ALBERT

Description: Allogeneic hematopoietic stem cell transplantation (allo-SCT) offers cure for a variety of conditions, in particular, but not limited to, hematologic malignancies. However, it can be associated with life-threatening complications, including graft-versus-host disease (GVHD) and infections, which are factors limiting its widespread use. Technical advances in the field of microbiome research have allowed for a better understanding of the microbial flora of the human intestine, as well as dissection of their interactions with the host immune system in allo-SCT and posttransplant complications. There is growing evidence that the commensal microbiome is frequently dysregulated following allo-SCT and that this dysbiosis can predispose to adverse clinical outcomes, especially including acute intestinal GVHD and reduced overall survival. In this review, we discuss the interactions between the microbiome and the components of the immune system that play a major role in the pathways leading to the inflammatory state of acute intestinal GVHD. We also discuss the microbiome-centered strategies that have been devised or are actively being investigated to improve the outcomes of allo-SCT patients in regard to acute intestinal GVHD.

Description: Summary In fields, such as ecology, microbiology and genomics, non-Euclidean distances are widely applied to describe pairwise dissimilarity between samples. Given these pairwise distances, principal coordinates analysis is commonly used to construct a visualization of the data. However, confounding covariates can make patterns related to the scientific question of interest difficult to observe. We provide adjusted principal coordinates analysis as an easy-to-use tool, available as both an R package and a Shiny app, to improve data visualization in this context, enabling enhanced presentation of the effects of interest. Availability and implementation The R package ‘aPCoA’ and Shiny app can be accessed at https://cran.r-project.org/web/packages/aPCoA/index.html and https://biostatistics.mdanderson.org/shinyapps/aPCoA/.

