Takuro Yukawa

Tumor-Infiltrating Foxp3+ Regulatory T Cells are Correlated with Cyclooxygenase-2 Expression and are Associated with Recurrence in Resected Non-small Cell Lung Cancer

Background: Cyclooxygenase-2 (COX-2) is constitutively overexpressed in a variety of epithelial malignancies and is usually associated with a poor prognosis. COX-2-derived prostaglandin E2 transforms CD4+CD25+ T regulatory (Treg) cells (Tregs), and Tregs are thought to moderate the antitumor immune response. Herein, we investigated the prognostic value of tumor-infiltrating Treg cells and their correlation with COX-2 expression in resected non-small cell lung cancer (NSCLC). Material and Methods: Intratumoral COX-2 and Treg expression were retrospectively assessed using immunohistochemistry in paraffin-embedded samples from 100 patients who had undergone complete resections for NSCLC. The expressions of COX-2 and Foxp3, which was most specific Treg cell marker, were compared with the clinicopathological variables, and the correlation between Foxp3+ Tregs and COX-2 expression was analyzed. Results: The recurrence-free survival (RFS) of patients with elevated COX-2 expression was significantly worse than that of patients without COX-2 expression. Tumor-infiltrating Foxp3-positive lymphocytes were positively correlated with COX-2 expression. The median count for Foxp3-positive lymphocytes was 3 (minimum-maximum, 0–24) in 10 high-power fields. The RFS of patients with tumors containing ≥3 Foxp3-positive cells (Foxp3 expression group) was significantly worse than that of patients with tumors containing <3 Foxp3-positive cells. In a multivariate analysis, only nodal status was an independent predictor of a significantly shorter RFS. However, in node-negative NSCLC, Foxp3 expression was an independent predictor of a significantly shorter RFS. Conclusions: Tumor-infiltrating Foxp3+ Tregs were positively correlated with intratumoral COX-2 expression and were associated with a worse RFS, especially among patients with node-negative NSCLC.

Post-recurrence survival of patients with non-small-cell lung cancer after curative resection with or without induction/adjuvant chemotherapy

OBJECTIVES Recently, the prognosis of patients with non-small-cell lung cancer (NSCLC) has improved, thanks to the standardization of adjuvant chemotherapy and the introduction of molecular-targeted drugs, notably epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors and other new anti-cancer agents. However, the survival characteristics and prognosis of patients with recurrent NSCLC after curative resection are not well understood. METHODS Of the 430 consecutive patients with NSCLC who underwent complete surgical resection at our institution between January 2004 and July 2011, we included 76 patients with recurrence whose post-recurrence treatment and outcome could be confirmed. We then retrospectively evaluated the effect of prognostic factors on post-recurrence survival. RESULTS There were 50 men and 26 women, and the median age at recurrence was 74.5 years. The median time from surgical resection to recurrence was 12.7 months. Thirty-eight of the 76 (50%) patients underwent multimodality treatment with surgery and preoperative and/or postoperative chemotherapy as their initial treatment. For recurrence, systemic chemotherapy was administered to 64 (84%) patients, and the disease control rate for first-line chemotherapy was 55%. The 1- and 2-year post-recurrence survival rates were 68.3 and 45.8%, respectively, and the median post-recurrence survival time was 17.7 months. Six independent prognostic factors were identified: wild-type EGFR, no adjuvant chemotherapy for the primary lung cancer, age ≥ 80 years at recurrence, a poor Eastern Cooperative Oncology Group performance status at recurrence, symptomatic at recurrence and no systemic chemotherapy for recurrence, which significantly decreased the post-recurrence survival. CONCLUSIONS The prognosis of patients with NSCLC recurrence after surgery is currently improving. Our results suggested two new prognostic factors, adjuvant chemotherapy and EGFR mutations, neither of which have been previously reported. Treatment strategies for postoperative recurrence should be established based on a more detailed subdivision of factors, such as histology and molecular markers, in the future.

