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Thrombotic thrombocytopenic purpura (TTP) is a rare thrombotic microangiopathy (TMA) characterized by the severe deficiency of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) activity (< 10%). Rapid ADAMTS13 testing is crucial for an early diagnosis and optimal management of acute TTP. We evaluated the performance of the HemosIL AcuStar ADAMTS13 activity assay (Instrumentation Laboratory, Bedford, Massachusetts, United States), a fully automated chemiluminescent immunoassay with an analytical time of 33 minutes. A method comparison study was performed on 176 samples from 49 healthy donors and 127 TMA patients (109 TTP, 7 atypical hemolytic uremic syndrome, 11 other TMAs), comparing this new assay with an in-house FRETS-VWF73 assay and a commercial enzyme-linked immunosorbent assay (ELISA) (TECHNOZYM ADAMTS-13 Activity, Technoclone GmbH, Vienna, Austria). Agreement between methods was assessed with focus on ADAMTS13 activity less than 10%, the medical decision level relevant for TTP diagnosis. The HemosIL AcuStar ADAMTS13 Activity showed good correlation with both the FRETS-VWF73 (r = 0.96) and ELISA (r = 0.96) methods. Slope of the Passing–Bablok regression was 1.05 for FRETS-VWF73 and 1.02 for ELISA, and absolute bias at the medical decision level was +0.1 and +0.3%, respectively. The study also revealed high agreement with FRETS-VWF73 (kappa 0.97) and ELISA (kappa 0.98) methods in classifying TTP patients with a severe deficiency of ADAMTS13 activity. Because of its short turnaround time and full automation, the HemosIL AcuStar ADAMTS13 activity assay might become the assay of choice to rapidly test ADAMTS13 activity in plasma and thus establish the diagnosis of acute TTP in emergency settings.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare, life-threatening thrombotic microangiopathy caused by severe ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin motifs 13) deficiency, recurring in 30&ndash, 50% of patients. The common human leukocyte antigen (HLA) variant rs6903608 was found to be associated with prevalent iTTP, but whether this variant is associated with disease relapse is unknown. To estimate the impact of rs6903608 on iTTP onset and relapse, we performed a case-control and cohort study in 161 Italian patients with a first iTTP episode between 2002 and 2018, and in 456 Italian controls. Variation in rs6903608 was strongly associated with iTTP onset (homozygotes odds ratio (OR) 4.68 (95% confidence interval (CI) 2.67 to 8.23), heterozygotes OR 1.64 (95%CI 0.95 to 2.83)), which occurred over three years earlier for each extra risk allele (&beta, &minus, 3.34, 95%CI &minus, 6.69 to 0.02). Of 153 survivors (median follow-up 4.9 years (95%CI 3.7 to 6.1)), 44 (29%) relapsed. The risk allele homozygotes had a 46% (95%CI 36 to 57%) absolute risk of relapse by year 6, which was significantly higher than both heterozygotes (22% (95%CI 16 to 29%)) and reference allele homozygotes (30% (95%CI 23 to 39%)). In conclusion, HLA variant rs6903608 is a risk factor for both iTTP onset and relapse. This newly identified biomarker may help with recognizing patients at high risk of relapse, who would benefit from close monitoring or intensified immunosuppressive therapy.

Sclerostin, an osteocyte-expressed negative regulator of bone formation, is one of the inhibitors of Wnt signaling that is a critical pathway in the correct process of osteoblast differentiation. It has been demonstrated that Wnt signaling through the secretion of Wnt inhibitors, such as DKK1, sFRP-2, and sFRP-3, plays a key role in the decreased osteoblast activity associated with multiple myeloma (MM) bone disease. We provide evidence that sclerostin is expressed by myeloma cells that are human myeloma cell lines and plasma cells (CD138(+) cells) obtained from the bone marrow (BM) of a large number of MM patients with bone disease. Moreover, we show that there are no differences in sclerostin serum levels between MM patients and controls. Thus, our data indicate that MM cells, as a sclerostin source in the BM, could create a microenvironment with high sclerostin concentration that could contribute toward inhibiting osteoblast differentiation.

