ALBERT

Description: Chronic Graft-versus-Host Disease (GVHD) is a major limitation to successful allogeneic bone marrow transplantation (BMT). Lipopolysaccharide (LPS), a cell-wall component of gram negative bacteria that signals through Toll-like receptor 4 (TLR4), is an established contributor to acute GVHD. Our lab has previously shown that splenocytes from mice with acute GVHD were hyper-responsive to CpG, unmethylated DNA with C-G repeats from bacteria and viruses, which induce rapid responses by dendritic cells, B cells, and monocytes through TLR9. As part of Children’s Oncology Group phase III randomized trial ASCT0031 evaluating the efficacy of hydroxychloroquine in the treatment of newly diagnosed chronic GVHD, peripheral blood was drawn for immune phenotype and functional studies in patients and control (no GVHD, 80–120 days post allogeneic transplant). The non-BMT group were healthy adult volunteers. After Ficoll isolation, lymphocytes were cultured with or without synthetic human immunostimulatory CpG 2006 (6μg/mL) or LPS (1μg/mL) for 48 hours and analyzed by flow cytometry. Cell surface marker expression was evaluated as fold difference between stimulated and unstimulated cells. In some samples, B cells were purified by negative selection to determine proliferative response and/or for the quantitation of TLR9 mRNA. Chronic GVHD samples had a 3.7 +/− 1.2 fold (n=11) increase in B cell CD40 mean channel fluorescence after CpG stimulation which was significantly greater than in the control group at 1.0 +/− 0.6 fold (n=3, Mann-Whitney U test p=0.02) and the non-BMT group at 1.5 +/− 0.9 fold (n=3, p=0.01). Similarly, chronic GVHD samples had a 9.1 +/− 5.7 fold (n=10) increase in the percentage of B cells expressing CD86 after CpG stimulation which was significantly greater than the control group at 2.1 +/− 2.1 fold (n=4, p=0.008) and the non-BMT group at 2.7 +/− 0.9 fold (n=5, p=0.008). Responses to LPS stimulation or culture without stimulation were not significantly different between the three groups. Purified B cells from GVHD samples (n=3) had enhanced mitogenic response to CpG compared to the control group (n=1), non-BMT group (n=2), and to non-stimulated cells. CpG response appears to be closely correlated with TLR9 mRNA expression (R2=0.82). These findings suggest that CpG responsiveness or TLR9 mRNA expression may potentially be useful as a biomarker for diagnosis or staging of chronic GVHD. Further evaluation on larger numbers of samples is required to confirm these observations.

