Ubiquitin-induced RNF168 condensation promotes DNA double-strand break repair.

Rapid accumulation of repair factors at DNA double-strand breaks (DSBs) is essential for DSB repair. Several factors involved in DSB repair have been found undergoing liquid-liquid phase separation (LLPS) at DSB sites to facilitate DNA repair. RNF168, a RING-type E3 ubiquitin ligase, catalyzes H2A.X ubiquitination for recruiting DNA repair factors. Yet, whether RNF168 undergoes LLPS at DSB sites remains unclear. Here, we identified K63-linked polyubiquitin-triggered RNF168 condensation which further promoted RNF168-mediated DSB repair. RNF168 formed liquid-like condensates upon irradiation in the nucleus while purified RNF168 protein also condensed in vitro. An intrinsically disordered region containing amino acids 460-550 was identified as the essential domain for RNF168 condensation. Interestingly, LLPS of RNF168 was significantly enhanced by K63-linked polyubiquitin chains, and LLPS largely enhanced the RNF168-mediated H2A.X ubiquitination, suggesting a positive feedback loop to facilitate RNF168 rapid accumulation and its catalytic activity. Functionally, LLPS deficiency of RNF168 resulted in delayed recruitment of 53BP1 and BRCA1 and subsequent impairment in DSB repair. Taken together, our finding demonstrates the pivotal effect of LLPS in RNF168-mediated DSB repair.

RAP80 phase separation at DNA double-strand break promotes BRCA1 recruitment.

RAP80 has been characterized as a component of the BRCA1-A complex and is responsible for the recruitment of BRCA1 to DNA double-strand breaks (DSBs). However, we and others found that the recruitment of RAP80 and BRCA1 were not absolutely temporally synchronized, indicating that other mechanisms, apart from physical interaction, might be implicated. Recently, liquid-liquid phase separation (LLPS) has been characterized as a novel mechanism for the organization of key signaling molecules to drive their particular cellular functions. Here, we characterized that RAP80 LLPS at DSB was required for RAP80-mediated BRCA1 recruitment. Both cellular and in vitro experiments showed that RAP80 phase separated at DSB, which was ascribed to a highly disordered region (IDR) at its N-terminal. Meanwhile, the Lys63-linked poly-ubiquitin chains that quickly formed after DSBs occur, strongly enhanced RAP80 phase separation and were responsible for the induction of RAP80 condensation at the DSB site. Most importantly, abolishing the condensation of RAP80 significantly suppressed the formation of BRCA1 foci, encovering a pivotal role of RAP80 condensates in BRCA1 recruitment and radiosensitivity. Together, our study disclosed a new mechanism underlying RAP80-mediated BRCA1 recruitment, which provided new insight into the role of phase separation in DSB repair.

Prognosis and immunotherapy efficacy in dMMR&MSS colorectal cancer patients and an MSI status predicting model.

The inconsistency between mismatch repair (MMR) protein immunohistochemistry (IHC) and microsatellite instability PCR (MSI-PCR) methods has been widely reported. We aim to investigate the prognosis and the effect of immunotherapy in dMMR by IHC but MSS by MSI-PCR (dMMR&MSS) colorectal cancer (CRC) patients. A microsatellite instability (MSI) predicting model was established to help find dMMR&MSS patients. MMR and MSI states were detected by the IHC and MSI-PCR in 1622 CRC patients (ZS6Y-1 cohort). Logistic regression analysis was used to screen clinical features to construct an MSI-predicting nomogram. We propose a new nomogram-based assay to find patients with dMMR&MSS, in which the MSI-PCR assay only detects dMMR patients with MSS predictive results. We applied the new strategy to a random cohort of 248 CRC patients (ZS6Y-2 cohort). The consistency of MMR IHC and MSI-PCR in the ZS6Y-1 cohort was 95.7% (1553/1622). Both pMMR&MSS and dMMR&MSS groups experienced significantly shorter overall survival (OS) than those in dMMR by IHC and MSI-H by MSI-PCR (dMMR&MSI-H) group (hazard ratio [HR] = 2.429, 95% confidence interval [CI]: 1.89-3.116, p < .01; HR = 21.96, 95% CI: 7.24-66.61, p < .01). The dMMR&MSS group experienced shorter OS than the pMMR&MSS group, but the difference did not reach significance (log rank test, p = .0686). In the immunotherapy group, the progression-free survival of dMMR&MSS patients was significantly shorter than that of dMMR&MSI-H patients (HR = 13.83, 95% CI: 1.508-126.8, p < .05). The ZS6Y-MSI-Pre nomogram (C-index = 0.816, 95% CI: 0.792-0.841, already online) found 66% (2/3) dMMR&MSS patients in the ZS6Y-2 cohort. There are significant differences in OS and immunotherapy effect between dMMR&MSI-H and dMMR&MSS patients. Our prediction model provides an economical way to screen dMMR&MSS patients.