There are a number of intriguing reports of lymphoproliferative disorders (LPDs) diagnosed during immunosuppressive treatment for underlying autoimmune disease, and spontaneously abated shortly after treatment discontinuation. Such LPDs, completely or partially regressing, occur in the clinical setting of "Methotrexate (MTX)-associated LPDs", recognized by the World Health Organization (WHO) among the "Immunodeficiency-associated LPDs". We identified 26 literature patients achieving spontaneous complete remission (CR) of their LPD, and eight others showing partial remission (PR). Most of them were affected by rheumatoid arthritis, received low-dose and long-term pulsed MTX alone or combined with other immunosuppressants, and developed a lymphoma. By reviewing the patients achieving CR, the following can be drawn: the absence of a unique type of LPD, the occurrence of an increased incidence of diffuse large B cell lymphoma as well as of frequent extranodal involvement, and EBV-infection. Further, CR mostly occurred within 4 weeks after discontinuation of immunosuppressant, and appeared to be persistent overtime. Conversely in the patients experiencing PR, the interval between discontinuation of immunosuppressive treatment and clinical response was mostly reported as longer than 4 weeks; moreover, in many cases the persistence of LPD or its progression induced to start cytotoxic therapy. Increased awareness is needed on the possible occurrence of LPD spontaneous remission following immunosuppressant discontinuation, after that it is therefore advisable to have a careful monitoring of the patient for some weeks, before starting cytotoxic therapy.

Schnitzler’s syndrome (SS) is defined by monoclonal gammopathy and chronic urticaria combined with at least two of the following features: fever, arthralgia or arthritis, bone pain, hepato- and/or splenomegaly, palpable lymph nodes, elevated ESR, and leukocytosis. We report a 49-year-old man with monoclonal IgM gammopathy and a 4-year history of recurrent urticarial rash, unexplained fever and arthralgias. The skin biopsy from an acute lesion revealed perivascular lymphocytic infiltrates consisting of CD4+ and CD8+ T lymphocytes. To our knowledge, this is the first report of an immunophenotypic characterization of skin infiltrates in SS. A lower CD4+/CD8+ ratio of circulating T lymphocytes was also detected. SS usually has a benign course, but in 15% of patients a lymphoproliferative disorder develops.

Background: the Registro Italiano Trombocitemie (RIT) was activated mainly to evaluate the diagnosis and therapy appropriateness in the thrombocythemic patients with Ph-negative myeloproliferative neoplasm (MPN) observed in the adhering centers. Objective: to evaluate how the diagnostic and therapeutic approach changed in the RIT patients diagnosed with thrombocythemic MPN in the last two decades. Methods: the RIT centers registered by a web-based system during the years 2005-2014 their thrombocythemic MPN patients, and semesterly updated their follow-up data. For patients diagnosed before 2005, the data on diagnosis and prior follow-up were retrospectively collected. The diagnostic process and the initial treatment (started into the first year after diagnosis) were comparatively analyzed in the patients diagnosed before and after 2005. Results: the RIT centers registered 2629 patients. 2388 of them, object of this analysis, were diagnosed between1995 and 2014: 1098 (46%) in the decade 1995-2004 (Group I), and 1290 (54%) in the decade 2005-2014 (Group II). The diagnostic process in the patients of Group II and Group I included bone marrow biopsy (BMB, performed into 1 year and before any cytoreduction): 85% vs 80%, p The patients of Group II, as compared with those of Group I, showed a similar gender distribution (M/F ratio 0.61 vs 0.65, p 0.452), and had at diagnosis: a higher age (median 60 vs 57 years, p 60 years in 50% vs 45% of cases, p Moreover, they had: a lower platelet (PLT) count (median 737 vs 775 x 109/L, p 10 x 109/L in 28% vs 26% cases); a similar median levels of hematocrit (HCT %, in females 41.4 vs 41.0; in males 44.7 vs 44.6) and hemoglobin (Hb g/dL, in females 13.8 vs 13.6; in males 15.0 vs 14.9). The BMB, revised according to the WHO 2008 criteria, showed a not different distribution (p 0.21) of ET (64% vs 61%), ep-PMF (16% vs 17%), PMF (3% vs 2%), PV (4% vs 4%), and U-MPN (13% vs 16%. The JAK2 V617F mutation in patients of Group II (at diagnosis) and of Group I (after diagnosis) was found in 62% and in 58% of tested cases (p 0.054), respectively. The patients at high standard thrombotic risk were 58% vs 52%, p 0.004. In the patients of Group II and Group I the distribution of the treatment started into the first year was significantly different (p The treatment CYT ± AntiPLT was started in the patients at high standard thrombotic risk with a rate of 81% vs 80%, respectively, and in the patients at low standard thrombotic risk in 37% vs 43% of cases, respectively. The initial treatment CYT±AntiPLT was related, in multivariate analysis, both in patients of Group II and Group I, with older age (>60 and 40-60 vs 1000 and 700-1000 vs Conclusion: in the studied thrombocythemic MPN patients the real-life diagnostic approach was improved after 2005 not only due to the routine use of JAK2 tests, but also due to the higher rate of BMB done (85% vs 80%). The appropriateness of the cytoreductive treatment (CYT±AntiPLT started into one year from diagnosis) remained high in patients at high standard thrombotic risk (over 80% of cases were treated). Concomitantly, the inappropriate use of the cytoreductive drugs in patients at low standard thrombotic risk appreciably decreased (from 43% to 37% of cases). Moreover, it has to be remarked that the therapeutic approach was significantly influenced not only by older age and prior thrombosis, but also by thrombocytosis (PLT count >700 x 109/L), disease-related symptoms, and inconstantly by leukocytosis and CVRFs. Table Table. Disclosures Gugliotta: SHIRE Co.: Honoraria. Gugliotta:Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.

