identified, 50.4% took action to get relief from their symptoms. The duration of over 6-month stay in the chamber was significantly associated with taking action (OR, 5.5, 95% CI: 2.3, 13.3). CONCLUSIONS: The TB control programme needs to further explore how to extend its services to such 'hard to reach' groups. Active case finding to ensure early diagnosis and treatment initiation amongst such groups needs consideration.

A pilot RCT of an intervention to reduce HIV sexual risk and increase self-acceptance among MSM in Chennai, India.

This is a 2-arm pilot randomized controlled trial (N = 96) of a behavioral intervention (4 group and 4 individual sessions) integrating risk reduction counseling with counseling to foster self-acceptance in MSM in India compared to enhanced standard of care (ESOC). Both conditions involved HIV and STI testing and counseling at baseline and 6-months, and assessments of condomless sex at baseline, 3-, and 6-months. A significant condition by time interaction suggested a difference in the rate of change in number of anal sex acts without condoms in the intervention versus ESOC (p < 0.0001). Post hoc contrasts suggested that the overall difference was due to intervention-response at 3-months. The incidence of bacterial STIs was 17.5 % in the intervention condition and a 28.6 % in ESOC. Addressing self-acceptance and related psychosocial concerns in the context sexual risk reduction counseling for MSM in India was feasible and acceptable. Testing the intervention for efficacy is justified.

Integrated single-nuclei and spatial transcriptomic analysis reveals propagation of early acute vein harvest and distension injury signaling pathways following arterial implantation.

Background: Vein graft failure (VGF) following cardiovascular bypass surgery results in significant patient morbidity and cost to the healthcare system. Vein graft injury can occur during autogenous vein harvest and preparation, as well as after implantation into the arterial system, leading to the development of intimal hyperplasia, vein graft stenosis, and, ultimately, bypass graft failure. While previous studies have identified maladaptive pathways that occur shortly after implantation, the specific signaling pathways that occur during vein graft preparation are not well defined and may result in a cumulative impact on VGF. We, therefore, aimed to elucidate the response of the vein conduit wall during harvest and following implantation, probing the key maladaptive pathways driving graft failure with the overarching goal of identifying therapeutic targets for biologic intervention to minimize these natural responses to surgical vein graft injury. Methods: Employing a novel approach to investigating vascular pathologies, we harnessed both single-nuclei RNA-sequencing (snRNA-seq) and spatial transcriptomics (ST) analyses to profile the genomic effects of vein grafts after harvest and distension, then compared these findings to vein grafts obtained 24 hours after carotid-cartoid vein bypass implantation in a canine model (n=4). Results: Spatial transcriptomic analysis of canine cephalic vein after initial conduit harvest and distention revealed significant enrichment of pathways (P < 0.05) involved in the activation of endothelial cells (ECs), fibroblasts (FBs), and vascular smooth muscle cells (VSMCs), namely pathways responsible for cellular proliferation and migration and platelet activation across the intimal and medial layers, cytokine signaling within the adventitial layer, and extracellular matrix (ECM) remodeling throughout the vein wall. Subsequent snRNA-seq analysis supported these findings and further unveiled distinct EC and FB subpopulations with significant upregulation (P < 0.00001) of markers related to endothelial injury response and cellular activation of ECs, FBs, and VSMCs. Similarly, in vein grafts obtained 24 hours after arterial bypass, there was an increase in myeloid cell, protomyofibroblast, injury-response EC, and mesenchymal-transitioning EC subpopulations with a concomitant decrease in homeostatic ECs and fibroblasts. Among these markers were genes previously implicated in vein graft injury, including VCAN (versican), FBN1 (fibrillin-1), and VEGFC (vascular endothelial growth factor C), in addition to novel genes of interest such as GLIS3 (GLIS family zinc finger 3) and EPHA3 (ephrin-A3). These genes were further noted to be driving the expression of genes implicated in vascular remodeling and graft failure, such as IL-6, TGFBR1, SMAD4, and ADAMTS9. By integrating the ST and snRNA-seq datasets, we highlighted the spatial architecture of the vein graft following distension, wherein activated and mesenchymal-transitioning ECs, myeloid cells, and FBs were notably enriched in the intima and media of distended veins. Lastly, intercellular communication network analysis unveiled the critical roles of activated ECs, mesenchymal transitioning ECs, protomyofibroblasts, and VSMCs in upregulating signaling pathways associated with cellular proliferation (MDK, PDGF, VEGF), transdifferentiation (Notch), migration (ephrin, semaphorin), ECM remodeling (collagen, laminin, fibronectin), and inflammation (thrombospondin), following distension. Conclusions: Vein conduit harvest and distension elicit a prompt genomic response facilitated by distinct cellular subpopulations heterogeneously distributed throughout the vein wall. This response was found to be further exacerbated following vein graft implantation, resulting in a cascade of maladaptive gene regulatory networks. Together, these results suggest that distension initiates the upregulation of pathological pathways that may ultimately contribute to bypass graft failure and presents potential early targets warranting investigation for targeted therapies. This work highlights the first applications of single-nuclei and spatial transcriptomic analyses to investigate venous pathologies, underscoring the utility of these methodologies and providing a foundation for future investigations.

