Katsuhiko Shimizu

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.

Preoperative neutrophil/lymphocyte ratio and prognostic nutritional index predict survival in patients with non-small cell lung cancer.

BackgroundThe immunological status, consisting of “inflammation status” and “nutritional condition,” is important for the survival of patients with various cancers, including non-small cell lung cancer (NSCLC). The neutrophil/lymphocyte ratio (NLR) reflects the inflammation status, and the prognostic nutritional index (PNI) reflects the immunological nutritional condition. In the present study, the correlation between the NLR and the PNI as well as the consistency and magnitude of the prognostic impact of the NLR and the PNI were investigated.MethodsWe conducted a retrospective review of data from 334 patients who had undergone a curative resection for NSCLC. The NLR and the PNI were calculated, which was routinely performed before surgery. The correlations between the NLR and the PNI and survival were then evaluated.ResultsA clear inverse correlation was observed between the NLR and the PNI. The NLR was associated with sex, smoking history, the CEA level, tumor size, and vascular invasion. The PNI was associated with sex, age, smoking history, tumor size, histological type, tumor differentiation, and vascular invasion. Patients with NLR ≥2.5 had a significantly poorer survival outcome, and patients with PNI <50 had a significantly poorer survival outcome. A multivariate analysis demonstrated that age, nodal metastasis, tumor differentiation, NLR, and PNI were independent predictors of disease-free and overall survival.ConclusionsOur study demonstrated a significant inverse correlation between the NLR and the PNI, and a high NLR and a low PNI were significantly associated with a poor survival among patients who had undergone a complete resection for NSCLC.

Heterogeneity of the EGFR mutation status between the primary tumor and metastatic lymph node and the sensitivity to EGFR tyrosine kinase inhibitor in non-small cell lung cancer

The purpose of this study was to clarify the distribution of epidermal growth factor receptor (EGFR) mutations between primary tumors (PT) and metastatic lymph node (MLN) in patients with resected non-small cell lung cancer (NSCLC) and to identify a better predictive marker of the response to EGFR tyrosine kinase inhibitor (EGFR-TKI). We conducted a retrospective review of the data of 70 lung cancer patients with lymph node metastasis who underwent surgical resection. Analysis to detect EGFR mutations was performed by a peptide nucleic acid–locked nucleic acid polymerase chain reaction clamp method. EGFR mutations were detected in 15.7xa0% of both the PT and MLN and in 14.3xa0% of the PT only. The response rate to EGFR-TKI tended to be higher in patients with EGFR mutations in the MLN, as all patients with EGFR mutations in the MLN showed disease control to treatment with EGFR-TKI. Our results demonstrated that the EGFR mutation status of MLN is a predictive marker of the response to EGFR-TKI therapy in patients with recurrent NSCLC after surgical resection.

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

OBJECTIVESnRecently, 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.nnnMETHODSnOf 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.nnnRESULTSnThere 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.nnnCONCLUSIONSnThe 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.

PD-L1 overexpression is partially regulated by EGFR/HER2 signaling and associated with poor prognosis in patients with non-small-cell lung cancer

Immunocheckpoint inhibitors targeting the programmed cell death-1 (PD-1) and PD-1 ligand 1 (PD-L1) axis have shown promising results in patients with non-small-cell lung cancer (NSCLC). Recent research has shown that epidermal growth factor receptor (EGFR) signaling affects PD-L1 expression in NSCLC cells; however, the mechanism regulating PD-L1 expression in tumor cells remains unclear. Using immunohistochemistry, we evaluated the impact of expression of PD-L1 and EGF family receptors EGFR and human epidermal growth factor receptor 2 (HER2) in tumor cells from 91 patients with pathological Stage IA–IIIA NSCLC. Overexpression of PD-L1 was observed in 14% of the resected tumors, and associated with poor recurrence-free survival (pu2009=u20090.021) and overall survival (pu2009=u20090.033). PD-L1 expression is positively correlated with EGFR expression and inversely correlated with HER2. NSCLC cell lines were treated in vitro with the EGFR ligand EGF with or without inhibition of EGFR or HER2, after which PD-L1 expression was evaluated using flow cytometry. Consistent with previous reports, PD-L1 expression was clearly enhanced by EGF. EGFR-tyrosine kinase inhibitors or EGFR small interfering RNA (siRNA) blocked EGF-induced PD-L1 overexpression in NSCLC cell lines, but HER2 siRNA did not. Moreover, our findings suggest that PD-L1 expression could be partially regulated via the PI3K/AKT and JAK/STAT pathways. We conclude that PD-L1 overexpression is associated with poor prognosis and is positively correlated with EGFR expression but inversely correlated with HER2 expression in NSCLC. We also showed that EGFR and HER2 have different effects on EGF-induced PD-L1 expression in NSCLC cell lines.

