Abstract Acute GVHD (aGVHD) is a major complication of allogeneic hematopoietic cell transplantation (alloHCT) associated with gut microbiota disruptions. However, whether therapeutic microbiota modulation prevents aGVHD is unknown. We conducted a randomized, placebo-controlled trial of third-party fecal microbiota transplantation (FMT) administered at the peak of microbiota injury in 100 patients with acute myeloid leukemia receiving induction chemotherapy and alloHCT recipients. Despite improvements in microbiome diversity, expansion of commensals, and shrinkage of potential pathogens, aGVHD occurred more frequently after FMT than placebo. Although this unexpected finding could be explained by clinical differences between the two arms, we asked whether a microbiota explanation might be also present. To this end, we performed multi-omics analysis of preintervention and postintervention gut microbiome and serum metabolome. We found that postintervention expansion of Faecalibacterium, a commensal genus with gut-protective and anti-inflammatory properties under homeostatic conditions, predicted a higher risk for aGVHD. Faecalibacterium expansion occurred predominantly after FMT and was due to engraftment of unique donor taxa, suggesting that donor Faecalibacterium-derived antigens might have stimulated allogeneic immune cells. Faecalibacterium and ursodeoxycholic acid (an anti-inflammatory secondary bile acid) were negatively correlated, offering an alternative mechanistic explanation. In conclusion, we demonstrate context dependence of microbiota effects where a normally beneficial bacteria may become detrimental in disease. While FMT is a broad, community-level intervention, it may need precision engineering in ecologically complex settings where multiple perturbations (e.g., antibiotics, intestinal damage, alloimmunity) are concurrently in effect. Significance: Post-FMT expansion of Faecalibacterium, associated with donor microbiota engraftment, predicted a higher risk for aGVHD in alloHCT recipients. Although Faecalibacterium is a commensal genus with gut-protective and anti-inflammatory properties under homeostatic conditions, our findings suggest that it may become pathogenic in the setting of FMT after alloHCT. Our results support a future trial with precision engineering of the FMT product used as GVHD prophylaxis after alloHCT.

Early post-FMT/placebo serum metabolomics and subsequent aGVHD. A, Logistic LASSO regression to find early posttreatment serum metabolomic predictors of grade II–IV aGVHD. The y axis has all the features evaluated in the model including metabolites and the GVHD prophylactic regimen. Stability (x axis) indicates the proportion of the 100 cross-validated runs in which a given feature remained a significant predictor of GVHD. Positive/negative values show metabolites positively/negatively associated with aGVHD. Other than one metabolite negatively associated with aGVHD, all features were insignificant and formed an almost straight line with 0 stability. B, Association between the novel metabolite in LASSO and grade II–IV aGVHD. C, sPLS-DA loading plot for significant contributors to the first latent component in classification of aGVHD status. Each variable's contribution is shown by a vector. Vectors pointing to the right versus left represent differentially abundant metabolites in early post-FMT/placebo samples not preceding versus preceding grade II–IV aGVHD, respectively. Longer vectors correspond to metabolites with more importance in group classification. D, Multi-omics correlation plot. Spearman correlation coefficients (color gradient) are shown for statistically significant (P < 0.05) correlations. The top five taxa in each cluster (along with the clusters as individual units) and the first 50 metabolites with the highest variance among samples (along with the novel metabolite) were used in this analysis. Each box in B shows the median (horizontal middle line) and interquartile range. Whisker lines indicate nonoutlier maximum and minimum values. A small jitter is included for better visualization. P value is from a Wilcoxon test. aGVHD: acute graft-versus-host disease.

In small series, third-party fecal microbiota transplantation (FMT) has been successful in decolonizing the gut from clinically relevant antibiotic resistance genes (ARGs). Less is known about the short- and long-term effects of FMT on larger panels of ARGs. We analyzed 226 pre- and post-treatment stool samples from a randomized placebo-controlled trial of FMT in 100 patients undergoing allogeneic hematopoietic cell transplantation or receiving anti-leukemia induction chemotherapy for 47 ARGs. These patients have heavy antibiotic exposure and a high incidence of colonization with multidrug-resistant organisms. Samples from each patient spanned a period of up to 9 months, allowing us to describe both short- and long-term effects of FMT on ARGs, while the randomized design allowed us to distinguish between spontaneous changes vs. FMT effect. We find an overall bimodal pattern. In the first phase (days to weeks after FMT), low-level transfer of ARGs largely associated with commensal healthy donor microbiota occurs. This phase is followed by long-term resistance to new ARGs as stable communities with colonization resistance are formed after FMT. The clinical implications of these findings are likely context-dependent and require further research. In the setting of cancer and intensive therapy, long-term ARG decolonization could translate into fewer downstream infections.

