ALBERT

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 1892 Poster Board I-915 There are conflicting results about quantitative modifications of V617F allele burden in patients (pts) with myeloproliferative neoplasms (MPN) who are either therapy-naive or are treated with hydroxyurea (HU). In a retrospective single center study in 48 pts with polycythemia vera (PV) or essential thrombocythemia (ET) the granulocyte JAK2V617F allele burden remained stable over time (median follow-up was 34 months for PV and 23 for ET) irrespective of the pts being treated or not with cytotoxic therapy (Theocharides A et al, Haematologica 2008). Conversely, another study in 25 patients reported a significant reduction of V617F allele burden (〉30% of baseline level) after HU therapy in 52% of the pts, becoming indetectable in 3 of them (Girodon F et al, Haematologica 2008). The aim of this study was to evaluate any modifications of JAK2V617F allele burden during long-term follow-up in patients with PV or ET and the effects of HU treatment. This two-center (Firenze and Bergamo) retrospective study concerned 172 patients with a diagnosis of PV or ET according to the WHO criteria. The only study inclusion criteria were the presence of JAK2V617F mutation and the availability of at least two sequential blood samples drawn at an interval time of at least 6 months. The JAK2-V617F allele load was measured by sensitive quantitative RT-PCR in granulocyte DNA according to the method of Lippert et al (Blood 2006). Differences between median values of JAK2 V617F allele burden were tested by the Wilcoxon matched-pairs signed-ranks test. Repeated measure test for JAK2 V617F mean change over time, irrespective of diagnosis, was also calculated to investigate a significant variance among ordered time measures. There were 103 pts with PV and 69 with ET; median age was 56 yr (range, 15–84), females were 49%. The median interval time between the baseline and follow-up sample in the whole pt population was 27 months (range 6–60), 26 and 28 months for ET and PV, respectively. The median patient follow-up was 3 years (range 0.5–25); no evolution to myelofibrosis or acute leukemia was recorded. According to previous reports, the mean V617F burden was significantly greater in PV than in ET pts (50 ± 26% and 32 ± 18%, respectively; p

Description: Abstract 1060 Poster Board I-82 Clofarabine has been shown to be effective in AML patients, either as single agent or, mainly, in association with intermediate dose cytarabine. On the basis of these reports, we conducted a preliminary study combining clofarabine, intermediate dose cytarabine and gemtuzumab ozogamicin (Mylotarg) in AML patients who relapsed or failed to respond to at least two induction therapies. We treated 20 patients affected by relapsed/refractory AML with a regimen including clofarabine at 22,5 mg/m2 daily on days 1-5, followed after three hours by cytarabine at 1 gr/m2 daily on days 1-5, with the addition of gemtuzumab ozogamicin 6 mg/m2 on day 6 (CLAC-Myl). Six patients received a further consolidation cycle with clofarabine at 22,5 mg/m2 and cytarabine at 1 gr/m2 day 1-4. Among the twenty patients, six were in first relapse, six in second relapse, eight with resistant disease. The mean age was 52 years (range 33-68 years), the white blood count at the accrual was 31.500 mcc (range 2140-153.000). 10/20 (50%) patients achieved a complete remission, 1/20 a partial response, 7/20 had resistant disease, 2/20 died of complications during the aplastic phase (a case of multiorgan failure an a septic shock caused by Pseudomonas Aeruginosa). The most frequent non hematologic adverse events were vomiting, diarrhea, transient liver toxicity (2/20 grade 3-4), febrile neutropenia (7/20), infections microbiologically documented (2/20 Pseudomonas Aeruginosa sepsis). Comparing with other salvage strategies, in this small cohort of patients we did not observe a significant delay in bone marrow recovery (median time to ANC recovery 31.5 days), except in a patient (female, 34 years old, relapsed after ABMT) that experienced an unexpected, irreversible aplasia after the consolidation course, complicated by an unusual HHV6 reactivation. Among the ten responding patients, three underwent allogeneic bone marrow transplantation, one patient still in CR after 7 months died for complications of an acute myocardial infaction occurred during the consolidation course, one relapsed after 6 months, and five not eligible for transplant procedures are still in complete remission with a median follow up of 6 months. These very preliminary results suggest that the CLAC-Myl regimen is effective in this particularly poor prognosis category of patients, with safety data consistent with previously reported salvage therapies. Further studies are warranted. Disclosures: Off Label Use: Clofarabine in relapsed/refractory AML.

