Masato Shingyoji

Phase II trial of gefitinib alone without radiation therapy for Japanese patients with brain metastases from EGFR-mutant lung adenocarcinoma

BACKGROUND Brain metastases (BM) are a common in patients with lung cancer. Although whole-brain radiation therapy (WBRT) is the standard therapy, it may have a risk of decline in cognitive function of patients. In this study, we evaluated the efficacy of gefitinib alone without radiation therapy for the treatment of patients with BM from lung adenocarcinoma. MATERIALS AND METHODS Eligible patients had BM from lung adenocarcinoma with epidermal growth factor receptor (EGFR) mutations. Gefitinib was given at 250 mg orally once a day until tumor progression or unacceptable toxicity. RESULTS Forty-one patients were enrolled. The response rate was 87.8%. No patient experienced grade ≥4 toxicity. The median progression-free survival time was 14.5 months (95% CI, 10.2-18.3 months), and the median overall survival time was 21.9 months (95% CI, 18.5-30.3 months). In compared with L858R, exon 19 deletion was associated with better outcome of patients after treatment with gefitinib in both progression-free (p = 0.003) and overall survival (p = 0.025). CONCLUSION Favorable response of BM to gefitinib even without irradiation was demonstrated. Exon 19 deletion was both a predictive and prognostic marker of patients with BM treated by gefitinib.

Comparison of 21-gauge and 22-gauge aspiration needle during endobronchial ultrasound-guided transbronchial needle aspiration

Background and objective:  Endobronchial ultrasound‐guided transbronchial needle aspiration (EBUS‐TBNA) has typically been performed using the 22gauge (G) dedicated TBNA needle. Recently a new 21G TBNA needle has been introduced. The efficacy of using a larger gauge biopsy needle during EBUS‐TBNA has not been reported. The purpose of this study was to compare the diagnostic yield and utility of 21G and 22G needles during EBUS‐TBNA.

Vascular Image Patterns of Lymph Nodes for the Prediction of Metastatic Disease During EBUS-TBNA for Mediastinal Staging of Lung Cancer

Introduction: The aims of this study were to establish and assess the utility of Power/Color Doppler-mode vascular image pattern classification of lymph nodes (LN) during mediastinal staging by endobronchial ultrasound (EBUS) for the prediction of metastasis in patients with lung cancer. Methods: One hundred and seventy-three LNs were retrospectively evaluated. The convex probe EBUS was used with the endoscopic ultrasound scanner. The vascular image patterns were graded as follows: Grade 0, no blood flow or small amounts of flow; Grade I, a few main vessels running toward the center of the LN from the hilum; Grade II, a few punctiforms or rod-shaped flow signals or a few small vessels found as a long strip of a curve; and Grade III, rich flow, more than four vessels found with different diameters or twist- or helical -low signal. The blood flow from the bronchial artery (BA) toward the LN was also recorded using Color Doppler imaging as a sign for BA inflow. Results: When we defined Grade 0 and I as “benign” and Grade II and III as “malignant,” the sensitivity, specificity, and diagnostic accuracy rate were 87.7%, 69.6%, and 78.0%, respectively. The accuracy of predicting metastasis solely from a positive BA inflow sign was 80.3%. Of the LNs, 84.5% were shown to be metastatic when they were determined as malignant, and 84.7% were proven nonmetastatic when it was determined as benign. Conclusions: Vascular image patterns of LN using Power/Color Doppler mode is helpful in the prediction of metastatic LN during EBUS-TBNA.

The significance and robustness of a plasma free amino acid (PFAA) profile-based multiplex function for detecting lung cancer

BackgroundWe have recently reported on the changes in plasma free amino acid (PFAA) profiles in lung cancer patients and the efficacy of a PFAA-based, multivariate discrimination index for the early detection of lung cancer. In this study, we aimed to verify the usefulness and robustness of PFAA profiling for detecting lung cancer using new test samples.MethodsPlasma samples were collected from 171 lung cancer patients and 3849 controls without apparent cancer. PFAA levels were measured by high-performance liquid chromatography (HPLC)–electrospray ionization (ESI)–mass spectrometry (MS).ResultsHigh reproducibility was observed for both the change in the PFAA profiles in the lung cancer patients and the discriminating performance for lung cancer patients compared to previously reported results. Furthermore, multivariate discriminating functions obtained in previous studies clearly distinguished the lung cancer patients from the controls based on the area under the receiver-operator characteristics curve (AUC of ROC = 0.731 ~ 0.806), strongly suggesting the robustness of the methodology for clinical use. Moreover, the results suggested that the combinatorial use of this classifier and tumor markers improves the clinical performance of tumor markers.ConclusionsThese findings suggest that PFAA profiling, which involves a relatively simple plasma assay and imposes a low physical burden on subjects, has great potential for improving early detection of lung cancer.

