Barbara Holler

Metagenomic analysis of the stool microbiome in patients receiving allogeneic stem cell transplantation: loss of diversity is associated with use of systemic antibiotics and more pronounced in gastrointestinal graft-versus-host disease.

Next-generation sequencing of the hypervariable V3 region of the 16s rRNA gene isolated from serial stool specimens collected from 31 patients receiving allogeneic stem cell transplantation (SCT) was performed to elucidate variations in the composition of the intestinal microbiome in the course of allogeneic SCT. Metagenomic analysis was complemented by strain-specific enterococcal PCR and indirect assessment of bacterial load by liquid chromatography-tandem mass spectrometry of urinary indoxyl sulfate. At the time of admission, patients showed a predominance of commensal bacteria. After transplantation, a relative shift toward enterococci was observed, which was more pronounced under antibiotic prophylaxis and treatment of neutropenic infections. The shift was particularly prominent in patients that developed subsequently or suffered from active gastrointestinal (GI) graft-versus-host disease (GVHD). The mean proportion of enterococci in post-transplant stool specimens was 21% in patients who did not develop GI GVHD as compared with 46% in those that subsequently developed GI GVHD and 74% at the time of active GVHD. Enterococcal PCR confirmed predominance of Enterococcus faecium or both E. faecium and Enterococcus faecalis in these specimens. As a consequence of the loss of bacterial diversity, mean urinary indoxyl sulfate levels dropped from 42.5 ± 11 μmol/L to 11.8 ± 2.8 μmol/L in all post-transplant samples and to 3.5 ± 3 μmol/L in samples from patients with active GVHD. Our study reveals major microbiome shifts in the course of allogeneic SCT that occur in the period of antibiotic treatment but are more prominent in association with GI GVHD. Our data indicate early microbiome shifts and a loss of diversity of the intestinal microbiome that may affect intestinal inflammation in the setting of allogeneic SCT.

Tryptophan catabolism is associated with acute GVHD after human allogeneic stem cell transplantation and indicates activation of indoleamine 2,3-dioxygenase

Induction of indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme in tryptophan degradation along the kynurenine pathway, acts as a potent immunoregulatory loop. To address its role in human allogeneic stem cell transplantation, we measured major tryptophan metabolites, such as quinolinic acid and kynurenine, in serial urine specimens from 51 patients by liquid chromatography-tandem mass spectrometry. Samples were collected between admission and day 90 after transplantation, and metabolite levels were correlated with early clinical events and outcome. In selected patients, IDO gene expression was assessed by quantitative RT-PCR in intestinal biopsies. Surviving patients had significantly lower metabolite levels on days 28, 42, and 90, respectively, compared with patients dying of GVHD and associated complications (n = 10). Kynurenine levels were directly correlated with severity and clinical course of GVHD: Mean urinary quinolinic acid levels were 4.5 ± 0.3 μmol/mmol creatinine in the absence of acute GVHD, 8.0 ± 1.1 μmol/mmol creatinine for GVHD grade 1 or 2, and 13.5 ± 2.7 μmol/mmol creatinine for GVHD grade 3 or 4 (P < .001), respectively. GVHD-dependent induction of IDO was further suggested by increased expression of IDO mRNA in intestinal biopsies from patients with severe GVHD. Our data indicate reactive release of kynurenines in GVHD-associated inflammation.

Risk Factors and Outcome of Chronic Graft-versus-Host Disease after Allogeneic Stem Cell Transplantation—Results from a Single-Center Observational Study

Chronic graft versus host disease (cGVHD) is the most frequent long-term complication after allogeneic stem cell transplantation (allo-SCT) and results in impaired quality of life and increased long-term morbidity and mortality. We analyzed 243 patients with cGVHD, documented according to the 2005 revised National Institutes of Health consensus criteria, to identify risk factors for the occurrence of cGVHD and outcomes for the patients with cGVHD. Patients without evidence of cGVHD (n = 147) were used as controls. Performing univariate and multivariate Cox regression analyses, we identified prior acute GVHD grades III or IV (hazard ratio [HR], 2.01; P = .005), use of peripheral blood stem cell graft (HR, 2.10; P = .03), and HLA-mismatched allo-SCT from unrelated donor (HR, 1.57; P = .02) as independent risk factors for cGVHD. Performing Kaplan-Meier analyses, progressive compared with de novo and quiescent onset of cGVHD and a platelet count of less than 100/nL compared with more than 100/nL at the time of cGVHD onset were associated with a significantly increased cumulative incidence of transplantation-related mortality (TRM) and significantly decreased overall survival. Furthermore, we found a significantly higher incidence of TRM in patients with severe cGVHD compared with patients without cGVHD (58% versus 11%, P < .0001). However, in subgroup analysis, patients with severe cGVHD and involvement of the lung, liver, or gastrointestinal (GI) tract had a 6.5-fold significantly higher incidence of TRM (72%), whereas patients with severe cGVHD lacking lung, liver, or GI involvement had only a 2.8-fold significantly higher incidence of TRM (31%) compared with patients without cGVHD (11%; P < .0001 and P = .03). Patients without lung, liver, or GI involvement did not have a significantly different TRM compared with patients with moderate cGVHD (31% versus 25%, P = .52). In conclusion, we confirm prior known risk factors for the occurrence of cGVHD and subsequent mortality and we provide evidence that the presence of lung, liver, or GI involvement in patients with severe cGVHD defines a subgroup with high mortality after allo-SCT; however, in the absence of these risk factors, the outcome appears not to be impaired compared with moderate cGVHD.

