Abstract: Introduction: Cellular immunotherapy with CD19-targeted chimeric antigen receptor (CAR) T cells has provided new therapeutic options for patients with high-risk hematologic malignancies. Following this therapy, patients may experience disease relapse or CAR-mediated toxicity due to cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS). Recent studies have confirmed that the intestinal microbiome can modulate the anti-tumor immune response to chemotherapy, immune checkpoint blockade, graft-versus-host disease after allogeneic hematopoietic cell transplantation, and adoptive cellular therapy. The contribution of the intestinal microbiome on the function of CAR T cells in vivo both with respect to their anti-tumor function and their propensity to induce toxicities is not known. Hence, in a multi-center study we analyzed the association between clinical outcomes and (1) antibiotic exposure prior to CAR T cell infusion and (2) the composition and diversity of the fecal microbiome. Methods and Results: We retrospectively collected clinical data and antibiotic exposures from patients with acute lymphoblastic leukemia (ALL, n=91) and non-Hodgkin lymphoma (NHL, n=137) treated with investigational or commercial CD19 CAR T cells at Memorial Sloan Kettering Cancer Center (MSK) and the University of Pennsylvania (Penn). We considered any antibiotic exposure between day -30 and the day of CAR T cell infusion. We focused our analysis on anaerobe-targeting antibiotics used in the setting of neutropenic fever: piperacillin-tazobactam, imipenem-cilastatin, and meropenem (here referred to as "P-I-M"). We found that forty-seven (20.6%) of 228 patients were exposed to P-I-M in the four weeks before CAR T cell infusion. Patient characteristics at the time of CAR T cell infusion were similar between the P-I-M-exposed and not-exposed groups, although a worse performance status was observed in patients with NHL treated with P-I-M. We found that overall survival (OS) was significantly decreased following CAR T cell infusion in patients exposed to P-I-M (Fig 1A; OS HR, 2.58; 95% CI, 1.68 - 3.98; p= & lt;0.001). A subgroup analysis of the patients with NHL also demonstrated decreased OS associated with P-I-M exposure whether the costimulatory domain was CD28 or 4-1BB (data not shown). Next, we queried whether patients exposed to P-I-M had more aggressive disease. We evaluated potential confounders for the findings in uni- and multi-variable models. Importantly, exposure to P-I-M remained a strong predictor of decreased OS (HR, 2.58; 95% CI, 1.55 - 4.3; p= & lt;0.001) (Table 1). Exposure to P-I-M was also associated with increased ICANS (p= 0.023) but not CRS (p= 0.058) in patients in the combined NHL and ALL cohort as well as in patients with NHL (CRS: p= 0.154, ICANS: p= 0.002) (data not shown). We also prospectively collected baseline fecal samples prior to cell infusion from CD19 CAR T cells recipients (n=48) at MSK and Penn. Samples were submitted for 16S RNA sequencing of the V4-V5 region on the Illumina MiSeq platform and the amplicon sequence variants (ASVs) were annotated according to the NCBI 16S database using BLAST. In comparison to healthy controls (n=30), we found that alpha-diversity was significantly lower in fecal samples from CAR T cell patients (p= 0.0023, Fig 1B) and the composition of fecal samples was significantly different (p= & lt;0.001, Fig 1C). Finally, linear discriminant analysis effect size (LEfSe) identified an increased abundance of Lachnospiraceae, Ruminococcaceae, and Bacteroidaceae in patients who achieved a Day 100 complete response (CR) and those who experienced CAR-mediated toxicity (data not shown). Conclusion: Our results suggest that exposure to antibiotics, in particular P-I-M, in the four weeks before therapy was associated with worse survival. Profiling of the baseline fecal microbiome samples by 16S revealed that CD19 CAR T cell patients presented with evidence of an altered fecal microbiome as measured by lower alpha-diversity and a composition that is distinct from that of healthy controls. Finally, we identified bacterial taxa that were associated with Day 100 CR and CAR-mediated toxicity. Our findings indicate that the intestinal microbiome can affect the efficacy of CD19 CAR T cell therapy and provides a rationale to target the intestinal microbiome to improve clinical outcomes of patients treated with cellular therapies. Figure 1 Figure 1. Disclosures Smith: Janssen: Consultancy, Honoraria. Gomes: Xbiome: Current Employment. Schluter: Postbiotics Plus LLC: Other: cofounder. Park: Kura Oncology: Consultancy; BMS: Consultancy; Servier: Consultancy; Autolus: Consultancy; Curocel: Consultancy; Artiva: Consultancy; Kite Pharma: Consultancy; Amgen: Consultancy; Novartis: Consultancy; Affyimmune: Consultancy; Intellia: Consultancy; Innate Pharma: Consultancy; Minerva: Consultancy; PrecisionBio: Consultancy. Palomba: Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Kite Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Jain: Targeted Healthcare Communications: Consultancy; Bristol Myers Squibb: Other: for advisory board participation; CareDx: Other: for advisory board participation; CTI Biopharma: Research Funding; Syneos Health: Research Funding. Pennisi: Gilead Sciences: Consultancy. Perales: Miltenyi Biotec: Honoraria, Other; Novartis: Honoraria, Other; Omeros: Honoraria; NexImmune: Honoraria; Bristol-Myers Squibb: Honoraria; Merck: Honoraria; Celgene: Honoraria; Takeda: Honoraria; Kite/Gilead: Honoraria, Other; Medigene: Honoraria; Nektar Therapeutics: Honoraria, Other; Cidara: Honoraria; Servier: Honoraria; Sellas Life Sciences: Honoraria; Karyopharm: Honoraria; MorphoSys: Honoraria; Equilium: Honoraria; Incyte: Honoraria, Other. Garfall: Amgen: Honoraria; CRISPR Therapeutics: Research Funding; GlaxoSmithKline: Honoraria; Janssen: Honoraria, Research Funding; Novartis: Research Funding; Tmunity: Research Funding. Landsburg: Triphase: Research Funding; Morphosys: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Other: DSMB member; Incyte: Membership on an entity's Board of Directors or advisory committees; ADCT: Membership on an entity's Board of Directors or advisory committees; Curis: Research Funding; Takeda: Research Funding. Gerson: Kite: Consultancy; Pharmacyclics: Consultancy; Abbvie: Consultancy; TG Therapeutics: Consultancy. Svoboda: Imbrium: Consultancy; Genmab: Consultancy; Astra Zeneca: Consultancy, Research Funding; Atara: Consultancy; BMS: Consultancy, Research Funding; Adaptive: Consultancy, Research Funding; Incyte: Research Funding; Merck: Research Funding; Pharmacyclics: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; TG: Research Funding. Giralt: AMGEN: Membership on an entity's Board of Directors or advisory committees; PFIZER: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; SANOFI: Membership on an entity's Board of Directors or advisory committees; CELGENE: Membership on an entity's Board of Directors or advisory committees; JAZZ: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; JENSENN: Membership on an entity's Board of Directors or advisory committees; Actinnum: Membership on an entity's Board of Directors or advisory committees. Gill: Interius Biotherapeutics: Current holder of stock options in a privately-held company, Research Funding; Novartis: Other: licensed intellectual property, Research Funding; Carisma Therapeutics: Current holder of stock options in a privately-held company, Research Funding. Rivière: FloDesign Sonics: Other: Provision of Services; Centre for Commercialization of Cancer Immunotherapy: Other: Provision of Services; Fate Therapeutics: Other: Provision of Services, Patents & Royalties; The Georgia Tech Research Corporation (GTRC): Other: Provision of Services (uncompensated); Juno Therapeutics: Patents & Royalties. Porter: Kite/Gilead: Membership on an entity's Board of Directors or advisory committees; Wiley and Sons Publishing: Honoraria; Tmunity: Patents & Royalties; Novartis: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; ASH: Membership on an entity's Board of Directors or advisory committees; DeCart: Membership on an entity's Board of Directors or advisory committees; Genentech: Current equity holder in publicly-traded company, Ended employment in the past 24 months; American Society for Transplantation and Cellular Therapy: Honoraria; National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees. Schuster: Abbvie: Consultancy, Research Funding; Acerta Pharma: Consultancy; AstraZeneca: Consultancy; Adaptive Biotechnologies: Research Funding; BeiGene: Consultancy; Celgene: Consultancy, Honoraria, Research Funding; DTRM: Research Funding; Genetech: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Incyte: Research Funding; Juno Theraputics: Consultancy, Research Funding; Loxo Oncology: Consultancy; Merck: Research Funding; Nordic Nanovector: Consultancy; Novartis: Consultancy, Honoraria, Patents & Royalties, Research Funding; Pharmaclcyclics: Research Funding; Tessa Theraputics: Consultancy; TG Theraputics: Research Funding. Sadelain: NHLBI Gene Therapy Resource Program: Other: Provision of Services (uncompensated); Fate Therapeutics: Other: Provision of Services (uncompensated), Patents & Royalties; Atara Biotherapeutics: Patents & Royalties; Ceramedix: Patents & Royalties; Mnemo Therapeutics: Patents & Royalties; Takeda Pharmaceuticals: Other: Provision of Services, Patents & Royalties; St. Jude Children's Research Hospital: Other: Provision of Services; Juno Therapeutics: Patents & Royalties; Minerva Biotechnologies: Patents & Royalties. Frey: Novartis: Research Funding; Kite Pharma: