ALBERT

Description: Key Points Donor treatment with AAT suppresses GVHD in the transplant recipient while enhancing the GVL effect. AAT effects are mediated via cell type–specific alterations of mitochondrial bioenergetics.

Description: Background: Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic cell transplantation (HCT). Severe acute GVHD of the gastrointestinal tract can be life threatening and is associated with a loss of acute phase proteins including alpha1-antitrypsin (AAT). AAT acts as a potent anti-inflammatory agent. We and others have shown in allogeneic murine models that administration of AAT during HCT suppresses serum levels of pro-inflammatory cytokines, interferes with GVHD and reduces transplant-related mortality. We embarked upon a phase I/II clinical study to evaluate the feasibility and safety of AAT given to HCT recipients with steroid non-responsive acute GVHD. We aimed at characterizing pharmacodynamic effects of AAT and assess clinical responses as determined by changes in stool volume and overall clinical improvement Methods: Seven patients (3 female, 4 male) with hematologic malignancies were enrolled in the first and second cohort. Patients were 35-59 (median 50.4) years old and were given transplants from HLA-matched siblings (n=5) or received cord blood (n=2) following high intensity (myeloablative) or reduced intensity conditioning with cyclophosphamide +TBI (n=2), Fludarabine+TBI (n=4), or busulfan +cyclophosphamide (n=1). All patients received cyclosporine and MMF for GVHD prophylaxis. Acute GVHD of grades III-IV developed at 49 to 71 (median of 48) days, and treatment with systemic methylprednisolone, 2 mg/kg/day was instituted. Patients showing no clinically satisfactory responses after 5 days were given AAT (GlassiaTM) at 90 mg/kg iv on day 1, followed by 30 mg/kg (first cohort) or 60 mg/kg (second cohort) every other day for a total of 8 doses (15 days). Results: As AAT levels vary considerably from person to person, we determined plasma AAT concentrations before and after administration of exogenous AAT. Pre-treatment AAT levels ranged from 1.25mg/ml to 2.3mg/ml (mean 1.76 mg/ml), while post-treatment AAT levels were 2.5 mg/ml to 5 mg/ml (mean 3.18 mg/ml), i.e. in the normal range. Concurrently, levels of Angptl4, IL10 and IL-15 protein increased in all patients, while TIM-3, a marker of lymphocyte activation, was down-regulated. RQ-PCR analysis of donor-derived cells showed up-regulation of IL-10 and IL-1Ra (50 log2 and 10 log2, respectively, compared to pre-treatment samples). FACS analysis of peripheral blood cells showed a 5-fold increase in CD4+CD24+FoxP3+ Treg cells and a 3 fold increase in CD8+CD205+ dendritic cells (DC) in comparison to pre-treatment samples in all 7 patients. Concurrently, the numbers of TIM-3 expressing CD4+ T lymphocytes decreased. While endoscopic evaluation of patients before treatment revealed severe intestinal GVHD with mucosal denudation, endoscopic evaluation one week after completion of AAT administration showed evidence of re-epithelialization of the bowel wall. Concurrently, five of the seven patients developed formed stools, associated with decreased intestinal loss of AAT. One patient required additional therapy and died with biopsy proven liver GVHD. With a median follow-up of 8 months (range 3.5-12 months), five of of seven patients are alive, no longer requiring treatment for GVHD. Summary and conclusions: Continuous administration of AAT as salvage therapy for steroid resistant gut GVHD is feasible without clinically relevant toxicity. Stool sampling showed a decrease in intestinal AAT loss, as measured by AAT clearance and endoscopic evaluation confirmed healing of the bowel mucosa. These data are encouraging, and further exploration of AAT therapy in extended phase II and randomized trials as therapy of steroid refractory acute GVHD or as first line therapy are warranted. Disclosures Off Label Use: Off label use of Alpha 1 Anti-Trypsin (AAT) as second line therapy for steroid-refractory gut GVHD..