Introduction: thrombotic thrombocytopenic purpura (TTP) is a rare, life-threatening thrombotic microangiopathy, with a reported incidence of 2-6 cases/million/year and a 10-20% mortality. TTP is associated with the severe deficiency of the von Willebrand factor cleaving protease, ADAMTS13, due to congenital defects in the ADAMTS13 gene (congenital TTP) or to the development of autoantibodies against ADAMTS13. The Milan TTP Registry is a digital database developed and curated at the Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico (Milan, Italy), including demographic, clinical and laboratory data of patients with a confirmed diagnosis of congenital or acquired thrombotic thrombocytopenic purpura (TTP). Aims: to report the natural history of patients affected with acquired TTP enrolled in the Milan TTP Registry, including demographic, clinical and laboratory data. Methods: acquired TTP patients enrolled in the Milan TTP Registry for an acute episode of TTP occurred between January 2002 and November 2015, and followed until May 2016, were included in this study. Acquired TTP was defined as the occurrence of thrombocytopenia and microangiopathic hemolytic anemia, in the absence of alternative causes. Patients with acquired TTP secondary to cancer and bone-marrow transplant were excluded from this analysis. Demographic and disease-related information were collected by a standardized clinical questionnaire. ADAMTS13-related measurements were performed in a centralized laboratory in Milan. Remission of an acute TTP episode was defined as two days after normalization of the platelet count (≥150 x 109/l). Results: 416 patients with a confirmed diagnosis of acquired TTP were included in the Milan TTP Registry, for a total of 837 acute events. Demographic, clinical and laboratory data are reported in Tables 1 and 2. The majority of patients were female (77%), of white ethnicity (98%) and Italian origin (82%). The median age at the first TTP episode was 40 years (interquartile range [IQR] 30-52). A history of other autoimmune diseases, cancer (non-active at the time of the TTP event), HIV infection and other diseases such as stroke, acute myocardial infarction and hypertension was reported in 34% of the patients. Among potential triggers of acute episodes (defined as occurring within three months before onset), infections were the most prevalent (22% of all patients), followed by the assumption of estroprogestinic drugs (5%). At TTP episode onset, systemic, bleeding and neurological signs and symptoms were present in 70%, 68% and 44% of acute events, respectively, whereas a lower prevalence of renal (19%) and cardiovascular (10%) signs and symptoms was observed. Clinical characteristics and hematologic laboratory parameters were less severe in relapsing episodes compared with first episodes (Table 2). Almost all acute events were treated by plasma exchange procedures and steroids. Rituximab therapy was administered in 12% of acute TTP episodes. In 197 acute TTP episodes with available ADAMTS13-related measurements at baseline, ADAMTS13 activity was severely reduced ( Conclusions: acquired TTP is a rare, potentially fatal disease, which may require a long hospitalization and presents in a recurrent form in nearly half of the patients. Digital registries, such as the Milan TTP Registry, represent a powerful and necessary tool to systematically collect epidemiologic, clinical and laboratory data which may ultimately improve our understanding and management of acquired TTP. Our registry, in collaboration with those of other international centers, might answer the most relevant questions in this field. Disclosures Peyvandi: Novo Nordisk: Other: research funding paid to Luigi Villa Foundation, Research Funding, Speakers Bureau; CSL Behring: Speakers Bureau; SOBI: Speakers Bureau; Biotest: Other: research funding paid to Luigi Villa Foundation, Research Funding, Speakers Bureau; Ablynx: Membership on an entity's Board of Directors or advisory committees, Other: research funding paid to Luigi Villa Foundation, Research Funding; Octapharma: Consultancy; Alexion: Other: research funding paid to Luigi Villa Foundation, Research Funding; Bayer: Speakers Bureau; Grifols: Speakers Bureau; LFB: Consultancy; Kedrion Biopharma: Consultancy, Other: research funding paid to Luigi Villa Foundation, Research Funding.

