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Patients with hematologic malignancies not in remission before allogeneic hematopoietic stem cell transplantation (HSCT) have a poor prognosis. To improve the antitumor activity of conditioning, we combined clofarabine with myeloablative doses of busulfan in a phase 1/2 study in nonremission hematologic malignancies. Forty-six patients were enrolled, including 31 patients with nonremission acute myelogenous leukemia (AML). Patients had a median age of 53 years, with a median comorbidity index of 3. Donors were unrelated, HLA mismatched, or both in 59% of patients. Common grade III to IV nonhematologic toxicities included transient transaminitis (50%), mucositis (24%), hand-foot syndrome (13%), transient hypoxia (13%), nausea/vomiting (9%), and diarrhea (9%). All patients engrafted. Complete remission was achieved in 80% of all patients by day +30 and in 100% of AML patients without prior hematopoietic stem cell transplantation. Two-year nonrelapse mortality for all patients was 31%, and overall survival was 28%. In AML, the overall survival was 48% at 1 year and 35% at 2 years. These data suggest that clofarabine combined with myeloablative doses of busulfan is well tolerated, secures engraftment, and possesses significant antitumor activity, particularly in nonremission AML. This study is registered at www.ClinicalTrials.gov under identifier NCT00556452.

The relationship between regulatory T cells (Tregs) and acute graft-versus-host disease (aGVHD) in clinical allogeneic bone marrow transplantation (BMT) recipients is not well established. We conducted a prospective analysis of peripheral blood Tregs as determined by the frequency of CD4(+)CD25(hi)FOXP3(+) lymphocytes in 215 BMT patients. Autologous BMT patients (N = 90) and allogeneic BMT patients without GVHD (N = 65) had similar Treg frequencies, whereas allogeneic patients with GVHD (N = 60) had Treg frequencies that were 40% less than those without GVHD. Treg frequencies decreased linearly with increasing grades of GVHD at onset, and correlated with eventual maximum grade of GVHD (P.001). In addition, frequency of Tregs at onset of GVHD predicted the response to GVHD treatment (P = .003). Patients with Treg frequencies less than the median had higher nonrelapse mortality (NRM) than patients with Tregs greater than the median, but experienced equivalent relapse mortality, resulting in an inferior survival at 2 years (38% versus 63%, P = .03). Treg frequency may therefore have important prognostic value as a biomarker of aGVHD.

Idiopathic pneumonia syndrome (IPS) refers to a diffuse, noninfectious, acute lung injury after hematopoietic stem cell transplantation. Historically, IPS is associated with respiratory failure and mortality rates exceeding 50%. Preclinical studies have implicated tumor necrosis factor-α as an important effector molecule in the development of disease. We studied the tumor necrosis factor-α inhibitor, etanercept, combined with corticosteroids in treating 15 patients (median age, 18 years; range, 1-60 years) with IPS. Eight of 15 patients required mechanical ventilation at therapy onset. Etanercept was administered subcutaneously at a dose of 0.4 mg/kg (maximum 25 mg) twice weekly, for a maximum of 8 doses. Therapy was well tolerated with no infectious pulmonary complications noted. Ten of 15 patients had a complete response, defined as the ability to discontinue supplemental oxygen support during study therapy. The median time to complete response was 7 days (range, 3-18 days), with a day 28 survival of 73%. IPS onset was associated with elevations of several inflammatory proteins in the bronchoalveolar lavage fluid and plasma, and response to therapy correlated with reductions in pulmonary and systemic inflammation. The combination of etanercept and corticosteroids is safe and is associated with high response rates and improved survival in patients with IPS.

Acute graft-versus-host disease (GVHD) remains a significant cause of mortality after hematopoietic cell transplantation (HCT). Tumor necrosis factor–alpha (TNF-α) mediates GVHD by amplifying donor immune responses to host tissues and by direct toxicity to target organs. We measured TNF receptor 1 (TNFR1) as a surrogate marker for TNF-α in 438 recipients of myeloablative HCT before transplantation and at day 7 after transplantation. Increases in TNFR1 levels more than or equal to 2.5 baseline correlated with eventual development of GVHD grade 2 to 4 (58% vs 32%, P < .001) and with treatment-related mortality (39% vs 17%, P < .001). In a multivariate analysis including age, degree of HLA match, donor type, recipient and donor sex, disease, and status at HCT, the increase in TNFR1 level at day 7 remained a significant predictor for outcome. Measurement of TNFR1 levels early after transplantation provides independent information in advance of important clinical outcomes, such as GVHD and death.