Indoximod-based chemo-immunotherapy for pediatric brain tumors: A first-in-children phase I trial.

BACKGROUND: Recurrent brain tumors are the leading cause of cancer death in children. Indoleamine 2,3-dioxygenase (IDO) is a targetable metabolic checkpoint that, in preclinical models, inhibits anti-tumor immunity following chemotherapy. METHODS: We conducted a phase I trial (NCT02502708) of the oral IDO-pathway inhibitor indoximod in children with recurrent brain tumors or newly diagnosed diffuse intrinsic pontine glioma (DIPG). Separate dose-finding arms were performed for indoximod in combination with oral temozolomide (200 mg/m2/day x 5 days in 28-day cycles), or with palliative conformal radiation. Blood samples were collected at baseline and monthly for single-cell RNA-sequencing with paired single-cell T cell receptor sequencing. RESULTS: Eighty-one patients were treated with indoximod-based combination therapy. Median follow-up was 52 months (range 39-77 months). Maximum tolerated dose was not reached, and the pediatric dose of indoximod was determined as 19.2 mg/kg/dose, twice daily. Median overall survival was 13.3 months (n = 68, range 0.2-62.7) for all patients with recurrent disease and 14.4 months (n = 13, range 4.7-29.7) for DIPG. The subset of n = 26 patients who showed evidence of objective response (even a partial or mixed response) had over 3-fold longer median OS (25.2 months, range 5.4-61.9, p = 0.006) compared to n = 37 nonresponders (7.3 months, range 0.2-62.7). Four patients remain free of active disease longer than 36 months. Single-cell sequencing confirmed emergence of new circulating CD8 T cell clonotypes with late effector phenotype. CONCLUSIONS: Indoximod was well tolerated and could be safely combined with chemotherapy and radiation. Encouraging preliminary evidence of efficacy supports advancing to Phase II/III trials for pediatric brain tumors.

A single-cell atlas characterizes dysregulation of the bone marrow immune microenvironment associated with outcomes in multiple myeloma.

Multiple Myeloma (MM) remains incurable despite advances in treatment options. Although tumor subtypes and specific DNA abnormalities are linked to worse prognosis, the impact of immune dysfunction on disease emergence and/or treatment sensitivity remains unclear. We established a harmonized consortium to generate an Immune Atlas of MM aimed at informing disease etiology, risk stratification, and potential therapeutic strategies. We generated a transcriptome profile of 1,149,344 single cells from the bone marrow of 263 newly diagnosed patients enrolled in the CoMMpass study and characterized immune and hematopoietic cell populations. Associating cell abundances and gene expression with disease progression revealed the presence of a proinflammatory immune senescence-associated secretory phenotype in rapidly progressing patients. Furthermore, signaling analyses suggested active intercellular communication involving APRIL-BCMA, potentially promoting tumor growth and survival. Finally, we demonstrate that integrating immune cell levels with genetic information can significantly improve patient stratification.

Pediatric T-cell acute lymphoblastic leukemia blast signature and MRD associated immune environment changes defined by single cell transcriptomics analysis.

