Cancer exosomal microRNAs from gefitinib-resistant lung cancer cells cause therapeutic resistance in gefitinib-sensitive cells.

PURPOSE: Exosomes and their cargo microRNAs play a significant role in various biological processes in cancer. We hypothesized that microRNAs in exosomes secreted by gefitinib-resistant lung cancer cells might induce resistant phenotypes in otherwise gefitinib-sensitive lung cancer cells. METHODS: We isolated exosomes generated by the gefitinib-resistant human lung adenocarcinoma cell line PS-9/ZD. PC-9, which is a gefitinib-sensitive cell line, was treated with the PC-9/ZD exosomes, and these PC-9 cells were analyzed for cell proliferation after treatment with gefitinib. miRNA arrays were analyzed in PC-9 and PC-9/ZD cells, and we isolated microRNAs that were expressed at elevated levels in PC-9/ZD cells. Furthermore, we transfected these microRNAs into PC-9 cells and analyzed the effects on the cells' sensitivity to gefitinib. RESULTS: Exosomes isolated from PC-9/ZD cells significantly increased the proliferation of PC-9 cells during gefitinib treatment. A microRNA array analysis showed that miR-564, miR-658, miR-3652, miR-3126-5p, miR-3682-3p and miR-6810-5p were significantly upregulated in PC-9/ZD cells. PC-9 cells transfected with miR-564 or miR-658 showed chemo-resistant phenotypes. CONCLUSION: Exosomal miR-564 and miR-658 derived from gefitinib-resistant lung cancer cells induce drug resistance in sensitive cells. Cell-to-cell interaction via exosomal microRNAs may be a novel mechanism and therapeutic target of resistance against gefitinib.

Clinicopathological features of small-sized peripheral squamous cell lung cancer.

Recent advances in imaging technology have enhanced the detection rate of small-sized peripheral lung cancers. The present study aimed to identify the clinicopathological differences between patients with small-sized peripheral squamous cell carcinoma (SCC) and adenocarcinoma (ADC). Patients with lung cancer who underwent radical surgical resection at Gunma University Hospital between July 2007 and October 2012 were retrospectively analyzed. Patients who exhibited small-sized peripheral tumors (pathological size, ≤2 cm) located within the outer-third of the lung field on preoperative computed tomography were enrolled in the present study. A total of 26 patients were diagnosed with SCC and 214 with ADC. The results revealed that patients with SCC exhibited higher rates of pleural invasion, vascular invasion and lymphatic invasion compared with ADC patients. Additionally, the rate of postoperative recurrence was higher in patients with SCC compared with ADC patients. Patients with ADC were subsequently into two groups: Solid ADCs (sADC) and non-solid ADCs (nsADC), which included pure ground glass nodules and part-solid ADCs. The results revealed that the incidence of pleural invasion, vascular invasion and lymphatic invasion, and the rate of postoperative recurrence in patients with sADCs were similar to those with SCC, but were also significantly higher when compared with nsADC patients. The present study concluded that patients with SCC and sADC may not be suitable candidates for sublobar resection, despite exhibiting small tumors that are located in the peripheral lung.

Carbonic anhydrase 9 expression is associated with poor prognosis, tumor proliferation, and radiosensitivity of thymic carcinomas.

INTRODUCTION: Thymic epithelial tumors (TETs) comprise several histologies of thymoma and thymic carcinomas (TCs), and TC frequently metastasizes and causes death. We therefore aimed here to identify key molecules closely related to prognosis and their biological roles in high-risk TETs, particularly TCs. RESULTS: RNA sequence analysis demonstrated that hypoxia-related genes were highly expressed in TETs. The expression of the hypoxia-related gene CA9 was noteworthy, particularly in TCs. Immunohistochemical analysis revealed that CA9 was expressed in 81.0% of TCs and 20.7% of all TET samples. CA9 expression was significantly associated with Masaoka stage, WHO classification, and recurrence-free survival after tumor resection (P = 0.005). The down-regulation of CA9 transcription in TC cell lines by small interfering RNAs significantly inhibited CA9 expression, which inhibited proliferation and increased sensitivity to irradiation. CONCLUSIONS: CA9 expression may serve as a significant prognostic marker of TETs and therefore represents a potential target for the development of novel drugs and radiation-sensitizing therapy designed to improve the outcomes of patients with TCs. MATERIALS AND METHODS: We performed comprehensive transcriptome sequencing of 23 TETs and physiologic thymic specimens to identify genes highly and specifically expressed in high-risk TETs, particulary TCs. We performed immunohistochemical analysis of 179 consecutive surgically resected TETs to evaluate the significance of the association of protein expression with clinicopathological features and prognosis. The biological significance of the most promising prognostic marker was further studied using the TC cell lines, Ty-82 and MP57.

