Supplementary figure S2. scRNA-seq of the colonic lamina propria in the syngeneic mice following FMT.

Background:Severe cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) remain frequent, life-threatening complications of CD19 chimeric antigen receptor (CAR) T-cell therapy and constrain its safety, scalability, and outpatient adoption. Existing predictive models lack sufficient external validation for routine clinical use, and pre-infusion biomarkers that capture host susceptibility before infusion are urgently needed. Methods:We applied unbiased mass-spectrometry-based proteomics to pre-infusion biofluids from 98 prospectively-followed adults with relapsed/refractory (r/r) lymphoma at two academic centers (MD Anderson Cancer Center, n = 39, plasma; Moffitt Cancer Center, n = 59, serum). Logistic regression with backward feature selection on the MD Anderson cohort yielded panels for severe (Grade ≥ 2) CRS and ICANS that were locked and tested without refitting on the Moffitt cohort. Patients were stratified into low-, intermediate-, and high-risk tertiles. Ingenuity Pathway Analysis defined upstream regulators and canonical pathways. The 17 CRS-associated and 21 ICANS-associated consensus proteins were classified into mechanistic themes for biological interpretation. Results:A 5-marker CRS panel (SCRIB, MYL6, MTHFD1L, HSP90B1, MMP2) achieved AUCs of 0.85 (95% CI 0.72-0.98) and 0.76 (0.63-0.89) in the discovery and validation cohorts, respectively. An expanded 8-marker ICANS panel (the CRS panel plus SPOCK2, SLC3A2, CD84) achieved AUCs of 0.91 (0.81-1.00) and 0.67 (0.51-0.84). In the combined dataset, high-risk-tertile patients were 13.84-fold (95% CI 4.21-56.26) and 8.59-fold (2.87-29.09) more likely to develop Grade ≥ 2 CRS and ICANS, respectively. Pathway analysis converged on AKT-driven inflammation and endothelial activation. Functional clustering of the consensus proteins partitioned into mechanistically coherent themes consistent with a dual-anatomy model: severe CRS reflected peripheral macrophage priming and endothelial activation with surplus complement amplification (HSP90B1▴, CSF1▴, MMP2▴, HEG1▴, C3▴) and endotheliopathic coagulation (PROC▾, F7▾), whereas severe ICANS reflected cerebrovascular junction and basement-membrane stripping (CDH5▾, ITGB1▾, FN1▾, brain-enriched SPOCK2▾), hepatic synthetic suppression (TTR▾, APOA2▾, IGFBP3▾), compromised plasma antioxidant capacity (GPX3▾, PON1▾), and inflammasome dis-restraint via DPP9▾. PGLYRP2 and SCRIB depletion were shared by both signatures and identified a common upstream priming substrate. Conclusions:Externally validated, pre-infusion proteomic panels predict severe CRS and ICANS following CAR T-cell therapy and define a coherent pre-infusion endothelial-immune priming axis (HSP90B1, MMP2, AKT) with mechanistically interpretable, druggable nodes. The dual-anatomy framework distinguishes peripheral CRS-biased from cerebrovascular ICANS-biased phenotypes downstream of a shared microbiome-host barrier priming substrate, providing a foundation for biomarker-guided risk stratification and cluster-matched prophylactic intervention to enhance the safety and outpatient feasibility of CAR T-cell therapy.

Supplementary figure S1. Effects of fecal microbiota transfer from Lcn2-deficient mice in various mouse models.

Differentially expressed genes in the myeloid cluster of the colonic lamina propria.

Differentially expressed genes in the tumor microenvironment.

