Haruhiro Saito

Novel heteroduplex method using small cytology specimens with a remarkably high success rate for analysing EGFR gene mutations with a significant correlation to gefitinib efficacy in non-small-cell lung cancer

We conducted a feasibility study to examine whether small numbers of cancer cells could be utilised for analysis of the EGFR gene status using the loop-hybrid mobility shift assay, which is a modified heteroduplex technique. Cytology specimens obtained by transbronchial abrasion were successfully used for analysis of the EGFR gene status in 50 of 52 (96.2%) patients diagnosed with class V non-small-cell carcinoma. Furthermore, the relationship between the EGFR gene status and clinical outcome was analysed in 25 patients treated with gefitinib. Overall, 10 of 11 patients with EGFR mutations in exon 19 or 21 showed tumour regression with gefitinib treatment, compared to only two of 14 patients with wild-type EGFR. The response rate was significantly higher in the EGFR mutation group than in the wild-type EGFR group (90.9 vs 14.3%, P=0.00014). Logistic regression analysis revealed that EGFR mutations in cytology specimens represented an independent predictor of the gefitinib response. The overall and progression-free survivals were significantly longer in the EGFR mutation group than in the wild-type EGFR group (P<0.05). In conclusion, cytology specimens could be useful for analysing the EGFR status in the majority of patients with non-small-cell lung cancer to determine whether they are likely to benefit from gefitinib treatment.

Diabetes is not a potent inducer of neuronal cell death in mouse sensory ganglia, but it enhances neurite regeneration in vitro.

We examined the effects of diabetes on the morphological features and regenerative capabilities of adult mouse nodose ganglia (NG) and dorsal root ganglia (DRG). By light and electron microscopy, no apoptotic cell death was detected in the ganglia obtained from either streptozotocin (STZ)-induced diabetic or normal C57BL/6J mice in vivo. Neurite regeneration from transected nerve terminals of NG and DRG explants in culture at normal (10 mM) and high (30 mM) glucose concentrations was significantly enhanced in the diabetic mice. Chromatolytic changes (i.e. swelling and migration of the nucleus to an eccentric position in the neurons, and a loss of Nissl substance in the neuronal perikarya) and apoptotic cell death (less than one-fifth of the neurons) in the cultured ganglia were present, but neither hyperglycemia in vivo nor high glucose conditions in vitro altered the morphological features of the ganglia or the ratios of apoptotic cells at 3 days in culture. By semiquantitative RT-PCR analysis, the mRNA expressions of ciliary neurotrophic factor (CNTF) in DRG from both mice were down-regulated at 1 day in culture. The expression in diabetic DRG, but not in control DRG, was significantly up-regulated at later stages (3 and 7 days) in culture. In summary, hyperglycemia is unlikely to induce cell death in the sensory ganglia, but enhances the regenerative capability of vagal and spinal sensory nerves in vitro. The up-regulation of CNTF mRNA expression during the culture of diabetic DRG may play a role in the enhanced neurite regeneration.

Initial findings and progression of lung adenocarcinoma on serial computed tomography scans.

To study the initial findings of lung adenocarcinoma revealed by computed tomography (CT) scanning and observe tumor progression and elucidate appropriate follow-up schedule of tumor diagnosis via CT findings of suspected lung adenocarcinoma. Method: We studied 59 patients who had undergone CT scanning twice or more at intervals of 3 months or longer before surgery. We evaluated the initial CT findings as well as all subsequent changes. The rate of tumor growth was estimated by tumor volume doubling time, using the original method of Schwartz. The histological classifications were evaluated according to the criteria of Noguchi et al (Cancer 1995;75:2844-2852). Result: The initial appearances of lung adenocarcinoma were divided into 4 types: (1) ground-glass opacity-like lesions, (2) bubble-like appearance, (3) small nodules, and (4) scar-like lesions. Ground-glass opacity-like lesions tended to increase in size over the years, with solid parts appearing in some lesions during follow-up examinations. Bubble-like appearance displayed characteristic CT findings and tended to increase over the years from the time of initial diagnosis, and we therefore tended to consider them as old inflammatory lesions. Small nodules tended to increase in size over the months more rapidly than in other types. Scar-like lesions tended to exist mainly in the lungs already damaged by lung fibrosis and/or emphysema and therefore were difficult to detect on initial CT scans. Conclusion: We categorized 4 types of initial findings of lung adenocarcinomas detected by CT. We determined that each type of lesion had its own unique characteristic growth patterns and required varying follow-up periods.

Enhanced autophagy is required for survival in EGFR-independent EGFR-mutant lung adenocarcinoma cells.

