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Microbiota Injury in Auto-HCT Is Frequent, Occurs across Geography, and Is Comparable to That Observed in Allo-HCT
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Abstracts / Biol Blood Marrow Transplant 25 (2019) S7 S75
busulfan conditioning (yes/no). We then examined the 5 variables with the highest correlation from the regression modeling as predictors of a patient eventually developing severe or very severe SOS. Results: 55 subjects were enrolled. Most patients were male (n=33, 55%) with a median age of 8 (range 0-20) years. 21 (35%) patients developed SOS, 15 (25%) had severe or greater disease by EBMT criteria. Busulfan use is associated with an average increase of 0.6 in EBMT grade, and a 100 cm/s decrease in main portal vein velocity is associated with an average increase of 1.3 in EBMT grade. Hepatomegaly, ascites, and higher SWE stiffness also appear to be associated with greater SOS severity (p < .10). The best predictor of patients developing severe SOS was increased SWE velocity (AUC = .90). A cutoff value of >1.95 m/s was 87.5% sensitive and 85% specific for severe or very severe SOS. Seven of eight patients with severe or very severe SOS had SWE >1.95 m/s two to thirteen days before the date of severe SOS grading. Conclusion: In a group of 12 clinical and ultrasound variables, SWE velocity was an excellent predictor of the patient developing severe SOS. SWE was able to predict severe SOS 2 to 13 days before the EBMT criteria with 87.5% sensitivity and 85% specificity.
Center, Sloan Kettering Institute, New York, NY; 3 Department of Medicine, Adult Bone Marrow Transplant Service, Memorial Sloan Kettering Cancer Center, New York, NY; 4 Division of Hematologic Malignancies and Cellular Therapy, Duke University Medical Center, Durham, NC; 5 Departments of Genomic Medicine and Stem Cell Transplantation Cellular Therapy, MD Anderson Cancer Center, University of Texas, Houston, TX; 6 Infectious Disease Service, Lucille Castori Center for Microbes, Inflammation and Cancer, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY Intestinal microbiota injuries in allo-HCT patients are characterized by loss of a-diversity and domination of microbial communities by single organisms. These injury patterns are associated with poor survival after allo-HCT and are likely attributable to antibiotic exposure, nutritional alterations, and regimen-related mucosal injury. As recipients of auto-HCT patients have similar exposures, we hypothesized that similar patterns of dysbiosis occur in auto-HCT patients. We present the first analysis of intestinal microbiota composition in autoHCT patients at two independent institutions. From a prospectively collected cohort, we retrospectively identified 365 patients (median age 60) who received auto-HCT (May 2009 to Feb. 2018) at two transplant centers (MSK n = 316; Duke n = 49) with heterogeneous conditioning regimens, pre-HCT remission status, and diagnoses: 179 (49%) myeloma, 153 (42%) lymphoma, and 33 (9%) other diseases. 857 samples collected approximately weekly peri-transplant were 16S sequenced (V4-V5 region, Illumina platform) at a central laboratory. Stool samples from 17 volunteers at MSK and a publicly available dataset of 313 subjects from the Human Microbiome Project (HMP) served as healthy-control cohorts. The median pre-auto-HCT a-diversity during day -10 to 0 (as measured by Simpson reciprocal index, S) at both centers was significantly lower than healthy controls (Fig A) (HMP vs MSK auto-HCT, S=12.05 vs. 9.19, p<0.005; HMP vs Duke auto-HCT,
Figure 1. ROC curve for SWE velocity for the diagnosis of severe or very severe SOS after pediatric HSCT. A cutoff value of >1.95 m/s was 87.5% sensitive and 85% specific for severe or very severe SOS.