Abstract Early-onset colorectal cancer (EOCRC) is rising globally and disproportionately affects populations at increased risk. The gut microbiome has been implicated in colorectal cancer development, yet its relationship to early- versus late-onset disease within these communities remains unclear. This study aimed to characterize microbiome differences across age-defined CRC subgroups using an AI-enabled, multi-domain analytical framework.We analyzed 2,715 colorectal cancer (CRC) tumor samples from patients in our NIH Cancer Moonshot COPECC PE-CGS Network and public data repositories. Within this cohort, stool samples were collected from 23 patients diagnosed with CRC. Microbial profiling was performed using 16S rRNA sequencing and complemented by whole-exome sequencing, RNA sequencing, clinical variables, and social determinants of health (SDOH). Mutation frequencies across different populations were evaluated using the AACR Project GENIE database. Conversational artificial intelligence platforms (AI-HOPE) were used to integrate and query multi-omics and SDOH data, enabling identification of patterns associated with age at onset.EOCRC cases demonstrated lower microbial richness compared with late-onset CRC. Distinct differences in microbial composition and relative abundance were observed when stratifying by genetic ancestry, mutation frequency, gene fusions, copy number variation, clinical features, and SDOH factors. AI-guided integration further highlighted age-specific microbial profiles that aligned with multi-omic alterations.These findings reveal notable microbiome differences between early- and late-onset CRC in populations at increased risk. This preliminary work underscores the utility of artificial intelligence-supported integrative analysis and highlights the need for larger comparative studies to determine whether specific microbial signatures contribute to variations in CRC onset and outcomes. Citation Format: Sophia Manjarrez, Francisco Carranza, Brigette Waldrup, Xinran Qi, Antonio L. Cruz Gomes, David O. Garcia, Adriana Maldonado, Jennifer Karmouch, Robert Jenq, PE-CGS Network, Enrique Velazquez-Villarreal. Artificial intelligence-integrated analysis of the gut microbiome in early- and late-onset colorectal cancer among populations at increased risk using clinical, genomic, and social determinants data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3993.

Although allogeneic haematopoietic cell transplantation (allo-HCT) is a curative therapy for various malignant diseases, severe complications such as graft-versus-host disease (GVHD) limit its use. The intestinal microbiome has long been known to modulate allo-HCT outcomes. Studies in the past two decades alone have uncovered a complex interplay between the microbial repertoire and the host immune system during allo-HCT. Preclinical studies have characterized the crosstalk between the microbiome and the immune response of the host, discovered associations between microbial taxa and the integrity of the mucosal intestinal barrier, and investigated the role of microbial metabolites in GVHD. Clinical studies have demonstrated that dysbiosis is an independent predictor of both transplantation-related and GVHD-related mortality, and ongoing trials are investigating microbiota-focused approaches to improve clinical outcomes and reduce GVHD severity after allo-HCT, paving the way for therapeutic applications of microbiome research. We anticipate that these insights will support the development of personalized therapies for patients receiving allo-HCT, integrating microbiome profiles with individual risk data. In this Review, we summarize current preclinical and clinical studies, providing a comprehensive account of translational efforts in this highly dynamic field.

LEfSe analysis of differential abundance across groups.

