After Allogeneic Stem Cell Transplantation Research Articles

Comparison of Infectious Complications in Patients Receiving High-Dose Cyclophosphamide as GvHD Prophylaxis After Transplantation From A 9/10 HLA-Matched Unrelated Donor with Standard GvHD Prophylaxis After Transplant From A Fully Matched Related Donor.

The aim of this study was to evaluate whether cyclophosphamide administered after allogeneic stem cell transplantation (ASCT) from 9/10 HLA-Matched Unrelated Donors (MMUD) increases the rates of bacterial, fungal, viral infections, complications (hemorrhagic cystitis (HC)), and infection-related mortality compared to allogeneic stem cell transplantation from matched related donors (MRD). This is a retrospective multicenter study. 45 MMUD ASCT patients who received posttransplant cyclophosphamide+methotrexate+calcineurin inhibitor compared with 45 MRD ASCT patients who received methotrexate+calcineurin inhibitor. Although there was a statistically significant prolongation of neutrophil engraftment time in the PTCy arm, there was no statistically significant difference in bacterial infection frequencies between the groups (PTCy; 9 (20%), control; 8 (17.8%), p=0.778). The distribution of CMV infection in the first 100 days was similar (p=0.827), but the distribution of CMV infection rate between the 100th and 365th days was observed more frequently in the control group (p=0.005). HC rates and their grades were similar in both groups (PTCy; 4 (8.8%), control; 6 (13.3%) p=0.502). The rates of VZV infection and invasive aspergillosis were similar in the PTCy and control groups (13.3% in the PTCy and 17.8% in the control group p=0.561). There is also no statistically significant difference in survival analysis (OS, LFS, GRFS, RI, IRM, NRM) between groups. However, the incidence of cGVHD was significantly higher in the control group (P=0.035). The addition of PTCy to standard GvHD prophylaxis in MMUD ASCT does not lead to an increase in CMV reactivation, bacterial infections, invasive fungal infection, viral hemorrhagic cystitis, or mortality.

Immunomodulatory Microbial Metabolites Identified in Allo-SCT Patients Protect from GvHD By Sting Dependent Modulation of Intestinal Tissue Homeostasis

Background: Graft-versus-host disease (GVHD) is a dreaded complication after allogeneic stem-cell transplantation (ASCT). Previously, we and others showed that activation of type I interferon (IFN-I) inducing pathways such as RIG-I/MAVS or cGAS/STING can promote the integrity of the intestinal barrier and limit GVHD. However, the signals that drive these protective IFN-I responses are poorly understood. Commensal microbiota can (i) have distant effects on immune responses through modulation of IFN-I signaling and (ii) predict mortality in ASCT patients. We hypothesized that microbiota-derived products such as microbial metabolites engage IFN-I signaling in immune and non-immune cells poising them for induction of protective responses. We established a prospective, multi-centric clinical study in patients newly diagnosed with acute leukemia and performed longitudinal stool sampling to track changes in microbial community composition and metabolites expression levels (Submitted as a separate abstract to ASH 2022 (Orberg & Meedt et al.)). We showed that patients with high metabolite expression are less likely to develop GVHD. In this study, we translated our clinical observations to mouse models of acute GVHD and human and mouse intestinal organoid models to uncover the molecular mechanisms of protective metabolites during ASCT. Methods: Stool samples from ASCT patients were obtained in Munich and Regensburg in accordance to IRB-approved study protocols. Patients were sampled at initial diagnosis (Dx), prior to conditioning and weekly after ASCT up to day 28. We analyzed samples by 16S rRNA sequencing and mass spectrometry. Next, we tested metabolites detected in patients in vitro in murine and human intestinal crypt-derived organoids and organoid/T cell coculture assays. To obtain a mechanistic understanding, we stimulated WT or IFN-I-signaling-impaired organoids or used small molecule inhibitors and analysed them regarding growth performance, cell composition and by scRNAseq. Finally, Metabolites were applied as treatment in preclinical intestinal damage models and GVHD mouse models. Outcomes were assessed by a novel organoid recovery assay in addition to established read-outs. Results: Here, we present expression profiles of metabolites including short-chain fatty acids, DAT, ICA and secondary bile acids. Following ASCT, and especially at the early time-points, metabolite expression declined drastically. We confirmed this trend in our multi-centric cohort of ASCT patients by comparing levels of DAT and ICA sampled at admission to the transplantation ward (Conditioning) versus at clinical diagnosis of GVHD: in patients with GVHD, metabolite levels were drastically reduced. Metabolite stimulation of mouse small intestinal organoids promoted organoid growth and required intact STING signaling. Additionally, organoids showed an increased stemness and resistance to T cell mediated damage. Both healthy donor and GvHD patient derived human colon organoids also responded to DAT and ICA, however the metabolite effect was lost when co-administered with the STING-inhibitor H151. Next, we prophylactically administered metabolites in a major mismatch mouse ASCT model. Metabolite-treated mice showed significantly better outcomes in barrier integrity and organoid based intestinal regeneration assays. Additionally, metabolite treatment was associated with an increased regulatory T cell infiltration. These effects were abrogated in STING-/- recipients, but not in IFNaR-/- recipients. Finally, metabolite treatment was protective in major mismatch GVHD mouse models. Conclusions: We identify that microbial-derived metabolites detected in patients can engage the STING pathway in humans and mice to confer resistance from immune damage. Thus, prophylactic administration of metabolite cocktails or bacterial consortia may reduce occurrence of GVHD in ASCT patients.