Tumor necrosis factor-α (TNF-α) is known to play a role in the pathogenesis of graft-versus-host disease (GVHD), a cause of significant morbidity and treatment-related mortality (TRM) after allogeneic hematopoietic stem cell transplantation (HCT). We measured the concentration of TNF-Receptor-1 (TNFR1) in the plasma of HCT recipients as a surrogate marker for TNF-α both prior to transplant and at day 7 in 82 children who underwent a myeloablative allogeneic HCT at the University of Michigan between 2000 and 2005. GVHD grade II–IV developed in 39% of patients at a median of 20 days after HCT. Increases in TNFR1 level at day 7 post-HCT, expressed as ratios compared to pretransplant baseline, correlated with the severity of GVHD (P = .02). In addition, day 7 TNFR1 ratios >2.5 baseline were associated with inferior 1-year overall survival (OS 51% versus 74%, P = .04). As an individual biomarker, TNFR1 lacks sufficient precision to be used as a predictor for the development of GVHD. However, increases in the concentration of TNFR1, which are detectable up to 2 weeks in advance of clinical manifestations of GVHD, correlate with survival in pediatric HCT patients.

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.

Graft-versus-host disease (GVHD) is a principal cause of morbidity following allogeneic hematopoietic cell transplantation (HCT). Standard therapy for GVHD, high-dose steroids, results in complete responses (CRs) in 35% of patients. Because tumor necrosis factor-α (TNFα) is an important effector of experimental GVHD, we treated patients with new-onset GVHD with steroids plus the TNFα inhibitor etanercept on a previously reported pilot trial (n = 20) and a phase 2 trial (n = 41). We compared their outcomes with those of contemporaneous patients with GVHD (n = 99) whose initial therapy was steroids alone. Groups were similar with respect to age, conditioning, donor, degree of HLA match, and severity of GVHD at onset. Patients treated with etanercept were more likely to achieve CR than were patients treated with steroids alone (69% vs 33%; P < .001). This difference was observed in HCT recipients of both related donors (79% vs 39%; P = .001) and unrelated donors (53% vs 26%; P < .001). Plasma TNFR1 levels, a biomarker for GVHD activity, were elevated at GVHD onset and decreased significantly only in patients with CR. We conclude that etanercept plus steroids as initial therapy for acute GVHD results in a substantial majority of CRs. This trial was referenced at www.clinicaltrials.gov as NCT00141713.