Description: Abstract 314 Background: Akt/mTOR activation has been found in association with dysregulated JAK2/STAT5 signaling due to JAK2V617F mutation. Increased phosphorylation of STAT5 and Akt was detected in patients (pts) with myeloproliferative neoplasms (MPN) (Grimwade LF, BJH 2009). We reported (Bogani C, ASH 2009) that RAD001, an oral inhibitor of mTOR, inhibited the proliferation of human and murine JAK2V617F-mutated cells and impaired colony formation by MPN progenitor cells, suggesting potential clinical activity. Methods: We will report final data from an investigator-initiated, multicenter phase I/II trial with RAD001 in myelofibrosis, primary (PMF) and post-polycythemia vera/essential thrombocythemia (PPV/PET MF). Inclusion criteria were intermediate/high risk score (Lille criteria) or need of treatment because of progressing splenomegaly. Phase I involved a 3+3 pts scheme in 3 sequential cohorts at 5.0, 7.5, and 10 mg daily for 3 mo to establish maximum tolerated dose (MTD). Phase II was a two-stage Simon design involving 16+14 pts at MTD for 4 mo. The protocol was approved by IRBs and pts provided informed consent. The study was supported by Agenzia Italiana per il Farmaco (AIFA) and AIRC-Gruppo Italiano Malattie Mieloproliferative (AGIMM). Results: Phase I: there was no DLT at 10 mg daily, considered as MTD; there were 2 major (MR), 3 moderate (MO) and 1 minor (MI) and 3 no (NR) responses. In part 1 of Phase II, 〉1/16 pts achieved MR; according to study design, 14 additional pts were enrolled in part 2 (n=30). 20 patients had low and 10 intermediate Lille score; according to IWG-MRT criteria, 5, 10, 8 and 7 were low, int-I, int-II and high risk score, respectively. There were 16 PMF, 8 PPV- and 6 PET-MF. 20 pts were JAK2V617Fpos, 3 were MPLW515pos. Patients evaluable for intention-to-treat (ITT) analysis as of aug 1st were 26; the remaining 4 pts will be updated at meeting. Therapy was discontinued in 5 pts: 1 pt's decision at d60 without evidence of any 〉grade-2 toxicity, 2 at d60 and d90 for physician's decision due to grade-2 toxicity, 1 death due to respiratory failure at d60, 1 at d30 for grade-3 acute renal failure. 21/26 pts (81%) were available for per-protocol analysis. Therapy was generally well tolerated; commonest toxicities were grade-2 mouth ulcers and grade-1/2 hypertrigliceridemia. Hematological toxicities: 3 grade-2 and 4 grade-3 reversible anemia, one grade-2 neuthropenia, one grade-2 and 1 grade-3 reversible thrombocytopenia. A reduction of spleen size consistent with CR, PR or NR was obtained in 8%, 46%, and 46% of the pts, respectively. 11 of 21 pts (52%) had complete resolution of systemic symptoms, and 14 of 19 pts (74%) reported disappearance of pruritus. Two pts achieved PR in anemia with decrease in transfusion requirement 〉50%, while in 3 pts Hb increased of 2g/dL. A CR in platelets was obtained in 3 of 19 pts and CR in leucocytes in 2 of 18 pts with abnormal blood count. Overall, according to ITT analysis: 6 major (23%), 6 moderate (23%), 2 minor (8%) and 12 NR (46%) (EUMNET criteria). According to IWG-MRT criteria, there were 6 (23%) clinical improvement. Per-protocol analysis: 33% major, 19% moderate, 5% minor, 43% NR; 24% clinical improvement. No disease progression. Changes induced by RAD001 in some biological parameters were preliminary evaluated and will be finally updated at meeting. Blood levels of CCDN1 mRNA, a target of mTOR, significantly decreased in responders (MR+MO) versus non-responders (P=0.02). In the 13 JAK2-mutated pts who completed the treatment, the V617F allele burden was 61.6+/−15.8 versus 66.6+/−19.6% at baseline. There was a trend (P=0.07) towards a reduction of granulocyte WT1mRNA copy number in responders versus non-responders. We found no correlation of circulating CD34+ cells or CXCR4 expression with clinical response. A set of 46 inflammatory protein markers and cytokines were quantified before and at d30 of treatment. Some, including IL-10 and MIP-1beta, showed significant decrease while others increased, including Factor VII, IL-8 and MMP-2. Levels of MIP-1 beta were significantly lower in responders (P=0.006). Conclusions: These data indicate that mTOR pathway targeting with RAD001 in MF pts induces an appreciable rate of response in splenomegaly, constitutional symptoms, and pruritus, with low-grade toxicity. However, effects on JAK2V617F mutational load were minimal, and should be better evaluated after longer trial duration. Disclosures: Vannucchi: Novartis: Membership on an entity's Board of Directors or advisory committees. Off Label Use: This is an investigator-initiated, non-sponsored, clinical trial with RAD001 in myelofibrosis. Paratore:Novartis: Employment.

