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Myeloproliferative neoplasms are clonal disorders characterized by the presence of several gene mutations associated with particular hematologic parameters, clinical evolution, and prognosis. Few therapeutic options are available, among which interferon α (IFNα) presents interesting properties like the ability to induce hematologic responses (HRs) and molecular responses (MRs) in patients with JAK2 mutation. We report on the response to IFNα therapy in a cohort of 31 essential thrombocythemia (ET) patients with CALR mutations (mean follow-up of 11.8 years). HR was achieved in all patients. Median CALR mutant allelic burden (%CALR) significantly decreased from 41% at baseline to 26% after treatment, and 2 patients even achieved complete MR. In contrast, %CALR was not significantly modified in ET patients treated with hydroxyurea or aspirin only. Next-generation sequencing identified additional mutations in 6 patients (affecting TET2, ASXL1, IDH2, and TP53 genes). The presence of additional mutations was associated with poorer MR on CALR mutant clones, with only minor or no MRs in this subset of patients. Analysis of the evolution of the different variant allele frequencies showed that the mutated clones had a differential sensitivity to IFNα in a given patient, but no new mutation emerged during treatment. In all, this study shows that IFNα induces high rates of HRs and MRs in CALR-mutated ET, and that the presence of additional nondriver mutations may influence the MR to therapy.

Extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) is thought to promote tumor angiogenesis mostly through its protease-inducing function and more recently by its ability to increase tumor cell expression of vascular endothelial growth factor (VEGF). In this study, we present evidence that EMMPRIN can promote angiogenesis by a direct effect on endothelial cells through a paracrine regulation of the VEGF/VEGF-receptor (VEGFR) system. Using human microvascular endothelial cell line–1 endothelial cells, we show that EMMPRIN selectively increased the soluble VEGF isoforms (121 and 165), but not the matrix-bound VEGF 189 form. In addition, EMMPRIN up-regulated the expression of VEGFR-2 without an effect on VEGFR-1. This increase in VEGFR-2 was responsible for the observed EMMPRIN stimulation of the migratory and tube formation capacity of endothelial cells. EMMPRIN′s effects, which were matrix metalloproteinase and urokinase-type plasminogen activator independent, were mediated primarily through hypoxia-inducible factor-2α expression, also up-regulated by EMMPRIN. VEGFR-2 increase was also observed in vivo in a mouse model of xenograph tumors overexpressing EMMPRIN. These results suggest that in addition to increasing protease production, EMMPRIN may contribute to the formation of a reactive stroma also through the up-regulation of hypoxia-inducible factor-2α, VEGFR-2, and the soluble forms of VEGF in endothelial cells, thus directly regulating the angiogenic process.

Short peptides isolated from fibrinogen and K-casein have been shown to inhibit platelet aggregation and fibrinogen binding to stimulated platelets. We studied the effects of synthetic peptides occurring in milk proteins (bovine K-casein, KNQDK, and human lactotransferrin, KRDS) and in fibrinogen (RGDS and L10) on subsequent binding of monoclonal antibodies (MoAb) against the glycoprotein (GP) Ilb-IIIa complex (AP2and P2) on adenosine diphosphate (ADP)-stimulated and unstimulated human platelets and megakaryocytes (MKs) by using an immunoperoxidase method to visualize antibody binding. Only KRDS (900 μmol/L) inhibited the binding of AP2and P2on ADP (5 μmol/L)-stimulated platelets, but not on unstimulated platelets. However, the binding of P2was considerably more inhibited than that of AP2as judged by immunoperoxidase intensity. Radiolabeled AP2binding was inhibited by 30% with KRDS on ADP-stimulated platelets as compared with platelets incubated in the absence of ADP. KRDS did not inhibit the binding of MoAbs against GP Ilia (SZ 21), GP lib (SZ 22), and GP lb (SZ 2) on ADP-stimulated human platelets. Inhibition of P2binding by KRDS was also observed in a section of MKs isolated from human bone marrow and stimulated by 15 or 20 Mm ADP. A lower concentration of ADP (5 or 10 Mmol/L) failed to produce any inhibition of binding. This indicates that MKs may not be equally responsive to agonists as platelets. Moreover, P2binding inhibition was observed in a larger (P<.001) percentage of mature MKs (29%) as compared with younger, maturing MKs (11%). The observations suggested that a functional ability possessed by platelets, namely, agonistinduced exposure of the site of interaction of KRDS, may occur at a late stage of MK development.

Rea: Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Kiladjian:BMS: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; AOP Orphan: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees. Benajiba:Gilead Foundation: Research Funding.