Faecalibacterium and aGVHD risk. A, log-ratio lasso regression to find early post-FMT/placebo, genus-level, gut microbiota predictors of grade II–IV aGVHD. The y axis shows taxa in the final model. The probability for each taxon of being selected in the final model is shown along the x-axis. This probability is calculated from 100 4-fold cross-validated runs. Positive/negative signs show positive/negative associations, and the value (indicated by a color gradient) represents the strength of association. B, Comparison between early post-FMT versus early post-placebo samples in Faecalibacterium abundance. C, Comparison between the groups developing versus not developing grade II–IV aGVHD in early post-FMT/placebo Faecalibacterium abundance. D,Faecalibacterium abundance at pre-FMT/placebo and early post-FMT/placebo timepoints. Samples from the same patient are connected, with line type indicating randomized treatment arm and line color showing future grade II–IV aGVHD status. E,Faecalibacterium abundance in FMT donor samples and patient samples according to treatment arm and timepoint. F, Relationship between Faecalibacterium abundance change (pre- to post-FMT) and ASV-level binary similarity of early post-FMT versus donor composition of this genus. The gray shaded area shows the 95% confidence interval. Higher post-FMT Faecalibacterium abundance was strongly associated with a greater similarity between post-FMT sample and donor composition in Faecalibacterium ASVs (a simple measure of engraftment). G,Faecalibacterium change from pre-FMT to early post-FMT timepoints per FMT donor. Each box in B, C, and G shows the median (horizontal middle line) and interquartile range. Whisker lines indicate nonoutlier maximum and minimum values. A small jitter is included for better visualization. P values in B and C are from a Wilcoxon test and in G from a Kruskal–Wallis test. aGVHD: acute graft-versus-host disease; FMT: fecal microbiota transplantation.

Microbiota clusters and aGVHD risk. A, Principal coordinates analysis of early post-FMT/placebo gut microbiota using topic model-based cluster abundances, with groups defined according to treatment arm. B, Same analysis as in A, but with groups defined according to subsequent development of grade II–IV aGVHD or not. C, Comparison between early post-FMT versus early post-placebo samples in topic model-based cluster abundances. D, Same analysis as in C, but with groups defined based on the occurrence of subsequent grade II–IV aGVHD. Samples are early post-FMT/placebo. E, Principal coordinates analysis of pre-FMT/placebo gut microbiota using topic model-based cluster abundances, with groups defined according to GVHD prophylaxis. P values in A, B, and E are from an adonis test with 999 permutations, with percent variation explained by each axis is shown in parentheses. 95% ellipses are shown. P values in C are from a Wilcoxon test after Bonferroni correction. Comparisons in D were not statistically significant. In C and D, each box shows the median (horizontal middle line) and interquartile range. Whisker lines indicate nonoutlier maximum and minimum values. A small jitter is included for better visualization. aGVHD: acute graft-versus-host disease; FMT: fecal microbiota transplantation; PC: principal coordinate; PTCy: posttransplantation cyclophosphamide.

Supplementary data S1: LASSO results

Representativeness of study participants

PURPOSE Gut microbiota injury in allogeneic hematopoietic cell transplantation (HCT) recipients and patients with AML has been associated with adverse clinical outcomes. Previous studies in these patients have shown improvements in various microbiome indices after fecal microbiota transplantation (FMT). However, whether microbiome improvements translate into improved clinical outcomes remains unclear. We examined this question in a randomized, double-blind, placebo-controlled phase II trial. METHODS Two independent cohorts of allogeneic HCT recipients and patients with AML receiving induction chemotherapy were randomly assigned in a 2:1 ratio to receive standardized oral encapsulated FMT versus placebo upon neutrophil recovery. After each course of antibacterial antibiotics, patients received a study treatment. Up to three treatments were administered within 3 months. The primary end point was 4-month all-cause infection rate. Patients were followed for 9 months. RESULTS In the HCT cohort (74 patients), 4-month infection density was 0.74 and 0.91 events per 100 patient-days in FMT and placebo arms, respectively (infection rate ratio, 0.83; 95% CI, 0.48 to 1.42; P = .49). In the AML cohort (26 patients), 4-month infection density was 0.93 in the FMT arm and 1.25 in the placebo arm, with an infection rate ratio of 0.74 (95% CI, 0.32 to 1.71; P = .48). Unique donor bacterial sequences comprised 25%-30% of the fecal microbiota after FMT. FMT improved postantibiotic recovery of microbiota diversity, restored several depleted obligate anaerobic commensals, and reduced the abundance of expanded genera Enterococcus, Streptococcus, Veillonella, and Dialister. CONCLUSION In allogeneic HCT recipients and patients with AML, third-party FMT was safe and ameliorated intestinal dysbiosis, but did not decrease infections. Novel findings from this trial will inform future development of FMT trials.

