ALBERT

Description: No validated biomarkers exist for acute graft-versus-host disease (GVHD). We screened plasma with antibody microarrays for 120 proteins in a discovery set of 42 patients who underwent transplantation that revealed 8 potential biomarkers for diagnostic of GVHD. We then measured by enzyme-linked immunosorbent assay (ELISA) the levels of these biomarkers in samples from 424 patients who underwent transplantation randomly divided into training (n = 282) and validation (n = 142) sets. Logistic regression analysis of these 8 proteins determined a composite biomarker panel of 4 proteins (interleukin-2-receptor-alpha, tumor-necrosis-factor-receptor-1, interleukin-8, and hepatocyte growth factor) that optimally discriminated patients with and without GVHD. The area under the receiver operating characteristic curve distinguishing these 2 groups in the training set was 0.91 (95% confidence interval, 0.87–0.94) and 0.86 (95% confidence interval, 0.79–0.92) in the validation set. In patients with GVHD, Cox regression analysis revealed that the biomarker panel predicted survival independently of GVHD severity. A panel of 4 biomarkers can confirm the diagnosis of GVHD in patients at onset of clinical symptoms of GVHD and provide prognostic information independent of GVHD severity.

Description: Experimental and clinical data suggest that acute graft vs. host disease (aGVHD) after allogeneic (allo-) stem cell transplantation (SCT) predominantly involves a Th1-type cytokine response. Paradoxically, the production of Interleukin 13 (IL-13), a typical Th2 cytokine, by donor T cells responding to host antigen in vitro was recently shown to correlate with the severity of clinical aGVHD. We used a well-established murine SCT model (BALB/c → B6) and mice deficient in IL-13 to more clearly investigate the role of IL-13 produced by donor cells in the pathogenesis of GVHD. In our first set of experiments, B6, BALB/c IL-13+/+ and BALB/c IL-13−/− T cells were stimulated with B6 splenic dendritic cells. BALB/c IL-13+/+ and IL-13−/− T cells proliferated equally to alloantigens (123718±9038 vs.100565±3083 cpm) compared to syngeneic controls (2411±349 cpm). In addition, IL-13+/+ T cells produced significant amounts of IL-13 (1882.5±301.9 pg/ml), whereas IL-13 levels in wells with IL-13−/− T cells were very low (37.7±2.1 pg/ml) and did not differ from syngeneic controls (52.9±7.4 pg/ml). Next, lethally irradiated B6 mice received bone marrow and T cells from either syngeneic B6, or allogeneic BALB/c IL-13+/+ or BALB/c IL-13−/− donors and serum cytokine levels were determined on day +7. As shown in table 1, serum levels of IL-13, IL-5, IFNγ and TNFa were all elevated in mice receiving allo-SCT from IL-13+/+ donors compared to syngeneic controls. By contrast, analysis of sera from IL-13−/− SCT recipients revealed significant reductions in IL-13 and IL-5 levels, no change in IFNγ and a significant increase in TNFa levels compared to allo controls. Subsequent experiments revealed that the altered cytokine milieu observed early after allo-SCT correlated with enhanced GVHD; day 100 survival in allo-recipients of IL13−/− SCT was significantly decreased when compared to mice transplanted with IL-13+/+ donor cells (table 1) although GVHD of the gut (table 1) and liver (data not shown) did not differ in animals surviving at day +42. In summary our data demonstrate that donor T cell production of IL-13 does not predict the severity of GVHD and may instead have a protective effect on mortality from GVHD in this system. The decrease in survival observed in recipients of IL-13−/− donor cells may be explained by 1) the significant decrease in Th2 cytokines early after SCT, 2) loss of the strong immuno-modulatory effects of IL-13 on macrophage function, and 3) the associated increase in TNFa levels observed in these animals. The previously described correlation between the in vitro production of IL-13 by donor T cells and clinical aGVHD may therefore reflect the overall activation status of these T cells rather than a direct functional link to GVHD pathophysiology. Table 1: Serum cytokines (pg/ml) 7 days after SCT IL-13 IL-5 IFN γ α TNF survival (day 100) gut pathology (day 42) (** p〈 0.01; * p 〈 0.05; ND = not detectable) syngeneic 28.0±16.9 32.1±7.9 ND 2.2±1.5 100% 15.3±3.2 allogeneic wt 161.2±29.0 115.0±21.3 2059±320 38.5±5.1 40% 26.0±1.1 allogeneic IL-13−/− 13.7±4.8** 53.5±9.4* 2042±313 70.5±11.0* 0% 26.2±1.2

