The Role of Proteinase Inhibitor 9 (PI-9) and Granzyme B in Peripheral Leukocytes for GVL and GVHD After Allogeneic Hematopoietic Stem Cell Transplantation

Abstract Abstract 1984 The serpin proteinase inhibitor 9 (PI-9) protects cells from serine protease granzyme B (GrB)- and perforin (PFR)-induced cytotoxicity and apoptosis by specifically inhibiting GrB. Graft-versus-leukemia (GVL) effect and graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT) are the reciprocal aspects of the established immunotherapeutic approach in hematopoietic malignancies, and are thought to be caused at least in part through GrB and PFR produced by donor-derived cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. However, GrB and PFR expressions with respect to a GVL effect, and the role of PI-9 in both GVHD and GVL, are unknown. In this study we analyzed the expression of GrB, PFR and PI-9 in acute leukemia patients to whom allogeneic HSCT was performed during remission. This study was performed under the approval of the ethical committee of Kobe University Graduate Schools of Medicine and Health Sciences, and all human samples were obtained from whom a written informed consent was obtained. We first investigated the age- and gender-related differences of expressions of PI-9 and GrB mRNA by quantitative RT-PCR in mononuclear and polymorphonuclear cells from healthy volunteers. GrB and PFR mRNAs were expressed almost exclusively in mononuclear cells that included CTLs and NK cells, while PI-9 was expressed both in mononuclear and polymorphonuclear cells. GrB and PFR expression levels were comparable among all age and gender groups. Intriguingly, however, the expression of PI-9 mRNA in lymphocytes gradually increased with age, and the PI-9 expression in the volunteer group aged 60 years old or older was significantly higher than the one in the group aged 20 to 39 years old (p=0.002). Meanwhile, the PI-9 expression in polymorphonuclear cells was comparable among all age groups, and there was no gender difference in PI-9 expression levels. Next, we analyzed the expressions of PI-9, GrB and PFR mRNAs in mononuclear and polymorphonuclear cells of 3 patients (male 2, female 1) with acute GVHD and 11 patients without acute GVHD (male 6, female 5). The GrB, PFR and PI-9 expression levels were comparable between patient groups with and without acute GVHD, both before transplantation and a week after engraftment. However, the GrB and PFR expressions prominently augmented when acute GVHD became overt in all 3 patients, while PI-9 expression level increased mildly. As a result, the ratio of GrB (or PFR) and PI-9 expressions was stable in patients with and without acute GVHD. Among them, 4 cohort patients eventually relapsed and 10 cohorts remained in remission. The GrB mRNA expression in lymphocytes one week after engraftment was 3-fold higher in 10 patients without relapse of primary disease than in 4 patients who eventually relapsed (P=0.044). This might suggest that GrB plays a role in eliciting a GVL effect as well as acute GVHD. On the other hand, PFR and PI-9 mRNA expressions were then relatively stable among patients both with and without later relapse (P=0.667; P=0.103), and the ratio of GrB (or PRF) and PI-9 expressions did not change. The expressions of GrB and PFR mRNAs in polymorphonuclear cells of all patients were under the detectable level throughout the course. Finally, we analyzed the expressions of GrB, PFR and PI-9 in leukocytes 6 months after HSCT, to see if these expressions might change in patients with chronic GVHD. Among 14 patients with chronic GVHD, the GrB expression increased (up to 6.2-fold) in 10 patients, but the PFR expression did not change. In contrast, the expression of PI-9 decreased profoundly in all of the cases both with and without chronic GVHD, relative to healthy volunteers (P=0.028). Importantly, the ratio of GrB and PI-9 expressions was significantly higher in patients with chronic GVHD than in those without GVHD (P = 0.035). In conclusion, (1) high PI-9 expression in elderly people might explain a mechanism of escape from tumor immunity in them, where PI-9 might inhibit functions of cytotoxic T cells and NK cells; (2) a high expression of GrB shortly after engraftment may be a novel biomarker for a GVL effect; and (3) a low level of PI-9 expression or an increased ratio of GrB/PI-9 later after allogeneic HSCT might be an important biomarker for cGVHD. Monitoring of both GrB and PI-9 mRNAs in peripheral blood after allogeneic HSCT could be useful for predicting the GVL effect, as well as for monitoring both acute and chronic GVHD. Disclosures: No relevant conflicts of interest to declare.

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Immunoglobulin Prophylaxis in Patients Undergoing Hematopoietic Stem Cell Transplantation - Systematic Review and Meta-Analysis.

Blood ◽

10.1182/blood.v110.11.4962.4962 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4962-4962

Author(s):

Pia Raanani ◽

Anat Gafter-Gvili ◽

Mical Paul ◽

Isaac Ben-Bassat ◽

Leonard Leibovici ◽

...

Keyword(s):

