High Expression of mRNA and Gene Amplification of Met In Myeloma Plasma Cells Characterize a More Aggressive Disease

Multiple myeloma (MM) cells growth is sustained by several stimuli derived from surrounding cells of bone marrow (BM) microenvironment. Besides the increase in growth factors production, a constitutive activation of several pathways determining protection from apoptosis and growth advantage have been reported too. Aberrant activation of Met/HGF (Hepatocyte Growth Factor) pathway has been described in several tumors causing cell proliferation, cell migration and neoplastic angiogenesis. A qualitative analysis demonstrated that Met transcription is present in MM plasma cells and increase plasmatic levels of HGF has been related to a bad prognosis subgroup of patients. However a comprehensive investigation of quantitative analysis on Met expression, plasmatic HGF values and correlation with clinical outcome on a large amount of MM patients treated with novel agents is still missing.

: to investigate the role of Met mRNA expression and HGF levels in a large panel of myeloma patients treated with novel drugs.

one hundred and five samples of purified plasma cells derived from newly diagnosed myeloma patients have been included in this study. Fifty two patients received 9 courses of Velcade-Melphalan-Prednisone (VMP) as part of the VMP-VMPT trial (Palumbo A, 2009 ASH Meeting, abs 128) while fifty three patients have been included in the PAD-MEL100-LP-L trial (Palumbo A, JCO 2010). Met and HGF mRNA quantitative expression have been investigated on both purified plasma cells (CD138+ bone marrow fraction) and on bone marrow CD138 negative fraction. mRNA expression has been evaluated using a quantitative Real-Time PCR (qRT-PCR) on Abi Prism 7900 (Applied Biosystems) with a relative quantification based on ΔΔCt approach. JUM2 cell line was used as calibrator and Gus as housekeeping gene. HGF serum level has been evaluated on 76 of those patients too. ELISA assay has been employed to determine HGF serum value. On 51 samples with higher levels of mRNA Met expression, a FISH analysis reaching for Met amplification has been performed. Purified plasma cells were treated with a dual-color FISH assay using a MET/CEP7 probe cocktail (Cytocell, Cambridge, UK).

Met mRNA expression was higher in CD138+ cells than in CD138- fractions (median 76,90 range 0,81-916,51 vs 11,03 range 0–243,88 respectively; p=0,0001). Similarly HGF mRNA expression was higher in CD138+ cells than in CD138- population (median 2,07 range 0–65,34 vs 0,49 range 0,11-3,22 respectively; p=0,03). Patients with high and low Met levels were divided using the median value of Met expression as cutoff. At a median follow up of 30 months, patients with low Met mRNA expression displayed a higher progression free survival (PFS) and overall survival (OS) compared with those with high Met levels (PFS 73% vs 42% respectively, p<0,001; OS 94% vs 78%, p=0,01). No differences in baseline albumin, β2-microglobulin, BM plasma cell infiltration and cytogenetic profile have been observed between patients with high and low Met mRNA levels. No differences in outcome were demonstrated when patients were divided using HGF mRNA level as parameter. No correlation between Met and HGF mRNA values have been found. Quantification of HGF levels in serum confirmed the increase of this growth factor in myeloma patients in comparison with age-matched healthy subjects (median 2082 pg/ml range 740–9000 vs median 1586 pg/ml, range 420–3079 respectively; p=0.03). FISH analysis revealed amplifications of Met gene in 20 samples out of 50 (40%). In 13 samples three copies of Met gene have been detected in more than 50% of cells, while in 7 samples 4 or more copies have been found in more than 50% of cells.

1) high Met mRNA expression levels identify a group of patients with worse prognosis even when treated with novel drugs containing regiments; 2) Met amplification has been described for the first time in myeloma cells and can determine high Met mRNA levels characterizing a more aggressive disease.

Cavallo:Celgene: Honoraria. Patriarca:Roche: Honoraria; Janseen-Cilag: Honoraria; Celgene: Honoraria; Merck: Membership on an entity's Board of Directors or advisory committees. Boccadoro:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janseen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Palumbo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janseen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees.