ALBERT

Description: Abstract 313 Background: While advanced PV and ET patients at high thrombotic risk are managed primarily with HU, patients who are intolerant or refractory to HU have limited therapeutic options. Identification of a dominant gain-of-function mutation in the JAK2 kinase, V617F, in myeloproliferative neoplasms (MPNs), including PV and ET, provided a key rationale for the development of a molecularly targeted therapy for these diseases. Long term follow-up data from an ongoing trial of INCB018424, a selective JAK 1/ JAK 2 inhibitor, in PV and ET patients refractory or intolerant to HU are presented. Methods: Study 18424-256 is an uncontrolled open-label Phase 2 study being conducted at 6 sites in the United States and Italy. An initial 8-week run-in evaluation established 10-mg and 25-mg twice daily as starting doses for expansion cohorts in PV and ET, respectively; dose adjustments for safety and efficacy are allowed so that each subject is titrated to their most appropriate dose. For PV, response is defined based on Hct control in the absence of phlebotomy, improvement or elimination of palpable splenomegaly when present, and normalization of leukocytosis and thrombocytosis. For ET, response is defined based on improvement or normalization of WBC and platelet counts and, when present, elimination of palpable splenomegaly. PV results (n=34; median 108 months from diagnosis): After a median follow-up of 15 months (range 8–21), 97% of enrolled subjects achieved Hct control to 15×109/L was present in 47% of subjects and improved (≤ 15×109/L) or normalized (≤ upper limit of normal) in 88% and 63%, respectively. Thrombocytosis 〉 600×109/L was present in 38% of subjects and improved (≤ 600×109/L) or normalized (≤ upper limit of normal) in 92% and 69%, respectively. 59% of subjects achieved phlebotomy independence, resolution of splenomegaly and normalization of leukocytosis and thrombocytosis. 6 patients discontinued therapy (3 due to AEs, 2 withdrew consent, 1 for no response). Grade 3 AEs potentially related to study medication included thrombocytopenia (2 patients), neutropenia (1), renal tumor (1), asthenia (1), viral infection (1), and atrial flutter (1). No Grade 4 drug-related AEs have occurred. ET results (n=39; median 84 months from diagnosis): After a median follow-up of 15 months (range 4–21), 49% of enrolled subjects normalized platelet counts to ≤ upper limit of normal after a median of 0.5 months and for a median duration of 3.5 months. 82% maintained platelet counts 〈 600×109/L, for a median duration of 9.8 months. Of 14 patients with baseline platelet counts 〉 1000×109/L, 13 have experienced 〉 50% reduction. 88% maintained normal WBC (median duration 14.5 months). Palpable spleens resolved in 3 of 4 subjects; 1 reduced 〉50% from baseline. 49% of subjects achieved normalization of WBC and platelet counts in the presence of non-palpable splenomegaly. 9 patients discontinued therapy (4 due to AEs, 2 withdrew consent, 3 for no response). Grade 3 AEs potentially related to study medication included leukopenia (2 patients), GI disorder (1), and peripheral neuropathy (1). No Grade 4 drug-related AEs have occurred. Both patient groups demonstrated reductions in patient-reported symptom scores for pruritus, night sweats, and bone pain. Of 26 PV patients reporting pruritus at baseline (median score of 6 on a 10-point scale), 24 reported scores of 0 after a median duration of 1 month and for a median duration of 7 months. 42% of PV and 56% of ET patients had at least a 20% decrease in JAK2V617F allele burden; 6% of PV and 12% of ET had 〉50% decrease. Clinical responses were unrelated to the presence/absence of JAK2V617F mutation at entry or to the allele burden changes following treatment. Conclusions: Rapid and durable clinical benefits (normalization of hematological parameters, resolution of splenomegaly and alleviation of symptoms) have been demonstrated in advanced PV and ET patients with 〉1 year of follow-up. In this study, INCB018424 continues to be a well tolerated, effective therapy in patients with PV and ET refractory or intolerant to hydroxyurea. Disclosures: Verstovsek: Incyte Corporation: Research Funding. Levy:Incyte Corporation: Employment, Equity Ownership. Bradley:Incyte Corporation: Employment. Garrett:Incyte Corporation: Employment. Vaddi:Incyte corporation: Employment. Huber:Incyte Corporation: Employment, Equity Ownership. Schacter:Pfizer Corporation: Employment. Vannucchi:Novartis: Membership on an entity's Board of Directors or advisory committees.

