Significant role of Psf3 expression in non‐small‐cell lung cancer

The GINS complex associates with cell division cycle (Cdc) protein 45 and mini‐chromosome maintenance (Mcm) proteins 2–7 to form the Cdc45–Mcm–GINS (CMG) complex, which is essential for DNA duplication. One member of the GINS complex is Psf3. We previously found that increased Psf3 expression was strongly associated with poor survival in lung adenocarcinoma. Here, we investigated the role of Psf3 expression in non‐small‐cell lung cancer (NSCLC). We verified Psf3 expression in human NSCLC tissues (180 patients) and cell lines. Immunohistochemical analysis revealed that the overexpression of Psf3 was significantly associated with vessel invasion (P = 0.016), lymphatic invasion (P = 0.002), and pleural invasion (P = 0.036). The overall survival rate in patients with Psf3 overexpression was significantly lower than that in patients without Psf3 overexpression (P = 0.006). Multivariate survival analysis revealed Psf3 expression to be an independent risk factor for an unfavorable outcome (P = 0.049). A proximal ligation assay showed interactions between Psf3 and other CMG components (such as Mcm2 and Cdc45) in both NSCLC specimens and cell lines, indicating that Psf3 acted as the CMG complex, which could lead to excessive proliferation. Knockdown of Psf3 inhibited the proliferation of both cell lines by delaying the S phase, which revealed that Psf3 played an important role in cancer proliferation. Thus, Psf3 acted as the CMG complex, promoting excessive proliferation. These results suggest that Psf3 inhibition might be a therapeutic target for NSCLC with Psf3 overexpression.     

Circulating exosomes contain protein biomarkers of metastatic non‐small‐cell lung cancer

The present study aimed to investigate the overall changes in exosomal proteomes in metastatic and non‐metastatic non‐small‐cell lung cancers (NSCLC) and healthy human serum samples, and evaluate the potential of serum exosomal biomarkers to predict NSCLC metastasis. Tandem mass tags combined with multidimensional liquid chromatography and mass spectrometry analysis were used for screening the proteomic profiles of serum samples. Quantitative proteome, significant pathway, and functional categories of patients with metastatic and non‐metastatic NSCLC and healthy donors were investigated. In total, 552 proteins of the 628 protein groups identified were quantified. Bioinformatics analysis indicated that quantifiable proteins were mainly involved in multiple biological functions, metastasis‐related pathways. Moreover, lipopolysaccharide‐binding proteins (LBP) in the exosomes were found to be well distinguished between patients with metastatic and patients with non‐metastatic NSCLC. Area under the curve (AUC) was 0.803 with a sensitivity of 83.1% and a specificity of 67% (P < .0001). Circulating LBP were also well distinguishable between metastatic and non‐metastatic NSCLC, the AUC was 0.683 with a sensitivity of 79.5% and a specificity of 47.2% (P = .005). This novel study provided a reference proteome map for metastatic NSCLC. Patients with metastatic and non‐metastatic NSCLC differed in exosome‐related proteins in the serum. LBP might be promising and effective candidates of metastatic NSCLC.     

Role of interleukin‐17 in lymphangiogenesis in non‐small‐cell lung cancer: Enhanced production of vascular endothelial growth factor C in non‐small‐cell lung carcinoma cells