There are no plasma biomarkers that are specific to any of the three target organs of acute graft versus host disease (GVHD): skin, GI tract and liver. We sought to identify a biomarker that is specific for GVHD of the skin in an initial discovery step using an intact proteomic analysis system. We compared plasma pooled from ten patients with GVHD only of the skin (sGVHD) to plasma pooled from ten patients with no GVHD to plasma pooled from ten patients with GVHD only of the GI tract. Of four candidate proteins that were both significantly elevated only in the plasma of sGVHD patients and that could be measured by ELISA, we selected elafin, an epidermal proteinase inhibitor that is induced by TNF-α and found in inflamed epidermis in diseases such as psoriasis. We therefore measured levels of elafin (expressed hereafter as mean ± SEM pg/ml) in individual samples of the discovery set, confirming that they were significantly higher in plasma from patients with sGVHD compared to patients presenting only with GI GVHD or without GVHD, respectively (13,312 ± 2456 vs. 3968 ± 537 vs. 3257 ± 332, p < 0.0001). We next analyzed a validation set of 429 plasma samples from allogeneic BMT patients transplanted at the University of Michigan from 2000–2008. We obtained samples at regular intervals from all patients until day 100 after BMT and at the first clinical signs of sGVHD. 275 (64%) patients had no GVHD and 154 (36%) had sGVHD. There were no statistically significant differences between patients with sGVHD and without GVHD with respect to age, underlying hematological malignancies and conditioning intensity. Recipients of grafts from donors who were not family members or who were less than 8/8 HLA identical matches were overrepresented in the sGVHD group. The median post-transplant day for sample collection was day 31 in GVHD-group and day 30 in sGVHD group. Elafin levels were two fold higher in plasma from patients with sGVHD as compared to plasma from patients without GVHD (7532 ± 488 vs. 3925 ± 112, p < 0.0001). We next analyzed whether elafin levels provided prognostic information regarding eventual maximum stage of GVHD skin, transplant-related mortality and overall survival. We divided the patients into 2 groups using a threshold level of elafin that provided 85% specificity (5500 pg/ml). The group with the high levels of elafin comprised 29% of the total patient population and developed more severe skin GVHD (maximum stage) than the group with low levels (p < 0.0001, Table 1). Patients with high elafin levels also experienced greater transplant-related mortality (TRM) at 1 year: 18% (95% CI, 11–25%) vs. 7% (95% CI, 4–11%) (p = 0.002). This difference remained significant when adjusted for age, family member donor, conditioning intensity and HLA mismatch (p = 0.01). Patients with high elafin levels also experienced lower overall survival (OS) at 1 year: 61% (95% CI, 52–71%) vs. 76% (95% CI, 71–82%) (p < 0.0001, adjusted for all the factors above) (Figure 1). Within the population of patients with sGVHD (n =154), patients with high elafin had a mortality-hazard ratio of 1.98 compared to patients with low elafin levels (p = 0.003). This hazard ratio of 1.98 remained significant (p = 0.017) after adjusting for stage of skin disease at onset, demonstrating that the association of mortality risk with elafin levels was independent of the GVHD clinical stage. In additional studies, we determined whether elafin levels measured 7 days prior to clinical onset of skin disease predicted a subsequent occurrence of sGVHD. In samples available from 133 patients, 7 days prior to sGVHD onset, elafin levels were 1.26 fold higher than in patients who did not later develop GVHD (4279 ± 222 vs. 3448 ± 108, p = 0.001). We conclude that elafin is a novel biomarker for sGVHD that can provide important diagnostic and prognostic information, including long term survival. Table 1: Correlating low/high elafin with maximum skin stage Maximum skin stage Maximum skin stage 0–1 2–4 p-value Low elafin 83% 17%

Abstract 675 We have previously identified five biomarker proteins that have diagnostic and prognostic value for acute graft-versus-host disease (GVHD) (Blood 113:273-278, Sci Transl Med 2:50-57). In order to determine whether biomarkers can predict GVHD before the appearance of clinical symptoms, we evaluated the three most informative biomarkers of the five (IL2-Receptor-α, TNFR1, elafin) in patient samples prospectively collected between 2000 and 2010 from 513 unrelated (URD) hematopoietic cell transplant (HCT) patients. We focused on URD HCT recipients because they are most likely to develop acute GVHD and could potentially benefit from a predictive laboratory assay and subsequent preemptive intervention. We measured biomarker plasma levels by sequential enzyme-linked immunosorbent assay on samples obtained prior to conditioning (pre-HCT), and at day +7 and day +14 after HCT. In designing the analytical approach, we took into consideration that median time to GVHD onset is delayed after reduced intensity conditioning, that there may be limited opportunity to preemptively intervene in patients on the verge of developing GVHD, and that there have been changes in GVHD prophylaxis agents over the past decade. Therefore, we randomly divided the patients into training (N=342) and validation (N=171) data sets that were balanced for (i) full intensity conditioning, (ii) onset of grade II-IV GVHD earlier than day +21 and (iii) HCT performed after 2005. In order to create a prediction model for acute GVHD, we used the biomarker levels in the training data set to simulate biomarker values for a hypothetical 50,000 patients using the following assumptions: (1) the incidence of GVHD by day 100 is 55%, (2) the median day of GVHD onset after full intensity URD HCT is day 21, with 10% of patients developing GVHD prior to day +7, and 3% developing GVHD after day +56. These assumptions were based on historical URD HCT data at our center. We used logistic regression to compute a predicted probability, p7, of developing grade II-IV GVHD for each of the 50,000 patients based upon the biomarker levels pre-HCT and at day +7. Any patient with p7≥0.64 was categorized as high risk for development of GVHD. For all low risk patients (p7 Disclosures: No relevant conflicts of interest to declare.