The importance of intermediate-dose Valacyclovir in primary CMV prophylaxis after Allogeneic-stem cell transplantation, and the advantages of step-wise pre-emptive treatment in CMV reactivation

Objective: Cytomegalovirus (CMV) reactivation and disease are still one of the most important causes of morbidity and mortality after allogeneic stem cell transplantation (ASCT). Letermovir prophylaxis has been clearly shown to be effective and well-tolerated. Drug interactions and cost are limitations. Alternative regimens such as Valacyclovir 3g-6g a day are of interest. In our study, we investigated the clinical results of intermediate dose (3 gr/d) valacyclovir after ASCT in primary CMV prophylaxis. Material and Methods: The data of 70 patients who underwent ASCT between 2019-2020 were retrospectively analyzed. Valacyclovir was given at a dose of 3 g/day to all patients for primary CMV prophylaxis after ASCT. If CMV reactivation developed during Valacyclovir prophylaxis, therapeutic oral Valganciclovir or parenteral Ganciclovir was gradually switched according to CMV DNA copy numbers. Results: The mean age of the patients included in the study was 45.5 years. The D+/R+ seropositivity was 97.2%. CMV reactivation developed in 37/70 (52.8%) patients within the first 100 days after transplantation. While CMV negativity could be achieved with oral VValganciclovir in 17 of the reactive patients (45.9%), hospitalization was required for parenteral ganciclovir use in 20 (28.1%) of them. The median PFS of patients with and without CMV reactivation was 10 months and 18 months, with a one-year PFS were 49.9% and 80.9%, respectively. One-year overall survival rates of patients with and without CMV reactivation were 52.9% and 92.9% respectively. Conclusion: It has become more important to prevent infections that may develop after ASCT with prophylaxis rather than treating. Post-transplant intermediate-dose Valacyclovir as primary prophylaxis has been shown to reduce CMV reactivation/disease rates at desired levels and reduce hospitalizations.

Microbial-Derived Metabolites Induce Epithelial Recovery Via the Sting Pathway in Mice and Men and Protect from Graft-Versus-Host Disease

Background: Graft-versus-host disease (GVHD) is a dreaded complication after allogeneic stem-cell transplantation (ASCT). Previously, we and others showed that activation of type I interferon (IFN-I) inducing pathways such as RIG-I/MAVS or cGAS-STING can promote the integrity of the intestinal barrier and limit GVHD (Fischer et al., Sci Transl Med, 2017). However, the signals that drive these protective IFN-I responses are poorly understood.Commensal microbiota can (i) have distant effects on immune responses through modulation of IFN-I signaling (Steed et al, Science, 2017; Swimm et al, Blood, 2018) and (ii) predict mortality in ASCT patients (Peled et al., N Engl J Med, 2020). We hypothesized that microbiota-derived products such as microbial metabolites engage IFN-I signaling in immune and non-immune cells poising them for induction of protective responses. We established a prospective, multi-centric clinical study in patients newly diagnosed with acute leukemia and performed longitudinal stool sampling to track changes in microbial community composition and metabolites expression levels. Submitted as a separate abstract to ASH 2021 (Orberg & Meedt et al.), we show that patients with high metabolite expression going into ASCT are less likely to develop GVHD. In this study, we translate our clinical observations to mouse models of acute GVHD and human and mouse intestinal organoids to uncover the molecular mechanisms via which metabolites protect the intestinal barrier during ASCT.Methods: Stool samples from ASCT patients were obtained in Munich and at Regensburg in accordance to IRB-approved study protocols. Patients were sampled at initial diagnosis (Dx), prior to conditioning and weekly after ASCT up to day 28. We analyzed samples by 16S rRNA sequencing and mass spectrometry to obtain a complete picture of microbiome composition and function. Next, we tested metabolites which we detected in patients (desaminotyrosine [DAT], indole-3-carboxaldehyde [ICA]) as treatment in preclinical models in ASCT and GVHD mouse models. Outcomes were assessed by a novel organoid recovery assay in addition to established read-outs. To obtain a mechanistic understanding of the signaling pathways involved, we stimulated WT or IFN-I-signaling-impaired mouse (incl. STING -/-, MAVS -/-, IFNαR -/-) as well as human intestinal crypt-derived organoids with metabolites.Results: Here, we present a 64-year-old female patient diagnosed with AML who received a 9/10 HLA-matched ASCT. At day 7, i.v. antibiotics were started due to fever and Enterococcus bacteraemia. At day 15, the patient developed skin and GI GVHD (Glucksberg III). At the timepoint initial diagnosis (Dx), i.e. the timepoint when we diagnosed AML but before therapy was initiated, we detected rich alpha diversity (Panel 1a) in the patient's stool. Flavonifractor plautii, a producer of the metabolite DAT, was detectable (Panel 1b). We observed high-level expression of metabolites including short-chain fatty acids, DAT, ICA and secondary bile acids (Panel 1c). Following ASCT, and especially at the early time-points day 0 and day 7, alpha diversity and metabolite expression declined drastically. We confirmed this trend in our multi-centric cohort of ASCT patients by comparing levels of DAT and ICA sampled at admission to the transplantation ward (Conditioning) versus at clinical diagnosis of GVHD: in patients with GVHD, metabolite levels were drastically reduced (Panel 2). Next, we prophylactically administered metabolites in a major mismatch mouse ASCT model. Metabolite-treated mice showed significantly showed better outcomes in our organoid recovery assay, which measures the ability of intestinal stem cells to recover after allogeneic injury (Panel 3). This effect that was abrogated in STING -/- recipients. Metabolite stimulation of mouse small intestinal organoids promoted organoid numbers and size, and required intact STING signaling (Panel 4a). Human colon organoids also responded to DAT and ICA, however the metabolite effect was lost when co-administered with the STING-inhibitor H151.Conclusions: We identify that microbial-derived metabolites detected in patients can engage the STING pathway in humans and mice to confer resistance from immune damage. Thus, prophylactic administration of metabolite cocktails or bacterial consortia that can produce these metabolites may reduce occurrence of GVHD in ASCT patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mixed Chimerism of Bone Marrow CD34+ Sorted Cells after Matched or Haploidentical Allogeneic Stem Cell Transplantation for Myeloid Malignancies Is Predictive of Relapse

▪IntroductionThe assessment of donor chimerism after allogeneic stem cell transplantation (ASCT) is a useful tool to monitor engraftment and predict relapse. The detection of mixed donor chimerism in CD34+ sorted cells from ASCT patients has been reported in small series in the literature (Bornhäuser M et al. Haematologica. 2009; Hoffmann JC et al. Ann Hematol. 2014) to be sensitive to detect relapse. Investigating these progenitors could indeed allow to suspect an upcoming relapse earlier than assessing chimerism in whole blood (WB) or sorted WB CD3+ T-cells. We report on the predictive impact on survival of chimerism assessment in CD34+ sorted cells from patients' bone marrow (BM) on day +90 after matched or haploidentical ASCT.Patients, materials and methodsAll ASCT recipients (n=168), between October 2009 and April 2017, treated for acute myeloblastic leukemia (n=115) or chronic myeloid disease (n=53) were reviewed, focusing on those having benefited from CD34+ chimerism assessment in BM around day +90 (50-150). The 20 cases of relapse before this evaluation were excluded. A cohort of 148 grafts was retrieved, and 145 were informative with enough cells for DNA extraction after magnetic CD34+ cell-enrichment.Chimerism was assessed by quantitative PCR using specific donor/recipient markers (Alizadeh et al. Blood, 2002). Data from chimerism assessed on CD3+ sorted cells or whole blood, performed at the same period, were also retrieved. Receiving Operator Characteristics (ROC) curves were drawn based on the presence or absence of relapse. No significant threshold appeared for CD3+ cells chimerism but a threshold of 95% was retained by convention. Conversely good specificity was obtained with a 95% threshold for whole blood (86%) and 80% for CD34+ sorted cells (97%). Event-free survival (EFS) was calculated from the date of the ASCT until the date of either relapse, progression, death or last news. Overall survival (OS) was calculated from the date of ASCT until the date of death or last news.ResultsThe whole cohort comprised 57% of males and had a median age of 60 years old (24-74) at the time of ASCT. Reduced-intensity (RIC), myeloablative (MAC) or sequential (SEQ) conditioning was used in 82%, 12% and 6% of the cases respectively. Donors were familial (73%), from registry (70%) or unrelated cord blood (5%). Post-transplant cyclophosphamide (PTCY) was used in 31 procedures (22 haploidentical-ASCT and 9 matched donors). The median follow-up of the cohort was 13 months (1-88) and the rates of relapse and death were 29% and 24% respectively.The 95% threshold for WB and WB CD3+ chimerisms had no impact on neither EFS nor OS.Conversely, full donor chimerism (>80%) was observed in CD34+ cells in 91% and 86% of all cases and PTCY procedures respectively (P=0.4) with 91% for haploidentical ASCT. Full CD34+ engraftment was observed in 91% of the cases after MAC, 93% after RIC and only 67% after SEQ (p=0.03, yet no difference between MAC and RIC). The presence of a mixed CD34+ chimerism was associated with more relapses (77% vs 25%, p<10-4) in the whole group as well as when considering only PTCY procedures (75% vs 31%) or haploidentical grafts (100% vs 30%) although this did not reach statistical significance because of the small size of these subgroups. The presence of a mixed CD34+ chimerism at day+90 was associated with a significantly worse EFS (median: 5 vs 79 months, HR 0.2 [95%CI: 0.06-0.6] p<10-4) and OS (median: 12 months vs not reached (NR), HR 0.4 [95%CI: 0.1-1.1] p=0.007). Of interest, patients who received ASCT while not in complete remission had a better EFS (median NR vs 5 months, HR 3.1 [95%CI: 1.1-9.2] p=0.005) if they achieved a full CD34+ chimerism at day +90.DiscussionIn this large series, the feasibility of BM CD34+ cells' sorting at day +90 after ASCT appears highly feasible with 98% of informative patients. BM CD34+ cells chimerism stands out as a more robust marker than CD3+ or whole blood chimerism to predict relapse when using the 80% threshold determined here. It could be of value to decide whether or not to prescribe donor lymphocytes infusions as ASCT consolidation. Moreover, since CD34+ mixed chimerism appears here of high predictive value for relapse, it could be used as a surrogate prognostic factor for patients without minimal residual disease molecular markers. Finally, of interest, this marker is valid whatever the type of donor, and can be used even after hapoloidentical ASCT and/or PTCY administration. DisclosuresMoreau:Amgen: Honoraria; Takeda: Honoraria; Novartis: Consultancy, Honoraria; Celgene, Janssen, Takeda, Novartis, Amgen, Roche: Membership on an entity's Board of Directors or advisory committees; Onyx Pharmaceutical: Consultancy, Honoraria; Bristol-Myers Squibb: Honoraria; Millennium: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Mohty:Sanofi: Honoraria, Speakers Bureau.

Prolonged Suppression of Butyrate-Producing Bacteria Is Associated With Acute Gastrointestinal Graft-vs-Host Disease and Transplantation-Related Mortality After Allogeneic Stem Cell Transplantation.

Butyrogenic bacteria play an important role in gut microbiome homeostasis and intestinal epithelial integrity. Previous studies have demonstrated an association between administration of short-chain fatty acids like butyrate and protection from acute graft-vs-host disease (GvHD) after allogeneic stem cell transplantation (ASCT). In the current study, we examined the abundance and butyrogenic capacity of butyrate-producing bacteria in 28 healthy donors and 201 patients after ASCT. We prospectively collected serial stool samples and performed polymerase chain reaction analysis of the butyrate-producing bacterial enzyme butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT) in fecal nucleic acid extracts. Our data demonstrate a strong and prolonged suppression of butyrogenic bacteria early in the course of ASCT. In a multivariable analysis, early use of broad-spectrum antibiotics before day 0 (day of transplantation) was identified as an independent factor associated with low BCoAT copy numbers (odds ratio, 0.370 [95% confidence interval, .175-.783]; P = .009). Diminished butyrogens correlated with other biomarkers of microbial diversity, such as low 3-indoxylsulfate levels, reduced abundance of Clostridiales and low inverse Simpson and effective Shannon indices (all P < .001). Low BCoAT copy numbers at GvHD-onset were correlated with GI-GvHD severity (P = .002) and associated with a significantly higher GvHD-associated mortality rate (P = .04). Furthermore, low BCoAT copy numbers at day 30 were associated with a significantly higher transplantation-related mortality rate (P = .02). Our results are consistent with the hypothesis that alterations in the microbiome play an important role in GvHD pathogenesis and that microbial parameters such as BCoAT might serve as biomarkers to identify patients at high risk of lethal GI-GvHD.

Donor Lymphocyte Infusion to Enhance the Graft-versus-Myeloma Effect

Donor lymphocyte infusion (DLI) has the potential to significantly deepen the response after allogeneic stem cell transplantation (ASCT) in multiple myeloma (MM). Subsequently, DLI offers the opportunity for long-term progression-free and, most importantly, overall survival for patients with MM. DLI application is a complex procedure, whereby many factors need to be considered (e.g., patient-oriented factors prior to application, disease-specific factors, as well as possible combinations with further therapies during and after DLI). There are two settings in which DLI can be given, they are as follows: as a salvage option in progressive disease or in the prophylactic setting for MM patients with resolved disease to further deepen the response. While the first studies used DLI in the salvage setting, results for prophylactic DLI appear to be associated with better and prolonged outcomes. Furthermore, DLI (both prophylactic and salvage) given earlier after ASCT (3–6 months) appear to be associated with better outcomes. The incorporation of novel agents showed similar responses and survival after DLI. However, updated and larger evaluations are urgently needed to determine the specific role of multiple variables in such a complex treatment environment of ASCT in an ever-evolving field of MM. This review underlines the rationale for DLI after ASCT, results in the salvage and prophylactic settings, patterns of disease progression after DLI, as well as avenues to further enhance the graft-versus-myeloma effect exerted by DLI.

Presence of Parvovirus B19 but Not Herpesvirus Genome in Acute Skin Rash after Allogeneic Stem Cell Transplantation Correlates with Outcome

Introduction: Skin rash is a first symptom of acute graft-versus-host disease (GvHD) after allogeneic stem cell transplantation (ASCT) but can also be caused by viruses. The relevance of virus DNA analyses in skin rash for diagnosis and clinical outcome is unknown. Objectives: To record the frequencies of detection of herpes and parvovirus B19 (ParvoB19) DNA in skin rash within 100 days after ASCT and to analyze their relevance for diagnosis, clinical course, and non-relapse mortality (NRM). Methods: We retrospectively identified 55 patients with virus DNA analysis for CMV, EBV, HHV6, HHV8, HSV, VZV, or ParvoB19. We assessed the rate of virus DNA detection and studied associations with histological diagnosis, virus DNA from concomitantly analyzed blood, clinical presentation, exanthema treatment, and NRM. Results: CMV, EBV, HHV6, HHV8, HSV, VZV and ParvoB19 DNA were detected in 12.5, 11.8, 10, 0, 0, 2.9, and 26.7% of exanthemas. Histopathological diagnosis was not associated with virus polymerase chain reaction (PCR) results. Detection of CMV, EBV, or HHV6 DNA but not ParvoB19 in skin and blood was associated with PCR results (p = 0.016; p < 0.001; p = 0.067; p = n.a.). Detection of CMV, EBV, HHV6, or ParvoB19 DNA in the skin was not significantly associated with patient, ASCT, or GvHD characteristics. Detection of ParvoB19 but not herpes virus DNA was associated with less immunosuppressive treatment (p = 0.015) and lower NRM (p = 0.041). In multivariate analyses, detection of ParvoB19 was associated with a lower NRM. Conclusions: Detection of ParvoB19 DNA in exanthema after ASCT might be associated with lower NRM.

A Phase 2 Study of Glasdegib for the Treatment of Acute Myeloid Leukemia and Myelodysplastic Syndrome Patients with High Risk of Post-Allogeneic Stem Cell Transplantation Relapse

A Phase 2 Study of Glasdegib for the Treatment of Acute Myeloid Leukemia and Myelodysplastic Syndrome Patients with High Risk of Post-Allogeneic Stem Cell Transplantation Relapse

PS1053 IMPACT OF CYTOMEGALOVIRUS REACTIVATION ON RELAPSE RATE AFTER ALLOGENEIC STEM CELL TRANSPLANTATION IN PATIENTS WITH ACUTE MYELOBLASTIC LEUKEMIA

