Comparison of skin dose in IMRT and VMAT with TrueBeam and Halcyon linear accelerator for whole breast irradiation.

With the increasing use of flattening filter free (FFF) beams, it is important to evaluate the impact on the skin dose and target coverage of breast cancer treatments. This study aimed to compare skin doses of treatments using FFF and flattening filter (FF) beams for breast cancer. The study established treatment plans for left breast of an anthropomorphic phantom using Halcyon's 6-MV FFF beam and TrueBeam's 6-MV FF beam. Volumetric modulated arc therapy (VMAT) with varying numbers of arcs and intensity modulated radiation therapy (IMRT) were employed, and skin doses were measured at five points using Gafchromic EBT3 film. Each measurement was repeated three times, and averaged to reduce uncertainty. All plans were compared in terms of plan quality to ensure homogeneous target coverage. The study found that when using VMAT with two, four, and six arcs, in-field doses were 19%, 15%, and 6% higher, respectively, when using Halcyon compared to TrueBeam. Additionally, when using two arcs for VMAT, in-field doses were 10% and 15% higher compared to four and six arcs when using Halcyon. Finally, in-field dose from Halcyon using IMRT was about 1% higher than when using TrueBeam. Our research confirmed that when treating breast cancer with FFF beams, skin dose is higher than with traditional FF beams. Moreover, number of arcs used in VMAT treatment with FFF beams affects skin dose to the patient. To maintain a skin dose similar to that of FF beams when using Halcyon, it may be worth considering increasing the number of arcs.

Clinical Application of a Customized 3D-Printed Bolus in Radiation Therapy for Distal Extremities.

In radiation therapy (RT) for skin cancer, tissue-equivalent substances called boluses are widely used to ensure the delivery of an adequate dose to the skin surface and to provide a radioprotective effect for normal tissue. The aim of this study was to develop a new type of three-dimensional (3D) bolus for RT involving body parts with irregular geometries and to evaluate its clinical feasibility. Two 3D-printed boluses were designed for two patients with squamous cell carcinoma (SCC) of their distal extremities based on computed tomography (CT) images and printed with polylactic acid (PLA). The clinical feasibility of the boluses was evaluated by measuring the in vivo skin dose at the tumor site with optically stimulated luminescence detectors (OSLDs) and comparing the results with the prescribed and calculated doses from the Eclipse treatment planning system (TPS). The average measured dose distribution for the two patients was 94.75% of the prescribed dose and 98.8% of the calculated dose. In addition, the average measured dose during repeated treatments was 189.5 ± 3.7 cGy, thus demonstrating the excellent reproducibility of the proposed approach. Overall, the customized 3D-printed boluses for the RT of distal extremities accurately delivered doses to skin tumors with improved reproducibility.

Safety and Efficacy of Allogeneic Natural Killer Cells in Combination with Pembrolizumab in Patients with Chemotherapy-Refractory Biliary Tract Cancer: A Multicenter Open-Label Phase 1/2a Trial.

BACKGROUND AND AIM: This study investigated the administration of combination therapy, allogeneic natural killer (NK) cells and pembrolizumab in the treatment of advanced biliary tract cancer to determine the safety and tolerability (phase 1) and the efficacy and safety (phase 2a). METHODS: Forty patients (phase 1, n = 6; phase 2a, n = 34) were enrolled between December 2019 and June 2021. The patients received highly activated allogeneic NK cells ("SMT-NK") on weeks 1 and 2 and pembrolizumab on week 1. This 3-week schedule (one cycle) was repeated until confirmed disease progression, intolerable adverse events (AEs), patient withdrawal, or finishing the maximum treatment schedule. The tumor response was evaluated after every three cycles. RESULTS: In phase 1, four patients (66.7%) experienced seven AEs, but no severe AE was observed. In phase 2a, 126 AEs occurred in 29 patients (85.3%). Severe AEs (≥grade 3) were reported in 16 patients (47.1%). The overall response rate (ORR) was 17.4% in the full analysis set and 50.0% in the per-protocol set. CONCLUSIONS: SMT-NKs plus pembrolizumab resulted in no severe AEs directly related to the drug combination. The combination therapy also exerted antitumor activity with improved efficacy compared to the recent monotherapy with pembrolizumab in patients with advanced biliary tract cancer.