Description: Chronic graft-vs-host disease (cGVHD) has previously been characterized by elevation of a number of inflammatory markers and autoantibodies. We hypothesized that evaluation of such markers would contribute to a biological classification of new onset cGVHD in children and possibly aid in early diagnosis. The COG study ASCT0031 is a phase III cGVHD therapeutic trial that included the biological evaluation of 52 newly diagnosed cGVHD pts and 34 time-matched post BMT control patients (2 – 18 yrs) with no clinical evidence of cGVHD. Chronic GVHD was divided into an early onset between 3 to 8 mos post BMT (n=37, mean 5.7, range 3 – 8 months ) and late onset ≥9 months post BMT (n=15, mean 14.3, range 9 to 30 months) groups and 34 non-cGVHD controls evaluated at 6 and 12 months. Soluble B-cell activating factor (sBAFF), soluble IL-2 receptor alpha (sIL-2Ralpha), transforming growth factor-beta (TGF-beta), interferon-alpha (IFN-alpha), monocyte chemoattractant protein-1 (MCP-1), IL-6, anti-nuclear antibody (ANA), anti-dsDNA antibody, anti-mitochondrial antibody and anti-cardiolipin antibody were evaluated by sandwich ELISA. We found an increased level of sBAFF in cGVHD patients with both early (7.2± 5.3 vs. 3.7 ± 2.5 ng/mL, p = 0.008, t-test) and late (3.2 ± 2.7 vs. 1.5 ± 0.8 ng/mL, p = 0.04) onset cGVHD compared with controls. Although the anti-dsDNA concentrations were considered to be in the normal range, they were significantly higher in both early (2.4 ± 2.5 vs. 0.1±0.3 IU/mL; p 〈 0.0001) and late (4.1 ± 3.1 vs. 0.2 ± 0.3 IU/mL; p 〈 0.0001) cGVHD compared to non-cGVHD controls. We found that early onset cGVHD compared to patients without cGVHD at 6 months had an elevation in sIL-2Ralpha (10.1±5.4 vs. 7.0±3.4 pg/mL; p 〈 0.0001) but not in late onset cGVHD. ANA, TGF-beta, IFNalpha, IL-6, MCP-1, and anti-cardiolipin antibodies were not associated with new onset cGVHD. Acute GVHD, a major factor for the development of cGVHD, did not correlate with any of the three markers. We found that the ANA (2.3±2.1 vs. 0.9±0.7 absorbance ratio, p = 0.0004) and anti-dsDNA (anti-dsDNA; 3.8±2.9 vs. 2.4±2.5 IU/mL, p = 0.04) were significantly elevated in de novo onset cGVHD patients compared to those with previous acute GVHD. Soluble BAFF and sIL-2Ralpha are associated with Th1 responses and B cell activation/expansion, whereas anti-dsDNA is associated with specific B cell responses. The elevation of sBAFF and sIL-2Ralpha are consistent with both high levels of T cell and B cell activation as part of early cGVHD. The association of later onset cGVHD with only sBAFF and anti-dsDNA suggests that B cell activation is more predominant in that setting and is consistent with our previous observation of an association of cGVHD with activated Toll-like receptor 9 expressing B cells. These findings suggest early and late cGVHD may be classified by different patterns of T and B cell activation. Early onset cGVHD (3–8 mo. Post BMT) Late Onset cGVHD (〉=9 mo. post BMT) Marker cGVHD (N=37) No cGVHD at 6 mo. (N=26) p value cGVHD (N=14) No cGVHD at 12 mo. (N=15) p value Anti-dsDNA (IU/mL) 2.4±2.5 0.1±0.3

Description: Background: NK alloreactivity based on KIR (Killer Immunoglobulin-like Receptor) expression by natural killer cells has been reported to decrease the risk of leukemia relapse after HLA-haploidentical stem cell transplantation (SCT). The use of donors with high KIR B-content score has been correlated with a decreased risk of relapse. Objective: We examined the outcome of leukemia relapse following CD34-positive selected T-cell depleted haploidentical donor peripheral blood SCT followed by donor lymphocyte infusion with methotrexate prophylaxis. Methods: Between 2009-2015, twenty patients underwent haploidentical donor CD34-selected, T cell-depleted peripheral blood SCT on an IRB-approved protocol. Nine patients had acute lymphoblastic leukemia (ALL), and eleven had myeloid leukemia (10 AML, 1 JMML). Among ALL patients, remission status was CR1 in 5, with either induction failure or MRD+ after consolidation, 3 were in CR2 and 1 was CR3. Four patients had T-cell disease. Among AML patients, 5 were in CR1, one had refractory disease and 5 were CR2 or beyond. Ligand-ligand NK alloreactivity based on HLA typing was available for all patients. Receptor-ligand NK alloreactivity based on immunophenotyping of KIR was available in 16 patients and KIR B-content score was available in twelve patients. Patients were considered to have receptor-ligand NK alloreactivity if immunophenotyping of the donor had 〉5 (cells/uL) expressing KIR2DL1 (recognizes C2 group) or KIR2DL1/2DL13 (recognizes C1 group). Patients with alloreactive KIR only recognizing BW4 were not considered alloreactive due to predicted weak alloreactivity. All patients received a uniform conditioning regimen including TBI 1200cGy, thiotepa 10mg/kg, fludarabine 200mg/m2, and rabbit-anti-thymocyte globulin (rATG) 6mg/kg, with no post-transplant immunosuppression. All patients received a planned donor lymphocyte infusion (DLI) 3-5 x 104 CD3+ cells/kg between days 30- 42 after transplant and received weekly IV methotrexate prophylaxis following DLI. The incidence of relapse and GVHD was evaluated. Results: Based on a ligand-ligand model of NK alloreactivity, only 4/20 patients had NK alloreactivity predicted. Seven patients expressed all 3 ligands and blocked all possible NK alloreactivity. Median follow-up was 306 days (range 45 days-5 years). Four of 20 patients relapsed (1/4 with predicted NK alloreactivity and 3/16 without). Median time to relapse was 110 days (range 124-455 days). NK alloreactivity based on receptor-ligand as defined above was present in 5/16 patients and relapse occurred in 2/5 with and 1/11 without. Donor KIR B-content was 〉=2 in 4 patients and 0-1 in 8 patients. Relapse occurred in 1/4 with 2 and 2/8 with 0-1. No donors had KIR B-content score of 3-4. The incidence of grade II-IV acute graft-versus-host disease after prophylactic DLI was 25%, with 2 grade II, 3 grade III and no grade IV GVHD. Three patients developed cGVHD, two mild and one severe by NIH consensus criteria. Conclusion: An approach using a DLI followed by methotrexate prophylaxis after a CD34-selected, T-cell depleted SCT for pediatric leukemia diminishes the effect of NK alloreactivity and KIR B-content. Disclosures No relevant conflicts of interest to declare.