Introduction. Nutritional intake during allogeneic hematopoietic cell transplantation (allo-HCT) may influence clinical outcomes and gut microbiome ecology, though longitudinal studies are limited. With a cohort of patients from our Center, enrolled on a Clostridium butyricum MIYAIRI 588 (CBM588) pilot trial (NCT03922035), we explored how calorie and protein intake during allo-HCT correlate with microbiome composition /and incidence of lower gastrointestinal (GI) graft-versus-host disease (GVHD). Methods. We evaluated trial participants (n=30) at our Center from 4/2018 to 1/2020 with dietary intake data collected from day -7 to day 30 relative to transplant. Patients enrolled on the trial underwent reduced-intensity allo-HCT and, after a 6 patient safety lead-in, were randomized to either the treatment (standard of care with daily CBM588 administration from admission to discharge) or control arm (standard of care alone). Daily oral calorie and protein intake were recorded by clinical dietitians and diet technicians using standardized inpatient calorie count procedures. Calorie and protein intake were evaluated at multiple timepoints: baseline (day-7 to 0), week 1 (day 0 to 7), week 2 (day 7 to 14), and week 3 (day 14 to discharge). Calorie and protein intake were evaluated relative to estimated energy and protein needs (calculated by dietitians using the Mifflin-St Jeor equation and adjustment factors based on age, gender, weight, height, BMI, and clinical status). Weekly stool samples (baseline, days 0, 7, 14, 21, and 28 post-HCT) were collected from the participants until discharge. Shotgun and 16S-amplicon sequencing were performed to estimate microbiome composition. Sample alpha-diversity was estimated by the Simpson's reciprocal (S) at genus level. Results. Oral nutritional intake was below 90% of estimated energy requirements for most participants most of the admission time for allo-HCT. At baseline, protein intake was below estimated needs for 90% of patients consuming less than 90% of their requirements regardless of treatment or control arm assignment. In contrast, the median baseline calorie intake met 95% of estimated needs, with 48% of patients consuming less than this threshold. Both calorie and protein intake declined significantly during HCT in both the control and treatment groups: calorie intake decreased from baseline to day 7 (from a median of 95% of estimated requirement to 40%; p=6.3e-07) and protein intake decreased (from a median of 55% of estimated requirement to 20%; p=1.997e-06). Lower calorie intake was observed in CBM588-treated patients compared to control at week 2 (p=5.8e-04) and week 3 (p=0.01) and lower protein intake at week 1 (p=0.038), week 2 (p=0.002), and week 3 (p=0.029). Baseline oral nutritional intake was associated with patient outcomes. A Kaplan Meier survival analysis revealed patients who consumed <95% estimated calorie requirements at baseline were more likely to develop lower GI-GVHD (p=0.023). Patients who consumed <50% estimated protein needs at baseline were more likely to develop lower GI-GVHD (p=0.048). We hypothesized that poor intake and consequent outcomes were due to comorbidity. However, a Cox proportional hazard ratio analysis showed that calorie (p=0.035, hazard ratio=0.034-0.88) and protein (p=0.019, hazard ratio=0.005-0.62) intake association with lower GI GVHD onset hold after controlling for patient comorbidity. We also evaluated the association between nutrition intake and patient microbiota. The median protein and calorie intake value in the microbiome sample window were correlated with alpha diversity (R=0.37; p=0.0034, and R=0.392; p=0.0019, respectively). When focusing on baseline calorie and protein intake, patients with greater intake (>95% of estimated calorie requirements and >50% of estimated protein) had increased alpha diversity at day 14 relative to transplant. Patients that consumed less than the median for protein at baseline were more likely to have Enterococcus domination at day 28 relative to transplant. Conclusions. Poor nutritional intake during allo-HCT, particularly pre-transplant and early peri-transplant, may contribute to the development of lower GI GVHD and microbial dysbiosis. These findings underscore the need to investigate nutritional intake and clinical outcomes in larger cohorts. It highlights the potential therapeutic opportunity of early nutritional intervention on microbiome and transplant outcomes.

Bronchiolitis obliterans (BOS) is a manifestation of pulmonary chronic graft-versus-host disease (cGVHD) and is a devastating complication of allogeneic hematopoietic stem cell transplantation (HCT). Early detection and treatment of BOS may improve outcomes, but biomarkers that accurately identify BOS early are lacking. We aimed to determine whether certain validated cGVHD markers could also accurately diagnose BOS as compared with patients without BOS and with or without extrapulmonary cGVHD. In addition, we sought to determine whether dysbiosis of the gut or oral microbiomes was associated with BOS or with inflammatory biomarkers. We enrolled 43 recipients of allogeneic HCT, 16 of whom had BOS. For each patient, we obtained pulmonary function tests, measured the levels of 9 serum biomarkers utilizing enzyme-linked immunosorbent assays, and analyzed both the oral and gut microbiome using microbial DNA amplification and sequencing. We compared biomarker levels to lung function, both at baseline and over time, as well as to microbiome diversity. Higher IL1RL1 (P = .002) and IL-17 (P = .041) at enrollment were negatively correlated with FEV1% (forced expiratory volume in 1 second) lung function over time. Increases in IL1RL1 (P = .035), IL-17 (P = .009), and WFDC2 (P = .045) levels over time were associated with worsened lung function/FEV1% over time. There were minimal correlations between gut microbiome diversity and lung function or serum biomarkers. Oral microbiome alpha diversity was lower in subjects with BOS than without (P = .00057), and oral beta diversity was associated with FEV1% and with levels of several biomarkers. Our pilot study suggests that certain serum cGVHD markers may identify recipients of allogeneic HCT at higher risk for pulmonary impairment over time and that these markers should be followed with robust, controlled studies.