Radiation-induced C-reactive protein triggers apoptosis of vascular smooth muscle cells through ROS interfering with the STAT3/Ref-1 complex.

Damage to normal tissue can occur over a long period after cancer radiotherapy. Free radical by radiation can initiate or accelerate chronic inflammation, which can lead to atherosclerosis. However, the underlying mechanisms remain unclear. Vascular smooth muscle cells (VSMCs) proliferate in response to JAK/STAT3 signalling. C-reactive protein (CRP) can induce VSMCs apoptosis via triggering NADPH oxidase (NOX). Apoptotic VSMCs promote instability and inflammation of atherosclerotic lesions. Herein, we identified a VSMCs that switched from proliferation to apoptosis through was enhanced by radiation-induced CRP. NOX inhibition using lentiviral sh-p22phox prevented apoptosis upon radiation-induced CRP. CRP overexpression reduced the amount of STAT3/Ref-1 complex, decreased JAK/STAT phosphorylation and formed a new complex of Ref-1/CRP in VSMC. Apoptosis of VSMCs was further increased by CRP co-overexpressed with Ref-1. Functional inhibition of NOX or p53 also prevented apoptotic activity of the CRP-Ref-1 complex. Immunofluorescence showed co-localization of CRP, Ref-1 and p53 with α-actin-positive VSMC in human atherosclerotic plaques. In conclusion, radiation-induced CRP increased the VSMCs apoptosis through Ref-1, which dissociated the STAT3/Ref-1 complex, interfered with JAK/STAT3 activity, and interacted with CRP-Ref-1, thus resulting in transcription-independent cell death via p53. Targeting CRP as a vascular side effect of radiotherapy could be exploited to improve curability.

Knockout of longevity gene Sirt1 in zebrafish leads to oxidative injury, chronic inflammation, and reduced life span.

Sirt1, a member of the sirtuin gene family, encodes the most conserved mammalian NAD+-dependent deacetylase enzyme responsible for removing acetyl groups from many proteins. The Sirt1 gene is known as a longevity gene whose knockout in mice leads to decreased lifespan relative to the wild type. This study aimed to explore phenotypic changes in zebrafish Sirt1-knockouts and to investigate the function of the Sirt1 gene. Targeted knockout of Sirt1 in zebrafish (Danio rerio) was achieved using the CRISPR-Cas9 genome editing system. We created a 4-bp insertion-homozygote Sirt1-knockout zebrafish. Although there was no evident difference in appearance in the early stages of development, a significant increase in reactive oxygen species and in the extent of apoptosis in Sirt1-knockout zebrafish was observed. Sirt1 knockout caused inflammatory genes, including IL-1b, IL-6 and TNF-α to be highly expressed. Additionally, the lack of Sirt1 caused chronic inflammation and intestinal atrophy, thereby increasing pro-apoptotic events, which ultimately reduced the lifespan of transgenic zebrafish. Overall, our data demonstrate that lack of Sirt1 caused a significantly increased generation of reactive oxygen species that resulted in chronic inflammation and regeneration. Continuous repetition of these events played an important role in accelerating aging, thereby decreasing lifespan. Our findings using the knockout zebrafish model confirmed the association of the Sirt1 gene to aging processes and lifespan. Furthermore, the Sirt1-knockout mutant zebrafish developed in our study will surely be a valuable model to explore the mechanism of chronic inflammation.

Iroquois Homeobox 1 Acts as a True Tumor Suppressor in Multiple Organs by Regulating Cell Cycle Progression.