Induction of filopodia formation by α-Actinin-2 via RelA with a feedforward activation loop promoting overt bone marrow metastasis of gastric cancer.

BACKGROUND: Bone marrow metastasis (BMM) is underestimated in gastric cancer (GC). GC with BMM frequently complicate critical hematological abnormalities like diffused intravascular coagulation and microangiopathic hemolytic anemia, which constitute a highly aggressive GC (HAGC) subtype. HAGC present a very poor prognosis with peculiar clinical and pathological features when compared with not otherwise specified advanced GC (NAGC). But the molecular mechanisms underlying BMM from GC remain rudimentary. METHODS: The transcriptomic difference between HAGC and NAGC were analyzed. Genes that were specifically upregulated in HAGC were identified, and their effect on cell migration and invasion was studied. The function of ACTN2 gene were confirmed by GC cell lines, bone-metastatic animal model and patients' tissues. Furthermore, the molecular mechanism of ACTN2 derived-BMM was explored by multiple immunofluorescence staining, western blot, chromatin immunoprecipitation, and luciferase reporter assays. RESULTS: We elucidated the key mechanisms of BMM depending on the transcriptomic difference between HAGC and NAGC. Five genes specifically upregulated in HAGC were assessed their effect on cell migration and invasion. The ACTN2 gene encoding protein α-Actinin-2 was detected enhanced the metastatic capability and induced BMM of GC cells in mouse models. Mechanically, α-Actinin-2 was involved in filopodia formation where it promoted the Actin filament cross-linking by replacing α-Actinin-1 to form α-Actinin-2:α-Actinin-4 complexes in GC cells. Moreover, NF-κB subunit RelA and α-Actinin-2 formed heterotrimers in the nuclei of GC cells. As a direct target of RelA:α-Actinin-2 heterotrimers, the ACTN2 gene was a positive auto-regulatory loop for α-Actinin-2 expression. CONCLUSIONS: We demonstrated a link between filopodia, BMM and ACTN2 activation, where a feedforward activation loop between ACTN2 and RelA is established via actin in response to distant metastasis. Given the novel filopodia formation function and the new mechanism of BMM in GC, we propose ACTN2 as a druggable molecular vulnerability that may provide potential therapeutic benefit against BMM of GC.

Effects of neoadjuvant radiochemotherapy for anorectal function in locally advanced rectal cancer patients: a study protocol for a prospective, observational, controlled, multicentre study.

