[Association between airborne particulate matter（PM 2.5） concentration and the incidence of allergic rhinitis in Shanghai].

Objective:To explore the impact of PM 2.5 concentration in Shanghai on the incidence of allergic rhinitis（AR） in the population, and provide strategies for early warning and prevention of AR. Methods:Collect daily average concentrations of atmospheric pollutants monitored in Shanghai from January 1, 2017 to December 31, 2019, and clinical data of AR patients from five hospitals in Shanghai during the same period. We used a time-series analysis additive Poisson regression model to analyze the correlation between PM 2.5 levels and outpatient attendance for AR patients. Results:During the study period, a total of 56 500 AR patients were included, and the daily average concentration of PM 2.5 was（35.28±23.07）µg/m³. There is a correlation between the concentration of PM 2.5 and the number of outpatient attendance for AR cases. There is a positive correlation between the daily average number of outpatient for AR and levels of PM 2.5 air pollution（（P<0.05）） . We found that every 10 µg/m³ increase in PM 2.5, the impact of on the number of AR visits was statistically significant on the same day, the first day behind, and the second day behind, with the strongest impact being the exposure on the same day. Every 10 µg/m³ increases in PM 2.5, the number of outpatient visits increased by 0.526% on the same day（95%CI 1.000 50-1.010 04）. Conclusion:The atmospheric PM 2.5 concentration in Shanghai is positively correlated with the number of outpatient for AR, and PM 2.5 exposure is an independent factor in the onset of AR. This provides an important theoretical basis for AR.

The association of maternal fat-soluble antioxidants in early pregnancy with gestational diabetes mellitus: a prospective cohort study.

INTRODUCTION: Oxidative stress is linked to the development of gestational diabetes mellitus (GDM). Maternal antioxidant vitamins in early pregnancy may play a role in GDM occurrence. We aimed to investigate the associations of vitamins A and E in early pregnancy with the risk of GDM and to explore whether these antioxidant vitamins can be biomarkers for the early prediction of GDM. METHODS: We carried out a prospective cohort study conducted in Beijing and enrolled pregnant women (n = 667) with vitamins A and E measurements at 9 weeks (IQR 8-10) of gestation and having one-step GDM screened with a 75-g oral glucose tolerance test between 24 and 28 weeks of gestation. RESULTS: The vitamin A levels in early pregnancy were significantly higher in women with GDM than in those without GDM (p < 0.0001) and positively correlated with fasting blood glucose. In multivariate models, vitamin A levels were significantly associated with GDM (OR, 1.46; 95% CI: 1.14-1.88; p = 0.0032) per SD. A significant trend of risk effect on GDM risk across quartiles of vitamin A was observed (ptrend = 0.016). No significant association of serum vitamin E with GDM was observed overall. However, a noted trend of protective effect on GDM risk across quartiles of vitamin E/cholesterol ratio was observed (ptrend = 0.043). In ROC analysis, the multivariate model consisting of vitamin A and other risk factors showed the best predictive performance (AUC: 0.760; 95% CI: 0.705-0.815; p < 0.001). CONCLUSIONS: Higher levels of vitamin A in early pregnancy were significantly associated with an increased risk of GDM. Vitamin A has the potential to be a biomarker indicating pathogenesis of GDM.

CSMD1 suppresses cancer progression by inhibiting proliferation, epithelial-mesenchymal transition, chemotherapy-resistance and inducing immunosuppression in esophageal squamous cell carcinoma.

Human CUB and Sushi multiple domains (CSMD1) is considered a crucial role in cancer progression, but the specific function in esophageal squamous cell carcinoma (ESCC) is not clear. Understanding the role of CSMD1 in ESCC progression may lead to a novel strategy for ESCC treatment. Here, we found that both CSMD1 mRNA and protein levels were downregulated in ESCC tissues. Reduced CSMD1 expression was correlated with a poor prognosis in ESCC patients. CSMD1 expression inhibited proliferation, migration and invasion in ESCC cell lines in vitro. CSMD1 deficiency in established xenografted tumors increases tumor size and weight. We further found that CSMD1-overexpression cells are more sensitive to chemotherapy. Moreover, we addressed the role of CSMD1 in the CD8+ T cell immune response. An in vitro killing assay showed that the cytotoxicity of CD8+ T cells was inhibited in CSMD1-overexpression tumor cells. In vivo, in CSMD1 deficiency tumor-bearing mice activation and expansion of CD8+ T cells were increased. Further investigation showed that CSMD1 expression on tumor cells was positively correlated with CD8+ T cells infiltration and cytokines secretion. These findings highlight that CSMD1 is a tumor suppressor gene in ESCC patients and a positive regulator of CD8+ T cells expansion and activation, and could increase cytokines secretion, indicating that tumor cell-associated CSMD1 might be a target for ESCC.