PURPOSE Intestinal microbiota disruptions early after allogeneic hematopoietic cell transplantation have been associated with increased risk for acute graft-versus-host disease (aGVHD). In our recent randomized phase 2 trial of oral, encapsulated, third-party fecal microbiota transplantation (FMT) versus placebo, FMT at the time of neutrophil recovery was safe and ameliorated dysbiosis. Here, we evaluated in post hoc analysis whether donor microbiota engraftment after FMT may protect against acute GVHD. PATIENTS AND METHODS We analyzed pre- and post-FMT stool samples and estimated donor microbiota engraftment (a pre-planned secondary endpoint) by determining the fraction of post-FMT microbiota formed by unique donor taxa (donor microbiota fraction; dMf). RESULTS dMf was higher in patients who later developed grade I or no aGVHD (median 33.9%, range 1.6-74.3%) than those who developed grade II-IV aGVHD (median 25.3%, range 2.2-34.8%) (P = 0.006). The cumulative incidence of grade II-IV aGVHD by day 180 was lower in the group with greater-than-median dMf than the group with less-than-median dMf (14.3% [95%CI, 2.1-37.5%] vs. 76.9% [95% CI, 39.7-92.8%], P = 0.008). The only determinant of dMf in cross-validated LASSO-regularized regression was the patient's pre-FMT microbiota diversity (Pearson's correlation coefficient -0.82, P = 1.6x10-9), indicating more potent microbiota modulation by FMT in patients with more severe dysbiosis. Microbiota network analysis revealed major rewiring including changes in the most central nodes, without emergence of keystone species, as a potential mechanism of FMT effect. CONCLUSIONS FMT may have protective effects against aGVHD, especially in patients with more severe microbiota disruptions.

AbstractPurpose: Intestinal microbiota disruptions early after allogeneic hematopoietic cell transplantation have been associated with increased risk for acute GVHD (aGVHD). In our recent randomized phase II trial of oral, encapsulated, third-party fecal microbiota transplantation (FMT) versus placebo, FMT at the time of neutrophil recovery was safe and ameliorated dysbiosis. Here, we evaluated in post hoc analysis whether donor microbiota engraftment after FMT may protect against aGVHD. Experimental Design: We analyzed pre- and post-FMT stool samples and estimated donor microbiota engraftment (a preplanned secondary endpoint) by determining the fraction of post-FMT microbiota formed by unique donor taxa (donor microbiota fraction; dMf). Results: dMf was higher in patients who later developed grade I or no aGVHD (median 33.9%; range, 1.6%–74.3%) than those who developed grade II–IV aGVHD (median 25.3%; range, 2.2%–34.8%; P = 0.006). The cumulative incidence of grade II–IV aGVHD by day 180 was lower in the group with greater-than-median dMf than the group with less-than-median dMf [14.3% (95% confidence interval, CI, 2.1–37.5) vs. 76.9% (95% CI, 39.7–92.8), P = 0.008]. The only determinant of dMf in cross-validated least absolute shrinkage and selection operator (LASSO)-regularized regression was the patient's pre-FMT microbiota diversity (Pearson correlation coefficient −0.82, P = 1.6 × 10−9), indicating more potent microbiota modulation by FMT in patients with more severe dysbiosis. Microbiota network analysis revealed major rewiring including changes in the most central nodes, without emergence of keystone species, as a potential mechanism of FMT effect. Conclusions: FMT may have protective effects against aGVHD, especially in patients with more severe microbiota disruptions.

