Quality assurance in a phase III, multicenter, randomized trial of POstmastectomy radioThErapy in Node posiTive breast cancer with or without Internal mAmmary nodaL irradiation (POTENTIAL): a planning benchmark case.

PURPOSE: To report the planning benchmark case results of the POTENTIAL trial-a multicenter, randomized, phase 3 trial-to evaluate the value of internal mammary nodal (IMN) irradiation for patients with high-risk breast cancer. METHODS: All participating institutions were provided the outlines of one benchmark case, and they generated radiation therapy plans per protocol. The plans were evaluated by a quality assurance team, after which the institutions resubmitted their revised plans. The information on beams arrangement, skin flash, inhomogeneity corrections, and protocol compliance was assessed in the first and final submission. RESULTS: The plans from 26 institutions were analyzed. Some major deviations were found in the first submission. The protocol compliance rates of dose coverage for the planning target volume of chest wall, supraclavicular fossa plus axilla, and IMN region (PTVim) were all significantly improved in the final submission, which were 96.2% vs. 69.2%, 100% vs. 76.9%, and 88.4% vs. 53.8%, respectively. For OARs, the compliance rates of heart Dmean, left anterior descending coronary artery V40Gy, ipsilateral lung V5Gy, and stomach V5Gy were significantly improved. In the first and final submission, the mean values of PTVim V100% were 79.9% vs. 92.7%; the mean values of heart Dmean were 11.5 Gy vs. 9.7 Gy for hypofractionated radiation therapy and 11.5 Gy vs. 11.0 Gy for conventional fractionated radiation therapy, respectively. CONCLUSION: The major deviations were corrected and protocol compliance was significantly improved after revision, which highlighted the importance of planning benchmark case to guarantee the planning quality for multicenter trials.

Variability of Target Volumes and Organs at Risk Delineation in Breast Cancer Radiation Therapy: Quality Assurance Results of the Pretrial Benchmark Case for the POTENTIAL Trial.

PURPOSE: To estimate the variations in clinical target volumes (CTVs) and organs at risk delineation within the quality assurance (QA) program of the POTENTIAL trial, which is a multicenter, randomized phase 3 trial evaluating postmastectomy radiation therapy (RT), with or without internal mammary nodal irradiation, for patients with high-risk breast cancer. METHODS AND MATERIALS: The simulating computed tomography scan data set of a benchmark case was sent to the participating centers, and the delineation of CTVs and organs at risk was required to be completed by the investigators following protocol guidelines. All submitted contours were reviewed and compared with the reference contours created by the QA team, using quantitative geometric analysis regarding volume and the Jaccard Index (JCI), Dice similarity coefficient, Geographic Miss Index, Discordance Index, and mean distance to agreement. In addition to the whole-volume analysis of all structures, the combination contour of the supraclavicular fossa and level III and II axilla (CTVsc + axIII + axII) was further analyzed on a slice-by-slice basis. RESULTS: The contours from 26 centers were reviewed and variations were observed between submission and reference. The variations of the CTV of the chest wall, contralateral breast, and heart were small, for which the mean JCI values were 0.62, 0.68, and 0.87, respectively. However, the mean JCI values of the CTV of the internal mammary nodal region, ipsilateral brachial plexus, left anterior descending coronary artery, and right coronary artery were 0.38, 0.21, 0.29, and 0.18, respectively, suggesting marked variations. In addition, marked under- and overoutlining variations were identified on 4 slices of CTVsc + axIII + axII on slice-by-slice analysis. CONCLUSIONS: There were residual contouring variations despite a detailed protocol being provided, confirming the importance of pretrial QA in RT and highlighting the need for education and consideration of a real-time central review of the target delineation before the trial participants begin RT.

Anti-PD-L1 Antibody Enhances T Cell Immune Responses and Reduces Resistance of Breast Cancer Cells to Radiotherapy.