Systematic Review ◽

Stem Cell ◽

Adverse Events ◽

Acute Gvhd ◽

Meta Analysis ◽

Hematopoietic Stem ◽

Allogeneic Hsct ◽

All Cause Mortality ◽

Autologous Hsct

Abstract Background: Many studies have evaluated the role of polyvalent immunoglobulins (IVIG) and CMV-hyperimmune IVIG (CMV-IVIG) prophylaxis in patients undergoing hematopoietic stem cell transplantation (HSCT), with inconsistent results and implications for practice. Objectives: To evaluate the role of IVIG and CMV- IVIG prophylaxis in HSCT. Methods: Systematic review and meta-analysis of randomized controlled trials comparing IVIG or CMV- IVIG with placebo or no intervention (control) for prophylaxis in HSCT. The Cochrane Library, MEDLINE, conference proceedings and references were searched until 2007. Primary outcome: all-cause-mortality ; Secondary outcomes: CMV infections, acute graft versus host disease (GVHD), interstitial pneumonitis (IP), veno-occlusive disease (VOD) and adverse events. Relative risks (RR) with 95% confidence intervals (CIs) were estimated and pooled. Results: Nineteen trials met inclusion criteria. IVIG was compared to control in 10 trials (7 IVIG in allogeneic HSCT, 2 allogeneic/autologous HSCT and 1 autologous HSCT). When IVIG was compared to control, there was no difference in all-cause mortality (RR 1.03; 95% CI 0.89–1.18, 6 trials, Fig.). There was a reduction in the number of CMV infections (RR 0.70; 95% CI 0.50–0.99, 4 trials) and in the number of episodes of IP (RR 0.61; 95% CI 0.43–0.87, 6 trials). There was no significant difference in the number of episodes of acute GVHD (RR 0.93; 95% CI 0.83–1.05, 6 trials). The risk for VOD was increased (RR 2.71; 95% CI 1.06–6.94, 3 trials) as were adverse events in the IVIG group (RR 8.12; 95% CI 3.15–20.9, 5 trials). CMV-IVIG was compared to control in 7 trials, all allogeneic HSCT. There was no difference in all-cause mortality (RR 0.86; 95% CI 0.63–1.16, 4 trials). In addition, there were no differences in the risk for developing CMV infections (RR 1.07; 95% CI 0.86–1.33, 7 trials), acute GVHD (RR 1.02; 95% CI 0.72–1.44, 5 trials) or IP (0.95; 95% CI 0.58–1.56, 5 trials). Conclusions: Our review demonstrates that IVIG prophylaxis for HSCT reduces the risk for CMV infections and IP without a significant influence on mortality. The beneficial effects should be weighed against the higher incidence of adverse events, VOD, and cost associated with the use of prophylactic IVIG in HSCT patients. Conversely, the use of CMV-IVIG was not associated with a change in any of the parameters. Outcomes Summary Outcome IVIG vs. control CMV-IVIG vs. control All Cause Mortality RR 1.03; 95% CI 0.89–1.18 RR 0.86; 95% CI 0.63–1.16 CMV infections RR 0.70; 95% CI 0.50–0.99 RR 1.07; 95% CI 0.86–1.33 acute GVHD RR 0.93; 95% CI 0.83–1.05 RR 1.02; 95% CI 0.72–1.44 IP RR 0.61; 95% CI 0.43–0.87 RR 0.95; 95% CI 0.58–1.56 VOD RR 2.71; 95% CI 1.06–6.94 No data Adverse events RR 8.12; 95% CI 3.15–20.9 RR 7.0; 95% CI 0.38–129.34 Figure Figure

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Azacitidine (Vidaza®, Aza) and Donor Lymphocyte Infusions (DLI) In Patients with Acute Myeloid Leukemia (AML) or Myelodysplastic Syndromes (MDS) Relapsing After Allogeneic Hematopoietic Stem Cell Transplantation (HSCT): Interim-Analysis From the AZARELA-Trial (NCT-00795548).

Blood ◽

10.1182/blood.v116.21.1294.1294 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1294-1294

Author(s):

Thomas Schroeder ◽

Akos Gabor Czibere ◽

Nicolaus Kröger ◽

Uwe Platzbecker ◽

Gesine Bug ◽

...

Keyword(s):

Overall Survival ◽

Missing Data ◽

Salvage Therapy ◽

Interim Analysis ◽

Research Funding ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Significant Activity ◽

Hematopoietic Stem ◽

Allogeneic Hsct

Abstract Abstract 1294 Background: Patients with AML or MDS who relapse after allogeneic HSCT have a poor prognosis and therapeutic options are limited. The DNA hypomethylating agent Aza has significant activity in patients (pts) with AML and MDS and retrospective analyses have recently shown encouraging results with the use of Aza +/− DLI in patients with AML and MDS, who relapsed after allogeneic HSCT (Czibere et al., 2010; Luebbert et al., 2010). In line with these clinical observations preclinical data suggest that Aza enhances a Graft-versus-Leukemia (GvL) effect while mitigating Graft-versus-Host Disease (GvHD). Design/Methods: To evaluate the activity and safety of Aza in combination with DLI as first salvage therapy in pts with AML or MDS relapsing after HSCT, we conducted a prospective, multicenter, single-arm phase-II trial. Pts were allowed to receive up to 8 cycles Aza (100 mg/m2/d d1-5, every 28 days) and 3 DLI with increasing dosages (1-5×106 – 1–5×108 cells/kg) after every 2nd Aza treatment cycle. Additional DLI were permitted. Results: Between January 2009 and May 2010, 30 pts from 6 German transplant centres were included into this trial. So far, 25 pts (15 female/10 male) were evaluable and are presented in this analysis: Of these, 23 (92%) suffered from AML (15 de novo/8 secondary following MDS), 1(4%) from a MDS (RAEB-1) and 1 (4%) from a myelodysplastic/myeloproliferative syndrome (MDS/MPS, CMML-1). Median age was 54 years (range 29–71). Conditioning was myeloablative in 24 pts (96%) and non-myeloablative in 1 patient (4%). Eight pts (35%) received a graft from a matched sibling donor, while 15 (65%) were transplanted with a matched unrelated donor (2 pts missing data). Peripheral blood stem cells (PBSC) were used in 24 pts (96%; 1 pt missing data). At the time of transplant 6 pts (24%) had primary induction failure, another 6 (24%) suffered from first or secondary relapse, 10 pts (40%) were in first or second complete remission (CR), while 3 pts (12%) were untreated. With regard to their molecular and genetic characteristics at diagnosis, 21 pts belonged to an adverse (9 pts) or intermediate (12 pts) group, whereas 2 pts were diagnosed with a favourable genetic phenotype (2 pts not performed). Prior to relapse 9 (36%) and 3 (12%) pts had episodes of acute GvHD and/or chronic GvHD, respectively. Relapse occurred in all pts after a median of 160 days (range 19–1199) following HSCT (median BM blasts: 34%, range 5–100%, median chimerism: 63% range-1-100%). At the time of relapse, karyotype was evaluable in 13 of 25 pts (52%). Of these 13 pts, 4 pts had a normal karyotype, while 9 had chromosomal aberrations including 6 pts with a complex karyotype. Patients received a median of 3 courses Aza (range 1–8) and 18 of 25 pts (72%) received DLI (median: 1, range: 1–4, median CD3 dose 5×106/kg/DLI, range: 1–207×106). Following treatment, overall response rate was 64% with 5 pts (20%) achieving a CR or CRi, 3 (12%) a partial remission (PR) and 8 (32%) a stable disease (SD). Median response duration was 266 days. Acute GvHD occurred in 6 pts (24%) (2 skin/6 liver/ 2 gut) after a median of 65 days (range 19–179) following the first DLI, while chronic GvHD was observed in 3 pts (12 %, all limited). Hematotoxicity (grade III-IV) was observed in 64% of all evaluated patients. Common adverse events were gastrointestinal side effects as well as infections. After a median follow-up of 100 days (range 25–485) 15 of 25 pts (60%) are currently alive. Median overall survival of all pts is 184 days (range 87–281). All pts, who achieved a CR/CRi, remained in ongoing remission for a median time of 229 days. Achieving a CR (CR: not reached vs. no CR: 117 days, p .008) or any type of response (CR/CRi, PR or SD) to the combination of Aza and DLI (any response: not reached vs. no response: 79 days, p .0001) were associated with a significantly longer overall survival. Conclusion: The combination of Aza and DLI as salvage treatment for patients with AML or MDS who relapse after allogeneic HSCT seems to be safe and shows significant anti-leukemic activity. Response, including CR rates, so far match those from retrospective analyses. Data presented in this interim-analysis suggest that salvage therapy with Aza and DLI is of substantial therapeutic benefit in these challenging patients. Disclosures: Platzbecker: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Bug:Celgene: Honoraria. Luft:Celgene: Research Funding. Fenk:Celgene: Research Funding. Kobbe:Celgene: Research Funding.