Description: Hepatic dysfunction following hematopoietic stem cell transplantation (SCT) is common, but making the correct diagnosis can be challenging.When the etiology cannot be determined by laboratory data, physical examination, and imaging studies, liver biopsies serve as an important diagnostic tool. In the immediate post SCT setting, cytopenias, coagulopathy, along with tissue injury and friability significantly increase the risk of bleeding and poor wound healing following percutaneous or "open" biopsies. At our institution, laparoscopic-guided liver biopsy (LGLB) is the preferred technique for pediatric SCT patients. Laparoscopy offers multiple benefits such as minimal invasiveness, faster recovery, better surgical hemostasis, superior visualization of the liver and peritoneal cavity, and the ability to assess for pathology confined to lobar segments. Little data exist on the feasibility of this technique in pediatric SCT patients. We reviewed the course of 11 consecutive patients (6 male, 5 female, age range 9–23 years) who received allogeneic SCT and subsequently underwent LGLB between 1998 and 2004 for hepatic dysfunction of unknown etiology. The time from SCT to LGLB ranged from 23 to 405 days (median 97 days) and 8 (73%) patients were biopsied within 106 days of SCT. Seven (64%) patients had platelet counts less than 100,000/mm3. Biopsies were performed using a single port technique. After visual inspection of the peritoneal cavity, a 16 or 18 gauge spring loaded biopsy gun (Monopty Biopsy instrument, CR Bard Inc., Covington, GA) was passed through a small stab wound in the right upper quadrant, and then directed to the right lobe of the liver. A total of 4 to 5 core biopsies were routinely obtained and specimens were subsequently sent fresh to the pathology department for processing and review. No intra- or post-operative complications were observed. The initial clinical diagnosis was confirmed in 5 patients. In one patient, veno-occlusive disease (VOD) was substantiated by evidence of bridging fibrosis, whereas in the other 4 patients, histopathologic findings were consistent with the diagnosis of GVHD. Of particular interest, biopsy results demonstrated that the initial working diagnosis was incorrect in the remaining 6 (55%) patients. In 4 of these 6 patients, the clinical diagnosis was hepatic GVHD, but histologic examination demonstrated that the actual causes of liver dysfunction were iron overload, VOD, cholestatic liver disease of unclear etiology, and hepatocyte injury secondary to drug toxicity or infection. In the two other patients, the clinical diagnosis was unresolved in one and the patient was found to have GVHD, whereas the other individual was initially diagnosed with infectious hepatitis, but was ultimately found to have hepatitis secondary to massive iron overload. Our results suggest that LGLB in pediatric patients experiencing liver dysfunction after SCT can be informative and safe. LGLB helped delineate the true cause of hepatic dysfunction and changed our approach to therapy in more than 50% of the patients reviewed. Results from LGLB thus maximized therapeutic efficiency and minimized the exposure of our patients to unnecessary toxicity including the profound risk of infection that accompanies immunosuppressive therapy.