Genetic and Immunohistochemical Studies Investigating the Histogenesis of Neuroendocrine and Carcinomatous Components of Combined Neuroendocrine Carcinoma.

BACKGROUND: Lung combined neuroendocrine carcinomas (NECs) comprise NEC and non-NEC components, such as adenocarcinoma and squamous cell carcinoma. Mutation of epidermal growth factor receptor (EGFR) often is observed in non-NEC but is very rare in sporadic NEC, which almost always has p53 mutation. Therefore, we hypothesized the following research concept: mutation analysis of EGFR and p53 in each component of combined NEC tissues can provide important information on whether such components originate from the same tumor cells or incidentally arise as collision cancers. METHODS: We compared the mutations of EGFR and p53 in laser-microdissected NEC and non-NEC from lungs of eight cases affected by combined NEC. We examined the expression of EGFR and NEC markers in the combined NECs by immunohistochemistry. RESULTS: Five of eight cases of combined NEC had the same mutations of EGFR and/or p53 in both non-NEC and NEC. One case had EGFR mutation in only the non-NEC component, and two cases did not have these mutations. Replacement transformation was observed in borderline areas between non-NEC and NEC. The signal of activated EGFR in non-NEC with the same EGFR mutation was more intense than that in NEC components. CONCLUSIONS: Our study suggests the mechanism behind the carcinogenesis of lung combined NEC, which is partially caused by the transformation from epithelial carcinoma of non-NEC to NEC.

Prognostic significance of PD-L1 expression and tumor infiltrating lymphocytes in large cell neuroendocrine carcinoma of lung.

OBJECTIVES: Since large cell neuroendocrine carcinoma (LCNEC) is a relatively rare histologic type of primary lung cancer, little is known about the immunological status of patients with LCNEC. We aimed to clarify the expression and prognostic impact of programmed cell death ligand 1 (PD-L1), CD8, CD4, and Forkhead box protein P3 (Foxp3) in LCNEC. METHODS: We retrospectively analyzed PD-L1, CD8, CD4, and Foxp3 expressions in 95 surgically resected LCNEC. PD-L1 positive staining was determined in tumors with more than 1% of tumor cells stained to any intensity, and CD8, CD4, and Foxp3 positivity was determined in tumors with more than 5% of lymphocytes stained. RESULTS: Positive expression of PD-L1, CD8, CD4, and Foxp3 was observed in 70 (74%), 52 (55%), 76 (80%), and 43 (45%) tumors, respectively. The expression of PD-L1 was significantly correlated with positive lymphatic permeation. Positive correlations were mutually observed among tumor infiltrating immune cells. Univariate and multivariate analyses showed that positive pleural invasion and Foxp3 negative expression were independent unfavorable prognostic factors for overall survival (OS). Advanced pathological stage, positive pleural invasion, CD4 negative expression in cancer stroma, and Foxp3 negative expression were identified as independent unfavorable prognostic factors for recurrence free survival (RFS). CONCLUSIONS: Foxp3 positive tumor infiltrating lymphocytes (TILs) were an independent favorable prognostic factor for both OS and RFS, whereas CD4 positive TILs were an independent significant unfavorable prognostic factor for RFS. The high frequency of PD-L1 expression could support the use of anti-programmed cell death 1 antibody in the treatment of LCNEC.

Ectopic thymoma in the paratracheal region of the middle mediastinum: a rare case report and literature review.