BACKGROUND: Neoadjuvant chemoradiotherapy (NCRT) and total mesorectal excision are standard treatment regimen for patients with locally advanced rectal cancer (LARC). This sphincter-saving treatment strategy may be accompanied by a series of anorectal functional disorders. Yet, prospective studies that dynamically evaluating the respective roles of radiotherapy, chemotherapy and surgery on anorectal function are lacking. PATIENTS/DESIGN: The study is a prospective, observational, controlled, multicentre study. After screening for eligibility and obtaining informed consent, a total of 402 LARC patients undergoing NCRT followed by surgery, or neoadjuvant chemotherapy followed by surgery, or surgery only would be included in the trial. The primary outcome measure is the average resting pressure of anal sphincter. The secondary outcome measures are maximum anal sphincter contraction pressure, Wexner continence score and low anterior resection syndrome (LARS) score. Evaluations will be carried out at the following stages: baseline (T1), after radiotherapy or chemotherapy (before surgery, T2), after surgery (before closing the temporary stoma, T3), and at follow-up visits (every 3 to 6 months, T4, T5……). Follow-up for each patient will be at least 2 years. DISCUSSION: We expect the program to provide more information of neoadjuvant radiotherapy and/or chemotherapy on anorectal function, and to optimize the treatment strategy to reduce anorectal dysfunction for LARC patients. TRIAL REGISTRATION: ClinicalTrials.gov (NCT05671809). Registered on 26 December 2022.

An artificial intelligence-based ecological index for prognostic evaluation of colorectal cancer.

BACKGROUND AND OBJECTIVE: In the tumor microenvironment (TME), the dynamic interaction between tumor cells and immune cells plays a critical role in predicting the prognosis of colorectal cancer. This study introduces a novel approach based on artificial intelligence (AI) and immunohistochemistry (IHC)-stained whole-slide images (WSIs) of colorectal cancer (CRC) patients to quantitatively assess the spatial associations between tumor cells and immune cells. To achieve this, we employ the Morisita-Horn ecological index (Mor-index), which allows for a comprehensive analysis of the spatial distribution patterns between tumor cells and immune cells within the TME. MATERIALS AND METHODS: In this study, we employed a combination of deep learning technology and traditional computer segmentation methods to accurately segment the tumor nuclei, immune nuclei, and stroma nuclei within the tumor regions of IHC-stained WSIs. The Mor-index was used to assess the spatial association between tumor cells and immune cells in TME of CRC patients by obtaining the results of cell nuclei segmentation. A discovery cohort (N = 432) and validation cohort (N = 137) were used to evaluate the prognostic value of the Mor-index for overall survival (OS). RESULTS: The efficacy of our method was demonstrated through experiments conducted on two datasets comprising a total of 569 patients. Compared to other studies, our method is not only superior to the QuPath tool but also produces better segmentation results with an accuracy of 0.85. Mor-index was quantified automatically by our method. Survival analysis indicated that the higher Mor-index correlated with better OS in the discovery cohorts (HR for high vs. low 0.49, 95% CI 0.27-0.77, P = 0.0014) and validation cohort (0.21, 0.10-0.46, < 0.0001). CONCLUSION: This study provided a novel AI-based approach to segmenting various nuclei in the TME. The Mor-index can reflect the immune status of CRC patients and is associated with favorable survival. Thus, Mor-index can potentially make a significant role in aiding clinical prognosis and decision-making.

Characterization of the tumor microenvironment and identification of spatially predictive biomarkers associated with beneficial neoadjuvant chemoradiotherapy in locally advanced rectal cancer.

Neoadjuvant chemoradiotherapy (nCRT) has become the standard treatment for patients with locally advanced rectal cancer (LARC). However, 20-40% of patients with LARC show little to no response to nCRT. Thus, comprehensively understanding the tumor microenvironment (TME), which might influence therapeutic efficacy, and identifying robust predictive biomarkers is urgently needed. Pre-treatment tumor biopsy specimens from patients with LARC were evaluated in detail through digital spatial profiling (DSP), public RNA sequencing datasets, and multiplex immunofluorescence (mIF). DSP analysis revealed distinct characteristics of the tumor stroma compared to the normal stroma and tumor compartments. We identified high levels of human leukocyte antigen-DR/major histocompatibility complex class II (HLA-DR/MHC-II) in the tumor compartment and B cells in the stroma as potential spatial predictors of nCRT efficacy in the Discovery cohort. Public datasets validated their predictive capacity for clinical outcomes. Using mIF in an independent nCRT cohort and/or the total cohort, we validated that a high density of HLA-DR/MHC-II+ cells in the tumor and CD20 + B cells in the stroma was associated with nCRT efficacy (all p ≤ 0.021). Spatial profiling successfully characterized the LARC TME and identified robust biomarkers with the potential to accurately predict nCRT response. These findings have important implications for individualized therapy.