Iroquois homeobox 1 (IRX1) belongs to the Iroquois homeobox family known to play an important role during embryonic development. Interestingly, however, recent studies have suggested that IRX1 also acts as a tumor suppressor. Here, we use homozygous knockout mutants of zebrafish to demonstrate that the IRX1 gene is a true tumor suppressor gene and mechanism of the tumor suppression is mediated by repressing cell cycle progression. In this study, we found that knockout of zebrafish Irx1 gene induced hyperplasia and tumorigenesis in the multiple organs where the gene was expressed. On the other hands, overexpression of the IRX1 gene in human tumor cell lines showed delayed cell proliferation of the tumor cells. These results suggest that the IRX1 gene is truly involved in tumor suppression. In an attempt to identify the genes regulated by the transcription factor IRX1, we performed microarray assay using the cRNA obtained from the knockout mutants. Our result indicated that the highest fold change of the differential genes fell into the gene category of cell cycle regulation, suggesting that the significant canonical pathway of IRX1 in antitumorigenesis is done by regulating cell cycle. Experiment with cell cycle blockers treated to IRX1 overexpressing tumor cells showed that the IRX1 overexpression actually delayed the cell cycle. Furthermore, Western blot analysis with cyclin antibodies showed that IRX1 overexpression induced decrease of cyclin production in the cancer cells. In conclusion, our in vivo and in vitro studies revealed that IRX1 gene functionally acts as a true tumor suppressor, inhibiting tumor cell growth by regulating cell cycle.

The pretreatment erythrocyte sedimentation rate predicts survival outcomes after surgery and adjuvant radiotherapy for extremity soft tissue sarcoma.

BACKGROUND: Systemic inflammation plays a critical role in cancer progression and oncologic outcomes in cancer patients. We investigated whether preoperative inflammatory biomarkers, including C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and neutrophil to lymphocyte ratio (NLR), could be surrogate biomarkers for predicting overall survival (OS) in soft tissue sarcoma (STS) patients treated with surgery and postoperative radiotherapy. METHODS: A series of 99 patients who presented with localized extremity STS were retrospectively reviewed. The preoperative CRP levels, ESR, and NLR were evaluated for associations with OS, disease-free survival (DFS), local recurrence-free survival (LRFS), and distant metastasis-free survival (DMFS). Cutoff values for CRP, ESR, and NLR were derived from receiver-operating characteristic curve analysis. RESULTS: Elevated CRP (> 0.14 mg/dL), ESR (> 15 mm/h), and NLR (> 1.95) levels were seen in 33, 44, and 45 patients, respectively. Of these three inflammatory biomarkers, elevated CRP and ESR were associated with a poorer OS (CRP: P = 0.050; ESR: P = 0.001), DFS (CRP: P = 0.023; ESR: P = 0.003), and DMFS (CRP: P = 0.015; ESR: P = 0.001). By multivariate analysis, an elevated ESR was found to be an independent prognostic factor for OS (HR 3.580, P = 0.025) and DMFS (HR 3.850, P = 0.036) after adjustment for other established prognostic factors. CONCLUSIONS: The preoperative ESR level is a simple and useful surrogate biomarker for predicting survival outcomes in STS patients and might improve the identification of high-risk patients of tumor relapse in clinical practice.

Treatment Outcome after Fractionated Conformal Radiotherapy for Hepatocellular Carcinoma in Patients with Child-Pugh Classification B in Korea (KROG 16-05).