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Outcome of Children Who Experience Disease Relapse Following Allogeneic Hematopoietic Stem Cell Transplantation for Hematologic Malignancies,

Blood ◽

10.1182/blood.v118.21.4101.4101 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4101-4101

Author(s):

Rajinder PS Bajwa ◽

Sandeep Soni ◽

Tal Schechter ◽

Adam Gassas ◽

John J Doyle ◽

...

Keyword(s):

Clinical Trials ◽

Salvage Therapy ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Hematologic Malignancies ◽

Hematopoietic Stem ◽

Allogeneic Hsct ◽

Conditioning Regimens ◽

Conditioning Therapy ◽

Biphenotypic Leukemia

Abstract Abstract 4101 Introduction: According to the Center for International Blood and Marrow Transplantation and Research (CIBMTR) data, relapse accounts for 41% of deaths after matched sibling donor hematopoietic stem cell transplantation (HSCT) and 34% of deaths after unrelated donor HSCT. Overall long term survival of 25–30% has been reported in patients who relapse after HSCT. Most of these data are from adult studies. In a recent study, survival following a second allogeneic HSCT ranged from 25–50%. There are limited data on the treatment outcome of children who relapse after an allogeneic HSCT for hematological malignancies. Methods: This multicenter retrospective study was done with the primary objective to examine the incidence and outcome of children who experience relapse after allogeneic HSCT. Patients with a primary diagnosis of acute lymphatic leukemia (ALL), acute or chronic myeloid leukemia (AML or CML), juvenile myelomonocytic leukemia (JMML), biphenotypic leukemia (BL), acute undifferentiated leukemia (AUL), and myelodysplastic syndrome (MDS) were included. After IRB approval, data was collected from review of the medical charts of patients who underwent HSCT at 5 institutions across North America. Results: Data on 532 patients was analyzed; these included 328 males and 204 females. These included ALL (n=254), AML (n=187), MDS (n=35), CML (n=26), JMML (n=3), sAML (n=12), Biphenotypic leukemia (n=10), AUL (n=3) and 1 each with APL and secondary ALL. The median age at HSCT was 9.7 years (range 0.44 to 24.98). Four hundred and fifty (85%) of the 532 patients were in complete remission (CR) at the time of HSCT. Five hundred and fifteen (97%) underwent a myeloablative (MA) HSCT and 16 (3%) had a non-myeloablative (NMA) HSCT. Total body irradiation (TBI) was part of the conditioning therapy in 334 (63%) while 198 (37%) had chemotherapy based regimens. Standard graft versus host disease (GvHD) prophylaxis with calcineurin inhibitors and methotrexate was used in 76% of patients. Grade 1–2 acute GvHD developed in 180 (34%) of patients and 82 (15%) had grade 3–4 acute GvHD. Chronic GvHD was seen in 118 (22%) cases with half having extensive chronic GvHD. After HSCT, 85 (16%) patients died of transplant related complications at 1 year, with infection as the leading (26%) cause of death. The 2, 5 and 10 year overall survival for the entire group was 58.5%, 46% and 44% respectively. Following HSCT, 131 (25%) patients relapsed at a median duration of 7.7 months (range 0.6 to 66.4 months). Of the 131 patients who relapsed 80 (61%) received salvage therapy, 39(30%) did not get any therapy and data were not available for another 12 (9%) patients. Salvage therapy included a combination of withdrawal of immunosuppression, ALL or AML re-induction therapy, or other remission inducing agents, clofarabine, and/or donor lymphocyte infusions (n=22). After salvage therapy 46 (57.5%) patients achieved CR, 26 (32.5%) had no response, 3 (4%) partial response, 2 unknown and 4 (5%) had marrow aplasia and no recovery of counts. Sixty-three (48%) of the relapsed patients underwent a second HSCT: 26 (41%) received a MA, 21 (33%) NMA, 3 (5%) with no conditioning and in 13 (21%) conditioning therapy was unknown. Nearly 22 different conditioning regimens were used for the second HSCT. After the second HSCT 19 (41%) patients died because of transplant related mortality (TRM) and 24 patients died because of relapse. Seventeen (27%) patients are alive and disease free. Conclusions: Relapse remains a major complication following allogeneic HSCT for hematologic malignancies. Our data shows that patients who relapse after HSCT have poor prognosis. In our study 30 different salvage therapies were used for these patients. Currently there is no standard acceptable salvage therapy and most cooperative group or industry sponsored clinical trials exclude patients who underwent HSCT. In addition there are no standard conditioning regimens designed specifically for the second HSCT, which could contribute to the high TRM seen during second HSCT. There is urgent need to develop prospective clinical trials to better understand the optimal therapy for this group of patients. Disclosures: No relevant conflicts of interest to declare.

