Association of NF2 Loss and MTOR Pathway Activation in Pleural Mesothelioma: Comprehensive Genetic Analysis of NF2 and Immunohistochemistry in 13 Cell Lines and 53 Tumors.
Marie Brevet, Matthew Bott, Qin Zhou, Valerie Rusch, Marc Ladanyi. Memorial Sloan-Kettering Cancer Center, New York, NY
Background: NF2 deletion has been described in >50% of pleural malignant mesotheliomas (MM) and NF2 inactivation has recently been causally linked to activation of the mTOR pathway in both MM and schwannomas, prompting renewed interest in defining NF2 status in MM and other cancers to guide targeted therapy with mTOR inhibitors.
Design: NF2 status was determined in 13 MM cell lines and 53 MM tumors using array CGH and sequencing. Immunohistochemistry (IHC) for NF2 (Sigma), mTOR, Phospho-mTOR (Ser2448), 4EBP1, Phosho-4EBP1 and Phosho-S6 (all from Cell Signaling) were performed on 47 tumors to look for a correlation between NF2 status and mTOR pathway activation.
Results: By array CGH, 4/13 (31%) MM cell lines showed NF2 loss and 6/13 (46%) MM cell lines showed absence of NF2 protein by western blot. Inactivating mutations were present in 5/13 cell lines (38%) (2 frameshift indels, 3 nonsense mutations). Two of 5 mutations co-occured with single copy NF2 loss (absence of NF2 protein by western in 1/2); the other 3 mutations were associated with normal NF2 copy number by aCGH but absence of NF2 protein by WB. By array CGH, 66% (35/53) of the tumors showed loss of NF2 and this was correlated with decrease of NF2 mRNA by expression microarray (p=0.002) and by IHC (p=0.007; for scoring, the % positive tumor cells was multiplied by the intensity (0 = no staining; 1 = faint staining; 2=moderate staining; 3= strong staining), giving a score between 0 and 300. Scores of triplicate TMA cores were averaged; a group comparison by ANOVA test was used). NF2 mutations were found in 11 patients (20%) (3 nonsense mutations, 1 missense mutation, 5 frameshift indels and 2 splice site mutations), of which 7 also showed single copy NF2 loss. By IHC, NF2 genomic loss was associated with increased Phospho-mTOR expression (p=0.002; similar scoring as NF2). The IHC combination of absent NF2 staining (score<100) and strong Phospho-mTOR (score > 200) was seen in 42% (13/31) of NF2-altered MM and in 12.5% of NF2-unaltered MM (2/16). We did not find significant correlations between NF2 loss and total-mTOR (p=0.08), phosho-4EBP1 (p=0.26), 4EBP1 (p=0.05) or phosho-S6 expression (p=0.22).
Conclusions: Our results confirm a high prevalence of NF2 inactivation in MM. Furthermore, we show an increase of phospho-mTOR expression in tumors lacking NF2 expression confirming the link between NF2 and the mTOR pathway. Staining for NF2 and Phospho-mTOR could be useful in selecting MM patients for trials targeting mTOR pathway activation due to NF2 loss.
Tuesday, March 1, 2011 9:30 AM
Poster Session III # 277, Tuesday Morning