Background:Cytomegalovirus (CMV) infection is a major complication after allogeneic stem cell transplantation (ASCT).Aims:The aim of the study was to evaluate the impact of CMV reactivation on the relapse rate after ASCT in patients with acute myeloid leukemia (AML).Methods:Retrospective study conducted in patients with AML who underwent ASCT from HLA identical sibling donor between January 2011 and December 2018. Conditioning regimen consisted of Busulfex and Cyclophosphamide (Bu/Cy) or Fludarabine and Busulfex (F/Bu). Graft‐versus‐host disease (GVHD) prophylaxis consisted of cyclosporine and a short course of methotrexate. Antiviral prophylaxis for CMV infection was assured by Acyclovir from day +1 to day +180. CMV detection was carried out once‐a‐week from engraftment to day +100 by either pp 65 antigenemia test or real‐time quantitative PCR.Results:Ninety‐three patients were enrolled (55 men and 38 women). Median age was 33 years (range, 5 ‐ 49 y). At the time of transplant, 73 patients (78.5%) were in CR1, 16 patients (17.2%) were in CR2 and 4 patients (4.3%) were in response failure. CMV serostatus for donor (D) and recipient (R) was available for 44 cases (47.3%). Serostatus of R+/D+ and R+/D− was observed in 38 patients (86.4%). Stem cell source were BM in 49 cases (52.7%) and PBSC in 44 cases (47.3%). No graft failure was observed. Acute GVHD grade II‐IV occurred in 19 patients (20.4%). Chronic GVHD was observed in 40 patients (45.4%). Twenty‐nine patients (31.2%) developed CMV reactivation. With a median follow‐up of 2 years (range 49 days – 7 years), the overall survival (OS) and the non‐relapse mortality (NRM) were not statistically significant between patients with CMV reactivation and those without CMV reactivation (74% vs 63%, p = 0.3 and 20.7% vs 7.8%, p = 0.08, respectively). Twenty‐four patients (25.8%) relapsed at a median of 6 months (range 2 ‐ 67 months). the rate of relapse among patients with CMV reactivation was significantly lower than in those without CMV reactivation (10.3% vs 32.8%, p = 0.02). In univariate analysis, the CMV reactivation was the only factor associated with a decreased risk of relapse (OR = 0.23, 95% CI: 0.06–0.87, p = 0.02).Summary/Conclusion:CMV reactivation was associated with decreased relapse risk after ASCT for patients with AML without a benefit in OS.

Intestinal Transplant-Associated Thrombotic Microangiopathy: Histopathological Review. a Single Center Experience

INTRODUCTIONTransplant-associated thrombotic microangiopathy (TMA) is a severe complication after allogeneic stem cell transplantation (ASCT) due to endothelial injury caused by many factors such Calcineurin-inhibitors. In most severe cases, TMA could affect different organs. Intestinal TMA could be fatal and missdiagnosed in many patients. Clinical and pathological criteria to differentiate from intestinal GVHD are needed in order to distinguish both entities with different therapeutical approach. The aim of this study was to review pathological TMA features in patients diagnosed of systemic TMA .PATIENTS AND METHODSWe analyzed the incidence of TMA in 527 pts who underwent ASCT in our institution between jan-2010 and apr-2018. Patients were identified if they had TMA according to probable TMA criteria by Ho. We do a pathological review in 96 samples from 18 of 42 patients in whom an endoscopy have been performed after the diagnosis of the TMA; endoscopy have been performed between 30 days before and 60 days after diagnosis of TMA for initial clinical diagnosis of GVHD. Review was performed by a pathologist expert. He examined the biopsies in search of features of GVHD, TMA or viral infection. Diagnosis of GVHD was stablished according to Mcdonald and Sales criteria, intestinal TMA diagnosis was perfomed by the 8 criteria summarized by Warren et al (perivascular mucosal hemorrhage, endothelial cell swelling, endothelial cell separation, intraluminal schistocytes, fibrin or microthrombi, loss of glands and mucosal denudation).RESULTSBaseline/transplant characteristics of patients with TMA are shown in table 1, TMA data in 2 and review hystological features of biopsies in 3.45 (8.5%) were diagnosed of TMA with a median time from transplant of 75 days. Median age was 49 (19-69). Prophylaxis of GVHD was: Calcineurin inhibitor-MTX in 16 (35.5%), Tacrolimus-Sirolimus in 21 (46.6%), Tacrólimus-MMF and Cyclophosphamide in 8 (17.6%). 42 (93%) had prior or simultaneous acute GVHD, half of them grade III-IV, and 80% with gastrointestinal GVHD. 42% had elevated levels of tacrolimus and 13.6% elevated levels of sirolimus, one week before the diagnosis of TMA.Gastrointestinal MAT have been reported only in 5 patients (28%) at diagnosis whereas when review based on Warren criteria was performed, in 16 patients (89%) the pathologist found at least 1 of the criteria of endothelial damage and 50 % of the patients met 3 or more Warren criteria. The most frequent features were endothelial cell swelling (13 patients, 73 %) and perivascular mucosal hemorrhage (12 patients, 66.7%). In 3 biopsies which we perfomed the inmunochemistry of C4d, an activation of classic way of complement biomarker, it was positive. 4 of the 18 patients (22.2%) presented refractory-hypertension and 3 of them (26.6%) more than 30 mg/d Lof proteinuria, both suggested of poor prognosis.Regarding GVHD, it was founded in 72% at diagnosis and in all patients (18) at pathological review. 13 had grade I, 1 grade II, 1 grade III and 3 histological grade IV).With a median follow-up of 5 months (2-25) 25 of the 45 (56%) are dead. 6 of the deaths (24%) were related to TMA (1 due to TMA, 3 due to TMA+GVHD, 2 due to TMA+infection). Other causes of death were progression (4), GVHD+Infection (7), GVHD (3), infection (2), sinusoidal obstruction síndrome (1) and other causes (2).CONCLUSIONTMA is a frequent complication, related with GVHD and underdiagnosed frequently. Only 5 of 18 patients were diagnosed of gastrointestinal TMA.In our study, we found that most of our patients had endotelial damage in the gastrointestinal biopsy pathological reviews. Although histological criteria of GVHD were present at review, in most of them it was only grade I; it contrasts with the severe clinical features. That lack of correlation would suggest that TMA and not GVHD is the main feature.Management of TMA and GVHD are different. To stress an appropiate diagnosis in gastrointestinal TMA is needed in order to offer the patients the best approach.REFERENCESWarren et al. A Complete Histologic Approach to Gastrointestinal Biopsy From Hematopoietic Stem Cell Transplant Patients With Evidence of Transplant-Associated Gastrointestinal Thrombotic Microangiopathy. Arch Pathol Lab Med.2017 Nov;141(11):1558-1566.Jodele S et al. A new paradigm: Diagnosis and management of HSCT-associated thrombotic microangiopathy as multi-system endothelial injury. Blood Rev. 2015;29(3):191-204. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Impact of ruxolitinib pretreatment on outcomes after allogeneic stem cell transplantation in patients with myelofibrosis.