ALK fusion gene positive lung cancer and 3 cases treated with an inhibitor for ALK kinase activity

BACKGROUND Anaplastic lymphoma kinase (ALK) fusion gene-positive lung cancer accounts for 4-5% of non-small cell lung carcinoma. A clinical trial of the specific inhibitor of ALK fusion-type tyrosine kinase is currently under way. METHODS ALK fusion gene products were analyzed immunohistochemically with the materials obtained by surgery or by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA). The echinoderm microtubule-associated protein-like 4(EML4)-ALK or kinesin family member 5B (KIF5B)-ALK translocation was confirmed by the reverse transcription polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH). After eligibility criteria were met and informed consent was obtained, 3 patients were enrolled for the Pfizer Study of Crizotinib (PF02341066), Clinical Trial A8081001, conducted at Seoul National University. RESULTS Out of 404 cases, there were 14 of EML4-ALK non-small cell carcinoma (NSCLC) and one KIF5B-ALK NSCLC case (8 men, 7 women; mean age, 61.9 years, range 48-82). Except for 2 light smokers, all patients were non-smokers. All cases were of adenocarcinoma with papillary or acinar subtypes. Three were of stage IA, 5 of stage IIIA, 1 of stage IIIB and 6 of stage IV. Ten patients underwent thoracotomy, 3 received chemotherapy and 2 only best supportive care (BSC). One BSC and 2 chemotherapy cases were enrolled for the clinical trial. Patients with advanced stages who received chemotherapy or best supportive care were younger (54.0±6.3) than those who were surgically treated (65.8±10.1) (p<0.05). The powerful effect of ALK inhibitor on EML4-ALK NSCLC was observed. Soon after its administration, almost all the multiple bone and lymph node metastases quickly disappeared. Nausea, diarrhea and the persistence of a light image were the main side effects, but they diminished within a few months. CONCLUSION ALK-fusion gene was found in 3.7% (15/404) NSCLC cases and advanced disease with this fusion gene was correlated with younger generation. The ALK inhibitor presented in this study is effective in EML4-ALK NSCLC cases. A further study will be necessary to evaluate the clinical effectiveness of this drug.

Endobronchial ultrasonography for positron emission tomography and computed tomography-negative lymph node staging in non-small cell lung cancer.

BACKGROUND Integrated positron emission tomography (PET) with computed tomography (CT) is a useful modality to investigate lymph node metastases for non-small cell lung cancer, but is less sensitive for normal-sized lymph nodes. We sometimes encounter cases with radiologically normal lymph nodes and unsuspected mediastinal metastases detected by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA). However, few studies have investigated staging in patients with radiologically normal mediastina, and the accuracy of EBUS-TBNA staging for radiologically normal mediastina and hila is unclear. METHODS This study was a retrospective, single-institution review of a prospectively maintained database at Chiba Cancer Center between May 1, 2008, and September 1, 2013. We analyzed 113 non-small cell lung cancer patients with both CT-negative and PET/CT-negative lymph nodes (N0) in preoperative nodal staging performed by EBUS-TBNA. After preoperative staging was performed, patients with either N0 or N1 clinical staging underwent surgery. Final N factors were determined by mediastinal lymphadenectomy. RESULTS In our study, the overall rate of N2 disease was 17.6% (20 of 113). For nodal staging by EBUS-TBNA, the sensitivity, specificity, negative predictive value, and diagnostic accuracy were 35.0% (7 of 20), 100% (93 of 93), 87.7% (93 of 106), and 88.4% (100 of 113), respectively. There were no severe complications from EBUS-TBNA staging. CONCLUSIONS The overall rate of unsuspected N2 was not low. EBUS-TBNA was accurate and feasible for preoperative mediastinal nodal staging of non-small cell lung cancer with both CT-negative and PET/CT-negative lymph nodes. The sensitivity of EBUS-TBNA for radiologically normal mediastina and hila was low. Further investigations are required.

Detection of epithelial growth factor receptor mutations in cerebrospinal fluid from patients with lung adenocarcinoma suspected of neoplastic meningitis.

Background: Neoplastic meningitis (NM) is a devastating neurological complication of cancer that needs to be diagnosed in the early stages of disease. Polymerase chain reaction detection of epithelial growth factor receptor (EGFR) mutations in cerebrospinal fluid (CSF), which are predictive markers for EGFR tyrosine kinase inhibitor therapy in lung cancer, might be important to diagnose and to treat NM in patients with lung cancer. In this study, we attempted to detect EGFR mutations in CSF and to compare EGFR status between CSF and primary or metastatic lesions in patients with lung adenocarcinoma suspected of NM. Methods: Twenty-nine patients with lung adenocarcinoma suspected of having NM underwent lumbar puncture. EGFR status of CSF was analyzed by direct DNA sequencing. EGFR mutations of primary or metastatic lesions (lymph nodes and bones) were analyzed in 20 cases. Results: EGFR mutations were detected in CSF of 13 (45%) of 29 patients. In 5 (31%) of 16 patients with negative CSF cytology, EGFR mutations were detected. In four patients, EGFR mutations were shown in CSF, but not in primary or metastatic lesions. Conversely, in two patients, EGFR mutations were shown in primary or metastatic lesions, but not in CSF despite positive CSF cytology. Conclusions: EGFR mutations, suggesting the existence of malignant cells, were detected in CSF, even in patients with non-small cell lung cancer with negative cytological results. EGFR mutations in CSF do not always reflect the same status as in primary or metastatic lesions.

