Abstract: Interleukin-7 (IL-7), a cytokine produced by stromal cells, is required for thymic development and peripheral homeostasis of most major subsets of T cells. We examined whether regulatory T (Treg) cells also required the IL-7 pathway by analyzing IL-7Rα−/− mice. We observed a striking reduction in cells with the Treg surface phenotype (CD4, CD25, GITR (glucocorticoid-induced tumor necrosis factor [TNF]-like receptor), CD45RB, CD62L, CD103) or intracellular markers (cytotoxic T-lymphocyte–associated antigen-4, CTLA-4, and forkhead box transcription factor 3, Foxp3). Foxp3 transcripts were virtually absent in IL-7Rα−/− lymphoid tissues, and no Treg cell suppressive activity could be detected. There are 2 known ligands for IL-7Rα: IL-7 itself and thymic stromal lymphopoietin (TSLP). Surprisingly, mice deficient in IL-7 or the other chain of the TSLP receptor (TSLPR) developed relatively normal numbers of Treg cells. Combined deletion of IL-7 and TSLP receptor greatly reduced Treg cell development in the thymus but was not required for survival of mature peripheral Treg cells. We conclude that Treg cells, like other T cells, require signals from the IL-7 receptor, but unlike other T cells, do not require IL-7 itself because of at least partially overlapping actions of IL-7 and TSLP for development of Treg cells.

Abstract: Background: The current standard of care for eligible newly diagnosed multiple myeloma (MM) patients is induction therapy with novel agents followed by high dose chemotherapy and autologous stem cell transplant (ASCT). The International Myeloma Working Group proposed the Revised International Staging System (R-ISS) based on the presence of adverse chromosomal abnormalities (CA) detected by FISH (t(4;14), t(14;16) or del17p), in combination with ISS and LDH at diagnosis. However, there are a limited number of studies that have validated this risk model in the transplant and novel agents setting. Aims: To determine whether R-ISS is an appropriate risk-model for estimating overall survival (OS) and progression free survival (PFS) for transplant-eligible MM patients. Patients and Methods: We retrospectively studied a cohort of 519 MM patients who received novel drugs in the induction and subsequently underwent ASCT at Mayo Clinic Arizona from 2005 to 2014. In all, 95 patients met the inclusion criteria: comprising complete data at diagnosis (ISS, serum LDH level, and CA by FISH). The primary endpoint was OS from SCT and the secondary end point was PFS from ASCT. R-ISS groups were defined as described by Palumbo et al. J Clin Oncol. 2015; 33(26):2863-2869. Results: There were 50 (52.6%) men and 45 (47.4%) women who underwent ASCT in this period, with a median age at the time of transplant of 66-years-old (range, 36-78). There were 27 patients (28.4%) with high-risk CA: 12 patients (12.6%) with del17p; 11 patients (11.6%) with t(4;14); and 6 (6%) with t(14;16). In addition, 8 patients (8.5%) had high LDH levels and 9 patients (9.5%) presented with renal impairment at diagnosis. The patients were staged at diagnosis according to the three R-ISS groups: 44 patients (46.3%) had stage I, 26 (27.4%) had stage II, and 25 (26.3%) had stage III. CyBorD was the preferred induction regimen which was received by 42 patients (44%). There were 14 patients (15%) who received at least 2 lines of induction. All patients were in at least partial response (PR) at the moment of transplant: 32 in complete response (CR), 32 in very good partial response (VGPR) and the remaining 31 in PR. The response achieved at day +100 after ASCT improved, with 54 patients (57%) in CR, and only 18 patients (19%) in PR. After a median follow-up of 61 months (range, 14-135), median OS from SCT was 108 months (95% CI: 85 - 132 months) and the median PFS was 45.4 months (95% CI: 31.1 - 53.8 months) in the whole series.MM patients with R-ISS III had a significantly shorter median OS compared to patients with R-ISS II or R-ISS I (32.1 months vs. 94.7 months vs. not reached, respectively, P 〈 0.0001) (Figure). No statistically significant differences in baseline characteristics were identified among these groups to explain the differences in OS observed. PFS among these groups was not statistically significant, only showing a trend towards shorter PFS in R-ISS III compared with either R-ISS I or II: median 22.1 months vs. 35.7 months, respectively, (P=0.2). Renal impairment at diagnosis, IgA subtype, ≥ 2 lines of induction treatment, and less than CR achieved at day +100 after ASCT were also associated with significantly inferior OS. Multivariate analysis selected R-ISS as an independent predictor for OS (HR: 2.3, 95% CI: 1.1-4.8; P=0.03), as well as ≥ 2 lines before ASCT. CR at day +100 after ASCT was the most important independent factor for predicting PFS (HR: 0.4; 95% CI: 0.2-0.6; P 〈 0.001). Conclusion: R-ISS assessed