Description: Acute graft versus host disease (aGVHD) is the major complication of allogeneic stem cell transplantation (allo-SCT) and limits the utility of this treatment strategy. The pathophysiology of aGVHD involves injury to host tissues by inflammatory cytokines and donor-derived cellular effectors, the recruitment of which is likely mediated by chemokine receptor: ligand interactions. CCR1 is expressed on various cell types such as T cells, monocytes and macrophages and binds to a group of CC chemokines that includes RANTES (CCL5) and Mip-1a (CCL3). In light of the previously described role for both donor T cells and monocytes in the development of aGVHD, we tested the hypothesis that CCR1 expression on donor leukocytes contributes to systemic and target organ inflammation that occurs in this setting by using a well established murine SCT model (B6→B6D2F1) and mutant mice deficient in CCR1. Lethally (1100cGy) irradiated B6D2F1 mice received SCT either from syngeneic (B6D2F1) or allogeneic (B6) CCR1+/+ or CCR1−/− donors. The severity of GVHD was assessed after SCT by survival and a clinical scoring system that incorporates changes in weight loss, fur texture, skin integrity, mobility and posture. As expected, syngeneic SCT recipients all survived and where indistinguishable from naïve, untransplanted controls, whereas animals receiving allo-SCT from CCR1+/+ donors developed significant GVHD and only 50% of animals survived by day 35. By contrast, allo-SCT with CCR1−/− donor cells resulted in significantly improved survival (92% vs. 50%) and less severe clinical GVHD (1.0±0.3 vs. 3.0±0.5) compared to allo-CCR1+/+ controls. In addition, serum TNFa levels were significantly decreased by day +7 following CCR1−/− SCT (4.7±2.7 vs. 55.3±14.4 pg/ml) and correlated with a significant reduction in intestinal histopathology both on day 7 (16.3±1.0 vs. 21.5±0.9) and on day 14 (15.8±1.8 vs. 23.8±1.0). GVHD injury to liver and skin was mild at these time points and did not differ between allo groups. Next we investigated the impact of CCR1 expression on allo-specific T cell responses in vivo and found that by day +7 after SCT both splenic T cell expansion (3.7±0.4 vs. 9.6±0.9 x 106 cells) and serum IFNγ levels (4561±559 vs. 10028±681 pg/ml) were significantly lower when CCR1 was absent on donor cells. In summary, we describe a heretofore unknown role for CCR1 on donor leukocytes in the development of aGVHD. The improvement in systemic and target organ disease observed after CCR1−/− SCT may be attributed to 1) alterations in leukocyte recruitment to the intestinal tract resulting in improved intestinal integrity, reduced translocation of endotoxin and decreased TNFa production and 2) modulation of donor T cell responses to host allo-antigens. Experiments are ongoing to determine whether strategies targeting CCR1 signalling can be exploited to prevent GVHD but maintain GVL effects and thereby improve overall survival after allo-SCT.

Description: SAA is a life-threatening hematopoietic stem cell disorder that is treated with either bone marrow transplantation (BMT) or immunosuppressive therapy (IST). BMT is the treatment of choice for young patients with a matched sibling donor. Those with acquired SAA lacking a matched sibling donor and older adults (〉 40yrs) generally receive IST because of the high toxicity of BMT in these patients. Roughly two-thirds will respond but up to 40% of patients eventually relapse or acquire a secondary clonal disease such as paroxysmal nocturnal hemoglobinuria (PNH) or myelodysplastic syndromes. Thus, a major challenge in treating SAA is the management of patients who are have relapsed after or are refractory to IST or who have acquired a secondary clonal disorder. BMT is the only curative therapy for patients with inherited SAA but problems with graft failure, graft-versus-host disease (GVHD), and lack of matched unrelated donors especially for ethnic minorities have limited the success of alternative donor BMT, especially for adult patients. PTCy after HLA-haplo BMT has been shown to facilitate engraftment and yield rates of GVHD comparable to those of matched sibling BMT in hematologic malignancies. We hypothesized that PTCy would improve outcomes for patients with refractory SAA by reducing the risk of GVHD and expanding the donor pool to include haplo related and mismatched unrelated donors (URD). Patients received an allogeneic BMT (alloBMT) for relapsed or refractory SAA. Relapsed/ refractory disease was defined as having failed previous course of IST. In the majority of the cohort, haplo donors were used due to lack of availability of suitable and timely URDs. Nine patients underwent haploBMT. A mismatched URD was used in one case of no suitably available haplo donor given the inherited nature of her disorder. The nonmyeloablative