Yohei Takumi

The prognostic impact of the platelet distribution width-to-platelet count ratio in patients with breast cancer

Activated platelets promote tumor cell growth, angiogenesis, and invasion. Platelet activity can be inferred by platelet volume indices (PVIs), which include platelet distribution width (PDW), mean platelet volume (MPV), platelet distribution width-to-platelet count ratio (PDW/P), and mean platelet volume-to-platelet count ratio. Platelets and platelet-related markers, such as the platelet-to-lymphocyte ratio, have been found to be significant prognostic factors in patients with breast cancer. However, the role of PVIs for predicting survival in breast cancer remains unknown; hence, we performed this retrospective analysis of 275 patients with breast cancer. PVIs were compared with clinicopathological variables, and were assessed to identify independent indicators associated with disease-free survival (DFS) using the Cox proportional hazards model. An elevated PDW/P significantly correlated with age and HER2 status. Univariate analysis revealed that elevated PDW, MPV, and PDW/P as well as tumor size, nuclear grade, and lymph node involvement were significantly associated with inferior DFS rates (tumor size: p<0.01; nuclear grade, lymph node involvement, PDW, MPV, and PDW/P: p<0.05). On multivariate analysis, a large tumor size and elevated PDW/P were significant prognostic factors for DFS, with hazard ratios of 3.24 (95% confidence interval [CI]: 1.24–8.47) and 2.99 (95% CI: 1.18–7.57), respectively (p<0.05). Our study is the first to reveal that an elevated PDW/P significantly reduces DFS in patients with breast carcinoma. Measuring the PDW/P is simple, relatively inexpensive, and almost universally available using routine blood counts; this makes it an attractive biomarker for improved risk assessment.

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

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.

Abstract 4107: Acquired resistance to EGFR-TKI in an uncommon G719S EGFR mutation

Background: Acquired resistance (AR) to EGFR-TKI is a common event and several underlying mechanisms, including T790M, MET amplification and PTEN downregulation have been reported for the common EGFR mutations: Deletion 19 and L858R. An EGFR G719X mutation is an uncommon mutation that was reported to show sensitivity to EGFR-TKIs in a series of clinical reports and experiments using transformed cultured cells. However, no established lung cancer or resistant cell lines harboring the EGFR G719X mutation have been reported in the literature. We established a lung adenocarcinoma cell line (G719S-GR) from the malignant pleural effusion of a patient whose tumor developed acquired resistance from initial treatment with gefitinib. Materials and methods: G719S-GR cells were established and maintained in RPMI1640 medium supplemented with 10%FBS and 10 μM ROCK inhibitor (Y-27632, Wako). The ROCK inhibitor was removed from the medium for the following experiments. Cell growth inhibition was examined with gefitinib and afatinib using CellTiter-Glo (Promega), and a comprehensive genomic analysis was performed using hybrid capture-based NGS (NCC oncopanel, Agilent; MiSeq, Illumina) for G719S-GR and MLPA (Salsa, MRC-holland) was used for the analysis of clinical tumor samples. Results: A cell growth inhibition test revealed EGFR-TKI resistance in G719S-GR cells with an LC50 of more than 100 μM for either gefitinib or afatinib, indicating that the G719S-GR cells are also resistant to EGFR-TKI in vitro. The NGS analysis showed that G719S-GR cells harbor EGFR mutations (G719S and E709A) as well as the amplification of EGFR, IL7R, MYC and the FGFR1 locus. The homozygous deletion of CDKN2A and the loss of PTEN and TSC1 were also detected. In order to estimate the mechanism underlying the development of EGFR-TKI resistance, copy number analyses of several tumor suppressor genes were performed by an MLPA using genomic DNA from G719S-GR and a tumor biopsy sample (obtained before gefitinib treatment). Losses of CDKN2A, PTEN and TSC1 were confirmed in G719S-GR cells. Interestingly, the loss of PTEN was not observed in the gefitinib-naive tumor sample. Discussion: Thus far, the mechanisms underlying the development of EGFR-TKI resistance in uncommon mutations have not been investigated. The newly established G719S-GR cell line could be a useful tool for investigating the mechanism underlying the development of AR in the G719X mutation; the loss PTEN could be one such mechanism. Further experiments are warranted. Citation Format: Atsushi Osoegawa, Tomonori Yamada, Takafumi Hashimoto, Yohei Takumi, Ryoji Kobayashi, Shuji Suehiro, Michiyo Miyawaki, Hideya Takeuchi, Tatsuro Okamoto, Kenji Sugio. Acquired resistance to EGFR-TKI in an uncommon G719S EGFR mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4107. doi:10.1158/1538-7445.AM2017-4107

Abstract 2227: Positive correlation between the gamma-H2AX and PD-L1 expressions in lung squamous cell carcinoma

Lung cancer is a leading cause of cancer death worldwide. Recently, molecular targeting therapy has been developed and it has shown promising results against advanced lung cancer. Among them, lung squamous cell carcinoma has fewer treatment options because it is not driven by oncogenic mutations, but by alterations in tumor suppressor genes and subsequent chromosomal instability. Programmed death-ligand 1 (PD-L1) is one of the immune checkpoint molecules that is expressed on the surface of tumor cells, where it inhibits activities of cytotoxic T cells. Recent studies revealed that the PD-1/PD-L1 blockade could improve the overall survival in lung squamous cell carcinoma, potentially because it carries high mutation burdens and neoantigens which could be targeted by cytotoxic T cells. We hypothesized that DNA damage could accumulate in tumors with high mutation burdens, thereby inducing the PD-L1 expression on tumor cells, sensitizing them to anti-PD-1 therapy. An immunohistochemical analysis of 41 consecutive lung squamous cell carcinoma cases, which received surgery at our institution between April 2013 and March 2014, revealed that a high PD-L1 expression was observed in 15 patients (37%) that was associated related with a poor recurrence-free survival (p = 0.028). The PD-L1 expression level was also positively associated with the γH2AX expression (a direct marker for DNA damage) (p = 0.02). The γH2AX expression in tumor cell nuclei was confirmed by immunofluorescent staining. It forms foci in tumor cell nuclei, indicating the incidence of DNA double strand breaks. Our findings demonstrate for the first time that the nuclear γH2AX expression in lung squamous cell carcinoma is positively associated with the PD-L1 expression. Further studies are warranted to investigate whether γH2AX could be a biomarker for PD-1 targeting therapy and whether combination therapy with PD-1 targeting therapy and a DNA-damaging agent has synergistic effects. Citation Format: Atsushi Osoegawa, Takafumi Hashimoto, Yohei Takumi, Miyuki Abe, Hitomi Hiraishi, Shuji Suehiro, Michiyo Miyawaki, Kenji Sugio. Positive correlation between the gamma-H2AX and PD-L1 expressions in lung squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2227.