Artificial intelligence for quantifying immune infiltrates interacting with stroma in colorectal cancer.

BACKGROUND: We proposed an artificial intelligence-based immune index, Deep-immune score, quantifying the infiltration of immune cells interacting with the tumor stroma in hematoxylin and eosin-stained whole-slide images of colorectal cancer. METHODS: A total of 1010 colorectal cancer patients from three centers were enrolled in this retrospective study, divided into a primary (N = 544) and a validation cohort (N = 466). We proposed the Deep-immune score, which reflected both tumor stroma proportion and the infiltration of immune cells in the stroma region. We further analyzed the correlation between the score and CD3+ T cells density in the stroma region using immunohistochemistry-stained whole-slide images. Survival analysis was performed using the Cox proportional hazard model, and the endpoint of the event was the overall survival. RESULT: Patients were classified into 4-level score groups (score 1-4). A high Deep-immune score was associated with a high level of CD3+ T cells infiltration in the stroma region. In the primary cohort, survival analysis showed a significant difference in 5-year survival rates between score 4 and score 1 groups: 87.4% vs. 58.2% (Hazard ratio for score 4 vs. score 1 0.27, 95% confidence interval 0.15-0.48, P < 0.001). Similar trends were observed in the validation cohort (89.8% vs. 67.0%; 0.31, 0.15-0.62, < 0.001). Stratified analysis showed that the Deep-immune score could distinguish high-risk and low-risk patients in stage II colorectal cancer (P = 0.018). CONCLUSION: The proposed Deep-immune score quantified by artificial intelligence can reflect the immune status of patients with colorectal cancer and is associate with favorable survival. This digital pathology-based finding might advocate change in risk stratification and consequent precision medicine.

The effect of recombinant human epidermal growth factor on radiation dermatitis in rectal and anal cancer patients: a self-controlled study.

BACKGROUND: Our previous study reported that recombinant human epidermal growth factor (rhEGF)-triggered EGFR internalization promoted radioresistance. Here, we aimed to evaluate the effect of rhEGF on the skin protection of rectal and anal cancer patients receiving radiotherapy. METHODS: One hundred and ninety-three rectal and anal cancer patients who received radiotherapy were prospectively enrolled from January 2019 to December 2020. To perform self-controlled study, the left and right pelvic skin area (separated by midline) were randomly assigned to the rhEGF and control side. The association between radiation dermatitis and factors including rhEGF, the dose of radiotherapy and tumor distance from anal edge were analyzed. RESULTS: Among 193 enrolled patients, 41 patients (21.2%) did not develop radiation dermatitis, and 152 patients (78.8%) suffered radiation dermatitis on at least one side of pelvic skin at the end of radiotherapy. For the effect on radiation dermatitis grade, rhEGF had improved effect on 6 (4.0%) patients, detrimental effect on 2 (1.3%) patients, and no effect on 144 (94.7%) patients. Whereas for the effect on radiation dermatitis area, rhEGF showed improved effect on the radiation dermatitis area of 46 (30.2%) patients, detrimental effect on 15 (9.9%) patients, and no effect on 91 (59.9%) patients. The radiation dermatitis area of rhEGF side was significantly smaller than that of control side (P = 0.0007). CONCLUSIONS: rhEGF is a skin protective reagent for rectal and anal cancer patients receiving radiotherapy. TRIAL REGISTRATION: Chinese Clinical Trial Registry identifier: ChiCTR1900020842; Date of registration: 20/01/2019.

Multiparametric MRI and Whole Slide Image-Based Pretreatment Prediction of Pathological Response to Neoadjuvant Chemoradiotherapy in Rectal Cancer: A Multicenter Radiopathomic Study.