preparatory regimen included antithymocyte globulin (0.5 mg/kg day-9 and 2 mg/kg on days -8,-7), fludarabine (30mg/m2/day on days - to -2), low dose CY (14.5 mg/kg on days -4, -5 ) and total body irradiation (200cGy day -1). After the unmanipulated marrow graft was infused on day 0, cyclophosphamide 50mg/kg/day IV on days +3,+4 post-transplant was administered. All patients received tacrolimus and mycophenolate mofetil (MMF) beginning day+5 through 35 (MMF) and day +5 through 1 year (tacrolimus) for additional graft versus host disease (GVHD) prophylaxis. Neutrophil engraftment was defined as an ANC 〉1.0 x109/L measured for three consecutive measurements on different days. Red cell engraftment was defined as days from last red blood cell transfusion. Platelet engraftment was defined as a platelet count 〉50,000 for 7 days without transfusion. Ten patients were treated according to this IRB approved protocol. Eight patients had documented acquired disease and received grafts from 5/10 related donors. Six had failed ATG-containing regimens previously and two relapsed after high dose cyclophosphamide as IST. Two patients had inherited syndromes: one with Diamond Blackfan who received a graft from 5/10 related donor and one with telomeres less than the first percentile in length with a presumed familial syndrome who received a 9/10 unrelated graft. Median follow-up time is 17.5 months (range 5-49). The median age was 34 years (range 17-54) with 6 patients greater than age 30 years, and 50% were males. Median time to neutrophil engraftment was 18 days (range 16-24). Median time to red cell engraftment was 25 days (range 16-48). Median time to platelet engraftment was 27.5 days (range 22-108). At the time of BMT, six patients had PNH clones, and all were eliminated. All patients are alive and well, fully engrafted with 100% donor chimerism in blood and marrow. Two patients had grade 1-2 skin acute GVHD. These two also had mild chronic GVHD of the skin/mouth requiring systemic steroids. One patient was able to come off all immunosuppression by 15 months and the other by 17 months. Nonmyeloablative alloBMT with PTCy appears promising in patients with refractory acquired and inherited SAA with acceptable rates of engraftment, eradication of pre-existing clonal diseases, and expansion of the donor pool. Given these promising results, this approach is being utilized in the next BMTCTN national trial for acquired refractory SAA. We believe this approach could also be considered as upfront therapy for SAA, and such a trial is in development at Hopkins Disclosures Brodsky: Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Description: Graft-vs-host disease (GVHD) remains the major cause of morbidity and treatment-related mortality (TRM) after allogeneic HSCT, but there are no independent laboratory tests to either predict or confirm the clinical diagnosis of GVHD. Pre-clinical studies have shown that tumor necrosis factor-α (TNF) plays an important role in the pathogenesis of GVHD and that TNF plasma levels rise often several weeks before clinical disease becomes apparent. We tested the hypothesis that rises in TNF (on day 7 following allogeneic HSCT) will predict the development of significant GVHD and treatment-related mortality (TRM). We measured soluble TNF receptor 1 (TNFR1) as a surrogate for TNF because TNF circulates as a ligand-receptor complex. We studied samples obtained under informed consent from 438 patients undergoing allogeneic HSCT following myeloablative conditioning at the University of Michigan between 2000 and 2005. The conditioning regimens were based on Busulfan (68%), BCNU (20%), or TBI (12%). The median age of the patients was 42y (range 0–65y). The distribution of donors by degree of HLA-match and type was: 6/6 HLA-matched related donors (n=247), 5/6 matched related donors (n=20), 6/6 matched unrelated donors (n=124), 5/6 matched unrelated donors (n=47). Hematologic malignancy was the indication for 95% of HSCT. The median day of onset of GVHD grade 2–4 for related donors was 30d and for unrelated donors was 20d. Because of the variablilty in baseline TNFR1 levels, we expressed the day 7 value as a ratio to pre-transplant baseline. The mean day 7 TNFR1 ratio strongly correlated with severity of GVHD. The mean TNFR1 ratio was 1.91±0.09 for pts with GVHD 0–1 (n=269), 2.32±0.20 for pts with GVHD 2 (n=83), and 2.92±0.26 for pts with GVHD 3–4 (n=86), p

