Keiko Shibuya

The Akt/mTOR Pathway Assures the Synthesis of HIF-1α Protein in a Glucose- and Reoxygenation-dependent Manner in Irradiated Tumors

Transcriptional activity of HIF-1 (hypoxia-inducible factor-1) has been reported to be up-regulated in solid tumors after ionizing radiation; however, the molecular mechanism underlying the response remains to be elucidated. In the present study, we performed a series of molecular imaging experiments using a HIF-1-dependent reporter gene, 5HREp-ODD-luc, and found an essential role of the Akt/mTOR pathway. Hypoxic tumor cells distant from blood vessels were dramatically reoxygenated at 24 h postirradiation, and HIF-1 activity increased as HIF-1α accumulated in the reoxygenated regions. The accumulation was inhibited with a nonmetabolizable glucose analog, 2-deoxy-d-glucose, through the suppression of radiation-induced phosphorylation of Akt in the reoxygenated regions. Akt knockdown and an mTOR inhibitor revealed the importance of the Akt/mTOR pathway in the postirradiation accumulation of HIF-1α. In vitro experiments confirmed that an increase in glucose availability induced Akt phosphorylation under reoxygenated conditions and consequently up-regulated HIF-1α translation. Moreover, both the accelerated translation and the previously reported reactive oxygen species-mediated stabilization of HIF-1α protein were essential to the activation of HIF-1. All of these results indicate that Akt/mTOR-dependent translation of HIF-1α plays a critical role in the postirradiation up-regulation of intratumoral HIF-1 activity in response to radiation-induced alterations of glucose and oxygen availability in a solid tumor.

Dose--volume metrics associated with radiation pneumonitis after stereotactic body radiation therapy for lung cancer.

PURPOSE To identify dose-volume factors associated with radiation pneumonitis (RP) after stereotactic body radiation therapy (SBRT) for lung cancer. METHODS AND MATERIALS This study analyzed 74 patients who underwent SBRT for primary lung cancer. The prescribed dose for SBRT was uniformly 48 Gy in four fractions at the isocenter. RP was graded according to the Common Terminology Criteria for Adverse Events (CTCAE) v.3. Symptomatic RP was defined as grade 2 or worse. Optimal cut-offs dividing the patient population into two subgroups based on the incidence of symptomatic RP were sought using the following dose-volume metrics: PTV volume (ml), mean lung dose (Gy), and V5, V10, V15, V20, V25, V30, V35, and V40 (%) of both lungs excluding the PTV. RESULTS With a median follow-up duration of 31.4 months, symptomatic RP was observed in 15 patients (20.3%), including 1 patient with grade 3. Optimal cut-offs for pulmonary dose-volume metrics were V25 and V20. These two factors were highly correlated with each other, and V25 was more significant. Symptomatic RP was observed in 14.8% of the patients with V25 <4.2%, and the rate was 46.2% in the remainder (p = 0.019). PTV volume was another significant factor. The symptomatic RP rate was significantly lower in the group with PTV <37.7 ml compared with the larger PTV group (11.1% vs. 34.5%, p = 0.020). The patients were divided into three subgroups (patients with PTV <37.7 ml; patients with, PTV ≥37.7 ml and V25 <4.2%; and patients with PTV ≥37.7 ml and V25 ≥4.2%); the incidence of RP grade 2 or worse was 11.1%, 23.5%, and 50.0%, respectively (p = 0.013). CONCLUSIONS Lung V25 and PTV volume were significant factors associated with RP after SBRT.

Significance of HIF-1-active cells in angiogenesis and radioresistance

Human solid tumors contain hypoxic regions that have considerably lower oxygen tension than the normal tissues. Hypoxia offers resistance to radiotherapy and anticancer chemotherapy, as well as predispose to increased tumor metastases. Furthermore, hypoxia induces hypoxia-inducible factor-1 (HIF-1), which in turn increases tumor angiogenesis. Thus, eradication of HIF-1-active/hypoxic tumor cells is very important for cancer therapy. We have previously reported that procaspase-3 fused with a von Hippel–Lindau (VHL)-mediated protein destruction motif of alpha subunit of HIF-1 (HIF-1α) containing Pro564, named TAT-ODD-procaspase-3 (TOP3), specifically induced cell death to hypoxic cells in vivo as well as in vitro. We now report that TOP3 also eradicates the radiation-induced HIF-1-active tumor cells. HIF-1 activity in the xenografts of human tumor cells, which express luciferase under the transcriptional control of HIF-1, were monitored and quantified daily with an in vivo bioluminescence photon-counting device. HIF-1 activity in tumors was more rapidly increased by ionizing radiation (IR) compared to untreated tumors. TOP3 efficiently decreased the HIF-1-activity in irradiated tumors as well as unirradiated ones, indicating TOP3 eradicated tumor cells with HIF-1-activity induced by IR as well as hypoxia. Eradication of HIF-1-active/hypoxic cells in the xenografts during irradiation exhibited significant suppression in angiogenesis and strong enhancement in a long-term growth suppression of tumor xenografts. These results further strengthen the argument that HIF-1-active/hypoxic cells play crucial roles in angiogenesis and radioresistance.

Prognostic Factors in Stereotactic Body Radiotherapy for Non–Small-Cell Lung Cancer

PURPOSE To investigate the factors that influence clinical outcomes after stereotactic body radiotherapy (SBRT) for non-small-cell lung cancer (NSCLC). METHODS AND MATERIALS A total of 101 consecutive patients who underwent SBRT with 48 Gy in 4 fractions for histologically confirmed Stage I NSCLC were enrolled in this study. Factors including age, maximal tumor diameter, sex, performance status, operability, histology, and overall treatment time were evaluated with regard to local progression (LP), disease progression (DP), and overall survival (OS) using the Cox proportional hazards model. Prognostic models were built with recursive partitioning analysis. RESULTS Three-year OS was 58.6% with a median follow-up of 31.4 months. Cumulative incidence rates of LP and DP were 13.2% and 40.8% at 3 years, respectively. Multivariate analysis demonstrated that tumor diameter was a significant factor in all endpoints of LP, DP, and OS. Other significant factors were age in DP and sex in OS. Recursive partitioning analysis indicated a condition for good prognosis (Class I) as follows: female or T1a (tumor diameter ≤20 mm). When the remaining male patients with T1b-2a (>20 mm) were defined as Class II, 3-year LP, DP, and OS were 6.8%, 23.6%, and 69.9% in recursive partitioning analysis Class I, respectively, whereas these values were 19.9%, 58.3%, and 47.1% in Class II. The differences between the classes were statistically significant. CONCLUSIONS Tumor diameter and sex were the most significant factors in SBRT for NSCLC. T1a or female patients had good prognosis.

Selective Killing of Hypoxia-Inducible Factor-1–Active Cells Improves Survival in a Mouse Model of Invasive and Metastatic Pancreatic Cancer

Purpose: Pancreatic cancer is characterized by intratumoral hypoxia, early and aggressive local invasion, and metastatic potential. Hypoxia-inducible factor-1 (HIF-1) is the major transcriptional activator of hypoxia-responsive genes and intratumoral hypoxia is associated with increased risk of metastasis. However, the behavior of the cells having HIF-1 activity during the malignant progression in pancreatic cancer has not been tested. Experimental Design: We orthotopically transplanted pancreatic cancer cells stably transfected with a HIF-1–dependent luciferase reporter gene and monitored HIF-1 activity in vivo in control and POP33-treated mice. POP33 is a novel prodrug, which has potential to increase caspase-3 activity and induce apoptosis in HIF-1–active/hypoxic cells. Results:In vivo optical imaging showed that HIF-1 activity proceeded along with local invasion, the peritoneal dissemination, and the liver metastasis. HIF-1–active hypoxic cells were selectively eradicated by POP33. Moreover, selective killing of HIF-1–active hypoxic cells significantly suppressed malignant progression, resulting in a significant improvement in survival rate. Conclusions: These results show that HIF-1–active cells constitute a large proportion of invading and metastatic cells and suggest that eradication of these cells may improve the outcome in advanced pancreatic cancer, a condition for which no effective therapy currently exists.

Salvage Lung Resection for Non-small Cell Lung Cancer After Stereotactic Body Radiotherapy in Initially Operable Patients

Background: Stereotactic body radiotherapy (SBRT) has emerged as a curative treatment for medically inoperable patients with early-stage non-small cell lung cancer (NSCLC). Since NSCLC recurs locally in 10% of the patients treated with SBRT, salvage lung resection after SBRT may be considered in these cases. To further understand the indications for salvage surgery and the pathogenesis of tumor recurrence in these patients, we retrospectively reviewed cases treated at our institution. Methods: SBRT has been performed in patients with early-stage NSCLC at Kyoto University Hospital. We encountered 5 patients who underwent salvage lung resection for NSCLC after SBRT. Results: All the patients were initially operable, but they chose SBRT. After SBRT, the tumors shrank initially in all patients, but increased in size within 18 months of SBRT in the case of 4 patients. During surgical extirpation, we did not find any significant SBRT-related adhesions in any of the patients. Conclusions: We have successfully treated 5 patients who underwent salvage lung resection for early-stage NSCLC after SBRT. We found that surgical resection was feasible after SBRT.

Simultaneous evaluation of radiation-induced apoptosis and micronuclei in five cell lines.