The RNA N6-Methyladenosine Demethylase FTO Promotes Head and Neck Squamous Cell Carcinoma Proliferation and Migration by Increasing CTNNB1.

OBJECTIVE: In this study, we aimed to investigate the role of RNA N6-methyladenosine demethylase fat mass and obesity-associated protein (FTO) in head and neck squamous cell carcinoma (HNSCC). METHODS: Clinical data downloaded from The Cancer Genome Atlas (TCGA) database were used to analyze the relationship between mRNA levels of FTO, METTL3, METTL14, and ALKBH5, and the overall survival in cancer and para-cancer datasets. FTO expression in tumor and normal tissues was compared using immunohistochemistry, and its relationship with overall survival was analyzed based on the Kaplan-Meier method. The FaDu cell line with high FTO levels was chosen from five HNSCC cell lines for further experiments. FTO was verified as an oncogene in HNSCC by in vitro loss-of-function and overexpression studies, cell proliferation assay, wound healing assay, and identification of expression changes of epithelial-mesenchymal transition (EMT)-related markers. Catenin beta 1 (CTNNB1) was confirmed as a downstream target gene of FTO with additional methods like the GEPIA online tool, qRT-PCR, Western blotting, and dot blot assay. RESULTS: We found that FTO expression was significantly upregulated in HNSCC datasets and tissues. Increased FTO expression indicated a trend towards poor prognosis and was found to promote disease proliferation and migration. Mechanistically, cell proliferation assay, wound healing assay, and identification of expression changes of EMT-related markers demonstrated that FTO could act as an oncogene in HNSCC. FTO expression was significantly correlated with CTNNB1 expression. Moreover, it exerted a tumorigenic effect by increasing CTNNB1 expression in an m6A-dependent manner. CONCLUSION: FTO promotes head and neck squamous cell carcinoma proliferation and migration by increasing CTNNB1 in an m6A-dependent manner.

Salvianolic acid B inhibits myofibroblast differentiation and extracellular matrix accumulation in nasal polyp fibroblasts via the TGF-ß1 signaling pathway.

In the process of nasal tissue remodeling, nasal fibroblasts serve an important role via myofibroblast differentiation and the production of extracellular matrix (ECM). Nasal fibroblast abnormalities can lead to conditions such as chronic rhinosinusitis. Salvianolic acid B (Sal B), a water-soluble active pharmaceutical compound extract from the root of the traditional Chinese medicine Salvia miltiorrhiza, displays antioxidative, antiproliferative and antifibrosis properties. The present study aimed to investigate the mechanism underlying the effects of Sal B on nasal polyp fibroblast (NPF) myofibroblast differentiation and ECM accumulation. Primary NPFs were obtained from nasal polyps of patients with chronic sinusitis. The proliferative and cytotoxic effects of Sal B on NPFs were evaluated by performing the Cell Counting Kit-8 assay. The Transwell assay was conducted to assess cell migration. α-smooth muscle actin (α-SMA), TGF-ß1 receptor (TßR)-I, TßR-II, Smad2/3 mRNA and protein expression levels and (p)-Smad2/3 phosphorylation levels were measured via reverse transcription-quantitative PCR and western blotting, respectively. Type III collagen and fibronectin levels were analyzed by ELISA. The results indicated that Sal B significantly downregulated TGF-ß1-induced α-SMA, fibronectin and collagen III expression levels in NPFs. Similarly, Sal B significantly decreased TGF-ß1-induced TßR-I, TßR-II, p-Smad2/3, MMP-2 and MMP-9 mRNA and protein expression levels in NPFs. Furthermore, Sal B significantly decreased TGF-ß1-induced NPF migration. Therefore, the present study indicated that Sal B inhibited myofibroblast differentiation and ECM accumulation in nasal fibroblasts, suggesting that Sal B may inhibit nasal polyp formation via certain mechanisms.

uPA affects the CRSsNP nasal mucosa epithelium apoptosis by regulating WIF1.