Abstract 514 JMM and XQ contributed equally to this work. Regulatory T cells (Treg) play an important role in the maintenance of tolerance after bone marrow transplantation (BMT) in experimental models. However, the relationship between Treg and acute graft-versus-host disease (GVHD) in allogeneic BMT recipients is not well established. We conducted a prospective analysis of Treg frequency in 215 BMT patients (125 allogeneic, 90 autologous) at the University of Michigan. Fresh peripheral blood samples were acquired prior to therapy within 24 hours of GVHD onset and at equivalent time points in patients without GVHD. Treg frequency was measured in triplicate by three color cytometry analysis to determine the frequency of CD4+CD25hiFOXP3+ cells within total lymphocytes. There were no significant differences between patients with and without GVHD for median age, nonmalignant disease, conditioning intensity, and median day of sample acquisition. Recipients of grafts from donors who were not family members or who were not HLA-matched were overrepresented in the GVHD group. Autologous BMT patients (N=90) and allogeneic BMT patients without GVHD (N=65) had the same mean Treg frequency (1.09% ± 0.10 vs. 1.09% ± 0.11, p = 0.54), showing that the use of calcineurin inhibitors did not affect Treg frequency. Patients with GVHD (N=60) had 40% fewer Treg (0.66% ± 0.07, p < 0.001) than those without GVHD. Frequencies of Tregs decreased in a linear fashion with each increasing grade of GVHD at onset, and were significantly reduced in patients with ≥ grade II compared to patients without GVHD (p < 0.001) (Figure 1). The calculated Receiver Operating Characteristic (ROC) curve for Treg frequency as an independent biomarker of GVHD was 0.69 (95%CI, 0.55-0.83). Treg frequency also correlated with the eventual maximum overall grade of GVHD (p < 0.001), suggesting a prognostic value for this measurement. Therefore, we evaluated whether Treg frequency would correlate with non relapse mortality (NRM) in the 60 patients with GVHD. Patients with low Treg frequency ( Disclosures: No relevant conflicts of interest to declare.

Background: The combination of fludarabine with a myeloablative dose of busulfan (FluBu4) has been shown to be well tolerated with reduced toxicity compared to other myeloablative regimens. Clofarabine is a second generation purine antimetabolite with potent anti-leukemia properties. We replaced fludarabine with clofarabine in a phase I/ II trial of Clofarabine-Busulfan x 4 (CloBu4) in an attempt to develop a pre-transplant conditioning regimen suitable for pts with refractory, non-remission hematologic malignancies at the time of transplant. Methods: All pts had a hematologic malignancy not in remission at the time of transplant. Busulfan was administered as a single daily dose of 3.2 mg/kg IV x 4d (days -5 to -2) and clofarabine as a single daily dose of 20, 30 or 40 mg/m2 IV x 5d (days -6 to -2) with the specific dose per pt determined by the Time to Event-Continuous Reassessment Method (TITE-CRM). All pts received dexamethasone 12 mg IV on the days of clofarabine to avoid capillary leak syndrome. GVHD prophylaxis was tacrolimus/MMF in19 pts and tacrolimus/methotrexate in 1. Results: To date, 20 pts are evaluable for toxicity, 19 for engraftment, and 16 for response. Diseases were AML (including 2 myeloid blast crisis of CML) (n=13), ALL (n=2), CLL (n=2), NHL (n=2), and multiple myeloma (n=1). Six pts had received previous stem cell transplantation (SCT), 2 auto SCT (range between SCT 295-1066 d) and 4 allo (range 75–1002 d). The clofarabine dose levels were: 20 mg/m2 (n=6), 30 mg/m2 (n=13) and 40 mg/ m2 (n=1). The median age of patients was 51 years (range 13–68). Donors were siblings (n=8) or unrelated volunteers (n=12). All 19 evaluable pts engrafted at a mean of 11 days for both neutrophils and platelets. Busulfan kinetics were evaluated in 14 patients with an average steady state level of 803 ng/ml (605–1132). Dose increase was required in 2 cases and decrease in 2 cases to maintain target range of 600–900 ng/ml. One pt who developed hypersensitivity to clofarabine with fever and joint pain after the first dose, received an additional 20 mg of dexamethasone with subsequent doses and finished all five doses of clofarabine. Other grade 3–4 toxicities attributable to the conditioning regimen include transient liver enzyme abnormalities (8/20), hypoxia (5/20), hypertension (2/20), seizure (1/20), and ascites (2/20). Neither case of ascites had elevated bilirubin, but one case showed pathologic findings compatible with veno-occlusive disease on liver biopsy. Both of these pts had pre-existing conditions including pretransplant liver damage (1) and enlarged spleen before SCT due to preceding myelofibrosis (1). Acute GVHD developed in 2/20 and proved fatal in one case. Peripheral blood CD3+ cell chimerism was evaluable in 10 pts on day 30; 1 pt had 100% donor CD3+ cells, whereas 9 pts had in mixed chimerism ( Conclusion: Clofarabine is well tolerated when given with full dose busulfan in this group of very high risk pts. All pts engrafted rapidly, and this combination shows promising anti-tumor activity in pts with relapsed/refractory diseases. The trial continues to accrue pts and updated results will be presented at the meeting. Figure Figure