Description: Abstract 123 Background: Lenalidomide is an effective agent in the treatment of MDS particularily in those possesing the deletion 5q (del5q) abnormality. The disease phenotype is partly related to loss of genetic material found in the commonly deleted region (CDR) on the long arm of chromosome 5. Recently implicated genes include miRNA-143, 145 and 146 all of which appear to play a role in innate immune signalling. In this study we examined whether upregulation of miRNAs in pretreatment CD34+ marrow cells from patients with MDS (both del5q and non-del5q) exposed to lenalidomide in vitro predicts for clinical response to the drug. Patients and Methods: In total 31 pre-treatment patient samples were obtained from three North American centres. 11 patients' samples were positive for del5q. All patients had received lenalidomide as per local protocols. Clinical response was defined as per the International Working Group 2000 criteria. Clinical data were available for 26 patients. Hematologic improvement (HI), specifically the eythroid response (HI-E), was examined as the main clinical endpoint. Changes in miR-143, miR-145, miR-146a and miR-146b expression in CD34+ cells after in vitro lenalidomide exposure (10uM for 48 hours) were examined. The CD34- fraction was also analyzed. dCT values were normalized to 5S. A ≥ 1.5 fold change in miRNA expression was considered significant. Results: For the entire cohort mean fold changes in miRNA-143 and 145 post lenalidomide exposure were 1.6 and 1.7, respectively. Little change was seen in miR-146a and miR-146b expression (mean fold change 1.1 and 1.2, respectively). No significant increase was seen in any of the examined miRNA in the CD34 - fraction (Figure 1a). When separated based on del5q status both del5q and non-del5q groups showed a 〉 1.5 fold increase in expression of miR-143 and miR-145. No significant change in either miR-146a or miR-146b expression was seen (Figure 1b). In the CD34- fraction no significant change was seen in any of the examined miRNAs, irrespective of del5q status (Figure 1c). Examining HI-E, major responses (MR) were seen in 42% of patients (87.5% in the the del 5q cohort (7/8 had a MR) and 22.2% for the non-del5q cohort (4/18 had a MR)). Correlation with clinical response and miRNA expression in the CD34+ fraction is shown in Table I. In the del5q cohort MR was associated with an increase in both miR-143 and miR-145. No correlation with miRNA expression and clinical response was seen in the non del5q cohort. Conclusion: Exposure of CD34+ cells from patients with MDS to lenalidomide results in up regulation of miR-143 and miR-145, irrespective of del5q status. Clinically, upregulation of miR-143 and miR-145 correlates with a HI-E in patients with del5q and may be predictive of response to therapy. Interestingly, the non-responder in the del5q group failed to upregulate miR-145. A similar gene induction pattern was seen in the non-del5q cohort, yet no correlation was seen with clinical outcome. Given that non-del5q patients do not harbor any abnormalities within the CDR of chromosome 5 these miRNAs are unlikely to play a role in their disease phenotype, nor in their response to lenalidomide. Disclosures: Nevill: Celgene: Honoraria. Karsan:Celgene: Research Funding.

Description: Abstract 522 Graft-versus-host disease (GVHD) is an important complication of allogeneic hematopoietic cell transplantation (HCT). The role of various cell populations, cytokines and chemokines, present pre and post-transplantation, in the development of GVHD has been studied extensively.We investigated the potential role of Interleukin (IL)-32 in alloreactivity and GVHD. IL-32 is the protein product of the NK4 transcript first reported in IL-2 activated T lymphocytes and natural killer cells. IL-32 has pro-inflammatory and pro-apoptotic properties and induces expression of TNF-α in several cell targets. We used one-way mixed lymphocyte cultures (MLC) as a simple in vitro model of GVHD to determine IL-32 expression upon alloactivation. The α and γ isforms of (IL)-32 protein were 2-fold upregulated in allogeneic MLC compared to autologous controls (n=4, p=0.037). Concurrently, the concentrations of TNF-α, IL-6 and IL-8 in the allogeneic MLC supernatants were, as expected, upregulated significantly. This finding led us to evaluate IL-32 expression as a potential marker for GVHD after allogeneic HCT in 45 patients with either active acute GVHD or chronic GVHD, and 16 patients who did not show clinical evidence of GVHD. IL-32 mRNA levels in peripheral blood unsorted white blood cells, correlated with acute GVHD (RT-PCR values expressed as mean +/− SEM of the ratio of expression of IL-32/GUS-B = 1.01, p=0.011, vs control values IL-32/GUS-B = 0.23) but not with chronic GVHD, (IL-32/GUS-B = 0.26, p=0.16) (Figure 1). As the serine protease neutrophil proteinase 3 (PR3) has been shown to serve as activator of IL-32, and to process several inflammatory cytokines, including IL-32, TNF-α and IL-8, we postulated that the addition of the serine protease inhibitor α-1 anti-trypsin (AAT), would interfere with the processing of IL-32 by PR3, and as a result would lead to decreased proliferation of cells in MLC, and reduced cytokine production. To test this hypothesis, MLCs were treated with AAT at concentrations of 1–20 ug/ml and expression of IL-32 and PR3 were determined. In MLCs treated with AAT at the optimal concentration of 5 ug/ml, added to cultures on alternate days for a period of 7 days T lymphocyte proliferation was suppressed when compared to vehicle-treated MLC, (mean CPM=33.000 versus CPM=67.000; p=0.012). Concurrently there was a 2.5 fold decrease in IL-32 and PR3 protein levels (n=4, p=0.023). CONCLUSION: IL-32 is upregulated in patients with acute GVHD. Determination of IL-32 in patients with suspected GVHD may support the diagnosis and the decision to treat.AAT interferred with T cell activation and the release of cytokines, including IL-32, suggesting that administration of AAT may have therapeutic potential in patients with acute GVHD. Disclosures: No relevant conflicts of interest to declare.