Influence of vascular endothelial growth factor single nucleotide polymorphisms on non-small cell lung cancer tumor angiogenesis

Vascular endothelial growth factor (VEGF) plays an important role in tumor angiogenesis. Several studies have reported that genomic VEGF polymorphisms may influence VEGF synthesis. To evaluate the role of VEGF single nucleotide polymorphisms (SNPs), we examined the expression of several angiogenesis-related proteins [VEGF, hypoxia-inducible factor-1α (HIF-1α) and delta-like ligand 4 (Dll4)] and the spread of microvessels in resected non-small cell lung cancer (NSCLC). Blood and tumor tissue from 83 patients with NSCLC were examined for VEGF −460T/C (rs833061) and VEGF +405G/C (rs2010963) SNPs using the SNaPshot method. Immunohistochemical staining was performed to measure protein expression and microvessel density (MVD). VEGF −460T/C and +405G/C SNPs showed no association with VEGF or HIF-1α expression and MVD. Patients with VEGF −460TT and the TC genotype had significantly higher MVD compared to those with the CC genotypes. Furthermore, patients with the VEGF −460TT genotype had significantly higher Dll4 expression compared to those with the TC or CC genotypes, while the VEGF +405G/C SNP displayed no association with Dll4 expression and MVD. These findings indicate that the VEGF −460T/C SNP may have a functional influence on tumor angiogenesis in NSCLC. We hypothesize that VEGF SNPs may influence angiogenesis through Dll4.

Contrasting Effects of the Cytotoxic Anticancer Drug Gemcitabine and the EGFR Tyrosine Kinase Inhibitor Gefitinib on NK Cell-Mediated Cytotoxicity via Regulation of NKG2D Ligand in Non-Small-Cell Lung Cancer Cells

Introduction Several cytotoxic anticancer drugs inhibit DNA replication and/or mitosis, while EGFR tyrosine kinase inhibitors inactivate EGFR signalling in cancer cell. Both types of anticancer drugs improve the overall survival of the patients with non-small-cell lung cancer (NSCLC), although tumors often become refractory to this treatment. Despite several mechanisms by which the tumors become resistant having been described the effect of these compounds on anti-tumor immunity remains largely unknown. Methods This study examines the effect of the cytotoxic drug Gemcitabine and the EGFR tyrosine kinase inhibitor Gefitinib on the expression of NK group 2 member D (NKG2D) ligands as well as the sensitivity of NSCLC cells to the NK-mediated lysis. Results We demonstrate that Gemcitabine treatment leads to an enhanced expression, while Gefitinib downregulated the expression of molecules that act as key ligands for the activating receptor NKG2D and promote NK cell-mediated recognition and cytolysis. Gemcitabine activated ATM and ATM- and Rad-3-related protein kinase (ATR) pathways. The Gemcitabine-induced phosphorylation of ATM as well as the upregulation of the NKG2D ligand expression could be blocked by an ATM-ATR inhibitor. In contrast, Gefitinib attenuated NKG2D ligand expression. Silencing EGFR using siRNA or addition of the PI3K inhibitor resulted in downregulation of NKG2D ligands. The observations suggest that the EGFR/PI3K pathway also regulates the expression of NKG2D ligands. Additionally, we showed that both ATM-ATR and EGFR regulate MICA/B via miR20a. Conclusion In keeping with the effect on NKG2D expression, Gemcitabine enhanced NK cell-mediated cytotoxicity while Gefitinib attenuated NK cell killing in NSCLC cells.

Maximal Standardized Uptake Value on FDG-PET Is Correlated With Cyclooxygenase-2 Expression in Patients With Lung Adenocarcinoma

BACKGROUND Cyclooxygenase-2 (COX-2) is constitutively overexpressed in a variety of epithelial malignancies and is usually associated with a poor prognosis. Fluorodeoxyglucose positron emission tomography (FDG-PET) has become an important tool for the diagnosis and staging of non-small-cell lung cancer. The maximal standardized uptake values (SUVmax) of primary tumors on FDG-PET have been shown to be correlated with some clinicopathologic factors. In this study, we investigated the prediction of intratumoral COX-2 expression by FDG-PET in cases of lung adenocarcinoma. METHODS We conducted a retrospective review of the data of 60 patients with lung adenocarcinoma measuring less than 3 cm in diameter. Immunohistochemical staining for COX-2 and other biological factors that might influence cancer progression was performed, and the correlations of the selective tumor marker expression with the SUVmax were evaluated. RESULTS A significant correlation was observed between the SUVmax and the expressions of COX-2, Ki-67, and vascular endothelial growth factor (VEGF). Multiple stepwise regression analysis revealed significant relationships between the SUVmax and the expression of COX-2 (p<0.001) and Ki-67 (p=0.016). Of the 2, COX-2 expression was the stronger determinant of the SUVmax, which increased in proportion to the score for COX-2 expression. The recurrence-free survival of patients with elevated COX-2 expression was significantly worse than that of patients not showing COX-2 expression. CONCLUSIONS The expression of COX-2 in primary tumors is as strongly correlated with a worse clinical outcome as is increased FDG uptake in cases of lung adenocarcinoma. These findings indicate that the SUVmax of primary tumors might reflect the biological malignant potential in lung adenocarcinomas.