Endobronchial Ultrasound Doppler Image Features Correlate with mRNA Expression of hif1-α and vegf-c in Patients with Non–Small-Cell Lung Cancer

Introduction: We attempted to assess the correlation between the Doppler mode image patterns during endobronchial ultrasound-guided (EBUS) transbronchial needle aspiration and the expression of angiogenesis-related molecules within lymph nodes in patients with non–small-cell lung cancer. Methods: Thirty-eight archived EBUS- transbronchial needle aspiration samples of lymph nodes (27 metastatic and 11 nonmetastatic) in patients with non–small-cell lung cancer with Doppler mode ultrasound image were analyzed. The Doppler mode image of the vasculature of the targeted lymph node was categorized into the following groups: normal blood flow, low blood flow (LBF), and high blood flow (HBF). Vascular index ratio (vascular area/lymph node area) of each metastatic lymph node was calculated. Total RNA and protein was extracted and analyzed for expression of HIF-1&agr;, VEGF-A, and VEGF-C by quantitative RT-PCR and enzyme-linked immunosorbent assay. Results: Within the 27 metastatic lymph nodes, eight were categorized into the LBF group and 19 into the HBF group. Vascular index ratio was significantly higher in HBF than LBF (p = 0.0003). mRNA expression of HIF-1&agr; and VEGF-A was significantly higher in metastatic lymph nodes than in benign lymph nodes (p < 0.0001). Compared with LBF and HBF, HIF-1&agr; mRNA expression was significantly higher in LBF (p = 0.01) and VEGF-C mRNA expression was significantly higher in HBF (p = 0.0315). There was no significant difference in protein expression by enzyme-linked immunosorbent assay analysis. Conclusions: The vascularity of metastatic lymph nodes observed by EBUS correlates with the mRNA expression of HIF-1&agr; and VEGF-C (not VEGF-A). This correlation is a clinical utility that needs to be evaluated further.

Zoledronic acid induces apoptosis and S-phase arrest in mesothelioma through inhibiting Rab family proteins and topoisomerase II actions

Zoledronic acid (ZOL), a nitrogen-containing bisphosphonate, produced anti-tumor effects through apoptosis induction or S-phase arrest depending on human mesothelioma cells tested. An addition of isoprenoid, geranylgeraniol but not farnesol, negated these ZOL-induced effects, indicating that the ZOL-mediated effects were attributable to depletion of geranylgeranyl pyrophosphates which were substrates for prenylation processes of small guanine-nucleotide-binding regulatory proteins (small G proteins). ZOL-treated cells decreased a ratio of membrane to cytoplasmic fractions in RhoA, Cdc42 and Rab6 but less significantly Rac1 proteins, indicating that these proteins were possible targets for ZOL-induced actions. We further analyzed which small G proteins were responsible for the three ZOL-induced effects, caspase-mediated apoptosis, S-phase arrest and morphological changes, using inhibitors for respective small G proteins and siRNA for Cdc42. ZOL-induced apoptosis is due to insufficient prenylation of Rab proteins because an inhibitor of geranlygeranyl transferase II that was specific for Rab family proteins prenylation, but not others inhibitors, activated the same apoptotic pathways that ZOL did. ZOL suppressed an endogenous topoisomerase II activity, which was associated with apoptosis and S-phase arrest in respective cells because we detected the same cell cycle changes in etoposide-treated cells. Inhibitors for geranlygeranyl transferase I and for RhoA produced morphological changes and disrupted actin fiber structures, both of which were similar to those by ZOL treatments. These data demonstrated that anti-tumor effects by ZOL were attributable to inhibited functions of respective small G proteins and topoisomerase II activity, and suggested that cellular factors were involved in the differential cell cycle changes.Cell Death and Disease advance online publication, 13 November 2014; doi:10.1038/cddis.2014.475

In vitro conversion of irinotecan to SN‐38 in human plasma

Irinotecan is an active cytotoxic agent for various cancers, and is converted to SN‐38, its most active metabolite, by carboxy‐lesterase converting enzyme (CCE) in vivo. Although the primary metabolic site is in the liver, ex vivo studies have proven that irinotecan is also converted to SN‐38 in intestines, plasma and tumor tissues. The present study attempted to elucidate the in vitro conversion efficiency in human plasma, and to examine possible inter‐individual variability and its clinical significance. Plasma samples were taken from 57 patients with lung cancer, 3 patients with benign pulmonary diseases and 9 healthy volunteers. After addition of 157 μM irinotecan to plasma, time courses of SN‐38 concentration, measured by high‐performance liquid chromatog‐raphy (HPLC), were investigated. All subjects showed linear increase in SN‐38 concentration during the first 60‐min period, followed by a plateau. Mean and standard deviation of the conversion rate in the first 60 min were 515.9±50.1 pmol/ml/h (n=69), with a coefficient of variation of 0.097. Although most of the subjects showed comparable conversion rates, 3 subjects had significantly higher conversion rates. In conclusion, the results of this study suggest that the enzyme activity of CCE in human plasma may show inter‐individual variability.