[Changes of immunohistochemical biomarkers before and after neoadjuvant chemotherapy in breast cancer and their prognosis].

Objective: To investigate changes in the expression of immunohistochemical (IHC) markers and factors associated with the effect of chemotherapy before and after neoadjuvant chemotherapy (NAC). Methods: A retrospective study included 200 breast cancer patients treated with NAC between January 2016 and December 2018. We analyzed the changes in the expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and Ki-67 in pre- and post-treated samples and the predictive factors of NAC. Results: Among the 200 cases, 16 cases were luminal A, 108 cases were luminal B, 36 cases were HER2+subtype, and 40 cases were basal-like. Twenty-five patients (12.50%) achieved pathological complete remission (PCR).There were significant differences in PR and Ki-67 before and after NAC but there were no differences in ER and HER2.In univariate analysis, factors associated with PCR were tumor less than 5 cm(P=0.009), non-luminal breast cancer (P=0.001), ER negative(P=0.001), PR negative (P=0.029) and HER2 positive(P=0.001). Tumor less than 5 cm [P=0.020, OR=2.581, 95%CI (1.207, 5.753)], ER negative [P=0.011, OR=2.264, 95%CI (1.207, 4.248)] and HER2 positive[P=0.007, OR=2.412, 95%CI (1.275, 4.561)] remained predictive variables in multivariate analysis after correction for the other variables. Conclusions: The expression of Ki-67 decreases after NAC. Negative PR and ER and positive HER2 status are related to the efficacy of pCR for breast cancer, and have guiding significance for the prognosis evaluation of NAC.

[Clinical study of cell sheets containing allogeneic keratinocytes and fibroblasts for the treatment of partial-thickness burn wounds].

Objective: To evaluate the efficacy and safety of cell sheets containing allogeneic keratinocytes and fibroblasts in the treatment of partial-thickness burn wounds. Methods: The cell sheets containing allogeneic keratinocytes and fibroblasts were constructed using polyurethane biofilm as carrier. Then gross observation and histological observation were conducted. From April 2016 to December 2017, Changhai Hospital of Naval Medical University recruited patients with acute partial-thickness burn wounds that met the inclusion criteria for this prospective and positively self-controlled clinical trial. Recruitment of 40 acute partial-thickness burn wounds were planned with each selected single wound being not smaller than 10 cm×10 cm and not more than 5% total body surface area (TBSA). Each wound was equally divided into two areas, which were recruited into cell sheet group and conventional treatment group according to the random number table. The wounds in cell sheet group were covered by cell sheet and then sterile gauze as secondary dressings. Depending on the wound healing and exudation, the sterile gauze was replaced every 1 to 3 day (s) after the treatment was started, and the cell sheet was replaced every 7 days (namely dressing changing). The wounds in conventional treatment group were covered by sulfadiazine silver cream gauze and then dressed with sterile gauze, with the dressings changed every 2 to 3 days depending on wound exudation. On treatment day 5, 7, 10, and 14, the wound healing rates in the two groups were calculated. The complete wound healing time, the total number of dressing changes, and the status of wound infection during treatment were recorded. The Visual Analogue Scale was used to score the pain at the first dressing change. Scar formation of patients was followed up for 6 to 12 months after injury. Safety indicators including vital signs, laboratory examination indexes, and adverse reactions during treatment were observed. Data were statistically analysed with Wilcoxon rank sum test and Bonferroni correction. Results: (1) Each prepared cell sheet had a diameter of about 8 cm and was about 49 cm(2) in size, containing 2 or 3 layers of keratinocytes and fibroblasts. (2) A total of 43 patients were enrolled, of whom 3 patients dropped out of the study. Of the 40 patients who completed the treatment, there were 22 males and 18 females who were aged 1 to 57 year (s), with total burn area of 2% to 26% TBSA. (3) On treatment day 5, 7, 10, and 14, the wound healing rates in cell sheet group were significantly higher than those in conventional treatment group (Z=4.205, 4.258, 3.495, 2.521, P<0.05 or P<0.01). The complete wound healing time in cell sheet group was 7 (6, 8) days, which was significantly shorter than 11 (7, 14) days in conventional treatment group (Z=4.219, P<0.01). The total number of wound dressing changes in cell sheet group was 1 (1, 2) times, which was significantly less than 6 (4, 7) times in conventional treatment group (Z=5.464, P<0.01). (4) The wounds in cell sheet group in 31 patients healed before the first dressing change. The pain score of wounds in the first dressing change in cell sheet group of 9 patients was 1 (0, 1) point, while the pain score of wounds in the first dressing change in conventional treatment group of 40 patients was 2 (1, 3) points. There was no obvious infection in the wounds in both groups of 40 patients before the wound healing. Nine patients completed the follow-up after the trial. In 6 patients, no scar formation was observed in cell sheet group or conventional treatment group. The color of wounds in cell sheet group was consistent with normal skin, and there was only a small amount of pigment deposition in the wounds of conventional treatment group. Three patients developed pigment deposition only in the wounds of cell sheet group but obvious scars in conventional treatment group. (5) The abnormal fluctuations of vital signs including body temperature, blood pressure, heart rate, respiratory rate, and laboratory examination indexes of all patients during treatment were alleviated through the process of burn wound healing. No obvious adverse reactions or abnormalities related to the treatment were observed. Conclusions: The cell sheet containing allogeneic keratinocytes and fibroblasts can reduce the number of dressing changes, accelerate wound epithelialization, shorten wound healing time, reduce pain during dressing change in the treatment of partial-thickness burn wounds, and it may reduce scar hyperplasia after wound healing because of accelerating wound epithelization. Its clinical application is simple, safe, and effective.