PURPOSE: There is limited data on radiotherapy (RT) for hepatocellular carcinoma (HCC) in patients with Child-Pugh classification B (CP-B). This study aimed to evaluate the treatment outcomes of fractionated conformal RT in HCC patients with CP-B. MATERIALS AND METHODS: We retrospectively reviewed the data of HCC patients with CP-B treated with RT between 2009 and 2014 at 13 institutions in Korea. HCC was diagnosed by the Korea guideline of 2009, and modern RT techniques were applied. Fraction size was ≤ 5 Gy and the biologically effective dose (BED) ≥ 40 Gy10 (α/ß = 10 Gy). A total of 184 patients were included in this study. RESULTS: Initial CP score was seven in 62.0% of patients, eight in 31.0%, and nine in 7.0%. Portal vein tumor thrombosis was present in 66.3% of patients. The BED ranged from 40.4 to 89.6 Gy10 (median, 56.0 Gy10). After RT completion, 48.4% of patients underwent additional treatment. The median overall survival (OS) was 9.4 months. The local progression-free survival and OS rates at 1 year were 58.9% and 39.8%, respectively. In the multivariate analysis, non-classic radiation-induced liver disease (RILD) (p < 0.001) and additional treatment (p < 0.001) were the most significant prognostic factors of OS. Among 132 evaluable patients without progressive disease, 19.7% experienced non-classic RILD. Normal liver volume was the most predictive dosimetric parameter of non-classic RILD. CONCLUSION: Fractionated conformal RT showed favorable OS with a moderate risk non-classic RILD. The individual radiotherapy for CP-B could be cautiously applied weighing the survival benefits and the RILD risks.

Clinical significance of the post-radiotherapy 18F-fludeoxyglucose positron emission tomography response in nasopharyngeal carcinoma.

OBJECTIVE: The aim of the present study was to evaluate the clinical significance of the post-radiotherapy 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET) response for detecting residual disease and predicting survival outcome in patients with nasopharyngeal cancer. METHODS: We reviewed 143 patients with nasopharyngeal cancer who underwent 18F-FDG PET within 6 months after completion of radiotherapy between 2001 and 2012. 18F-FDG PET findings at the primary tumor (T-) and regional lymph nodes (N-) were separately assessed and considered negative [PET (-)] or positive [PET (+)] depending on the remaining focal increased uptake of 18F-FDG that was greater than that of the surrounding muscle or blood vessels. The standard of reference was histopathological confirmation or clinical/imaging follow-up. Overall survival (OS), distant metastasis-free survival (DMFS), and locoregional recurrence-free survival (LRRFS) rates were estimated from the date of the start of radiotherapy. RESULTS: The median follow-up period was 73 months (range, 9-182 months). Overall, 83 and 66% of patients achieved T-PET (-) and N-PET (-) responses, and the negative-predictive values (NPVs) for T- and N- were 100 and 99%, respectively. The sensitivity, specificity, and positive-predictive value were 100, 84, and 8% for T-, and 67, 80, and 7% for N-, respectively. The 5-year OS, DMFS, and LRRFS rates were 83, 83, and 87%, respectively, and patients with N-PET (+) with SUVmax >2.5 showed significantly inferior 5-year OS and DMFS rates than patients with N-PET (-) or N-PET (+) with SUVmax ≤2.5 (44 vs 86%, p = 0.004; 36 vs 85%, p < 0.001). CONCLUSION: In patients that have received definitive (chemo)radiotherapy for nasopharyngeal cancer, 18F-FDG PET within 6 months of completion of treatment has a high NPV for predicting residual disease and is prognostic for long-term treatment outcomes. Patients with remaining focal increased uptake of 18F-FDG at lymph nodes may benefit from more aggressive treatments, and further studies are needed to validate the clinical significance of post-radiotherapy 18F-FDG PET. ADVANCES IN KNOWLEDGE: We found that post-radiotherapy 18F-FDG PET findings have a high NPV for detecting residual disease and are a significant prognostic factor for treatment outcomes.

Long-term outcomes of the 2-week schedule of hypofractionated radiotherapy for recurrent hepatocellular carcinoma.