Description: Systemic mastocytosis (SM) is a rare myeloproliferative neoplasm characterized by proliferation and hyperactivation of clonal mast cells. Clinical manifestations are heterogeneous and encompass cutaneous lesions, gastrointestinal alterations, osteoporosis, anaphylaxis and involvement of bone marrow and other organs due to neoplastic mast cells (MC) infiltration. As consequence, diagnosis may be difficult and patients (pts) are often evaluated by different specialists before the disease is recognized. To date, only few studies (Lim 2009, Escribano 2009, Cohen 2014) described relatively large series of pts with SM. We performed a multicentre retrospective study to evaluate clinical and biological features and therapeutic management in a large series of pts from 10 Italian centres experienced in management of SM and organized in multidisciplinary groups of specialists. We collected 455 pts diagnosed with SM according to WHO criteria. Additionally 26 pts with mastocytosis in the skin (MIS) evaluated with BM examination did not fulfil criteria for SM, leading to diagnosis of Cutaneous Mastocytosis (CM); however 2/26 pts with CM had both cKITD816V mutation and CD2/CD25 expression on MC in BM, additional 3 showed either cKITD816V or CD2/CD25. Moreover, we found 22 pts without MIS but with features of monoclonal mast cell activation syndrome. Of the 455 pts with WHO-SM (male 56%), 252 (55%) had MIS: median age at MIS diagnosis (dg) was 37 years (y) (range 0-79), while at SM dg it was 46.5 (range 18-82). Time from onset of MIS to dg of SM was 9 y (range 0-43). In 18/252 pts (7%) MIS occurred before age of 18 y (median 9, range 0-17) and persisted over childhood. Median age at dg of SM without MIS (203/455 pts, 45%) was older: 54 y, range 19-79 (p

Description: Abstract 307 Constitutive and enhanced activation of JAK/STAT pathway is found in patients (pts) with myeloproliferative neoplasms (MPN), particularly but not exclusively in those with mutation in the Janus tyrosine kinase 2 gene (JAK2V617F) or the MPL gene (MPLW515L/K/A). Activation of the Erk and PI3K/Akt pathways have also been described in cells harboring the JAK2V617F mutation. In in-vitro studies performed in our laboratory (AM Vannucchi et al, ASH 2009, abstract submitted) we observed that RAD001, a specific inhibitor of the serine/threonine kinase mTOR (mammalian target of rapamycin) that is a downstream target of PI3K, remarkably inhibited proliferation of JAK2V617F mutant cell lines and reduced the clonogenic activity of CD34+ cells from MPN pts. The PI3K/Akt/mTOR pathway has become an important focus for cancer therapy, and mTOR inhibitors have demonstrated activity against solid tumors and lymphomas. A phase I/II trial of RAD001 is being conducted in pts with PMF and PPV/PET MF, including JAK2V617F or MPL mutated as well as wild-type pts with intermediate/high risk score according to Lille criteria. Diagnosis of PMF and of PPV/PET-MF was made following the WHO and International Working Group for Myelofibrosis Research and Treatment (IWG-MRT) criteria, respectively. Phase I is a dose escalation of RAD001 involving 3 sequential cohorts of 3 pts each at 5.0, 7.5, and 10 mg daily, orally, for 3 months, to define maximum tolerated dose (MTD). Phase II involves the use of MTD for 4 months, according to a two-stage Simon design, aimed at assessing efficacy of the drug. Responses are evaluated using the EUMNET criteria for treatment response in myelofibrosis (Barosi G et al, Blood 2005). At the time of writing, 8 of 9 pts included in the Phase I have completed the 3-month study period without dose-limiting toxicities or other significant adverse events; the ninth pt at 10 mg is completing the last month of treatment. There were seven pts with PMF and 2 with PPV-MF. Main toxicities were represented by grade 2 mouth ulcers in 3 pts and transient grade 1/2 hypertrigliceridemia in 5 pts, that occurred irrespective of the dose. One pt at 10 mg presented transient muscular weakness and arthralgia. The initial dose of 5.0 mg resulted in a reduction in spleen size consistent with a complete response (CR) in one pt (from 4 to 0 cm below the left costal margin) and partial responses (PR) in two pts (from 10 and 13 cm to 1 and 9 cm, respectively). In