Infection with antimicrobial-resistant (AR) pathogens is a leading cause of morbidity and mortality among patients with hematological malignancies; however, little is known about the gut microbiome dynamics in acute myeloid leukemia patients and its impact on AR infections (ARI) and/or colonization with AR pathogens (ARC). Longitudinal stool samples collected from 154 patients undergoing induction chemotherapy were analyzed using 16S rRNA sequencing, selective and differential media culturing, MALDI-TOF, and VITEK2 to identify patients with ARC or ARI and to isolate AR infectious and colonizing bacterial strains. Shotgun metagenomic sequencing of baseline stool samples revealed taxa abundances, resistome features, and KEGG pathways associated with AR-events. Baseline observed species were lower in patients with AR-events (p = 0.01). Although several baseline taxa were more abundant in AR-event patients, they were not statistically significant when they were corrected for false discovery. Functional analysis revealed that penicillin and cephalosporin biosynthesis pathways were significantly enriched in patients with ARC. In summary, identifying the baseline microbiome, resistome, and functional pathway biomarkers may forecast an increased risk of ARI and/or ARC, thereby informing antimicrobial treatment strategies in AML patients.

Introduction: Immune checkpoint inhibitors have revolutionized cancer therapy; however, adverse events from an unchecked immune system such as immune checkpoint inhibitor mediated diarrhea and colitis (IMDC) can develop. Fecal microbiota transplantation (FMT) remains an option for patients with refractory colitis, but has not been tested in an upfront setting. Methods: From an open-label, phase I/II clinical trial (NCT0403861) starting June 2021, we report an analysis of adult patients with IMDC treated with upfront FMT. We performed fecal shotgun metagenomic sequencing, metabolomic, transcriptomic and immunofluorescence profiling pre-FMT and post-FMT and and plasma biomarkers of inflamamtion and immune response pre-FMT to predict response. Results: 13 patients were treated with FMT, of which 11 (84.6%) achieved clinical response with a median time to clinical improvement of 1(1-5) days. Among responders sequenced with baseline and follow-up (n = 8), 6 patients (75%) had an increase in alpha diversity post-FMT. Notably, in responders Lacrimispora amygdalina and Alistipes shahii increased independently at both 2 and 4 weeks post FMT. Interestingly, aspartic and propionic acid decreased post-FMT (p < 0.05). Using multiplex immunohistochemical immunofluorescence staining of samples obtained at presentation, we determined that total CK+ and CK+Ki67+ cell populations were reduced in non-responder patients. Additionally, there was a trend toward lower pre- and post-FMT CD20+, CD20+Ki67+, CD4+FOXP3+, and CD8+FOXP3+ cell populations in non-responder patients. Immune cell-type abundance scores showed increased plasma cells, neutrophils, (M1 and M2) macrophages, memory activated and resting memory CD4+ T cells , CD8+ T cells,T follicular helper (Tfh) cells, regulatory T cells (Treg), in pre-FMT samples which all decreased dramatically in post-FMT samples, likely related to response to FMT. Non-responders had lower baseline plasma GDF2, TLR3, CCL22, and FGF21 and higher baseline plasma CXCL14, OSM, IL-1β and IFNG. Conclusion: FMT is a promising front-line therapeutic option for patients who develop IMDC with high efficacy and favorable safety profile. Additional microbiome, blood and tissue analysis provides insights in future directions of potential mechanisms and targets for development of novel therapeutic intervention.