BACKGROUND: Thymomas generally arise from the thymus in the anterior mediastinum. Ectopic thymomas arising in the middle mediastinum are rare. We present a case of a thymoma arising from the ectopic thymic tissue in the right paratracheal region. CASE PRESENTATION: The patient was a 67-year-old male who underwent an enhanced-computed tomography examination as preoperative staging for colon cancer. A 20-mm nodule in the right paratracheal region was found incidentally. Fluorodeoxyglucose (FDG) accumulation was detected in this solitary nodule by FDG-positron emission tomography, mimicking an enlarged, possibly malignant lymph node. The tumor was removed by thoracoscopic surgery, and a postoperative pathological diagnosis of type AB thymoma was made. Foci of ectopic thymic tissues were found adjacent to the thymoma. The patient was disease-free and without recurrence 2 years postoperatively. CONCLUSIONS: Including the present case, 13 cases of ectopic paratracheal thymoma have been reported in the English literature, all of which were found on the right side of the paratracheal region. Although ectopic thymomas in the paratracheal region are rare, thymomas may be considered as a differential diagnosis for a paratracheal nodule.

Development of single nanometer-sized ultrafine oxygen bubbles to overcome the hypoxia-induced resistance to radiation therapy via the suppression of hypoxia-inducible factor­1α.

Radiation therapy can result in severe side-effects, including the development of radiation resistance. The aim of this study was to validate the use of oxygen nanobubble water to overcome resistance to radiation in cancer cell lines via the suppression of the hypoxia-inducible factor 1-α (HIF­1α) subunit. Oxygen nanobubble water was created using a newly developed method to produce nanobubbles in the single-nanometer range with the ΣPM-5 device. The size and concentration of the oxygen nanobubbles in the water was examined using a cryo-transmission electron microscope. The nanobubble size was ranged from 2 to 3 nm, and the concentration of the nanobubbles was calculated at 2x1018 particles/ml. Cell viability and HIF-1α levels were evaluated in EBC­1 lung cancer and MDA­MB­231 breast cancer cells treated with or without the nanobubble water and radiation under normoxic and hypoxic conditions in vitro. The cancer cells grown in oxygen nanobubble-containing media exhibited a clear suppression of hypoxia-induced HIF­1α expression compared to the cells grown in media made with distilled water. Under hypoxic conditions, the EBC­1 and MDA­MB231 cells displayed resistance to radiation compared to the cells cultured under normoxic cells. The use of oxygen nanobubble medium significantly suppressed the hypoxia-induced resistance to radiation compared to the use of normal medium at 2, 6, 10 and 14 Gy doses. Importantly, the use of nanobubble media did not affect the viability and radiation sensitivity of the cancer cell lines, or the non­cancerous cell line, BEAS­2B, under normoxic conditions. This newly created single-nanometer range oxygen nanobubble water, without any additives, may thus prove to be a promising agent which may be used to overcome the hypoxia-induced resistance of cancer cells to radiation via the suppression of HIF-1α.

Clinical and Radiological Discrimination of Solitary Pulmonary Lesions in Colorectal Cancer Patients.

OBJECTIVES: The lung is one of the most common organs of metastasis from colorectal cancer (CRC), and we have encountered lung cancer patients with a history of CRC. There have been few studies regarding methods used to discriminate between primary lung cancer (PLC) and pulmonary metastasis from CRC (PM-CRC) based only on preoperative findings. We retrospectively investigated predictive factors discriminating between these lesions in patients with a history of CRC. METHODS: Between 2006 and 2015, 117 patients with a history of CRC (44 patients with 47 PLC and 73 patients with 102 PM-CRC) underwent subsequent or concurrent resection of pulmonary lesions. We compared the clinical and radiological characteristics of 100 patients with solitary lesions (43 PLC and 57 PM-CRC). Using univariate and multivariate analyses, we examined predictive factors for discrimination of these two lesions. RESULTS: All tumors with findings of ground-glass opacity (GGO) were PLC (n = 19). In a multivariate analysis of 81 radiologically solid tumors, two factors were found to be significant independent predictors of PLC: a history of stage I CRC and presence of pleural indentation. All tumors in 26 patients with either GGO or both a stage I CRC history and pleural indentation were PLC, while most tumors in patients without all three factors were PM-CRC (43/44; 97.7%). CONCLUSIONS: The presence or absence of GGO, pathological CRC stage, and pleural indentation could be useful factors to distinguish between PLC and PM-CRC.