Ruxolitinib is the first approved drug for treatment of myelofibrosis, but its impact of outcome after allogeneic stem cell transplantation (ASCT) is unknown. We reported on 159 myelofibrosis patients (pts) with a median age of 59years (r: 28-74) who received reduced intensity ASCT between 2000 and 2015 in eight German centers from related (n=23), matched (n=86) or mismatched (n=50) unrelated donors. Forty-six (29%) patients received ruxolitinib at any time point prior to ASCT. The median daily dose of ruxolitinib was 30mg (range 10-40mg) and the median duration of treatment was 4.9months (range 0.4-39.1months). Primary graft failure was seen in 2 pts (4%) in the ruxolitinib and 3 (2%) in the non-ruxolitinib group. Engraftment and incidence of acute GVHD grade II to IV and III/IV did not differ between groups (37% vs 39% and 19% vs 28%, respectively), nor did the non-relapse mortality at 2years (23% vs 23%). A trend for lower risk of relapse was seen in the ruxolitinib group (9% vs 17%, P=.2), resulting in a similar 2year DFS and OS (68% vs 60% and 73% vs 70%, respectively). No difference in any outcome variable could be seen between ruxolitinib responders and those who failed or lost response to ruxolitinib. These results suggest that ruxolitinib pretreatment in myelofibrosis patient does not negatively influence outcome after allogeneic stem cell transplantation.

A pilot study of single nucleotide polymorphisms in the interleukin-6 receptor and their effects on pre- and post-transplant serum mediator level and outcome after allogeneic stem cell transplantation.

Interleukin (IL)-6 is an important regulator of immunity and inflammation in many diseases. Single nucleotide polymorphisms (SNPs) in the IL-6 gene influence outcome after allogeneic stem cell transplantation (ASCT), but the possible importance of SNPs in the IL-6 receptor has not been examined. We therefore investigated whether SNPs in the IL-6R gene influenced biochemical characteristics and clinical outcomes after ASCT. We examined the IL-6 promoter variant rs1800975 and the IL-6R SNPs rs4453032, rs2228145, rs4129267, rs4845374, rs4329505, rs4845617, rs12083537, rs4845618, rs6698040 and rs4379670 in a 101 population-based cohort of allotransplant recipients and their family donors. Patients being homozygous for the major alleles of the IL-6R SNPs rs2228145 and rs4845618 showed high pretransplant CRP serum levels together with decreased sIL-6R levels; the decreased IL-6R levels persisted 6 months post-transplant. In contrast, patients being homozygous for the minor allele of the IL-6R SNP rs4379670 showed decreased pretransplant CRP levels. Furthermore, the IL-6R rs4845618 donor genotype showed an association with severe acute graft-versus-host disease (GVHD), whereas the donor genotype of the IL-6 SNP rs1800795 was associated with decreased survival 100 days post-transplant. Finally, the recipient genotype of the IL-6R SNP rs4329505 showed a strong association with 2-years non-relapse mortality, and this effect was also highly significant in multivariate analysis. IL-6 and IL-6R SNPs influence the clinical outcome after allogeneic stem cell transplantation.

The association between acute graft-versus-host disease and antimicrobial peptide expression in the gastrointestinal tract after allogeneic stem cell transplantation.

Intestinal microbiota disruption is associated with acute gastrointestinal (GI) Graft-versus-Host Disease (GvHD) and poor outcome after allogeneic stem cell transplantation (ASCT). Here, in a retrospective analysis of 200 patients undergoing ASCT at the Regensburg University Medical Center, we assessed the relative expression of Paneth cell antimicrobial peptides (AMPs), Human Defensins (HD) 5 and 6 and regenerating islet-derived 3α (Reg3α), in 292 human intestinal biopsies as well as Reg3α serum levels in relation to acute GI GvHD. In the absence of GI GvHD, the relative expression of Paneth cell AMPs was significantly higher in the small intestine (duodenum to ileum) than in the stomach and large intestine (cecum to rectum) for Reg3α (p≤0.001), HD5 (p≤0.002) and HD6 (p≤0.02). Acute stage 2–4 GI GvHD was associated with reduced expression of AMPs in the small intestine (p≤0.01) in comparison to stage 0–1 disease, accompanied by a decrease in Paneth cell count in case of severe acute GI GvHD (p<0.001). The opposite held true for the large intestine as we found stage 2–4 GI GvHD correlated with significantly higher expression of HD5, HD6, and Reg3α compared to mild or no acute GI GvHD (p≤0.002). Severe GI GvHD in both the lower and the upper GI tract also correlated with higher serum concentrations of Reg3α (p = 0.002). As indirect markers of intestinal microbiome diversity low levels of urinary 3-indoxyl sulfate levels were associated with severe stages of acute GI GvHD compared to mild stage or no acute GI GvHD (p = 0.05). In conclusion, acute GI GvHD correlates with intestinal expression of HD5, HD6 and Reg3α as well as Reg3α serum levels and is associated with intestinal dysbiosis.

