Integrated mutation, copy number and expression profiling in resectable non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is the commonest cause of cancer death in Western communities. Current treatments offer the potential of cure only to the small number of patients who present with early stage NSCLC, whilst outcomes for those with advanced disease remain poor. Recent advances including adjuvant chemotherapy and targeted biological therapies have lead to modest improvements in survival for small subgroups of patients. Clearly new treatment approaches are required to substantially improve outcome. As has been the case for other tumour types, molecular profiling techniques have the potential to provide benefit through improved understanding of disease pathogenesis, identification of subgroups in whom current therapies are most likely to be effective and in the development of novel therapies.

Genetic heterogeneity is a feature of NSCLC, with varying combinations of multiple molecular alterations contributing to tumour development [1]. A key challenge for high-throughput molecular profiling techniques is to distinguish between genes whose expression is altered directly by heritable changes in gene function and those where changes are an inevitable down-stream consequence of primary changes to genes directly involved in disease pathogenesis. Correlation of transcriptional and genomic data allows more focussed analysis of the large number of genetic alterations identified by molecular profiling. This study incorporates the results of both transcriptional and genomic profiling for clinically relevant subgroups of NSCLC to identify genes of potential predictive or pathogenic importance in this deadly disease.

Molecular and clinical data was available for 69 patients who underwent surgery for NSCLC between May 1999 and July 2006. Demographic and pathologic data are included in table 1. Median follow-up for surviving patients exceeds 35 months. After a median follow-up of 36 months (1 - 80) for all patients, 28/68 patients developed recurrent disease, and 23/68 patients died of NSCLC (one patient with disseminated disease at diagnosis excluded from analysis). Comparable to other series of early stage NSCLC [9], five year overall survival rates approximated 55%.

The clinical, demographic and pathologic characteristics of this NSCLC cohort are consistent with the published literature. The transcriptional and genomic profiles identified in this study should therefore be generalisable to other patients with early-stage NSCLC. The tumour samples analysed demonstrated substantial genomic instability, with comparisons between subgroups failing to demonstrate any significant difference. Previous studies of copy number changes in NSCLC have found no association between age, gender, histology, stage or tumour grade and the degree of genomic instability [19‐21]. The absence of difference in the degree of genomic abnormalities between KRAS mutant and wt tumours is interesting, as both our transcriptional and genomic data imply enhanced activity of genes involved in chromosome structure and organisation in KRAS mutant tumours. We recognise that there were a small number of KRAS mutant tumours available for comparison and this may have limited our analysis.

Consistent with previously reported studies [9‐16], the major differences in copy number and gene expression profiles between AC and SCC of the lung involved chromosome 3q. The strong independent correlation with amplification and over-expression at this locus suggests a causal relationship in SCC for genes in this region which warrant further investigation. These include TP73L, a gene extensively implicated in SCC, whose expression was most strongly correlated with the SCC phenotype, and which has been previously reported to be a putative oncogene [22‐32]. While the role of TP73L in squamous cell pathogenesis remains unclear, a recent study of SiRNA mediated TP73L inhibition in SCC resulted in reduced cell survival with maintenance of squamous characteristics [28]. These results suggest that TP73L is important in SCC cell survival. Other genes previously shown to be over-expressed in SCC were included in our differentiating list (CSTA [33, 34], FGFBP1), and warrant functional validation.

The differential copy number and expression levels between AC and SCC of TNFSF10/TRAIL and ABCC5, which have roles in apoptosis and chemoresistance respectively, may have implications for treatment of NSCLC. Recently published clinical data [35] suggest there are histotype-specific differences in response to systemic therapies. Validation of the differential activity of the roles of these genes and sensitivity to conventional and novel chemotherapeutic agents may be an area for future research.

Published data on the relationship between TP53 mutations and histotype in NSCLC is conflicting [36, 37]. SCC were associated with more frequent TP53 mutations than AC in our dataset. Cigarette smoking is a causal factor for both SCC phenotype and TP53 mutation [37]. However, we also observed a correlation between 3q amplification and TP53 mutations in AC samples. This suggests that the apparent association between TP53 mutations and SCC may be mediated by the relationship between TP53 mutations and amplification of regions of 3q. We caution that this study is underpowered to draw strong conclusions regarding the role of TP53 in NSCLC pathogenesis.

Tumours possessing mutations of KRAS express genes playing key roles in cell growth, chromosome organisation and gene regulation. As previously reported, we identified amplification and over-expression of COX2 in KRAS mutant tumours. KRAS mutant tumours did not demonstrate mutations in EGFR consistent with previous reports in both NSCLC and colon carcinoma which suggest that KRAS mutations predict resistance to EGFR antagonists [29, 38‐41]. Several reports link NCAM1 to Ras-dependent activation of ERK MAPK's [42, 43]. Reduced copy number and expression of NCAM1 in tumours bearing KRAS mutations, as seen in our data, has not previously been reported. Further research into a KRAS mutation profile may yield simple and reliable immunohistochemical markers of KRAS mutation, thereby significantly reduce the cost of determining KRAS status in clinical practice.

The gene expression profile observed in 'metastatic' tumours is consistent with a growing body of literature implicating deregulated protein synthesis in the development and metastatic potential of human cancers [44, 45]. Increased mRNA translation is a critical downstream function of many cancer related genes, and many gene products with roles in metastasis are not mutated but inappropriately expressed in malignant cells (e.g. VEGF, c-Myc, fos, Her2Neu, PDGF) [18]. Opportunities for therapeutic intervention currently in development include oncolytic viruses that require deregulated protein translation for their replication [18], or agents that inhibit mTOR, an integral factor in protein translation (eg. temsirolimus (CCI-779), everolimus (RAD001) and deforolimus (AP23573)).

While the small number of recurrences and deaths due to NSCLC in our tumour-set makes it difficult to draw strong conclusions, transcriptional profiles linked to tumour recurrence suggest KRAS pathway activation. This may be due to a higher proportion of AC and LCC vs. SCC in the 'recurrent' group. Other regions of copy number change demonstrate genomic gains in the region of c-Myc and losses in the region of p16 (INK4a, CDNK2A) in recurrent or non-survivor tumours, supporting a prognostic association of the Myc:CDNK2A ratio in NSCLC, as has been described in head and neck SCC [46]. Specific genes linked to recurrence or survival include SMARCA2 (implicated in the regulation of gene expression cell cycle control and oncogenesis), MINK (linked to the JNK MAP kinase pathway) [47] and RECK, which has putative roles in the suppression of tumour growth, invasion, angiogenesis and metastasis [48]. KRAS mutation has been associated with reduced expression of RECK in NSCLC [49], consistent with the clinical observation of poor outcome in patients with KRAS mutation bearing NSCLC. Activation of the Ras pathway may reduce RECK expression and thereby increase tumour recurrence. Importantly, our prognostic gene signature was validated in an independent test set, suggesting that these findings may eventually yield prognostic markers in resected early-stage NSCLC to better select patients for adjuvant treatments.

Several molecular alterations have been identified in association with NSCLC histotype, KRAS mutation, TP53 mutation, metastatic potential, disease recurrence and survival. Although the size of the current study is small, our findings are in many cases consistent with those of previous studies, and have been validated in the case of the prognostic classifier in an independent test set. In addition, several novel molecular changes associated with clinically relevant endpoints have been demonstrated. It is hoped that these results will contribute to identifying new predictive markers and targets for novel therapies to improve treatment selection and better outcomes for patients with this deadly disease.