Tesshi Yamada

Alternative Mammalian Target of Rapamycin (mTOR) Signal Activation in Sorafenib-resistant Hepatocellular Carcinoma Cells Revealed by Array-based Pathway Profiling

Sorafenib is a multi-kinase inhibitor that has been proven effective for the treatment of unresectable hepatocellular carcinoma (HCC). However, its precise mechanisms of action and resistance have not been well established. We have developed high-density fluorescence reverse-phase protein arrays and used them to determine the status of 180 phosphorylation sites of signaling molecules in the 120 pathways registered in the NCI-Nature curated database in 23 HCC cell lines. Among the 180 signaling nodes, we found that the level of ribosomal protein S6 phosphorylated at serine residue 235/236 (p-RPS6 S235/236) was most significantly correlated with the resistance of HCC cells to sorafenib. The high expression of p-RPS6 S235/236 was confirmed immunohistochemically in biopsy samples obtained from HCC patients who responded poorly to sorafenib. Sorafenib-resistant HCC cells showed constitutive activation of the mammalian target of rapamycin (mTOR) pathway, but whole-exon sequencing of kinase genes revealed no evident alteration in the pathway. p-RPS6 S235/236 is a potential biomarker that predicts unresponsiveness of HCC to sorafenib. The use of mTOR inhibitors may be considered for the treatment of such tumors.

Distinct outcome of stage I lung adenocarcinoma with ACTN4 cell motility gene amplification

BACKGROUND Even if detected at an early stage, a substantial number of lung cancers relapse after curative surgery. However, no method for distinguishing such tumors has yet been established. PATIENTS AND METHODS The copy number of the actinin-4 (ACTN4) gene was determined by fluorescence in situ hybridization on tissue microarrays comprising 543 surgically resected adenocarcinomas of the lung. RESULTS Amplification (an increase in the copy number by ≥ 2.0 fold) of the ACTN4 gene was detected in two of seven lung adenocarcinoma cell lines and 79 (15%) of 543 cases of pathological stage I-IV lung adenocarcinoma. Multivariate analysis revealed that ACTN4 gene amplification was the most significant independent factor associated with an extremely high risk of death (hazard ratio, 6.78; P = 9.48 × 10(-5), Cox regression analysis) among 290 patients with stage I lung adenocarcinoma. The prognostic significance of ACTN gene amplification was further validated in three independent cohorts totaling 1033 patients. CONCLUSIONS Amplification of the ACTN4 gene defines a small but substantial subset of patients with stage I lung adenocarcinoma showing a distinct outcome. Such patients require intensive medical attention and might benefit from postoperative adjuvant chemotherapy.

Soluble interleukin‐6 receptor is a serum biomarker for the response of esophageal carcinoma to neoadjuvant chemoradiotherapy

Preoperative chemoradiotherapy has been shown to improve the outcome of patients with esophageal cancer, but because response to this therapy varies, it is desirable to identify in advance individuals who would be unlikely to benefit, in order to avoid unnecessary adverse drug effects. The serum profiles of 84 cytokines and related proteins were determined in 37 patients with esophageal squamous cell carcinoma who received identical neoadjuvant preoperative chemoradiotherapy regimens and underwent surgical resection. Histological response to this therapy was assessed in surgically resected specimens. The serum soluble interleukin‐6 receptor (sIL6R) level was significantly higher in 30 patients who failed to achieve a histological complete response (P = 0.005). Multivariate analysis revealed that the increased level of sIL6R was one of several significant independent predictors of an unfavorable outcome (hazard ratio, 2.87; P = 0.017). The increased level of this cytokine in patients who did not obtain a complete response was reproducibly observed in an independent cohort of 34 patients. Esophageal squamous cell carcinoma patients with an increased serum level of sIL6R are predicted to respond poorly to preoperative chemoradiotherapy, therefore, their exclusion from this treatment may be considered. Persistent systemic inflammation is implicated as a possible mechanism of resistance to this therapy.

