Data presented herein have shown that lung adenocarcinomas in the intrinsic molecular subtypes have significantly different alterations in gene sequence mutations, chromosomal instability, regional DNA copy number, DNA methylation, and integrated combinations. We strengthen the evidence for subtype-alteration associations by a priori hypothesis testing in a previously-uncharacterized cohort from our institution, which provided substantial confidence that these associations are robust. Our results indicate that the intrinsic, naturally-occurring molecular subtypes are not only a gene expression phenomenon but also a representation of different variants of LAD disease defined by different genomic alterations.
Whether in clinical management or in laboratory models, LAD is primarily classified by morphology, mutations, or clinical characteristics. The LAD intrinsic molecular subtypes capture many clinically-relevant phenotypes from these separate classifications. Bronchioid is represented by patients who are female and nonsmoking, who have a superior survival outcome, and who present with well-differentiated, bronchioloalveolar morphology, early stage and EGFR
mutated cancers 
. High smoking exposure, poor survival outcome and late-stage presenting patients are common in the other two subtypes: Magnoid and Squamoid. Magnoid has a high prevalence of patients who are male and have KRAS
. Squamoid includes patients who present with poorly differentiated and solid morphology cancers 
This comprehensive genomic analysis provides significant insights into subtype-specific alterations. For example not only does Bronchioid have the most EGFR
mutations, this subtype also has the most patients with integrated mutation, amplification and overexpression of EGFR
. Although this combination has been observed 
, this is the first evidence that it predominantly occurs in one molecular subtype. Development of the Bronchioid subtype seems to be uniquely dependent on mutant EGFR
, while the rare EGFR
mutants in other subtypes usually have concurrent TP53
mutation. Finally, Bronchioid had the most BRAF
mutants, suggesting a second less common Bronchioid driver, although this mutation is too rare to confirm by our analysis.
In addition to having the most TP53
mutations, Magnoid tumors also have severe genomic alterations including the greatest CIN, the most regional CN alterations, DNA hypermethylation, and the greatest genomewide mutation rate. Magnoid's overexpression of DNA repair genes suggests that these tumors are actively repairing their heavily damaged genome, possibly in response to these patients' heavy smoke exposure. Magnoid has the most genes with concurrent alterations in the same patients (TP53
), unlike Bronchioid which typically had EGFR
as its sole sequence mutation as discussed above. This again suggests that excessive DNA damage occurred in these tumors, perhaps due to smoking exposure, and has driven multiple gene mutations. Magnoid's concurrency of TP53
alterations and high CIN is a novel association within an LAD patient subgroup and could be explained by TP53
alteration inducing high CIN, similar to recently reported cell culture studies 
. Finally, the Magnoid subtype exhibited increased hypermethylation, a phenomenon similar to the CpG-Island-Methylator-Phenotype (“CIMP”) observed in other cancers 
Lastly, the Squamoid subtype displayed the fewest distinctive alterations that included only regional CN alterations. Adenosquamous features were most prevalent in Squamoid, which is the first association with a molecular subtype to date. Squamoid had the most PTEN mutations and loss of its locus, 10q22–q26, suggesting this may be a Squamoid-specific driver; however, PTEN mutation was too rare to confirm by our analysis. Interestingly, the Squamoid subtype, which presents in patients as a poorly differentiated solid morphology cancer and predicts poor survival, had the fewest distinctive genomic alterations.
This study reports associations using retrospective cohorts and determination of causation is not possible. However, because cancers stratified by the molecular subtypes have different genomic alterations and because genomic alterations cause cancer 
, we infer that cancers stratified by the molecular subtypes have arisen by different genomic alterations, so called ‘molecular pathogenesis’. But beyond subtype and genomic alterations co-associating in cancers, is there more to the nature of this relationship? We offer several possible explanations. One model is that genomic alterations change a cancer's gene expression and that differences in genomic alterations directly cause the three molecular subtypes we observe. Supporting evidence is provided by mouse studies with activated cancer genes producing tumors with varied gene expression. However, it remains unlikely that alterations by themselves control cancer gene expression. Therefore, an alternative model is that subtypes and alterations are both caused by additional factors, such as the cancer's cell type of origin, patient behavior such as smoking, and/or patient germline sequence. Properties of a cancer's original cell type may promote specific genomic alterations due to physical mutability or the selective advantage that a specific mutation confers on a specific cell type. Considering that lung adenocarcinoma and lung squamous cell carcinoma have very different mutational profiles and are believed to originate from distinct cell types 
, differences in cell of origin seems to be a reasonable model. This model is also supported by observations of increased EGFR
mutation prevalence in the terminal respiratory units of the lung compared to other areas 
. We surmise that associations between subtypes and alterations can be explained by differences in cell type of origin that incur different alterations, promoted by different patient characteristics, which combined results in a different molecular subtype and patient outcome.
The LAD molecular subtypes and their associated alterations have clear translational significance. This study represents a second validation of the survival advantage for the Bronchioid subtype of LAD, a disease with few clinically implemented biomarkers. Additionally, we present data suggesting that molecular subtypes have relevance in predicting response both to cytotoxic chemotherapy and targeted EGFR inhibitory therapy beyond the established role of EGFR mutation status. These observations were derived from retrospective clinical data and surrogate response markers. Future prospective clinical trial and model systems studies are needed to confirm and more deeply describe the genomic basis of these findings.
In conclusion, we demonstrated that lung adenocarcinomas in different molecular subtypes have grossly distinct genomic alterations, clinical phenotypes, and clinical outcomes.