BACKGROUND: The 2-week schedule of hypofractionated radiotherapy as a salvage treatment for hepatocellular carcinoma (HCC) has previously exhibited promising results; this study aimed to assess its long-term clinical outcomes in patients with recurrent HCC ineligible for curative treatments. METHODS: We retrospectively enrolled 77 patients (84 lesions) with HCC who were treated with hypofractionated radiotherapy between December 2008 and July 2013. Primary inclusion criteria were HCC unsuitable for curative treatments and HCC located within 2 cm of a critical normal organ. We administered 3.5-5 Gy/fraction for 2 weeks, resulting in a total dose of 35-50 Gy. RESULTS: The median follow-up period was 33.6 (range, 4.8-78.3) months. The 3- and 5-year overall survival rates were 52.3% and 40.9%, respectively, and local control rates were 79.5% and 72.6% in all treated lesions, respectively. The 5-year local control rate was better in the higher radiation dose group than in the lower radiation dose group (50 Gy: 79.7% vs. < 50 Gy: 66.1%); however, the difference was not statistically significant (P = 0.493). We observed grade ≥ 3 hepatic toxicity in 2 (2.6%) patients and grade 3 gastrointestinal bleeding in 1 (1.3%) patient. However, grade ≥ 4 toxicity was not observed after hypofractionated radiotherapy. CONCLUSIONS: The 2-week schedule of hypofractionated radiotherapy for recurrent HCC exhibited good local control and acceptable treatment-related toxicity during the long-term follow-up period. Thus, this fractionation schedule can be a potential salvage treatment option for recurrent HCC, particularly for tumors located close to a radiosensitive gastrointestinal organ.

In Vivo Study of Natural Killer (NK) Cell Cytotoxicity Against Cholangiocarcinoma in a Nude Mouse Model.

BACKGROUND/AIM: Natural killer (NK) cells are one of the lymphocytes clinically used for various cancer types. Cytotoxicity of NK cells to cholangiocarcinoma (CC), however, has not yet been studied. Nor NK cell therapy against CC has been clinically applied. In this study, relevance of NK cell therapy for anti-tumor efficacy against CC was pre-clinically investigated. MATERIALS AND METHODS: Human HuCCT-1 cells, an intrahepatic CC cell line, were xenografted into nude mice. The HuCCT-1 tumor-bearing nude mice then received multiple infusions of ex vivo-expanded human NK cells (SMT01) and in vivo cytotoxic activity of the NK cells against the CC cells was evaluated. RESULTS: SMT01 infusion resulted in significant inhibition of the CC tumor growth. Body weight of the mice administrated with chemotherapy was found to be maintained at the lowest level among all treatment groups while all the SMT01 infusion groups well maintained their body weight. CONCLUSION: The present in vivo study demonstrates that NK cells contain cytolytic activity against cholangiocarcinoma and show beneficial effect of NK cell therapy in relevance to quality of life. Further investigation of the NK cell-based immunotherapy can be useful to determine cancer therapeutics for the specific tumor.

High-dose radiotherapy is associated with better local control of bone metastasis from hepatocellular carcinoma.

We evaluated the pain and radiologic response, time to progression, and dose-response relationship after palliative radiotherapy for bone metastasis from hepatocellular carcinoma. We retrospectively reviewed the medical records of 91 patients between January 2004 and August 2012. The reviewed medical records included data on changes in pain, local tumor progression, and radiologic response evaluated via follow-up images. The radiologic response was assessed based on the Response Evaluation Criteria In Solid Tumors. The pain response was defined according to the International Bone Metastases Consensus Working Party palliative radiotherapy endpoints. Median radiation dose was 40 Gy (range, 20-66 Gy), with various fraction sizes (range, 2.0-6.0 Gy). Pain response rate was 81.4%. During the follow-up periods, radiologic local tumor progression was found in 42 patients (46.2%). The median time to progression was 14.1 months. When the patients were divided into two groups according to their radiation dose (< 55 Gy10 vs. ≥ 55 Gy10), the pain response rates of the high- and low-dose groups did not differ significantly (p = 0.728). However, the radiologic response rate and the time to progression showed significant differences between the two groups (p = 0.009 and p = 0.018, respectively). With dose escalation, higher radiologic response rates and a longer time to progression were achieved in patients with mass-forming bone metastases from hepatocellular carcinoma.

Impaired Lymphocytes Development and Xenotransplantation of Gastrointestinal Tumor Cells in Prkdc-Null SCID Zebrafish Model.