Diagnostic and prognostic significance of the alternatively spliced ACTN4 variant in high-grade neuroendocrine pulmonary tumours

BACKGROUND High-grade neuroendocrine tumours (HGNTs) of the lung manifest a wide spectrum of clinical behaviour, but no method for predicting their outcome has been established. MATERIALS AND METHODS We newly established a monoclonal antibody specifically recognizing the product of the alternatively spliced ACTN4 transcript (namely, variant actinin-4), and used it to examine the expression of variant actinin-4 immunohistochemically in a total of 609 surgical specimens of various histological subtypes of lung cancer. RESULTS Variant actinin-4 was expressed in 55% (96/176) of HGNTs, but in only 0.8% (3/378) of non-neuroendocrine (NE) lung cancers. The expression of variant actinin-4 was significantly associated with poorer overall survival in HGNT patients (P=0.00021, log-rank test). Multivariate analysis using the Cox proportional hazards model showed that the expression of variant actinin-4 was the most significant independent negative predictor of survival in HGNT patients (hazard ratio (HR), 2.15; P=0.00113) after the presence of lymph node metastasis (HR, 2.25; P=0.00023). CONCLUSIONS The expression of variant actinin-4 is an independent prognostic factor for patients with HGNTs. This protein has a high affinity for filamentous actin polymers and likely promotes aggressive behaviour of cancer cells. The present clinical findings clearly support this notion.

Copy number increase of ACTN4 is a prognostic indicator in salivary gland carcinoma

Copy number increase (CNI) of ACTN4 has been associated with poor prognosis and metastatic phenotypes in various human carcinomas. To identify a novel prognostic factor for salivary gland carcinoma, we investigated the copy number of ACTN4. We evaluated DNA copy number of ACTN4 in 58 patients with salivary gland carcinoma by using fluorescent in situ hybridization (FISH). CNI of ACTN4 was recognized in 14 of 58 patients (24.1%) with salivary gland carcinoma. The cases with CNI of ACTN4 were closely associated with histological grade (P = 0.047) and vascular invasion (P = 0.033). The patients with CNI of ACTN4 had a significantly worse prognosis than the patients with normal copy number of ACTN4 (P = 0.0005 log‐rank test). Univariate analysis by the Cox proportional hazards model showed that histological grade, vascular invasion, and CNI of ACTN4 were independent risk factors for cancer death. Vascular invasion (hazard ratio [HR]: 7.46; 95% confidence interval [CI]: 1.98–28.06) and CNI of ACTN4 (HR: 3.23; 95% CI: 1.08–9.68) remained as risk factors for cancer death in multivariate analysis. Thus, CNI of ACTN4 is a novel indicator for an unfavorable outcome in patients with salivary gland carcinoma.

ACTN4 copy number increase as a predictive biomarker for chemoradiotherapy of locally advanced pancreatic cancer

Background:Several clinical trials have compared chemotherapy alone and chemoradiotherapy (CRT) for locally advanced pancreatic cancer (LAPC) treatment. However, predictive biomarkers for optimal therapy of LAPC remain to be identified.We retrospectively estimated amplification of the ACTN4 gene to determine its usefulness as a predictive biomarker for LAPC.Methods:The copy number of ACTN4 in 91 biopsy specimens of LAPC before treatment was evaluated using fluorescence in situ hybridisation (FISH).Results:There were no statistically significant differences in overall survival (OS) or progression-free survival (PFS) of LAPC between patients treated with chemotherapy alone or with CRT. In a subgroup analysis of patients treated with CRT, patients with a copy number increase (CNI) of ACTN4 had a worse prognosis of OS than those with a normal copy number (NCN) of ACTN4 (P=0.0005, log-rank test). However, OS in the subgroup treated with chemotherapy alone was not significantly different between patients with a CNI and a NCN of ACTN4. In the patients with a NCN of ACTN4, the median survival time of PFS in CRT-treated patients was longer than that of patients treated with chemotherapy alone (P=0.049).Conclusions:The copy number of ACTN4 is a predictive biomarker for CRT of LAPC.

