Mapping of actionable mutations to histological subtype domains in lung adenocarcinoma: implications for precision medicine.

Precision medicine depends on the accurate identification of actionable mutations in a tumor sample. It is unknown how heterogeneous the distribution of such mutations can be in a tumor. Morphological (i.e. histopathological) heterogeneity is well described in lung adenocarcinoma and has been specifically recognized in the most recent official clinico-pathological classification. The most predominant subtype present is now used to classify each lung adenocarcinoma. No molecular profile exists to explain the intratumoral differences in lung adenocarcinoma morphology, despite the consistently observed association between specific predominant subtypes and poorer survival. Given a recent proposal stratifying lung adenocarcinoma into subtypes of differing metastatic potential, we questioned the assumption that major mutations are present uniformly throughout tumors; especially those showing discrete different subtypes. We selected formalin-fixed paraffin embedded lung adenocarcinoma specimens that showed discrete areas of different subtypes, extracted subtype DNA samples from those areas and screened for mutations in hotspot regions of the EGFR, KRAS and BRAF genes using high resolution melting. Sanger sequencing was used to confirm all identified mutations. Chromogenic in situ hybridization (CISH) was used to identify mutant allele specific imbalances in tumors with EGFR mutations. Interestingly, we found that KRAS and BRAF mutations could be confined to morphological domains of higher grade. On the other hand, EGFR mutations were found through all histological subtypes in each tumor consistent with the driver status of this mutation. Intratumoral heterogeneity has major implications for tumorigenesis, chemoresistance and the role of histopathology in molecular screening for precision medicine. This study not only confirms that intratumoral mutational heterogeneity does occur, but also that it is associated with morphologically distinct regions in some tumors. From a practical perspective, small biopsies may not adequately represent a tumor's full mutational profile, particularly for later arising but prognostically important mutations such as those in the KRAS and BRAF genes.