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Chronic graft-versus-host disease (cGVHD) is the most common cause for non-relapse mortality postallogeneic hematopoietic stem cell transplant (HSCT). However, there are no well-defined biomarkers for cGVHD or late acute GVHD (aGVHD). This study is a longitudinal evaluation of metabolomic patterns of cGVHD and late aGVHD in pediatric HSCT recipients. A quantitative analysis of plasma metabolites was performed on 222 evaluable pediatric subjects from the ABLE/PBMTC1202 study. We performed a risk-assignment analysis at day + 100 (D100) on subjects who later developed either cGVHD or late aGVHD after day 114 to non-cGVHD controls. A second analysis at diagnosis used fixed and mixed multiple regression to compare cGVHD at onset to time-matched non-cGVHD controls. A metabolomic biomarker was considered biologically relevant only if it met all 3 selection criteria: (1) P ≤ .05; (2) effect ratio of ≥1.3 or ≤0.75; and (3) receiver operator characteristic AUC ≥0.60. We found a consistent elevation in plasma α-ketoglutaric acid before (D100) and at the onset of cGVHD, not impacted by cGVHD severity, pubertal status, or previous aGVHD. In addition, late aGVHD had a unique metabolomic pattern at D100 compared with cGVHD. Additional metabolomic correlation patterns were seen with the clinical presentation of pulmonary, de novo, and progressive cGVHD. α-ketoglutaric acid emerged as the single most significant metabolite associated with cGVHD, both in the D100 risk-assignment and later diagnostic onset analysis. These distinctive metabolic patterns may lead to improved subclassification of cGVHD. Future validation of these exploratory results is needed. This trial was registered at www.clinicaltrials.gov as #NCT02067832.

Chronic graft-versus-host disease (cGVHD) and late acute graft-versus-host disease (L-aGVHD) are understudied complications of allogeneic hematopoietic stem cell transplantation in children. The National Institutes of Health Consensus Criteria (NIH-CC) were designed to improve the diagnostic accuracy of cGVHD and to better classify graft-versus-host disease (GVHD) syndromes but have not been validated in patients