Chronic rhinosinusitis without nasal polyps (CRSsNP) is the main type of Chronic rhinosinusitis (CRS) and is a common otorhinolaryngologic disease worldwide. However, the mechanisms of CRSsNP remain poorly understood. In this study, C57BL/6J wild-type and urokinase-type plasminogen activator (uPA) gene knockout (uPA-/-) mice were used to construct the CRSsNP model. Primary human nasal epithelial cells (HNEC) were isolated from CRSsNP patient and treated with uPA knockdown/overexpression lentivirus. CCK-8 and Annexin-V/PI staining were used to detected cell proliferation and apoptosis. In vivo, we found that uPA depletion alleviated mucosal inflammation in the CRSsNP mice model. Wnt inhibitory factor 1 (WIF1) was upregulated in the uPA-/- CRSsNP mice model. In vitro, inhibition of uPA increased cell proliferation and decreased cell apoptosis. Mechanistically, uPA depletion upregulated WIF1 and BCL2 expression, and reduced the expression level of BAX in CRSsNP HNEC. In contrast, decreased cell proliferation and increased cell apoptosis were observed after uPA overexpression. Consistently, a reduction in WIF1 and BCL2 expression levels and an increase in the BAX expression level were observed upon uPA ectopic expression. Furthermore, WIF1 overexpression rescued the effects caused by uPA overexpression in vitro. In conclusion, uPA affects the CRSsNP nasal mucosal epithelium cell apoptosis by upregulating WIF1. To our knowledge, this is the first study to explore the role of uPA in CRSsNP to date.

Does hypermethylation of CpG island in the promoter region of the E-cadherin gene increase the risk of lung cancer? A meta-analysis.

BACKGROUND: Hypermethylation of the CpG island in the promoter regions of tumor suppressor genes is common in the cancer tissue of non-small cell lung cancer (NSCLC) patients. Epithelial cadherin (E-cadherin) is a classic tumor suppressor gene of the cadherin superfamily and its promoter region is usually hypermethylated in malignant carcinomas. However, whether hypermethylation of the CpG island in the promoter region of E-cadherin increases the risk of lung cancer is unknown. We conducted a meta-analysis of E-cadherin gene promoter methylation status in cancer tissue (CT) and autologous controls (AC). METHODS: Electronic databases were searched for E-cadherin gene promoter methylation and NSCLC. The hypermethylation status between CT and AC of NSCLC patients were compared and pooled by random or fixed effect models according to statistical heterogeneity across the included studies. RESULTS: Eleven publications relevant to E-cadherin gene promoter hypermethylation and lung cancer risk were identified and included. E-cadherin gene promoter hypermethylation frequency in CT and AC was 0.32 (95% confidence interval [CI] 0.22-0.41) and 0.12 (95% CI 0.04-0.20), respectively, with statistical difference (P < 0.05). Significant statistical heterogeneity was found across the 11 studies (I2 = 54.5, P < 0.05). The data was pooled through a random effect model with an odds ratio of 4.21 (95% CI 2.33-7.58) in CT compared to AC. CONCLUSION: The frequency of E-cadherin promoter hypermethylation in CT is significantly higher than in AC, indicating that promoter hypermethylation of E-cadherin plays an important role in NSCLC carcinogenesis.

[The effect of MDR1 (P-gp) and ABCG2 on the drug resistance in Hep 2 cells].

OBJECTIVE: To study the effect of MDR1 (P-gp) and ABCG2 on the drug resistance in Hep 2 cells. METHOD: Flow cytometry was used to detect the variations of the antitumor drugs accumulation and discharging, and activity variations when MDR1 and ABCG2 inhibitors were used in Hep-2. RESULT: The accumulation and discharging of mitoxantrone was significantly higher than the control group when ABCG2 inhibitor FTC was used in Hep-2 (P<0. 05). In contrast, P-gp did not appear similar case; To the mitoxantrone and cisplatin, there was no statistical correlation about activity of Hep-2 between P-gp or ABCG2 antagonist and the control; To the doxorubicin, combining FTC and P-gp, the activity of Hep-2 was higher than the control and difference was significant (P<. 05), In contrast, FTC and P-gp did not appear similar case when used alone; To the 5-FU, when PGP used, the activity of Hep-2 was higher than that in the control and difference was significant (P<0. 05), In con- trast, FTC and FTC+P-gp did not appear similar case; To the paclitaxel, when P-gp or FTC+P-gp used, the activity of Hep-2 was higher than that in the control and difference was significant(P<0. 05). CONCLUSION: ABCG2 may lead to drug resistance mainly by changing the ability of cell in accumulating and discharging chemotherapy drugs. P-gp has other way. P-gp and ABCG2 play different roles in different drug resistance.