Lung cancers harboring epidermal growth factor receptor (EGFR) mutations depend on constitutive activation of the kinase for survival. Although most EGFR-mutant lung cancers are sensitive to EGFR tyrosine kinase inhibitors (TKIs) and shrink in response to treatment, acquired resistance to TKI therapy is common. We demonstrate here that two EGFR-mutated lung adenocarcinoma cell lines, HCC827 and HCC4006, contain a subpopulation of cells that have undergone epithelial-to-mesenchymal transition and survive independent of activated EGFR. These EGFR-independent cancer cells, herein termed gefitinib-resistant (GR) cells, demonstrate higher levels of basal autophagy than their parental cells and thrive under hypoxic, reduced-serum conditions in vitro; this somewhat simulates the hypoxic environment common to cancerous tissues. We show that depletion of the essential autophagy gene, ATG5, by small interfering RNA (siRNA) or chloroquine, an autophagy inhibitor, markedly reduces GR cell viability under hypoxic conditions. Moreover, we show a significant elevation in caspase activity in GR cells following knockdown of ATG5. These results suggest that GR cells can evade apoptosis and survive in hostile, hypoxic environments with constant autophagic flux. We also show the presence of autophagosomes in some cancer cells from patient samples, even in untreated EGFR-mutant lung cancer tissue samples. Together, our results indicate that autophagy inhibitors alone or in combination with EGFR TKIs may be an effective approach for the treatment of EGFR-mutant lung cancers, where basal autophagy of some cancer cells is upregulated.

Comparison of thin-section CT and pathological findings in small solid-density type pulmonary adenocarcinoma: Prognostic factors from CT findings

OBJECTIVE We divided pulmonary adenocarcinoma of ≤ 20 mm into air-containing and solid-density types based on a percentage reduction of the maximum tumor diameter in the mediastinal window image compared to the area in the lung window image on thin-section (TS) CT of ≥ 50% (air-containing type) and <50% (solid-density type). No relapse occurred in patients with air-containing type. The prognosis of solid-density type may be poor even when the tumor size is 20mm or smaller. We investigated whether CT findings for these tumors could serve as prognostic factors. METHODS The subjects were 105 patients with solid-density type pulmonary adenocarcinoma that was identified on TSCT and found to have a diameter of 20mm or smaller after surgical resection during the period from April 1997 to November 2004. Notches, air bronchogram, pleural retraction, spiculation, venous involvement, and ground glass opacity were examined on TSCT, and their associations with pathological findings (i.e., pleural invasion, lymphatic permeation, vascular invasion, lymph node metastasis, and Noguchis classification) and relapse were investigated using chi-square test and Cox proportional hazards model. RESULTS The incidence of relapse was significantly higher in cases with notches. The incidence of notches increased with tumor growth and notches were frequent in Noguchi type D tumors, reflecting poorly differentiated adenocarcinoma. Lymphatic permeation and type D cases were independent factors associated with a poor prognosis using Cox proportional hazards model. CONCLUSIONS TSCT findings may be useful for prediction of the prognosis of solid-density type pulmonary adenocarcinoma.

Prognostic impact of survivin, cyclin D1, integrin β1, and VEGF in patients with small adenocarcinoma of stage I lung cancer

The purpose of this study was to investigate the impact of survivin, cyclin D1, integrin β1, and vascular endothelial growth factor (VEGF) in tumor on survival of patients with small adenocarcinoma of the lung. Seventy-two patients with pathologic stage I resected tumors <2 cm in diameter were entered into the study. Each patient underwent curative surgical resection for lung cancer between July 1992 and November 1999. The resected tumors were subjected to immunostaining for each gene. Thirty-five, 26, 6, and 16 patients had tumors with >10% survivin-, >20% cyclin D1-, >10% integrin β1-, and >10% VEGF-positive cells, respectively. When the survival of 72 patients was compared according to each gene expression, the overall survival of patients with positive expression of survivin, cyclin D1, and integrin β1 was significantly worse than that of individuals whose tumors had negative expression of each gene. By multivariate analysis controlling for each gene expression, no gene expression was an independent marker of poor prognosis, however, the overall survival of the complex gene expression (2 or more gene-positive) group (n = 35) was significantly worse than that of 0 or 1 gene-positive group (n = 37; log-rank test, P = 0.0011; Wilcoxon test, P = 0.0011). When the association between survival and pathologic factors, including lymphatic invasion, venous invasion, type of bronchioalveolar carcinoma, and complex gene positive expression was analyzed, only complex gene-positive expression was found to be a significant independent factor (hazard ratio = 0.085, P = 0.0299). It can be concluded that multiple increased expression of oncogene is a poor prognostic factor in patients with small adenocarcinoma of the lung.