Interleukin‐17 (IL‐17), a potent pro‐inflammatory cytokine, plays an active role in inflammation and cancer. Recently, we found that increased IL‐17‐producing cells correlate with poor survival and increased lymphangiogenesis in non‐small‐cell lung cancer (NSCLC), but the mechanism is unknown. Here, we show that IL‐17 promotes lymphangiogenesis via inducing vascular endothelial growth factor‐C (VEGF‐C) production by lung cancer cells. We found that IL‐17 receptor (IL‐17R) is expressed on the surface of Lewis lung carcinoma (LLC) cells but not on lymphatic endothelial cells (LEC). Moreover, LEC chemotaxis and tube formation (measures of net lymphangiogenic potential) were increased by conditioned medium from recombinant mouse IL‐17 (rmIL‐17)‐stimulated LLC but not by rmIL‐17. Interleukin‐17 increased production of VEGF‐C in lung cancer cell lines. The enhanced chemotaxis and endothelial cord formation in the presence of LLC/rmIL‐17 was inhibited by addition of recombinant mouse VEGF R3/Fc chimera. Treatment of the A549 cells with rIL‐17 significantly increased VEGF‐C expression, which was extracellular signal‐regulated protein kinase 1/2 (ERK 1/2) dependent. Importantly, we found significant correlations between IL‐17 expression, VEGF‐C expression and lymphatic vascular density (LVD) in NSCLC. We conclude that IL‐17 is involved in lymphangiogenesis in NSCLC by enhancing production of VEGF‐C, and IL‐17 may be an important target for the treatment of NSCLC. (Cancer Sci 2010; 101: 2384–2390)     

Polymorphisms of EpCAM gene and prognosis for non‐small‐cell lung cancer in Han Chinese

The epithelial cell adhesion molecule (EpCAM) is overexpressed in a wide variety of human cancers and is associated with patient prognosis, including those with lung cancer. However, the association of single nucleotide polymorphisms (SNPs) in the EpCAM gene with the prognosis for non‐small‐cell lung cancer (NSCLC) patients has never been investigated. We evaluated the association between two SNPs, rs1126497 and rs1421, in the EpCAM gene and clinical outcomes in a Chinese cohort of 506 NSCLC patients. The SNPs were genotyped using the Sequenom iPLEX genotyping system. Multivariate Cox proportional hazards model and Kaplan–Meier curves were used to assess the association of EpCAM gene genotypes with the prognosis of NSCLC. We found that the non‐synonymous SNP rs1126497 was significantly associated with survival. Compared with the CC genotype, the CT+TT genotype was a risk factor for both death (hazard ratio, 1.40; 95% confidence interval [CI], 1.02–1.94; P = 0.040) and recurrence (hazard ratio, 1.34; 95% CI, 1.02–1.77; P = 0.039). However, the SNP rs1421 did not show any significant effect on patient prognosis. Instead, the AG+GG genotype in rs1421 was significantly associated with early T stages (T1/T2) when compared with the AA genotype (odds ratio for late stage = 0.65; 95% CI, 0.44–0.96, P = 0.029). Further stratified analysis showed notable modulating effects of clinical characteristics on the associations between variant genotypes of rs1126497 and NSCLC outcomes. In conclusion, our study indicated that the non‐synonymous SNP rs1126497 may be a potential prognostic marker for NSCLC patients.     

Clinical outcomes of carbon‐ion radiotherapy for locally advanced non‐small‐cell lung cancer

The efficacy and safety of carbon‐ion radiotherapy (CIRT) for locally advanced non‐small‐cell lung cancer (LA‐NSCLC) remain unclear. We reported the clinical outcomes of CIRT for LA‐NSCLC. Data for 141 eligible patients who received CIRT between 1995 and 2015 were retrospectively analyzed. Local control (LC), locoregional control (LRC), progression‐free survival (PFS) and overall survival (OS) were calculated using the Kaplan‐Meier method. The median age was 75.0 years. Overall, 21 (14.9%), 57 (40.4%), 43 (30.5%) and 20 (14.2%) patients had T1, T2, T3 and T4 disease, respectively. Moreover, 51 (36.2%), 45 (31.9%), 40 (28.4%) and 5 (3.5%) patients had N0, N1, N2 and N3 disease, respectively. Furthermore, 34 (24.1%), 42 (29.8%), 45 (31.9%) and 20 (14.2%) patients had stages IIA, IIB, IIIA and ΙΙΙB disease, respectively. Overall, 62 (44.0%), 60 (42.6%), 8 (5.7%) and 11 (7.8%) patients had adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and others, respectively. The median dose was 72.0 Gy (relative biological effectiveness). No patient received concurrent chemotherapy. Median follow‐up periods were 29.3 (1.6‐207.7) and 40.0 (10.7‐207.7) months for all patients and survivors, respectively. Two‐year LC, PFS and OS rates were 80.3%, 40.2% and 58.7%, respectively. Overall, 1 (0.7%), 5 (3.5%) and 1 (0.7%) patient developed Grades 4 (mediastinal hemorrhage), 3 (radiation pneumonitis) and 3 (bronchial fistula) toxicities, respectively. Multivariate analysis showed adenocarcinoma and N2/3 classification as significant poor prognosticators of PFS. CIRT is an effective treatment with acceptable toxicity for LA‐NSCLC, especially for elderly patients or patients with severe comorbidities who cannot be treated with surgery or chemoradiotherapy.     