Severe combined immunodeficiency (SCID) mice have widely been used as hosts for human tumor cell xenograft study. This animal model, however, is labor intensive. As zebrafish is largely emerging as a promising model system for studying human diseases including cancer, developing efficient immunocompromised strains for tumor xenograft study are also demanded in zebrafish. Here, we have created the Prkdc-null SCID zebrafish model which provides the stable immune-deficient background required for xenotransplantation of tumor cell. In this study, the two transcription activator-like effector nucleases that specifically target the exon3 of the zebrafish Prkdc gene were used to induce a frame shift mutation, causing a complete knockout of the gene function. The SCID zebrafish showed susceptibility to spontaneous infection, a well-known phenotype found in the SCID mutation. Further characterization revealed that the SCID zebrafish contained no functional T and B lymphocytes which reflected the phenotypes identified in the mice SCID model. Intraperitoneal injection of human cancer cells into the adult SCID zebrafish clearly showed tumor cell growth forming into a solid mass. Our present data show the suitability of using the SCID zebrafish strain for xenotransplantation experiments, and in vivo monitoring of the tumor cell growth in the zebrafish demonstrates use of the animal model as a new platform of tumor xenograft study.

Feasibility of normal tissue dose reduction in radiotherapy using low strength magnetic field.

PURPOSE: Toxicity of mucosa is one of the major concerns of radiotherapy (RT), when a target tumor is located near a mucosal lined organ. Energy of photon RT is transferred primarily by secondary electrons. If these secondary electrons could be removed in an internal cavity of mucosal lined organ, the mucosa will be spared without compromising the target tumor dose. The purpose of this study was to present a RT dose reduction in near target inner-surface (NTIS) of internal cavity, using Lorentz force of magnetic field. MATERIALS AND METHODS: Tissue equivalent phantoms, composed with a cylinder shaped internal cavity, and adjacent a target tumor part, were developed. The phantoms were irradiated using 6 MV photon beam, with or without 0.3 T of perpendicular magnetic field. Two experimental models were developed: single beam model (SBM) to analyze central axis dose distributions and multiple beam model (MBM) to simulate a clinical case of prostate cancer with rectum. RT dose of NTIS of internal cavity and target tumor area (TTA) were measured. RESULTS: With magnetic field applied, bending effect of dose distribution was visualized. The depth dose distribution of SBM showed 28.1% dose reduction of NTIS and little difference in dose of TTA with magnetic field. In MBM, cross-sectional dose of NTIS was reduced by 33.1% with magnetic field, while TTA dose were the same, irrespective of magnetic field. CONCLUSION: RT dose of mucosal lined organ, located near treatment target, could be modulated by perpendicular magnetic field.

Predominant Activation of JAK/STAT3 Pathway by Interleukin-6 Is Implicated in Hepatocarcinogenesis.

Chronic inflammation is an important process leading to tumorigenesis. Therefore, targeting and controlling inflammation can be a promising cancer therapy. Inflammation is often caused by a variety of inflammatory cytokine such as the interleukin (IL)-6, a pleiotrophic cytokine known to be involved in the tumorigenesis. In this study, an in vivo hepatic tumorigenesis model of zebrafish was generated to demonstrate a direct consequence of the human IL6 expression causing hepatocarcinogenesis. To do this, an elevated expression of the hIL6 gene was established to specifically target the zebrafish hepatocytes by transgenesis. Interestingly, the elevated hIL6 expression caused the chronic inflammation which results in a massive infiltration of inflammatory cells. This eventually resulted in the generation of various dysplastic lesions such as clear cell, small cell, and large cell changes, and also eosinophilic and basophilic foci of hepatocellular alteration. Hepatocellular carcinoma was then developed in the transgenic zebrafish. Molecular characterization revealed upregulation of the downstream components involved in the IL6-mediated signaling pathways, especially PI3K/Akt and JAK/STAT3 pathways. Further investigation indicated that PI3K was the most reactive to the infiltrated inflammatory cells and dysplasia with large cell change, whereas STAT3 was heavily activated in the region with dysplastic foci, suggesting that the JAK/STAT3 pathway was mainly implicated in the hepatic tumorigenesis in the current model. Our present study provides an in vivo evidence of the relationship between chronic inflammation and tumorigenesis and reinforces the pivotal role of IL6 in the inflammation-associated hepatocarcinogenesis.