Gene and microRNA expression reveals sensitivity to paclitaxel in laryngeal cancer cell line.

Paclitaxel is a widely used chemotherapy drug for advanced laryngeal cancer patients. However, the fact that there are 20-40% of advanced laryngeal cancer patients do not response to paclitaxel makes it necessary to figure out potential biomarkers for paclitaxel sensitivity prediction. In this work, Hep2, a laryngeal cancer cell line, untreated or treated with lower dose of paclitaxel for 24 h, was applied to DNA microarray chips for gene and miR expression profile analysis. Expression of eight genes altered significantly following paclitaxel treatment, which was further validated by quantitative real-time PCR. Four up-regulated genes were ID2, BMP4, CCL4 and ACTG2, in which ID2 and BMP4 were implicated to be involved in several drugs sensitivity. While the down-regulated four genes, MAPK4, FASN, INSIG1 and SCD, were mainly linked to the endoplasmic reticulum and fatty acid biosynthesis, these two cell processes that are associated with drug sensitivity by increasing evidences. After paclitaxel treatment, expression of 49 miRs was significantly altered. Within these miRs, the most markedly expression-changed were miR-31-star, miR-1264, miR-3150b-5p and miR-210. While the miRs putatively modulated the mRNA expression of the most significantly expression-altered genes were miR-1264, miR-130a, miR-27b, miR-195, miR-1291, miR-214, miR-1277 and miR-1265, which were obtained by miR target prediction and miRNA target correlation. Collectively, our study might provide potential biomarkers for paclitaxel sensitivity prediction and drug resistance targets in laryngeal cancer patients.

miRNA expression pattern associated with prognosis in elderly patients with advanced OPSC and OCC.

The long-term survival for elderly patients with advanced ovarian papillary serous carcinoma (OPSC) does not exceed 30%, and the incidence and prognosis rise continuously after menopause. The aim of this study was to identify the differences in key miRNAs and their potential regulators through miRNA microarray analysis, functional target prediction, and clinical outcome between the elderly patients with advanced OPSC and ovarian clear cell carcinoma (OCC) who all suffered poor prognosis, to identify the pathogenetic basis, and to improve the understanding of the molecular basis of advanced OPCS in elderly patients. Through microarray analysis, we found 52 unique miRNAs with significant fold­change in expression levels, of which 9 were upregulated, whereas 43 were downregulated in OCC patients compared to elderly OPSC patients with advanced stage. Among these prediction miRNAs, miR-30a, miR-30e and miR-505 were found to have some common cancer-related targets. Lower expression of these three miRNAs of advanced OPSC in elderly patients, all associated with significantly poorer survival rate, strongly suggesting that they could be critical oncogenes and take important roles in OPSC etiology in elderly patients with advantaged stage. Functional analyses support the above hypothesis. Their targets, ATF3, STMN1 and MYC suggest that OPSC also has aggressive biological behavior when presented with advanced stage, and support the epidemiology results that incidence and mortality of advanced OPSC increases continuously. miR-30a, miR-30e and miR-505 may be important pathogenetic factors for OPSC in elderly patients with advanced stage. Age could be regarded as a continuous covariate in this process. This may improve the understanding of molecular underpinnings of advanced OPSC in elderly patients, and provide improved diagnostic, prognostic and therapeutic approaches.

An empirical study of modified frontolateral partial laryngectomy without tracheotomy.