Introduction The intestinal microbiota modulates the host immune-system and is particularly relevant in immune-related diseases. Molecular signals from the intestinal microbiota are perceived by the immune system and amplified by the release of cytokines. Depending on the cytokine signal, the immune response may become more pro-inflammatory or more regulatory. Multiple studies have identified the importance of the intestinal microbiota in the outcome of allogeneic hematopoietic cell transplantation (allo-HCT) patients, particularly in graft-versus-host disease (GVHD) incidence. Although specific microbiome features, such as alpha-diversity, Enterococcus expansion and the metabolism of short chain fatty acids (SCFA) have shown association with patient outcomes and causal role in animal models, the interaction between microbiome signatures, cytokine profile and HCT outcomes have been under-investigated. Methods The cohort consisted of 30 patients; half received Clostridium butyricum MIYAIRI 588 (CBM588) and half received placebo treatment as part of a pilot clinical trial investigating the safety of CBM588 administration in transplant recipients undergoing reduced intensity conditioning regimen (NCT03922035). Stool and serum samples were collected at baseline and weekly follow-ups until day +28 post-HCT. Shotgun sequencing was performed to evaluate microbiome composition. Alpha diversity, as well as Enterococcus and Clostridium genera abundance, were chosen for downstream evaluation due to their relevance in literature. The genus Phocaeicola was also evaluated due to its high prevalence in the data and fermentative metabolism. Cytokine profiling consisted of the measurement of 23 cytokines or cytokine receptors. Cytokines were normalized from a value of 0 to 1 to enable proper comparisons, where 1 represents the maximum measured value for that cytokine. Eight SCFAs were measured and evaluated from stool samples. Results We performed hierarchical clustering to investigate the cytokine profiles of patients. We identified five clusters that were associated with different patterns of cytokine concentration. Cluster 1 was particularly high in inflammatory cytokines IL-6, IL-8, and receptors TNF-R1 and IL-2aR. Cluster 2 was high in IL-2aR, the anti-inflammatory cytokine IL-10, and the chemokines ST2 and IP-10. The other three clusters had more diffuse and moderate patterns. We evaluated the association between cytokine patterns and the microbiota and observed that Cluster 1 had the lowest alpha diversity, the highest Enterococcus relative abundance, and the lowest Phocaeicola relative abundance. Nine patients showed skin or GI GVHD of grade 2 or more. By evaluating the association between cytokine abundance and the incidence of acute GVHD, we noticed that at day 7 after transplant the abundance of cytokines IL-8, IL-6, and the receptors TNF-R1 and IL-2aR were enriched in patients who developed aGVHD. We found that alpha diversity was not associated with the abundance of these cytokines. However, Enterococcus abundance was positively associated (Pearson's correlation ranging from 0.22 to 0.4) with the abundance of each of these cytokines. Interestingly, the genus Phocaeicola was highly negatively correlated with the same cytokines (Pearson's correlation ranging from -0.26 to -0.5). We did not find a significant correlation between aGVHD cytokine markers and the Clostridium genus. Finally, we found that the abundance of SCFA around day 7 and day 14 after allo-HCT were significantly lower in patients that developed aGVHD for all measured SCFAs, with the strongest signal for propionate and butyrate. Conclusion The link between intestinal microbiota and the incidence of aGVHD could be related with an increase in pro-inflammatory cytokines, such as IL-8 and IL-6, after allo-HCT. Understanding the molecular mechanisms that link specific taxonomic groups, such as Enterococcus and Phocaeicola species, with the triggering of cytokine release could help identify strategies to mitigate the incidence of aGVHD.

Successful hematopoietic cell transplant requires immunosuppression to prevent graft-versus-host disease (GVHD), a lethal, T-cell-mediated post-transplant complication. The phase 3 BMT CTN 1703 trial demonstrated superior GVHD-free/relapse-free survival for post-transplant cyclophosphamide (PT-Cy)-based GVHD prophylaxis versus tacrolimus/methotrexate (Tac/MTX), but did not improve overall survival. To compare T-cell biology between GVHD prophylaxis regimens, 324 patients were co-enrolled onto BMT CTN 1801 (NCT03959241). We quantified T-cell immune reconstitution using multi-modal analysis, including T-cell receptor (TCR) sequencing of 2,359 longitudinal samples (180,432,350 T-cells). Compared to Tac/MTX, PT-Cy was associated with an early, substantial reduction in TCR diversity that was sustained for 2 years. PT-Cy led to a T-cell reconstitution bottleneck, including reduced thymic output and virus-associated TCRs. Decreased D+14 TCR diversity predicted prevention of chronic GVHD, but also correlated with increased moderate-to-severe infections. This study reveals how distinct immunosuppression strategies have significant effects on the global immune repertoire, underpinning post-transplant clinical outcomes.