at diagnosis was an independent predictor for OS after ASCT in our series, with median OS for the different R-ISS groups comparable to those reported by Palumbo et al. in their subgroup of younger patients. Thus, this study lends further support for the R-ISS as a reliable prognostic tool for estimating OS in transplant-eligible MM patients. In addition, new treatment approaches are needed for the high-risk patients (R-ISS III) with a median OS of 2.5 years. Figure Figure. Disclosures Reeder: Millennium: Research Funding; BMS: Research Funding; Celgene: Research Funding; Novartis: Research Funding. Mikhael:Abbvie: Research Funding; Onyx: Research Funding; Sanofi: Research Funding; Celgene: Research Funding. Bergsagel:Amgen, BMS, Novartis, Incyte: Consultancy; Novartis: Research Funding. Stewart:celgene: Consultancy. Fonseca:Janssen: Consultancy; Celgene: Consultancy; Sanofi: Consultancy; Novartis: Consultancy; Bayer: Consultancy; AMGEN: Consultancy; AMGEN: Consultancy; AMGEN: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; AMGEN: Consultancy; Millennium, a Takeda Company: Consultancy; Janssen: Consultancy; Sanofi: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Novartis: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Millennium, a Takeda Company: Consultancy; Millennium, a Takeda Company: Consultancy; Bayer: Consultancy; Celgene: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; BMS: Consultancy; Millennium, a Takeda Company: Consultancy; BMS: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms.

Abstract: Abstract 2208 Diagnosis of von Willebrand disease (VWD) currently relies on two assays of von Willebrand factor (VWF), the VWF antigen ELISA (VWF:Ag) and the VWF ristocetin cofactor activity assay (VWF:RCo). The latter exploits the capacity of ristocetin to induce VWF – platelet interactions as a measure of VWF function. Ristocetin, however, is a non-physiologic agonist as shear stress is the physiologic stimulus inducing VWF to bind platelets in vivo. Recently we have reported that the VWF:RCo/VWF:Ag ratio is decreased in individuals with an A1 domain polymorphism, D1472H. The lack of bleeding in subjects with this polymorphism suggests D1472H does not create a physiologic defect in VWF – platelet interactions. D1472H is directly adjacent to a known ristocetin-binding area in the VWF A1 region (Leu 1457 – Pro 1471), supporting the hypothesis that D1472H affects the ability of ristocetin to bind VWF. Similarly, a heterozygous sequence change leading to P1467S (located in the same ristocetin binding domain) resulted in an undetectable VWF:RCo but no bleeding symptoms were noted in affected subjects. To further investigate the cause of this observation, we developed a method to study the binding of ristocetin to VWF directly. Maleic anhydride microtiter plates were used to capture ristocetin via its amine groups. A VWF source, either plasma or recombinant VWF (rVWF), was then added, wells washed, and VWF binding detected using anti-VWF antibodies. Both plasma and rVWF bound to the captured ristocetin similarly with ristocetin plating concentrations ranging from 0.01 to 1 mg/ml. Specificity of ristocetin dependent VWF binding was confirmed, as preincubation of ristocetin with rVWF decreased binding of rVWF to immobilized solid-phase ristocetin. No detectable binding was present for a full length rVWF construct with the entire A1 loop deleted (del 1242–1478) or a construct missing part of the A1 loop (del 1392–1402). VWF binding to ristocetin was inhibited by both monoclonal and polyclonal antibodies directed against the VWF A1 loop. VWF A1 loop constructs with the A1 domain polymorphisms D1472H and P1467S showed decreased binding to ristocetin when compared to a wild-type A1 loop construct. Wild-type A1 loop binding to ristocetin in our assay was 0.98 while 1472H A1 loop binding was reduced at 0.77 (p 〈 0.001 compared to wild-type) and 1467S A1 loop binding was 0.45 (p 〈 0.001 compared to wild-type). No difference was seen when full length VWF constructs with these polymorphisms were studied, perhaps due to other ristocetin recognition sequences present in VWF as reported by Scott and colleagues (JBC 1991). Binding of VWF to vancomycin, a glycopeptide antibiotic similar to ristocetin that does not induce platelet agglutination, was normal. The ristocetin-specific interaction with VWF may require a specific structural conformation of the A1 loop. These results suggest that VWF A1 domain polymorphisms, or the disruption of the entire A1 loop, have the ability to interfere with VWF – ristocetin interactions independent from VWF – platelet interactions. Clinical assays of VWF function that depend on ristocetin may therefore provide misleading results for subjects with VWF polymorphisms affecting ristocetin binding. Disclosures: Montgomery: GTI Diagnostics, Inc: Consultancy.