Different driver mutations and/or chromosomal aberrations and dysregulated signaling interactions between leukemia cells and the immune microenvironment have been implicated in the development of T-cell acute lymphoblastic leukemia (T-ALL). To better understand changes in the bone marrow microenvironment and signaling pathways in pediatric T-ALL, bone marrows collected at diagnosis (Dx) and end of induction therapy (EOI) from 11 patients at a single center were profiled by single cell transcriptomics (10 Dx, 5 paired EOI, 1 relapse). T-ALL blasts were identified by comparison with healthy bone marrow cells. T-ALL blast-associated gene signature included SOX4, STMN1, JUN, HES4, CDK6, ARMH1 among the most significantly overexpressed genes, some of which are associated with poor prognosis in children with T-ALL. Transcriptome profiles of the blast cells exhibited significant inter-patient heterogeneity. Post induction therapy expression profiles of the immune cells revealed significant changes. Residual blast cells in MRD+ EOI samples exhibited significant upregulation (P < 0.01) of PD-1 and RhoGDI signaling pathways. Differences in cellular communication were noted in the presence of residual disease in T cell and hematopoietic stem cell compartments in the bone marrow. Together, these studies generate new insights and expand our understanding of the bone marrow landscape in pediatric T-ALL.

The Simple prEservatioN of Single cElls method for cryopreservation enables the generation of single-cell immune profiles from whole blood.

Introduction: Current multistep methods utilized for preparing and cryopreserving single-cell suspensions from blood samples for single-cell RNA sequencing (scRNA-seq) are time-consuming, requiring trained personnel and special equipment, so limiting their clinical adoption. We developed a method, Simple prEservatioN of Single cElls (SENSE), for single-step cryopreservation of whole blood (WB) along with granulocyte depletion during single-cell assay, to generate high quality single-cell profiles (SCP). Methods: WB was cryopreserved using the SENSE method and peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved using the traditional density-gradient method (PBMC method) from the same blood sample (n=6). The SCPs obtained from both methods were processed using a similar pipeline and quality control parameters. Further, entropy calculation, differential gene expression, and cellular communication analysis were performed to compare cell types and subtypes from both methods. Results: Highly viable (86.3 ± 1.51%) single-cell suspensions (22,353 cells) were obtained from the six WB samples cryopreserved using the SENSE method. In-depth characterization of the scRNA-seq datasets from the samples processed with the SENSE method yielded high-quality profiles of lymphoid and myeloid cell types which were in concordance with the profiles obtained with classical multistep PBMC method processed samples. Additionally, the SENSE method cryopreserved samples exhibited significantly higher T-cell enrichment, enabling deeper characterization of T-cell subtypes. Overall, the SENSE and PBMC methods processed samples exhibited transcriptional, and cellular communication network level similarities across cell types with no batch effect except in myeloid lineage cells. Discussion: Comparative analysis of scRNA-seq datasets obtained with the two cryopreservation methods i.e., SENSE and PBMC methods, yielded similar cellular and molecular profiles, confirming the suitability of the former method's incorporation in clinics/labs for cryopreserving and obtaining high-quality single-cells for conducting critical translational research.

Single-cell RNA sequencing distinctly characterizes the wide heterogeneity in pediatric mixed phenotype acute leukemia.