Rea: Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Kiladjian:AOP Orphan: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Benajiba:Gilead Foundation: Research Funding.

Introduction:

Myeloproliferative neoplasm (MPN)-associated myelofibrosis (MF) can occur as a de novo (Primary MF, PMF), or as secondary MF (SMF) resulting from transformation of Polycythemia Vera or Essential Thrombocythemia. In all cases, altered hematopoiesis results in abnormal blood counts (either hyperproliferation or cytopenias), splenomegaly and general symptoms related to inflammation, all of which are the intended targets of treatments.

No relevant conflicts of interest to declare.

Kiladjian: Novartis: Honoraria, Research Funding; AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding.

No relevant conflicts of interest to declare.

Off Label Use: Interferon alpha was used off-label in selected ET patients according to local and international guidelines (Barbui et al., J Clin Oncol. 2011 Feb 20;29(6):761-70).

No relevant conflicts of interest to declare.

Off Label Use: 18F-FLT is an investigational isotope not approved for the imaging of myelofibrosis. Patients treated off-label with interferon were included in the study..

Positron emission tomography (PET) generally employing fluorodeoxyglucose (FDG) combined with high-resolution structural imaging using computed tomography (CT) is regularly used in the diagnosis, staging and monitoring of treatment response in clinical oncology. 3′-18Fluoro-3′-deoxy-L-thymidine (18F-FLT) is a nucleoside analog that quickly accumulates in proliferating cells, more recently evaluated in various cancers including hematologic malignancies like acute leukemias or lymphomas as a PET radiotracer offering non invasive assessment of cell proliferation in vivo. Published results suggest that this technique could be useful to assess bone marrow (BM) activity and extramedullary hematopoiesis (EMH). However, to our knowledge, only 3 patients (pts) with myelofibrosis (MF) have been explored with 18F-FLT PET/CT (FLT PET). This pilot study aimed to establish proof-of-concept that FLT PET could be a new non invasive technique useful for MF management, in terms of diagnosis, staging and for monitoring response to therapy.Pts were evaluated using 2 different techniques. First, conventional BM scintigraphy (BMS) was performed: on day 1, pts were injected with 99mTechnetium-nanocolloids and a planar image of the reticuloendothelial system was performed 30 min after injection; pts were then injected with 111Indium-Cl3 and planar imaging of the erythroid BM was performed after 48h. Secondly, FLT PET was performed 1 hour after injection of 18F-FLT (provided by AAA), and consisted in a whole-body acquisition. Images were interpreted in a blinded fashion independently by two nuclear physicians, qualitatively and according to a visual scale for both examinations. In addition, 18F-FLT uptake was quantified using standardized uptake value (SUV) in several sites of the skeleton, spleen and liver.15 pts (9 men, 6 women, mean age: 62 years) were included between Apr 2011 and Jul 2012 (14 evaluable at time of abstract submission). 7 pts (47%) had primary (PMF), 4 post-polycythemia vera (PV), and 4 post-essential thrombocythemia (ET) MF, respectively (WHO criteria). All the pts had a BM biopsy with quantification of fibrosis. 11 pts (73%) had a JAK2V617F mutation, 1 a MPL515 mutation, and 3 had neither of these mutations. Therapies included hydroxyurea (n=1), androgens (n=1), interferon (n=4) and ruxolitinib (n=5); 4 pts had no specific therapy for MF.Three distinct patterns of FLT PET images were observed. 3 pts showed a marked reduced hematopoietic activity in the central compartment of the skeleton but a high uptake in spleen, suggesting the existence of myeloid metaplasia (Fig 1A). 8 pts had a rather normal pattern of BM activity in the central skeleton associated with marked expansion of BM activity to distal extremities and intense uptake of the tracer in the spleen (Fig 1B). 3 pts showed a relatively normal pattern of BM activity in the central skeleton, a mild expansion to distal extremities with no splenic abnormality (Fig 1C). FLT interpretation in myeloid malignancies is not standardized and we used comparisons with BMS to establish interpretation guidelines. Qualitative FLT PET results were equivalent to the 111In-Cl3 imaging in most cases, but in 2 pts FLT uptake was normal when BMS showed reduced 111In-Cl3 uptake. Compared to BMS, PET will also provide much more information including: (i) quantitative analyses of 18F-FLT uptake using SUV (preliminary results show that SUV ranges are [1.8 – 18.4] and [2.3 – 19.8] in BM and spleen, respectively); (ii) precise evaluation of malignant myelopoiesis in the different anatomical sites using coupled CT images. These analyses, and correlation with clinical and biological characteristics, BM histopathology and type of therapy received are ongoing.FLT PET is a new, convenient non invasive technique for evaluation of malignant hematopoiesis in MF, including BM activity and EMH. Distinct patterns of FLT uptake may help in the diagnosis and staging of MF. In addition, ongoing correlation studies with histological BM fibrosis could provide evidence for a role of this non invasive technique in the assessment of the evolution of fibrosis over time without the need for sequential biopsies. A subsequent clinical trial will determine in a larger cohort of MF pts the usefulness of PET for evaluation of tumor response to therapy and prediction of early response using sequential evaluation of FLT uptake in BM and spleen.Off Label Use: 3′-18Fluoro-3′-deoxy-L-thymidine (18F-FLT) is a nucleoside analog tested as a PET radiotracer in patients with myelofibrosis.

