ALBERT

Description: Through an immune-mediated graft-versus-leukemia effect, allogeneic hematopoietic stem cell transplantation (HSCT) affords durable clinical benefits for many patients with hematologic malignancies. Nonetheless, subjects with high-risk acute myeloid leukemia or advanced myelodysplasia often relapse, underscoring the need to intensify tumor immunity within this cohort. In preclinical models, allogeneic HSCT followed by vaccination with irradiated tumor cells engineered to secrete GM-CSF generates a potent antitumor effect without exacerbating the toxicities of graft-versus-host disease (GVHD). To test whether this strategy might be similarly active in humans, we conducted a Phase I clinical trial in which high-risk acute myeloid leukemia or myelodysplasia patients were immunized with irradiated, autologous, GM-CSF-secreting tumor cells early after allogeneic, nonmyeloablative HSCT. Despite the administration of a calcineurin inhibitor as prophylaxis against GVHD, vaccination elicited local and systemic reactions that were qualitatively similar to those previously observed in nontransplanted, immunized solid-tumor patients. While the frequencies of acute and chronic GVHD were not increased, 9 of 10 subjects who completed vaccination achieved durable complete remissions, with a median follow-up of 26 months (range 12–43 months). Six long-term responders showed marked decreases in the levels of soluble NKG2D ligands, and 3 demonstrated normalization of cytotoxic lymphocyte NKG2D expression as a function of treatment. Together, these results establish the safety and immunogenicity of irradiated, autologous, GM-CSF-secreting leukemia cell vaccines early after allogeneic HSCT, and raise the possibility that this combinatorial immunotherapy might potentiate graft-versus-leukemia in patients.

Description: Abstract 4471 Background: Patient-Reported Outcome (PRO) measures help clinicians and researchers monitor symptoms, HRQOL, satisfaction, and adherence related to cancer treatment. Symptoms affect HRQOL, and when both are reported frequently and longitudinally, a patient-reported data stream emerges that reflects physiological functioning and complements traditional laboratory and clinician-based assessments. Such data could significantly enhance risk prediction and safety monitoring in patients undergoing HCT. This study evaluates the feasibility of collecting daily and weekly PRO measurements to inform our ability to capture variation in patient experiences over time. Patients and methods: We enrolled 32 patients undergoing planned HCT (10 autologous, 11 myeloablative allogeneic, 11 reduced intensity allogeneic) in a feasibility study of frequent HRQOL and symptom surveillance following HCT. All surveys were administered electronically though patients could opt for pen and paper. PRO measures were derived from the NIH PROMIS and PRO-CTCAE measures, which have not been previously used extensively or at all in HCT patients. All patients completed a 10-question HRQOL measure (PROMIS-Global Health) and a 34-question symptom measure (a pre-selected subset of the 83-question PRO-CTCAE, with 7-day recall period) prior to HCT and weekly until D+100. Auto patients completed a daily 21-question symptom measure (a pre-selected subset of the weekly symptom surveys, with 24-hour recall period) until hospital discharge, and allo patients completed daily symptom surveys until 100 days after stem cell infusion (D+100). Kruskal-Wallis tests were used to compare groups. Median age of the sample was 55 years (range 18–70). 16 patients (50%) were female. Most auto patients had myeloma (N=8, 80%) and most allo patients had acute leukemia (16, 72%); other diagnoses included NHL (4), CML, MDS and AA. Twenty-six (81%) patients were Caucasian, 4 (12.5%) were African American, 2 were other (6.2%). Thirteen (41%) had a high school education or lower. Results: Median daily survey completion percentages prior to hospital discharge for surviving patients were 94% among auto patients, 90% among reduced intensity allo patients and 70% among myeloablative allo patients (p=0.07). Prior to D+100, median daily survey completion percentages were 87% among reduced intensity allo patients and 58% among myeloablative allo patients (p=0.004). Median weekly survey completion percentages prior to hospital discharge were 100% in all cohorts. Prior to D+100, these were 100% in auto and reduced intensity allo cohorts, and 80% among myeloablative allo patients (p=0.002). Daily surveys were completed in a median of 3 minutes, and longer weekly surveys in a median of 4.3 minutes. 