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Heterogeneous Components of Lung Adenocarcinomas Harbour Distinct EGFR Mutations and PD-L1 Expression

This study aimed to determine what patterns of histopathological areas within heterogenous NSCLCs may contribute to resistance to EGFR TKIs and immune blockade therapies.

Lung cancer causes the most cancer-related deaths worldwide. Lung Adenocarcinoma (LAC), the most prevalent type of non-small cell lung cancer (NSCLC), can occur with various heterogeneity as assessed histologically and can contain acinar, papillary, micropapillary, lepidic and solid histological types at varying percentages. This intratumoural heterogeneity can result in sporadic and unpredictable responses to therapies.

NSCLCs are frequently driven by activation of EGFR and the development of anti-EGFR tyrosine kinase inhibitors (TKIs) has been met with optimism. The demonstration that EGFR-driven NSCLCs had increased activity of the PD1/PDL1 pathway facilitating immune escape led to increased research into combination therapy with EGFR TKIs and immune checkpoint blockade. Treatment strategies using combination treatment become complex due to the expression of EGFR mutants that can downregulate PD-L1 expression. It is hypothesised that tumour heterogeneity also results in mixed responses (MR) to chemo and EGFR TKI therapies.

In this study the authors investigated the effects of tumour heterogeneity using laser capture microdissection of distinct histological regions within lung tumours and their expression of EGFR mutants and PDL1.

  • 261 patients with LAC were enrolled in the study, 96 at stage I, 21 at stage II, 79 at stage IIIA and 65 at stage IIB and IV. FFPE sections were taken from distinct histopathological areas and assessed for mutant EGFR and PD-L1 expressions.
  • PD-L1 staining in FFPE sections of non-LCM tissue shows diffuse localisation. Solid areas had high levels of staining whereas in ordinary areas there was very little staining. A three tiered tumour proportion scoring (TPS) system was developed to assess PD-L1 staining. TPS <1, 1-49 or >50%
  • In LACs where the PD-L1 staining TPS>50%, LACs with solid areas of tumour had higher PD-L1 staining compared to those without
  • More than 50% of cancer cells stained positively for PD-L1 in later stage tumours compared to the early stage tumours and progression free survival time was shorter in those tumours with high PD-L1 TPS.
  • Digital droplet PCR was used to detect hotspot mutations in the E19del/L858R TKI sensitising hotspot and the T790M hotspot that confers resistance to EGFR TKIs. In 18 patients, both of these mutations were detected. In LCM sections, these mutations were associated with papillary, lepidic and micropapillary areas, whilst wild type EGFR was more commonly associated with acinar tissue.
  • In mixed tissue types, both mutations were observed in the papillary and micropapillary areas but absent from the acinar and solid components. The heterogeneity of these mutations may result in inconsistent response to EGFR TKIs.
  • Amongst the 18 patients with the TKI resistant mutation T790M, 6 also expressed PD-L1. Nine LACs had mixed areas, had heterogenous areas of mutant EGFR and Wild type EGFR and expressed PD-L1. PD-L1 TPS was not associated with the mutation status of EGFR.

This study aimed to determine what patterns of histopathological areas within heterogenous NSCLCs may contribute to resistance to EGFR TKIs and immune blockade therapies. The authors demonstrated that different areas of the same LAC tissue can harbour both sensitising EGFR mutations and those mutations that confer resistance to TKIs. The expression of PD-L1 correlated with the progression free survival time for patients and the authors also demonstrated that some areas such as the solid histopathological areas were more inclined to have higher PD-L1 expression. The authors conclude that these areas in combination with mutations in EGFR could be informative for patient treatment. For example, Gefitinib could be useful for the treatment of patients with micropapillary or papillary LAC. However, Osimertinib would be better for LACs with EGFR mutations.



At Epistem we have years of experience isolating RNA from target tissues using laser capture microdissection. We have isolated fibrotic, specific tumour regions and infiltrating lymphocytes for downstream analysis using our gene expression analysis platforms. We have leveraged over 15 years of histology and IHC expertise to develop RNA-friendly stains for specific cell types making them amenable for gene expression studies using LCM. We have recently optimised our protocols to isolate RNA from FFPE tissue for analysis on our NextSeq sequencing platform and have available a Fluidigm Biomark HD qPCR platform for SNP analysis by ddPCR. All of our gene expression analysis is GCLP compliant and we also have GCLP compliant histology labs for further biomarker analysis.