Lung cancer is the leading cause of cancer-related deaths in the western world [1]. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of the cases and represents a heterogeneous group mainly consisting of adenocarcinoma (ADC), large cell carcinoma (LCC) and squamous cell carcinoma (SCC). The incidence of subtypes has changed in the last decades with increasing incidence of ADC. Moreover, while smoking remains the major risk factor for lung cancer, a subgroup of patients develop lung ADC without smoking history. It is not clear whether lung cancer in non-smokers is increasing in western countries but it is obvious that it has particular clinical and biological features. Population based studies showed that lung cancer in non-smokers occurs preferentially in women with almost 20% of non-smoker lung cancer diagnosed in women versus 2.5% in men [2]. Different studies have shown that genetic abnormalities can be specifically identified in cancer from non-smokers. Indeed we and others showed that KRAS mutations were linked to tobacco consumption whereas EGF receptor (EGFR) mutations were found in non smokers [3–5]. The development of EGFR targeted therapies demonstrated that patients with major clinical response were those that had never smoked, had ADC with bronchioloalveolar component, were women and had EGFR mutations [6–8]. The biology underlying the pathogenesis of the disease may be different from that of smokers and risk factors have not been clearly identified although environmental etiologies are suspected especially in Asians [9]. Transformation of a normal phenotype into a malignant phenotype requires accumulation of multiple genetic and-or epigenetic changes resulting in growth advantage.

The genetic alteration proved to be linked with ADC from non-smokers is the presence of EGFR activating mutations. In order to improve our knowledge of lung cancer biology in non-smokers, one of the first questions to answer is: what are the molecular alterations associated to EGFR mutations in lung cancer?

Lung cancer develops as a result of multiple genetic alterations. Loss of heterozygosity (LOH) and gains of chromosome segments are common mechanisms of disease progression. Recently, high-density oligonucleotide-based single polymorphism have been used to quantify chromosome copy number and has been proved to be efficient [10–12]. In an attempt to identify genetic alterations associated with EGFR mutations, we used genome wide SNP assay covering 50000 SNP loci to screen for regions of allelic imbalance (amplified or LOH regions) in a panel of 13 EGFR mutated ADC and 11 non-EGFR mutated ADC.

Then, in a second part, we focused on cell cycle genes that were found to be differentially involved between groups and screened a large series of 98 NSCLC for genetic alterations at CCND1, CCNE1, CDKN2A and RB1. Alterations were studied according to other known mutations (EGFR, ERBB2, BRAF, KRAS, TP53 and STK11). This work led to the characterization of two different models of oncogenic cooperation one linked to smoking and the other not. Moreover, we identified four chromosome regions at 14q and 7p specifically amplified in EGFR mutated ADC.

Patients with primary lung cancers were enrolled in this study according to French laws and have been previously described [4]. Briefly, patients had surgery for non-small cell lung cancer, no neoadjuvant treatment and were managed to the Georges Pompidou European Hospital in Paris, France from 2003 to 2004. All tumors but numbers 134, 135, 177 and 246 had been characterized for mutations in EGFR, KRAS, BRAF, ERBB2, ERBB3, PIK3CA, TP53 and STK11 [4]. STK11 mutations have not been previously published. Patient characteristics are summarized in Table 3. DNAs were extracted after pulverization in liquid nitrogen and protein kinase digestion using Qiamp tissue kit (Qiagen, Les Ulis, France). Twenty-four DNAs (13 with classic EGFR mutations, 11 without) and 6 non-tumor DNAs were selected for SNP array analysis. All tumors selected were from women.

In lung cancer, EGFR inhibitors have been shown to be efficient in tumors with activating mutations of the receptor. This molecular alteration defines a subgroup of patients with specific clinicopathological features. The most striking one is the fact that patients are mostly non-smokers. Understanding the carcinogenesis pathway that drives lung carcinogenesis in non-smokers is therefore of major interest. The present work represents the first comparative study of genome wide allelic imbalance between EGFR mutated lung ADC and KRAS mutated ADC. In our work, EGFR mutated tumors are from non-smoking women with ADC, the control group was selected among smoking women with ADC and without EGFR mutations. As most of them have KRAS mutations, we chose to consider for the SNP array only tumors with KRAS mutations in order to have a homogenous control group. Moreover, it was already suggested that KRAS and EGFR mutations were hallmarks of tobacco and non-tobacco induced lung carcinogenesis [17]. Different reports showed that SNP array technology provided the opportunity to assess DNA copy number and LOH through the entire genome [12, 18–20]. The overall pattern of alterations seen in this study is reliable with previous lung ADC studies. As already noted, frequent chromosome gains are found at 1q, 5p, 7p/q, 8q and 14q and losses at 6q, 8p, 9p, 13q, 18q and 19p [11, 20–22]. But new regions of homozygous deletion have been found in EGFR mutated tumors especially at 18q involving the DCC gene and a cluster of desmosomal proteins. New regions of focal amplification at 7p and 14q have also been delineated that encodes potential oncogenes [22].