Graft-versus-host disease (GVHD) is a principal cause of morbidity and mortality following allogeneic hematopoietic cell transplant (HCT). Standard therapy for GVHD, high dose steroids, results in complete responses in only 35% of patients. Because tumor necrosis factor-α (TNFα) is an important effector of GVHD in animal models we treated 61 pts with new onset GVHD with methlyprednisolone 2 mg/kg/d plus etanercept, a TNFα inhibitor. All pts continued their GVHD prophylaxis agent, usually tacrolimus, at therapeutic dosing. Etanercept was given subcutaneously twice weekly for 8 wks at a dose of 0.4 mg/kg/dose (maximum dose 25 mg). The outcomes in these 61 pts were compared to those of 99 contemporaneous pts with GVHD whose initial therapy was steroids alone. Both groups of pts were similar with respect to age, transplant conditioning intensity, donor type and degree of HLA-match, and severity of GVHD at onset. Pts treated with etanercept plus steroids were significantly more likely to achieve a complete response 4 wks later than were pts treated with steroids alone [69% (95% CI: 57%, 81%) vs 33% (95% CI: 24%, 42%), p Pts treated with etanercept plus steroids were significantly more likely to be alive 100 days from the initiation of GVHD treatment than pts treated with steroids alone (82% vs 66%, p=0.04). The infection rates in the first 100d from initiation of GVHD treatment were not different between pts treated with etanercept plus steroids or treated with steroids alone for bacterial, invasive fungal or viral infections Blood samples obtained at onset of GVHD and four wks later were analyzed for plasma levels of TNF-receptor 1 (TNFR1) as a biomarker of GVHD activity. At the onset of GVHD the mean plasma TNFR1 levels were significantly elevated compared to levels at a similar timepoint from a control group of pts without GVHD; 4 wks later mean plasma TNFR1 levels were decreased significantly only in pts with CR. Although not a randomized phase III trial, these large differences between treatment groups strongly suggest that etanercept plus steroids as initial therapy for acute GVHD results in improved complete response rates compared to steroids alone. Figure Figure Complete Response Rates at Four Weeks According to Treatment Group Steroids alone Etanercept plus steroids p value Overall 33/99 (33%) 42/61 (69%)

There are no independent laboratory diagnostic tests for acute GVHD. We first identified 7 potential plasma biomarkers (IL-2R-α, CRP, IL-8, TIMP-1, TNFR1, HGF, CA-19.9) of acute GVHD using a combination of proteomic approaches and antibody microarrays. We next conducted a retrospective analysis using plasma samples from 424 patients at the University of Michigan under IRB approval. We obtained samples at the first clinical signs of acute GVHD prior to treatment and at equivalent time points in patients without GVHD (Table 1). The median duration of follow-up was 420 days with a minimum follow-up of 180 days. Patients with veno-occlusive disease, idiopathic pneumonia syndrome, or septic shock were not included. We measured plasma levels of the 7 proteins by sequential ELISA. Logistic regression models with and without leave-one-out-cross-validation (LOOCV) tested the correlation of the laboratory values with the diagnosis of acute GVHD using area under the receiver-operating-characteristic (ROC) curves (AUC). The training set consisted of 282 randomly selected patients; the validation set included the remaining 142 patients. The final optimal fingerprint of four proteins excluded CRP because of its association with non-specific inflammation and included IL-2R-α, TNFR1, IL-8 and HGF, with AUCs of 0.91 and 0.89 in the training set (without and with LOOCV, respectively) and 0.86 in the validation set. The fingerprint had a strong association with grade of GVHD (p Figure Figure Table 1: Patients characteristics GVHD- (N=242) GVHD+ (N=182) Age-yr Median (range) 45 (1–69) 49 (1–71) Donor type (%) MRD: 169 (70%) MRD: 105 (58%) URD: 73 (30%) URD: 77 (42%) Conditioning regimen Intensity (%) Full: 182 (75%) Full: 114 (63%) Reduced: 60 (25%) Reduced: 68 (37%) Day after BMT of samples : median (range) 30 (7–104) 29 (5–119) Grade at GVHD Onset (%) Grade 0 Grade 1 Grade 2 Grade 3–4 242 (57%) 48 (12%) 100 (24%) 34 (7%) Organ Target at GVHD Onset (%) n/a Skin Gut Liver Combined 119 (65%) 38 (21%) 7 (4%) 18 (10%)