Sclerostin, an osteocyte-expressed negative regulator of bone formation, is one of the inhibitors of Wnt signaling that is a critical pathway in the correct process of osteoblast differentiation. It has been demonstrated that Wnt signaling through the secretion of Wnt inhibitors, such as DKK1, sFRP-2, and sFRP-3, plays a key role in the decreased osteoblast activity associated with multiple myeloma (MM) bone disease. We provide evidence that sclerostin is expressed by myeloma cells that are human myeloma cell lines and plasma cells (CD138(+) cells) obtained from the bone marrow (BM) of a large number of MM patients with bone disease. Moreover, we show that there are no differences in sclerostin serum levels between MM patients and controls. Thus, our data indicate that MM cells, as a sclerostin source in the BM, could create a microenvironment with high sclerostin concentration that could contribute toward inhibiting osteoblast differentiation.

Wingless-type (Wnt) signaling through the secretion of Wnt inhibitors Dickkopf1, soluble frizzled-related protein-2 and -3 has a key role in the decreased osteoblast (OB) activity associated with multiple myeloma (MM) bone disease. We provide evidence that another Wnt antagonist, sclerostin, an osteocyte-expressed negative regulator of bone formation, is expressed by myeloma cells, that is, human myeloma cell lines (HMCLs) and plasma cells (CD138+ cells) obtained from the bone marrow (BM) of a large number of MM patients with bone disease. We demonstrated that BM stromal cells (BMSCs), differentiated into OBs and co-cultured with HMCLs showed, compared with BMSCs alone, reduced expression of major osteoblastic-specific proteins, decreased mineralized nodule formation and attenuated the expression of members of the activator protein 1 transcription factor family (Fra-1, Fra-2 and Jun-D). Moreover, in the same co-culture system, the addition of neutralizing anti-sclerostin antibodies restored OB functions by inducing nuclear accumulation of β-catenin. We further demonstrated that the upregulation of receptor activator of nuclear factor κ-B ligand and the downregulation of osteoprotegerin in OBs were also sclerostin mediated. Our data indicated that sclerostin secretion by myeloma cells contribute to the suppression of bone formation in the osteolytic bone disease associated to MM.

Abstract 2961 Reduced osteoblast activity contributes to the development of multiple myeloma-bone disease. Wingless-type (Wnt) signalling pathway is critical in osteoblastogenesis, and it is negatively regulated by molecules such as frizzled-related proteins (sFRPs), Dickkopf proteins (DKKs) and sclerostin. Myeloma cells are known to induce inhibition of osteoblastogenesis through Wnt antagonists such as DKK-1 and sFRP-2 and -3 whereas the role of sclerostin, an osteocyte-expressed negative regulator of bone formation, has not been yet investigated. We provide novel evidence showing sclerostin expression by myeloma cells from patients with multiple myeloma-bone disease and human myeloma cell lines (HMCLs). By means of a co-culture system of bone marrow stromal cells (BMSCs) and HMCLs, we demonstrated that sclerostin expression by myeloma cells and HMCLs is responsible for reduced expression of major osteoblastic specific proteins namely bone-specific alkaline phosphatase, collagen-type I, bone sialoprotein II and osteocalcin as well as decreased mineralized nodule formation and attenuated expression of member of the AP-1 transcription factor family (i.e. Fra-1, Fra-2 and Jun-D). The addition of a neutralizing anti-sclerostin antibody to our co-culture system can restore the above parameters, through the intranuclear accumulation of β-catenin in BMSCs. On the other hand, we demonstrated that sclerostin is also involved in inducing increased receptor activator of nuclear factor-k B ligand (RANKL) and decreased osteoprotegerin (OPG) expression in osteoblasts, contributing to the enhanced osteoclast activity occurring in patients with multiple myeloma-bone disease. Our data suggest that myeloma cells contribute to the suppression of bone formation through sclerostin secretion. Disclosures: No relevant conflicts of interest to declare.