Introduction: Graft Failure (GF) is a rare but devastating outcome of HCT. Primary GF (PGF), defined as a failure of neutrophil recovery by Day+28, is straightforward to diagnose. Diagnosing secondary GF (SGF) is more challenging, with its broad time-range, multiple confounding diagnoses, and lack of predictive biomarkers. In BMT CTN1703/1801, we analyzed patients receiving RIC HCT for heme malignancies with either Tac/MTX (n=159) or PT-Cy (n=165) GVHD prophylaxis. SGF was defined as donor chimerism <5% after initial donor engraftment. With Tac/MTX, there were 3 PGF and 1 SGF diagnoses. With PT-Cy, there were 4 PGF and 6 SGF. Median SGF diagnosis was Day +64 (range: Day+28-215). There were too few Tac/MTX patients to analyze SGF, but sufficient events with PT-Cy. To identify PT-Cy SGF predictors, we leveraged lymphocyte, T, B, and NK cell reconstitution analysis. In SGF we found an early, profound deficit in the reconstitution of all major lymphocyte populations, including total lymphocytes, T, B, and NK cells, as early as Day+28. This enabled the modeling of a SGF risk classifier based on the Absolute Lymphocyte Count (ALC). Methods: Clinical ALC measurements were performed on all SGF patients (n=6) and on non-GF controls with ALCs available (146 of 155 non-GF patients). Flow cytometry was performed on all PT-Cy SGF patients (n = 6) and from a subset of non-GF controls (n = 18: controls were chosen as patients without relapse or severe GVHD, to reduce confounders introduced by immunologic interventions, and for whom all samples, including from the graft infusion, were available). T, B, and NK counts, as well as T cell subsets, were compared (using Welch's T test) on Days 7, 14, 21, 28, 42, 63, 98, 180, 270, 365, 730, with SGF patients censored on the day of GF diagnosis. To interrogate the optimal ALC cutoff, the cumulative incidence of SGF was computed at Days +28 and +42, with death without GF as a competing risk. Results: We have previously demonstrated that, compared to Tac/MTX, PT-Cy patients exhibited an early decrease in reconstitution of all T cell populations (with normal NK and B cell reconstitution). Here we focused specifically on PT-Cy patients with or without SGF. Graft CD34 counts/kg were not different between SGF patients and non-GF patients (mean CD34/kg = 1.38 x10e6 (SGF) vs 0.67 x10e6 (non-GF, p = 0.61) However, even amidst the overarching early suppression in T cell reconstitution with PT-Cy, SGF patients could be easily distinguished from the larger PT-Cy cohort, based on more profound deficits in ALC, T, B and NK cells reconstitution, measured using 2 strategies: (1) SGF patients demonstrated significant early (Day +28) quantitative defects in the reconstitution of the ALC (mean +/- SEM 262+/-32 cells/µL (non-GF) vs 60 +/- 25 (SGF, p<0.0001), CD4 T cells (62 +/- 12 cells/µL vs 19+/-15, p=0.048), CD8 T cells (15 +/-3 cells/µL vs 3.5 +/-1 p=0.0009), all CD8 T cell subpopulations, as well as NK cells (116+/-31 cells/µL vs 2.9+/-1.4 cells/µL, p= 0.002) and B cells (3.5 +/-1.4 cells/µL vs 0.16+/-0.07 p =0.03). (2) In SGF, there was a significant deficit in the rate of rise of all major lymphocyte populations between Days 28-42-60 vs non-GF, including CD4 T cells (p<0.0001), CD8 T cells (p = 0.0002), B cells (p<0.0001), and NK cells (p =0.048). These discoveries suggested that a classifier could be identified to risk-stratify patients for SGF. We explored an ALC cutpoint, amenable to standard clinical lab analysis. A statistically significant threshold was identified at both Days+28 and +42, with Day+42 being most predictive: A threshold of 120 cells/µL was identified as optimal, with landmark analysis documenting a SGF rate of 34.7% below the cutpoint, and SGF of 1% above it (HR = 45.9, 95% CI 5.7 - 366). Conclusions: Despite the small number of events, PT-Cy patients with SGF demonstrated a distinctive reconstitution trajectory that encompassed an early, substantial, and sustained deficit in all lymphocyte counts, as well as a lack of their longitudinal expansion. This enabled the discovery of a candidate ALC biomarker cutpoint at Day+42 that could distinguish patients who were more likely to develop SGF. If confirmed, these data could generate a predictive biomarker for SGF, which would enable the design of trials evaluating early interventions (e.g. CD34+ boosts, DLI, modification of immunosuppression) to improve outcomes for these patients.