Description: Aplastic anemia (AA), a potentially fatal disease, may be cured with marrow transplantation. Survival in pediatric patients has been excellent early after transplantation, but only limited data are available regarding late effects. This study evaluates late effects among 152 patients followed 1-38 years (median, 21.8 years). Transplantation-preparative regimes were mostly cyclophosphamide with or without antithymocyte globulin. Survival at 30 years for the acquired AA patients is 82%, and for the Fanconi anemia patients it is 58% (P = .01). Multivariate analysis demonstrated that chronic GVHD (P = .02) and Fanconi anemia (P = .03) negatively impacted survival. Two Fanconi patients and 18 acquired AA patients developed a malignancy that was fatal for 4. There was an increased incidence of thyroid function test abnormalities among those who received total body irradiation. Cyclophosphamide recipients demonstrated normal growth, basically normal development, and pregnancies with mostly normal offspring. Quality-of-life studies in adult survivors of this pediatric transplantation cohort indicated that patients were comparable with control patients except for difficulty with health and life insurance. These data indicate that the majority of long-term survivors after transplantation for AA during childhood can have a normal productive life.

Description: Building on a successful non-myeloablative conditioning regimen developed in Seattle (Blood 2003), Luznik and O´Donnell et al created a protocol that incorporates post-transplant cyclophosphamide (CY) after human leukocyte antigen (HLA)-haploidentical hematopoietic cell transplantation (HCT) (BBMT 2008). This method both promotes engraftment while selectively-depleting alloreactive donor T cells to prevent graft-versus-host disease (GVHD). We have previously shown that Fanconi Anemia (FA) patients can be treated with CY 60 mg/kg in a conditioning regimen with minimal toxicity (BBMT 2007), thus we adapted this post-HCT CY strategy for in vivo T-cell depletion in patients with FA. Between 2008 and 2012, four patients from three North American centers with FA and severe marrow failure in the absence of HLA-matched donors underwent HLA-haploidentical HCT. All four patients were referred for transplantation with minimal to no transfusion burden and all were in excellent clinical condition with HCT-CI scores of 0-2 and Lansky scores of 90-100%. Median age at transplant was 9.7 (6.9-11.9) years old. Patients were transplanted at a median of 1.6 (range, 0.6 -7.1) years after FA diagnosis. Conditioning consisted of fludarabine (150 mg/m2) and 2 Gy total body irradiation; one patient also received CY (10 mg/kg), which was deleted in subsequent patients to decrease the risk of mucositis. Marrow was infused on day 0, followed by post-grafting immunosuppression with CY (25 mg/kg/day, days +3, +4), mycophenolate mofetil, and cyclosporine, the latter two beginning at day +5 with plans to continue until days +35 and +180, respectively. Full donor engraftment was seen in all patients. Two patients developed acute grade I GVHD and none of the four patients has developed chronic extensive GVHD to date. With a follow-up of 5 years, 1 year, 11 months, and 9 months, all four patients are alive with stable, full donor chimerism, and are transfusion independent. While two patients required cyclosporine beyond day +180, only one patient currently remains on low-dose immunosuppression for treatment of limited chronic skin GVHD, which has now resolved. Our results confirm that modulated post-HCT CY can be used in patients with FA to promote engraftment across histocompatibility barriers. Despite concerns for both excessive toxicity related to CY and severe GVHD related to minimizing the dose of post-transplant CY, none of the FA patients in our small series experienced these problems. Our findings also suggest that transplant should not be delayed when there is lack of an HLA-matched donor. FA patients with few comorbidities and minimal transfusion burden can successfully undergo this HLA-haploidentical HCT approach. Disclosures: Off Label Use: MMF.

