Takaomi Koga

Platelet-derived growth factor-AA is an essential and autocrine regulator of vascular endothelial growth factor expression in non-small cell lung carcinomas.

It is widely accepted that angiogenesis is required for tumor progression. Vascular endothelial growth factor (VEGF) is a key molecule for tumor angiogenesis; however, its expressional regulation is not well understood during all stages of tumorigenesis. Using cell lines and surgical specimens of human non-small cell lung cancers (NSCLCs), we here show that platelet-derived growth factor-AA (PDGF-AA) is an essential autocrine regulator for VEGF expression. To directly assess the expression of PDGF-AA-dependent VEGF and its roles in tumorigenesis, we stably transfected established cell lines with their antisense genes. In addition, the levels of PDGF-AA and VEGF expression in surgical sections were measured and compared with clinicopathologic findings such as tumor size and patient prognosis. PDGF-AA tightly regulated VEGF expression and had a greater effect on tumor size and patient prognosis than did VEGF in both cell lines and surgical sections. PDGF-AA expression was not seen in the atypical adenomatous hyperplasia at all, whereas VEGF was occasionally seen. Furthermore, the frequency of VEGF expression was higher in advanced NSCLCs than in precancerous lesions, which was tightly correspondent to the results for PDGF-AA. These results indicate that PDGF-AA is an important regulator of the frequency and level of VEGF expression during the transition from a precancerous lesion to advanced cancer. The PDGF-AA/VEGF axis, therefore, may be a ubiquitous autocrine system for enhancing angiogenic signals, and PDGF-AA, and its related pathways could be a more efficient target of antiangiogenic therapy for cancers than VEGF and its pathways.

CK20 expression, CDX2 expression, K‐ras mutation, and goblet cell morphology in a subset of lung adenocarcinomas

There are data in the literature that suggest a close relationship between the expression of CK20 and CDX2, K‐ras mutations, and goblet cell morphology. The present study has examined these factors in a cohort of 264 non‐small cell lung cancers. Thirteen of 212 adenocarcinomas expressed CK20; 29 expressed CDX2; K‐ras mutation was identified in 28; and goblet cell features were present in 19. These four factors correlated with each other in a complex way and therefore a logistic regression model was constructed. Significant correlations were found between CK20 and CDX2 expression, and between K‐ras mutation and goblet cell morphology, and there was a marginal correlation between CDX2 immunoreactivity and goblet cell morphology. These four features have also been commonly detected in colorectal, pancreato‐biliary, and ovarian mucinous carcinomas, suggesting that these adenocarcinomas may be prototypical, independent of the organ of origin. Furthermore, as high and uniform expression of CDX2 was characteristic of metastatic colorectal cancer, weak and/or focal CDX2 expression should alert surgical pathologists to the possibility of primary lung adenocarcinoma, especially in the presence of goblet cell morphology. However, some lung adenocarcinomas may express CDX2 strongly: in this case, CK20 also tends to be positive. Copyright

Expression of TrkB and BDNF is associated with poor prognosis in non-small cell lung cancer

High expression levels of TrkB and BDNF are associated with aggressive malignant behavior in tumor cells and a poor prognosis in patients with various types of cancer. In this study, we aimed to identify the relationship between TrkB and BDNF expression and clinicopathological variables and prognosis in non-small cell lung cancer (NSCLC). We evaluated TrkB and BDNF expression in the tumor cells of 102 NSCLC patients by immunohistochemistry. Out of all clinicopathological factors examined, only vascular invasion was significantly correlated with TrkB (P=0.010) and BDNF (P=0.015) expression. TrkB-positive tumors had significantly worse disease-free survival (P=0.0094) and overall survival (P=0.0019) than TrkB-negative tumors, and TrkB expression was an independent prognostic factor for disease-free survival (HR 3.735, 95% CI 1.560-11.068, P=0.002) and overall survival (HR 4.335, 95% CI 1.534-15.963, P=0.004) in multivariate analysis. Finally, our analysis revealed that co-expression of TrkB and BDNF conferred poorer prognosis compared with overexpression of either protein alone. Our results indicate that expression of TrkB and BDNF is associated with poor prognosis in NSCLC patients.