56 Microbiota Injury in Auto-HCT Is Frequent, Occurs across Geography, and Is Comparable to That Observed in Allo-HCT Niloufer Khan MD1, Jonathan U. Peled MD, PhD1, Antonio Gomes PhD1, Sean M. Devlin PhD1, Carlos Rondon Clavo MD2, Annelie Clurman B.A.1, John Slingerland B.S.1, Ann E. Slingerland B.S.1, Molly A. Maloy M.S.3, Anthony D. Sung MD4, Nelson J. Chao MD, MBA4, Kate Ann Markey MBBS, PhD1, Craig S. Sauter MD3, Heather Landau MD3, Doris M. Ponce MD3, Boglarka Gyurkocza MD3, Gunjan L. Shah MD, MS3, Michael Scordo MD3, Parastoo B Dahi MD3, Miguel-Angel Perales MD1, Robert R. Jenq MD5, Sergio A. Giralt MD1, Eric G. Pamer MD6, Marcel R.M. van den Brink MD, PhD1. 1 Memorial Sloan Kettering Cancer Center, New York, NY; 2 Memorial Sloan Kettering Cancer
Figure 1. Microbiota injury auto-HCT, is common and comparable across geography. A: The media Simpson reciprocal diversity index (S) of pretransplant (days-10 to 0) samples of auto-HCT and allo-HCT patients from two centers, as well as two cohorts of healthy volunteers, was plotted and a pairwise Wilcoxon test was performed, with p values as indicated. B (S) ws plotted against time for samples collected from day -10 to +30. Larger values greater diversity. C: Taxa contributing to monodomination events in auto-HCT patients.
Abstracts / Biol Blood Marrow Transplant 25 (2019) S7 S75
S=12.05 vs 6.91, p<0.005, Wilcoxon test). Pre-HCT diversity was comparable in between auto-HCT and allo-HCT at each center (MSK auto-HCT vs MSK allo-HCT, S = 12.05 vs 8.74, p=0.53). Overall (days -10 to +30) diversity decreased comparably after auto-HCT and allo-HST across both centers, while auto-HCT patients demonstrated a slightly more rapid recovery at day +30 compared to allo-HCT patients (Fig 1B). Monodomination was observed in the samples (Fig 1C), with Streptococcus and Enterococcus as the most common genus, as defined by any single taxon comprising >30% of bacterial abundance. The cumulative incidence of intestinal domination by any organism was >50% by day 0 and was >75% by day +14. Microbial diversity is reduced prior to transplant in both autoHCT and allo-HCT patients compared with healthy volunteers. Loss of diversity after auto-HCT occurs across transplant centers and the degree of injury is comparable to the dysbiosis observed in allo-HCT patients. We previously reported that intestinal monodomination increases the risk of bacteremia with corresponding organisms in allo-HCT patients. We now report these events are a common occurrence after auto-HCT, which suggests prevention or repair of microbiota injury as a strategy to reduce the toxicity of auto-HCT.
57 Metatranscriptomic Evaluation of Pulmonary Complications after Pediatric Hematopoietic Cell Transplantation Reveals Pathogenic Microbes Linked to Dysregulated Human Immunologic Gene Expression Matt S. Zinter MD1, Caroline A Lindemans MD, PhD2, Madeline Y Mayday BA3, Birgitta Versluys MD4, Marina Sirota PhD5, Christopher C. Dvorak MD6, Jaap-Jan Boelens MD, PhD7, Joseph L DeRisi PhD8, Chan Zuckerberg Biohub9. 1 Pediatric Critical Care Medicine, UCSF Benioff Children's Hospitals, San Francisco, CA; 2 Princess Maxima Centre for Pediatric Oncology, Utrecht, Netherlands; 3 Pediatrics, University of California, San Francisco, San Francisco, CA; 4 Pediatric Blood and Marrow Transplantation Program, Prinses Maxima Centrum/University Medical Center, Utrecht, Netherlands; 5 Institute for Human Genetics, University of California, San Francisco, San Francisco, CA; 6 Division of Pediatric Allergy, Immunology, and Blood and Marrow Transplantation, UCSF Benioff Children's Hospital, San Francisco, CA; 7 Department of Pediatrics, Bone Marrow Transplant Service, Memorial Sloan Kettering Cancer Center, New York, NY; 8 Biochemistry & Biophysics, University of California, San Francisco, San Francisco, CA; 9 San Francisco, CA Introduction: Pulmonary complications (PCs) after pediatric hematopoietic cell transplant (HCT) are heterogeneous in nature and are strongly associated with transplant-related