Based on array-CGH data in NSCLC, it has been shown that multiple molecular carcinogenesis pathways exist that are most likely related to gender and smoking habits 
. Furthermore, it was shown that there is a large overlap between aberrations observed in the adenocarcinoma and squamous cell carcinoma subtype, except for 3q gains which seem to be more specific for the squamous cell carcinoma subtype 
. Various gene expression signatures have been correlated to survival of NSCLC patients 
. In addition, molecular studies have allowed the development of personalized treatment approaches in several tumor types 
. However, it is likely that many cancer-related target genes have not been identified yet. In this regard, integrated genome wide screening of copy number changes and gene expression using microarrays has been recently carried out in various tumor types to identify genes whose expression is affected by gene dosage 
. These studies aim to identify novel cancer-related genes and to define novel biomarkers for response or prognostic signatures. In both NSCLC and ductal pancreatic cancer, two focal amplifications of 8p12 and 20q11 have been studied in detail leading to two candidate genes (WHSC1L1 and TPX2) important in these diseases 
We here performed an integrated genome wide screening of gene copy number changes and gene expression in 32 radically resected NSCLC patients, in order to identify novel NSCLC-related genes. By using ACE-it, a novel informatics tool for integration of gene dosage and gene expression data, we identified 359 transcripts to be significantly affected by copy number. A cox survival analysis on all 359 genes revealed no significant relation after multiple testing. The top list of genes related to survival mainly included genes residing on gained regions 3q and 5p. These regions cover many genes and to pinpoint the gene of most importance is a challenging task. Further investigations should elucidate the importance of these genes in relation to NSCLC, in particular of those in the top list of correlation with survival such as SLC45A2, WDR70 and NIPBL (see supplementary table S1
). In this paper we focused on the recurrent deletion on chromosome 14 and the gene HSP90, which was also in the top list of relation with survival and not previously investigated in detail. Deletion of region 14q32.2-33 was correlated with improved survival, further suggesting that it may contain one or more genes related to NSCLC progression. Deletion of this region has been previously described by one group reporting genomic aberrations in NSCLC, but was not investigated in further detail 
. Out of the 109 genes mapping to the 14q32.2-33 region, HSP90 was the only gene with significantly lower expression in patients harboring the 14q32.2-33 deletion. In the initial series of 29 patients (3 patients excluded from survival analysis), we observed improved survival in patients with lower levels of HSP90. The association between HSP90 expression levels and NSCLC patient prognosis was confirmed to be significant in three independent validation sets of NSCLC patients. Multivariate analysis including stage, histology, age and gender showed that HSP90 remained independently related to survival.
A critical issue in defining “low” and “normal” expression is the choice of an appropriate cut off value. In the initial analyses we used the median of expression ratios as cut off between “low” and “normal” expression, since the low-risk and high-risk separation of patients was equal. However, in the validation sets the low-risk and high-risk survival groups were not equally balanced (two thirds versus one third). Consequently the cut off values used in these data sets was not the median value, but the 33-percentile.
In this study, using a genome wide integrative analysis of gene copy number and expression we were able to identify expression of HSP90 as an important gene in early stage NSCLC patients. HSP90 is a chaperone protein involved in the stabilization of multiple oncoproteins such as EGFR, Her-2 and Akt 
. Recent work has shown that HSP90 plays a role in maintaining the active conformation of EGFR and in particular EGFR mutants 
. We show here that several NSCLC cell lines bearing wild-type EGFR are sensitive to HSP90 inhibition, indicating that the inhibitory effect of 17-AAG can not be solely attributed to mutant EGFR. HSP90 has been recently recognized as a potential cancer therapeutic target and investigations of HSP90 inhibitors are ongoing 
. In this regard, glioblastoma cells overexpressing EGFR, but resistant to inhibition by EGFR kinase inhibitors, were sensitive to HSP90 inhibition 
. Therefore, while Hsp90 may be required to stabilize over-expressed or mutated EGFR, our data support a more wide-ranging and complex role for Hsp90 in mediating NSCLC growth and survival. The low nanomolar sensitivity observed in the 4 cell lines tested in our experiments, is in agreement with other published reports of highly 17-AAG sensitive tumor cell lines 
. These concentrations are readily achievable in patients for prolonged time periods using current scheduling and dosing regimens 
In summary, the observation that HSP90 expression level is a prognostic factor for NSCLC patient survival (independent of EGFR mutational status), coupled with the extreme sensitivity of EGFR wild type NSCLC cells to the Hsp90 inhibitor 17-AAG, suggests that Hsp90 inhibitors may have greater clinical utility in NSCLC than has been previously considered and warrants further investigation of the dependence of other proto-oncogenes on this chaperone protein in NSCLC.