93% of respondents were satisfied with survey length and 85% of respondents were satisfied with the electronic self-report system. Median weekly total symptom scores (higher scores indicated greater symptom severity) prior to conditioning were 16 in autos, 12 in myeloablative allos, and 5 in reduced intensity allos (p=0.3) and at D+7 were 23 in autos, 40 in myeloablative allos and 18 in reduced intensity allos (p=0.01). For the physical health subscale of the PROMIS measure (lower scores indicated greater impairment), baseline mean weekly HRQOL scores were 47.7 in autos, 50.8 in myeloablative allos and 50.8 in reduced intensity allos (p=0.9). By D+7, mean HRQOL scores were 37.4 in autos, 37.4 in myeloablative allos and 52.5 in reduced intensity allos (p=0.005). Conclusion: Frequent symptom and HRQOL surveillance is feasible and acceptable to HCT patients, and survey data correlates with toxicity and physiological function after transplant. Compliance rates were lower in myeloablative allo patients, especially for daily surveys, perhaps reflecting the higher burden of critical illness in this population. Future studies may be enhanced by caregiver-reported proxy data. Analyses of weekly symptom and HRQOL surveys beyond D+7, daily surveys, symptom clusters, biologic correlates and individualized profiles are ongoing. Larger studies are warranted to explore and develop risk prediction models based on this technique. Disclosures: No relevant conflicts of interest to declare.

Description: Investigation of the effects of rituximab (anti-CD20) on B-cell-activating factor of the tumor necrosis factor family (BAFF) and B cells would better define the significance of B-cell homeostasis in chronic graft-versus-host disease (cGVHD) pathophysiology. We studied 20 cGVHD patients at a median of 25 months after rituximab treatment when most patients had recovered total B-cell numbers. A total of 55% of patients had stable/improved cGVHD, and total B-cell numbers in these patients were significantly higher compared with rituximab-unresponsive patients. Although total B-cell number did not differ significantly between cGVHD groups before rituximab, there was a proportional increase in B-cell precursors in patients who later had stable/improved cGVHD. After rituximab, BAFF levels increased in all patients. Coincident with B-cell recovery in the stable/improved group, BAFF/B-cell ratios and CD27+ B-cell frequencies decreased significantly. The peripheral B-cell pool in stable/improved cGVHD patients was largely composed of naive IgD+ B cells. By contrast, rituximab-unresponsive cGVHD patients had persistent elevation of BAFF and a predominance of circulating B cells possessing an activated BAFF-RLoCD20Lo cell surface phenotype. Thus, naive B-cell reconstitution and decreased BAFF/B-cell ratios were associated with clinical response after rituximab in cGVHD. Our findings begin to delineate B-cell homeostatic mechanisms important for human immune tolerance.

Description: Key Points NOTCH2 activation confers a marked increase in BCR responsiveness by cGVHD patient B cells that associates with increased BLNK. ATRA increases the IRF4-to-IRF8 ratio and blocks aberrant NOTCH2-BCR activation without affecting cGVHD patient B-cell viability/function.

Description: Approximately 35% to 50% of patients otherwise cured of hematologic malignancies after allogeneic hematopoietic stem cell transplantation will develop the pleomorphic autoimmune-like syndrome known as chronic graft-versus-host disease (cGVHD). Since in 2005, National Institutes of Health (NIH) consensus panels have proposed definitions and classifications of disease to standardize treatment trials. Recently, the first agent was approved by the US Food and Drug Administration for steroid-refractory cGVHD. Despite these advances, most individuals do not achieve durable resolution of disease activity with initial treatment. Moreover, standardized recommendations on how to best implement existing and novel immunomodulatory agents and taper salvage agents are often lacking. Given the potential life-threatening nature of cGVHD, we employ in our practice patient assessment templates at each clinic visit to elucidate known prognostic indicators and red flags. We find NIH scoring templates practical for ongoing assessments of these complex patient cases and determination of when changes in immunosuppressive therapy are warranted. Patients not eligible or suitable for clinical trials have systemic and organ-directed adjunctive treatments crafted in a multidisciplinary clinic. Herein, we review these treatment options and offer a management and monitoring scaffold for representative patients with cGVHD not responding to initial therapy.