Lung Adenocarcinoma With Bronchioloalveolar Carcinoma Component Is Frequently Associated With Foci of High-Grade Atypical Adenomatous Hyperplasia

We assessed the occurrence of atypical adenomatous hyperplasia (AAH) in whole lung lobes with primary cancer lesions. Following surgical resection, tissue specimens were sliced to a thickness of 4 mm (3,641 specimens from 61 cases; mean = 59.7 specimens per case). A total of 119 AAH foci were found and an association was evident in 25 (57%) of 44 adenocarcinomas, 3 (30%) of 10 squamous cell carcinomas, and 2 (29%) of 7 other lung cancers. Histologic evaluation showed that 108 AAH foci were categorized as low-grade and the other 11 as high-grade AAH. These 11 foci of high-grade AAH were present in 7 patients with adenocarcinoma, and in 1 patient there was a synchronous double primary lung adenocarcinoma. High-grade AAH was closely associated with bronchioloalveolar carcinoma (BAC) type adenocarcinoma, and low-grade AAH with non-BAC adenocarcinoma. The mean +/- SD Ki-67 labeling index in high-grade AAH (3.5%+/-2.9%) was significantly higher than for the low-grade index (1.4%+/-1.6%). We propose that foci of high- but not low-grade AAH may be potential precursor lesions of lung adenocarcinoma, especially with the BAC component.

Role and Relevance of TrkB Mutations and Expression in Non―Small Cell Lung Cancer

Purpose: TrkB has been involved in poor cancer outcome. TrkB mutations have been reported in non–small cell lung cancer. In this study, we aimed at characterizing the role of three potentially sensitizing TrkB mutations previously reported in lung cancer. Experimental Design: We characterized three activation loop mutants of TrkB (M713I, R715G, and R734C) in terms of pathway activation/phosphorylation, migration, anchorage-independent growth, and sensitivity to a Trk inhibitor, using NIH3T3 cells and Baf3 cells. We also sequenced the tyrosine kinase domain of TrkB in a large number of lung cancer samples of East-Asian origin and cell lines. Results: None of the mutants were constitutively active in NIH3T3 transformation and migration assays. M713I and R734C mutants showed low levels of autophosphorylation in comparison with wild-type TrkB. Although R715G showed similar level of autophosphorylation to wild-type TrkB on brain-derived neurotrophic factor stimulation, the mutant was not as competent as wild-type TrkB in supporting interleukin-3–independent growth of Baf3 cells. In addition, the Trk inhibitor AZD6918 inhibited wild-type TrkB-induced cell migration and cell growth, whereas the mutants were relatively resistant to the Trk inhibitor compared with wild-type TrkB. We could not confirm the presence of nonsynonymous mutation in 78 lung cancer samples and 29 cell lines. Conclusions: Wild-type, but not mutant, TrkB enhances cell migration and transformation. Our study suggests that TrkB mutations should not be used for selection of patients with lung cancer treated with Trk inhibitors. High expression of wild-type TrkB might be beneficial for studies of Trk inhibitors. Clin Cancer Res; 17(9); 2638–45. ©2011 AACR.

Aurora-B overexpression is correlated with aneuploidy and poor prognosis in non-small cell lung cancer

Aurora-B is a key regulator of mitosis, and the overexpression has been detected in a variety of solid tumors. The Aurora-B overexpression has been suggested to correlate with clinical aggressiveness and aneuploidy in vitro, however, the frequency of overexpression of Aurora-B protein, the association with clinicopathologic parameters and aneuploidy remain poorly defined in non-small-cell lung cancer (NSCLC). Using 157 surgical specimens of human NSCLC, we here show that overexpression of Aurora-B proteins are significantly correlated with aneuploidy and poor outcomes in NSCLC. We examined immunohistochemical protein expression of Aurora-B, and DNA ploidy by laser scanning cytometry in 157 NSCLC cases. Aurora-B overexpression was found in 83 cases (53%) of NSCLC, and was significantly correlated with vascular invasion (p=0.012), poor differentiation (p<0.001), larger tumor size (p=0.010) and lymph node metastasis (p=0.05) and poor prognosis (p=0.011). Aneuploidy was found in 87 cases (57%), and was significantly correlated with Aurora-B overexpression (p=0.0065). Logistic multivariate analysis revealed overexpression of Aurora-B protein to be significant risk factors for aneuploidy compared with other factors. These results indicate that Aurora-B overexpression may contribute to malignant potential and increased aneuploidy in NSCLC. Thus, Aurora-B may serve as a new therapeutic target in against patients with NSCLC, although further studies will be necessary.

Heterogeneous distribution of P53 immunoreactivity in human lung adenocarcinoma correlates with MDM2 protein expression, rather than with P53 gene mutation.