The aim of this study was to validate the feasibility of modified frontolateral partial laryngectomy without tracheotomy using animal experiments. The glottic area before and after surgery of 6 excised canine larynges and 10 canine larynges in vivo were compared to observe whether the glottic area following modified frontolateral partial laryngectomy without tracheotomy is adequate for breathing. Significant differences were observed between the glottic areas of the excised larynges in the initial state and following modified frontolateral partial laryngectomy with the cartilage closed. However, no significant differences were observed between the glottic areas of the excised larynx in the initial state and following modified frontolateral partial laryngectomy with the cartilage open. The glottic area of the larynges in vivo in the initial state and following right chordectomy via laryngofissure were not observed to be significantly different. Furthermore, no significant differences were observed between the glottic areas of the larynges in vivo in the initial state and following modified frontolateral partial laryngectomy without tracheotomy. In conclusion, modified frontolateral partial laryngectomy without tracheotomy is a feasible and efficacious means of eradicating early and selected invasive carcinomas of the larynx, which is supported by animal experiments.

Association between GSTM1 polymorphism and nasopharyngeal cancer susceptibility: a meta-analysis.

BACKGROUND/AIMS: Glutathione S-transferase M1 (GSTM1) is a multifunctional enzyme that plays a critical role in the detoxification of varieties of carcinogenic metabolites. Many studies have been conducted to investigate the association between GSTM1 polymorphism and nasopharyngeal cancer (NPC) risk, but the findings among those studies are inconsistent. To assess this relationship more precisely, we performed a meta-analysis of all available studies on the subject. METHODS: Case-control studies were identified by searching Pubmed, Embase, ISI Web of Science, and Wanfang databases through September 6, 2012. We used the pooled odds ratio (OR) with its corresponding 95% confidence interval (95%CI) to evaluate the association of GSTM1 polymorphism with NPC susceptibility. Subgroup analyses by pathological types, sex and smoking status were performed to further identify the association. RESULTS: Overall, 11 published studies with 1,513 cases and 2,802 controls were finally included into this meta-analysis according to the inclusion criteria. Meta-analysis of total studies showed that the null genotype of GSTM1 was significantly associated with increased risk of NPC, when comparing with the non-null genotype (OR=1.51, 95%CI=1.33-1.72, POR<0.001). The association was still statistically significant in subgroup analysis of patients with nasopharyngeal squamous cell carcinoma (OR=1.73, 95%CI=1.24-2.42, POR=0.001). Males with the null genotype of GSTM1 were more likely to subject to NPC than females. In addition, the association between the null genotype of GSTM1 and NPC risk was strongest in individuals with exposure to smoking. Sensitivity analysis by sequential omission of any individual studies one at a time further demonstrated the significant association. CONCLUSIONS: The findings suggest that the null genotype of GSTM1 is a risk factor for NPC, and there is a gene- smoking interaction in this association.

Lack of association between glutathione S-transferase T1 gene polymorphism and laryngeal cancer susceptibility: a meta-analysis based on 2,124 cases and 2,059 controls.

Studies investigating the association between glutathione S-transferase T1 (GSTT1) gene polymorphism and laryngeal cancer susceptibility have reported conflicting results. The aim of the present study was to conduct a meta-analysis assessing the possible association of GSTT1 gene polymorphism with laryngeal cancer risk. The relevant studies were identified through a search of PubMed, Embase, ISI Web of Knowledge and Chinese National Knowledge Infrastructure until May 2011. Twelve studies were included in the present meta-analysis, which described a total of 2124 laryngeal cancer cases and 2059 controls. The overall odds ratio (OR) for GSTT1 null genotype was 1.40 (95% CI=0.90-2.16). When stratifying for race, the pooled ORs for GSTT1 null genotype were 1.07 (95% CI=0.81-1.41) in Caucasians and 5.63 (95% CI=1.00-31.83) in Asians. The pooled ORs for GSTT1 null genotype were 1.03 (95% CI=0.71-1.49) in population-based studies and 2.39 (95% CI=0.73-7.86) in hospital-based studies, stratifying for study design. This meta-analysis suggested that there was lack of association between GSTT1 gene polymorphism and laryngeal cancer risk. However, larger scale primary studies are still required to further evaluate the interaction of GSTT1 gene polymorphism with laryngeal cancer risk.

[Clinical analysis of the diagnosis of laryngeal tuberculosis].