Clinical outcome of fiducial-less CyberKnife radiosurgery for stage I non-small cell lung cancer.

PURPOSE: To evaluate the treatment results in early stage non-small cell lung cancer patients who have undergone fiducial-less CyberKnife radiosurgery (CKRS). MATERIALS AND METHODS: From June 2011 to November 2013, 58 patients underwent CKRS at Asan Medical Center for stage I lung cancer. After excluding 14 patients, we retrospectively reviewed the records of the remaining 44 patients. All analyses were performed using SPSS ver. 21. RESULTS: The median age at diagnosis was 75 years. Most patients had inoperable primary lung cancer with a poor pulmonary function test with comorbidity or old age. The clinical stage was IA in 30 patients (68.2%), IB in 14 (31.8%). The mean tumor size was 2.6 cm (range, 1.2 to 4.8 cm), and the tumor was smaller than 2 cm in 12 patients (27.3%). The radiation dose given was 48-60 Gy in 3-4 fractions. In a median follow-up of 23.1 months, local recurrence occurred in three patients (2-year local recurrence-free survival rate, 90.4%) and distant metastasis occurred in 13 patients. All patients tolerated the radiosurgery well, only two patients developing grade 3 dyspnea. The most common complications were radiation-induced fibrosis and pneumonitis. Eight patients died due to cancer progression. CONCLUSION: The results showed that fiducial-less CKRS shows comparable local tumor control and survival rates to those of LINAC-based SABR or CKRS with a fiducial marker. Thus, fiducial-less CKRS using Xsight lung tracking system can be effectively and safely performed for patients with medically inoperable stage I non-small cell lung cancer without any risk of procedure-related complication.

Glioma is formed by active Akt1 alone and promoted by active Rac1 in transgenic zebrafish.

BACKGROUND: Ongoing characterization of glioma has revealed that Akt signaling plays a crucial role in gliomagenesis. In mouse models, however, Akt alone was not sufficient to induce glioma. METHODS: We established transgenic zebrafish that overexpressed dominant-active (DA) human Akt1 or Rac1(G12V) (DARac1) at ptf1a domain and investigated transgenic phenotypes and mechanisms leading to gliomagenesis. RESULTS: Transgene expressions were spatiotemporally restricted without any developmental abnormality of embryos and persisted at cerebellum and medulla in adult zebrafish. DAAkt1 alone induced glioma (with visible bumps at the head), with incidences of 36.6% and 49% at 6 and 9 months, respectively. Histologically, gliomas showed various histologic grades, increased proliferation, and frequent invasion into the fourth ventricle. Preferential location of small tumors at periventricular area and coexpression of Her4 suggested that tumors originated from Ptf1a- and Her4-positive progenitor cells at ventricular zone. Gliomagenesis was principally mediated by activation of survival pathway through upregulation of survivin genes. Although DARac1 alone was incapable of gliomagenesis, when coexpressed with DAAkt1, gliomagenesis was accelerated, showing higher tumor incidences (62.0% and 73.3% at 6 and 9 months, respectively), advanced histologic grade, invasiveness, and shortened survival. DARac1 upregulated survivin2, cyclin D1, ß-catenin, and snail1a but downregulated E-cadherin, indicating that DARac1 promotes gliomagenesis by enhancing proliferation, survival, and epithelial-to-mesenchymal transition. On pharmacologic tests, only Akt1/2 inhibitor effectively suppressed gliomagenesis, inhibited cellular proliferation, and induced apoptosis in established gliomas. CONCLUSIONS: The zebrafish model reinforces the pivotal role of Akt signaling in gliomagenesis and suggests Rac1 as an important protein involved in progression.