mortality. Microbial pathogens play a role in the evolution of many post-HCT PCs and may contribute to or develop in the setting of an altered inflammatory microenvironment in the lungs. Objectives: To improve characterization of the pulmonary microbiome and transcriptome in pediatric HCT patients with PCs. Methods: Bronchoalveolar lavage (BAL) was performed at the University Medical Center in Utrecht between April 2004 and November 2016 for all children <18 years of age prior to undergoing HCT and again in select patients after HCT. In this study, n=257 BALs (n=210 obtained pre-HCT and n=47 obtained post-HCT) underwent metatranscriptomic sequencing to a median depth of 41.5 million read-pairs per sample followed by alignment to human and microbial genomes. Pathogenic bacteria and fungi were identified if RNA was quantified
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2 standard deviations above the pre-HCT cohort mean (Zscore 2). Pathogenic and non-pathogenic viruses were noted when quantified 10 and 1 reads per million total reads, respectively. Diversity of the bacterial microbiome was considered abnormal if the Simpson’s Diversity Index was 0.6. Differential gene expression was calculated using DEseq2 with FDR-adjusted p-values <0.05, followed by Gene Ontology analysis. Results: Relative to pre-HCT patients, post-HCT patients were more likely to have abnormal quantities of RNA aligning to pathogenic microbes (59.6% vs. 40.5%, p=0.017), including RNA aligning to pathogenic bacteria (23.4% vs. 11.0%, p=0.023) and pathogenic viruses (48.9% vs 29.0%, p=0.009), but not pathogenic fungi (6.4% vs 7.1%, p=0.854). In addition, post-HCT patients were also more likely to have depressed bacterial diversity (23.4% vs. 10.0%, p=0.012) and to harbor non-pathogenic viruses (27.7% vs. 7.1%, p<0.001). Relative to pre-HCT patients without an identified pathogen, post-HCT patients with a pathogen demonstrated at least 396 differentially expressed genes in the biological processes of immunity (leukocyte transendothelial migration; neutrophil degranulation; T-cell receptor activation, IL-1, IL-10 and TNF/NF-kb mediated signaling; regulation of type 1 IFN production) and cellular response to stress (regulation of HIF-1, response to hypoxia, and regulation of apoptosis and programmed cell death). Conclusions: As detection of pathogenic pulmonary microbes is exceedingly common pre-HCT and increases after HCT, incorporation of host gene expression profiles along with standard diagnostic criteria for pulmonary complications may improve classification accuracy and facilitate patient-targeted therapies. Further investigation into the microbiologic and human transcriptional heterogeneity within these high-risk patients is required.
58 Thrombotic Microangiopathy after Pediatric Allogeneic Stem Cell Transplant: Potential Early Markers to Predict Individuals at High-Risk Christopher E Dandoy MD, MSc1, Audrey Stegman1, Abigail R Pate BS1, Ava Stendahl1, Priscila Badia Alonso MD1, Sonata Jodele MD2, Adam Lane PhD1, Stella M Davies MBBS, PhD1. 1 Bone Marrow Transplantation and Immune Deficiency, Cincinnati Children's Hospital Medical Center, Cincinnati, OH; 2 Children's Hospital of Los Angeles, Los Angeles, CA Background: Transplant associated thrombotic microangiopathy (TMA) is a severe complication of stem cell transplant (SCT), characterized by endothelial damage leading to microangiopathic hemolytic anemia, thrombocytopenia and organ damage. Mild cases of TMA do not require treatment, but severe cases are associated with organ damage and mortality. Soluble suppression of tumorigenicity-2 (ST2) is a member of the interleukin 1 receptor family and has been associated with cardiac as well as endothelial injury. Elevated soluble terminal complement complex (sC5b-9) has recently been shown to be associated with high-risk TMA. We hypothesize that both complement activation (indicated by increased Sc5b9) and endothelial injury (indicated by increased ST2) occur early in the transplant process, setting up later tissue injury and organ damage, and these markers may be used to predict individuals at high-risk of later TMA, perhaps even before the start of the conditioning regimen. Methods: We evaluated 276 consecutive pediatric allogeneic SCT patients at our center. Patients were diagnosed with highrisk TMA if they received eculizumab therapy and demonstrated laboratory and clinical markers of TMA on at least two