Immune escape is a frequent occurrence, which limits the duration of antitumor immune responses to radiotherapy. Here, we aimed to ascertain the roles and underlying mechanisms of programmed death ligand 1 (PD-L1) in tolerance of breast cancer (BC) to radiotherapy. We first quantified microRNA-21 (miR-21) and PD-L1 expression in BC tissues and cells, followed by identification of the interactions between miR-21, PD-L1, and programmed cell death protein 4 (PDCD4). miR-21 knock-in mice were used to construct tumor-bearing models, which were then treated with anti-PD-L1 antibody and irradiation, followed by measurement of tumor growth and tumor immune escape. Finally, we evaluated the synergistic effects of radiotherapy and anti-PD-L1 antibody in vivo. The results showed increased miR-21 expression in BC tissues and cells, which was positively correlated with PD-L1 expression. The treatment with radiotherapy or anti-PD-L1 antibody in the miR-21 knock-in mice diminished tumor weight and volume, along with decreased CD3+CD8+ positive cells, serum IL-2 and IFN-γ levels, and lower PD-L1 expression, but augmented apoptosis of T and BC cells. Moreover, miR-21 significantly augmented PD-L1 expression via PI3K/Akt pathway activation by targeting PDCD4 in BC cells. Thus, radiotherapy and anti-PD-L1 antibody synergistically accelerated the therapeutic effect against BC in mice, thereby implicating a close interplay between radiotherapy, T cells, and the miR-21/PDCD4/PI3K/Akt/PD-L1 axis.

Effects of microRNA-217 on proliferation, apoptosis, and autophagy of hepatocytes in rat models of CCL4-induced liver injury by targeting NAT2.

Liver injury is an important cause of serious liver disease. This study aims to explore the effects of miR-217 targeting NAT2 on hepatocyte proliferation, apoptosis, and autophagy following carbon tetrachloride (CCL4)-induced liver injury. Rat models of CCL4-induced liver injury were established. Healthy Wistar rats were randomized into the normal, blank, negative control (NC), microRNA-217 (miR-217) mimic, miR-217 inhibitor, small interfering RNA (siRNA)-N-acetyltransferase 2 (NAT2), and miR-217 inhibitor + siRNA-NAT2 groups. NAT2 activity was evaluated with reversed-phase high-performance liquid chromatographic method. Immunohistochemistry was used to detect NAT2 protein positive rate. Reverse transcription quantitative polymerase chain reaction and western blot analysis were used to examine expressions of miR-217, NAT2, Bcl-2, Bax, p35, LC3-II, Becline-1, and the ratio of caspase-3/cleaved caspase-3. Autophagy, proliferation, and cell cycle distribution were determined by electron microscope, CCK-8, and flow cytometry. NAT2 protein positive rate and miR-217, NAT2, Bcl-2, and p35 expressions were higher and Bax, LC3-II, and Becline-1 expressions and the ratio of caspase-3/cleaved caspase-3 lower in the normal group than the other six groups. Compared with the blank and NC groups, in the miR-217 mimic and siRNA-NAT2 groups, Bax, LC3-II, and Becline-1 expressions and the ratio of caspase-3/cleaved caspase-3, and hepatocyte apoptosis and autophagy increased, while NAT2, Bcl-2, and p35 expressions and hepatocyte proliferation decreased; opposite results were observed in the miR-217 inhibitor group. Collectively, miR-217 targeting NAT2 inhibits proliferation and promotes apoptosis and autophagy of hepatocytes in CCL4-induced liver injury.

Stomach wall structure and vessels imaging by acoustic resolution photoacoustic microscopy.

