In the WHO diagnostic classification, prefibrotic myelofibrosis (pre-MF) is included in the category of primary myelofibrosis (PMF). However, strong evidence for this position is lacking.
We ...investigated whether pre-MF may be aligned along a clinical and biological continuum in 683 consecutive patients who received a WHO diagnosis of PMF.
As compared with PMF-fibrotic type, pre-MF (132 cases) showed female dominance, younger age, higher hemoglobin, higher platelet count, lower white blood cell count, smaller spleen index and higher incidence of splanchnic vein thrombosis. Female to male ratio and hemoglobin steadily decreased, while age increased from pre-MF to PMF- fibrotic type with early and to advanced bone marrow (BM) fibrosis. Likely, circulating CD34+ cells, LDH levels, and frequency of chromosomal abnormalities increased, while CXCR4 expression on CD34+ cells and serum cholesterol decreased along the continuum of BM fibrosis. Median survival of the entire cohort of PMF cases was 21 years. Ninety-eight, eighty-one and fifty-six percent of patients with pre-MF, PMF-fibrotic type with early and with advanced BM fibrosis, respectively, were alive at 10 years from diagnosis.
Pre-MF is a presentation mode of PMF with a very indolent phenotype. The major consequences of this contention is a new clinical vision of PMF, and the need to improve prognosis prediction of the disease.
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.
Myeloproliferative neoplasms (MPNs) are clonal disorders characterized by excessive production of mature cells. In most of the classic Philadelphia-negative MPNs—polycythemia vera (PV), essential ...thrombocythemia (ET), and MPN-associated myelofibrosis (MPN-MF)—oncogenic mutations affecting JAK2 or MPL lead to constitutive activation of cytokine-regulated intracellular signalling pathways. The traditional therapy for PV and ET is the prevention of thrombotic events with antiproliferative agents in association with aspirin. New drugs such as pegylated interferon and anti-JAK agents are candidates for slowing the evolution to myelofibrosis or leukemia. Conventional therapy for MPN-MF is driven by clinical needs, primarily anemia and splenomegaly. Lenalidomide and pomalidomide are new candidates for treating anemia. JAK2 ATP-competitive inhibitors or drugs that indirectly inhibit the JAK-STAT pathway, like RAD001, are the new candidates for splenomegaly in MPN-MF, but in spite of their strong rationale, they have shown only a palliative benefit. Allogeneic stem cell transplantation remains the only potentially curative approach.
Abstract 1743
WHO diagnostic criteria of myeloproliferative neoplasms (MPNs) identify a preMF variant including patients having absence or early grade fibrosis, dual myeloid and megakaryocytic ...dominance, and clusters of atypical, ectopic, variable in size megakaryocytes in the bone marrow (BM). Many issues on the variant are critical, such as its distinction from essential thrombocythemia, recognisability, biological identity and true place among MPNs. Here we describe the clinical presentation of patients with preMF collected among a large cohort of patients diagnosed with PMF according WHO and for whom long-term follow-up data were available. The aim is to investigate whether there is an underlying structure of the patients' characteristics that allows to assign them to a distinct variant of MPNs or to align them along a continuum in the realm of PMF.
We included in this study patients with PMF who were referred to the Center for the Study of Myelofibrosis of IRCCS Policlinico S. Matteo Foundation, Italy from 1990 to 2011. In all patients the BM biopsy taken at diagnosis was reviewed (pathologist: UM) and the diagnosis of preMF was established adopting the stringent criterion that, besides typical BM cellularity and megakaryocyte morphology, BM had less than grade 1 reticulin fibrosis (EUMNET criteria).