The human microbiota is a complex ecosystem that dynamically interacts with the host systemically. Perturbations in the delicate balance of this ecological niche, termed dysbiosis, can make individuals susceptible to a multitude of diseases, including cancer. Specific microbes have been implicated in carcinogenesis through direct effects, modulation of the host immune system, and by promoting inflammation. Furthermore, the microbiota alters the response to and efficacy of anti-cancer therapeutics. Here, we highlight mechanisms by which dysbiosis contributes to cancer development, progression, and therapy as well as how the microbiota can be targeted to enhance cancer outcomes.

Nivolumab alone and in combination with ipilimumab demonstrated durable clinical benefit in patients with previously treated microsatellite instability-high/mismatch repair-deficient metastatic colorectal cancer in the phase 2 CheckMate 142 study. Here, we report exploratory biomarker analyses from CheckMate 142 evaluating associations between various tissue biomarkers and the efficacy of nivolumab monotherapy and nivolumab plus ipilimumab combination in these patients. Higher expression of inflammation-related gene expression signatures is associated with improved response per investigator assessment and survival benefit with nivolumab monotherapy. In contrast, higher tumor mutational burden, tumor indel burden, and degrees of microsatellite instability are associated with improved response per investigator assessment and survival benefit with nivolumab plus ipilimumab. While interpretation is limited by the exploratory nature of these analyses, they suggest that tumor antigenicity rather than baseline tumor inflammation might be important for the combinatorial efficacy. Validation of these findings in larger, randomized studies is necessary.

PURPOSE:Sarcopenia is a hallmark of cancer cachexia. Chimeric antigen receptor (CAR) T-cell therapy is associated with an inflammatory state that may exacerbate sarcopenia. The relationship among CAR T-cell therapy, sarcopenia, and metabolism is poorly understood. EXPERIMENTAL DESIGN:In 83 patients with large B-cell lymphoma, the skeletal muscle index was measured from clinical images obtained at baseline and days 30 and 90 after therapy. Serum metabolomics (n = 57 patients) was performed in the first 4 weeks. RESULTS:Baseline sarcopenia was present in more than half of patients and associated with shorter median overall survival than for non-sarcopenic patients (10.5 vs. 34.3 months; P = 0.006). This reduction was due to increased nonrelapse mortality with all six nonrelapse mortality events occurring in patients with baseline sarcopenia. In the first 30 days after CAR T-cell therapy, one of three patients experienced skeletal muscle loss greater than 10%. Muscle loss was associated with higher tumor burden and neurotoxicity but was not significantly associated with long-term survival. Serum metabolomics revealed an early (weeks 1-2) increase in purine metabolites, followed by a later (weeks 3-4) increase in triglyceride levels. The serum metabolite with the highest fold-increase from baseline was adipic acid, attributed to the inpatient hospital menu of jello and other tart beverages. CONCLUSIONS:Skeletal muscle loss after CAR T-cell therapy is common and is associated with fatty acid catabolism. Patients with baseline sarcopenia have poor tolerance and reduced survival. Future studies of dietary and exercise interventions may improve CAR T-cell therapy outcomes.