Pediatric chronic graft-versus-host disease (cGvHD) contributes to poor quality of life and increased morbidity and mortality in long term survivors of hematopoietic stem cell transplant (HSCT). Given the insidious nature of cGvHD, diagnosing cGvHD in children according to the National Institutes of Health Consensus Criteria (NIH-CC) can be challenging, particularly in the early phase of disease development. Diagnostic cGvHD biomarkers could aid in diagnosis. The Applied Biomarkers of Late Effects / Pediatric Blood and Marrow Transplant Consortium (ABLE / PBMTC 1202) study was a prospective, multi-institution study at 27 pediatric HSCT centers that evaluated biomarkers of cGvHD. Detailed clinical evaluation of cGvHD, including central adjudication when necessary, occurred as per the NIH-CC. Of the 302 enrolled children, 233 were included in this diagnostic cGvHD biomarker analysis, including 43 with cGvHD and 190 non-cGVHD controls. Peripheral blood was drawn into a STRECK tube at the onset of cGvHD and before escalation of immune suppression, sent overnight to a central laboratory, and evaluated by flow cytometry. Five flow cytometric antibody panels consisting of 76 combinations of cell surface markers were used to delineate subpopulations of T, Regulatory T (Tregs, CD4+CD127LowCD25+), B, NK, and myeloid cells. We analyzed each marker in two ways. First, to control for normal post-HSCT immune reconstitution, we divided cGvHD subjects into early- (day 0-120), mid- (day 121-240), and late-onset (>day 240) and compared their marker values against non-cGVHD controls at day 100, 6-months, and 12-months, respectively, using fixed effect linear regression models. Second, we compared the marker values of all cGvHD subjects at diagnosis against the marker values of non-cGvHD controls at all three time points and used mixed effect linear regression models to account for within-subject correlations. A cellular subpopulation was considered a clinically relevant and potential diagnostic cGvHD biomarker if all 4 criteria were met: a) the effect ratio (mean marker value of cGvHD subjects over non-cGvHD controls) was ≥1.3 or ≤0.75, b) the p-value was ≤.05, c) the receiver-operating characteristic area under the curve was ≥0.60, and d) the marker was detected by both the mixed effect model and in the same direction across all measurement time points in the fixed effect models. Using these criteria, 8 cellular subpopulations arose as potential diagnostic biomarkers of pediatric cGvHD (Table). Key subsets identified in cGvHD patients included decreased proportions of naïve helper T cells (CD4+CD45RA+, PD1-CD4+CD45RA+, CCR7+CD4+CD45RA+, CD27+CD4+CD45RA+), decreased naïve and recently emigrated Tregs (CD31+CD45RA+, PD1-CD45RA+), and decreased non-cytolytic regulatory NK cells (CD56BrightCD335High, CD56BrightPerforinLow). CD4+ recent thymic emigrants (CD4+CD31+CD45RA+), markers of thymopoeisis, were also significantly decreased in cGvHD (p=0.01) but did not meet all the required biomarker criteria. Evaluation of specific subsets of pediatric cGvHD showed that CD21LowCD19+ B cells were lower in patients with pulmonary (n=12) compared to a mucocutaneous predominant phenotype (n=31) (p=0.048). Cases of progressive cGvHD (n=18), characterized by concurrent acute GvHD features (overlap syndrome), had decreased proportions of CD19 B cells, T3 transitional B cells (CD38LowCD10-) and mature naïve B cells (IgD+CD27-), decreased naïve helper T cells (CCR7+CD4+CD45RA+) and increased activated NK cells (CD56dimCD69+) (all p Using a well characterized cohort of pediatric patients with cGvHD, the ABLE / PBTMC 1202 study showed that a variety of subpopulations of naïve helper T cells, Tregs, and regulatory NK cells were associated with cGvHD. Additional B-cell markers were associated with progressive cGvHD in children. These data support alterations in thymopoeisis and decreases in regulatory T- and NK-cell populations underlying pediatric cGvHD. Future work includes the addition of non-cellular plasma markers into a clinically applicable diagnostic biomarker algorithm to aid clinicians in the accurate diagnosis of pediatric cGvHD, as well as further validation in the ABLE 2.0 / Pediatric Transplant Cellular Therapy Consortium 1901 study. Disclosures Wahlstrom: Pharmacyclics: Current Employment. Pulsipher:Mesoblast: Honoraria; Adaptive: Research Funding; Miltenyi: Honoraria, Research Funding; Bellicum: Honoraria; Jasper: Honoraria; Novartis: Honoraria.