Consultancy; Sana Biotechnology: Consultancy; Syndax Pharmaceuticals: Consultancy. Brentjens: Gracell Biotechnologies, Inc: Consultancy, Ended employment in the past 24 months; BMS: Consultancy, Patents & Royalties, Research Funding; sanofi: Patents & Royalties; Caribou: Patents & Royalties. June: AC Immune, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Consultancy; Novartis: Patents & Royalties; Tmunity, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Current equity holder in publicly-traded company. Pamer: Diversigen: Other: Advisory board; Bristol Myers Squibb, Celgene, Seres Therapeutics, MedImmune, Novartis and Ferring Pharmaceuticals: Honoraria. Peled: DaVolterra: Consultancy; MaaT Pharma: Consultancy; CSL Behring: Consultancy; Seres Therapeutics: Research Funding. Ruella: BMS, BAYER, GSK: Consultancy; Novartis: Patents & Royalties; AbClon: Consultancy, Research Funding; Tmunity: Patents & Royalties; viTToria biotherapeutics: Research Funding. van den Brink: WindMILTherapeutics: Honoraria; Pluto Therapeutics: Current holder of stock options in a privately-held company, Other: has consulted, received honorarium from or participated in advisory boards ; Priothera: Research Funding; Forty-Seven, Inc.: Honoraria; MagentaTherapeutics: Honoraria; GlaskoSmithKline: Other: has consulted, received honorarium from or participated in advisory boards; Ceramedix: Other: has consulted, received honorarium from or participated in advisory boards ; Merck & Co, Inc: Honoraria; Synthekine (Spouse): Other: has consulted, received honorarium from or participated in advisory boards; Kite Pharmaceuticals: Other; Amgen: Honoraria; Frazier Healthcare Partners: Honoraria; Seres: Other: Honorarium, Intellectual Property Rights, Research Fundingand Stock Options; Rheos: Honoraria; Therakos: Honoraria; Jazz Pharmaceuticals: Honoraria; Notch Therapeutics: Honoraria; Nektar Therapeutics: Honoraria; Wolters Kluwer: Patents & Royalties; Juno Therapeutics: Other; DKMS (nonprofit): Other; Pharmacyclics: Other; Da Volterra: Other: has consulted, received honorarium from or participated in advisory boards; Novartis (Spouse): Other: has consulted, received honorarium from or participated in advisory boards; Lygenesis: Other: has consulted, received honorarium from or participated in advisory boards .

Abstract: Rapid advancements in cancer genomics and in the development of targeted therapies provide expanding opportunities to use genomic profiling to improve patient outcomes. However, most patients do not have access to clinical genomic profiling platforms, and currently available assays capture a small set of known mutations or translocations tailored to specific tumor types. The spectrum of somatic alterations in leukemia, lymphoma, and myeloma includes substitutions, insertions/deletions (indels), copy number alterations (CNAs) and gene fusions; no current assay captures the different types of alterations in a single clinical genomic test. We developed a novel, CLIA-certified next-generation sequencing-based assay designed to provide targeted assessment of the genomic landscape of hematologic malignancies, including identification of all classes of genomic alterations using archived FFPE, blood and bone marrow aspirate samples with high accuracy in a clinically relevant timeframe. Methods DNA and RNA were successfully extracted from 350/362 (96%) specimens from 319 patients, including 57 FFPE samples, 150 blood samples and 142 bone marrow aspirates. The initial sample cohort included 20 ALL, 83 AML, 53 CLL, 57 DLBCL, 48 MDS, 32 MPN and 57 multiple myeloma samples. Adaptor ligated sequencing libraries were captured by solution hybridization using a custom bait-set targeting 374 cancer-related genes and 24 frequently rearranged genes by DNA-seq, and 258 frequently-rearranged genes by RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq) in a CLIA-certified laboratory (Foundation Medicine), averaging 590x for DNA and 〉 20M total pairs for RNA, to enable the sensitive and specific detection of substitutions, indels, CNAs and gene fusions. Results Sufficient tumor content (≥20%) was present in 317/350 (91%) of the samples (289/319 patients), and a total of 885 alterations were identified (3.1 alterations per sample), including 555 base substitutions, 213 indels, 36 splice mutations, 51 CNAs and 36 fusions/rearrangements. The most frequent alterations across all hematologic malignancies included mutations in TP53 (9%), ASXL1, KRAS, NRAS, IDH2, TET2, SF3B1, JAK2, MLL2, DNMT3A, RUNX1, and SRSF2 (2-5% each); FLT3 ITDs (2%); MLL PTDs (1%); homozygous loss of CDKN2A/B (3%); and focal amplification of REL (1%). Rearrangements in BCL2/6, MYC, MLL, MLL2, NOTCH2, ABL1 and ETV6 were identified using DNA and RNA targeted sequencing, demonstrating the ability of this platform to