AIM: To image stomach wall blood vessels and tissue, layer-by-layer. METHODS: We built up the acoustic resolution photoacoustic microscopy (AR-PAM) system for imaging layered tissues, such as the stomach wall. A tunable dye laser system was coupled to a fiber bundle. The fibers of the bundle were placed in nine directions with an incident angle of 45° around a high-frequency ultrasound transducer attached to the acoustic lens. This structure formed a dark field on the tissue surface under the acoustic lens and the nine light beams from the fibers to be combined near the focal point of the acoustic lens. The sample piece was cut from a part of the porcine stomach into a petri dish. In order to realize photoacoustic depth imaging of tumor, we designed a tumor model based on indocyanine green (ICG) dye. The ICG solution (concentration of 129 µM/mL) was mixed into molten gel, and then a gel mixture of ICG (concentration of 12.9 µM/mL) was injected into the stomach submucosa. The injection quantity was controlled by 0.1 mL to make a small tumor model. RESULTS: An acoustic resolution photoacoustic microscopy based on fiber illumination was established and an axial resolution of 25 µm and a lateral resolution of 50 µm in its focal zone range of 500 µm has been accomplished. We tuned the laser wavelength to 600 nm. The photoacoustic probe was driven to do B-scan imaging in tissue thickness of 200 µm. The photoacoustic micro-image of mucosa and submucosa of the tissue have been obtained and compared with a pathological photograph of the tissue stained by hematoxylin-eosin staining. We have observed more detailed internal structure of the tissue. We also utilized this photoacoustic microscopy to image blood vessels inside the submucosa. High contrast imaging of the submucosa tumor model was obtained using ICG dye. CONCLUSION: This AR-PAM is able to image layer-by-layer construction and some blood vessels under mucosa in the stomach wall without any contrast agents.

NDRG2 suppresses proliferation, migration, invasion and epithelial-mesenchymal transition of esophageal cancer cells through regulating the AKT/XIAP signaling pathway.

N-Myc downstream-regulated gene 2 (NDRG2) has recently revealed as a candidate tumor suppressor gene. To inhibit tumor growth and decrease morbidity of esophageal cancer (EC), this study aims to test the hypothesis that the upregulation of NDRG2 may suppress proliferation, invasion, migration and epithelial-mesenchymal transition (EMT) of EC cells by regulating the AKT/XIAP signaling pathway. Immunohistochemistry was conducted for the identification of NDRG2, protein kinase B (p-AKT), X-linked inhibitor of apoptosis protein (XIAP) in EC tissues. To identify the regulatory mechanism of NDRG2 on the AKT/XIAP signaling pathway and EMT in EC, over-expressed lentiviral vector and shRNA were applied for up-regulating and interfering NDRG2 expression, and a series of determinations on the biological behavior of EC cells were performed to validate this regulation action. The results of immunohistochemistry showed NDRG2 was lowly expressed in EC tissues while p-AKT and XIAP are highly expressed. Over-expression of NDRG2 suppresses the proteins related to AKT/XIAP signaling pathway and EMT. Besides, a series of determinations shows the proliferation, migration and invasion of TE-13 cells were suppressed by over-expressed NDRG2, while the cell cycle progression was blocked and cell apoptosis was promoted. And in vivo experiment also demonstrated NDRG2 could inhibit tumor growth. Our findings demonstrate over-expression of NDRG2 works as tumor suppressive role in EC through its effects on inhibition of cell migration, invasion, and EMT by inhibiting the AKT/XIAP signaling pathway.

MicroRNA-183 Acts as a Tumor Suppressor in Human Non-Small Cell Lung Cancer by Down-Regulating MTA1.