Introduction Acute graft versus host disease (aGvHD) still remains one of the major causes of procedure-related morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Information on the outcome of paediatric patients experiencing this complication is limited. For this reason, we conducted a retrospective registry-based analysis on 2519 children who developed grade III-IV acute GVHD and were reported to the European Blood and Marrow Transplantation (EBMT) registry. Patients and methods Included in the study were children below age of 18 years who were transplanted between 2004 and 2014. Median year at transplantation was 2009. Of these children, 826 children had a non-malignant disorder, while 1689 were affected by malignancies. The donor was an HLA-identical sibling in 707 cases and an unrelated donor in 1081 cases. Umbilical cord blood (UCBT) was employed as stem cell source in 396 cases, while a relative other than a compatible sibling was utilized in 202 cases. Overall, 1281 patients were given bone marrow (BM), while 800 received peripheral blood stem cells (PBSC). Results Grade III acute GvHD occurred in 1607 patients (64% of the overall population), while grade IV acute GvHD was diagnosed in 908 (36%). Chronic GvHD occurred in 649 patients (26 % of the overall number of children who developed grade III-IV aGVHD). It was extensive in 333 (13%) and of limited severity in 269 (11%). At time of last follow-up, 1341 patients were alive (53%), while 1178 were dead (47 %). Fifty-seven patients were lost to the follow-up. Relapse of the original disorder occurred in 219 children (19.7% of patients experiencing grade III-IV aGvHD). Transplant-related causes were responsible for the death of 902 patients (76.5%), while 6 (0.5%) patients developed a secondary malignancy. The 3-year Kaplan-Meyer probability of overall survival (OS) was 46.7% (confidence interval 95, 44.1-49.5) and 50.9% (confidence interval 95, 47.1-54.9) in patients affected by malignant and non-malignant disorders, respectively. Patients who received as stem cell source BM had a better outcome in comparison to those who received PBSC (3-year OS, 53.2 vs 40.4%, P Conclusion These data indicate that the occurrence of grade III-IV aGVHD is associated with a dismal outcome also in paediatric patients. The main cause of fatality is represented by non-relapse mortality, while leukemia recurrence affected outcome of a lower number of children. Although the outcome of children experiencing grade III-IV aGvHD is improving over time, strategies aimed at preventing this immune-mediated complication and at optimizing its treatment are desirable. Disclosures Bader: Novartis, Medac, Amgen, Riemser, Neovii: Consultancy, Honoraria, Research Funding.

In hematopoietic stem cell transplant (HSCT) recipients, the recognition of polymorphic antigens by the donor-derived immune system is an important mechanism underlying both graft-versus-host disease and graft-versus-leukemia (GVL) effect. Here we show that a subset of HSCT recipients (13.9%, n = 108) have antibodies directed to surface molecules of dendritic cells. We have used one such serum in conjunction with retroviral expression cloning to identify the highly polymorphic surface molecule immunoglobulin-like transcript 5 (ILT5) as one of the targets of dendritic cell-reactive antibodies. ILT5 reactive antibodies were found in 5.4% of HSCT patients but not in solid organ transplantation recipients, patients with collagen diseases, multiparous women, or polytransfused or healthy persons. We show that ILT5-specific antibodies can mediate killing of ILT5-bearing cells and furthermore demonstrate ILT5 expression in some leukemic cells, indicating that it might be a target for GVL effects. Thus, our results represent the first description of potent allogeneic antibody responses to a non–major histocompatibility complex cell surface molecule in hematopoietic stem cell transplanted patients and warrant further studies to elucidate the role of antibodies to polymorphic cell surface molecules in GVL and graft-versus-host responses.

Adenovirus (AdV) infection in the course of allogeneic stem cell transplantation (SCT) is associated with high transplant-related morbidity and mortality. Disseminated AdV disease is lethal in most instances. Early detection of AdV infection and identification of patients carrying a high risk of disseminated disease therefore remain a major challenge. In view of the large number of existing AdV types, we have established real-time polymerase chain reaction (PCR) assays permitting sensitive detection and quantification of all 51 currently known human AdV serotypes. In a series of 132 consecutive pediatric patients undergoing SCT, more than 5000 samples derived from peripheral blood (PB), stool, urine, and throat were screened for adenovirus infection by PCR during the posttransplantation period. Thirty-six patients (27%) tested positive by PCR, revealing AdV types of the subgenera A, B, C, D, and F. Except for enteritis in some patients with AdV positivity in stool, detection of the virus at sites other than PB was not associated with clinical signs of virus disease, and transplant-related mortality was not significantly different from AdV-negative patients. By contrast, 82% of patients who had detectable AdV in PB died from infectious complications (P < .001). Monitoring of PB specimens by real-time PCR permitted early diagnosis of invasive AdV infection in all instances. In patients who developed disseminated AdV disease, detection of the virus in PB preceded onset of clinical symptoms by a median of more than 3 weeks. The observation of AdV in peripheral blood may therefore serve as a basis for early initiation of preemptive antiviral treatment.