reliably identify gene fusions with immediate clinical relevance. Overall high accuracy of the assay for substitutions, indels and CNAs was previously demonstrated by extensive validation studies achieving 95-99% across alteration types with high specificity (PPV 〉 99%) [Frampton et al, Nat Biotech, in press]. Comparison of detected alterations to previous molecular testing for JAK2, NPM1, IDH2, FLT3 and CEBPA in MPN/AML samples demonstrated 97% sensitivity (33/34) in our ability to identify known mutations in these clinical samples. We identified additional clinically relevant mutations that were not detected using standard clinical assays, including alterations in JAK2, FLT3 and IDH2, which can inform therapeutic decisions. The use of our content rich sequencing platform allowed us to identify clinically actionable mutations in hematologic malignancies, including IDH1/2 mutations in a spectrum of myeloid/lymphoid malignancies, recurrent BRAF mutations in refractory CLL and myeloma, and mutations in the JAK-STAT signaling pathway in diffuse-large B cell lymphoma. These results demonstrate that a targeted sequencing platform which includes a large set of known disease alleles/therapeutic targets can identify mutations with therapeutic relevance in disease contexts where gene-specific assays are not currently performed in the clinical setting. Conclusions We have developed a sensitive, high throughput assay to detect somatic alterations in hundreds of genes known to be deregulated in hematologic malignancies, which can be used for clinical sequencing of frozen/paraffin samples. We demonstrate that targeted DNA and RNA sequencing can be used to identify all classes of genomic alterations in genes known to be therapeutic targets in a broad spectrum of hematologic malignancies. Disclosures: Lipson: Foundation Medicine, Inc: Employment, Equity Ownership. Nahas:Foundation Medicine, Inc: Employment, Equity Ownership. Otto:Foundation Medicine, Inc: Employment, Equity Ownership. Yelensky:Foundation Medicine, Inc: Employment, Equity Ownership. Wang:Foundation Medicine, Inc: Employment, Equity Ownership. He:Foundation Medicine, Inc: Employment, Equity Ownership. Rampal:Foundation Medicine: Consultancy. Brennan:Foundation Medicine, Inc: Employment, Equity Ownership. Brennan:Foundation Medicine, Inc: Employment, Equity Ownership. Young:Foundation Medicine, Inc: Employment, Equity Ownership. Donahue:Foundation Medicine, Inc: Employment, Equity Ownership. Sanford:Foundation Medicine, Inc: Employment, Equity Ownership. Greenbowe:Foundation Medicine, Inc: Employment, Equity Ownership. Frampton:Foundation Medicine, Inc: Employment, Equity Ownership. Fichtenholtz:Foundation Medicine, Inc: Employment, Equity Ownership. Young:Foundation Medicine, Inc: Employment, Equity Ownership. Erlich:Foundation Medicine, Inc: Employment, Equity Ownership. Parker:Foundation Medicine, Inc: Employment, Equity Ownership. Ross:Foundation Medicine, Inc: Employment, Equity Ownership. Stephens:Foundation Medicine, Inc: Employment, Equity Ownership. Miller:Foundation Medicine, Inc: Employment, Equity Ownership. Levine:Foundation Medicine, Inc: Consultancy.

Abstract: Background Fecal microbiota composition is associated with important outcomes after allo-HCT including survival, relapse, GVHD, and infections. We previously demonstrated in a multicenter observational study that HCT patients present with fecal microbiota configurations that have lower diversity and are distinct from those of healthy individuals, and that pre-HCT microbiota injury predicts poor overall survival. Here, we hypothesized that pre-HCT fecal microbiota features predict development of critical illness post-HCT. Methods We analyzed 828 adults who received a first allo-HCT from 2009 to 2017 at a single institution who had an evaluable fecal sample in our biobank collected within the 10 days prior to cell infusion. The patients were heterogeneous with respect to transplant indication, conditioning intensity, graft source (cord blood, peripheral blood, marrow) and graft manipulation (CD34-selection). The V4-V5 regions of 16S rRNA genes of DNA extracted from fecal samples were amplified and annotated taxonomically. The outcome of interest was time to ICU admission, which was assessed using survival-analysis methods. The reason for admission to the ICU was evaluated for each subject. Results Seventy-five (9%) patients were admitted to the intensive care unit (ICU) between the day of cell infusion and day +50; the peak incidence of ICU admission occurred on day +10. The most common indications for ICU admission were respiratory failure (65%) and infection (27%). Patients were stratified based on fecal microbiota diversity, as assessed by 16S sequencing of stool samples collected prior to transplantation, into high (inverse Simpson index ≥4) and low ( 〈 4) diversity groups following a previously-published cutoff. Patients with low diversity pre-HCT had a strikingly higher risk of ICU admission than those with high diversity (HR 2.38 [95% CI 1.5-3.7], p 〈 0.001, see the Figure). This association remained significant in a multivariate Cox proportional hazard model that accounted for conditioning intensity, graft source, graft manipulation, and the HCT-CI comorbidity index (multivariate p = 0.003). HCT-CI score was also an independent predictor of ICU admission. The association between pre-HCT fecal diversity and ICU admission was also significant when the outcome definition was limited to ICU transfers for reason of respiratory failure or sepsis (to the exclusion of such indications as hemorrhage, anaphylaxis, or isolated dysfunctions of the cardiac, renal, or neurological systems). Conclusion Pre-transplant fecal microbial diversity is an independent predictor of intensive-care-requiring critical illness in the post-HCT period. These observations highlight the pre-HCT period as a window of opportunity to (a) assess microbiota injury in conjunction with comorbidity evaluation, (b) inform selection of antibiotic prophylaxis, gut-decontamination, GVHD-prophylaxis, or conditioning regimens, and (c) intervene with microbiota injury-remediation or prevention strategies. Figure Disclosures Brereton: Seres Therapeutics: Other: Salary Support. Clurman:Seres Therapeutics: Research Funding. Slingerland:Seres Therapeutics: Other: Salary supported by Seres funding. Shah:Janssen Pharmaceutica: Research Funding; Amgen: Research Funding. Scordo:McKinsey & Company: Consultancy; Angiocrine Bioscience, Inc.: Consultancy. Politikos:Angiocrine Bioscience Inc: Research Funding. Gyurkocza:Actinium Pharmaceuticals: Research Funding. Barker:Angiocrine Bioscience Inc: Research Funding; Gamida Cell: Research Funding; Merck: Research Funding. Perales:Kyte/Gilead: Research Funding; Miltenyi: Research Funding; MolMed: Membership on an entity's Board of Directors or advisory committees; NexImmune: Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Nektar Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Omeros: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria; Medigene: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees. Giralt:Amgen: Consultancy, Research Funding; Spectrum Pharmaceuticals: Consultancy; Actinium: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Kite: Consultancy; Johnson & Johnson: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; Miltenyi: Research Funding; Takeda: Consultancy, Research Funding; Novartis: Consultancy. van den Brink:Merck & Co, Inc.: Consultancy, Honoraria; Acute Leukemia Forum (ALF): Consultancy, Honoraria; Magenta and DKMS Medical Council: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Other: Licensing; Amgen: Consultancy, Honoraria; Therakos: Consultancy, Honoraria; Seres Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Flagship Ventures: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Evelo: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria. Pamer:Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Seres Therapeutics: Honoraria, Patents & Royalties; MedImmune: Honoraria; Novartis: Honoraria; Ferring Pharmaceuticals: Honoraria. Peled:Seres Therapeutics: Research Funding.

Abstract: To obtain the large amount of T cells required for adoptive immunotherapy in a clinical setting, T-cell lifespan extension by human telomerase reverse transcriptase (hTERT) transduction is of particular interest. However, constitutive expression of hTERT is associated with malignant transformation and thus warrants a detailed evaluation of the safety of hTERT-transduced T cells before clinical application. In view of this, we performed an extensive cytogenetic analysis of hTERT-transduced MART-1 (melanoma antigen recognized by T cell 1)–and human papillomavirus type 16 (HPV16) E7–specific human CD8+ cytotoxic T lymphocytes (CTLs), reactive against melanoma and cervical carcinoma, respectively. Our results, obtained by (spectral) karyotyping and array comparative genomic hybridization, showed the development of minor chromosomal aberrations in an hTERT-transduced MART-1–specific CTL clone, whereas severe clonal aberrations were detected in an hTERT-transduced HPV16 E7–specific CTL clone. Furthermore, hTERT transduction did not protect CTLs from immunosenescence, because the HPV16 E7–specific, hTERT-transduced CTL clone showed a decreased functional activity on prolonged culture. Although the general frequency of major chromosomal aberrations in hTERT-transduced CTLs and the in vivo significance of our observations remain still unclear at this point, the currently available data suggest that clinical application of hTERT-transduced CTLs should proceed with caution.