Abstract 1741

Background:Only 4 randomized clinical trials in PV have directly compared cytoreductive therapies (32P, busulfan, chlorambucil, HU, and Pi), all published after a median follow-up of 10 years or less. Thrombosis was the main cause of mortality and morbidity in all trials. Cumulative incidence of evolution to myelofibrosis (MF), and acute myeloid leukemia or myelodysplastic syndromes (AML/MDS) was about 8%, and 6%, respectively. In 1 of those trials, conducted between 1980 and 1996 by the French Polycythemia Study Group (FPSG) in 292 PV patients (pts) younger than 65 yrs, no difference between HU and Pi was found in terms of survival, risk of thrombosis, and AML/MDS/MF evolution at the time of first analysis with a median follow up of 7 years (Najean, Blood , 1997, 90:3370).

Since the description in 2005 of the recurrent V617F JAK2 gene mutation in MPD patients, we have tested more than 1500 patients each year and confirmed the relative frequency of this mutation in distinct MPD subclasses and its usefulness in classyfying diseases such early PV, MIF, ET and thrombosis such Budd Chiari syndrome. In the case of erythrocytosis we notably observed that among patients with true polycythemia (red cell mass measurement increased by 25%), the presence of the mutation significantly distinguished between PV patients (95% V617F positive) and patients with Idiopathic Erythrocytosis (IE, 0% positive); In ET patients about 50% were positive ; In thrombosis, we demonstrated that the V617F mutation is present in about half of the patients with deep vein thrombosis (Budd Chiari syndrome or portal vein thrombosis), reinforcing the guess that occurrence of thrombosis in these patients is caused by an ignored MPD. The description in 2007 of novel recurrent mutations in the exon 12 of the JAK2 gene led us to test whether these new alterations could help in classifying some of the V617F negative patients. We have tested patients with PV (n= 25) of whom 10 had a post PV myelofibrosis, deep vein thrombosis (n= 20) and IE (n= 21). PV patients fulfilled either PVSG or WHO criteria. IE patients were diagnosed on the basis of an elevation of the hematocrit (median hematocrit of 54% (range: 49% to 56%)), a raised red cell mass (median excess of red cell mass was +35% (range: +25% to +104%); no identifiable cause of secondary erythrocytosis (serum Epo level was under or within the normal range in all patients); and absence of PV according to PVSG or WHO criteria (no splenomegaly, median WBC (×109/L) of 6180 (range: 3500–8300) ; median platelet count (x109/L) of 240 (range: 174–358). DNA extracted from peripheral blood has been tested using the allele specific PCR described in Scott et al., NEJM 2007, allowing to detect all of the described mutations. We failed to identify any mutation in patients with IE or deep vein thrombosis. However, 2 female patients diagnosed with PV were identified to harbour the N542-E543del mutation. With the exception of their relative younger age (41 and 45 respectively) and a very elevated hematocrit (58 and 59%) it was not possible to find any common points to these 2 patients that could allow individualizing patients susceptible to harbour exon 12 mutations. One patient had very low Epo level and splenomegaly, while the other patient had increased WBC but no splenomegaly. None of them had myelofibrosis. In conclusion, only 2 out of 25 PV patients harboured JAK2 exon 12 mutations, thus around 5% of PV patients that fulfilled PV criteria are still negative for any mutation. The fact that IE patients are always negative let the question unsolved of whether this entity is a MPD or not. The search for exon 12 JAK2 gene mutation should be restricted to patients which fulfil PV diagnosis criteria and do not exhibit the “classical” V617F mutation. In these cases, the presence of the mutation is very useful in order to well classify these patients as MPD patients.