BACKGROUND: Mixed phenotype acute leukemia (MPAL), a rare subgroup of leukemia characterized by blast cells with myeloid and lymphoid lineage features, is difficult to diagnose and treat. A better characterization of MPAL is essential to understand the subtype heterogeneity and how it compares with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Therefore, we performed single-cell RNA sequencing (scRNAseq) on pediatric MPAL bone marrow (BM) samples to develop a granular map of the MPAL blasts and microenvironment landscape. METHODS: We analyzed over 40,000 cells from nine pediatric MPAL BM samples to generate a single-cell transcriptomic landscape of B/myeloid (B/My) and T/myeloid (T/My) MPAL. Cells were clustered using unsupervised single-cell methods, and malignant blast and immune clusters were annotated. Differential expression analysis was performed to identify B/My and T/My MPAL blast-specific signatures by comparing transcriptome profiles of MPAL with normal BM, AML, and ALL. Gene set enrichment analysis (GSEA) was performed, and significantly enriched pathways were compared in MPAL subtypes. RESULTS: B/My and T/My MPAL blasts displayed distinct blast signatures. Transcriptomic analysis revealed that B/My MPAL profile overlaps with B-ALL and AML samples. Similarly, T/My MPAL exhibited overlap with T-ALL and AML samples. Genes overexpressed in both MPAL subtypes' blast cells compared to AML, ALL, and healthy BM included MAP2K2 and CD81. Subtype-specific genes included HBEGF for B/My and PTEN for T/My. These marker sets segregated bulk RNA-seq AML, ALL, and MPAL samples based on expression profiles. Analysis comparing T/My MPAL to ETP, near-ETP, and non-ETP T-ALL, showed that T/My MPAL had greater overlap with ETP-ALL cases. Comparisons among MPAL subtypes between adult and pediatric samples showed analogous transcriptomic landscapes of corresponding subtypes. Transcriptomic differences were observed in the MPAL samples based on response to induction chemotherapy, including selective upregulation of the IL-16 pathway in relapsed samples. CONCLUSIONS: We have for the first time described the single-cell transcriptomic landscape of pediatric MPAL and demonstrated that B/My and T/My MPAL have distinct scRNAseq profiles from each other, AML, and ALL. Differences in transcriptomic profiles were seen based on response to therapy, but larger studies will be needed to validate these findings.

Single-cell analysis reveals altered tumor microenvironments of relapse- and remission-associated pediatric acute myeloid leukemia.

Acute myeloid leukemia (AML) microenvironment exhibits cellular and molecular differences among various subtypes. Here, we utilize single-cell RNA sequencing (scRNA-seq) to analyze pediatric AML bone marrow (BM) samples from diagnosis (Dx), end of induction (EOI), and relapse timepoints. Analysis of Dx, EOI scRNA-seq, and TARGET AML RNA-seq datasets reveals an AML blasts-associated 7-gene signature (CLEC11A, PRAME, AZU1, NREP, ARMH1, C1QBP, TRH), which we validate on independent datasets. The analysis reveals distinct clusters of Dx relapse- and continuous complete remission (CCR)-associated AML-blasts with differential expression of genes associated with survival. At Dx, relapse-associated samples have more exhausted T cells while CCR-associated samples have more inflammatory M1 macrophages. Post-therapy EOI residual blasts overexpress fatty acid oxidation, tumor growth, and stemness genes. Also, a post-therapy T-cell cluster associated with relapse samples exhibits downregulation of MHC Class I and T-cell regulatory genes. Altogether, this study deeply characterizes pediatric AML relapse- and CCR-associated samples to provide insights into the BM microenvironment landscape.

Cross center single-cell RNA sequencing study of the immune microenvironment in rapid progressing multiple myeloma.

Despite advancements in understanding the pathophysiology of Multiple Myeloma (MM), the cause of rapid progressing disease in a subset of patients is still unclear. MM's progression is facilitated by complex interactions with the surrounding bone marrow (BM) cells, forming a microenvironment that supports tumor growth and drug resistance. Understanding the immune microenvironment is key to identifying factors that promote rapid progression of MM. To accomplish this, we performed a multi-center single-cell RNA sequencing (scRNA-seq) study on 102,207 cells from 48 CD138- BM samples collected at the time of disease diagnosis from 18 patients with either rapid progressing (progression-free survival (PFS) < 18 months) or non-progressing (PFS > 4 years) disease. Comparative analysis of data from three centers demonstrated similar transcriptome profiles and cell type distributions, indicating subtle technical variation in scRNA-seq, opening avenues for an expanded multicenter trial. Rapid progressors depicted significantly higher enrichment of GZMK+ and TIGIT+ exhausted CD8+ T-cells (P = 0.022) along with decreased expression of cytolytic markers (PRF1, GZMB, GNLY). We also observed a significantly higher enrichment of M2 tolerogenic macrophages in rapid progressors and activation of pro-proliferative signaling pathways, such as BAFF, CCL, and IL16. On the other hand, non-progressive patients depicted higher enrichment for immature B Cells (i.e., Pre/Pro B cells), with elevated expression for markers of B cell development (IGLL1, SOX4, DNTT). This multi-center study identifies the enrichment of various pro-tumorigenic cell populations and pathways in those with rapid progressing disease and further validates the robustness of scRNA-seq data generated at different study centers.