BACKGROUND: The aim of this work is to combine radiological and pathological information of tumor to develop a signature for pretreatment prediction of discrepancies of pathological response at several centers and restage patients with locally advanced rectal cancer (LARC) for individualized treatment planning. PATIENTS AND METHODS: A total of 981 consecutive patients with evaluation of response according to tumor regression grade (TRG) who received nCRT were retrospectively recruited from four hospitals (primary cohort and external validation cohort 1-3); both pretreatment multiparametric MRI (mp-MRI) and whole slide image (WSI) of biopsy specimens were available for each patient. Quantitative image features were extracted from mp-MRI and WSI and used to construct a radiopathomics signature (RPS) powered by an artificial-intelligence model. Models based on mp-MRI or WSI alone were also constructed for comparison. RESULTS: The RPS showed overall accuracy of 79.66-87.66% in validation cohorts. The areas under the curve of RPS at specific response grades were 0.98 (TRG0), 0.93 (≤ TRG1), and 0.84 (≤ TRG2). RPS at each grade of pathological response revealed significant improvement compared with both signatures constructed without combining multiscale tumor information (P < 0.01). Moreover, RPS showed relevance to distinct probabilities of overall survival and disease-free survival in patients with LARC who underwent nCRT (P < 0.05). CONCLUSIONS: The results of this study suggest that radiopathomics, combining both radiological information of the whole tumor and pathological information of local lesions from biopsy, could potentially predict discrepancies of pathological response prior to nCRT for better treatment planning.

Demographic trends and KRAS/BRAFV600E mutations in colorectal cancer patients of South China: A single-site report.

The incidence of colorectal cancer (CRC) is increasing in China. Here, we aimed to evaluate the latest demographic trends and KRAS/BRAF mutations status of Chinese CRC. Five thousand five hundred and forty-six CRC patients diagnosed from 2010 to 2017 were involved. KRAS exon 2 and BRAFV600E mutations were detected by Sanger sequencing and high-resolution melting analysis or allelic-specific probe method. Gene mutation profiles and clinicopathologic characteristics of 5495 patients were analyzed. The joinpoint regression model was used to predict the demographic data in 2018. We found KRAS exon 2 and BRAFV600E mutation rates were 37.7 and 2.8% in CRC patients. Tumors with KRAS exon 2 mutations were more likely to be present in female and patients aged older than 75 years, right colon and have well-differentiated histology. Tumors with BRAFV600E mutations were more likely to be present in the female, right colon and have poorly differentiated histology. From 2010 to 2017, the percentage of colon cancer and tubular adenocarcinoma in CRC increased substantially (from 39.3 to 51.8%, from 78.6 to 93.4%, respectively). The percentage of right colon cancer increased from 18.3 to 20.5%, which predictively may keep at 22.6% in 2018. The rise trends for patients with moderate differentiation tumor or KRAS exon 2 mutated tumor were apparent (from 50.3 to 78.6%, from 32.8 to 39.7%, respectively). In conclusion, in recent 8 years, there is a shift to the colon, especially right colon in the incidence of Chinese CRC. Moreover tubular adenocarcinoma is becoming the primary histology type.

A comparability study of immunohistochemical assays for PD-L1 expression in hepatocellular carcinoma.

Programmed death ligand 1 (PD-L1) protein expression by immunohistochemistry is a promising biomarker for PD-1/PD-L1 blockade in hepatocellular carcinoma. There are a number of commercially available PD-L1 assays. Our study aimed to compare the analytical performance of different PD-L1 assays and evaluate the reliability of pathologists in PD-L1 scoring. Consecutive sections from tumor samples from 55 patients with surgically resected primary hepatocellular carcinoma were stained with four standardized PD-L1 assays (22C3, 28-8, SP142, and SP263). We also correlated the PD-L1 protein level by immunohistochemistry with the mRNA level of those genes associated with tumor immune microenvironment by the NanoString platform. Five pathologists independently assessed PD-L1 expression on tumor cells [tumor proportion score] together with tumor-infiltrating immune cells (combined positive score). The 22C3, 28-8, and SP263 assays had comparable sensitivity in detecting PD-L1 expression, whereas the SP142 assay was the least sensitive assay. The inter-assay agreement measured by intraclass correlation coefficients for the tumor proportion score and combined positive score were 0.646 and 0.780, respectively. The inter-rater agreement was good to excellent (the overall intraclass correlation coefficient for the tumor proportion score and combined positive score was 0.946 and 0.809, respectively). Pathologists were less reliable in scoring combined positive score than tumor proportion score, particularly when using the SP142 assay. Up to 18% of samples were misclassified by individual pathologists in comparison to the consensus score at the cutoff of combined positive score ≥ 1. The combined positive score by the 22C3 assay demonstrated the strongest correlation with immune-related gene mRNA signatures, closely followed by combined positive scores by the 28-8 and SP263 assays. In conclusion, the 22C3, 28-8, and SP263 assays are highly concordant in PD-L1 scoring and suggest the interchangeability of these three assays. Further improvement of the accuracy in assessing PD-L1 expression at a low cutoff is still necessary.