Of the 659 patients with PMF, 126 fulfilled the criteria we established for the diagnosis of preMF, thus accounting for 19.1% of the cohort. Female sex accounted for 59.5% of the preMF cohort while it accounted for 33.8% of PMF fibrotic type (P<0.0000). The mean age at diagnosis of patients with preMF was 39.2 years (range, 6 to 78 years), while it was 54.6, range 6 to 90 years (P<0.0000) in patients with PMF fibrosis type. Patients with preMF showed higher hemoglobin (14.0 vs. 11.9 g/dL; P=0.0004), higher platelet count (647 vs. 435 × 109/L; P<0.0000), lower WBC count (9.0 vs. 10.7 x109/L; P=0.02) and smaller spleen index (120 vs. 182; P<0.0000) than patients with MF fibrotic type. As a consequence, preMF displayed a greater frequency of patients with grade 0 IWG prognostic score ( 88.7% vs. 56.9%, P<0.0000). PreMF patients displayed a higher frequency of thrombotic events at the time of the diagnosis or in the year preceding the diagnosis (30.1% vs. 6.4%; P<0.0000). Both in prefibrotic and fibrotic type MF, the great majority of the thrombotic episodes were portal vein thrombosis or Budd-Chiari syndrome (89.4% in preMF and 79.4% in PMF fibrotic type). The proportion of JAK2 V617F mutated patients was not significantly different in the two categories of patients (65.2% vs. 62.1%; P=NS).
In order to assign patients with PMF to groups with different degrees of BM fibrosis, we further categorized MF fibrotic type into early MF (BM fibrosis grade 1) and advanced MF (BM fibrosis grade 2 or 3). Female frequency steadily varied from 59.5% to 43.2% and 25.6% in the 3 categories of BM fibrosis, respectively. Age varied from 39.2, 50.4, 57.0 years, respectively. Hemoglobin and platelet count steadily decreased (Hb=14.0, 13.3, 11.2 g/dL, respectively; platelet count=647, 554, 346 x109/L, respectively). Spleen size steadily increased along with increasing of BM fibrosis (spleen index=120, 152, 200). All these parameters exhibited a statistically significant trend along the continuum of BM fibrosis grade (ANOVA, P<0.0001). During 993 cumulative person-years of follow-up, 4% of patients with preMF died. During 1372 cumulative person-years of follow-up, 15.4% of patients with early MF died. During 1446 cumulative person-years of follow-up, 24.5% of patients with advanced MF died. The cause of death was blast transformation in 80, 53 and 41.7% of cases, respectively. 94%, 80% and 54% of patients with preMF, early MF and advanced MF were alive at 10 years from the diagnosis. Overall, these results speak in favour to preMF belonging to a continuum of epidemiological, clinical and histological phenotypes in the realm of PMF. Splanchnic vein thrombosis, on the contrary, clusters in the category of preMF, suggesting that young age, female sex and a very indolent hematological phenotype are associated with biological characteristics that predispose to thrombosis.
The BM picture of preMF identifies a group of patients with PMF associated with a very indolent hematologic phenotype, female dominance and very young age at presentation characterized by high incidence of splanchnic vein thrombosis.
Barosi:Novartis: Membership on an entity's Board of Directors or advisory committees.
Abstract 314
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.
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).
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).
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.
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.
In patients with hepatocellular carcinoma (HCC) receiving sorafenib, drug resistance is common. HCC develops in a microenvironment enriched with extracellular matrix proteins including laminin ...(Ln)‐332, produced by hepatic stellate cells (HSCs). Ln‐332 is the ligand of α3β1 and α6β4 integrins, differently expressed on the HCC cell surface, that deliver intracellular pathways. The aim of this study was to investigate the effect of Ln‐332 on sorafenib's effectiveness. HCC cells were challenged with sorafenib in the presence of Ln‐332 and of HSC conditioned medium (CM). Sorafenib impaired HCC cell proliferation and induced apoptosis. HSC‐CM or Ln‐332 inhibited sorafenib's effectiveness in HCC cells expressing both α3β1 and α6β4. Inhibiting α3 but not α6 integrin subunit using blocking antibodies or small interfering RNA abrogated the protection induced by Ln‐332 and HSC‐CM. Hep3B cells expressing α6β4 but lacking the α3 integrin were insensitive to Ln‐332 and HSC‐CM protective effects. Hep3B α3‐positive, but not wild‐type and scramble transfected, cells acquired protection by sorafenib when plated on Ln‐332‐CM or HSCs. Sorafenib dephosphorylated focal adhesion kinase (FAK) and extracellular signal‐regulated kinases 1/2, whereas Ln‐332 and HSC‐CM partially restored the pathways. Silencing FAK, but not extracellular signal‐regulated kinases 1/2, abrogated the protection induced by Ln‐332 and HSC‐CM, suggesting a specific role for FAK. Sorafenib down‐regulated total FAK, inducing its proteasomal degradation, while Ln‐332 and HSC‐CM promoted the escape of FAK from ubiquitination, probably inducing a preferential membrane localization. Conclusion: This study unveils a novel mechanism of sorafenib resistance depending on the α3β1/Ln‐332 axis and requiring FAK ubiquitination, providing new insights into personalizing therapy for patients with HCC. (Hepatology 2016;64:2103‐2117).