Abstract: The intestinal microbiota undergoes major perturbations during allogeneic hematopoietic stem cell transplantation (allo-HCT), and low microbiota diversity during this period is associated with an increased risk of graft-versus-host disease and mortality. Identifying the environmental variables that might impact intestinal microbiota could inform strategies to maintain and restore a healthy microbiota state. However, understanding microbial dynamics is challenging due to the high-dimensional nature of microbiota data. Here, we simplified complex microbiota communities into clusters and investigated the dynamics under different conditions in terms of transition probabilities in a large dataset of allo-HCT fecal specimens (Fig. a). The bacterial compositions of 7,930 samples from 1,076 allo-HCT patients were determined by 16S rRNA deep-sequencing. Samples were then clustered into 10 distinct states by k-means clustering of a Bray-Curtis β-diversity matrix (Fig. b). These clusters captured variations in diversity and microbiota compositions (Fig. c-d). Cluster 1 represented a high-diversity state, and Lachnospiraceae and other Clostridiales were the most commonly observed taxa in this cluster. The low-diversity clusters 9 and 10 consisted mostly of Streptococcus-dominated and Enterococcus-dominated samples, respectively. We utilized a regression-based predictive approach to model cluster transition probabilities in terms of a weight for remaining in the same cluster over time (self-weight) and a weight for attracting transitions from other clusters over time (attractor-weight). Controlling for the effect of time, the weights measured the contribution of different environmental exposures to intestinal microbial behaviors. A negative parameter coefficient indicates cluster destabilization or decreased cluster transition likelihood in the case of self-weights and attractor-weights, respectively. We evaluated the impact of the 3 most commonly used non-prophylactic antibacterial drugs using 2359 daily samples from 385 allo-HCT patients collected between day -14 to 7 relative to transplant. High-diversity cluster 1 was significantly destabilized by piperacillin-tazobactam (pip-tazo) exposure (β = -0.87, P & lt; 0.05). Meanwhile, exposure to cefepime and meropenem did not have a significant effect on cluster 1 stability (Fig. e). Exposure to pip-tazo also increased the transition probability to the Streptococcus-dominated cluster 9 (β = 1.83, P & lt; 0.001), while cefepime (β = 2.69, P & lt; 0.05) and meropenem (β = 1.96, P & lt; 0.01) exposure favored transitions to the Enterococcus-dominant cluster 10. These results suggest that antibiotic exposures are associated with different composition outcomes depending on patient microbiota states during transplant period. In a small subset of 242 daily samples from 46 allo-HCT patients with detailed daily dietary information, we observed that an increase in total protein intake (range = 0-137.4g; median = 36g) was associated with low self-maintenance of cluster 1 (β = -1.29, P & lt; 0.05), while an increase in total fat intake (range = 0-183.3g; median = 34.5g) improved cluster 1 stability (β = 1.44, P & lt; 0.05). Overall, dietary intakes could also modulate transition probabilities between microbial communities in allo-HCT patients. While prior studies have assessed specific bacterial taxa or diversity indices as biomarkers of clinical outcomes, here we considered the entire intestinal communities and demonstrated that various environmental exposures were associated with changes in microbiota composition during allo-HCT. Using a regression-based approach that predicts cluster transitions in response to environmental conditions, we found that pip-tazo exposure was associated with destabilization of a high-diversity state and increased transitions to a Streptococcus-dominated state, while cefepime and meropenem exposure did not disrupt high-diversity microbial community. Furthermore, increased protein intake was also associated with disruption to the high-diversity cluster, while increased fat intake strengthened the maintenance of a diverse and healthy microbial community. Ultimately, this computation framework aims to inform strategies to optimize treatment plans for allo-HCT patients to maximize a healthy gut microbiota state and clinical outcomes. Disclosures Gomes: Seres Therapeutics: Other: Part of Salary. Peled:Seres Therapeutics: Research Funding. Slingerland:Seres Therapeutics: Other: Salary supported by Seres funding. Clurman:Seres Therapeutics: Research Funding. Giralt:Celgene: Consultancy, Research Funding; Takeda: Consultancy; Sanofi: Consultancy, Research Funding; Amgen: Consultancy, Research Funding. Perales:Bristol-Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Honoraria, Membership on an entity's Board of Directors or advisory committees; NexImmune: Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Nektar Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Omeros: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria; Medigene: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Kyte/Gilead: Research Funding; Miltenyi: Research Funding; MolMed: Membership on an entity's Board of Directors or advisory committees. Pamer:MedImmune: Honoraria; Seres Therapeutics: Honoraria, Patents & Royalties; Bristol Myers Squibb: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Ferring Pharmaceuticals: Honoraria. van den Brink:Acute Leukemia Forum (ALF): Consultancy, Honoraria; Seres Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Flagship Ventures: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Evelo: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria; Therakos: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Merck & Co, Inc.: Consultancy, Honoraria; Juno Therapeutics: Other: Licensing; Magenta and DKMS Medical Council: Membership on an entity's Board of Directors or advisory committees.