Description: Allogeneic (allo) bone marrow transplantation (BMT) is the only curative option for many patients with malignant and non-malignant diseases. Acute graft versus host disease (GVHD) is the major complication of allo-BMT and limits the utility of this treatment strategy. The induction of GVHD fundamentally depends upon the activation of donor T cells by host antigen presenting cells (APCs), and the prevailing hypothesis is that these critical interactions occur in secondary lymphoid organs, such as lymph nodes (LN) Peyer’s patches (PP), and spleen (SP). We tested this hypothesis by using a well established, MHC disparate, murine SCT system (Balb/c → B6) and homozygous aly/aly (alymphoplasia) mice that are deficient in all LN and PP and heterozygous aly/+ littermate controls. Lethally irradiated, splenectomized, aly/aly mice (LN/PP/SP −/ −) and aly/+ sham mice (LN/PP/SP +/+) received BMT either from syngeneic (aly/aly) or allo (Balb/c) donors. In some experiments, wild-type B6 recipients of B6 or Balb/c BMT served as additional negative and positive GVHD controls respectively. The severity of GVHD was assessed by survival and well-described scoring systems of both clinical and target organ disease. As expected, greater than 95% of syngeneic (syn) BMT recipients survived and were indistinguishable from naïve, un-transplanted controls, whereas LN/PP/SP +/+ mice receiving allo-BMT showed significant signs of GVHD with ~40% mortality by day 49. All LN/PP/SP −/ − allo-BMT recipients also survived, but surprisingly, examination demonstrated that they too developed significant clinical GVHD compared to syn controls (score: 3.2 vs. 0.85) that was comparable in severity to LN/PP/SP +/+ mice (3.1). Moreover, histopathologic analysis demonstrated that LN/PP/SP −/ − allo-BMT recipients developed significantly greater GVHD target tissue damage in the liver, intestinal tract and skin compared to syn controls. In fact, LN/PP/SP −/ − allo-BMT recipients developed more severe hepatic GVHD compared to allo littermate (LN/PP/SP +/+) controls (30.8±1.9 vs. 20.7±2.2; p 〈 0.01). Similar differences in liver GVHD was also seen between allo groups as early as day 7 (16.0±2.2 vs. 7.3±0.9; p 〈 0.01). We next tested the ability of host aly/aly and aly/+ APCs to stimulate donor Balb/c T cells in vitro. No differences in proliferation, IFN γ production or CTL generation were detected, thus showing that the allo-stimulatory capacity of host APCs was not different between groups. In order to ascertain what extra-lymphoid host tissues might serve as initial sites for allo-antigen exposure, we examined donor T cell expansion (CD3+), activation (CD69+) and proliferation (CFSE) in the bone marrow compartment 3 days after BMT. We found that in each case, LN/PP/SP −/ − allo-BMT recipients had significantly higher numbers / divisions compared to allo, littermate, (LN/PP/SP +/+) controls. Collectively, these data challenge the paradigm that secondary lymphoid tissues are required for GVHD induction, and suggest that the bone marrow may represent an alternative site for allo-antigen recognition and donor T cell activation.

Description: HCT entails substantial TRM justifying continued efforts to better risk stratify patients. Single institutional studies have suggested several clinically available biomarkers of CRP, ferritin and albumin influence TRM if not overall survival (OS). We sought to confirm the independent prognostic value of these biomarkers in a large multi-institutional cohort. The study population consisted of 784 adults with AML in remission (80%) or MDS (20%) undergoing unrelated donor HCT between 2008 and 2010 and available cryopreserved samples through the Center for International Blood and Marrow Transplant Research (CIBMTR) repository. CRP and ferritin were centrally quantified by ELISA from cryopreserved plasma whereas albumin levels obtained from center reported data. Correlation studies for biomarkers required log transformation of CRP and ferritin to produce a normal distribution. Multivariate models were fitted separately for each biomarker applying protocol specified thresholds generated from the literature of CRP 〉 10 mg/L, ferritin 〉 2500 ng/mL, albumin 〈 3.5 g/dL on TRM. Further analysis explored optimal cutpoints for this cohort for all significant clinical variables for TRM. HCT characteristics included a median age of 50 (range 18-78) years, HCT-CI (co-morbidity index) 3 or more in 35%, single allele/antigen mismatch (7/8) in 23%, PB as stem cell source in 83%, and myeloablative conditioning in 72%. Biomarker data were available in 783, 781 and 695 cases for CRP, ferritin and albumin respectively. The median values and ranges for each biomarker were as follows: 5.0 mg/L (0.3 - 316) for CRP, 1148 ng/mL (51 – 14,298) for ferritin and 3.6 g/dL (0.6-5.3) for albumin. Log transformed CRP and ferritin showed a modest correlation (r=0.35, P2500 ng/mL 93 (12) 1.26 0.84 – 1.88 0.27 1.15 0.86 – 1.54 0.35 Albumin 〈 3.5 g/dL 290 (42) 1.48 1.10 – 2.0 0.010 1.39 1.13 – 1.72 0.002 Optimal threshold, combined model ** CRP 〉3.67 mg/L 497 (64) 1.56 1.11 – 2.19 0.010 1.24 1.00 – 1.55 0.054 Log(Ferritin), linear n/a 1.20 0.99 – 1.46 0.07 1.20 1.04 – 1.38 0.010