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Impact of the Inhibitory KIR-HLA Receptor-Ligand Model on Unrelated Hematopoietic Stem Cell Transplantation in Patients with Leukemia.

Blood ◽

10.1182/blood.v110.11.5063.5063 ◽

2007 ◽

Vol 110 (11) ◽

pp. 5063-5063

Author(s):

Jun He ◽

Zi-xing Chen ◽

Xiao-jing Bao ◽

De-pei Wu ◽

Xiao-ni Yuan ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

Nk Cells ◽

Cell Transplantation ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Receptor Ligand ◽

Hematopoietic Stem ◽

Ligand Model

Abstract The interaction between killer cell immunoglobin-like receptor (KIR) and HLA molecule has particular relevance to the unrelated hematopoietic stem cell transplantation (HSCT). The presence of KIR on donor’s NK cells and the absence of the corresponding KIR ligand in the recipient’s HLA repertoire as a receptor-ligand model can be used to predict the outcome of HSCT. To investigate the effect of KIR-HLA receptor-ligand mismatching and the expression of inhibitory KIR (iKIR) on the unrelated Allo-HSCT of leukemia, the following study has been carried out. The KIR genotypes of 36 patients of ALL (n=18), AML (n=10), and CML (n=8) as well as their unrelated donors were obtained from the Database of China Marrow Donor Program. KIR genotyping was performed with PCR-SSP. The KIR and HLA mismatching status (receptor-ligand mismatch model) between recipients and donors was analyzed thereafter. The expression of iKIR was determined by flow cytometry on recipients after HSCT. All cases were followed up closely until July, 2007. Among all the donor/recipient pairs analyzed, 15 pairs displayed the matched pattern with respect to the KIR genotyping though, graft-versus-host (GVH) KIR ligand-mismatched in 13 pairs, and host-versus-graft (HVG) KIR ligand-mismatched in 8 pairs. 26 recipients were lack of HLA-Cw2,4,5,6,15 ligands although iKIR/2DL1 was expressed on their corresponding donors. Similarly, 35 donors with iKIR/2DL2/L3 on their NK cells could recognized HLA- Cw1,3,7,8,12,14 ligands in their recipients. iKIR/3DL1 was expressed on 9 donors although the correlated HLA-Bw4 ligand was absent on the recipients. iKIR/3DL2 was expressed on 24 donors although the correlated HLA-A11 ligand was absent on the recipients. Except for one case, 35 patients were successfully transplanted, in which seven patients were dead, giving the survival rate of 80.0%. The cause of death was either acute/chronic GVHD or relapse. The frequency of acute GVHD (60.0%, 9/15), leading to death (26.7%, 4/15), was the highest in KIR receptor-ligand matched model. The incidence of acute GVHD and death was 30.8% (4/13) and 23.1% (3/13) in the GVH KIR ligand-mismatching, respectively; while the incidence of chronic GVHD was 37.5% (3/8) and no death happened in HVG KIR ligand-mismatching pattern. The expression of iKIR/2DL1 could be detected on ALL (13.0%∼16.6%) and CML (1.8∼6.2%) patients four month after HSCT. The expression of both iKIR/2DL1 (4%) and iKIR/2DL2 (12%) was found only in one patient with CML three month after HSCT, increased to 33.4% and 32.6% accompanied by cGVHD, and dropped to 7.6% and 10.3% one year after HSCT. These results suggested that the deficiency in iKIR/2DL1 based on the KIR-HLA receptor-ligand mismatching model together with the expression of a number of activating KIR in recipients increases the occurrence of GVHD, relapse, and death in unrelated HSCT. Analysis on KIR-HLA gene expression profiling could be useful in predicting the clinical outcome of unrelated Allo-HSCT in leukemia patients. The overall and disease-free survival after HSCT could be improved by preventing the development of GVHD.