[Preliminary effect observation on the application of micro-negative pressure in children with small-area deep partial-thickness burn].

Objective: To preliminarily observe the effects of application of micro-negative pressure in children with small-area deep partial-thickness burn. Methods: From January 2016 to August 2018, 64 children with small-area deep partial-thickness burn who were admitted to the Department of Burn Surgery of the First Affiliated Hospital of Naval Medical University were recruited in this prospective randomized controlled study. According to the random number table, they were divided into negative pressure group [18 boys and 14 girls, aged (3.9±1.6) years with total burn area of (5.5±2.2)% total body surface area (TBSA)] and conventional group [20 boys and 12 girls, aged (3.8±1.7) years with total burn area of (5.8±1.6)% TBSA], with 32 patients in each group. After admission, simple debridement was performed in the patients of 2 groups. After that, the children in negative pressure group were treated with micro-negative pressure with negative pressure material replaced every 3 to 5 days. Children in conventional group were treated with silver sulfadiazine cream with dressing change every other day. On post injury day (PID) 14 and 21, general wound observation was performed, the wound healing rate was calculated, the exudates from the wounds were cultured and the positive detection rate was calculated. The number of patients requiring surgical skin grafting was recorded and the rate of surgical skin grafting was calculated, and the complete wound healing time was recorded in the patients of 2 groups. Scar formation was evaluated by the Vancouver Scar Scale (VSS) in 3, 6, and 12 months after wound healing. Data were processed with chi-square test, t test, Bonferroni correction, and analysis of variance for repeated measurement. Results: (1) On PID 14, all the necrotic tissue in the wounds of patients in negative pressure group was removed, with few exudates, and most of the wounds had been epithelialized; most of necrotic tissue in the wounds of patients in conventional group was removed, with more exudates and smaller wound healing area than those in negative pressure group. On PID 21, most of the wounds of patients in negative pressure group were healed, and the exudates were rare, while the wound healing area of patients in conventional group was significantly smaller than that in negative pressure group with more exudates. (2) On PID 14 and 21, the wound healing rates [(49.8±3.3)% and (95.8±2.4)%] of patients in negative pressure group were significantly higher than those in conventional group [(40.0±3.2)% and (75.3±2.5)%, t=11.899, 33.461, P<0.01]. (3) On PID 14 and 21, the positive detection rates of wound bacteria of patients in negative pressure group were significantly lower than those in conventional group (χ(2)=6.275, 5.741, P<0.05). (4) The rate of surgical skin grafting of patients in negative pressure group was significantly lower than that in conventional group (χ(2)=5.333, P<0.05). (5) The complete wound healing time of patients in negative pressure group [(23.9±2.3) d] was significantly shorter than that in conventional group [(27.9±1.8) d, t=-7.806, P<0.01]. (6) In 3, 6, and 12 months after wound healing, the VSS scores [(6.9±1.8), (5.6±1.4), (3.4±1.5) points] of patients in negative pressure group were significantly lower than those in conventional group [(9.0±1.5), (7.4±2.0), (5.7±1.6) points, t=-4.987, -4.127, -5.988, P<0.01]. Conclusions: In comparison with routine dressing change, the treatment of application of micro-negative pressure in children with small-area deep partial-thickness burn can significantly improve the wound healing rate and rate of surgical skin grafting, decrease the wound infection rate, shorten the wound healing time, and improve the wound healing quality.