Abstract: Von Willebrand disease is a bleeding disorder with reduced or abnormal function of VWF. While VWF has at least three functions (binding to FVIII, platelet GPIb, and collagen), the function of VWF has been quantified primarily using techniques in which ristocetin promotes the binding of VWF to platelet GPIb. It is recognized that the physiologic agonist for VWF-platelet interactions is shear but ristocetin facilitates a change in VWF structure to induce binding to platelet GPIb. For most individuals, this assessment of VWF function is valid, but over the past several years our group, the Zimmerman Program for the Molecular and Clinical Biology of VWD, has identified “polymorphisms” that do not affect VWF function but interfere with ristocetin binding to VWF in those laboratory tests of VWF function that utilize ristocetin. The two “polymorphisms” are P1467S and D1472H. P1467S is a rare sequence variant not seen in 12,900 alleles in the 1000-genome project. In contrast, the D1472H is a polymorphism seen in 63% of African Americans and in 17% of Caucasians. Since these two sequence variations are physically located in close proximity just C-terminal from the A1 loop, we initiated a systematic study of the adjacent N- and C-terminal sides of the A1 domain by alanine scanning mutagenesis. We concentrated on the sequence between p.C1237 – p.C1272 and p.C1458 – p.K1491 and replaced each amino acid with an alanine. These sequences were then transferred into a full-length expression construct. The individual altered VWF proteins were studied by GPIb-binding in two solid phase assays. The first assay, VWF:RCo, is an ELISA using an antibody-captured recombinant GPIb-alpha in which ristocetin-induced bound VWF is detected using an anti-VWF monoclonal antibody. The second assay, VWF:IbCo, is a similar assay, but the expressed GPIb contained two mutations, D235Y and M239V, that confer spontaneous binding to VWF without the addition of ristocetin as our laboratory has previously published. Where possible all assays were done at similar concentrations of VWF antigen and expressed as a ratio of VWF:IbCo/VWF:RCo compared to VWF:Ag concentration. An increased ratio indicates a reduction in ristocetin-induced binding. A pronounced difference was identified at several amino acids between p.D1459 and p.P1471. Interestingly, the D1472H polymorphism was not abnormal by this approach (D1472A) – thereby indicating that alanine substitution for the aspartic acid does not confer the same conformation that the histidine does in African Americans. Although earlier studies by Mohri and coworkers (JBC 1988) suggest a second binding site, only a minimal signal was identified for the region between p.C1237 –p.P1251. Hot spots were identified with VWF:IbCo/VWF:RCo scores 7- to 30-fold higher than background. To date, natural sequence variations have not yet been identified in these positions. Studies by Scott and coworkers (JBC 1991), suggested that ristocetin binding to proteins was proportional to the presence of X-P-G-X’ beta-turns. The region of VWF that we are studying contains one of these sequences and is associated with a major effect on the VWF:IbCo/VWF:RCo signal. Since ristocetin dimerizes at a concentration of 1 mg/ml and the dose used in VWF:RCo is usually 1 mg/ml, these studies support the concept that alterations in ristocetin binding brought about by sequence variations may give the false impression that VWF function is profoundly affected when it is only the in vitro assay that is abnormal. Alanine scanning mutagenesis provides further substantiation of the role of the VWF-A1-loop in the binding of ristocetin to VWF. “Potential” Type 2M VWD subjects with sequence variations in this region may not have VWF functional abnormalities. Therefore, such individuals may require additional studies – particularly if bleeding symptoms are not identified in the presence of significantly abnormal laboratory testing. Disclosures: No relevant conflicts of interest to declare.