Masafumi Takeshita

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.

Nicotine Induces Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor by α1 Nicotinic Acetylcholine Receptor–Mediated Activation in PC9 Cells

Introduction: Nicotine, the major component among the 4000 identified chemicals in cigarette smoke, binds to nicotinic acetylcholine receptors (nAChRs) on non–small-cell lung cancer (NSCLC) cells and regulates cellular proliferation by activating mitogen-activated protein kinases [AQ: MAPK has been expanded to mitogen-activated protein kinases. Please approve.]and PI3K/Akt pathways. In patients with smoking-related lung cancer who continue smoking, the anticancer effect of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) is weaker than that in nonsmokers; however, the precise reason for this difference remains unclear. We investigated the role of &agr;1 nAChR subunit in this phenomenon. Methods: We screened for &agr;1 nAChR mRNA in three NSCLC cell lines and analyzed the protein in resected primary NSCLC tissues. We used Western blot and RNA interference (siRNA) methodology to confirm the results. Results: We determined that &agr;1 nAChR plays an essential role in nicotine-induced cell signaling and nicotine-induced resistance to EGFR-TKI. In addition, we showed that silencing of &agr;1 nAChR subunit in NSCLC may suppress the nicotine-induced resistance to EGFR-TKI. Conclusions: These results further implicate nicotine in lung carcinogenesis, and suggest that &agr;1 nAChR may be a biomarker for EGFR-TKI treatment and also a personalizing target molecule for patients with smoking-related lung cancer.

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.

CHFR expression is preferentially impaired in smoking-related squamous cell carcinoma of the lung, and the diminished expression significantly harms outcomes.

Loss of tumor suppressors and activation of oncogenes lead to carcinogenesis. Abnormal expression of CHFR, a novel checkpoint gene, or of Aurora kinases, key regulators of mitosis, has been detected in a variety of solid tumors. Recently, CHFR has been revealed to ensure chromosomal stability by controlling the expression level of Aurora‐A in vitro. However, the frequency of aberrant expression of these proteins and the association with clinicopathologic parameters remain poorly defined in nonsmall‐cell lung cancer (NSCLC). In this study, we investigated the immunohistochemical protein expression of CHFR and Aurora‐A in 157 NSCLC cases and evaluated the association between clinicopathologic parameters statistically. The relationship between CHFR protein and mRNA levels and the association between this relationship and promoter methylation of the CHFR gene were also examined in 20 frozen sections of NSCLC. Overexpression of Aurora‐A and reduced expression of CHFR were found in 94 cases (59.8%) and 62 cases (39%) of NSCLC, respectively, and those were significantly correlated with tumor differentiation and size. Moreover, diminished CHFR expression was significantly associated with smoking‐related squamous cell carcinoma cases and poor prognosis. Multivariate analysis revealed that CHFR expression was an independent prognostic factor. A statistical correlation was evident between CHFR protein and mRNA expression. In conclusion, our results suggest the aberrant expression of Aurora‐A and/or of CHFR contributed to the increase in the malignant potential of NSCLC. We also revealed that CHFR expression was predominantly impaired in smoking‐related squamous cell carcinoma and might be a useful prognostic marker in NSCLC.

Acquisition of the T790M resistance mutation during afatinib treatment in EGFR tyrosine kinase inhibitor–naïve patients with non–small cell lung cancer harboring EGFR mutations

The T790M secondary mutation of the epidermal growth factor receptor (EGFR) gene accounts for 50% to 60% of cases of resistance to the first-generation EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib. The prevalence of T790M in EGFR mutation-positive patients who acquire resistance to the irreversible, second-generation EGFR-TKI afatinib has remained unclear, however. We here determined the frequency of T790M acquisition at diagnosis of progressive disease in patients with EGFR-mutated non-small cell lung cancer (NSCLC) treated with afatinib as first-line EGFR-TKI. Among 56 enrolled patients, 37 individuals underwent molecular analysis at rebiopsy. Of these 37 patients, 16 individuals (43.2%) had acquired T790M, including 11/21 patients (52.4%) with an exon 19 deletion of EGFR and 5/13 patients (38.5%) with L858R. None of three patients with an uncommon EGFR mutation harbored T790M. T790M was detected in 14/29 patients (48.3%) with a partial response to afatinib, 1/4 patients (25%) with stable disease, and 1/4 patients (25%) with progressive disease as the best response. Median progression-free survival after initiation of afatinib treatment was significantly (P = 0.043) longer in patients who acquired T790M (11.9 months; 95% confidence interval, 8.7-15.1) than in those who did not (4.5 months; 95% confidence interval, 2.0-7.0). Together, our results show that EGFR-mutated NSCLC patients treated with afatinib as first-line EGFR-TKI acquire T790M at the time of progression at a frequency similar to that for patients treated with gefitinib or erlotinib. They further underline the importance of rebiopsy for detection of T790M in afatinib-treated patients.The T790M secondary mutation of the epidermal growth factor receptor (EGFR) gene accounts for 50% to 60% of cases of resistance to the first-generation EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib. The prevalence of T790M in EGFR mutation–positive patients who acquire resistance to the irreversible, second-generation EGFR-TKI afatinib has remained unclear, however. We here determined the frequency of T790M acquisition at diagnosis of progressive disease in patients with EGFR-mutated non–small cell lung cancer (NSCLC) treated with afatinib as first-line EGFR-TKI. Among 56 enrolled patients, 37 individuals underwent molecular analysis at rebiopsy. Of these 37 patients, 16 individuals (43.2%) had acquired T790M, including 11/21 patients (52.4%) with an exon 19 deletion of EGFR and 5/13 patients (38.5%) with L858R. None of three patients with an uncommon EGFR mutation harbored T790M. T790M was detected in 14/29 patients (48.3%) with a partial response to afatinib, 1/4 patients (25%) with stable disease, and 1/4 patients (25%) with progressive disease as the best response. Median progression-free survival after initiation of afatinib treatment was significantly (P = 0.043) longer in patients who acquired T790M (11.9 months; 95% confidence interval, 8.7–15.1) than in those who did not (4.5 months; 95% confidence interval, 2.0–7.0). Together, our results show that EGFR-mutated NSCLC patients treated with afatinib as first-line EGFR-TKI acquire T790M at the time of progression at a frequency similar to that for patients treated with gefitinib or erlotinib. They further underline the importance of rebiopsy for detection of T790M in afatinib-treated patients.