Impact of Pre-Transplant Ruxolitinib in Myelofibrosis Patients on Outcome after Allogeneic Stem Cell Transplantation

▪IntroductionRuxolitinib is the first approved drug for treatment of myelofibrosis. Major effects are reduction in spleen size and improvement of constitutional symptoms. Because spleen size and constitutional symptoms may influence outcome after allogeneic stem cell transplantation (ASCT), ruxolitinib is recommended before stem cell transplantation in order to reduce therapy-related morbidity and mortality and improve outcome (EBMT/ELN recommendation, Leukemia 2015) The aim of this retrospective study was to evaluate the impact of pretreatment with ruxolitinib in comparison to transplantation of ruxolitinib-naïve MF patients with regard to outcome after ASCT.Patients and methodsWe included 171 myelofibrosis patients (pts) with a median age of 59 years (r: 28 - 74) who received ASCT between 2000 and 2015 from related (n = 25), matched (n=94) or mismatched (n=52) unrelated donor. Stem cell source were more peripheral blood stem cells (n = 167) than bone marrow (n = 4). All patients received busulfan-based reduced intensity conditioning. While 113 pts (66%) did not receive ruxolitinib, 58 pts (34%) received ruxolitinib at any time point prior to ASCT. The median daily dose of ruxolitinib was 30mg (range 10-40mg) and the median duration of treatment was 28 days (range 12-159 days). In 11 pts ruxolitinib was stopped before stem cell transplantation because of no response or loss of response, while in 35 pts ruxolitinib was given until start of conditioning and in 12 pts ruxolitinib was given until stable engraftment. GvHD prophylaxis consisted of CNI plus short course MTX or MMF and anti-lymphocyte globulin. According to dynamic IPSS (DIPSS) (n = 170) the patients were either low (n = 2), intermediate-1 (n = 37), intermediat-2 (n = 81), or high risk (n = 38). 74 patients (43%) were transfusion dependent.ResultsAs the median follow up was shorter for patients treated with ruxolitinib (15 vs 73 months, p<0.001), we analyzed only 2 years RFS, OS, NRM and relapse incidence. Primary graft failure was seen in 2 pts in the ruxolitinib and 3 in the non-ruxolitinib group. The median leukocyte engraftment was 13 days (r., 9-32) in the ruxolitinib and 14 days (r., 7-34) in the non ruxolitinib group (p=0.7). The median age in the ruxolitinib group was slightly older ( 62 vs 58 years , p= 0.09). The incidence of acute GvHD grade I to IV was significantly lower in the ruxolitinib group (49% vs 64%, p=0.05), while aGvHD grade II-IV (33% vs 44%, p=0.14) and grade III/IV (23% vs 25%, p=0.48), did not differ significantly. The CI of NRM at 1 year was 18% (95% CI: 6-30%) for the ruxolitinib group and 22% (95% CI: 14-30%) for the non-ruxolitinib group (p=0.58), and the CI of relapse at 2 years was 8% (95% CI: 0-16%) vs 20% (95%CI: 12-28%, p=0.25). The 2 years RFS and OS was 66% (95%CI: 50-82%) and 69% (95%CI: 51-87) for the ruxolitinib group and 59% (95% CI: 49-69%) and 70% (95% CI:62-78%) for the non-ruxolitinib group (p=0.29 and p=0.45, respectively). Within the ruxolitinib group (n=53), 24 pts responded to ruxolitinib (more than 25% spleen size reduction), while 29 pts did not respond or lost response prior to stem cell transplantation. Here, no significant difference could be seen between the responding and non-responding group for NRM (19% vs 17%, p=0.69), Relapse (4% vs 13%, p=0.62), RFS (61% vs 72%, p=0.81) and OS (63% vs 75%, p=0.89). In a multivariate analysis including ruxolitinib treatment as variable there was a non-significant trend in favor for ruxolitinib pretreatment regarding NRM (HR 0.79; 95%CI: 0.38-1.66, p=0.54), relapse (HR 0.48; 95%CI: 0.18-1.31, p=0.15), RFS (HR 0.55; 95%CI: 0.29-1.03, p=0.06) and OS (HR 0.83; 95%CI: 0.41-1.67, p=0.61).ConclusionsThese results suggest that ruxolitinib pretreatment in myelofibrosis patient does not negatively influence outcome after allogeneic stem cell transplantation. To confirm the observed favorable trend in outcome after ruxolitinib treatment more patients and a longer follow-up is needed. DisclosuresCrysandt:Novartis: Other: Travel grant. Stelljes:Pfizer: Consultancy. Kröger:Novartis: Honoraria, Research Funding.

Targeting polo-like kinase 1 suppresses essential functions of alloreactive T cells

Acute graft-versus-host disease (aGvHD) is still a major cause of transplant-related mortality after allogeneic stem cell transplantation (ASCT). It requires immunosuppressive treatments that broadly abrogate T cell responses including beneficial ones directed against tumor cells or infective pathogens. Polo-like kinase 1 (PLK1) is overexpressed in many cancer types including leukemia, and clinical studies demonstrated that targeting PLK1 using selective PLK1 inhibitors resulted in inhibition of proliferation and induction of apoptosis predominantly in tumor cells, supporting the feasibility of PLK1 as target for anticancer therapy. Here, we show that activation of alloreactive T cells (Tallo) up-regulate expression of PLK1, suggesting that PLK1 is a potential new candidate for dual therapy of aGvHD and leukemia after ASCT. Inhibition of PLK1, using PLK1-specific inhibitor GSK461364A selectively depletes Tallo by preventing activation and by inducing apoptosis in already activated Tallo, while memory T cells are preserved. Activated Tallo cells which survive exposure to PLK1 undergo inhibition of proliferation by induction of G2/M cell cycle arrest, which is accompanied by accumulation of cell cycle regulator proteins p21(WAF/CIP1), p27(Kip1), p53 and cyclin B1, whereas abundance of CDK4 decreased. We also show that suppressive effects of PLK1 inhibition on Tallo were synergistically enhanced by concomitant inhibition of molecular chaperone Hsp90. Taken together, our data suggest that PLK1 inhibition represents a reasonable dual strategy to suppress residual tumor growth and efficiently deplete Tallo, and thus provide a rationale to selectively prevent and treat aGvHD.