BACKGROUNDS/AIMS: MicroRNAs (miRs) often contribute to the progression of non-small cell lung cancer (NSCLC) via regulation of mRNAs that are involved in lung homeostasis. We conducted a study aimed at exploring the roles of miR-183 in the proliferation, epithelial-mesenchymal transition (EMT), invasion and migration of human NSCLC cells via targeting MTA1. METHODS: NSCLC and adjacent normal tissues were collected from 194 patients with NSCLC. Positive expression of MTA1 protein was detected by immunohistochemistry. The highest levels of expression of miR-183 were detected using RT-qPCR in SPC-A-1 cells, which were selected and assigned to the following groups: blank, negative control (NC), miR-183 mimic, miR-183 inhibitor, siRNA-MTA1, and miR-183 inhibitor + siRNA-MTA1. The expression of miR-183 and the mRNA and protein expression of MTA1, E-cadherin, Vimentin, Snail, PCNA, Bax and Bcl-2 in tissues and transfected cells were measured using RT-qPCR and western blot analysis. Cell proliferation, apoptosis, migration and invasion were evaluated by CCK-8, flow cytometry, scratch tests and Transwell assays. Tumor xenografts were conducted in nude mice to determine tumor growth. RESULTS: SPC-A-1 cells with the highest levels of miR-183 expression were selected. Compared with adjacent normal tissues, the expression of miR-183 and the mRNA and protein expression of E-cadherin and Bax were decreased in NSCLC tissues, while mRNA and protein expression of MTA1, Vimentin, snail, PCNA and Bcl-2 were increased. MiR-183 was over-expressed in the miR-183 mimic group and under-expressed in the miR-183 inhibitor and miR-183 inhibitor + siRNA-MTA1 groups. In the miR-183 mimic and siRNA-MTA1 groups, the mRNA and protein expression of E-cadherin and Bax, as well as cell apoptosis, were enhanced, while the expression levels of MTA1, Vimentin, snail, PCNA and Bcl-2 mRNA and protein, cell proliferation, migration, invasion and tumor growth were reduced relative to the blank and NC groups. The miR-183 inhibitor group exhibited an opposite trend. CONCLUSION: Our study indicates that miR-183 down-regulates MTA1 to inhibit the proliferation, EMT, migration and invasion of human NSCLC cells.

Esophageal/gastric cancer screening in high-risk populations in Henan Province, China.

OBJECTIVE: To summarize the endoscopic screening findings in high-risk population of esophageal and gastric carcinoma and analyze influential factors related to screening. METHODS: In seven selected cities and counties with high incidences of esophageal carcinoma, people at age of 40-69 were set as the target population. Those with gastroscopy contradictions were excluded, and all who were voluntary and willing to comply with the medical requirements were subjected to endoscopic screening and histological examination for esophageal, gastric cardia and gastric carcinoma in accordance with national technical manual for early detection and treatment of cancer. RESULTS: In three years, 36,154 people were screened, and 16,847 (46.60%) cases were found to have precancerous lesions. A total of 875 cases were found to have cancers (2.42%), and among them 739 cases had early stage with an early diagnosis rate is 84.5%. Some 715 patients underwent prompt treatment and the success rate was 81.8%. CONCLUSIONS: In a high-risk population of esophageal and gastric carcinoma, it is feasible to implement early detection and treatment by endoscopic screening. Screening can identify potential invasive carcinoma, early stage carcinoma and precancerous lesions, improving efficacy through early detection and treatment. The exploratory analysis of related influential factors will help broad implementation of early detection and treatment for esophageal and gastric carcinoma.

Glucocorticoid receptors in the basolateral nucleus of amygdala are required for postreactivation reconsolidation of auditory fear memory.

It is well known that initial consolidation requires de novo gene transcription and protein synthesis in order for memory to become stable. The consolidated memory again becomes labile and temporarily sensitive to disruption when retrieved, requiring a reconsolidation process to become permanent. Although it is well established that glucocorticoid receptors (GR) in the basolateral nucleus of amygdala (BLA) are required for consolidation of fear memory, little is known about their role in reconsolidation of fear memory. In the present study, we first examined the effect of a GR antagonist on postconditioning consolidation of auditory fear memory (AFM). Intra-BLA infusion of the GR antagonist RU486 0 h postconditioning impaired long-term AFM, leaving short-term AFM intact. RU486 had no effect if infusion was performed 6 h postconditioning. We then investigated the effect of the RU486 treatment on postretrieval reconsolidation of AFM. Severe amnesia took place when RU486 was infused into the BLA 0 h postretrieval (reactivation) of AFM, regardless of whether the retrieval was performed 1 day or 10 days postconditioning. RU486 produced no amnesia if the memory retrieval was omitted or if the drug was administered 6 h postretrieval. Treatment with RU486 0 h postretrieval produced no deficit in postretrieval short-term memory but impaired postretrieval long-term memory, and the amnesia exhibited no spontaneous recovery 6 days after retrieval. The present results provide strong evidence that glucocorticoid receptors in the BLA are required for reconsolidation as well as consolidation of AFM.