Microvascular imaging of asymptomatic MCA steno-occlusive patients using ultra-high-field 7T MRI.

It is common that physicians are faced with patients who have severe steno-occlusion of intracranial arteries in the absence of clinical symptoms. This study is to determine whether a 7T magnetic resonance angiogram (MRA) could provide the improved depiction of microvessels of asymptomatic patients with steno-occlusive middle cerebral artery (MCA) and possible clues for diagnosis of their clinical symptoms. All the patients who were identified as severe MCA steno-occlusions underwent a 7T MRA study. Three of these also underwent conventional angiography (TFCA). The vascular densities around the steno-occlusive MCA and posterior cerebral artery (PCA) branches as control vessels were measured and the difference between the median values of 7T and 3T MRA data was also analyzed. The results of 7T MRA revealed numerous microvessels not visible by conventional MRA. These 7T MRA images were also comparable to those obtained by an invasive conventional angiography (TFCA). The median values of vascular densities observed by 7T MRA were significantly higher than those by 3T MRA (5.28 at 7T vs. 1.45 at 3T for steno-occlusive MCA, p = 0.012, while 3.39 at 7T vs. 3.32 at 3T for normal PCA, p = 0.093). Ultra-high-field 7T MRA is a totally noninvasive angiographic technique that is capable of visualizing microvascular circulation that is usually difficult with conventional MRA. This visualization technique, therefore, could provide an important avenue on the diagnosis of steno-occlusion as well as the search of the roles of the microvessel, especially collaterals, in the preservation of normal blood circulation in patients with asymptomatic MCA steno-occlusion.

Aberrant Hedgehog ligands induce progressive pancreatic fibrosis by paracrine activation of myofibroblasts and ductular cells in transgenic zebrafish.

Hedgehog (Hh) signaling is frequently up-regulated in fibrogenic pancreatic diseases including chronic pancreatitis and pancreatic cancer. Although recent series suggest exclusive paracrine activation of stromal cells by Hh ligands from epithelial components, debates still exist on how Hh signaling works in pathologic conditions. To explore how Hh signaling affects the pancreas, we investigated transgenic phenotypes in zebrafish that over-express either Indian Hh or Sonic Hh along with green fluorescence protein (GFP) to enable real-time observation, or GFP alone as control, at the ptf1a domain. Transgenic embryos and zebrafish were serially followed for transgenic phenotypes, and investigated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), in situ hybridization, and immunohistochemistry. Over-expression of Ihh or Shh reveals virtually identical phenotypes. Hh induces morphologic changes in a developing pancreas without derangement in acinar differentiation. In older zebrafish, Hh induces progressive pancreatic fibrosis intermingled with proliferating ductular structures, which is accompanied by the destruction of the acinar structures. Both myofibroblasts and ductular are activated and proliferated by paracrine Hh signaling, showing restricted expression of Hh downstream components including Patched1 (Ptc1), Smoothened (Smo), and Gli1/2 in those Hh-responsive cells. Hh ligands induce matrix metalloproteinases (MMPs), especially MMP9 in all Hh-responsive cells, and transform growth factor-ß1 (TGFß1) only in ductular cells. Aberrant Hh over-expression, however, does not induce pancreatic tumors. On treatment with inhibitors, embryonic phenotypes are reversed by either cyclopamine or Hedgehog Primary Inhibitor-4 (HPI-4). Pancreatic fibrosis is only prevented by HPI-4. Our study provides strong evidence of Hh signaling which induces pancreatic fibrosis through paracrine activation of Hh-responsive cells in vivo. Induction of MMPs and TGFß1 by Hh signaling expands on the current understanding of how Hh signaling affects fibrosis and tumorigenesis. These transgenic models will be a valuable platform in exploring the mechanism of fibrogenic pancreatic diseases which are induced by Hh signaling activation.