ObjectiveThis paper aims to use the digital muscle function assessment system Myoton PRO to assess the correlation between muscle tension，clinical characteristics， and traditional Chinese medicine（TCM） syndromes in patients with hepatolenticular degeneration ［also known as Wilson disease（WD）］. MethodsA total of 104 patients with WD accompanied by abnormal muscle tension（increased or decreased，hereinafter the same） who were hospitalized in the Brain Disease Centre of the First Affiliated Hospital of Anhui University of Chinese Medicine from April 2021 to November 2023 were selected，all of whom were subjected to TCM syndrome diagnosis and Myoton PRO for the measurement of F value of muscle tension，Goldstein， and UWDRS-N scales. The age of onset of the disease and disease duration were analyzed，and the differences and correlations of the above indexes in different TCM syndromes of WD were analyzed ResultsAmong the 104 patients with WD ，the phlegm and stasis syndrome was the most common（60 patients），followed by the damp-heat syndrome（33 patients），and the least common was the liver-kidney Yin deficiency syndrome（11 patients）. The F value of the phlegm and stasis syndrome group was higher than that of the liver-kidney Yin deficiency syndrome group and the damp-heat syndrome group（P<0.01）. The F value of the damp-heat syndrome group was higher than that of the liver-kidney Yin deficiency syndrome group（P<0.05），and the F value of the lower limbs of each group was higher than that of the upper limbs（P<0.01）. Goldstein and UWDRS-N scores of the patients in the phlegm and stasis syndrome group were higher than those in the damp-heat syndrome group and the liver-kidney Yin deficiency syndrome group（P<0.05）. There was no significant difference between the Goldstein and UWDRS-N scores of patients in the liver-kidney Yin deficiency syndrome group and the damp-heat syndrome group. Correlation analysis revealed that the age of onset and duration of the disease were positively correlated with the F values of the lower limbs（r=0.20，P<0.05，r=0.38，P<0.01）and had no significant correlation with those of the upper limbs. The F value levels of muscle tension of all limbs in the three groups of patients were positively correlated with the Goldstein and UWDRS-N scores（muscle tension of the upper limbs in the phlegm and stasis syndrome group，r=0.36，P<0.01，r=0.42，P<0.01. muscle tension of the lower limbs in the phlegm and stasis syndrome group，r=0.70，P<0.01，r=0.60，P<0.01. muscle tension of the upper limbs in the damp-heat syndrome group，r=0.64，P<0.01，r=0.53，P<0.01. muscle tension of the lower limbs in the damp-heat syndrome group，r=0.59，P<0.01，r=0.70，P<0.01. muscle tension of the upper limbs in the liver-kidney Yin deficiency syndrome group，r=0.70，P<0.01，r=0.74，P<0.01. muscle tension of the lower limbs in the liver-kidney Yin deficiency syndrome group，r=0.85，P<0.01，r=0.62，P<0.01）. Conlusion： The digital muscle function assessment system Myoton PRO can objectively evaluate the muscle tension level of patients with WD accompanied by abnormal muscle tension. This level has significant differences among different TCM syndromes and can evaluate the patient's condition to some extent.

Background Mercury, as a global heavy metal pollutant, poses a serious threat to human health. The toxicity of mercury depends on its chemical form. Distinguishing the forms of mercury in the environment is of great significance for mercury management and reducing human mercury exposure risks. Objective To establish a non-targeted metallomics method based on synchrotron radiation X-ray fluorescence (SRXRF) spectroscopy combined with machine learning to screen inorganic mercury (IHg) or methylmercury (MeHg) exposed rice plants. Methods Rice seeds were exposed to ultra-pure water (control group), 0.1 mg·L⁻¹ IHg (IHg group) or MeHg (MeHg group) solutions, respectively. After germination, the seedlings were cultured for 21 d, and rice leaves were collected, dried, weighed, and pressed. The content of metallome in rice leaves was determined by SRXRF. Machine learning models including soft independent modeling cluster analysis (SIMCA), partial least squares discriminant analysis (PLS-DA), and logistic regression (LR) were used to classify the SRXRF full spectra of different groups and find the best model to distinguish rice exposed to IHg or MeHg. Besides, characteristic elements were selected as input parameters to optimize the model by improving computing speed and reducing model calculation. Results The SRXRF spectral intensities of the control group, IHg group, and MeHg group were different, indicating that exposure to IHg and MeHg can interfere the homeostasis of metallome in rice leaves. The results of principal component analysis (PCA) of SRXRF spectra showed that the control group could be well distinguished from the mercury exposed groups, but the IHg group and the MeHg group were mostly overlapped. The accuracy rates of the three models (PLS-DA, SIMCA, and LR) were higher than 98% for the training set, higher than 95% for the validation set, and higher than 94% for the cross-validation set. Besides, the accuracy of the LR model was higher than that of the PLS-DA model and the SIMCA model. Furthermore, the accuracy was 92.05% when using characteristic elements K, Ca, Mn, Fe, and Zn selected by LR to distinguish the IHg group and the MeHg group. Compared with the full spectra model, although the prediction accuracy of the characteristic spectral model decreased, the input parameters of the model decreased by 99.51%, and precision, recall, and F1 score were above 84.48%, indicating that the model could distinguish rice exposed to different mercury forms. Conclusion Non-targeted metallomics method based on SRXRF and machine learning can be applied for high-throughput screening of rice exposed to different forms of mercury and thus decrease the risks of people being exposed to mercury.