MicroRNA Expression and Functional Profiles of Osteosarcoma

Objective: Osteosarcoma (OS) is the most frequent primary malignant bone tumor in children and young adults. Although the introduction of combined neoadjuvant chemotherapy has significantly prolonged survival, the outcome for OS patients showing a poor response to chemotherapy is still unfavorable. In order to develop new therapeutic approaches, elucidation of the entire molecular pathway regulating OS cell proliferation would be desirable. Methods: MicroRNA (miRNA) are highly conserved noncoding RNA that play important roles in the development and progression of various other cancers. Using miRNA microarrays capable of detecting a known number of 933 miRNA, 108 miRNA were found to be commonly expressed in 24 samples of OS tissue and subjected to a cell proliferation assay. Results: We found that inhibition of 5 let-7 family miRNA (hsa-let-7a, b, f, g and i) significantly suppressed the proliferation of OS cells. Using a quantitative shotgun proteomics approach, we also found that the let-7 family miRNA regulated the expression of vimentin and serpin H1 proteins. Conclusions: Our present results indicate the involvement of let-7 family miRNA in regulation of the cell proliferation as well as epithelial-mesenchymal transition of OS. Thus, let-7 family miRNA may potentially provide novel targets for the development of therapeutic strategies against OS.

The peripheral immune status of granulocytic myeloid-derived suppressor cells correlates the survival in advanced gastric cancer patients receiving cisplatin-based chemotherapy

BACKGROUND The prognostic significance of peripheral immune status in patients with advanced gastric cancer (AGC) remains unclear. RESULTS From July 2013 through December 2014, 37 patients were enrolled. Among patients with 25 subsets of immune cells, patients in the high group of granulocytic myeloid-derived suppressor cells (Gr-MDSCs) showed significantly shorter progression-free survival (PFS) than those in the low group (3.98 vs. 8.78 months; hazards ratio (HR), 2.61; p = 0.01). In multivariate analysis, the high Gr-MDSCs value was also associated with shorter PFS (HR, 4.60; 95% confidence interval (CI), 1.79-11.8; p = 0.001). Although significant difference was not found in univariate analysis, the high Gr-MDSCs group was associated with shorter overall survival (OS) (HR, 2.89; 95% CI, 1.23-6.80; p = 0.015) in multivariate analysis. MATERIALS AND METHODS In this explorative prospective study, peripheral blood samples were collected from AGC patients before initiating first-line cisplatin-based chemotherapy (S-1 + cisplatin or S-1 + cisplatin + docetaxel). Peripheral blood mononuclear cells were analyzed for 25 immune subsets by multicolor flow cytometry. PFS and OS were compared between the patients divided into high and low (≥ and < median, respectively) groups based on the median value for each immune cell subset. CONCLUSIONS The peripheral immune status of Gr-MDSCs appears to affect the prognosis in AGC. Further research is needed to confirm the clinical value of the level of circulating Gr-MDSCs as a prognostic and/or predictive marker in AGC.Background The prognostic significance of peripheral immune status in patients with advanced gastric cancer (AGC) remains unclear. Results From July 2013 through December 2014, 37 patients were enrolled. Among patients with 25 subsets of immune cells, patients in the high group of granulocytic myeloid-derived suppressor cells (Gr-MDSCs) showed significantly shorter progression-free survival (PFS) than those in the low group (3.98 vs. 8.78 months; hazards ratio (HR), 2.61; p = 0.01). In multivariate analysis, the high Gr-MDSCs value was also associated with shorter PFS (HR, 4.60; 95% confidence interval (CI), 1.79−11.8; p = 0.001). Although significant difference was not found in univariate analysis, the high Gr-MDSCs group was associated with shorter overall survival (OS) (HR, 2.89; 95% CI, 1.23–6.80; p = 0.015) in multivariate analysis. Materials and Methods In this explorative prospective study, peripheral blood samples were collected from AGC patients before initiating first-line cisplatin-based chemotherapy (S-1 + cisplatin or S-1 + cisplatin + docetaxel). Peripheral blood mononuclear cells were analyzed for 25 immune subsets by multicolor flow cytometry. PFS and OS were compared between the patients divided into high and low (≥ and < median, respectively) groups based on the median value for each immune cell subset. Conclusions The peripheral immune status of Gr-MDSCs appears to affect the prognosis in AGC. Further research is needed to confirm the clinical value of the level of circulating Gr-MDSCs as a prognostic and/or predictive marker in AGC.

