Abstract 2200: Intratumoral heterogeneity of copy number variation in lung cancer harboring L858R via immunohistochemical heterogeneous staining

Background: Acquired resistance to EGFR-TKI is an inevitable event for advanced lung cancer patients harboring the EGFR-activating mutation. Although intratumoral heterogeneity is commonly observed in several cancers, such as renal cell carcinoma, few studies have shown its presence in EGFR-mutated lung cancer. We analyzed the intratumoral heterogeneity in EGFR-mutated (L858R) lung cancer through immunohistochemistry (IHC) for the primary site and targeted sequencing for specific cases in order to clarify the mechanism of acquired resistance. Methods: Twenty resected primary lung cancers known to harbor EGFR L858R were analyzed. IHC was performed using an L858R mutant-specific rabbit monoclonal antibody (clone43B2; Japan) and the samples were scored by staining intensity (0-3) and proportion. For cases with heterogeneous L858R protein expression, the nucleic acids were extracted from each differently stained lesion, and targeted sequencing (Oncomine Cancer Research Panel) was performed. Single-nucleotide variations (SNVs) and copy number variations (CNVs) from each site were then analyzed. The cell proliferation and apoptosis were also evaluated by the ki-67 labeling index and TUNEL staining. Results: Among 20 cases, 3 (15%) showed heterogeneous staining, defined as having both an L858R IHC-positive part and an IHC-negative one. Genetic analyses for cases with heterogeneous staining revealed an increase in the copy number of EGFR in the IHC-positive part (CNV 19.54) compared to the negative part (CNV 3.03), and an increase in the copy number of CCNE1 was observed in the IHC-positive part (CNV 18.59) compared to the negative part (CNV 2.59) in one case (case 1). In another case (case 2), an increase in the copy number of EGFR was observed in the IHC-positive part (CNV 8.21) compared to the negative part (CNV 2.90), and an increase in the copy number of MDM2 was observed in the IHC-positive part (CNV 6.65) compared to the negative part (CNV 1.37). Case 3 did not show any CNV. In three cases, no SNV changes were observed. An increase in the ki-67 labeling index in the L858R-positive part in case 1 and increased apoptosis in the L858R-positive part in case 2 were observed, suggesting the functional significance of CNV changes. Conclusion: These cases exhibiting L858R IHC intratumoral heterogeneity suggest a heterogeneous effect on the cell activity due to CNV heterogeneity. Citation Format: Takafumi Hashimoto. Intratumoral heterogeneity of copy number variation in lung cancer harboring L858R via immunohistochemical heterogeneous staining [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2200.