Description: Recent studies have compelled further interest in the potential pathological role of B cells in chronic graft-versus-host disease (cGVHD). In patients with cGVHD, B cells are activated and primed for survival via B-cell activating factor and B-cell receptor–associated pathways. Understanding the signaling pathways that drive immune pathology in cGVHD will facilitate the development of new strategies to selectively target aberrantly activated B cells and restore normal B-cell homeostasis after allogeneic stem cell transplantation.

Description: While Notch signaling is being well studied with regard to T cell pathology and graft-versus host disease (GVHD) (Tran IT et al., 2013. J. Clin. Invest.), the role of Notch receptors in the development and activation of B cell subsets in chronic GVHD (cGVHD) genesis remains unknown. We previously identified a subset of Ôpre-germinal centerÕ B cells within the peripheral blood of cGVHD patients that is largely absent in patients without cGVHD. In addition to cell surface characteristics, this extrafollicular B cell subset has potential functional characteristics of marginal zone (MZ)-like B cells, including increased responsiveness to surrogate antigen stimulation. Along with increased proliferative responses to BCR stimulation, B cells from patients with active cGVHD had significantly increased signaling via proximal B cell receptor (BCR) molecules, including Syk and BLNK. In murine models with lymphopenic environments, Notch 2 binds the ligand Delta-like 1 (DLL1/Dll1) and drives maturation of MZ-like B cells. Also, healthy human B cells have increased Notch receptor responsiveness after BCR stimulation. Together previous studies allowed us to hypothesize that a Notch 2 signaling axis underpins B cell hyper-responsiveness in human cGVHD. We found that limiting dose BCR stimulation with surrogate antigen in the presence of Notch ligand over-expressing cells (OP9-DL1) resulted in maintenance of cell surface Notch 2 expression at significantly higher levels on B cells from patients with active cGVHD compared to patients without cGVHD, as assessed by flow cytometry analysis (P 〈 0.01). We also found that in the presence of Notch ligand, B cells from patients with active cGVHD responded to minimal BCR stimulation with surrogate antigen. Using nearly 100x less surrogate antigen than was required to induce proliferation without Notch ligand, cGVHD B cells proliferated to a significantly greater degree than B cells from patients with no cGVHD, as evaluated by Ki-67 staining using flow cytometry (P 〈 0.001 in a two-sided t-test, Figure 1A). Likewise, concomitant BCR- Notch activation of active cGVHD patient B cells was associated with significantly increased B-cell size compared to patients without disease (P 〈 0.01). BLNK expression in active cGVHD B cells was also maintained at higher levels under these conditions, suggesting a mechanistic link between the BCR and Notch pathways in cGVHD. Strikingly, targeting Notch 2 with an antagonistic monoclonal antibody (mAb) (Wu Y et al., 2010. Nature; kindly provided by Genentech, Inc.) completely abrogated the BCR-Notch axis hyper-responsiveness of active cGVHD B cells without affecting B-cell survival (P 〈 0.001, Figure 1B). In this in vitro system, using nanoString Technologies¨ gene profiling, we found that two, well-defined effector genes downstream of Notch signaling were significantly decreased in active cGVHD B cells after exposure to the anti-Notch 2 mAb (P = 0.0006 and P 〈 0.02, respectively, compared to isotype control mAb). Also consistent with a Notch 2-driven activation pathway, the expression of multiple genes involved in homeostasis/cell cycle regulation were altered in active cGVHD B cells exposed to anti-Notch 2 mAb (P 〈 0.01). Finally, ongoing in vivo analyses of the Notch 2 mAb in a pre-clinical mouse model of cGVHD indicates that Notch 2 blockade does not negatively impact early B cell recovery following bone marrow transplantation. These results may reveal that therapeutic targeting of Notch 2 alone would be sufficient to quell B cell hyper-responsiveness in active cGVHD, while preserving protective humoral immunity. In summary, our data suggest a working model in which Notch-mediated aberrant B cell maturation contributes to cGVHD pathophysiology. In this model, Notch 2 stimulation along with a combination of complex B-cell selection and tolerance mechanisms afford production of pathological B cells. Given that Notch 2 is a cell surface receptor expressed by activated B cell subsets of pathological relevance, and Notch 2 blockade has been shown to be well-tolerated in pre-clinical models, our findings support an important clinical opportunity: Targeting Notch 2 on B cells in active cGVHD represents a viable future therapeutic strategy worthy of continued investigation. This work was supported by National Institutes of Health grant 5K08-HL107756, and a Translational Research Program grant from the Leukemia & Lymphoma Society. Figure 1. Figure 1. Disclosures Rizzieri: Teva: Other: ad board, Speakers Bureau; Celgene: Other: ad board, Speakers Bureau.

Description: Abstract 216 High BAFF levels correlate with the presence of activated B cells in patients who develop cGVHD after hematopoietic stem cell transplantation. B cell reconstitution in these patients occurs under constant exposure to alloantigens, and we previously showed that B Cell Receptor (BCR) stimulated CD27+ B cells in cGVHD patients are activated, capable of spontaneous IgG production without requirement of further BCR or second signal stimulation. B cell survival is dependent on both BCR and BAFF signaling. BAFF is known to attenuate B cell apoptosis by counteracting pro-apoptotic Bcl-2-interacting mediator of cell death (Bim) protein, but it is not known whether BAFF can provide survival signals to activated B cells in cGVHD. Therefore, we examined the survival rates of B cells in cGVHD. CD19+ B cells were purified (〉95% purity) by magnetic bead sorting. Rates of death were measured by flow cytometry staining with propidium iodide and Annexin V of unmanipulated B cells cultured without addition of cytokines over time. After 24 and 48 hours, the frequency of cells undergoing apoptosis (Annexin V+) B cells was significantly lower in samples from patients with cGVHD compared to those without cGVHD and to healthy individuals (one-way ANOVA p=0.007, Figure 1). In addition to Annexin V + cells, total death rates as measured by propidium iodide of unmanipulated purified CD27+ B cells were lower in cGVHD compared to healthy individuals. Importantly, we found that the frequency of propidium iodide stained CD27+ B cells did not increase 24 hours ex vivo if BAFF was added in cGVHD, but not in healthy, CD27+ B cells, consistent with BAFF mediated survival in these cells (Table 1). Further examination of CD27+ B cell subsets ex vivo was performed to determine if subpopulations we previously identified to uniquely circulate in cGVHD patients were more viable. The morphology of pre-germinal center (GC) CD27+IgD+CD38Hi cells and the antigen-inexperienced, most recent bone marrow emigrants, transitional CD27NegIgD+CD38Hi cells was compared. Unlike transitional cells, the pre-GC cells were enlarged, adherent and viable, consistent with an activated state. While the Bim isoforms are upregulated after BCR activation or by apoptosis-inducing drugs, Bim is degraded in response to BAFF signaling. Since steroids (previously shown to increase Bim in lymphocytes) are the only standard therapy for cGVHD and unfortunately often clinically ineffective, we first performed in vitro assays with dexamethasone and BAFF. Ninety-five percent of healthy CD19+ purified B cells were induced to apoptose (94.9% Annexin V+) with dexamathasone at 24 hours. Addition of BAFF blocked dexamethasone-induced apoptosis to the baseline levels found in untreated B cells (27.3% Annexin V+). Next, to determine whether in vivo BAFF survival signaling of B cells occurred in cGVHD patients, we examined protein levels of Bim by immunoblotting cell lysates from freshly purified unmanipulated CD19+ cells. B cells from healthy individuals did not generate the long form of Bim (BimL), likely due to the lack of BCR activation in these B cells, while 80% of patients with inactive cGVHD had increased BimL. In contrast, 75% of B cells from patients with active cGVHD lacked BimL. Thus, loss of BimL in cGVHD is likely BAFF-driven and may contribute to improved survival of potentially allo- or autoreactive B cells in cGVHD. Potential upstream activators of Bim degradation and inhibition such as mitogen-activated protein kinase/ERK activating kinase (MEK) or NFkB, respectively, are currently being investigated. In addition to characterizing a potential therapeutic role for MEK and NFkB inhibitors, since Bim has been shown to be increased with