Description: Hydroxychloroquine (HCQ), a lysosomotropic amine, is an immunosuppressive agent presently being evaluated in bone marrow transplant patients to treat graft-versus-host disease. While its immunosuppressive properties have been attributed primarily to its ability to interfere with antigen processing, recent reports demonstrate HCQ also blocks T-cell activation in vitro. To more precisely define the T-cell inhibitory effects of HCQ, the authors evaluated T-cell antigen receptor (TCR) signaling events in a T-cell line pretreated with HCQ. In a concentration-dependent manner, HCQ inhibited anti-TCR–induced up-regulation of CD69 expression, a distal TCR signaling event. Proximal TCR signals, including inductive protein tyrosine phosphorylation, tyrosine phosphorylation of phospholipase C γ1, and total inositol phosphate production, were unaffected by HCQ. Strikingly, anti-TCR-crosslinking–induced calcium mobilization was significantly inhibited by HCQ, particularly at the highest concentrations tested (100 μmol/L) in both T-cell lines and primary T cells. HCQ, in a dose-dependent fashion, also reduced a B-cell antigen receptor calcium signal, indicating this effect may be a general property of HCQ. Inhibition of the calcium signal correlated directly with a reduction in the size of thapsigargin-sensitive intracellular calcium stores in HCQ-treated cells. Together, these findings suggest that disruption of TCR-crosslinking–dependent calcium signaling provides an additional mechanism to explain the immunomodulatory properties of HCQ.

Description: Hydroxychloroquine (HCQ), a lysosomotropic amine, is an immunosuppressive agent presently being evaluated in bone marrow transplant patients to treat graft-versus-host disease. While its immunosuppressive properties have been attributed primarily to its ability to interfere with antigen processing, recent reports demonstrate HCQ also blocks T-cell activation in vitro. To more precisely define the T-cell inhibitory effects of HCQ, the authors evaluated T-cell antigen receptor (TCR) signaling events in a T-cell line pretreated with HCQ. In a concentration-dependent manner, HCQ inhibited anti-TCR–induced up-regulation of CD69 expression, a distal TCR signaling event. Proximal TCR signals, including inductive protein tyrosine phosphorylation, tyrosine phosphorylation of phospholipase C γ1, and total inositol phosphate production, were unaffected by HCQ. Strikingly, anti-TCR-crosslinking–induced calcium mobilization was significantly inhibited by HCQ, particularly at the highest concentrations tested (100 μmol/L) in both T-cell lines and primary T cells. HCQ, in a dose-dependent fashion, also reduced a B-cell antigen receptor calcium signal, indicating this effect may be a general property of HCQ. Inhibition of the calcium signal correlated directly with a reduction in the size of thapsigargin-sensitive intracellular calcium stores in HCQ-treated cells. Together, these findings suggest that disruption of TCR-crosslinking–dependent calcium signaling provides an additional mechanism to explain the immunomodulatory properties of HCQ.