Distant Metastasis Risk Definition by Tumor Biomarkers Integrated Nomogram Approach for Locally Advanced Nasopharyngeal Carcinoma.

Identifying metastasis remains a challenge for death control and tailored therapy for nasopharyngeal carcinoma (NPC). Here, we addressed this by designing a nomogram-based Cox proportional regression model through integrating a panel of tumor biomarkers. A total of 147 locally patients with advanced NPC, derived from a randomized phase III clinical trial, were enrolled. We constructed the model by selecting the variables from 31 tumor biomarkers, including 6 pathological signaling pathway molecules and 3 Epstein-Barr virus-related serological variables. Through the least absolute shrinkage and selection operator (LASSO) Cox proportional regression analysis, a nomogram was designed to refine the metastasis risk of each NPC individuals. Using the LASSO Cox regression model, we constructed a 9 biomarkers-based prognostic nomogram: Beclin 1, Aurora-A, Cyclin D1, Ki-67, P27, Bcl-2, MMP-9, 14-3-3σ, and VCA-IgA. The time-dependence receiver operating characteristic analysis at 1, 3, and 5 years showed an appealing prognostic accuracy with the area under the curve of 0.830, 0.827, and 0.817, respectively. In the validation subset, the concordance index of this nomogram reached to 0.64 to identify the individual metastasis pattern. Supporting by this nomogram algorithm, the individual metastasis risk might be refined personally and potentially guiding the treatment decisions and target therapy against the related signaling pathways for patients with locally advanced NPC.

Preoperative radiomic signature based on multiparametric magnetic resonance imaging for noninvasive evaluation of biological characteristics in rectal cancer.

OBJECTIVES: To develop and validate radiomic models in evaluating biological characteristics of rectal cancer based on multiparametric magnetic resonance imaging (MP-MRI). METHODS: This study consisted of 345 patients with rectal cancer who underwent MP-MRI. We focused on evaluating five postoperative confirmed characteristics: lymph node (LN) metastasis, tumor differentiation, fraction of Ki-67-positive tumor cells, human epidermal growth factor receptor 2 (HER-2), and KRAS-2 gene mutation status. Data from 197 patients were used to develop the biological characteristics evaluation models. Radiomic features were extracted from MP-MRI and then refined for reproducibility and redundancy. The refined features were investigated for usefulness in building radiomic signatures by using two feature-ranking methods (MRMR and WLCX) and three classifiers (RF, SVM, and LASSO). Multivariable logistic regression was used to build an integrated evaluation model combining radiomic signatures and clinical characteristics. The performance was evaluated using an independent validation dataset comprising 148 patients. RESULTS: The MRMR and LASSO regression produced the best-performing radiomic signatures for evaluating HER-2, LN metastasis, tumor differentiation, and KRAS-2 gene status, with AUC values of 0.696 (95% CI, 0.610-0.782), 0.677 (95% CI, 0.591-0.763), 0.720 (95% CI, 0.621-0.819), and 0.651 (95% CI, 0.539-0.763), respectively. The best-performing signatures for evaluating Ki-67 produced an AUC value of 0.699 (95% CI, 0.611-0.786), and it was developed by WLCX and RF algorithm. The integrated evaluation model incorporating radiomic signature and MRI-reported LN status had improved AUC of 0.697 (95% CI, 0.612-0.781). CONCLUSION: Radiomic signatures based on MP-MRI have potential to noninvasively evaluate the biological characteristics of rectal cancer. KEY POINTS: • Radiomic features were extracted from MP-MRI images of the rectal tumor. • The proposed radiomic signatures demonstrated discrimination ability in identifying the histopathological, immunohistochemical, and genetic characteristics of rectal cancer. • All MRI sequences were important and could provide complementary information in radiomic analysis.