Cyclooxygenase-2 expression is a prognostic biomarker for non-small cell lung cancer patients treated with adjuvant platinum-based chemotherapy

BackgroundAdjuvant chemotherapy after the resection of stage IB-IIIA non-small cell lung cancer (NSCLC) is now the standard of care based on large-scale phase III trials and a meta-analysis. However, chemotherapy has plateaued in terms of its efficacy, and the search for treatment prediction biomarkers is imperative for the further identification of treatable subgroups. Therefore, we investigated the significance of cyclooxygenase-2 (Cox-2) expression and the applicability of a Cox-2 inhibitor in patients who had received adjuvant chemotherapy.MethodsWe conducted a retrospective review of data from 97 patients who had received adjuvant chemotherapy. The adjuvant chemotherapy consisted of an oral tegafur agent (OT) or platinum-based chemotherapy (PB). The criteria for regimen selection were based on a discussion among the cancer board and enrollment in a clinical trial. Immunohistochemical staining (IHC) for Cox-2 was performed, and the correlation between Cox-2 expression and disease-free survival (DFS) was evaluated.ResultsIHC showed that 56 cases (57.7%) were positive for Cox-2. The rate of Cox-2 expression was similar for the PB and OT groups. Among the patients who received PB, the DFS of the patients with Cox-2 expression was significantly poorer than that of the patients without Cox-2 expression (P = 0.017), but there was no significant difference among the patients who received OT (P = 0.617). In a multivariate analysis, Cox-2 expression and lymph node metastasis were independent predictors of DFS among patients who received PB.ConclusionsCox-2 expression was a powerful predictor of DFS among patients who received PB as an adjuvant chemotherapy. Further study investigating the use of a Cox-2 inhibitor for adjuvant chemotherapy is needed.

Unknown primary large cell neuroendocrine carcinoma (LCNEC) in the mediastinum

Unknown primary large cell neuroendocrine carcinoma (LCNEC) in the mediastinum is extremely rare. In this report, we present a case of a 53-year-old man with superior vena cava (SVC) syndrome who developed LCNEC in the middle mediastinum. His chief complaint was facial edema. Chest X-ray revealed an abnormal shadow in the right upper mediastinum. Computed tomography (CT) scan of the chest revealed a 67-mm mass in the middle mediastinum. Tumor invasion caused constriction of the SVC. The patient underwent induction chemoradiotherapy with vinorelbin and cisplatin and concurrent radiation therapy. After induction therapy, the tumor size decreased remarkably and was resected completely. The pathological diagnosis was LCNEC.

Prognostic nutritional index before adjuvant chemotherapy predicts chemotherapy compliance and survival among patients with non-small-cell lung cancer

