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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%

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

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.