the 7.5 mg cohort, there were one CR (from 4 to 0 cm) and one PR (from 16 to 8 cm) in splenomegaly; none of the 2 pts at 10 mg had a response in splenomegaly. In 1 pt CR in splenomegaly was maintained at +4 mo after suspension of drug while in the other responding pts there was a progressive re-enlargment of the spleen to baseline level. A complete resolution of systemic symptoms was achieved in 2 of 3 pts while aquagenic pruritus disappeared in all the 5 pts who complained of it. Overall, there were 2 major and 3 moderate responses, while 3 pts did not show a measurable response. At the end of therapy, the absolute count of circulating CD34+ cells had decreased in all but one pt of a median value of 39% from baseline (range 16% to 72%). There was no significant decrease in the burden of granulocyte V617F allele at the end of treatment in the 8 mutated pts. Single-colony JAK2V617F genotyping was performed in 4 pts, 2 each from the 5.0 and 7.5 mg cohort. In one patient receiving 7.5 mg the percentage of homozygous BFU-E and CFU-GM decreased from 14% and 45%, respectively, at baseline to 0 and 18% at the end of therapy; in the other 3 pts changes were minimal. In conclusion, initial results showed rapid reduction of splenomegaly and symptomatic improvement, without relevant toxicity, in pts with myelofibrosis receiving RAD001. The drug also reduced the circulating CD34+ cell count and V617F-homozygous progenitor cells appeared to be decreasing at least in some patients, although the allelic burden measured in granulocytes did not modify. Updated results on current patients and future patients enrolled in Phase II will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2914 Poster Board II-890 The JAK2 and MPL somatic mutations in chronic myeloproliferative neoplasms (MPN) result in constitutive activation of JAK2/STAT pathway; in-vitro JAK2 inhibitors reduced the proliferation of JAK2V617F mutant cells while in early clinical trials in patients (pts) with myelofibrosis resulted in clinical improvement, although the effects on the burden of JAK2-mutant clone were less impressive than anticipated. On the other hand, little is known about other signalling pathways, such as PI3K/Akt and ERK, that also resulted activated in cells harboring the JAK2V617F mutation. In this study we focused on the mammalian target of rapamycin (mTOR), a downstream target of key signalling molecules including Akt, PI3K, GTPase Ras and Rheb; mTOR is often abnormally activated in cancer cells and represents an attractive target for anticancer therapy, as shown by the effects of mTOR inhibitors in solid tumors and lymphomas. First, we documented constitutive phosphorylation of mTOR and of the eukaryotic initiation factor 4E-binding protein 1 (4EBP1), a downstream target of mTOR, in Ba/F3 cells transduced with JAK2 V617F (kindly provided by R. Skoda); conversely, phosphorylation of both mTOR and 4EBP1 in wild-type (wt) Ba/F3 cells was largely dependent on exposure to IL3. Then, to assess the effects of mTOR inhibition on the growth of JAK2V617F mutant cells we used the specific mTOR inhibitor RAD001. We observed that day-14 clonogenic growth of JAK2V617F mutant cells HEL, UKE-1 (a gift of W. Fiedler) and SET2 was potently inhibited by RAD001 compared to BCR-ABL-pos K562 cells: IC50 values were 85±30 nM, 60±20 nM, 330±50nM and 12,000±340 nM, respectively (all P

Description: Most patients with polycythemia vera (PV) carry a mutation in the Janus tyrosine kinase 2 gene (JAK2V617F); in 20–30% of them, mitotic recombination leads to homozygosity. It has been suggested that phenotype may be in part dependent on the mutant allele burden and the residual amount of wild-type (wt) allele, since wt JAK2 exherted a dominant negative effect during co-transfection experiments. However, simply differentiating heterozygotes from homozygotes may be misleading, since it does not distinguish between true heterozygosity or homozygosity in a mixed normal background, nor does it provide any measure of the relative contribution of the two alleles; consistently, homozygous progenitors have been found almost invariably in PV patients. The aim of this study was to assess the influence of JAK2V617F mutant allele burden on hematologic and clinical