Alternative efficacy-predicting markers for paclitaxel instead of CHFR in non-small-cell lung cancer

Experiments using cancer cell lines have revealed that CHFR methylation correlates with sensitivity to microtubule inhibitors. However, this marker may not benefit actual clinical cases because it is difficult to detect CHFR methylation without surgically resected samples. Thus, a more easily accessible marker that correlates with sensitivity to microtubule inhibitors might be useful in NSCLC, especially in advanced cases. In this study, we show that EGFR gene status and smoking are predict the efficacy of treatment with microtubule inhibitors in NSCLC. Chemosensitivity to paclitaxel and six other chemotherapeutic agents was evaluated using the succinate dehydrogenase inhibition (SDI) method in 69 NSCLC cases, consisting of 48 adenocarcinomas, 20 squamous cell carcinomas and 1 large cell carcinoma. Next, we evaluated the relationships between CHFR or EGFR status and clinicopathologic data. Methylation-specific PCR (MSP) and direct DNA sequencing were performed to detect aberrant methylation of CHFR and EGFR mutations, respectively. CHFR gene promoter methylation and EGFR gene mutation were observed in 11 cases (15.9%) and 7 cases (10.1%), respectively. The SDI method revealed that CHFR gene methylation was significantly related to high sensitivity to paclitaxel (p

CHFR aberrant methylation involves a subset of human lung adenocarcinoma associated with poor clinical outcomes

Excluding epidermal growth factor receptor (EGFR) mutation, v-Ki-ras2/Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation, and echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion, the genetic alterations involved in lung adenocarcinogenesis, especially those linked to poor clinical outcomes, are still unknown. In this study, we analyzed abnormal checkpoint gene with forkhead-associated domain and ring finger (CHFR) methylation along with the above 3 mutations in 165 lung adenocarcinomas, evaluated the spectrum of each molecular abnormality, and correlated the results with clinical and pathologic variables. Reverse transcription-polymerase chain reaction assay, reverse transcription-polymerase chain reaction followed by direct DNA sequencing, and methylation-specific polymerase chain reaction were performed to detect these 3 mutations and CHFR hypermethylation. The EML4-ALK transcript or CHFR hypermethylation was found in 11 (6.7%) or 16 (10%) adenocarcinomas, respectively, whereas EGFR or KRAS mutation was detected in 48 (29%) or 13 (8%) cases, respectively. EGFR mutations occurred in patients who were negative for both CHFR hypermethylation and KRAS mutation. Among the 4 genetic or epigenetic abnormalities, only CHFR hypermethylation was significantly correlated with poor prognosis and lymphatic vessel invasion (P = .024). Histopathologically, the molecular abnormality that correlated with alveolar-destructive growth was the CHFR hypermethylation rather than the EGFR mutation (P = .03). Our results demonstrate that CHFR hypermethylation maybe one of the molecular abnormalities involved in a subset of lung adenocarcinomas with poor prognoses that might be induced by destructive growth and lymphatic vessel invasion of carcinoma cells. Thus, CHFR abnormality might be pursued as a novel therapeutic target against lung adenocarcinoma without an already-known mutation.

A Case of Lung Adenocarcinoma Resistant to Crizotinib Harboring a Novel EML4-ALK Variant, Exon 6 of EML4 Fused to Exon 18 of ALK

3 weeks after completion of therapy. Unfortunately, the patient progressed before the operation and died 3 months later. Sorafenib is a multikinase inhibitor, and it is used for the treatment of metastatic disease of hepatocellular carcinoma, renal cell carcinoma, and thyroid carcinoma. Few data are available regarding the role of sorafenib in thymoma and thymic carcinoma. Only a few case reports so far have reported that sorafenib is effective for the treatment of thymoma in cases of refractory disease. Considering the available literature, sorafenib could be biologically active in some malignant thymomas and thymic carcinomas; further studies are required to better define the real efficacy of sorafenib and identify a possible biomarker of response.