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Prospective Validation of a Chronic GvHD-Specific Proteome Pattern (cGvHD-MS14) Post Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood.v118.21.1970.1970 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1970-1970

Author(s):

Eva M. Weissinger ◽

Daniel Wolff ◽

Jochen Metzger ◽

Christiane E Dobbelstein ◽

Stefanie Buchholz ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Hematopoietic Stem Cell ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Urine Samples ◽

Hematopoietic Stem ◽

Allogeneic Hsct

Abstract Abstract 1970 Introduction: Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment for many hematologic malignancies and non-malignant hematopoietic disorders, but is associated with significant morbidity and mortality with focus on acute and chronic graft-versus-host disease (GvHD). Chronic GvHD (cGvHD) occurs with increasing frequency, hampering quality of life of patients post-allogeneic HSCT and leading to increased morbidity and mortality even years after allogeneic HSCT. Diagnosis of chronic GvHD is based on clinical features and histology. Here we present the generation of cGvHD-specific proteomic pattern (cGvHD-MS14) using capillary electrophoreses and mass spectrometry to analyze urine sample collected prospectively after allogeneic HSCT. Methods: A proteomic pattern (cGvHD-MS14) was developed in order to diagnose cGvHD, to differentiate acute versus cGvHD, and to predict onset and severity of cGvHD prior to clinical diagnosis of cGVHD as a non-invasive, unbiased laboratory test for diagnosis of cGvHD. This pattern was prospectively evaluated on 329 patients (1034 urine samples) after allogeneic HSCT at MHH and 3 collaborating transplant centers. The majority of the patients had acute leukemias prior to transplantation (n=210) and were transplanted from matched unrelated or related donors (MUD n=134; MRD n=125). Reduced intensity conditioning regimens were used in about 75% of all patients and the majority (80%) received ATG (anti-thymocyte globulin) as GVHD-prophylaxis prior to transplantation and a calcineurin-inhibitor based prophylaxis afterwards. Results: Prospective and blinded evaluation revealed the correct classification of patients developing cGvHD with a sensitivity 78% and specificity of about 71% at time of diagnosis. Differentiation between late onset acute GvHD and chronic GvHD was achieved in 3 patients in this validation set. Acute GvHD prior to day 100 is not recognized by cGvHD-MS14, since aGvHD-specific peptides had been excluded during cGvHD-pattern generation. The pattern consists of 14 differentially excreted peptides, differentiating chronic GvHD from tolerant patients. Four of 14 peptides have been sequenced to date, 2 are fragments from collagen 1, 1 is from inter-alpha trypsin inhibitor heavy chain 4 and 1 is a fragment from the fibrinogen ß-chain. Conclusions: The proteomic pattern of urine proteomics enables diagnosis of cGvHD as well as differentiation of acute versus chronic GvHD. Further prospective evaluation of the cGvHD-specific pattern cGvHD-MS14 for organ specificity as well as severity prediction is currently ongoing. Taken together our results indicate that diagnosis of cGvHD is possible using CE/MS analysis of prospectively collected urine samples with high sensitivity and specificity. Disclosures: Metzger: mosaiques-diagnostics GmbH: Employment. Krons:mosaiques-diagnostics GmbH: Employment.

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Prognostic Value Of Plasma Biomarkers Compared To Urinary Proteome Patterns

Blood ◽

10.1182/blood.v122.21.4593.4593 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4593-4593

Author(s):

Christin Human ◽

Sylvia Borchers ◽

Michael Stadler ◽

Helmut Diedrich ◽

Jürgen Krauter ◽

...

Keyword(s):

Clinical Diagnosis ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Phase Iii ◽

Test Set ◽

Hematopoietic Stem ◽

Allogeneic Hsct ◽

Plasma Biomarkers ◽

Time Points ◽

Validation Set

Introduction Graft-versus-host disease (GvHD) is a major complication and cause of morbidity and mortality after hematopoietic stem cell transplantation (HSCT). Prediction of acute GvHD is essential to optimize treatment. An effective method for early investigator independent diagnosis of aGvHD is under investigation in a phase III clinical trial. A urinary proteomic pattern – aGvHD-MS17 – is evaluated in a preemptive treatment trial, administering steroids at the time of pattern positivity. Here we present our data for early diagnosis of GvHD by monitoring easily accessible samples like urine for the presence of a specific proteom pattern and compare the data to published plasma biomarkers. Methods Plasma and urine were collected from patients after HSCT at defined time points (Weissinger et al., Blood 2007). Capillary electrophoresis and mass spectometry (CE-MS) analyses were done according to Weissinger et al., Blood 2007 and Leukemia, 2013. Two peptides found to be highly specific in urine for development of aGvHD were β2-microglobulin (β2m) and CD99. For both, antibodies are available and thus they can be analyzed in plasma samples from the same patients with ready-to-use ELISA-kits (Pasnecey et al., 2010). Plasma biomarkers were first analyzed in a test set based on 34 patients, followed by validation in 31 patients. As a further contribution to harmonization, published plasma biomarkers elafin, soluble IL-2-receptor alpha (IL-2sRα), REG3α, ST2 (IL-1 receptor 4) and sTNFRI were added to the ELISA-panel. Using the angiogenesis panel for bioluminex analyses, IL-8 and hepatocyte growth factor (HGF) were analyzed in addition and the outcomes were compared to the urinary proteomic analyses. Results & Discussion While the urine derived CE-MS pattern was able to predict an upcoming GvHD prior to the development of clinical signs, most of the plasma biomarkers could not predict pending GvHD. The test set for analysis of plasma consisted of 20 patients with aGvHD, from whom samples from the time of clinical diagnosis of aGvHD were used, and 14 controls without aGvHD at any time. Receiver operated characteristic curves were generated, areas under the curve (AUC) were more than 80% for β2m, CD99, sIL2Rα and sTNFRI (p<0.01). The validation set consisted of samples from 31 patients at different time points (7 samples each patient). In the validation set some markers did not reach their established cut-off point for aGvHD-diagnosis prior to or at the day of clinical diagnosis of aGvHD. Especially REG3α and elafin did not turn positive in any samples of patients with acute GvHD. Both markers presumably are organ-specific, but also limiting the analysis to patients with isolated GvHD of the intestine (REG3α) or skin (elafin) did not increase the sensitivity of these markers. CD99 and β2m discriminated patients with aGvHD from controls and CD99 was even specific for chronic GvHD and could distinguish between patients with acute and chronic GvHD with high sensitivity (89%) and specificity (95%); AUC was 86% (p<0.001). Additional problems with the ELISA-technique were the need to dilute samples prior to testing due to highly diverse concentrations of the biomarkers. The concentrations sometimes varied by the factor of 10 or more which required preparation of each sample in different dilutions. This made diagnosis based on ELISA data more time consuming and costly as envisioned. Urine on the other hand has shown to be stable and to provide a reliable proteome pattern. Furthermore, urinary proteom analysis can be normalized within each sample by using internal standards. Conclusion and future perspectives Prediction of pending aGvHD in patients post-allogeneic HSCT is possible with urine monitoring using CE-MS analyses with a sensitivity of 82% and specificity of 77% for aGvHD (Weissinger et al., Leukemia 2013). In contrast, published plasma biomarkers in our hands lack reliable sensitivity and specificity in the validation set. In addition, the markers detected in plasma do not appear to predict development of aGvHD. Biomarkers identified in urine (CD99 and β2m) can be found in plasma as well and are predictive for aGvHD-development. CD99 is also predictive for cGvHD development which can be explained by its function as an activation marker of (donor) T-cells. In conclusion, our results demonstrate that plasma monitoring post-allogeneic HSCT seems less reliable than urinary proteomics for prediction of aGvHD. Disclosures: No relevant conflicts of interest to declare.