proteasome inhibitor and BH3 mimetic induced cell death, these findings begin to delineate immunologic rationale for the therapeutic use of these agents to target B cells in cGVHD. Taken together, our data suggest that activated and potentially pathologic B cells in cGVHD utilize distinct survival pathways. Thus, activated B cells represent novel therapeutic targets in cGVHD.Table 1.CD27+ B Cell Source% PI at Time 0 (mean +/-SD)% PI at 24 Hours (mean +/-SD)% PI at 48 Hours (mean +/-SD)Healthy12.8 +/- 10.7 (n=3)34.9 +/- 8.7 (n=2)34.3 +/- 9.6 (n=3)Healthy +BAFF33.1 +/- 9.1 (n=2)42.2 +/- 11.4 (n=2)Yes cGVHD13.5 +/- 5.9 (n=3)20.5 +/- 3.1 (n=2)34.8 +/- 4.0 (n=3)Yes cGVHD +BAFF14.8 +/- 5.4 (n=2)29.7 +/- 2.7 (n=2) Disclosures: Off Label Use: NFkB inhibitor, MEK inhibitor, BH3 mimetic, bortezomib for chronic graft versus host disease.

Description: Background: A major advance in allogeneic stem cell transplant (SCT) has been the establishment of effective and less toxic reduced intensity regimens as alternative therapeutic approaches for patients with certain hematologic malignancies. The focus of these approaches has been to reduce treatment related mortality (TRM) while providing sufficient host immunosuppression to permit the achievement of complete donor chimerism that leads to a graft versus tumor effect, resulting in long-term disease control and cure. In this study we report overall survival (OS), relapse rate (RR), and non-relapse mortality (NRM) in patients receiving reduced intensity conditioning (RIC) with a 48 hour continuous infusion of busulfan with fludarabine followed by methotrexate (MTX) alone or with lympho-depleting anti-CD52 monoclonal antibody (mAb), alemtuzumab, or T cell depleting immunoglobulins, anti-thymocyte globulin (ATG) given as graft versus host disease (GVHD) prophylaxis. Methods: Patients with histologically confirmed hematologic malignancies, who were over the age of 55, or otherwise ineligible for more intensive busulfan-based therapy were enrolled on a Phase II protocol, LCCC 0306, at the University of North Carolina. All patients received IV fludarabine 30 mg/m2/day on days-7 through-3; busulfan 6.4 mg/kg by continuous infusion over 48 hours on days-6 through-5 and tacrolimus from day-1 plus either MTX alone, ATG or alemtuzumab alone, or a combination of these agents depending on disease risk and donor status. Results: 71 patients were enrolled. The median age was 58 (range 28 - 69). 58% were men and 42% women. 37 patients received HLA-matched related donor (MRD) stem cell grafts, and 34 patients received either matched unrelated donor (MUD) or HLA-mismatched grafts. The median HCT-Comorbidity Index (CI) score was 3 (range 0 - 8, HCT-CI score: 0 = 12 pts, 1 - 2 = 22 pts, ⪴ 3 = 36 pts). The majority of pts were intermediate risk by Disease Risk Index (DRI) score (9 low, 40 int, 19 high, 1 very high, 2 undetermined). The DRI low/int group had an estimated 18 month OS of 65% (CI 0.50 - 0.77), and DRI high/very high OS was 35% (CI 0.16 - 0.55). Estimated median survival time based on DRI for low/int was 1674 days (CI 646 - 2920) versus 375.5 days (CI 136 - 1018) (p = 0.003) for DRI high/very high pts. OS at 18 months in the MTX alone arm (n = 20) was 70% (CI 0.45 - 0.83), ATG (+/- MTX, n = 27) was 52% (CI 0.32 - 0.69), and alemtuzumab (+/- MTX, n = 24) was 46% (CI 0.26 - 0.64); (p = 0.17). The 18 month relapse rate for the MTX alone arm was 50% (CI 0.27 - 0.69), 41% in ATG (CI 0.37 - 0.76), and 41% in alemtuzumab (CI 0.36 - 0.77); (p = 0.85). The 18 month NRM in the MTX alone arm was 7% (CI 0.61 - 0.99), alemtuzumab (+/- MTX) was 32% (CI 0.42 - 0.85), and ATG (+/- MTX) was 27% (CI 0.52 - 0.86); (p = 0.05). Much of the increased NRM in the alemtuzumab and ATG arms was