Impact of Minimal Residual Disease after Allografting Detected By JAK2V617F, MPL or Calreticulin in Myelofibrosis Patients

▪Introduction:Residual minimal disease (MRD) in hematological malignancies has become a valid tool to predict clinical relapse after allogeneic stem cell transplantation (ASCT).Methods and Patients:We screened 154 patients with myelofibrosis who underwent ASCT for molecular residual disease by qPCR or next-generation sequencing (NGS) for JAK2V617F, MPLW515L and MPLW515K or Calreticulin (L367fs*46, and K385fs*47) mutations in peripheral blood (PB) on days +100 and +180 after transplantation. Out of 154 pts, 103 were JAK2V617F (n=101), 31 Calreticulin (CALR), and 4 MPL mutated, while 13 pts were triple negative. 136 pts could be followed after ASCT with one molecular marker. The median age of the pts was 58 years (range, 32-75 y). Patients had either primary myelofibrosis (n= 90), post ET/PV myelofibrosis (n=40), myelofibrosis in acceleration or were transformed to AML (n=6). Conditioning mainly relied on a busulfan-based reduced-intensity regimen. Donor were HLA-identical sibling (n=26), matched unrelated (n=71) or mismatched unrelated (n=39). JAK2V617F, MPL, and CALR mutations were measured by usage of Taqman PCR or in case of CALR Type 2 mutation by digital PCR on day +100 and day +180 from PB as described elsewhere.Results:After a median follow up of 78 months (range, 49-101 months) the 5-year estimated overall survival was 60% (95% CI: 50-70%) and the cumulative incidence of relapse at 5 years was 26% (95% CI: 18-34%) for the entire study population. On days +100 and +180 after transplantation in 27% and 12% of the patients the underlying mutation was still detectable in peripheral blood. The percentage of complete clearance was higher in CALR-mutated patients (96%) in comparison to MPL (75%) and JAKV2617F (70%) mutated pts. Whereas there was a trend for better survival for CALR-mutated patients in comparison to JAK2-mutated patients (71% vs 57%; p=0.48), once a patient achieved molecular remission post-transplant the risk of relapse remained low independently of the underlying mutation. Patients who were alive and without relapse at days +100 or +180 but with still detectable mutation in PB had a significantly higher risk of relapse than those who were molecular negative (62% vs 10%, p<0.001; and 70% vs 10%, p<0.001, respectively). In a multivariate analysis only high-risk disease status (HR 2.5; 95% CI: 1.18-5.25, p=0.016) and detectable MRD at day 180 (HR 8.36, 95% CI: 2.76-25.30, p<0.001) were significant factors for a higher risk of relapse.Conclusions:JAK2V617F, CALR and MPL genetic lesions allow to monitor MRD in around 90% of the myelofibrosis patients after ASCT by qPCR or digital PCR in the PB. Persistence of MRD on days +100 or +180 in peripheral blood can be used to taper immunosuppressive drugs or to apply donor lymphocyte infusion to prevent clinical relapse. DisclosuresNo relevant conflicts of interest to declare.

Low urinary indoxyl sulfate levels early after transplantation reflect a disrupted microbiome and are associated with poor outcome

AbstractIndole, which is produced from l-tryptophan by commensal bacteria expressing tryptophanase, not only is an important intercellular signal in microbial communities, but also modulates mucosal barrier function and expression of pro- and anti-inflammatory genes by intestinal epithelial cells. Here, we hypothesized that decreased urinary excretion of 3-indoxyl sulfate (3-IS), the major conjugate of indole found in humans, may be a marker of gut microbiota disruption and increased risk of developing gastrointestinal (GI) graft-versus-host-disease. Using liquid chromatography/tandem mass spectrometry, 3-IS was determined in urine specimens collected weekly within the first 28 days after allogeneic stem cell transplantation (ASCT) in 131 patients. Low 3-IS levels within the first 10 days after ASCT were associated with significantly higher transplant-related mortality (P = .017) and worse overall survival (P = .05) 1 year after ASCT. Least absolute shrinkage and selection operator regression models trained on log-normalized counts of 763 operational taxonomic units derived from next-generation sequencing of the hypervariable V3 region of the 16S ribosomal RNA gene showed members of the families of Lachnospiraceae and Ruminococcaceae of the class of Clostridia to be associated with high urinary 3-IS levels, whereas members of the class of Bacilli were associated with low 3-IS levels. Risk factors of early suppression of 3-IS levels were the type of GI decontamination (P = .01), early onset of antibiotic treatment (P = .001), and recipient NOD2/CARD15 genotype (P = .04). In conclusion, our findings underscore the relevance of microbiota-derived indole and metabolites thereof in mucosal integrity and protection from inflammation.

Extramedullary relapses after allogeneic stem cell transplantation for leukemia: Clinical characteristics, cumulative incidence and risk factors.

e18005 Background: In acute leukemia (AL) relapse after allogeneic stem cell transplantation (ASCT) is a devastating complication. Although this problem has been well studied in the literature ther...

Transformation of an Unclassified Myeloproliferative Neoplasm with a Rare BCR-JAK2 Fusion Transcript Resulting from the Translocation (9;22)(p24;q11).

BCR-ABL1 negative myeloproliferative neoplasms (MPNs) are known to contain alterations of the tyrosine kinase JAK2 (located on 9p24) that result in constitutive activation of the encoded protein. JAK2 fusions are reported in acute and chronic leukemias of myeloid and lymphoid phenotypes. Here, we report an unclassified case of MPN (MPN-U) showing a t(9;22)(p24;q11), which generates a BCR-JAK2 fusion gene by fusing the BCR at intron 13 to JAK2 at intron 17 on the derivative chromosome 22. Most reported JAK2 fusions cases reveal an aggressive clinical course and long-term remissions have only been achieved after allogeneic stem cell transplantation (ASCT). To the best of our knowledge, this is the thirteenth case reported worldwide to describe a BCR-JAK2 fusion transcript in MPN-U. The present report revealed a sustained complete clinical, hematologic, and cytogenetic remission 35 months after diagnosis and ~24 months after ASCT. Regarding BCR-ABL1 negative MPN patients this case report provides strong support for a role of JAK2 activation in the oncogenesis and suggests a possible diagnostic and therapeutic target that should be investigated.