Protein kinase Cɛ inhibition restores megakaryocytic differentiation of hematopoietic progenitors from primary myelofibrosis patients.

Among the three classic Philadelphia chromosome-negative myeloproliferative neoplasms, primary myelofibrosis (PMF) is the most severe in terms of disease biology, survival and quality of life. Abnormalities in the process of differentiation of PMF megakaryocytes (MKs) are a hallmark of the disease. Nevertheless, the molecular events that lead to aberrant megakaryocytopoiesis have yet to be clarified. Protein kinase Cɛ (PKCɛ) is a novel serine/threonine kinase that is overexpressed in a variety of cancers, promoting aggressive phenotype, invasiveness and drug resistance. Our previous findings on the role of PKCɛ in normal (erythroid and megakaryocytic commitment) and malignant (acute myeloid leukemia) hematopoiesis prompted us to investigate whether it could be involved in the pathogenesis of PMF MK-impaired differentiation. We demonstrate that PMF megakaryocytic cultures express higher levels of PKCɛ than healthy donors, which correlate with higher disease burden but not with JAK2V617F mutation. Inhibition of PKCɛ function (by a negative regulator of PKCɛ translocation) or translation (by target small hairpin RNA) leads to reduction in PMF cell growth, restoration of PMF MK differentiation and inhibition of PKCɛ-related anti-apoptotic signaling (Bcl-xL). Our data suggest that targeting PKCɛ directly affects the PMF neoplastic clone and represent a proof-of-concept for PKCɛ inhibition as a novel therapeutic strategy in PMF.

Mutations and long-term outcome of 217 young patients with essential thrombocythemia or early primary myelofibrosis.

We investigated the influence of molecular status on disease characteristics and clinical outcome in young patients (⩽ 40 years) with World Health Organization (WHO)-defined essential thrombocythemia (ET) or early/prefibrotic primary myelofibrosis (early-PMF). Overall, 217 patients with ET (number 197) and early-PMF (number 20) were included in the analysis. Median follow-up time was 10.2 years. The cumulative incidence of thrombosis, hemorrhages and disease evolution into myelofibrosis/acute leukemia were 16.6%, 8.6% and 3% at 15 years, respectively. No differences were detectable between ET and early-PMF patients, although the latter cohort showed a trend for worse combined-event free survival (EFS). Mutation frequency were 61% for JAK2V617F, 25% for CALR and 1% for MPLW515K, and were comparable across WHO diagnosis; however, JAK2V617F allele burden was higher in the early-PMF group. Compared with JAK2V617F-positive patients, CALR-mutated patients displayed higher platelet count and lower hemoglobin level. CALR mutations significantly correlated with lower thrombotic risk (9.1% versus 21.7%, P = 0.04), longer survival (100% versus 96%, P = 0.05) and better combined-EFS (86% versus 71%, P = 0.02). However, non-type 1/type 2 CALR mutations ('minor' mutations) and abnormal karyotype were found to correlate with increased risk of disease evolution. At last contact, six patients had died; in five cases, the causes of death were related to the hematological disease and occurred at a median age of 64 years (range: 53-68 years). Twenty-eight patients (13%) were unmutated for JAK2, CALR and MPL: no event was registered in these 'triple-negative' patients.

Lenalidomide, melphalan, prednisone and thalidomide (RMPT) for relapsed/refractory multiple myeloma.

This multicenter, open-label, non-comparative phase II trial evaluated the safety and efficacy of salvage therapy with lenalidomide, melphalan, prednisone and thalidomide (RMPT) in patients with relapsed/refractory multiple myeloma (MM). Oral lenalidomide (10 mg/day) was administered on days 1-21, and oral melphalan (0.18 mg/kg) and oral prednisone (2 mg/kg) on days 1-4 of each 28-day cycle. Thalidomide was administered at 50 mg/day or 100 mg/day on days 1-28; six cycles were administered in total. Maintenance included lenalidomide 10 mg/day on days 1-21, until unacceptable adverse events or disease progression. Aspirin (100 mg/day) was given as thromboprophylaxis. A total of 44 patients with relapsed/refractory MM were enrolled and 75% achieved at least a partial response (PR), including 32% very good PR (VGPR) and 2% complete response (CR). The 1-year progression-free survival (PFS) was 51% and the 1-year overall survival (OS) from study entry was 72%. Grade 4 hematologic adverse events included neutropenia (18%), thrombocytopenia (7%) and anemia (2%). Grade 3 non-hematologic adverse events were infections (14%), neurological toxicity (4.5%) and fatigue (7%). No grade 3/4 thromboembolic events or peripheral neuropathy were reported. In conclusion, RMPT is an active salvage therapy with good efficacy and manageable side effects. This study represents the basis for larger phase III randomized trials.