Abstract: Introduction: Peripheral T-cell lymphomas (PTCL) comprise a heterogenous group of mature T-cell neoplasms with generally an unfavorable prognosis. Presentation of PTCL with stage I(E) disease, according to the Ann Arbor classification, is uncommon. Clinical trials in diffuse large B-cell lymphoma (DLBCL) support the use of an abbreviated treatment with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone combined with radiotherapy (combined modality therapy (CMT)) in case of stage I(E) disease. CMT in stage I(E) PTCL has been adapted in daily practice, but clinical trials are lacking. A recent population-based study conducted in Scandinavia indicated that the outcome in patients with limited stage PTCL is as poor as in patients with extensive disease. However, the outcomes of different treatment modalities were not analyzed. Aim: The aim of this nationwide population-based cohort study is to describe first-line treatment and outcome of patients with stage I(E) PTCL in comparison to advanced stage PTCL. Methods: All newly diagnosed patients ≥ 18 years with stage I(E) PTCL who were diagnosed in 1989-2018 were identified in the Netherlands Cancer Registry (NCR). Survival follow-up was available through February 1, 2021. The PTCL subgroups analyzed included anaplastic large cell lymphoma (ALCL), angioimmunoblastic T-cell lymphoma (AITL), enteropathy associated T-cell lymphoma (EATL) and peripheral T-cell lymphoma NOS (PTCL NOS). Patients were categorized according to treatment regimen, i.e. chemotherapy, chemotherapy followed by autologous stem cell transplantation (ASCT), radiotherapy, CMT, and no/other therapy. Calendar period analyses (1989-1999 and 2000-2018) were conducted to assess trends in primary therapy and overall survival (OS) over time. The calendar periods were defined according to the implementation of CMT in patients with limited stage DLBCL from approximately 2000 onward in the Netherlands. The primary endpoint was OS, defined as all-cause-death post-diagnosis. Results: From 1989 to 2018, 854 patients with a median age of 62 years were diagnosed with stage I(E) PTCL, accounting for 19% of all PTCL diagnoses. In stage I(E), the predominant PTCL subtype was PTCL NOS (40%, n=343). Furthermore, 26% (n=222) of patients were diagnosed with ALCL, 3% (n=28) with AITL, 11% (n=93) with EATL and 20% (n=168) with other histological subtypes. In contrast, for patients with advanced stage disease, 42% was diagnosed with PTCL NOS, 23% with ALCL, 24% with AITL, 6% with EATL and 5% with other histological subtypes . To evaluate treatment and survival outcome in patients with stage I(E) PTCL, patients with ALCL, AITL, PTCL NOS and EATL were included for further analyses (n=686). Patients with ALCL, AITL and PTCL NOS were most commonly treated with CMT (n=164; 28%) or chemotherapy only (n=154; 26%). Only 10 patients (1%) received chemotherapy followed by ASCT. The remaining patients were treated with either radiotherapy only (n=116; 20%) or received other/no therapy (n=149; 25%). More patients were treated with CMT in 2000-2018 as compared to 1989-1999 (36% versus 17%, p & lt;0.01). Patients with EATL were most commonly treated with either chemotherapy only or other/no treatment (both n=45; 48%), while 3 patients (4%) received chemotherapy in combination with ASCT (n=2) or CMT (n=1). In EATL, no differences in treatment strategy were observed over time. Overall, 5-year OS for all stage I(E) PTCL was 52%. There was no significant difference in outcome between the two time periods (53% vs. 52%, p=0.92). EATL had a worse prognosis when compared to ALCL, AITL and PTCL NOS (5-year OS 15% vs. 58%, respectively; p & lt;0.01). Five-year OS was significantly higher for patients with ALCL, AITL and PTCL NOS treated with CMT (71%) as compared to patients treated with either chemotherapy alone or with radiotherapy alone (52%, and 53%, respectively; p & lt;0.01). Independent predictors for poor prognosis were higher age, male gender and EATL subtype whereas CMT was associated with a lower risk of mortality when evaluated for in multivariable analysis. Conclusions: For stage I(E) ALCL, AITL and PTCL NOS, 5-year OS is 58%. This compares favorably to the reported outcomes in advanced stage disease. EATL, even when presenting with limited stage disease, is associated with a very poor prognosis. CMT is associated with superior OS when compared to either chemotherapy or radiotherapy alone. Disclosures Van Der Poel: Roche, Janssen, Abbvie: Honoraria. Kersten: Kite/Gilead: Consultancy, Honoraria, Research Funding; BMS/Celgene: Consultancy; Miltenyi Biotech: Consultancy; Novartis: Consultancy, Honoraria; Roche: Honoraria; Takeda: Consultancy. Mutsaers: BMS: Consultancy; AstraZeneca: Research Funding. Woei-a-Jin: University Hospitals Leuven, Belgium: Current Employment; Recordati: Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding; Kyowa Kirin: Research Funding.