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.

MHC class I chain-related molecule A and B expression is upregulated by cisplatin and associated with good prognosis in patients with non-small cell lung cancer

Abstract MHC class I chain-related molecule A and B (MICA/B) are NK group 2 member D (NKG2D) ligands, which are broadly expressed in transformed cells. Both DNA damage-induced ataxia-telangiectasia-mutated (ATM)- and ATM and Rad3-related protein kinases (ATM–ATR) signaling and oncogene-induced PI3K–AKT signaling regulate the expression of NKG2D ligands, which promote NK cell-mediated cytotoxicity via NKG2D–NKG2D ligand interactions. NKG2D ligand overexpression was recently reported to be correlated with good prognosis in several types of cancer. However, the prognostic significance of NKG2D ligands in non-small cell lung cancer (NSCLC) remains unclear. Here, MICA/B expression was evaluated based on immunohistochemistry of 91 NSCLC samples from patients following radical surgery. In addition, expression of MICA/B was assessed in NSCLC cell lines treated with cisplatin in order to evaluate the regulatory mechanisms of MICA/B expression. Overall, 28 out of 91 (30.8xa0%) specimens showed high expression level of MICA/B, which was associated with low 18F-fluorodeoxyglucose uptake and manifestation of adenocarcinoma. After a median follow-up of 48.2xa0months, high MICA/B expression was associated with good recurrence-free survival (pxa0=xa00.037). In vitro assays using cell lines revealed that MICA/B expression was upregulated by cisplatin via ATM–ATR signaling, resulting in enhanced NK cell-mediated cytotoxicity. Upregulated MICA/B expressions in patients with radically resected NSCLC are predictive of good disease prognosis. Cisplatin-induced MICA/B upregulation is possibly an indirect mechanism by which the innate immune system eliminates tumor cells. NKG2D–NKG2D ligand-targeting therapy is a promising avenue for future immune-chemotherapy development.

Clinical significance of the tumor microenvironment in non-small cell lung cancer.

Several biomarkers have been reported as predictors of survival and recurrence in non-small cell lung cancer. Recently, several groups have demonstrated that the immune microenvironment of the primary tumors is a prognostic factor. These immunological biomarkers in the tumor microenvironment are useful predictors of prognosis as well as promising targets for novel therapeutic approaches. Especially, tumor-infiltrating Treg cells are a powerful immunological biomarker, and possible mechanisms involved in the induction of tumor-infiltrating Treg cells are the expression of Cox-2, IL-12Rβ2 or the lack of IL7R on the tumor cells. These findings may pave the way for individualized immunomodulatory therapies to deplete tumor-infiltrating Treg cells from the tumor microenvironment.

Membrane-bound estrogen receptor-α expression and epidermal growth factor receptor mutation are associated with a poor prognosis in lung adenocarcinoma patients

BackgroundThe purpose of this study is to clarify the correlations between the expression of membrane-bound estrogen receptor-α (mERα) and epidermal growth factor receptor (EGFR) mutation and clinicopathological factors, especially in relation to the prognosis, in patients with lung adenocarcinoma.MethodsWe conducted a retrospective review of the data of 51 lung adenocarcinoma patients with tumors measuring less than 3u2009cm in diameter. Immunohistochemical staining for mERα expression and detection of the EGFR mutation status were performed.ResultsAmong the 51 patients, the tumors in 15 showed both mERα expression and EGFR mutation. (double positive) Significant associations between double positive and vascular invasion, vascular endothelial growth factor expression, and Ki-67 expression were observed. A multivariate analysis revealed that only double positive was an independent risk factor influencing the recurrence-free survival.ConclusionsPresence of mERα expression together with EGFR mutation was found to be an independent prognostic factor for survival in patients with lung adenocarcinoma, suggesting cross-talk between mERα and EGFR mutation.

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.