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Anaerobic Antibiotics and the Risk of Graft-Versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood-2019-125080 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1992-1992

Author(s):

John Tanaka ◽

Rebecca Young ◽

Lisa Spees ◽

Kirsten Jenkins ◽

Anthony D. Sung ◽

...

Keyword(s):

Stem Cell ◽

Febrile Neutropenia ◽

Research Funding ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Research Support ◽

Hematopoietic Stem ◽

Allogeneic Hsct ◽

Graft Versus Host ◽

Grade Ii

Background: The gut microbiota interacts extensively with the host immune system and thus may modify the risk of graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). During the post-transplant neutropenic period, the majority of allogeneic HSCT recipients receive empirical broad-spectrum antibiotics for febrile neutropenia. We hypothesized that receipt of an antibiotic regimen with an anaerobic spectrum of activity is associated with a higher risk of grade II-IV acute GVHD than receipt of a non-anaerobic antibiotic regimens. Methods: In this single-center retrospective cohort study, we evaluated associations between peri-transplant receipt of antibiotics with an anaerobic spectrum of activity and the risk and severity of GVHD among 877 adults who received an allogeneic HSCT between January 1, 2005 and December 31, 2016. We identified 609 patients who developed febrile neutropenia after HSCT and compared GVHD risk and mortality among patients who received anaerobic antibiotics (piperacillin-tazobactam or carbapenems; n=333) to patients who received only antibiotics with minimal activity against anaerobes (aztreonam, ceftazidime, or cefepime; n=276). Antibiotics received by patients between 7 days before and 28 days after allogeneic HSCT and GVHD diagnoses were verified via manual review of medication orders and provider notes in electronic medical records. Results: Receipt of anaerobic antibiotics was associated with an increased risks of grade II-IV acute GVHD (hazard ratio (HR): 1.41; 95% confidence interval (CI): 1.10-1.79; P=0.01) and acute GVHD mortality (HR: 1.87; 95% CI: 1.13, 3.11; P=0.02). This hazard was primarily associated with acute GVHD of the gut or liver (HR: 1.38; 95% CI: 1.06, 1.79; P=0.02). The association remained with even short (<7 days) courses of anaerobic antibiotics. Anaerobic antibiotic exposure was not associated with acute skin GVHD (HR: 0.97; 95% CI: 0.69, 1.37; P=0.88), chronic GVHD diagnosis (HR: 0.93; 95% CI: 0.70, 1.23; P=0.43), or chronic GVHD mortality (HR: 0.89; 95% CI: 0.44, 1.81; P=0.76). Conclusions: Receipt of anaerobic antibiotics for febrile neutropenia post-HSCT is associated with an increased risks of acute GVHD of the gut or liver and acute GVHD mortality. Limiting use of antibiotics with an anaerobic spectrum of activity after allogeneic HSCT may reduce acute GVHD incidence and mortality. Disclosures Sung: Novartis: Research Funding; Merck: Research Funding; Seres: Research Funding. Martin:Novartis Pharmaceuticals: Other: research support; Jazz Pharmaceuticals: Other: research support.

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Sirolimus-Containing Graft-Versus-Host Disease Prophylaxis and High- Resolution HLA Typing Improves the Outcome of Mismatched Unrelated Donor Allogeneic Hematopoietic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v112.11.2216.2216 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2216-2216

Author(s):

Ronald J. Maggiore ◽

Dennis L. Cooper ◽

Francine M. Foss ◽

Warren D. Shlomchik ◽

Stuart E. Seropian

Keyword(s):