Correlation of 18F-fluorodeoxyglucose uptake on positron emission tomography with Ki-67 index and pathological invasive area in lung adenocarcinomas 30 mm or less in size

BACKGROUND (18)F-fluorodeoxyglucose positron emission tomography (FDG-PET) is commonly used to distinguish benign from malignant lesion. Recently, maximum standardized uptake value (SUVmax) on FDG-PET has found to have prognostic value. We examined the relationship between SUVmax and proliferative activities as indicated by maximum diameter of tumor opacity on mediastinal-window images (TOM), Ki-67 index, and diameter of the pathological invasive area in lung adenocarcinomas <or=30 mm. METHODS Thin-section computed tomography (TS-CT) and FDG-PET were performed on 140 patients with resectable lung adenocarcinomas <or=30 mm between March 2006 and May 2008. Tumors were classified as air-type or solid-type based on TS-CT findings. In all resected specimens, diameter of the pathological invasive area and Ki-67 index were assessed. RESULTS SUVmax was significantly lower for air-type than for solid-type tumors (0.97 vs. 3.96, p<0.0001). In solid-type tumors, SUVmax correlated with diameter of TOM (r=0.450, p<0.0001), Ki-67 index (r=0.567, p<0.0001), and diameter of the pathological invasive area (r=0.672, p<0.0001). In multiple regression analysis, SUVmax correlated significantly with Ki-67 index and diameter of the pathological invasive area but not with diameter of TOM. The cut-off value of SUVmax for predicting invasive area >5mm was determined as 2.15 by ROC analysis, with sensitivity of 88.3% and specificity of 84.6%. CONCLUSIONS SUVmax correlated significantly with Ki-67 index and diameter of the pathological invasive area. The present results suggest the potential role of FDG-PET in predicting adenocarcinomas with invasive characteristics.

Prognosis of small adenocarcinoma of the lung based on thin-section computed tomography and pathological preparations.

Objective: We investigated the relationship between findings from tumor opacity in the mediastinal window image and solid lesions in pathological preparations and related the results to tumor recurrence. Methods: The subjects were 115 patients with a lung adenocarcinoma of 20 mm or smaller who underwent surgical resection. The proportion of the reduction in the tumor opacity in the mediastinal window image maximum diameter to the maximum diameter of the tumor opacity was calculated as the reduction percentage, and the proportion of the maximum solid lesions in pathological preparation diameter to the maximum tumor diameter was calculated as the pathological ratio. Results: The incidence of relapse was significantly higher in patients with a reduction percentage of less than 50% and in patients with a pathological ratio of less than 50%. Conclusions: Measurement of the reduction percentage and the pathological ratio may allow prediction of prognosis of small adenocarcinoma of the lung.

Enhanced neural regeneration from transected vagus nerve terminal in diabetic mice in vitro.

This study examined the effect of diabetes on neural regeneration in vitro. Nodose ganglia (NG) with vagal nerve fibers, dissected from streptozotocin-induced diabetic and normal C57BL/6J mice were embedded in collagen gel. After 3 and 7 days in culture, the numbers of regenerating neurites from transected nerve terminals of NG in diabetic mice were significantly greater than those in controls. Although many studies have revealed diabetes-associated impairment in neural regeneration, the results in the present study suggest that experimental diabetes could induce the potential to enhance regenerative capability of vagal sensory nerves after axotomy.

Phase II study of nedaplatin and irinotecan with concurrent thoracic radiotherapy in patients with locally advanced non-small-cell lung cancer

Background:Current international guidelines recommend the use of platinum-based chemotherapy with thoracic radiotherapy (TRT) for patients with locally advanced non-small-cell lung cancer (NSCLC).Methods:Patients with unresectable stage IIIA or IIIB NSCLC were treated with nedaplatin (NP) at 50 mg m−2 and irinotecan (CPT) at 60 mg m−2 on days 1 and 8 every 4 weeks for two to four cycles with concurrent TRT (2 Gy per day, total 60 Gy).Results:All 35 patients were able to receive a total of 60 Gy. Adverse effects and events in chemotherapy with TRT were grade 3 or 4 anaemia, neutropenia and thrombocytopenia, which occurred in 3.0%, 32.8% and 6.0% of patients, respectively. There was no grade 3 pneumonitis or oesophagitis. Adverse effects and events in chemotherapy alone were mild. There was no treatment-related death. An overall response rate was 94.3%. The median progression-free and overall survivals were 13.0 and 36.0 months, respectively. The 5-year disease-free and overall survival rates were 25.7% and 40.0%, respectively.Conclusion:NP and CPT treatment with concurrent TRT is effective and safe for patients with unresectable, locally advanced NSCLC.