Differentiation between malignant and benign breast masses: combination of semi-quantitative analysis on DCE-MRI and histogram analysis of ADC maps.

AIM: To investigate the performance of combined semi-quantitative analysis on dynamic contrast enhanced (DCE) magnetic resonance imaging (MRI) and histogram analysis of diffusion-weighted imaging (DWI) for distinguishing malignant from benign breast masses. MATERIALS AND METHODS: This study included 178 patients with breast masses (benign:malignant=88:9) who underwent both DCE-MRI and DWI. The semi-quantitative parameters, derived from DCE-MRI, included maximum slope of increase (MSI), signal intensity slope (SIslope), initial percentage of enhancement (Einitial), percentage of peak enhancement (Epeak), early signal enhancement ratio (ESER), and second enhancement percentage (SEP). Histogram parameters derived from apparent diffusion coefficient (ADC) maps included ADCmin, ADCmax, ADCmean, ADC10, ADC25, ADC50, ADC75, ADC90, skewness, and kurtosis. All parameters were compared between malignant and benign groups, and their differences were tested using independent-samples t-test or Mann-Whitney test. Receiver operating characteristic (ROC) curves were used to determine the diagnostic value of each significant parameter. RESULTS: Among semi-quantitative parameters, SIslope exhibited the best diagnostic performance in predicting malignancy (cut-off value, 0.096; ROC, 0.756; sensitivity, 86.7%; specificity, 61.4%). Among histogram parameters, ADC10 exhibited the best diagnostic performance in predicting malignancy (cut-off value, 1.051; ROC, 0.885; sensitivity, 86.7%; specificity, 84.1%). The optimal diagnostic performance of combined ADC10 and SIslope (area under curve [AUC], 0.888; sensitivity, 82.2%; specificity, 95.5%) was significantly better than SIslope alone (p<0.001). Moreover, the combination showed higher AUC (0.888 versus 0.885) than ADC10 alone, but the difference was not statistically significant (p=0.914). CONCLUSION: SIslope and ADC10 are significant predictors for breast malignancy. The combination of DCE-MRI and DWI improves differentiating performance.

HOI-02 induces apoptosis and G2-M arrest in esophageal cancer mediated by ROS.

Reactive oxygen species (ROS) are chemically reactive molecules that perform essential functions in living organisms. Accumulating evidence suggests that many types of cancer cells exhibit elevated levels of ROS. Conversely, generation of ROS has become an effective method to kill cancer cells. (E)-3-hydroxy-3-(4-(4-nitrophenyl)-2-oxobut-3-en-1-yl) indolin-2-one, which is an NO2 group-containing compound designated herein as HOI-02, generated ROS and, in a dose-dependent manner, decreased esophageal cancer cell viability and inhibited anchorage-independent growth, followed by apoptosis and G2-M arrest. Moreover, results of an in vivo study using a patient-derived xenograft mouse model showed that HOI-02 treatment suppressed the growth of esophageal tumors, without affecting the body weight of mice. The expression of Ki-67 was significantly decreased with HOI-02 treatment. In addition, the phosphorylation of c-Jun, and expression of p21, cleaved caspase 3, and DCFH-DA were increased in the HOI-02-treated group compared with the untreated control group. In contrast, treatment of cells with (E)-3-(4-(4-aminophenyl)-2-oxobut-3-en-1-yl)-3-hydroxyindolin-2-one, which is an NH2 group-containing compound designated herein as HOI-11, had no effect. Overall, we identified HOI-02 as an effective NO2 group-containing compound that was an effective therapeutic or preventive agent against esophageal cancer cell growth.

Whole genome and transcriptome sequencing of matched primary and peritoneal metastatic gastric carcinoma.

Gastric cancer is one of the most aggressive cancers and is the second leading cause of cancer death worldwide. Approximately 40% of global gastric cancer cases occur in China, with peritoneal metastasis being the prevalent form of recurrence and metastasis in advanced disease. Currently, there are limited clinical approaches for predicting and treatment of peritoneal metastasis, resulting in a 6-month average survival time. By comprehensive genome analysis will uncover the pathogenesis of peritoneal metastasis. Here we describe a comprehensive whole-genome and transcriptome sequencing analysis of one advanced gastric cancer case, including non-cancerous mucosa, primary cancer and matched peritoneal metastatic cancer. The peripheral blood is used as normal control. We identified 27 mutated genes, of which 19 genes are reported in COSMIC database (ZNF208, CRNN, ATXN3, DCTN1, RP1L1, PRB4, PRB1, MUC4, HS6ST3, MUC17, JAM2, ITGAD, IREB2, IQUB, CORO1B, CCDC121, AKAP2, ACAN and ACADL), and eight genes have not previously been described in gastric cancer (CCDC178, ARMC4, TUBB6, PLIN4, PKLR, PDZD2, DMBT1and DAB1).Additionally,GPX4 and MPND in 19q13.3-13.4 region, is characterized as a novel fusion-gene. This study disclosed novel biological markers and tumorigenic pathways that would predict gastric cancer occurring peritoneal metastasis.