Acute Gvhd ◽

Chronic Gvhd ◽

Disease Recurrence ◽

Hla Typing ◽

Unrelated Donor ◽

Prophylactic Regimen ◽

Hematopoietic Stem ◽

Allogeneic Hsct ◽

Kaplan Meier ◽

Gvhd Prophylaxis

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) from unrelated donors can cure a variety of hematologic diseases; however, decreased survival and graft versus host disease (GVHD) are associated with mismatches at HLA class I and class II loci. Beginning in 2004, we have employed a modified three-drug sirolimus-based GVHD prophylactic regimen (Antin JH et al., Blood2003;102:1601) for all patients undergoing unrelated donor allogeneic HSCT with any antigen or allele mismatch at HLA A, B, C, DR, or DQ in the GVHD direction. Outcomes of 22 patients undergoing mismatched unrelated donor (MMUD) allogeneic HSCT (sirolimus(+) group) were retrospectively reviewed in comparison to 14 patients receiving MMUD allografts between 2000–2004 who received a tacrolimus-based two-drug GVHD prophylactic regimen (sirolimus(−) group). Median age (45 vs. 51), disease status, donor gender, and type of transplant conditioning (reduced-intensity, 9 vs. 8) were similar between the two groups. Seven of 14 patients in the sirolimus (−) group received bone marrow grafts whereas all patients in the sirolimus + group received peripheral blood stem cells. Nine patients in the sirolimus – group had antigen-level typing at HLA A, B, C and DQ loci and allele level typing at HLA DRB1 and 5 patients had allele-level typing at all loci. Patients in the sirolimus+ group had allele-level typing at all loci. One subject was mismatched at HLA DRB1 and DQ at the allele level; the remaining subjects had a single allele or antigen mismatch. GVHD prophylaxis for the sirolimus(−) group included tacrolimus and mycophenolate (n=11) or tacrolimus and low-dose methotrexate x 3 doses (n=3). GVHD prophylaxis for the sirolimus (+) group included sirolimus, tacrolimus and post- transplant methotrexate for 2 or 3 doses, totaling 15mg/m2. Median follow-up for surviving patients was 1921 days (1781–2295) in the sirolimus-group and 447 days (53–1122) in the sirolimus(+) group. The day +100 cumulative incidence of grades II-IV and III-IV acute GVHD were, respectively, 71% and 43% in the sirolimus(−) group, and 14% and 5% in the sirolimus(+) groups (p=0.002; p=0.02). Kaplan-Meier estimates of the probability of chronic GVHD or late onset acute GVHD at two years were 51% in the sirolimus(–) group and 70% in the sirolimus(+) group. Graft failure occurred in 2 patients each in both groups and was successfully treated in two patients by retransplantation or pentostatin plus donor lymphocyte infusion. Adverse effects of sirolimus included 8 patients with renal dysfunction (36%), 4 with thrombotic microangiopathy (TMA) (18%), and 3 with dyslipidemia requiring medical therapy (14%). Deaths in the sirolimus(−) group were due to disease recurrence (n=3), graft failure (n=1), and GVHD (n=7). Deaths in the sirolimus(+) group were due to disease recurrence (n=2), TMA (n=1) and GVHD (n=4). The Kaplan-Meier estimates of disease free (DFS) and overall survival (OS) at 3 years were 21% and 21% for the sirolimus(−) group and 53% and 56% for the sirolimus(+) group. The Kaplan-Meier estimates of non-relapse mortality (NRM) at day +100 and day +365 were 22% and 50% for the sirolimus (−) group and 5% and 13% for the sirolimus(+) group. Summary: High-resolution HLA typing and GVHD prophylaxis with sirolimus, tacrolimus, and low-dose methotrexate in patients undergoing MMUD allogeneic HSCT is associated with low rates of severe acute GVHD and non-relapse mortality. Renal dysfunction and TMA appear more common with sirolimus, and, as in prior studies with this regimen, a lower rate of acute GVHD did not result in less chronic GVHD. Nonetheless, DFS and OS appear to compare favorably to recently reported registry data for MMUD allograft recipients (Lee SJ et al., Blood;110:4576), and a prospective trial has been initiated to further study this regimen in MMUD recipients.

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Role of Il-17 Varies at Different Periods After Hematopoietic Stem Cell Transplantation: Protection From Acute Graft-Versus-Host Disease and Exacerbation of Chronic Graft-Versus-Host Disease.

Blood ◽

10.1182/blood.v116.21.3741.3741 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3741-3741 ◽

Cited By ~ 1

Author(s):

Rie Kuroda ◽

Ryosei Nishimura ◽

Katsuaki Sato ◽

Hideaki Maeba ◽

Kazuhito Naka ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

Immune Cells ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Hematopoietic Stem ◽

Graft Versus Host

Abstract Abstract 3741 Th17 is a newly identified T cell lineage that secretes the proinflammatory cytokine IL-17. Th17 cells have been shown to play a crucial role in mediating autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE), arthritis, and colitis. Anti-IL-17 therapy for some autoimmune diseases in clinical settings has been started and promising results have been reported. However the role of IL-17 on developing acute and chronic GVHD in hematopoietic stem cell transplantation (HSCT) is not yet fully understood. Interaction between IL-17 and IL-17 receptor is complicated because IL-17 is produced in various kinds of immune cells other than CD4+ T-cells, and IL-17 receptors express on not only immune cells but also various epithelial cells, including lung and intestine, both of which are target organs of GVHD. To explore the role of host derived or donor derived IL-17 separately in acute GVHD, lethally irradiated wild type (WT) or IL-17 knockout (KO) Balb/c (H-2d) were given WT or IL-17 KO C57BL/6 (H-2b) bone marrow (BM) cells with WT splenocytes to induce acute GVHD. Infused cell number of WT splenocytes in this study induced acute GVHD, but not lethal in IL-17 WT host mice. In contrast, IL-17 KO host mice receiving WT BM plus WT splenocytes developed severe acute gut GVHD and finally half of them died (p<0.05). To exclude the possibility that alloreactivity of host IL-17 KO derived dentritic cells (DCs) could be much more than that of WT DCs, mixed leukocyte reaction (MLR) was performed using stimulators from WT or IL-17 KO DCs and responders from WT CD4+ T-cells. No significant differences were observed between WT DCs and IL-17 KO DCs in thymidine uptake and percentage of responder cells producing IFN-g or TNF-a. Taken together, host-derived IL-17 has a protective effect against acute GVHD. Moreover similar results were observed when IL-17 KO Balb/c mice were given BM cells from another strain B10.D2 plus splenocytes shown in the figure below (p<0.05). Next, we compared the development of chronic GVHD between the lethally irradiated WT Balb/c mice given IL-17 KO C57BL/6 BM cells or WT BM cells with low dose WT splenocytes to induce sublethal acute GVHD and chronic GVHD subsequently. After day 60 the mice receiving WT BM cells plus WT splenocytes experienced weight loss accompanied by skin histological changes (p<0.05, shown in the figure below), while mice receiving IL-17 KO BM plus WT splenocytes showed minimal signs of GVHD as well as mice receiving IL-17 KO BM or WT BM alone. Increased number of donor-BM derived IL-17 producing cells was observed in the mice showing chronic GVHD compared to BM control (p<0.05). Moreover, a significant increase of T-cell proliferation was observed by adding rIL-17 into MLR culture (p<0.05). These results suggest that donor BM derived IL-17 producing cells involved in the pathogenesis of chronic GVHD by exacerbating the alloimmune response in part. In conclusion IL-17, especially from host-derived, has a protective effect against acute GVHD. On the contrary, donor BM derived IL-17 exacerbates chronic GVHD. Neutralizing IL-17 would be a potent strategy only for preventing chronic GVHD, not for acute GVHD. Disclosures: No relevant conflicts of interest to declare.