Role of Angiogenesis in Chronic Radiation Proctitis: New Evidence Favoring Inhibition of Angiogenesis Ex Vivo.

BACKGROUND: Chronic radiation proctitis (CRP), a common complication after radiotherapy for pelvic malignancies, compromises patient quality of life. Vascular damage and aberrant angiogenesis in the mucosal layer are essential histological features, but changes to the submucosal layer are unclear. Thus, we evaluated the histological characteristics and distribution changes of key angiogenic factors in full-layered human CRP samples. METHODS: Thirty paraffin-embedded CRP and twenty-nine non-CRP tissues were used to evaluate histopathological changes. Immunohistochemistry with anti-CD34 antibody was performed to calculate microvascular density (MVD). Frozen tissues from eight CRP patients and five non-CRP controls were collected and analyzed by antibody array, which contained sixty human angiogenesis-related factors. Quality controls with positive and negative controls were performed during antibody array analysis. Two differentially expressed factors were confirmed by ELISA. RESULTS: CRP lesions showed vasculopathy, fibrosis, mucosal ulceration, edema, and inflammatory cell infiltration. Human angiogenesis antibody array and ELISA confirmed the increased angiostatin in CRP lesions. Immunohistochemical staining showed dispersed distribution of angiostatin throughout the mucosal and submucosal layers in CRP lesions, while angiostatin accumulated within the vessel lumens in non-CRP tissues. MVD significantly decreased in the submucosal layer of CRP, suggesting a potential association with increased angiostatin. CONCLUSIONS: Angiostatin increased and had a distinct distribution in CRP lesions. Compensatory telangiectasia in the mucosa, vessel stenosis, and reduced MVD might attenuate blood flow in the submucosa and contribute to CRP progression. Restoration of vascular functionality by promoting angiogenesis in the submucosal layer may help alleviate CRP in clinical practice.

A Novel Immune Marker Model Predicts Oncological Outcomes of Patients with Colorectal Cancer.

BACKGROUND: The purpose of this study was to develop an in situ immune marker model to predict postoperative oncological outcomes in patients with colorectal cancer (CRC). METHODS: Immunohistochemistry for 13 immune cell markers was performed on tumor tissue microarrays from 300 CRC patients who underwent curative resection from January 2000 to January 2006. Genetic algorithm was applied for the construction of an in situ immune marker model. RESULTS: The infiltration of CD3+ cells, CD45RO+ cells, and FOXP3+ cells, but not the infiltration of Tryptase+ cells, in the tumor was significantly associated with better clinical outcome in overall survival (OS) and disease-free survival (DFS) of CRC patients, as assessed by univariate analysis (P < 0.05). Based on the genetic algorithms, a total of 6 markers, including CD3, CD45RO, IL17, CD15, Tryptase, and FOXP3, were selected to construct an immune marker model. Our model was identified to have an independent predictive capability for both OS and DFS in Cox multivariable model (P < 0.001). This was further confirmed by the ROC analysis (area under curve: OS, 0.669; DFS, 0.684). CONCLUSIONS: The in situ immune marker model constructed in this study provides a novel approach to identify CRC patients who were at an increased risk for poor oncological outcomes.

Epigenetic silencing of TPM2 contributes to colorectal cancer progression upon RhoA activation.