High STMN1 Expression is Associated with Cancer Progression and Chemo-Resistance in Lung Squamous Cell Carcinoma.

BACKGROUND: Known as a microtubule-destabilizing protein, STMN1 (gene symbol: STMN1) regulates the dynamics of microtubules, cell cycle progress, and chemo-resistance against taxane agents. It is highly expressed in various human cancers and involved in cancer progression as well as poor prognosis. METHODS: Expression of STMN1 was examined by immunohistochemistry using FFPE tissue sections from 186 patients with lung squamous cell carcinoma (LSCC). Analysis of STMN1 suppression was performed for STMN1 small interfering RNA (siRNA)-transfected LSCC cell lines to determine the change in proliferation, invasive and apoptosis abilities, and paclitaxel sensitivity. RESULTS: The cytoplasmic STMN1 expression in LSCC was higher than in normal tissues. The high expression was significantly associated with vascular invasion (P = 0.0477) and poor prognosis. In addition, the proliferating and invasive abilities were decreased, and the apoptosis ability and paclitaxel sensitivity were increased in STMN1-suppressed LSCC cells compared with control cells. CONCLUSION: The results suggest that STMN1 is a prognostic factor that also is associated with caner progression and chemo-resistance. Therefore, STMN1 could be a predictor for poor prognosis and a potential therapeutic target in LSCC.

Elevated expression of ΔNp63 in advanced esophageal squamous cell carcinoma.

This study aims to explore the expression level of ΔNp63 in esophageal squamous cell carcinoma (ESCC). To investigate the association between ΔNp63 (p40) expression and ESCC biology, we compared the levels of ΔNp63 expression in normal and tumor tissues, with a specific focus on the diagnostic value of ΔNp63 in ESCC. We analyzed 160 consecutive patients with ESCC who underwent surgical resection without neoadjuvant chemotherapy at Gunma University Hospital (Maebashi, Japan) between September 2000 and January 2010. The clinicopathological characteristics and survival of patients were subclassified based on the expression of ΔNp63 as determined by immunohistochemistry, indicating that ΔNp63 was highly expressed in 75.6% (121/160) of ESCC patients. Clinicopathological analysis of ΔNp63 expression showed that ΔNp63-positive tumors significantly correlated with two important clinical parameters: T factor (P = 0.0316) and venous invasion (P = 0.0195). The 5-year overall survival rates of advanced ESCC patients with positive and negative expression of ΔNp63 were 35.6% and 71.7%, respectively. Multivariate analysis revealed that the expression of ΔNp63 was identified as an independent prognostic factor (P = 0.0049) in advanced ESCC. In line with this, ΔNp63α-transduced ESCC cell lines increased tumor growth in a soft agar colony formation assay. We report here for the first time that ΔNp63 expression increases the oncogenic potential of ESCC and is an independent marker for predicting poor outcome in advanced ESCC. Our findings suggest that ΔNp63 could serve as a new diagnostic marker for ESCC and might be a relevant therapeutic target for the treatment of patients with this disease.

Src kinase-associated phosphoprotein2 expression is associated with poor prognosis in non-small cell lung cancer.

BACKGROUND/AIM: Non-small cell lung cancer (NSCLC) is among the leading causes of cancer-related deaths worldwide. In certain human cancer types, Src is associated with cancer progression and refractory cancer. To improve the prognoses of NSCLC patients, we evaluated Src kinase-associated phosphoprotein 2 (SKAP2), a factor associated with integrin-stimulated cytoskeletal rearrangement, as a new therapeutic target. MATERIALS AND METHODS: We performed immunohistochemistry for SKAP2 in 99 NSCLC samples and evaluated the relationship between SKAP2 expression, clinicopathological factors and prognosis. RESULTS: Higher SKAP2 expression was detected in cancerous tissues and was predominantly expressed in the cytoplasm. Elevated SKAP2 expression levels were associated with poor prognosis (p=0.007) and shorter survival time after recurrence (p=0.035). High SKAP2 expression was an independent prognostic factor in NSCLC patients (p=0.027). CONCLUSION: High SKAP2 expression levels in NSCLC tissues could be a powerful biomarker of poor prognosis. Therefore, SKAP2 is a promising candidate molecular target for NSCLC treatment.