In patients with hepatocellular carcinoma (HCC) receiving sorafenib, drug resistance is common. HCC develops in a microenvironment enriched with extracellular matrix proteins including laminin ...(Ln)-332, produced by hepatic stellate cells (HSCs). Ln-332 is the ligand of alpha 3 beta 1 and alpha 6 beta 4 integrins, differently expressed on the HCC cell surface, that deliver intracellular pathways. The aim of this study was to investigate the effect of Ln-332 on sorafenib's effectiveness. HCC cells were challenged with sorafenib in the presence of Ln-332 and of HSC conditioned medium (CM). Sorafenib impaired HCC cell proliferation and induced apoptosis. HSC-CM or Ln-332 inhibited sorafenib's effectiveness in HCC cells expressing both alpha 3 beta 1 and alpha 6 beta 4. Inhibiting alpha 3 but not alpha 6 integrin subunit using blocking antibodies or small interfering RNA abrogated the protection induced by Ln-332 and HSC-CM. Hep3B cells expressing alpha 6 beta 4 but lacking the alpha 3 integrin were insensitive to Ln-332 and HSC-CM protective effects. Hep3B alpha 3-positive, but not wild-type and scramble transfected, cells acquired protection by sorafenib when plated on Ln-332-CM or HSCs. Sorafenib dephosphorylated focal adhesion kinase (FAK) and extracellular signal-regulated kinases 1/2, whereas Ln-332 and HSC-CM partially restored the pathways. Silencing FAK, but not extracellular signal-regulated kinases 1/2, abrogated the protection induced by Ln-332 and HSC-CM, suggesting a specific role for FAK. Sorafenib down-regulated total FAK, inducing its proteasomal degradation, while Ln-332 and HSC-CM promoted the escape of FAK from ubiquitination, probably inducing a preferential membrane localization. Conclusion: This study unveils a novel mechanism of sorafenib resistance depending on the alpha 3 beta 1/Ln-332 axis and requiring FAK ubiquitination, providing new insights into personalizing therapy for patients with HCC. (Hepatology 2016; 64:2103-2117).
Eosinophilic granulomatosis with polyangiitis (EGPA) is a rare systemic vasculitis. Cardiac specific involvement (CSI) is caused by coronary artery vasculitis, but also by myocardial eosinophilic ...infiltration. To date, the prevalence of CSI associated with EGPA is unresolved. Aim of this study was to systematically assess the prevalence and clinical impact of CSI in a consecutive outpatient EGPA population.
Between October 2018 and July 2019, we prospectively enrolled 52 consecutive EGPA patients. All underwent comprehensive evaluation including a standardized questionnaire, physical examination, 12-lead-ECG, echocardiography. Cardiac magnetic resonance and 24 h-Holter were performed as deemed clinically appropriate. Cardiac abnormalities were defined as CSI based on the likelihood of their relation to EGPA vasculitis, after exclusion of alternative diagnoses.
52 enrolled patients, mean age 59±1 years. Thirteen of the 52 patients (25%) were classified as CSI+. CSI was characterized by myocarditis in four patients, non-scar-related regional wall motions abnormalities (RWMA) in three, apical thrombosis in two (one also had RWMA), pericarditis in three and non-atherosclerotic coronary disease (Prinzmetal angina and coronaritis) in 2. Five (38%) of the 13 CSI+ patients, presented an apical aneurysm. Peak eosinophil count at diagnosis was higher in CSI+: 8000 /μl vs CSI-: 3000 /μl, p = 0.017. Overall, 2 patients had severe LV dysfunction, 5 required urgent hospitalization and 8 required long-term cardioactive therapy.
CSI was present in one-quarter of patients, often associated with high peak eosinophils. Myocarditis, RWMA and apical aneurysms were the most common manifestations. Although rarely severe and life-threatening, CSI often required long-term cardioactive treatment.
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