Description: Introduction: Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy has improved survival outcomes in patients with relapsed/refractory (r/r) large B-cell lymphoma (LBCL). However, many patients experience systemic toxicities such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) and about 60% relapse. Recent studies indicate that the gut microbiome can modulate tumor response and also influence toxicities associated with immune checkpoint inhibitor therapy. However, the influence of gut microbiome has not been well-studied in patients receiving CAR T-cell therapy. Here, we analyzed the association between gut microbiome composition and diversity metrics with survival outcomes and toxicities in r/r LBCL patients treated with anti-CD19 CAR T-cell therapy. Methods: Baseline stool samples were collected from r/r LBCL patients within 3 days of CAR T-cell infusion (days -3 to +3). Taxonomic profiling was performed on all samples using targeted ribosomal 16S RNA gene sequencing of the V4 region. The anti-tumor response was assessed as per Lugano 2014 criteria. Bacterial community alpha diversity was calculated using the inverse Simpson Index (ISI). Results were correlated with toxicity outcomes, including CRS and ICANS, as well as efficacy outcomes, including response, progression-free (PFS), and overall survival (OS). Results: We analyzed baseline stool samples from 33 r/r LBCL patients (24 DLBCL, 7 transformed follicular lymphoma, and 2 double-hit lymphomas) treated with standard of care axicabtagene ciloleucel (n =30) or tisagenlecleucel (n=3). The median age of the cohort (23 males, 10 females) was 54 (range 28 - 84) years. The number of patients with ECOG performance status (PS) 0-1, Ann-Arbor stage ≥3, and International Prognostic Index (IPI) of ≥3 at the time of apheresis were 29 (88%), 33 (100%), and 15 (46%), respectively. The median number of prior lines of therapy was 3 (range 2-6). The median follow-up from CAR-T infusion was 7.3 (range 0.8 -13.6) months. The best overall and complete response (CR) rates were 82% (27/33) and 58% (19/33), respectively. All patients were evaluable for toxicity assessment and 29 were evaluable for response assessment at 3 months (3 patients died of toxicity before 3 months and 1 was lost to follow-up). Since ongoing response at 3 months was previously shown to be associated with long-term durability (Locke et al, Lancet Oncol 2019), we analyzed differences in baseline gut microbiome markers in patients with or without ongoing CR at 3 months post-CAR-T infusion. ISI was significantly higher in patients with ongoing CR at 3 months (N=13) compared to those without ongoing CR (NoCR) group (N=16) (Fig.1A, p=0.045). However, beta diversity using weighted UniFrac distances by principal coordinate analysis was not significantly different. ISI did not correlate with age, sex, IPI score, ECOG PS, serum LDH, number of lines of therapy or disease status (primary refractory vs. relapsed). We found an increased relative abundance of several bacterial families in patients with ongoing CR vs. NoCR group. We also analyzed the impact of gut microbial diversity on PFS and OS by stratifying the patients according to the tertile of ISI. The PFS of patients in the highest tertile of ISI values (n=11, median PFS not reached) was significantly higher compared to those with intermediate (n=11, median PFS = 2.82 months, HR 12.7, 95% CI 3.61 to 44.77, log-rank, p=0.001) or low (n=11, median PFS = 2.43 months, HR 12.9, 95% CI 3.68 to 45.75, log-rank, p=0.001) ISI values (Fig. 1B). High ISI values also correlated positively and significantly with OS (Fig. 1C). We did not observe any significant association between microbial diversity, either alpha or beta, and toxicities including CRS [(grade 0-1 (N=22) vs ≥2 (N=11)] and ICANS [(grade 0-2 (N=23) vs ≥3 (N=10)]. Additional details including impact of microbial composition will be presented at the meeting. Conclusion: Our results suggest that the baseline gut bacterial diversity may serve as a predictive biomarker for efficacy of anti-CD19 CAR T-cell therapy in r/r LBCL patients, with high diversity favoring improved outcomes. If confirmed in larger studies, future investigations should explore if the microbiome could be a modulator of CAR T-cell response in order to determine whether developing therapeutic interventional strategies to favorably alter the microbiome are warranted. Disclosures Nastoupil: TG Therapeutics: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Merck: Research Funding; Celgene: Honoraria, Research Funding; Gamida Cell: Honoraria; Gilead/KITE: Honoraria; Bayer: Honoraria; Pfizer: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Genentech, Inc.: Honoraria, Research Funding; LAM Therapeutics: Research Funding; Karus Therapeutics: Research Funding. Westin:Janssen: Consultancy, Research Funding; Amgen: Consultancy; Kite: Consultancy, Research Funding; Curis: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; BMS: Consultancy, Research Funding; 47: Research Funding; Morphosys: Consultancy, Research Funding; Astra Zeneca: Consultancy, Research Funding. Lee:Guidepoint Blogal: Consultancy; Celgene: Research Funding; Seattle Genetics: Research Funding; Oncternal Therapeutics: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Aptitude Health: Speakers Bureau; Takeda: Research Funding. Kebriaei:Kite: Other: Served on advisory board; Novartis: Other: Served on advisory board; Ziopharm: Other: Research Support; Amgen: Other: Research Support; Pfizer: Other: Served on advisory board; Jazz: Consultancy. Shpall:Takeda: Other: Licensing Agreement; Adaptimmune: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Zelluna: Membership on an entity's Board of Directors or advisory committees; Magenta: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Jain:Cellectis: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Precision Bioscienes: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Research Funding; Aprea Therapeutics: Research Funding; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Pfizer: Research Funding; ADC Therapeutics: Research Funding; Incyte: Research Funding; TG Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees. Green:KDAc Therapeutics: Current equity holder in private company. Flowers:Kite: Research Funding; Pharmacyclics/Janssen: Consultancy; Spectrum: Consultancy; Gilead: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Eastern Cooperative Oncology Group: Research Funding; AbbVie: Consultancy, Research Funding; Leukemia and Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Karyopharm: Consultancy; V Foundation: Research Funding; National Cancer Institute: Research Funding; Bayer: Consultancy; Denovo Biopharma: Consultancy; Cancer Prevention and Research Institute of Texas: Research Funding; Millennium/Takeda: Consultancy, Research Funding; BeiGene: Consultancy; OptumRx: Consultancy; Genentech, Inc./F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Burroughs Wellcome Fund: Research Funding; TG Therapeutics: Research Funding. Champlin:Genzyme: Speakers Bureau; DKMS America: Membership on an entity's Board of Directors or advisory committees; Cytonus: Consultancy; Omeros: Consultancy; Johnson and Johnson: Consultancy; Actinium: Consultancy; Takeda: Patents & Royalties. Wargo:Imedex, Dava Oncology, Omniprex, Illumina, gilead, PeerView, PET, MedImmune and Bristol-Myers Squibb: Honoraria, Speakers Bureau; Merck: Consultancy; Microbiome DX: Consultancy; GlaxoSmithKline: Consultancy, Research Funding; Roche/Genentech: Consultancy, Research Funding; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Astrazeneca: Consultancy; Biothera Pharmaceuticals: Consultancy. Neelapu:Celgene: Other: personal fees, Research Funding; Pfizer: Other: personal fees; Allogene Therapeutics: Other: personal fees, Research Funding; Cell Medica/Kuur: Other: personal fees; Novartis: Other: personal fees; N/A: Other; Precision Biosciences: Other: personal fees, Research Funding; Incyte: Other: personal fees; Bristol-Myers Squibb: Other: personal fees, Research Funding; Acerta: Research Funding; Takeda Pharmaceuticals: Patents & Royalties; Poseida: Research Funding; Cellectis: Research Funding; Karus Therapeutics: Research Funding; Calibr: Other; Legend Biotech: Other; Adicet Bio: Other; Merck: Other: personal fees, Research Funding; Kite, a Gilead Company: Other: personal fees, Research Funding; Unum Therapeutics: Other, Research Funding.