Description: This study was conducted to elucidate the influence of immunosuppressive treatment (IST) and GVHD on risk of recurrent malignancy after allogeneic hematopoietic cell transplantation (HCT). The study cohort included 2656 patients who received allogeneic HCT after high-intensity conditioning regimens for treatment of hematologic malignancies. Rates and hazard ratios of relapse and mortality were analyzed according to GVHD and IST as time-varying covariates. Adjusted Cox analyses showed that acute and chronic GVHD were both associated with statistically similar reductions in risk of relapse beyond 18 months after HCT but not during the first 18 months. In patients with GVHD, resolution of GVHD followed by withdrawal of IST was not associated with a subsequent increase in risk of relapse. In patients without GVHD, withdrawal of IST was associated with a reduced risk of relapse during the first 18 months, but the risk of subsequent relapse remained considerably higher than in patients with GVHD. In summary, the association of GVHD with risk of relapse changes over time after HCT. In patients without GVHD, early withdrawal of IST might help to prevent relapse during the first 18 months, but other interventions would be needed to prevent relapse at later time points.

Description: In patients with myelodysplastic syndromes (MDS), apoptosis in hematopoietic cells is up-regulated in low-grade disease, whereas advanced disease is characterized by apoptosis resistance. We have shown that marrow stroma–derived signals convey sensitivity to tumor-necrosis-factor alpha (TNF-α)–mediated apoptosis in otherwise-resistant KG1a myeloid cells and CD34+ cells from MDS marrow. Here, we used a PhosphoScan proteomic liquid chromatography–mass spectrometry method to identify signals relevant for this effect. The transcription factor DJ-1/PARK-7 (DJ-1) was highly phosphorylated in KG1a cells cultured without stroma but dephosphorylated after stroma coculture, whereas expression of p53 increased significantly, suggesting a stroma contact-dependent effect of DJ-1 on p53. In CD34+ marrow cells from advanced MDS, expression of DJ-1 was up-regulated, whereas p53 levels were low, resulting in significantly greater DJ-1/p53 ratios than in patients with low-grade MDS (P = .01). DJ-1 levels were correlated with increasing International Prognostic Scoring System scores (P = .006). Increasing DJ-1/p53 ratios were associated with an increased risk of mortality, although the correlation did not reach statistical significance (P = .18). These data suggest that DJ-1/p53 interactions contribute to apoptosis resistance in clonal myeloid cells and may serve as a prognostic marker in patients with MDS.

Description: Patients with low-grade myelodysplastic syndromes (MDS) show high levels of tumor necrosis factor α (TNFα) and up-regulation of apoptosis in the marrow. In contrast, marrow cells in advanced MDS are typically resistant to TNFα-induced apoptosis but are rendered apoptosis-sensitive on coculture with stroma. The present studies show that CD34+ marrow cells in advanced MDS express high levels of TWIST, a basic helix-loop-helix transcription factor that opposes p53 function. TWIST levels correlated with disease stage (advanced 〉 low grade; P = .01). Coculture with HS5 stroma resulted in down-regulation of TWIST and increased apoptosis in response to TNFα in CD34+ cells from advanced MDS; the same effect was achieved by TWIST-specific RNA interference in CD34+ cells. In primary MDS marrow stroma TWIST expression was lower than in healthy controls; suppression of TWIST in stroma interfered with induction of apoptosis sensitivity in cocultured CD34+ cells. Stroma cells so modified expressed reduced levels of intercellular adhesion molecule-1 (ICAM1; CD54); blockade of ICAM1 in unmodified stroma was associated with reduced apoptosis in cocultured CD34+ MDS marrow cells. These data suggest role for dysregulation of TWIST in the pathophysiology of MDS. Conceivably, TWIST or components in the signaling pathway could serve as therapeutic targets for patients with MDS.