Long‐term results of concurrent chemoradiotherapy using cisplatin and vinorelbine for stage III non‐small‐cell lung cancer

Concurrent chemoradiotherapy is the standard treatment for unresectable stage III non‐small cell lung cancer (NSCLC). The long‐term feasibility and efficacy of vinorelbine and cisplatin with concurrent thoracic radiotherapy were investigated. Eighteen patients received cisplatin (80 mg/m²) on day 1 and vinorelbine (20 mg/m² in level 1, and 25 mg/m² in level 2) on days 1 and 8 every 4 weeks for four cycles in a phase I trial. Ninety‐three patients received the same chemotherapy regimen except for the fixed vinorelbine (20 mg/m²) dosage and consolidation therapy with docetaxel (60 mg/m², every 3 weeks). The thoracic radiotherapy consisted of a single dose of 2 Gy once daily to a total dose of 60 Gy. A total of 111 patients were analyzed in the present study: male/female, 91/20; median age, 60 years; stage IIIA/IIIB, 50/61; and squamous/non‐squamous histology, 26/85. The 3‐, 5‐, and 7‐year overall survival rates (95% CI) were 43.2% (33.9–52.2), 25.2% (17.6–33.5), and 23.2% (15.8–31.4), respectively. The median progression‐free survival and median survival time (95% CI) were 13.5 (10.1–16.7) months and 30.0 (24.3–38.8) months, respectively. Four patients (4%) experienced Grade 5 pulmonary toxicities from 4.4 to 9.4 months after the start of treatment. In conclusion, approximately 15% of patients with unresectable stage III NSCLC could be cured with chemoradiotherapy without severe late toxicities after 10 months of follow‐up. Although based on the data from highly selected population participated in phase I and phase II trial, this analysis would strengthen and confirm the previous reports concerning concurrent chemoradiotherapy with third generation cytotoxic agents. (Cancer Sci 2013; 104: 93–97)     

The immune response‐related mutational signatures and driver genes in non‐small‐cell lung cancer

Immune checkpoint blockade (ICB) therapy has achieved remarkable clinical benefit in non‐small‐cell lung cancer (NSCLC), but our understanding of biomarkers that predict the response to ICB remain obscure. Here we integrated somatic mutational profile and clinicopathologic information from 113 NSCLC patients treated by ICB (CTLA‐4/PD‐1). High tumor mutation burden (TMB) and neoantigen burden were identified significantly associated with improved efficacy in NSCLC immunotherapy. Furthermore, we identified apolipoprotein B mRNA editing enzyme, catalytic polypeptide‐like (APOBEC) mutational signature was markedly associated with responding of ICB therapy (log‐rank test, P = .001; odds ratio (OR), 0.18 [95% CI, 0.06‐0.50], P < .001). The association with progression‐free survival remained statistically significant after controlling for age, sex, histological type, smoking, PD‐L1 expression, hypermutation, smoking signature and mismatch repair (MMR) (HR, 0.30 [95% CI, 0.12‐0.75], P = .010). Combined high TMB with APOBEC signature preferably predict immunotherapy responders in NSCLC cohort. The CIBERSORT algorithm revealed that high APOBEC mutational activity samples were associated with increased infiltration of CD4 memory activated T cells, CD8⁺ T cells and natural killer (NK) cells, but reduced infiltration of regulatory T cells. Besides, individual genes mutation of IFNGR1 or VTCN1 were only found in responders; however, the PTEN mutation was only found in non‐responders (Fisher's exact test, all P < .05). These findings may be applicable for guiding immunotherapy for patients with NSCLC.     