Although the tumor suppressor p53 protein (P53) immunoreactivity and its gene (p53) mutation were reported to be significant prognostic indicators for human lung adenocarcinomas, little is known regarding the relationship between the heterogeneous distribution of P53 and its genetic status in each tumor focus and the clinicopathological significance. To determine how P53 is heterogeneously stabilized in patients, we compared P53 expression to both the p53 allelic mutation in exon 2 ∼ 9 by polymerase chain reaction‐single strand conformation polymorphism using microdissected DNA fractions, and the immunohistochemical MDM2 expression. Of the 48 positive to P53 in 118 lung adenocarcinomas examined, 10 with heterogeneous P53 expression were closely examined. The higher P53 expression foci in 7 of 10 cases were less differentiated, histologically in respective cases, and were frequently associated with fibrous stroma. Two had genetic mutations in exon 7 of the p53 gene in both the high and low P53 expression foci of cancer tissue indicating no apparent correlation between heterogeneous P53 expression and the occurrence of gene mutation. Immunohistochemical expression of MDM2 was significantly lower in high P53 expression areas (p < 0.05, the mean labeling indices of high and low P53 expression areas being 4.2 ± 5.4% and 13.6 ± 12.2%, respectively). In addition, among all the 118 cases examined, MDM2 expression was significantly suppressed in cases of p53 gene mutation, simultaneously with P53 overexpression, as compared with cases without both the p53 mutation and expression (p < 0.001). These findings suggest that the heterogeneous stabilization of P53 in human lung adenocarcinomas could be partly due to suppressed MDM2 expression. The overexpression of non‐mutated P53 may afford a protective mechanism in human lung adenocarcinomas.

Histological evolution of pleuroparenchymal fibroelastosis

To investigate the histological evolution in the development of pleuroparenchymal fibroelastosis (PPFE).

High podoplanin expression in cancer cells predicts lower incidence of nodal metastasis in patients with lung squamous cell carcinoma.

Podoplanin is expressed in a variety of malignant cells, and is generally regarded as a factor promoting tumor progression in conventional studies. Conversely, a recent clinicopathological study has revealed that low podoplanin in cancer cells was correlated with poor prognosis of patients with stage IB lung squamous cell carcinoma (LSCC). We here evaluated the clinicopathological relationship between cancer-cell podoplanin expression and clinicopathological parameters in 40 cases of LSCC (stage I-III). Immunohistochemical podoplanin expression significantly correlated with N classification and pathological stage, but not with other clinicopathological parameters. Notably, all 16 cases with high podoplanin expression unexceptionally exhibited pathological N0 status. Cases without nodal metastasis showed a significantly higher podoplanin-positive score. Furthermore, patients with high podoplanin expression exhibited a significantly longer survival time and disease-free time. These findings suggest that immunohistochemical analysis for podoplanin may serve as a marker of risk of nodal metastasis and prognosis in patients with LSCC.

CHFR hypermethylation and EGFR mutation are mutually exclusive and exhibit contrastive clinical backgrounds and outcomes in non-small cell lung cancer

Aberrant promoter methylation of the checkpoint gene with forkhead‐associated domain and ring finger (CHFR) gene is frequently detected in human cancer. We previously demonstrated that diminished CHFR expression was significantly correlated with both poor prognosis and heavy smoking in nonsmall cell lung cancer (NSCLC). Conversely, epidermal growth receptor (EGFR) mutation is detected in NSCLC among those who have never smoked or smoked lightly. To address the frequency of CHFR hypermethylation as well as differences in the distributions and clinicopathologic backgrounds against EGFR mutation in NSCLC, we investigated a large group of 208 NSCLC patients, including 165 with adenocarcinoma (ADC), 40 with squamous cell carcinoma and three others. We found that CHFR hypermethylation and EGFR mutation are mutually exclusive and have contrastive clinicopathologic backgrounds in NSCLC. Methylation‐specific polymerase chain reaction (MSP) and direct DNA sequencing were performed to detect CHFR hypermethylation and EGFR mutation, respectively. CHFR hypermethylation was found in 29 cases (14%) (16 ADC (8%), 12 SCC (6%) and one adenosquamous carcinoma), while EGFR mutation was detected in 48 (23%) cases, all of which were ADC. CHFR hypermethylation and EGFR mutation were mutually exclusive (p = 0.004). NSCLC with altered CHFR was significantly correlated with smoking history, poor differentiation, lymphatic invasion, and poor prognosis; this contrasted sharply with EGFR mutation, which had statistically better clinical outcomes. Our results demonstrate that CHFR loss might be critical for the tumorigenesis of NSCLC in patients with a history of smoking and induces tumors of a more malignant phenotype than the EGFR mutation. Thus, CHFR alteration should be considered a therapeutic target against NSCLC in patients with poor prognoses.