Description: We have previously described clinically relevant reductions in fecal microbiota diversity of patients undergoing allogeneic hematopoietic cell transplantation (allo-HCT). Recipients of high-dose chemotherapy and autologous hematopoietic cell transplantation (auto-HCT) incur similar antibiotic exposures and nutritional alterations. To characterize the fecal microbiota in the auto-HCT population, we analyzed 1,161 fecal samples collected from 534 adult recipients of auto-HCT for lymphoma, myeloma and amyloidosis in an observational study conducted at two transplant centers in the United States. Using 16S ribosomal gene sequencing, we assessed fecal microbiota diversity as measured by the inverse Simpson index, and composition. At both centers, early pre-transplant fecal microbiota diversity was lower than in healthy control subjects and decreased further during the course of transplantation. Loss of diversity and domination by specific bacterial taxa occurred during auto-HCT in patterns similar to allo-HCT. Above-median fecal intestinal diversity in the peri-engraftment period was associated with decreased risk of death or progression (PFS HR 0.46 [95% CI, 0.26-0.82], P=0.008), adjusting for disease and disease status. This suggests that further investigation into the health of the intestinal microbiota in auto-HCT patients and post-transplant outcomes should be undertaken.

Description: Background Identification of features is a critical task in microbiome studies that is complicated by the fact that microbial data are high dimensional and heterogeneous. Masked by the complexity of the data, the problem of separating signals (differential features between groups) from noise (features that are not differential between groups) becomes challenging and troublesome. For instance, when performing differential abundance tests, multiple testing adjustments tend to be overconservative, as the probability of a type I error (false positive) increases dramatically with the large numbers of hypotheses. Moreover, the grouping effect of interest can be obscured by heterogeneity. These factors can incorrectly lead to the conclusion that there are no differences in the microbiome compositions. Results We translate and represent the problem of identifying differential features, which are differential in two-group comparisons (e.g., treatment versus control), as a dynamic layout of separating the signal from its random background. More specifically, we progressively permute the grouping factor labels of the microbiome samples and perform multiple differential abundance tests in each scenario. We then compare the signal strength of the most differential features from the original data with their performance in permutations, and will observe a visually apparent decreasing trend if these features are true positives identified from the data. Simulations and applications on real data show that the proposed method creates a U-curve when plotting the number of significant features versus the proportion of mixing. The shape of the U-Curve can convey the strength of the overall association between the microbiome and the grouping factor. We also define a fragility index to measure the robustness of the discoveries. Finally, we recommend the identified features by comparing p-values in the observed data with p-values in the fully mixed data. Conclusions We have developed this into a user-friendly and efficient R-shiny tool with visualizations. By default, we use the Wilcoxon rank sum test to compute the p-values, since it is a robust nonparametric test. Our proposed method can also utilize p-values obtained from other testing methods, such as DESeq. This demonstrates the potential of the progressive permutation method to be extended to new settings.

Description: Efforts to improve the prognosis of steroid-resistant gut acute graft-versus-host-disease (SR-Gut-aGVHD) have suffered from poor understanding of its pathogenesis. Here we show that the pathogenesis of SR-Gut-aGVHD is associated with reduction of IFN-γ+ Th/Tc1 cells and preferential expansion of IL-17−IL-22+ Th/Tc22 cells. The IL-22 from Th/Tc22 cells causes dysbiosis in a Reg3γ-dependent manner. Transplantation of IFN-γ-deficient donor CD8+ T cells in the absence of CD4+ T cells produces a phenocopy of SR-Gut-aGVHD. IFN-γ deficiency in donor CD8+ T cells also leads to a PD-1-dependent depletion of intestinal protective CX3CR1hi mononuclear phagocytes (MNP), which also augments expansion of Tc22 cells. Supporting the dual regulation, simultaneous dysbiosis induction and depletion of CX3CR1hi MNP results in full-blown Gut-aGVHD. Our results thus provide insights into SR-Gut-aGVHD pathogenesis and suggest the potential efficacy of IL-22 antagonists and IFN-γ agonists in SR-Gut-aGVHD therapy.