Abstracts / Biol Blood Marrow Transplant 25 (2019) S7 S75
busulfan conditioning (yes/no). We then examined the 5 variables with the highest correlation from the regression modeling as predictors of a patient eventually developing severe or very severe SOS. Results: 55 subjects were enrolled. Most patients were male (n=33, 55%) with a median age of 8 (range 0-20) years. 21 (35%) patients developed SOS, 15 (25%) had severe or greater disease by EBMT criteria. Busulfan use is associated with an average increase of 0.6 in EBMT grade, and a 100 cm/s decrease in main portal vein velocity is associated with an average increase of 1.3 in EBMT grade. Hepatomegaly, ascites, and higher SWE stiffness also appear to be associated with greater SOS severity (p < .10). The best predictor of patients developing severe SOS was increased SWE velocity (AUC = .90). A cutoff value of >1.95 m/s was 87.5% sensitive and 85% specific for severe or very severe SOS. Seven of eight patients with severe or very severe SOS had SWE >1.95 m/s two to thirteen days before the date of severe SOS grading. Conclusion: In a group of 12 clinical and ultrasound variables, SWE velocity was an excellent predictor of the patient developing severe SOS. SWE was able to predict severe SOS 2 to 13 days before the EBMT criteria with 87.5% sensitivity and 85% specificity.
Center, Sloan Kettering Institute, New York, NY; 3 Department of Medicine, Adult Bone Marrow Transplant Service, Memorial Sloan Kettering Cancer Center, New York, NY; 4 Division of Hematologic Malignancies and Cellular Therapy, Duke University Medical Center, Durham, NC; 5 Departments of Genomic Medicine and Stem Cell Transplantation Cellular Therapy, MD Anderson Cancer Center, University of Texas, Houston, TX; 6 Infectious Disease Service, Lucille Castori Center for Microbes, Inflammation and Cancer, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY Intestinal microbiota injuries in allo-HCT patients are characterized by loss of a-diversity and domination of microbial communities by single organisms. These injury patterns are associated with poor survival after allo-HCT and are likely attributable to antibiotic exposure, nutritional alterations, and regimen-related mucosal injury. As recipients of auto-HCT patients have similar exposures, we hypothesized that similar patterns of dysbiosis occur in auto-HCT patients. We present the first analysis of intestinal microbiota composition in autoHCT patients at two independent institutions. From a prospectively collected cohort, we retrospectively identified 365 patients (median age 60) who received auto-HCT (May 2009 to Feb. 2018) at two transplant centers (MSK n = 316; Duke n = 49) with heterogeneous conditioning regimens, pre-HCT remission status, and diagnoses: 179 (49%) myeloma, 153 (42%) lymphoma, and 33 (9%) other diseases. 857 samples collected approximately weekly peri-transplant were 16S sequenced (V4-V5 region, Illumina platform) at a central laboratory. Stool samples from 17 volunteers at MSK and a publicly available dataset of 313 subjects from the Human Microbiome Project (HMP) served as healthy-control cohorts. The median pre-auto-HCT a-diversity during day -10 to 0 (as measured by Simpson reciprocal index, S) at both centers was significantly lower than healthy controls (Fig A) (HMP vs MSK auto-HCT, S=12.05 vs. 9.19, p<0.005; HMP vs Duke auto-HCT,
Figure 1. ROC curve for SWE velocity for the diagnosis of severe or very severe SOS after pediatric HSCT. A cutoff value of >1.95 m/s was 87.5% sensitive and 85% specific for severe or very severe SOS.