Wilms tumor is the most common pediatric renal malignancy. While developed countries have had excellent survival, it remains poorer by comparison in developing countries. The aim was to analyze the clinical outcome of children with Wilms tumor managed in a developing country from 2004 to 2014 by the SIOP WT 2001 protocol.Fifty-nine children with Wilms tumor managed by a SIOP WT 2001 regimen from 2004 to 2014 were analyzed.The median age at presentation was 36 months, and 59% were boys. The average size of the tumor at presentation was 523 mL. Inferior vena cava thrombus was present in 11, distant metastases in 18, and bilateral tumors in six. Preoperative chemotherapy was given to all children after a diagnostic core needle biopsy. Preoperative chemotherapy reduced the tumor size to a mean of 208 mL and resolved venacaval thrombus in eight. Fifty-five children underwent definitive surgery while two children died during preoperative chemotherapy and two remained inoperable. All surviving children received adjuvant chemotherapy with 17 receiving radiotherapy as well. The overall survival (OS) was 80% and the event-free survival (EFS) was 73% after a mean follow up of 42 months after completion of therapy.The tumor volumes at presentation and the incidence of venous tumor thrombosis in our cohort were much higher than those reported from developed countries. The incidence of metastatic disease at diagnosis (30.5%) was significantly higher than the 10–12% reported in Western data, but similar to that reported from various developing countries (14.1–31%). The OS in our cohort was 80% and the EFS was 73% with there being no events after 28 months. Although the survival rate for localized disease is similar to that in developed countries, the OS for metastatic disease was significantly less (50% vs. 75%). We also found that using an upfront posterior flank core biopsy was safe and beneficial for differentiating Wilms tumor from other pediatric renal tumors that are less chemosensitive.In a resource-restricted environment such as ours, the SIOP WT 2001 protocol has been found to show excellent results.

Intestinal colonization with antibiotic-resistant bacteria has been associated with increased risk of systemic infections after allogeneic hematopoietic cell transplantation (HCT) and anti-leukemia induction chemotherapy. Fecal microbiota transplantation (FMT) has been used with success in gut decolonization from select antibiotic-resistant pathogens in small series. Large-scale data using expanded panels of antibiotic-resistance genes (ARGs) and long-term effects of FMT are unavailable. We performed a correlative analysis of samples from a prospective trial to address this knowledge gap. We conducted a randomized double-blind trial of oral, encapsulated, third-party fecal microbiota transplantation (FMT) versus placebo at the time of neutrophil recovery in two independent cohorts of patients with AML undergoing induction chemotherapy or allogeneic transplant recipients, with the primary objective of reducing infections (ClinicalTrials.gov identifier: NCT03678493; J. Clin. Oncol 2023 May 26). 100 patients (HCT: 74, AML: 26) were randomized between FMT vs. placebo, each given as 5 oral capsules taken at once a the time of neutrophil engraftment. FMT was safe and ameliorated intestinal dysbiosis, but the trial did not meet its primary endpoint. Longitudinal pre- and post-treatment stool samples were collected at the following timepoints: baseline (before starting conditioning; T0), pre-dose 1 (T1), day 10 post-dose 1 (T2), day 28 post-dose 1 (T3), and at the end of study follow up (9 months; T4). All stool samples underwent 16S rRNA gene sequencing for the pre-planned microbiota endpoints of the trial. 226 samples with remaining DNA were used for microfluidic qPCR to simultaneously detect and quantify 47 ARGs including those for beta-lactams, quinolones, vancomycin, and carbapenem resistance. All samples were run in duplicate and their average gene content for each ARG (gene copies/microL DNA) was calculated using standard curves of the respective genes. Both qualitative (presence/absence; Fisher's exact test) and quantitative (Wilcoxon's test) analyses were performed, with a 0.05 threshold for P values defining statistical significance. The small number of T3 samples (n = 27) did not allow a meaningful comparison between the two arms; these samples were not included in analysis. As expected from the randomized design, there was no difference between the FMT and placebo arms in the number of ARGs at T0 or T1 (median [range]; T0: 29 [1-45] vs. 31 [13-39], P = 0.33; T1: 25 [5-40] vs. 26 [9-43], P = 0.52). There was a trend for more ARGs at T2 in the FMT arm (28 [1-42] vs. 22 [12-36], P = 0.07). At T4, there were fewer ARGs in the FMT arm compared to placebo (23 [9-38] vs. 32 [20-40], P < 0.01; Fig. 1 left panel). In quantitative analysis comparing the two arms at each post-treatment timepoint, sul1 (sulfonamide), floR (chloramphenicol), ctxm32 (beta-lactam), qnrB (quinolone), tetS (tetracycline), and aadA5 (aminoglycoside) were more abundant in the FMT arm at T2. At T4, blaKPC (carbapenem) and aadD (aminoglycoside) were more abundant in the placebo arm ( Fig. 1 right panel). Given our findings at T2, we evaluated whether ARGs in post-treatment samples were also present in pre-treatment samples from the same patient. In the case of FMT, de novo appearance of an ARG post-treatment could indicate ARG transmission via FMT. Although donor stool was screened for a panel of clinically relevant ARGs and pathogens, some ARGs may be found in healthy individuals and could have been present in the FMT product. Using a per-ARG Fisher's exact test, no ARG appeared de novo post-FMT more frequently than after placebo, arguing against ARG transmission via FMT. Findings from this largest randomized trial of FMT in AML patients and HCT recipients to date indicate long-term efficacy of FMT in antibiotic-resistant gut decolonization. A temporary early phase of reduced colonization resistance after FMT, possibly due to drastic, rapidly changing rewiring of microbiota networks, seems to occur, followed by formation of more stable communities resistant to ARG colonization. Clinical translation of these findings requires further research.