RAC1 inhibition as a therapeutic target for gefitinib‐resistant non‐small‐cell lung cancer

Although epidermal growth factor receptor (EGFR)‐tyrosine kinase inhibitors (EGFR‐TKI), including gefitinib, provide a significant clinical benefit in non‐small‐cell lung cancer (NSCLC) patients, the acquisition of drug resistance has been known to limit the efficacy of EGFR‐TKI therapy. In this study, we demonstrated the involvement of EGF‐EGFR signaling in NSCLC cell migration and the requirement of RAC1 in EGFR‐mediated progression of NSCLC. We showed the significant role of RAC1 pathway in the cell migration or lamellipodia formation by using gene silencing of RAC1 or induction of constitutive active RAC1 in EGFR‐mutant NSCLC cells. Importantly, the RAC1 inhibition suppressed EGFR‐mutant NSCLC cell migration and growth in vitro, and growth in vivo even in the gefitinib‐resistant cells. In addition, these suppressions by RAC1 inhibition were mediated through MEK or PI3K independent mechanisms. Collectively, these results open up a new opportunity to control the cancer progression by targeting the RAC1 pathway to overcome the resistance to EGFR‐TKI in NSCLC patients.     

BOK displays cell death‐independent tumor suppressor activity in non‐small‐cell lung carcinoma

As the genomic region containing the Bcl‐2‐related ovarian killer (BOK) locus is frequently deleted in certain human cancers, BOK is hypothesized to have a tumor suppressor function. In the present study, we analyzed primary non‐small‐cell lung carcinoma (NSCLC) tumors and matched lung tissues from 102 surgically treated patients. We show that BOK protein levels are significantly downregulated in NSCLC tumors as compared to lung tissues (p < 0.001). In particular, we found BOK downregulation in NSCLC tumors of grades two (p = 0.004, n = 35) and three (p = 0.031, n = 39) as well as in tumors with metastases to hilar (pN1) (p = 0.047, n = 31) and mediastinal/subcarinal lymph nodes (pN2) (p = 0.021, n = 18) as opposed to grade one tumors (p = 0.688, n = 7) and tumors without lymph node metastases (p = 0.112, n = 51). Importantly, in lymph node‐positive patients, BOK expression greater than the median value was associated with longer survival (p = 0.002, Mantel test). Using in vitro approaches, we provide evidence that BOK overexpression is inefficient in inducing apoptosis but that it inhibits TGFβ‐induced migration and epithelial‐to‐mesenchymal transition (EMT) in lung adenocarcinoma‐derived A549 cells. We have identified epigenetic mechanisms, in particular BOK promoter methylation, as an important means to silence BOK expression in NSCLC cells. Taken together, our data point toward a novel mechanism by which BOK acts as a tumor suppressor in NSCLC by inhibiting EMT. Consequently, the restoration of BOK levels in low‐BOK‐expressing tumors might favor the overall survival of NSCLC patients.     

Treatment of small‐cell lung cancer in elderly patients

Small‐cell lung cancer (SCLC) represents 15% to 20% of all lung carcinomas. Approximately 30% to 40% of these cases are diagnosed in patients older than 70 years of age. Staging of SCLC classifies patients as having either limited or extensive‐stage disease. The standard treatment for limited‐stage disease is platinum‐based chemotherapy, combined with external‐beam thoracic radiotherapy, whereas platinum‐based regimens alone represent the standard of care for extensive‐stage disease. In the elderly population, treatment of SCLC is more challenging given the decline in physiological organ reserve and the presence of comorbidities. The majority of data are drawn from retrospective studies, which are likely to suffer from selection bias. However, limited prospective data are available to guide treatment decisions in that special population. Nonetheless, these data demonstrate that standard approaches are feasible in carefully selected elderly patients. The purpose of this article is to review the currently available evidence on treatment of SCLC in patients older than 65‐70 years of age. Cancer 2010. © 2010 American Cancer Society.     