Background Adjuvant chemotherapy after the complete resection of non-small-cell lung cancer (NSCLC) is now the standard of care. To improve survival, it is important to identify risk factors for the continuation of adjuvant chemotherapy. In this study, we analyzed chemotherapy compliance and magnitude of the prognostic impact of the prognostic nutritional index (PNI) before adjuvant chemotherapy. Methods We conducted a retrospective review of data from 106 patients who had received adjuvant chemotherapy. The adjuvant chemotherapy consisted of an oral tegafur agent (OT) or platinum-based chemotherapy (PB). The correlations between the PNI values and recurrence-free survival (RFS) were then evaluated. Results In the PB group, the percentage of patients who completed the four planned cycles of chemotherapy was not correlated with the PNI. In the OT group, however, a significant difference was observed in the percentage of patients who completed the planned chemotherapy according to the PNI before adjuvant chemotherapy. The RFS of patients with a PNI <50 before adjuvant chemotherapy was significantly poorer than that of the patients with a PNI ≥50. A multivariate analysis showed that nodal metastasis and PNI before chemotherapy were independent predictors of the RFS. However, PNI before surgery was not a predictor of the RFS. In the subgroup analysis, PNI before chemotherapy was independent predictor of the RFS in the OT group (P=0.019), but not in the PB group (P=0.095). Conclusion The PNI before adjuvant chemotherapy influenced the treatment compliance with the planned chemotherapy in the OT group, but not the PB group. In addition, a low PNI before adjuvant chemotherapy was associated with a poor RFS in a multivariate analysis, especially in the OT group.

Lapatinib enhances trastuzumab-mediated antibody-dependent cellular cytotoxicity via upregulation of HER2 in malignant mesothelioma cells.

EGFR/HER2 are frequently expressed in MPM tissues, however, no studies have shown the clinical benefit of using EGFR/HER2-targeting drugs in patients with malignant pleural mesothelioma (MPM). It was reported that the tyrosine kinase inhibitor (TKI) lapatinib enhanced trastuzumab-mediated antibody-dependent cellular cytotoxicity (ADCC) in HER2-positive breast cancer, suggesting that this combination is a promising strategy for MPM treatment. The aim of the present study was to explore the possibility of a TKI combined with trastuzumab to enhance ADCC in MPM cells. Five MPM cell lines were used to test the effects of TKIs targeting EGFR (gefitinib, afatinib and lapatinib) on cell proliferation and the expression of the HER family receptor. The combined effects of TKI with trastuzumab on ADCC were evaluated using the LDH release assay. Additionally, MPM cells were isolated from patients and evaluated for lapatinib-induced upregulation of HER family receptors and trastuzumab- or cetuximab-mediated ADCC. In MPM cell lines, HER2 expression was upregulated by lapatinib, downregulated by afatinib and unaffected by gefitinib. As expected, more trastuzumab bound to MPM cells pretreated with lapatinib than untreated cells, resulting in the enhancement of trastuzumab-mediated ADCC in MPM cells. In patient-derived MPM cells, both HER2 and EGFR were upregulated by lapatinib, resulting in the enhancement of both trastuzumab- and cetuximab-mediated ADCC. Of the three TKIs, only lapatinib enhanced trastuzumab-mediated ADCC via the upregulation of HER2 expression in MPM cells, suggesting that sequential combination of lapatinib and trastuzumab may be a promising strategy for MPM treatment.

Cyclooxygenase-2 genetic variants influence intratumoral infiltration of Foxp3-positive regulatory T cells in non-small cell lung cancer

The immune microenvironment of primary tumors has been reported to be a prognostic factor. We previously reported that the tumor-infiltrating regulatory T cell (Treg) count was positively correlated with the intratumoral cyclooxygenase-2 (COX-2) expression level and was associated with a poor survival among patients with non-small cell lung cancer (NSCLC). Recently, numerous single nucleotide polymorphisms (SNPs) in the COX-2 gene have been identified, and these SNPs may contribute to differential gene expression and enzyme activity levels. However, whether COX-2 genetic variants influence the functions of COX-2 in NSCLC remains unclear. Eighty NSCLC patients who underwent a complete resection at our institute were enrolled. We extracted DNA from the peripheral blood and identified five different COX-2 SNPs. The correlations between the COX-2 SNPs and the expression levels of COX-2, Tregs and Ki-67 were studied. The prognostic significance of the COX-2 SNPs was also evaluated. COX-2 SNPs were not correlated with the expression of COX-2. However, for the COX-2 -1195G/A polymorphism, the AA genotype group had a significantly higher Treg score. Furthermore, the AA group had a significantly higher Treg score regardless of the COX-2 expression level. The COX-2 -1195AA genotype group tended to have a shorter disease-free survival period than the GA/GG group. In conclusion, the COX-2 -1195G/A polymorphism influences the infiltration of Tregs into NSCLC, and the COX-2 SNP factor may be a prognostic factor reflecting Treg infiltration in NSCLC.