Description: Abstract 431 Background. Primary myelofibrosis (PMF) has the worst prognosis among myeloproliferative neoplasms with median overall survival (OS) of 4.6y in the International Prognostic Scoring System (IPSS) series (Cervantes F, Blood 2009;113:2895) and 6.5y in patients (pts) seen more recently (Cervantes F, JCO, 2012 in press). OS is predicted by the four risk categories of IPSS, dynamic-IPSS and IPSS-plus system, and these scores are used for therapeutic choices particularly allogeneic stem cell transplantation. Nevertheless, pts heterogeneity still remains within these categories, necessitating improved risk stratification. A number of molecular abnormalities have been reported in PMF pts, but their prognostic relevance is incompletely understood, particularly with regard to transformation to leukemia (AL). The aim of this work was to analyze the prognostic impact of known mutations detected close to diagnosis in an international series of 429 pts. Patients and methods. PMF diagnosis had to satisfy the 2008 WHO criteria. Mutations in JAK2V617F, MPLW515, EZH2, ASXL1, TET2, IDH1/2, DNMT3A, CBL, SRSF2 were genotyped in whole blood or granulocytes using allele specific RTQ-PCR, HRM and direct sequencing; all mutations were confirmed at least twice. Missense, nonsense and frameshift mutations were considered; in case of novel mutations, SNPs were excluded by database searching and when feasible by germline DNA genotyping. The prognostic value of the molecular variables with regard to overall survival (OS) was analyzed by Cox regression and adjusted for the IPSS category. The association of molecular features with the risk of progression to AL was investigated in the framework of competing risks by the Fine & Gray regression method. Replicability of the prognostic models for both OS and progression to AL was assessed by replication in 1000 bootstrap samples randomly taken from the original series. Results. Patient median age was 60y. Median follow-up was 3.7y (95% CI, 0.02–27.9), death occurred in 157 pts (32%). Frequency of pts with constitutional symptoms was 28%, splenomegaly 74%, anemia 27%, leukocytosis 8%, 〉1% blasts 16%, thrombocytopenia 12%. Abnormal karyotype was found in 24% (of 229 evaluated). IPSS risk category: low-risk 35%, Int-1 30%, Int-2 21%, High-risk 14%. Frequency of mutations was: JAK2V617F 59.8% with 49% of pts having

Description: Abstract 5055 Classical chronic myeloproliferative neoplasms (MPNs) are amongst the best-characterized neoplasms associated with one of a set of specific somatic mutations, the most common of which is in the JAK2 gene. MPN is a relatively rare condition, and it could be surmised that it develops preferentially in people who have an increased tendency to somatic mutations, i. e. an increased somatic mutation rate (μ). Since measuring μ is rather labor-intensive, in this study we have measured instead the frequency of peripheral blood granulocytes that have inactivating mutations in a reporter gene, namely the gene PIG-A, a gene whose protein product is required for numerous glycosyl-phosphatidylinositol-anchored proteins to become surface bound. Since several such proteins are displayed by normal granulocytes, mutant cells can be numbered accurately by flow cytometry; and the frequency (ƒ) of such mutant cells may be a good surrogate of μ (Peruzzi et al. Mutation Research. 2010, 705:3). We have determined ƒ in 74 patients with MPN (including 29 with polycythemia vera (PV), 17 with essential thrombocythemia (ET), 16 with primary myelofibrosis (PMF) 12 with post-PV/post-ET (PPV/PET) MF. Overall the proportion of those with a JAK2(V617F) mutation was 90%. In a subset of 59 of these patients, who had “uncomplicated” MPNs, the median of ƒ was no different compared to 142 healthy controls (4. 74×10−6vs. 4. 87×10−6: Figure 1). However, in a subset of 15, consisting of patients who in addition to MPN had at least one primary malignancy (2 had lymphomas and 13 had one of several types of solid tumors), the median value of ƒ was 15. 22×10−6, i. e. significantly elevated (Mann Whitney test: MPNs vs. MPNs with a second neoplasm, p