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HLA-DRB4 Mismatch Is Associated with Worse Outcomes in Unrelated Allogeneic Hematopoietic Stem Cell Transplantation for Hematological Malignancies

Blood ◽

10.1182/blood.v120.21.4540.4540 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4540-4540 ◽

Cited By ~ 1

Author(s):

Marie Y. Detrait ◽

Ibrahim Yakoub-Agha ◽

Valerie Dubois ◽

Françoise Dufossé ◽

Myriam Labalette ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Unrelated Donor ◽

Hematopoietic Stem ◽

Allogeneic Hsct ◽

Stem Cell Source ◽

Cell Source

Abstract Abstract 4540 Introduction The impact of HLA DRB3 and DRB4 allele mismatch after allogeneic HSCT using unrelated donors is unclear. We therefore examined retrospectively the outcome of 35 patients who received HLA-10/10 unrelated hematopoietic stem cell transplantation with a DRB3 or DRB4 mismatch between 2005 and 2011. This cohort of 35 patients was a part of a cohort of 132 consecutive patients who underwent allogeneic HSCT between 2005–2011 with a 10/10-HLA matched donor. There were 18 males (51.4%) and 17 females (48.6%) with a median age of 48 years (range, 6–64), there were 13 (37%) AML, 9 (26%) ALL, 4 (11.5%) MDS, 3 (8.5%) multiple myeloma and 6 (5.7%) other (CML, CLL, NHL). Twenty patients (57%) received a myeloablative conditioning (MAC) and 15 (43%) received a reduced intensity conditioning (RIC). At transplantation, 21 patients (60%) were in complete remission (CR), 4 patients (11.5%) in partial remission (PR) and 10 (28.5%) in relapse; 13 (37%) patients received peripheral blood stem cell (PBSC) and 22 (63%) received bone marrow (BM). Twelve (34%) patients had a mismatched DRB4 donor and 23 (66%) patients had a mismatched DRB3 donor. In the remains of 97 patients, there were 55 male (57%) and 42 female (43%), 28 (29%) patients received a MAC and 69 (71%) a RIC as regimen before allogeneic HSCT. The stem cell source was BM for 32 (34%) patients and PBSC for 65 (66%). At transplantation, 34 (35%) patients are in CR and 63 (65%) were in PR. The distribution of diagnosis was acute leukaemia and MDS for 44 (45%), CLL for 2 (2.5%) and other diagnosis (aplastic anemia, NHL, CML, MPS) for 51 patients (52.5%). Results After HSCT, 124 (94%) patients engrafted. After a median follow-up of 11.5 months (range, 0–76), the cumulative incidence of acute GvHD≥2 at 3 months was 20% (95%CI,16.5–24) and the cumulative incidence of chronic GvHD at one year was 19 % (95%CI, 15–22). In univariate analysis, the mismatch DRB3 or DRB4 had no effect on engraftment and no effect on acute GvHD (p=0.08) or chronic GvHD (p=0.63). There was no impact of DRB3 or DRB4 mismatch on relapse (p=0.33 and p=0.53, respectively) and on PFS (p=0.63 and p=0.07, respectively). We found an impact of the DRB4 mismatching (p=0.016) on overall survival. The median survival for patient without DRB3 or DRB4 mismatch was 23 months (14-NR), for patients with DRB3 mismatch 32 months (12-NR), and for DRB4 mismatched patients 5 months (3-NR). The probability of survival at 24 months, for patients without mismatch DRB3 or DRB4 is 47% (36–61), for patients with DRB3 mismatch 51% (32–82) and for DRB4 mismatched patients 19% (6–66%). (figure1). The multivariate analysis that studied age, type of disease, DRB3 or DRB4 mismatch, sexmatching, TBI, ATG, disease status at transplantation and type of conditioning and stem cell source showed a significant impact of mismatch DRB4 on survival (HR= 2.5 [95%CI, 1.2–5.5] p=0.019); there was no impact for DRB3 mismatch (HR= 1.3 (95%CI,0.5–3.9 p=0.58). We found also an impact of the DRB4 mismatch on TRM (HR= 3.5; [95%CI, 1.6 –8] p= 0.026). The incidence of TRM at 24 months for patients without DRB3 or DRB4 mismatch is 29% (24–34), for patients with DRB3 mismatch 17% (9–26%) and for DRB4 mismatched patients 50% (34–66%). (figure 2). Conclusion The HLA DRB4 matching donor is relevant for survival of patients who undergo allo-HSCT from unrelated donor in the HLA-10/10 matching settings. In view of the important impact of these loci mismatches on clinical outcome, it seems to be important to consider this matching loci in the unrelated donor selection. Disclosures: No relevant conflicts of interest to declare.

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