Prolonged survival after resection and radiotherapy for solitary brain metastases from non‐small‐cell lung cancer

Selected patients with brain metastases from non‐small‐cell lung cancer benefit from aggressive treatment. This report describes three patients who developed solitary brain metastases after previous resection of primary adenocarcinoma of the lung. Each underwent surgical resection of their brain metastasis followed by cranial irradiation and remain disease free 10 or more years later. Two patients developed cognitive impairment approximately 8 years after treatment of their brain metastasis, which was felt to be due to their previous brain irradiation. Here we discuss the treatment of solitary brain metastasis, particularly the value of combined method approaches in selected patients and dose–volume considerations.     

Overexpression of yes‐associated protein contributes to progression and poor prognosis of non‐small‐cell lung cancer

Yes‐associated protein (YAP), the nuclear effector of the Hippo pathway, is a key regulator of organ size and a candidate human oncogene. This study aimed to assess the clinical significance and biological functions of YAP in non‐small‐cell lung cancer (NSCLC). We investigated the expression of YAP in 92 cases of NSCLC tissue by immunohistochemistry and found that YAP was expressed in 66.3% (61/92) cases and predominantly presented in the nucleus. The expression of YAP in NSCLC was significantly correlated with p‐TNM stage (P = 0.0037) and lymph node metastasis (P = 0.0093). Importantly, YAP expression was associated with short overall survival. Further study in NSCLC cell lines in which YAP was either overexpressed or depleted confirmed that YAP markedly promoted cell proliferation and invasion. These results indicate that YAP plays an important role in NSCLC and might be a useful therapeutic target of NSCLC. (Cancer Sci 2010; 101: 1279–1285)     

Phase I study of salazosulfapyridine in combination with cisplatin and pemetrexed for advanced non‐small‐cell lung cancer

Spliced variant isoforms of CD44 (CD44v) are a marker of cancer stem cells in solid tumors. They stabilize the xCT subunit of the transporter system xc(–) and thereby promote synthesis of the antioxidant glutathione. Salazosulfapyridine (SASP) is an inhibitor of xCT and suppresses the proliferation of CD44v‐positive cancer cells. Chemotherapy‐naïve patients with advanced non‐squamous non‐small‐cell lung cancer were enrolled in a dose‐escalation study (standard 3 + 3 design) of SASP in combination with cisplatin and pemetrexed. The primary end‐point was the percentage of patients who experience dose‐limiting toxicity. Fifteen patients were enrolled in the study. Dose‐limiting toxicity was observed in one of six patients at a SASP dose of 1.5 g/day (elevation of aspartate and alanine aminotransferase levels, each of grade 3), two of five patients at 3 g/day (hypotension or pneumonitis, each of grade 3), and two of three patients at 4.5 g/day (anorexia of grade 3). The maximum tolerated dose was thus 3 g/day, and the recommended dose was 1.5 g/day. The overall response rate was 26.7% and median progression‐free survival was 11.7 months, much longer than that for cisplatin–pemetrexed alone in previous studies. Exposure to SASP varied markedly among individuals according to ABCG2 and NAT2 genotypes. The serum concentration of free CD44v protein was increased after the first cycle of treatment, possibly reflecting death of cancer stem cells. Salazosulfapyridine was thus given safely in combination with cisplatin–pemetrexed, with the addition of SASP tending to prolong progression‐free survival. This trial is registered in the UMIN Clinical Trials Registry as UMIN000017854.