parameters in 116 PV patients diagnosed according to the WHO criteria, for whom a blood sample, collected at, or within six months from, the diagnosis was available. To measure the ratio between mutated and wt JAK2, we employed an ARMS-PCR procedure on RNA, purified from ≥95% pure preparations of PB granulocytes; mutation-specific amplicons were resolved and quantitated using capillary electrophoresis. Detectable amount of JAK2V617F RNA in the granulocytes was found in 96 patients (83%), with a median value of JAK2V617F/JAK2WT ratio of 38% (range, 1 to 100%). JAK2V617F patients were divided into four classes based on the relative amount of mutant allele: 32 patients (28%) had JAK2V617F/JAK2WT ratio of 1–25%, 24 (21%) of 26–50%, 17 (16%) of 51–75%, and 23 (20%) had 76–100% ratio. There was no difference among these classes as concerned the relative frequency of patients, to indicate that the extent of mutant allele at diagnosis is heterogeneous and that acquisition of the highest allele burden is not necessarily a time-dependent event. The hematocrit, leukocyte count, LDH and ALP values were directly related to the relative amount of JAK2V617F RNA ( P

Description: Philadelphia chromosome–negative myeloproliferative neoplasms (MPNs) are clonal myeloid disorders with increased production of terminally differentiated cells. The disease course is generally chronic, but some patients show disease progression (secondary myelofibrosis or accelerated phase) and/or leukemic transformation. We investigated chromosomal aberrations in 408 MPN samples using high-resolution single-nucleotide polymorphism microarrays to identify disease-associated somatic lesions. Of 408 samples, 37.5% had a wild-type karyotype and 62.5% harbored at least 1 chromosomal aberration. We identified 25 recurrent aberrations that were found in 3 or more samples. An increased number of chromosomal lesions was significantly associated with patient age, as well as with disease progression and leukemic transformation, but no association was observed with MPN subtypes, Janus kinase 2 (JAK2) mutational status, or disease duration. Aberrations of chromosomes 1q and 9p were positively associated with disease progression to secondary myelofibrosis or accelerated phase. Changes of chromosomes 1q, 7q, 5q, 6p, 7p, 19q, 22q, and 3q were positively associated with post-MPN acute myeloid leukemia. We mapped commonly affected regions to single target genes on chromosomes 3p (forkhead box P1 [FOXP1]), 4q (tet oncogene family member 2 [TET2]), 7p (IKAROS family zinc finger 1 [IKZF1]), 7q (cut-like homeobox 1 [CUX1]), 12p (ets variant 6 [ETV6]), and 21q (runt-related transcription factor 1 [RUNX1]). Our data provide insight into the genetic complexity of MPNs and implicate new genes involved in disease progression.

Description: In addition to dysregulated JAK/STAT signaling, activation of the AKT/mTOR pathway occurs in myelofibrosis, a myeloproliferative neoplasm with no approved therapies. We conducted a phase 1/2 study with everolimus, an mTOR inhibitor, in 39 high- or intermediate-risk primary or postpolycythemia vera/postessential thrombocythemia myelofibrosis subjects. Responses were evaluated in 30 patients of phase 2. No dose-limiting toxicity was observed in phase 1 up to 10 mg/d. When this dose was used in phase 2, grade ≥ 3 toxicities were infrequent; the commonest toxicity was grade 1-2 stomatitis. Rapid and sustained splenomegaly reduction of 〉 50% and 〉 30% occurred in 20% and 44% of subjects, respectively. A total of 69% and 80% experienced complete resolution of systemic symptoms and pruritus. Response in leukocytosis, anemia, and thrombocytosis occurred in 15%-25%. Clinical responses were not associated with reduced JAK2V617F burden, circulating CD34+ cells, or cytokine levels, whereas CCDN1 mRNA and phospho-p70S6K level, known targets of mTOR, and WT1 mRNA were identified as possible biomarkers associated with response. Response rate was 60% when European Network for Myelofibrosis criteria were used (8 major, 7 moderate, 3 minor responses) or 23% when IWG-MRT criteria (1 partial response, 6 clinical improvements) were used. These results provide proof-of-concept that targeting mTOR pathway in myelofibrosis may be clinically relevant.