Pancreatic neuroendocrine tumors: a single-center 20-year experience with 100 patients

BACKGROUND/OBJECTIVES Pancreatic neuroendocrine neoplasms (NENs) are rare tumors, exhibiting several morphological, functional, and behavioral characteristics. However, only few reports have evaluated large case series of pancreatic NEN. METHODS We conducted a retrospective review of 100 consecutive patients with pancreatic NEN diagnosed pathologically and treated at the National Cancer Center Hospital between 1991 and 2010. RESULTS The study included 48 males and 52 females (median age: 55 years). Fourteen patients had clinical symptoms caused by excess hormone secretion at diagnosis. Twelve patients were diagnosed with neuroendocrine tumor (NET) G1, 54 with NET G2, and 32 with neuroendocrine carcinoma (NEC) as per the 2010 World Health Organization classification. Distant metastases were observed in 25%, 43%, and 84% of the patients with NET G1, NET G2, and NEC, respectively. Serum levels of neuron-specific enolase and lactate dehydrogenase significantly increased in patients with NEC compared with those in patients with NET G1/G2. The 5-year survival rates of patients with NET G1, NET G2, and NEC were 91%, 69%, and 10%, respectively. Good performance status (PS), lower stage, and histopathological grade were identified as independent favorable prognostic factors. CONCLUSIONS Patients with NET G1/G2 treated with surgical resection had a good prognosis. Most patients with NEC exhibited distant metastases and had a poor prognosis. Staging classification and the WHO 2010 grading are important factors for selecting the appropriate treatment strategy and predicting prognosis for patients with pancreatic NEN.

1613PNEXT GENERATION SEQUENCING OF CIRCULATING TUMOR CELLS ISOLATED FROM THE PERIPHERAL BLOOD OF PATIENTS WITH GASTROINTESTINAL CANCER. CIRCLE-1 TRIAL

ABSTRACT Aim: Monitoring of tumor biology provides pivotal information for suitable therapy. Circulating tumor cells (CTCs) released into the blood stream from primary tumor and metastases may reflect current tumor status. The ClearCell FX system (Clearbridge Biomedics, Singapore), which uses a label-free inertial microfluidics approach based on biomechanical properties, is able to capture CTCs independent of EpCAM expression. We investigated the tumor biology by performing genomic profiling of CTCs using next-generation sequencing (NGS). Methods: Patients with advanced gastric and colorectal cancer were enrolled in this study. We collected 10mL of blood from each patient. CTCs were extracted by using the Clearcell FX system. DNA was prepared immediately after isolation, after a weeks culture with RepCellTM (CellSeed) which was a temperature-responsive cell cultureware. Whole genome amplification (WGA) was performed by using REPLI-g Single Cell Kit (Qiagen), and NGS was performed by using Ion Ampliseq (LifeTechnology). Cells were enumerated using cytokeratin staining and immunocytochemical analysis. At the same time, we prepared DNA not only from peripheral blood mononuclear cells but also from plasma (cell-free DNA). Results: 8 patients were enrolled; 6 had colorectal cancer and 2 had gastric cancer. Median of CTC count was 6/mL (ranges 3 to 14). On the other hand, the number of CTCs collected by CellSearch ranged from 0 to 1/7.5mL. In these 8 cases, either right after isolation or after 1 week of culture, DNA derived from CTCs was successfully prepared by WGA. NGS was successfully performed with WGA treated DNA from CTCs and related materials. Conclusions: By using ClearCell FX Systems label-free technology, we were able to capture a larger number of CTCs from patients with gastric and colorectal cancer than with using the CellSearch system. NGS from CTCs and related materials can be performed successfully. Disclosure: A. Kakimoto: I am a employee of SRL Inc.; T. Sakuma: I am a employee of Mitsui Knowledge Industry Co and Ltd.; S. Fang: I am a employee of Clearbridge Accelarator; A. Wu: I am a employee of Clearbridge Bio Medics; C.T. Lim: I have stock of Clearbridge Biomedics Pte Ltd. All other authors have declared no conflicts of interest.