PURPOSE This study was conducted to clarify the relationship among the frequencies of micronuclei (MN) and apoptosis, and clonogenic cell survival after irradiation. MATERIALS AND METHODS The frequencies of MN and apoptosis were compared in the surviving fraction in three human tumour cell lines and two rodent cell lines at various irradiation doses. RESULTS The SHIN-3, DU-145 and CHO-K1 cells showed dose-dependent increases of MN per binucleate cell and an excellent correlation between the MN frequency and surviving fraction after irradiation. The F9 and COLO 320DM cells did not show this correlation. The number of apoptotic cells increased according to the increase in radiation dose in the F9 and COLO 320DM cells, but not in the SHIN-3, DU-145 or CHO-K1 cells. CONCLUSIONS The detection of the MN frequency alone is insufficient to measure cellular intrinsic radiosensitivity. The simultaneous use of the MN assay and the detection of apoptotic cells would be more reliable as a method for predicting cell survival after radiation.

Impact of Pretreatment Interstitial Lung Disease on Radiation Pneumonitis and Survival after Stereotactic Body Radiation Therapy for Lung Cancer

Introduction: To investigate the impact of pre-existing radiological interstitial lung disease (ILD) findings on the incidence of radiation pneumonitis (RP) and clinical outcomes after stereotactic body radiation therapy (SBRT) for stage I non–small-cell lung cancer. Methods: We included 157 consecutive patients who underwent SBRT alone for stage I non–small-cell lung cancer and whose pretreatment lung computed tomography images were available for retrospective review. The pretreatment computed tomography images were evaluated retrospectively for the presence of ILD. The incidence of RP, overall survival (OS) rate, and the incidence of disease progression and local progression were evaluated between patients with ILD (ILD[+]) and without ILD (ILD[−]). Results: Pre-existing ILD was identified in 20 patients. The median follow-up period was 39.5 months. The incidences of RP worse than grade 2 (≥ Gr2 RP) and worse than grade 3 (≥ Gr3 RP) were significantly higher in ILD(+) than ILD(−) (1 year ≥ Gr2 RP rate, 55.0% versus 13.3%; p < 0.001 and 1year ≥ Gr3 RP rate 10.0% versus 1.5%; p = 0.020). Multivariate analysis also indicated that ILD(+) was a risk factor for ≥ Gr2 and ≥ Gr3 RP, and the volume of the irradiated lung. The OS rate tended to be worse in ILD(+) than ILD(−) (3-year OS, 53.8% versus 70.8%; p = 0.28). No difference was observed in the disease progression or local progression rates. Conclusions: Pre-existing ILD was a significant risk factor for symptomatic and severe RP. Prescreening for ILD findings is important for determining the radiation pneumonitis risk when planning SBRT.

Characterization of FDG-PET images after stereotactic body radiation therapy for lung cancer.

BACKGROUND AND PURPOSE The purpose was to characterize (18)F-fluorodeoxyglucose-positron emission tomography (FDG-PET) findings after stereotactic body radiation therapy (SBRT) for lung cancer. MATERIALS AND METHODS This was a retrospective review of 32 FDG-PET scans from 23 patients who underwent SBRT for lung cancer and who showed no evidence of local recurrence. The FDG uptake by lesions was assessed visually using a 3-point scale (0, none or faint; 1, mild; or 2, moderate to intense), and the demarcation (ill- or well-defined) was evaluated. For semi-quantitative analysis, the maximum standardized uptake value (SUVmax) was calculated. RESULTS Grade 2 intensity was observed in 70%, 33%, 30%, and 0% of PET scans performed <6, 6-12, 12-24, and >24 months, respectively, after SBRT; well-defined demarcation was observed in 80%, 33%, 40%, and 17%, respectively, and the respective means of the SUVmax were 4.9, 2.6, 3.0, and 2.3. The SUVmax was significantly higher for scans performed at <6 months than at 6-12 or >24 months. CONCLUSIONS FDG uptake tended to be intense and well-defined at early times after SBRT, especially within 6 months, and was faint and ill-defined at later periods. Moderate to intense FDG uptake observed soon after SBRT does not always indicate a residual tumour.

Current status of stereotactic body radiotherapy for lung cancer

Stereotactic radiotherapy (SRT) for extracranial tumors has been recently performed to treat lung and liver cancers, and has subsequently been named stereotactic body radiotherapy (SBRT). The advantages of hypofractionated radiotherapy for treating lung tumors are a shortened treatment course that requires fewer trips to the clinic than a conventional program, and the adoption of a smaller irradiated volume allowed by greater setup precision. This treatment is possible because the lung and liver are considered parallel organs at risk. The preliminary clinical results, mostly reported on lung cancer, have been very promising, including a local control rate of more than 90%, and a relatively low complication rate. The final results of a few clinical trials are awaited. SBRT may be useful for the treatment of stage I lung tumors.