Description: Abstract 3805 Background: Mastocytosis is a rare disease characterized by an abnormal proliferation and accumulation of mast cells (MC) in several organs and tissues such as skin, bone marrow, liver, gastrointestinal tract and lymphnodes. The reported prevalence of mastocytosis (cutaneous or systemic) is lower than 1/50,000. Mastocytosis encompasses a wide range of clinical entities, extremely heterogeneous for symptoms, clinical course and prognosis. The heterogeneity and the complexity of its clinical signs lead to the definition of mastocytosis as a multidisciplinary pathology, involving different specialists such as hematologists, internists, dermatologists, immunologists and pediatricians. Mastocytosis is a MC clonal disease associated to a somatic mutation (D816V) of the proto-oncogene c-kit (KIT), which codifies for the stem cell factor (SCF) receptor. SFC is the main factor stimulating the proliferation, chemotaxis and activation of human mast cells. Different KIT mutations have been found in 15% of patients. Clinical signs and symptoms of mastocytosis mainly depend on the liberation of chemical mediators produced by the mast cells, on the tissue infiltration of the mast cells and on other associated hematological diseases. Aim of the Registry: promoting studies on mastocytosis in Italy aimed at investigating the epidemiology of the disease, its prognostic factors and health technology assessment (HTA) aspects associated to the management of a rare “orphan” disease. Methods: The Italian Mastocytosis Registry was constituted in 2009, with the aim of promoting communication between specialists and collecting data about patients diagnosed with mastocytosis at a national level. Anagraphical, anamnestic, clinical, biological, treatment and follow-up data of patients with mastocytosis are being routinely collected in 15 Italian centers after written informed consent. An on-line database (www.registroitalianomastocitosi.it) has been set up for this purpose. The collected data will allow specialists to: Results: At present, data on 175 patients have been collected. Seventy-nine (45%) have been diagnosed with systemic mastocytosis and 40 (50%) of them progressed from a cutaneous disease. Ninety-four (54%) are females; 81 (46%) are males. Among 49 patients for whom data on familiarity were available, 12 (24%) reported familiar cases of autoimmune diseases (n=3), allergies (n=5) or interestingly mastocytosis (n=4). Sixty-one (35%) patients reported allergies. Of 121 reported lines of therapy, 37 (31%) were described as not specified anti chemical mediators, 51 (42%) anti-H1, 21 (17%) anti-H2, 30 (25%) corticosteroids, 22 (18%) phototherapy, 7 (6%) alpha-interpheron, 8 (7%) chemotherapy and 13 (11%) tyrosine-kinase inhibitors (total exceeds 100% because multiple choice is allowed). As to the histological findings, 82 (47%) patients have data on bone marrow biopsy: 48 (59%) had a positive finding, with a median mast cells infiltrate of 30% (range 3–90%). Among 43 patients tested for tryptase serum level, 41 (95%) had levels above normal values (12.5 ng/ml). Conclusions: This is the first spontaneous observational study on mastocytosis in Italy. The on-line database is a useful tool for data collection at a national level. The Registry is an opportunity to carry out epidemiological studies aimed at estimating occurrence and geographical distribution of the disease. It will also allow specialists to investigate possible prognostic factors and provide a starting point for the research into ad hoc therapies and HTA studies. It will hopefully provide a link with other international registries to improve understanding of this disease. Last but not least, the Italian Registry may support a National Government policy to provide assistance by the Public Health System to patients with mastocytosis. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4093 Background: In a previous study (Verrucci M et al, J. Cell Physiol, 2010, Epub May10) we reported that the cyclic depsipeptide Aplidin®, a potent cytotoxic agent currently in phase II/III clinical trials for solid and hematologic neoplasia, displayed activity in a murine model of myelofibrosis, the GATA1-low mice. In fact, Aplidin improved the proliferation of Gata1-low hematopoietic cells, corrected abnormal microvessel density and reduced bone marrow fibrosis. These effects were largely attributed to improved maturation of megakaryocytes, as suggested by the increased platelet count in thrombocytopenic Gata1-low mice. It has been previously shown that a low expression of p27 in tumor cells might correlate with their response to Aplidin; of interest, GATA1-low megakaryocytes expressed reduced levels of p27. Overall, these results suggested that Aplidin could have the potential to alter the course of myelofibrosis-like disease in Gata1-low mice and could be useful for the treatment of myelofibrosis. Aims: to evaluate activity and targets of Aplidin in cellular models of myeloproliferative neoplasms (MPN). Methods: We measured the effect of Aplidin on the proliferation of JAK2V617F-mutated cell lines (UKE-1, HEL, SET2) and of primary cells from MPN patients in liquid cultures and semisolid medium, the rate of apoptosis using Annexin V flow cytometry analysis, and the cell cycle by propidium iodide staining. Expression of mRNA was quantified by real-time PCR, while protein level was measured by western blotting. Gene expression profiling was accomplished with Agilent Whole Human Genome Oligo Microarrays (44K). Results: Aplidin reduced the proliferation of all human V617F-mutated cell lines in the low nanomolar range, with IC50 from 0.5+/−0.03nM for UKE-1 to 1.5+/−0.05nM for HEL cells. Aplidin increased the proportion of cells in the G1/G0 phase of cell cycle, up to a mean of 80+/−5% from 60+/−3% in control cells (P

Description: Introduction. Ruxolitinib is a JAK inhibitor (JAKi) approved for myelofibrosis (MF) patients (pts), while other JAKi are being tested in clinical trials. The IWG-MRT response criteria (RC) used to evaluate drug efficacy were revised on 2013 and adapted to take into account efficacy in specific aspects of the disease, duration of response and toxicities (Blood 2013;122:1395). Methods. We retrospectively evaluatedthe response rate in MF pts treated for at least 3 months (mo) with JAKi in phase II/III and expanded access trials at University of Florence and Pavia, according to IWG-MRT 2013 RC. We also collected molecular data, last follow up and reasons of discontinuation. Changes in spleen size (SS) were evaluated only by palpatory speen length measurement from the left costal margin without confirmation by MRI or CT scan and symptomatic improvement evaluation was present/absent (no grading). Results. We collected 83 pts, 66 treated with ruxolitinib (79.5%), 12 fedratinib (14.5%), 3 pacritinib (3.6%), 2 gandotinib/LY2784544 (2.4%). At enrolment, the 4 groups did not differ for gender, age, MF diagnosis (primary or secondary to polycythemia vera or essential thrombocythemia), DIPSS, DIPSS-plus, JAK2V617F, MPLW515 and CALR mutational status, and JAK2V617F allele burden. 64 cases (77%) were evaluated for high molecular risk status (HMR; at least one mutation in EZH2, ASXL1, IDH1/2 and SRSF2 genes): 22/64 (34.4%) were HMR with no differences among groups. Of the 3 pacritinib pts, one carried SRSF2 and one both EZH2 and ASXL1 mutation. At baseline, median hemoglobin (Hb, g/dL) levels were lower for fedratinib and pacritinib group: 11.5 (range 7-15.5) for ruxolitinib, 10 (7.4-13.3) for fedratinib, 8.1 (8-8.3) for pacritinib and 11.9 ( 11.9-12) for gandotinib (p=0.012). All pacritinib treated pts were red blood cell transfusion dependent, compared to 10.6% in ruxolitinib (7/66), 8.3% in fedratinib (1/12) and none in gandotinib (p