Suppression of tumor angiogenesis by targeting the protein neddylation pathway.

Inhibition of protein neddylation, particularly cullin neddylation, has emerged as a promising anticancer strategy, as evidenced by the antitumor activity in preclinical studies of the Nedd8-activating enzyme (NAE) inhibitor MLN4924. This small molecule can block the protein neddylation pathway and is now in clinical trials. We and others have previously shown that the antitumor activity of MLN4924 is mediated by its ability to induce apoptosis, autophagy and senescence in a cell context-dependent manner. However, whether MLN4924 has any effect on tumor angiogenesis remains unexplored. Here we report that MLN4924 inhibits angiogenesis in various in vitro and in vivo models, leading to the suppression of tumor growth and metastasis in highly malignant pancreatic cancer, indicating that blockage of angiogenesis is yet another mechanism contributing to its antitumor activity. At the molecular level, MLN4924 inhibits Cullin-RING E3 ligases (CRLs) by cullin deneddylation, causing accumulation of RhoA at an early stage to impair angiogenic activity of vascular endothelial cells and subsequently DNA damage response, cell cycle arrest and apoptosis due to accumulation of other tumor-suppressive substrates of CRLs. Furthermore, we showed that inactivation of CRLs, via small interfering RNA (siRNA) silencing of its essential subunit ROC1/RBX1, recapitulates the antiangiogenic effect of MLN4924. Taken together, our study demonstrates a previously unrecognized role of neddylation in the regulation of tumor angiogenesis using both pharmaceutical and genetic approaches, and provides proof of concept evidence for future development of neddylation inhibitors (such as MLN4924) as a novel class of antiangiogenic agents.

Characterization of hTERT-immortalized caprine mammary epithelial cells.

The aim of this article is to demonstrate and characterize caprine mammary epithelial cells (CMC) immortalized with human telomerase reverse transcriptase (hTERT) gene. Five immortalized CMCs were assigned to either myoepithelial or luminal epithelial groups based on their morphology and expression of cell lineage-specific intermediate filaments. Telomeric repeat amplification protocol revealed various telomerase activities in CMCs associated with their distinct proliferation potential. Karyotypic analysis showed three CMCs retained their modal Capra hircus chromosome number (2n = 60), whereas the remaining two CMCs were abnormal at 2n = 19 and 2n = 36. CMCs with abnormal karyotypes lost p53 protein after chemical-induced DNA damage and showed anchorage-independent growth in soft agar assay. In terms of functional differentiation, luminal CMCs organized into alveolus-like structures when grown in Matrigel. Furthermore, αs1- and ß-casein gene was induced in luminal CMCs in response to lacto-hormones stimulation. Together these results showed that hTERT-immortalized CMCs retained major characteristics of mammary epithelial cells, and stability of the genome is required for maintaining normal mammary epithelium function. Application of CMCs can provide valuable models to study alveologenesis and lactogenesis of mammary epithelium and test the feasibility of recombinant constructs designed for the generation of transgenic livestock.

[Studies on preparation of herba epimedii total flavonoids phytosomes and their pharmaceutics].

OBJECTIVE: To optimize preparation techniques for Herba Epimedii flavonoid phytosomes (EFP) and explore their suitable pharmaceutics. METHODS: To optimize the preparation conditions by means of uniform design and step regression, prepare Herba Epimedii total flavonoid phytosomes by means of solvent evaporation and investigate the accumulative dissolution of different ratios of EFP-PVP precipitates by means of dissolution release. RESULT: The optimized preparation conditions are as follows: solvent-tetrahydrofuran, lecithin to PVP--2.5 times, temperature--40 degrees C and reaction--3 hours. Oil/water apparent partition coefficient of icariin was enhanced more than 4 times by phospholipid. The accumulative dissolution of Herba Epimedii flavonoids of EFP-PVP precipitate was significantly higher than that of its physical mixture and Herba Epimedii extract tablet. CONCLUSION: Phospholipid can effectively enhance the oil/water apparent partition coefficient of icariin, and PVP can improve the dissolution of Herba Epimedii phytocomes, but the pharmacokinetics needs further study.