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Disseminated adenovirus (AdV) infection is associated with high mortality in allogeneic stem cell transplant recipients. We have demonstrated that molecular detection of AdV in peripheral blood (PB) precedes the onset of life-threatening virus disease (Lion et al., Blood 102:1114–20, 2003). In most instances, detection of AdV in stool preceded the onset of viremia, thus raising the possibility that intestinal infections represent a common source of virus dissemination. To address this question, we have investigated 170 consecutive allogeneic transplantations in 138 pediatric patients transplanted at our center. Patients were monitored for the presence and the load of AdV in stool and in PB by a real-time PCR approach covering the entire spectrum of known human AdV serotypes. Fifty one patients (37%) tested positive in serial stool samples, revealing adenoviruses of nearly all subgenera, with strong predominance of serotypes 2 and 1 of species C. The overall risk of developing viremia in patients displaying or lacking intestinal AdV infection was 31% and 0%, respectively (p

Viral infections remain a major cause for transplant related mortality. The introduction of PCR-based assays permits rapid, specific and highly sensitive detection of various viral infections prior to clinical symptoms and thus facilitates preemtive treatment strategies. However a broad routine screening by PCR during the early post-transplant period is cost-intensive. Clinical benefit of broad vs restriced viral screening by PCR was evaluated in total of 98 consecutive pediatric SCT-patients(pts). The first 56 consecutive pts underwent broad viral screening including CMV, ADV, EBV, HSV, VZV, HHV6, HHV7, HHV8, PVB19, RSV, BKV, Enterovirus, Influenza A and B, and Parainfluenza 1–3. PCR-screening was performed twice weekly until day +28 and once weekly until day +100 in peripheral blood (PB), stool, urine and mouth wash. A total of 15675 samples were analyzed. HSV-IgG positive pts. received prophylactic acyclovir. All pts. received preemptive treatment for CMV viremia (gancyclovir) and ADV viremia (cidofovir). Pts with EBV-viremia exceeding 103 copies per ml PB received gancyclovir. In pts. undergoing the broad screening 50/56 pts were positive for at least one virus. The overall incidence of viremia was 61%. HHV6 was detectable in 66% (39% in PB), HHV7 in 16% (11% in PB), CMV in 36% (all in PB, in 23% additionally positive in other sites), EBV in 39% (35% in PB), ADV in 27% (5% in PB), BKV in 13% (all in urine) and PVB19 in one pts. Although the first detection of a viral infection occured before day +120 in all children, PCR positivity persisted throughout the first year post-transplant in many cases. There was no significant difference in the incidence of overall viral infection, viremia or multiple viremia between pts. with T-cell depleted grafts and those with unmanipulated grafts. However T-cell depletion was associated with significantly more lethal viral infections (32% vs 6%, p=0,0127). There was no significant difference in the incidence of viral infections, viremia and lethal viral disease between pts receiving myeloablative conditioning and those receiving reduced intensity conditioning. Isolated HHV6 or HHV7 viremia was not associated with any clinical symptoms and there was no difference concerning the incidence of CMV viremia, lethal viral disease or TRM between pts with and without HHV6 viremia. Of the 20 pts with CMV viremia, 6 pts had lethal CMV disease. 10% of the patients with EBV viremia were symptomatic, the disease being lethal in one case. The three pts who developed ADV viremia had previously been ADV positive in stool. All three pts died from disseminated ADV disease despite treatment with cidofovir. All pts with BKV positive urine had hemorrhagic cystitis. On the basis of these results, the viral screening for the subsequent 42 pts PCR screening was restricted to ADV, CMV and EBV in PB, ADV in stool and BKV in urine once weekly until day +28 and every two weeks thereafter, reducing the cost of the viral screening to 13%. For the patients undergoing restricted screening there was no difference concerning the incidence of CMV, EBV, ADV viremia and the incidence of lethal viral disease (18% vs 14%). We conclude that broad and cost intensive PCR screening for viral infections is of no clinical benefit.