Using this technology, we showed that genome wide allelic imbalance patterns are different in tumors from non-smokers when compared to that of smokers. Indeed, more LOH and amplified regions were found in the EGFR mutated group when considering either all regions > 5 SNPs or entire chromosome arm losses or amplifications. This fact has already been suggested in a previous work using microsatellite markers and showing that tumors in non-smokers had more alterations than that of smokers [23]. The mechanisms leading to this increased chromosomal instability is not yet understood. In this series TP53 mutations were equally distributed in both groups therefore the impact of TP53 on genome stability cannot be discussed. Other mechanism such as repair defects could be involved in increased chromosomal instability in non-smokers.

In parallel to DNA assays, RNA expression profiles also demonstrated differences between ADC from smokers when compared with non-smokers [24, 25]. In a recent paper dealing with expression profiling of epidermal growth factor receptor/KRAS pathway activation in lung cancer two groups of ADC were individualized, one being a bronchial-type, the other an alveolar-type [26]. Unsupervised classification failed to detect any specific group of tumors that had EGFR mutations but they showed 26 genes preferentially expressed in EGFR mutated group. Among them, three genes EGFR, PTK7 and HMOX2 were found in our series located in focal regions of amplifications. Finally, a genetic classification of lung ADC by CGH array showed that EGFR mutated tumors could be individualized as a specific cluster [27]. All together, these results tend to prove that lung ADC in non-smokers forms a distinct disease at least at a molecular point of view. SNP arrays allow the identification of many loci with either LOH or amplification. Some of which may represent background alterations with no specific involvement in the carcinogenesis process. The identification of regions that are critical for tumor cell proliferation is of major importance. In order to go further in the identification of alterations linked to EGFR mutations we focused on genes implicated in cell cycle regulation and validated SNP array information on a large series of NSCLC. Previous genome analysis using SNP Arrays also showed CDKN2A homozygous deletions and CCNE1 amplifications in lung cancers [12]. We found a link between CDKN2A homozygous deletion and the non-smoking status as already suggested by Kraunz et al. [28]. Moreover, in our series CDKN2A deletion was associated with EGFR mutation and maybe with an activation of the EGFR/PI3K/AKT/mTOR transduction pathway. Indeed, 7 out of 8 tumors with CDKN2A homozygous deletion have EGFR, STK11 or PIK3CA mutations. Activation of this signal transduction pathway can lead to enhance transcription of cell cycle genes as CCND1 and it is not surprising that no cyclin amplification was observed in this group. For this group of tumors, it seems that proliferation is under the dependence of an activation of cell cycle through EGFR/PI3K/AKT/mTOR signaling and an inactivation of the cell cycle inhibitor CDKN2A preventing cells to down-regulate proliferation. The CDKN2A locus at 9p21 encodes two genes, one inhibits CDK4 mediated RB phosphorylation (CDKN2A/p16) and the other binds MDM2 leading to TP53 stabilization (p14/ARF). qPCR experiments reported here, analyzed homozygous deletions at 9p21 and therefore co-deletion of both genes. A recent paper showed that the p14/ARF protein was frequently down regulated in lung cancers with EGFR mutation or in tumors with ERBB2 mutations. As in our work, down regulation of the CDKN2A/p14/ARF locus could be linked to PI3K/AKT/mTOR activation as both EGFR and ERBB2 activate this pathway [29]. Although, regarding our results, it is very surprising that in their experiments, CDKN2A expression measured by immunohistochemistry remained positive when p14/ARF expression was extinguished. It suggested that p14/ARF could be down regulated independently of CDKN2A by mutation or promoter hypermethylation in EGFR mutated tumors. Promoter hypermethylation has indeed been shown to turn off CDKN2A locus in cancer but CDKN2A hypermethylation was linked to heavy smoking and squamous cell cancer which represents a different subgroup of tumors [30]. Here we suggest that CDKN2A/p14/ARF locus homozygous deletion could be an alternative mechanism to down regulate cell cycle inhibitors in ADC from non-smoking patients. In our series, Illumina GoldenGate Assay for methylation was used to quantify CDKN2A methylation, in the 24 tumors analyzed by SNP array. No difference in methylation status was found between EGFR, KRAS mutated tumors and non-tumor tissues (data not shown). Two other papers reported a negative correlation between EGFR mutation and CDKN2A methylation status in NSCLC [31, 32]. Cyclin amplifications were in opposite related to tobacco and associated to TP53 mutations. In this case, cell cycle is activated through direct amplification of cyclins and TP53 inactivating mutations unable cells to repress proliferation. An association between CCND1 and TP53 was already suggested by protein expression analysis [33].

Although these models are not to generalize since other mechanisms such as mutations epigenetic alteration and protein overexpression can activate oncogene or inactivate tumor suppressor, our work enlightened two different carcinogenesis pathways in NSCLC. One is tobacco independent and driven by CDKN2A inactivation and EGFR mutations, the other is smoking dependent and driven by cyclin amplification and TP53 mutation (Figure 2).

Although this work concerned a limited series of tumors, we focused on a comparative analysis and showed that patterns of genome wide genetic alterations are different between ADC with and without EGFR mutation. More alterations and a higher frequency of large alterations (entire chromosome arms deletion or amplification) were found in the EGFR mutated group suggesting that carcinogenesis pathways are different. Indeed, for a subset of tumors specific involvement of cell cycle genes were identified and an oncogenic cooperation between EGFR mutations and CDKN2A homozygous deletion was identified.