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 When treated as a continuous variable, change in TNFR1 on day 7 strongly correlated with both the likelihood of GVHD 2–4 (p TNFR1 ratio - All patients (n=438) GVHD 2–4 1y TRM TNFR1 ratio ≤2.5 (n=328) 32% 17% TNFR1 ratio >2.5 (n=110) 58% 39% p2.5 (n=57) 50% 29% p2.5 (n=53) 65% 49% p=0.001 p=0.01 Patients with a day 7 TNFR1 ratio ≤2.5 were more likely to be alive at 1yr post-transplant (64% vs 50%, p=0.008). We conclude that even within risk groups stratified by donor source the magnitude of rise in early post-transplant TNFR1 ratios helps to predict the subsequent severity of GVHD, TRM and survival. A single threshhold at this early timepoint identifies roughly a quarter of patients who are at approximately twice the risk of significant GVHD and death. These informative changes are detectable often two to three weeks in advance of clinical manifestations of GVHD and may therefore provide the basis for development of a predictive laboratory test.

Summary. A monoclonal antibody, designated M148, produced by the hybridoma technique from spleen cells of mice immunized with human medulloblastoma, was found by indirect immunofluorescence to bind to normal human platelets (both PlAl positive and PlAl negative) and megakaryocytes, as well as to some medulloblastoma and neuroblastoma cells and cell lines and certain other solid tumours. No binding was observed to other marrow constituents, nor to any other normal tissue examined. The antibody bound to platelets from a patient with the Bernard-Soulier syndrome but not to thrombasthenic platelets. It immunoprecipitated glycoproteins IIb and IIIa from 125I-labelled normal platelet membranes, and completely inhibited ADP-induced fibrinogen binding and aggregation of platelets. Aggregation was also inhibited in response to adrenaline, collagen, thrombin, sodium arachidonate and the ionophore A23187; clot retraction was partially inhibited. The antibody was without effect on thromboxane formation or 5-hydroxytryptamine (5HT) secretion in response to thrombin, but inhibited 5HT secretion in response to arachidonate. It did not inhibit factor VIII binding or agglutination in response to ristocetin, but completely inhibited factor VIII binding in response to thrombin. These findings suggest that the epitopes are close to the fibrinogen and factor VIII binding sites on glycoproteins IIb/IIIa, and that the lack of these glycoproteins is sufficient explanation for the pattern of dysfunction observed in thrombasthenic platelets, without invoking any other membrane abnormality.

Clinical and preclinical data indicate that tumor necrosis factor (TNF)–α is an important mediator of acute graft-versus-host disease (aGVHD) after allogeneic bone marrow transplantation. We completed a study using etanercept, a fusion protein capable of neutralizing TNF-α, for the initial treatment of aGVHD. Etanercept (25 mg subcutaneously) was administered twice weekly for 16 doses, along with methylprednisolone (2 mg/kg) and tacrolimus for biopsy-proven aGVHD. Twenty patients with a median age of 47 years (range, 8–63 years) were enrolled. Fourteen patients with grade II aGVHD (11 family donors and 3 unrelated donors) and 6 patients with grade III aGVHD (3 family donors and 3 unrelated donors) were treated. Twelve patients completed 16 doses of therapy, and 8 received 5 to 15 doses. Reasons for not completing all doses of etanercept included progression of aGVHD (n = 4), relapsed leukemia (n = 2), progression of pulmonary and central nervous system lesions (n = 1), and perforated duodenal ulcer (n = 1). Fifteen (75%) of 20 patients had complete resolution of aGVHD within 4 weeks of therapy. Increasing levels of soluble TNF receptor 1 plasma concentration during the first 4 weeks of therapy indicated progression of aGVHD in 5 patients. In contrast, for 15 responding patients, soluble TNF receptor 1 plasma concentration levels returned to baseline. These data demonstrate the feasibility of using cytokine blockade in the early treatment of aGVHD.