56 Microbiota Injury in Auto-HCT Is Frequent, Occurs across Geography, and Is Comparable to That Observed in Allo-HCT Niloufer Khan MD1, Jonathan U. Peled MD, PhD1, Antonio Gomes PhD1, Sean M. Devlin PhD1, Carlos Rondon Clavo MD2, Annelie Clurman B.A.1, John Slingerland B.S.1, Ann E. Slingerland B.S.1, Molly A. Maloy M.S.3, Anthony D. Sung MD4, Nelson J. Chao MD, MBA4, Kate Ann Markey MBBS, PhD1, Craig S. Sauter MD3, Heather Landau MD3, Doris M. Ponce MD3, Boglarka Gyurkocza MD3, Gunjan L. Shah MD, MS3, Michael Scordo MD3, Parastoo B Dahi MD3, Miguel-Angel Perales MD1, Robert R. Jenq MD5, Sergio A. Giralt MD1, Eric G. Pamer MD6, Marcel R.M. van den Brink MD, PhD1. 1 Memorial Sloan Kettering Cancer Center, New York, NY; 2 Memorial Sloan Kettering Cancer
Figure 1. Microbiota injury auto-HCT, is common and comparable across geography. A: The media Simpson reciprocal diversity index (S) of pretransplant (days-10 to 0) samples of auto-HCT and allo-HCT patients from two centers, as well as two cohorts of healthy volunteers, was plotted and a pairwise Wilcoxon test was performed, with p values as indicated. B (S) ws plotted against time for samples collected from day -10 to +30. Larger values greater diversity. C: Taxa contributing to monodomination events in auto-HCT patients.
Abstracts / Biol Blood Marrow Transplant 25 (2019) S7 S75
S=12.05 vs 6.91, p<0.005, Wilcoxon test). Pre-HCT diversity was comparable in between auto-HCT and allo-HCT at each center (MSK auto-HCT vs MSK allo-HCT, S = 12.05 vs 8.74, p=0.53). Overall (days -10 to +30) diversity decreased comparably after auto-HCT and allo-HST across both centers, while auto-HCT patients demonstrated a slightly more rapid recovery at day +30 compared to allo-HCT patients (Fig 1B). Monodomination was observed in the samples (Fig 1C), with Streptococcus and Enterococcus as the most common genus, as defined by any single taxon comprising >30% of bacterial abundance. The cumulative incidence of intestinal domination by any organism was >50% by day 0 and was >75% by day +14. Microbial diversity is reduced prior to transplant in both autoHCT and allo-HCT patients compared with healthy volunteers. Loss of diversity after auto-HCT occurs across transplant centers and the degree of injury is comparable to the dysbiosis observed in allo-HCT patients. We previously reported that intestinal monodomination increases the risk of bacteremia with corresponding organisms in allo-HCT patients. We now report these events are a common occurrence after auto-HCT, which suggests prevention or repair of microbiota injury as a strategy to reduce the toxicity of auto-HCT.
57 Metatranscriptomic Evaluation of Pulmonary Complications after Pediatric Hematopoietic Cell Transplantation Reveals Pathogenic Microbes Linked to Dysregulated Human Immunologic Gene Expression Matt S. Zinter MD1, Caroline A Lindemans MD, PhD2, Madeline Y Mayday BA3, Birgitta Versluys MD4, Marina Sirota PhD5, Christopher C. Dvorak MD6, Jaap-Jan Boelens MD, PhD7, Joseph L DeRisi PhD8, Chan Zuckerberg Biohub9. 1 Pediatric Critical Care Medicine, UCSF Benioff Children's Hospitals, San Francisco, CA; 2 Princess Maxima Centre for Pediatric Oncology, Utrecht, Netherlands; 3 Pediatrics, University of California, San Francisco, San Francisco, CA; 4 Pediatric Blood and Marrow Transplantation Program, Prinses Maxima Centrum/University Medical Center, Utrecht, Netherlands; 5 Institute for Human Genetics, University of California, San Francisco, San Francisco, CA; 6 Division of Pediatric Allergy, Immunology, and Blood and Marrow Transplantation, UCSF Benioff Children's Hospital, San Francisco, CA; 7 Department of Pediatrics, Bone Marrow Transplant Service, Memorial Sloan Kettering Cancer Center, New York, NY; 8 Biochemistry & Biophysics, University of California, San Francisco, San Francisco, CA; 9 San Francisco, CA Introduction: Pulmonary complications (PCs) after pediatric hematopoietic cell transplant (HCT) are heterogeneous in nature and are strongly associated with transplant-related mortality. Microbial pathogens play a role in the evolution of many post-HCT PCs and may contribute to or develop in the setting of an altered inflammatory microenvironment in the