Mesenchymal Stromal Cells Protect Endothelial Cells from Cytotoxic T Lymphocyte‐Induced Lysis

The integrity of the vasculature plays an important role in the success of allogeneic organ and haematopoietic stem cell transplantation. Endothelial cells (EC) have previously been shown to be the target of activated cytotoxic T lymphocytes (CTL) resulting in extensive cell lysis. Mesenchymal stromal cells (MSC) are multipotent cells which can be isolated from multiple sites, each demonstrating immunomodulatory capabilities. They are explored herein for their potential to protect EC from CTL‐targeted lysis. CD8+ T cells isolated from human PBMC were stimulated with mitotically inactive cells of a human microvascular endothelial cell line (CDC/EU.HMEC‐1, further referred to as HMEC) for 7 days. Target HMEC were cultured in the presence or absence of MSC for 24 h before exposure to activated allogeneic CTL for 4 h. EC were then analysed for cytotoxic lysis by flow cytometry. Culture of HMEC with MSC in the efferent immune phase (24 h before the assay) led to a decrease in HMEC lysis. This lysis was determined to be MHC Class I restricted linked and further analysis suggested that MSC contact is important in abrogation of lysis, as protection is reduced where MSC are separated in transwell experiments. The efficacy of multiple sources of MSC was also confirmed, and the collaborative effect of MSC and the endothelium protective drug defibrotide were determined, with defibrotide enhancing the protection provided by MSC. These results support the use of MSC as an adjuvant cellular therapeutic in transplant medicine, alone or in conjunction with EC protective agents such as defibrotide.

Potential Role of the PD-1/PD-L1 Axis in the Immune Regulation of Chronic GVHD

Background: Antibodies blocking the PD-1/PD-L1 axis have been shown to have substantial antitumor effects also in the treatment of Hodgkins lymphoma (HL) relapsing after conventional chemotherapy or even autologous hematopoietic stem cell transplantation (autoHSCT). In the case of allogeneic HSCT (alloHSCT), this treatment bears the risk of inducing graft-versus-host disease (GVHD). So far, only a small number of patients who developed acute GVHD after PD-1 antibody administration are described in the literature. Case Reports: We herein report the cases of 2 HL patients after alloHSCT who both responded well to the therapy; however, 1 patient developed chronic GVHD (cGVHD) within 3 days of administration of nivolumab. This patient already had a history of cGVHD and interestingly showed manifestations at the very same sites. The other patient never showed any signs of cGVHD, even with the administration of 13 cycles of anti-PD-1 therapy and large doses of donor lymphocytes. Conclusion: The rapid reappearance of cGVHD after blockade of PD-1 implies an important role of PD-1/PD-L1 in peripheral immune tolerance in cGVHD after alloHSCT and warrants further investigation.

The lung function score and its components as predictors of overall survival and chronic graft-vs-host disease after allogeneic stem cell transplantation.

Aim To retrospectively assess if the modified lung function score (LFS) and/or its components, forced expiratory volume within the first second (FEV1) and diffusion capacity for carbon monoxide corrected for hemoglobin level (cDLCO), predict overall survival (OS) and chronic graft-vs-host-disease (cGvHD). Methods We evaluated 241 patients receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT) at the University of Regensburg Transplant Center between June 1998 and July 2005 in relation to their LFS, FEV1 and cDLCO, before and after HSCT. Results Decreased OS after allo-HSCT was related to decreased pre-transplantation values of FEV1<60% (P = 0.040), cDLCO<50% of predicted value (P = 0.025), and LFS≥III (P = 0.037). It was also related to decreased FEV1 at 3 and 12 months after HSCT (P < 0.001 and P = 0.001, respectively) and increased LFS at 3 and 12 months after HSCT (P = 0.028 and P = 0.002, respectively), but not to changes of cDLCO. A higher incidence of cGvHD was related to decreased FEV1 at 6, 12, and 18 months (P = 0.069, P = 0.054, and P = 0.009, respectively) and increased LFS at 12 months (P = 0.002), but not to changes in cDLCO. Conclusions OS was related to both LFS and FEV1, but cGvHD had a stronger relation to FEV1 than to cDLCO or LFS. FEV1 alone offered more information on the outcome after allo-HSCT than LFS or cDLCO, suggesting limited value of LFS for the patients’ assessment after allo-HSCT.

Total nodal irradiation in patients with severe treatment-refractory chronic graft-versus-host disease after allogeneic stem cell transplantation: Response rates and immunomodulatory effects.

BACKGROUND AND PURPOSE The use of total nodal irradiation (TNI) has been reported as an immunomodulatory therapy for different diseases including chronic graft-versus-host disease (cGVHD). MATERIAL AND METHODS We retrospectively analyzed 13 patients with treatment-refractory cGVHD receiving TNI with 1×1Gy from 2001 to 2014. In 10 of 13 patients immunomodulatory effects of TNI were measured. RESULTS At time of TNI all patients had severe cGVHD (involving the skin: n=12), fascia (n=6), oral mucosa (n=8), eye (n=8), and lung (n=5). Nine of 13 patients had corticosteroid-refractory cGVHD. In 7 of 13 patients (54%) a partial response (PR) could be achieved. In 3 patients (23%) cGVHD manifestations remained stable, 2 patients progressed. One patient was not evaluable due to follow-up <1 month. At 3 months after TNI, best responses could be achieved in skin, and oral involvement including steroid sparing activity. TNI was well tolerated with adverse effects limited to reversible thrombocytopenia and neutropenia. Immunomodulatory effects on peripheral blood cells could be demonstrated including an increase of CD4+ T cells in the group of responders. CONCLUSIONS TNI represents an effective immunomodulating therapy in treatment-refractory cGVHD.