attributable to increased rates of fatal infectious complications: 1 in the MTX arm, 3 in the ATG arm and 8 in the alemtuzumab arm. No patients receiving MTX alone, 3 receiving alemtuzumab and 6 receiving ATG developed grade 3-4 acute GVHD. 5 of the 20 surviving patients have extensive chronic GVHD. Conclusion: Continuous infusion, reduced dose, busulfan and fludarabine can be safely administered to patients who are ineligible for myeloablative conditioning either because of age or comorbidities. The use of MTX alone with this regimen results in a median survival of over 4 years. Patients with high DRI or who received treatment with alemtuzumab or ATG had higher NRM and poorer OS irrespective of donor status. Disclosures Wood: Best Doctors: Consultancy; Inform Genomics: Consultancy.

Description: Abstract 1167 Poster Board I-189 Alloantibody responses to HY antigens have been well described in patients with chronic graft versus host disease (cGVHD), but specific B cell reactivity to autosomal minor histocompatibility antigens has not been described. Recently, genetic polymorphisms in hemophiliacs treated with recombinant factor VIII (F8) have been associated with an increased risk of developing F8 antibody inhibitors (Viel KR NEJM 2009), highlighting the possibility that F8 is an alloantigen target in cGVHD. From a patient with cGVHD manifesting with a clinically significant antibody inhibitor to F8, we identified a novel non-synonymous mutation resulting in a glycine (G) to arginine (R) substitution at codon 2076 (G2076R) in the patient/recipient's F8 gene not present in the donor. To visualize the G to R substitution, homology modeling was performed using the SWISS-MODEL server. The G2076R substitution was found to alter the surface of the C1 domain. The homology model is expected to be particularly reliable in this case because the side chain is on the surface, making conformational changes in peptide backbone structure unlikely. Enzyme-Linked Immunosorbent Assays (ELISA) using peptides synthesized with or without the G2076R mutation were employed to measure direct IgG binding. The donor had pre-existing alloantibodies to the mutated peptide, while the recipient, prior to hematopoietic stem cell transplantation (HSCT), did not have a pre-existing allo-IgG response to the mutated peptide. The recipient had no IgG response to native or mutated peptide or whole, unmutated F8 before HSCT or at 1,2,3,6 or 9 months after HSCT, but a specific IgG response to the G2076R containing peptide developed at 11 months post-HSCT just prior to clinically evident coagulopathy. Negative control amino acid substitutions (G2076A) did not result in IgG binding and negative control G2076R peptide (oriented on the ELISA so that R was not available for IgG binding) also did not result in IgG binding. An IgG response to whole, unmutated recombinant F8 protein as measured by ELISA and Western Blot was detected by 11 months post-HSCT. The recipient was treated with a 4 week course of rituximab and corticosteroids and had a complete clinical response with normalization of coagulation parameters. Alloreactivity in the recipient was no longer detectable 6 months later, although IgG responses to unmutated F8 protein remained, suggesting that rituximab resulted in removal of the alloantibody producing B cell. Persistence of IgG reactive to unmutated whole F8 also suggests epitope spreading occurred, resulting in the development of antibodies to other F8 epitopes that contribute to the autoreactivity found in this patient. Thus, we demonstrate ‘proof of principal’ that B cell responses to autosomal minor histocompatibility antigens occur in human cGVHD. Disclosures: Off Label Use: rituximab use in chronic graft versus host disease.