OBJECTIVE: To explore the clinical characteristic and diagnosis of modern laryngeal tuberculosis. METHOD: A retrospective research among 33 patients of laryngeal tuberculosis, with definitive pathological result, had been done in the laryngeal-endoscope room in our department. RESULT: The main symptoms of laryngeal tuberculosis are hoarseness and sore throat. Most of the patients did not accompany with pulmonary tuberculosis (TB) (63.6%). Few of them accompany with low-grade fever, night sweat and malnutrition (24.2%). There are three types in the laryngeal-endoscope vision: edema type, hyperplasia type and ulcer exudation type. The positive rates of PPD and phlegm bacteria examination are respectively 87.9% and 68.4%. There can be seen in the pathological slides the interstitial phagocytes and giant cell reaction. Granuloma consisting of epithelioid cell and Langhans' cells is coexistent with necrotic tissue. Cheese necrosis is the typical characteristic of the disease. CONCLUSION: Severe local symptoms of laryngeal with slightly general symptoms are the clinical characteristics of modern laryngeal tuberculosis. The diagnosis of it depends mainly on the process of the disease, laryngeal-endoscope examination and PPD examination combined with phlegm bacteria examination. And the final diagnosis is based on the pathological biopsy and tubercular bacillus culturing.

[Expression of S-100 positive dendritic cell, TIMP-1 and p63 and its significance in the primary laryngeal carcinoma].

OBJECTIVE: To evaluate the predictive role of S-100 positive dendritic cell, tissue inhibitor of metalloproteinases-1 (TIMP-1) and p63 gene in primary laryngeal carcinoma with epidemiology (smoking and drinking), histological grading, surgical treatment, TNM stage and prognosis by the tissuechip technology. METHODS: We studied the expression of dendritic cell (S-100), TIMP-1 and p63 gene on a series of 85 primary laryngeal carcinoma patients who had ever received in our hospital between 1992 and 2000 by the tissuechip technology and SP method. The correlation of each score according to the intensity and percentage of labeled cells or intercellular substance with relevant clinical dada was statistically analyzed. RESULT: Some cases were lost or boasted no tumor tissue in our tissuechip. In available 79 patients, the rate of expressing S-100 positive dendritic cell is 59.5% (47/79), and the average percentage of its labeled cells in them is 8.71%. S-100 positive dentritic cells showed significant difference among different pathological grade group, early and late stage( P < 0.05). The rate is 55.7% (44/79) of the specimens whose basal membrane and extracellular matrix was strongly stained by TIMP-1; There was statistical significant in TIMP-1 protein demonstration between early and late stages, lymph node metastasis and 3-year survival rate ( P < 0.05) by chi-square test, but no relation with smoking, drinking, gender, age and histological classes (P > 0.05). There was wo statistical significant in p63 protein demonstration between TNM stages, lymph node metastasis, 3-year survival rate, smoking, drinking, gender, age and histological classes (P > 0.05). CONCLUSION: The tissue microarray technique spent shorter time and less expense, and showed higher consistency in our essays. And the present study suggests TIMP-1 and S-100 could be the clinical discriminators in laryngeal carcinoma.

Application of tissue microarray: evaluation of the expression of S-100-positive dendritic cells, tumor suppressor gene p63 and tissue inhibitor of metalloproteinase-1 in laryngeal carcinoma.

OBJECTIVE: To evaluate the predictive role of S100-positive dendritic cells, tissue inhibitor of metalloproteinase-1 (TIMP1) and p63 gene in laryngeal carcinoma using a tissue chip. MATERIAL AND METHODS: The expression of dendritic cells, TIMP1 and p63 gene in a series of 85 primary laryngeal carcinoma patients who had undergone operations in our hospital between 1992 and 2000 was studied immunohistochemically using the streptavidin-biotin peroxidase-conjugated method. RESULTS: Some cases were lost or showed no tumor tissue in the tissue chip. The rate of expression of dendritic cells was 59.5% and there were significant differences in expression between the differential degrees of laryngeal squamous carcinoma and the different clinical stages (p <0.05). The basal membrane and extracellular matrix of the specimens were strongly stained by TIMP1 in 55.7% of cases. There were statistically significant correlations between TIMP1 protein expression and early- and advanced-stage tumors, lymph node metastasis and 3-year survival rate (p <0.05). No statistically significant correlations were found between p63 protein expression and any of the clinical parameters. CONCLUSIONS: The tissue microarray technique used in this study is quick, inexpensive and very consistent. It is suggested that TIMP1 and S100 should be used as clinical discriminators of laryngeal carcinoma.