Description: Because of the variable and often protean manifestations of chronic GVHD, its diagnosis is difficult and based solely on clinical features. As part of a Children’s Oncology Group multi-institutional clinical trial (ASCT0031 - a randomized study of hydroxychloroquine plus standard therapy for children with newly-diagnosed extensive chronic GVHD), we prospectively evaluated laboratory markers that have been previously reported in single-institution studies to be associated with chronic GVHD. Fifty-four patients were enrolled: median age 12 (1–21) yrs: 37M/17F. The diagnosis of extensive chronic GVHD was made at a median of 210 days (range 82–914) following transplant. Eighteen of 45(40%) patients had eosinophilia (absolute eosinophil count (AEC)≥500/μL). Twelve of 43 (28%) patients had an anti-nuclear antibody (ANA) titer ≥1:80 and 9 of these (21%) had a titer ≥1:160. Finally, 11 of 48 (23%) patients had an elevated IgG level. At presentation, 67% of patients had one or more of eosinophilia, ANA≥1:80, and hyper-IgG. A Th-2 predominance is suggested by eosinophilia (↑ IL-5) and hyper-IgG and positive ANA (↑ IL-4). Of note, of 9 patients with sclerodermatous chronic GVHD, 6 had hyper-IgG and 5 had eosinophilia. These results support a Th2 predominance in a major subset of children presenting with chronic GVHD. These results emphasize the importance of including evaluation of eosinophil count, ANA, and IgG level as tests supportive of the diagnosis of chronic GVHD in children.

Description: There is no standard therapy for steroid-refractory chronic graft-versus-host disease (GVHD). This problem is particularly daunting in children with chronic GVHD, whereby the effects of the disease and its treatment may impair normal growth and development. Children are also particularly vulnerable to failure and/or toxicity of therapy; for example, joint contractures or joint damage may result in life-long disability. The Pediatric Blood and Marrow Transplant Consortium performed a phase 2 trial of pentostatin for steroid-refractory chronic GVHD in 51 children (median age, 9.8 years) from 24 institutions. Overall response was 53% (95% confidence interval, 40%-64%), with a response of 59% (95% confidence interval, 42%-75%) in sclerosis. Thirteen subjects (25%) had toxicity requiring them to stop pentostatin. The drug had a significant steroid-sparing effect in those that responded. A trend was also observed toward increased survival at 3 years in responders versus nonresponders (69% vs 50%; P = .06). The intravenous administration of the drug ensures compliance in a patient group in which oral therapy is difficult to monitor. Pentostatin has activity in refractory chronic GVHD in children, and future studies, including treatment of children newly diagnosed with high-risk chronic GVHD, are warranted. The trial was registered at www.Clinicaltrials.gov as #NCT00144430.

Description: Numerous chronic graft-versus-host disease (cGVHD) biomarkers have been identified in limited, single-institution studies without validation. We hypothesized that plasma-derived biomarkers could diagnose, classify, and evaluate response in children with cGVHD. We performed a concomitant analysis of a number of known and predicted peripheral blood cGVHD biomarkers from a Children's Oncology Group (COG) phase 3 cGVHD therapeutic trial. A total of 52 newly diagnosed patients with extensive cGVHD were compared for time of onset after blood and marrow transplantation (BMT) (early, 3-8 months; late, ≥ 9 months) with 28 time-matched controls with no cGVHD (early, 6 months after BMT; late, 12 months after BMT). Soluble B-cell activation factor (sBAFF), anti-dsDNA antibody, soluble IL-2 receptor alpha (sIL-2Rα), and soluble CD13 (sCD13) were elevated in patients with early-onset cGVHD compared with controls. sBAFF and anti-dsDNA were elevated in patients with late-onset cGVHD. Some of the biomarkers correlated with specific organ involvement and with therapeutic response. These 4 biomarkers had high specificity with higher sensitivity in combination. Changes in biomarker concentrations with immune reconstitution after transplantation significantly affected interpretation of results. The identified biomarkers have the potential for improved classification, early response evaluation, and direction of cGVHD treatment, but require validation in larger studies. This study is registered at www.cancer.gov/clinicaltrials as no. COG-ASCT0031.