Beta-tropomyosin (ß-tropomyosin, TPM2) has been found to be downregulated in colorectal cancer (CRC) in previous studies. In this study, we aimed to investigate the mechanisms and potential biological consequences of the downregulation of TPM2 in colorectal cancer. TPM2 expression in colorectal cancer was assessed by qRT-PCR and immunostaining. The biological functions of TPM2 were assessed in cell lines either overexpressing or underexpressingTPM2. Aberrant DNA methylation in the promoter region is associated with suppression of TPM2 expression in primary colorectal cancer tissue samples. Treatment with the demethylation agent 5-AZA can induceTPM2 expression in colorectal cancer cell lines. Reconstitution of TPM2 suppresses cell proliferation and migration in colorectal cancer cell lines, whereas the loss of TPM2 expression is associated with increased tumor proliferation and migration in vitro, which was accompanied by RhoA activation. In summary, our findings indicate that TPM2 appears to be commonly silenced by aberrant DNA methylation in colon cancer. TPM2 loss is associated with RhoA activation and tumor proliferation.

Early prediction of intestinal mucosal barrier function impairment by elevated serum procalcitonin in rats with severe acute pancreatitis.

OBJECTIVES: The aim of this study was to evaluate serum procalcitonin (PCT) levels as a prognostic indicator of intestinal barrier function impairment in rats with severe acute pancreatitis (SAP). METHODS: Thirty-six male Sprague Dawley rats were randomly grouped into SAP group (injected sodium taurocholate via biliopancreatic duct), Gln group (gavaged with glutamine after modeling), and control group. Blood, pancreatic, and terminal ileum tissues were obtained from the rats after 6 h of modeling. Serum amylase (Amy) levels were determined using an automatic biochemical detector, while endotoxin (ET), diamine oxidase (DAO), and PCT levels were measured by ELISA test. The pathology of pancreatic and small intestine tissues were observed. PCT protein expression in intestinal tissues were detected by immunohistochemistry and western blot. RESULT: Pancreatic and intestinal injuries in Gln group were significantly lower than SAP group. Serum amylase, DAO, and PCT levels in SAP and Gln groups differed greatly and were significantly higher than control group. Immuno-histochemistry and western blot results showed that PCT protein expression levels in small intestine tissues of SAP group were higher than Gln group and control group. Serum PCT levels had a significant correlation with serum endotoxin, DAO levels and intestinal mucosal injury scores. CONCLUSION: PCT expression in serum and intestinal tissues in SAP rats increased significantly in the early stages of SAP, and was closely related to the onset and degree of intestinal barrier function impairment. Thus, our results showed that measuring serum PCT can be used to predict intestinal mucosal barrier function impairment in SAP rats.

Relationship between biofilms and clinical features in patients with sinus fungal ball.

The presence of bacterial biofilms (BBF) and fungal biofilms (FBF) is associated with greater disease severity in chronic rhinosinusitis. However, researches on biofilms in fungal rhinosinusitis are rare. This study investigated the relationship between biofilms and clinical features in patients with sinus fungal ball (SFB). Sixty-four SFB patients undergoing endoscopic sinus surgery and 21 controls were enrolled in this study. Mucosal samples from nasal sinuses were collected for biofilm detection under confocal scanning laser microscopy (CSLM). The general clinical data, Lund-Mackay computed tomography (CT) score, Lund-Kennedy endoscopy score, Global Osteitis Scoring Scale (GOSS) score, Sinonasal Outcome Test (SNOT)-22 score and visual analog scale (VAS) score were recorded. Associations between these parameters and biofilms were assessed. Under CSLM, the positive rates of BBF and FBF were 45.3 % (29/64) and 21.9 % (14/64), respectively in the SFB group but none in controls. When sub-classified according to biofilm status, the BBF-positive subgroup had significantly higher Lund-Mackay score and GOSS score than the BBF-negative one, but there were no differences in demographic characteristics, health-related quality-of-life and endoscopic inflammatory severity. BBF and FBF coexisted on the sinus mucosa of the patients with SFB. BBF was associated with more severe disease, but the distribution of FBF did not affect the severity of SFB.