Jaccard index showing similarity between sets of the most central nodes in FMT and placebo arms at pre-treatment and day 28 post-treatment timepoints

Bacteremia is a rare complication of Clostridium tetani infection. To our knowledge, there are only two case reports to date of C. tetani bacteremia, both hypothesized to be secondary to a gastrointestinal source. Herein, we report a case of an elderly man with genome sequence-proven C. tetani bacteremia from a possible cutaneous source without neuromuscular symptoms.

Loss of function of senataxin (SETX), a bona-fide RNA/DNA helicase, is associated with neuronal degeneration leading to Ataxia and Ocular Apraxia (AOA) in human patients. SETX is proposed to promote transcription termination, DNA replication, DNA repair, and to unwind deleterious RNA:DNA hybrids in the genome. In all the above-mentioned mechanisms, SETX unwinds transcription complex-associated nascent RNA which is then degraded by the RNA exosome complex. Here we have used B cells isolated from a SETX mutant mouse model and compared genomic instability and immunoglobulin heavy chain locus (IgH) class switch recombination (CSR) to evaluate aberrant and programmed genomic rearrangements, respectively. Similar to RNA exosome mutant primary B cells, SETX mutant primary B cells display genomic instability but a modest decrease in efficiency of CSR. Furthermore, knockdown of Setx mRNAs from CH12-F3 B-cell lines leads to a defect in IgA CSR and accumulation of aberrant patterns of mutations in IgH switch sequences. Given that SETX mutant mice do not recapitulate the AOA neurodegenerative phenotype, it is possible that some aspects of SETX biology are rescued by redundant helicases in mice. Overall, our study provides new insights into the role of the SETX/RNA exosome axis in suppressing genomic instability so that programmed DNA breaks are properly orchestrated.

The distribution of sense and antisense strand DNA mutations on transcribed duplex DNA contributes to the development of immune and neural systems along with the progression of cancer. Because developmentally matured B cells undergo biologically programmed strand-specific DNA mutagenesis at focal DNA/RNA hybrid structures, they make a convenient system to investigate strand-specific mutagenesis mechanisms. We demonstrate that the sense and antisense strand DNA mutagenesis at the immunoglobulin heavy chain locus and some other regions of the B cell genome depends upon localized RNA processing protein complex formation in the nucleus. Both the physical proximity and coupled activities of RNA helicase Mtr4 (and senataxin) with the noncoding RNA processing function of RNA exosome determine the strand-specific distribution of DNA mutations. Our study suggests that strand-specific DNA mutagenesis-associated mechanisms will play major roles in other undiscovered aspects of organismic development.

We have ablated the cellular RNA degradation machinery in differentiated B cells and pluripotent embryonic stem cells (ESCs) by conditional mutagenesis of core (Exosc3) and nuclear RNase (Exosc10) components of RNA exosome and identified a vast number of long non-coding RNAs (lncRNAs) and enhancer RNAs (eRNAs) with emergent functionality. Unexpectedly, eRNA-expressing regions accumulate R-loop structures upon RNA exosome ablation, thus demonstrating the role of RNA exosome in resolving deleterious DNA/RNA hybrids arising from active enhancers. We have uncovered a distal divergent eRNA-expressing element (lncRNA-CSR) engaged in long-range DNA interactions and regulating IgH 30 regulatory region super-enhancer function. CRISPR-Cas9-mediated ablation of lncRNA-CSR transcription decreases its chromosomal looping-mediated association with the IgH 30 regulatory region super-enhancer and leads to decreased class switch recombination efficiency. We propose that the RNA exosome protects divergently transcribed lncRNA expressing enhancers by resolving deleterious transcription-coupled secondary DNA structures, while also regulating long-range super-enhancer chromosomal interactions important for cellular function.

The mechanisms by which B cells somatically engineer their genomes to generate the vast diversity of antibodies required to challenge the nearly infinite number of antigens that immune systems encounter are of tremendous clinical and academic interest. The DNA cytidine deaminase activation-induced deaminase (AID) catalyzes two of these mechanisms: class switch recombination (CSR) and somatic hypermutation (SHM). Recent discoveries indicate a significant promiscuous targeting of this B-cell mutator enzyme genome-wide. Here we discuss the various regulatory elements that control AID activity and prevent AID from inducing genomic instability and thereby initiating oncogenesis.