Background: A randomized multicenter trial (BMT CTN 1703) demonstrated that after reduced intensity conditioning HCT, PT-Cy-based GVHD prophylaxis led to significant reductions in acute and chronic GVHD, but was associated with significantly more Grade 2 (moderate) infections and similar overall survival vs Tac/MTX. A linked mechanistic study (BMT CTN 1801) found that PT-Cy led to pan-T cell depletion and reduced T cell receptor (TCR) repertoire diversity vs Tac/MTX. To discover the molecular mechanisms driving the association of PT-Cy with increased infections, we examined pathogen-specific T cell reconstitution in BMT CTN 1801 patients (n=165 PT-Cy, n=159 Tac/MTX). Methods: We performed deep TCR sequencing on 2,369 blood samples collected pre-HCT (recipient baseline) and on Days +7, 14, 28, 63, 100, 180, 270, 365, 730, and from the graft infusion product. We then applied a novel set of TCR classifiers (Adaptive Biotechnologies) capable of identifying pathogen-specific clones associated with CMV, EBV, SARS-CoV-2, HSV-1, HSV-2, parvovirus, RSV, influenza A, rhinovirus, adenovirus, HHV-6B, norovirus and Toxoplasma. Single-cell (sc) TCR/RNA-sequencing on a sample subset (n=26) was performed to determine the phenotype of pathogen-specific T cells. Results. By linking scRNA-seq with pathogen-specific TCR barcoding, we identified CD4 and CD8 memory T cells as their predominant T cell reservoir. We found that the total number of pathogen-specific singleton TCRs was significantly lower with PT-Cy, starting early post-HCT and persisting through 2 years. For example, at Day +14, PT-Cy patients had 7.8-fold fewer pathogen-specific singleton TCRs vs Tac/MTX (mean of 6.9 vs 54.1, p<0.001). Consistent differences between PT-Cy and Tac/MTX were observed for TCRs specific for each pathogen individually. Patients who developed Grade 3 (severe) viral infections had significantly fewer pathogen-specific singleton TCRs at Day +14 compared to those who had no viral infections (mean of 4.0 vs 32.5, p=0.03). Leveraging intrinsic TCR barcoding of the HCT infusion (“graft”) and of the patient's baseline samples (“recipient”), we could assign the graft/recipient origin of the pathogen-specific cells with high fidelity. This analysis revealed pronounced differences in the origin of pathogen-specific T cells with PT-Cy vs Tac/MTX, especially prior to Day +100. The absolute number of pathogen-specific TCRs originating from the graft was significantly higher with Tac/MTX vs PT-Cy. This difference was observed from day +7 through 6 months post-HCT, with up to 7.1-fold more graft-derived pathogen-specific TCRs in Tac/MTX vs PT-Cy (p<0.001 at each timepoint before day +100). Within patients who received Tac/MTX, the balance of graft vs recipient pathogen-specific clones significantly favored graft clones (for example, a mean of 14.9 graft vs 8.6 recipient pathogen-specific TCRs at Day+14, p=0.02). By contrast, with PT-Cy, in addition to having substantially fewer pathogen-specific TCRs overall, the proportion of these TCRs was not significantly different between graft and recipient clones (mean of 2.1 graft vs 3.3 recipient pathogen-specific TCRs at Day+14, p=0.42). These data uncover that, in addition to the reduction in pathogen-specific T cells with PT-Cy, there was a relative skewing of the anti-microbial T cell reservoir towards recipient cells that persisted after conditioning. Conclusions: PT-Cy led to in vivo T cell depletion across all T cell subsets, which prominently included clones tracked from the allograft itself into the HCT recipient, with a relative sparing of recipient T cell clones that survived transplant conditioning. While the mechanism for relative sparing of recipient cells has not yet been determined, these conditioning-exposed cells may be less proliferative than newly infused graft T cells. The depletion of graft T cells included CD4 and CD8 memory cells specific for infections, which may have contributed to relative deficits in protective immunity against infection in patients receiving PT-Cy. Importantly, these data suggest that with PT-Cy, persisting recipient TCRs may play a key role in preserving anti-infectious immunity. Taken together, these results provide a mechanistic basis for key post-transplant clinical outcomes of the landmark BMT CTN 1703 study. They also underscore the importance of recipient immune status with PT-Cy, creating the opportunity for targeted infection risk assessment in these patients.