lungs. Objectives: To improve characterization of the pulmonary microbiome and transcriptome in pediatric HCT patients with PCs. Methods: Bronchoalveolar lavage (BAL) was performed at the University Medical Center in Utrecht between April 2004 and November 2016 for all children <18 years of age prior to undergoing HCT and again in select patients after HCT. In this study, n=257 BALs (n=210 obtained pre-HCT and n=47 obtained post-HCT) underwent metatranscriptomic sequencing to a median depth of 41.5 million read-pairs per sample followed by alignment to human and microbial genomes. Pathogenic bacteria and fungi were identified if RNA was quantified
S45
2 standard deviations above the pre-HCT cohort mean (Zscore 2). Pathogenic and non-pathogenic viruses were noted when quantified 10 and 1 reads per million total reads, respectively. Diversity of the bacterial microbiome was considered abnormal if the Simpson’s Diversity Index was 0.6. Differential gene expression was calculated using DEseq2 with FDR-adjusted p-values <0.05, followed by Gene Ontology analysis. Results: Relative to pre-HCT patients, post-HCT patients were more likely to have abnormal quantities of RNA aligning to pathogenic microbes (59.6% vs. 40.5%, p=0.017), including RNA aligning to pathogenic bacteria (23.4% vs. 11.0%, p=0.023) and pathogenic viruses (48.9% vs 29.0%, p=0.009), but not pathogenic fungi (6.4% vs 7.1%, p=0.854). In addition, post-HCT patients were also more likely to have depressed bacterial diversity (23.4% vs. 10.0%, p=0.012) and to harbor non-pathogenic viruses (27.7% vs. 7.1%, p<0.001). Relative to pre-HCT patients without an identified pathogen, post-HCT patients with a pathogen demonstrated at least 396 differentially expressed genes in the biological processes of immunity (leukocyte transendothelial migration; neutrophil degranulation; T-cell receptor activation, IL-1, IL-10 and TNF/NF-kb mediated signaling; regulation of type 1 IFN production) and cellular response to stress (regulation of HIF-1, response to hypoxia, and regulation of apoptosis and programmed cell death). Conclusions: As detection of pathogenic pulmonary microbes is exceedingly common pre-HCT and increases after HCT, incorporation of host gene expression profiles along with standard diagnostic criteria for pulmonary complications may improve classification accuracy and facilitate patient-targeted therapies. Further investigation into the microbiologic and human transcriptional heterogeneity within these high-risk patients is required.
58 Thrombotic Microangiopathy after Pediatric Allogeneic Stem Cell Transplant: Potential Early Markers to Predict Individuals at High-Risk Christopher E Dandoy MD, MSc1, Audrey Stegman1, Abigail R Pate BS1, Ava Stendahl1, Priscila Badia Alonso MD1, Sonata Jodele MD2, Adam Lane PhD1, Stella M Davies MBBS, PhD1. 1 Bone Marrow Transplantation and Immune Deficiency, Cincinnati Children's Hospital Medical Center, Cincinnati, OH; 2 Children's Hospital of Los Angeles, Los Angeles, CA Background: Transplant associated thrombotic microangiopathy (TMA) is a severe complication of stem cell transplant (SCT), characterized by endothelial damage leading to microangiopathic hemolytic anemia, thrombocytopenia and organ damage. Mild cases of TMA do not require treatment, but severe cases are associated with organ damage and mortality. Soluble suppression of tumorigenicity-2 (ST2) is a member of the interleukin 1 receptor family and has been associated with cardiac as well as endothelial injury. Elevated soluble terminal complement complex (sC5b-9) has recently been shown to be associated with high-risk TMA. We hypothesize that both complement activation (indicated by increased Sc5b9) and endothelial injury (indicated by increased ST2) occur early in the transplant process, setting up later tissue injury and organ damage, and these markers may be used to predict individuals at high-risk of later TMA, perhaps even before the start of the conditioning regimen. Methods: We evaluated 276 consecutive pediatric allogeneic SCT patients at our center. Patients were diagnosed with highrisk TMA if they received eculizumab therapy and demonstrated laboratory and clinical markers of TMA on at least two