Takafumi Hashimoto1, Atsushi Osoegawa2, Yohei Takumi3, Miyuki Abe4, Ryoji Kobayashi5, Michiyo Miyawaki6, Hideya Takeuchi7, Tatsuro Okamoto8, Kenji Sugio9. 1. Department of Thoracic and Breast Surgery, Oita University, Faculty of Medicine, Yufu, Oita, 879-5593 Japan. Electronic address: t-hashimoto@oita-u.ac.jp. 2. Department of Thoracic and Breast Surgery, Oita University, Faculty of Medicine, Yufu, Oita, 879-5593 Japan. Electronic address: osoegawa-ths@oita-u.ac.jp. 3. Department of Thoracic and Breast Surgery, Oita University, Faculty of Medicine, Yufu, Oita, 879-5593 Japan. Electronic address: ytakumi@oita-u.ac.jp. 4. Department of Thoracic and Breast Surgery, Oita University, Faculty of Medicine, Yufu, Oita, 879-5593 Japan. Electronic address: abemiyuki2d2@oita-u.ac.jp. 5. Department of Thoracic and Breast Surgery, Oita University, Faculty of Medicine, Yufu, Oita, 879-5593 Japan. Electronic address: rkobayashi@oita-u.ac.jp. 6. Department of Thoracic and Breast Surgery, Oita University, Faculty of Medicine, Yufu, Oita, 879-5593 Japan. Electronic address: miyawak@oita-u.ac.jp. 7. Department of Thoracic and Breast Surgery, Oita University, Faculty of Medicine, Yufu, Oita, 879-5593 Japan. Electronic address: takeuchi@oita-u.ac.jp. 8. Department of Thoracic and Breast Surgery, Oita University, Faculty of Medicine, Yufu, Oita, 879-5593 Japan. Electronic address: tokamoto@oita-u.ac.jp. 9. Department of Thoracic and Breast Surgery, Oita University, Faculty of Medicine, Yufu, Oita, 879-5593 Japan. Electronic address: ksugio@oita-u.ac.jp.
Abstract
BACKGROUND: Although intratumoral heterogeneity is commonly observed in several cancers, few studies have shown its presence in EGFR-mutated lung cancer. We performed immunohistochemistry to analyze the intratumoral heterogeneity in EGFR-mutated (L858R) lung cancer and performed targeted sequencing in specific cases. We discuss the effects of intratumoral heterogeneity and acquired resistance to EGFR-TKI. METHODS: Twenty resected primary lung cancers known to harbor EGFR L858R were analyzed. IHC was performed using an L858R mutant-specific rabbit monoclonal antibody 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 was performed. Single nucleotide variations (SNVs) and copy number variations (CNVs) 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 showed heterogeneous staining. Genetic analyses for cases with heterogeneous staining revealed an increase in the copy number of EGFR in the IHC-positive part compared to the negative part, and an increase in the copy number of CCNE1 was observed in the IHC-positive part compared to the negative part 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 compared to the negative part, and an increase in the copy number of MDM2 was observed in the IHC-positive part compared to the negative part. 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.
BACKGROUND: Although intratumoral heterogeneity is commonly observed in several cancers, few studies have shown its presence in EGFR-mutated lung cancer. We performed immunohistochemistry to analyze the intratumoral heterogeneity in EGFR-mutated (L858R) lung cancer and performed targeted sequencing in specific cases. We discuss the effects of intratumoral heterogeneity and acquired resistance to EGFR-TKI. METHODS: Twenty resected primary lung cancers known to harbor EGFRL858R were analyzed. IHC was performed using an L858R mutant-specific rabbit monoclonal antibody 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 was performed. Single nucleotide variations (SNVs) and copy number variations (CNVs) 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 showed heterogeneous staining. Genetic analyses for cases with heterogeneous staining revealed an increase in the copy number of EGFR in the IHC-positive part compared to the negative part, and an increase in the copy number of CCNE1 was observed in the IHC-positive part compared to the negative part 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 compared to the negative part, and an increase in the copy number of MDM2 was observed in the IHC-positive part compared to the negative part. 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.