Human myeloma cells express the bone regulating gene Runx2/Cbfa1 and produce osteopontin that is involved in angiogenesis in multiple myeloma patients.

Osteopontin (OPN) is a multifunctional bone matrix glycoprotein that is involved in angiogenesis, cell survival and tumor progression. In this study we show that human myeloma cells directly produce OPN and express its major regulating gene Runx2/Cbfa1. The activity of Runx2/Cbfa1 protein in human myeloma cells has also been demonstrated. Moreover, using small interfering RNA (siRNA) to silent Runx2 in myeloma cells, we suppressed OPN mRNA and protein expression. OPN production in myeloma cells was stimulated by growth factors as IL-6 and IFG-1 and in turn OPN stimulated myeloma cell proliferation. In an 'in vitro' angiogenesis system we showed that OPN production by myeloma cells is critical for the proangiogenic effect of myeloma cells. The expression of OPN by purified bone marrow (BM) CD138(+) cells has also been investigated in 60 newly diagnosed multiple myeloma (MM) patients, finding that 40% of MM patients tested expressed OPN. Higher OPN levels have been detected in the BM plasma of MM patients positive for OPN as compared to controls. Moreover, significantly higher BM angiogenesis has been observed in MM patients positive for OPN as compared to those negative. Our data highlight that human myeloma cells with active Runx2/Cbfa1 protein directly produce OPN that is involved in the pathophysiology of MM-induced angiogenesis.

Upfront high-dose sequential therapy (HDS) versus VACOP-B with or without HDS in aggressive non-Hodgkin's lymphoma: long-term results by the NHLCSG.

BACKGROUND: There is not univocal concordance for using high-dose sequential therapy (HDS) as first-line treatment for aggressive non-Hodgkin's lymphoma (NHL). We designed this study to evaluate the usefulness of HDS followed by high-dose therapy (HDT) with autologous stem cell transplantation as front-line treatment in different subsets of aggressive NHL. PATIENTS AND METHODS: Among 223 patients aged 15-60 years with aggressive, advanced stage NHL, 106 patients were randomized to VACOP-B (etoposide, doxorubicin, cyclophosphamide, vincristine, prednisone, bleomycin) for 12 weeks (plus HDS/HDT in case of persistent disease) (arm A), and 117 patients to VACOP-B for 8 weeks plus upfront HDS/HDT (arm B). RESULTS: According to the intention-to-treat analysis, the complete response rate was 75% for arm A and 72.6% for arm B. With a median follow-up of 62 months there was no difference in 7-year probability of survival (60% and 57.8%; P = 0.5), disease-free survival (DFS) (62% and 71%; P = 0.2) and progression-free survival (PFS) (44.9% and 40.9%; P = 0.7) between the two arms. Subgroup analyses confirmed that the best results in terms of survival, DFS and PFS were achieved by patients with large B-cell NHL without bone marrow (BM) involvement, independently of the treatment arm. Results were poorer in other categories of patients and poorest in patients with BM involvement. CONCLUSIONS: Aggressive NHL patients do not benefit from upfront HDS/HDT.

Vinorelbine and prednisone in frail elderly patients with intermediate-high grade non-Hodgkin's lymphomas.

BACKGROUND: Frail patients with non-Hodgkin's lymphoma (NHL) are generally excluded from clinical trials and not even treated. The aim of this study was to evaluate the efficacy and tolerability of vinorelbine and prednisone in frail elderly patients with NHL. PATIENTS AND METHODS: Thirty consecutive frail elderly patients were entered in a phase II study with vinorelbine 25 mg/m2 i.v. on days 1 and 8 and oral prednisone 30 mg total dose on days 1-8 for six cycles. Criteria of frailty were age > or =80 years, or age > or =70 years and three or more comorbidities of grade 3 or at least one comorbidity of grade 4 according to the Cumulative Illness Rating Scale (CIRS), or not self-sufficient or the presence of one or more geriatric syndromes. RESULTS: Of 30 evaluable patients, three (10.0%) achieved a complete response (CR), nine (30.0%) showed a partial response (PR), while 10 presented with stable disease and eight with progressive disease. The median duration of CR was 29 months (range 5-36 months), and the median duration of PR was 1 month (range 1-22 months). Three patients had grade 3 neutropenia and one had grade 4. One grade 4 neurotoxicity was observed. Three patients died because of heart failure within 28 days of therapy, and one patient died after 4 days because of rapid progression. The median overall survival was only 10 months. CONCLUSION: Vinorelbine and prednisone is a relatively non-toxic combination with modest activity in frail patients with NHL. If initial aggressive chemotherapy has been excluded, this combination could be tried to obtain a temporary palliation.