Abstract: Studies of the relationship between the gastrointestinal microbiota and outcomes in allogeneic hematopoietic stem cell transplantation (allo-HCT) have thus far largely focused on early complications, predominantly infection and acute graft-versus-host disease (GVHD). We examined the potential relationship of the microbiome with chronic GVHD (cGVHD) by analyzing stool and plasma samples collected late after allo-HCT using a case-control study design. We found lower circulating concentrations of the microbe-derived short-chain fatty acids (SCFAs) propionate and butyrate in day 100 plasma samples from patients who developed cGVHD, compared with those who remained free of this complication, in the initial case-control cohort of transplant patients and in a further cross-sectional cohort from an independent transplant center. An additional cross-sectional patient cohort from a third transplant center was analyzed; however, serum (rather than plasma) was available, and the differences in SCFAs observed in the plasma samples were not recapitulated. In sum, our findings from the primary case-control cohort and 1 of 2 cross-sectional cohorts explored suggest that the gastrointestinal microbiome may exert immunomodulatory effects in allo-HCT patients at least in part due to control of systemic concentrations of microbe-derived SCFAs.

Abstract: To ascertain the therapeutic potential of non-TBI-based conditioning for CD34+ HPC-selected , T cell-depleted allografts, we conducted a trial comparing our standard regimen, arm (A) 1375cGy HFTBI+ thiotepa,5 mg/kg/day x 2 days + cyclophosphamide 60 mg/kg/day x 2 days vs. arm (B) Busulfex 0.8 mg/kg/6h x12 (dose adjusted) + melphalan 70 mg/kg/day x 2 + fludarabine 25 mg/m2/day x5 and arm (C) Clofarabine 20 mg/m2/day x 5 + melphalan 70 mg/m2/day x 2 + thiotepa 5 mg/kg/day x2, as preparation for T-cell depleted CD34+ PBSC transplants from GCSF-mobilized leukocytes fractionated with the CliniMACS device. Primary endpoints were engraftment, GVHD, transplant-related mortality (TRM) and 2 yr OS and DFS (Confer Table). Stratification of pts to arms A (standard), B or C was based on the patient’s disease, disease stage and clinical factors such as age, prior therapy or comorbidities enhancing risks of TBI. Arm B was the non-TBI arm predominantly used for myeloid and Arm C for lymphoid malignancies. Prior to transplant, recipients of HLA-matched or non-identical transplants received rabbit thymoglobulin at 2.5 mg/kg/day x2 or 3 days respectively, to prevent graft failure. No GVHD drug prophylaxis was given post transplant. A total of 181 consecutive patients, accrued between 5/13/2010 and 6/12/2013, were analyzed (81 in arm A, 78 in arm B, 22 in arm C). These pts have been followed for a median of 12.1 months. Donors were related or unrelated and HLA-matched for 74% of the patients and 1-2 HLA allele disparate for 26%. Median age for the entire group was 50.5 years, with older pts predominating in the non-TBI groups (medians arm A ,31.9 yrs; arm B , 61.9 yrs; arm C, 44.6 yrs). The CD34+ PBSC transplants provided a mean dose of 9.7x106 CD34+ progenitors/Kg (range 1.4-89.7) and 4.5x103 CD3+ T-cells/Kg (range 0.6-25.3). All pts engrafted; but 2 pts (2.5%) in arm B experienced late graft failure, one of whom was reconstituted after a secondary graft. Overall the incidence of grade II-IV acute GVHD was 18%, and 14% for recipients of HLA-matched grafts. TRM at 1 year was 10% in Arm A, and 15% in Arms B and C. Two year OS and DFS for each arm are: arm A, 66.7% and 58.4%; arm B 62.3% and 59.5%; arm C 52% and 53%. For the 101 pts who received standard risk transplants (i.e., pts with high risk forms of AML, ALL or NHL in 1o CR, AML in 2o CR, MDS RA/RCMD, CML in 1o CP or MM in CR1, VGPR or first PR ), 2 year OS and DFS are: arm A 68% and 62%; arm B 67% and 66%; arm C 86% and 86%, with relapse rates at 2 yrs of: arm A 23%, arm B 15%, and arm C 14%. These results thus identify two non-TBI-based conditioning regimens that secure consistent engraftment of rigorously T-cell depleted allogeneic HSCT and can yield favorable long-term DFS and OS with low incidences of GVHD and relapse. Table 1 Overall Results Graft 1 Year Acute GVHD II – IV 2 Year PTs ENG Failure TRM ALL HLA-Matched O.S. DFS ARM A 81 81 0 10% 23% 17% 66.7% 58.4% ARM B 78 78 2 15% 12.3% 13% 62.3% 59.5% ARM C 22 22 0 15% 27% 20% 52% 53% Disclosures: No relevant conflicts of interest to declare.