Objective:Trigeminal neuralgia(TN)is a severe chronic neuropathic pain that mainly affects the distribution area of the trigeminal nerve with limited treating efficacy.There are numerous treatments for TN,but currently the main clinical approach is to suppress pain by carbamazepine(CBZ).Brain-derived neurotrophic factor(BDNF)is closely related to chronic pain.This study aims to determine the effects of CBZ treatment on BDNF expression in both the trigeminal ganglion(TG)and serum of TN via a chronic constriction injury of the infraorbital nerve(ION-CCI)rat model. Methods:The ION-CCI models were established in male Sprague-Dawley rats and were randomly divided into a sham group,a TN group,a TN+low-dose CBZ treatment group(TN+20 mg/kg CBZ group),a TN+medium-dose CBZ treatment group(TN+40 mg/kg CBZ group),and a TN+high-dose CBZ treatment group(TN+80 mg/kg CBZ group).The mechanical pain threshold in each group of rats was measured regularly before and after surgery.The expressions of BDNF and tyrosine kinase receptor B(TrkB)mRNA in TGs of rats in different groups were determined by real-time PCR,and the expression of BDNF protein on neurons in TGs was observed by immunofluorescence.Western Blotting was used to detect the protein expression of BDNF,TrkB,extracellular regulated protein kinases(ERK),and phospho-extracellular regulated protein kinases(p-ERK)in TGs of rats in different groups.The expression of BDNF in the serum of rats in different groups was detected by enzyme-linked immunosorbent assay(ELISA). Results:The results of mechanical pain sensitivity showed that there was no significant difference in the mechanical pain threshold in the right facial sensory area of the experimental rats in each group before surgery(all P>0.05).From the 3rd day after operation,the mechanical pain threshold of rats in the TN group was significantly lower than that in the sham group(all P<0.01),and the mechanical pain threshold of rats in the TN+80 mg/kg CBZ group,the TN+40 mg/kg CBZ group,and the TN+20 CBZ mg/kg group was higher than that in the TN group(all P<0.05).The BDNF and TrkB mRNA and protein expressions in TGs of rats in the TN group were higher than those in the sham group(all P<0.05),and those in the TN+80 mg/kg CBZ group,the TN+40 mg/kg CBZ group,and the TN+20 mg/kg CBZ group were lower than the TN group(all P<0.05).The p-ERK levels in TG of rats in the TN+80 mg/kg CBZ group,the TN+40 mg/kg CBZ group,and the TN+20 mg/kg CBZ group were significantly decreased compared with the TN group(all P<0.05).The BDNF and neuron-specific nuclear protein(NeuN)were mainly co-expressed in neuron of TGs in the TN group and they were significantly higher than those in the sham group(all P<0.05).The co-labeled expressions of BDNF and NeuN in TGs of the TN+ 80 mg/kg CBZ group,the TN+40 mg/kg CBZ group,and the TN+20 mg/kg CBZ group were lower than those in the TN group(all P<0.05).The results of ELISA showed that the level of BDNF in the serum of the TN group was significantly higher than that in the sham group(P<0.05).The levels of BDNF in the TN+80 mg/kg CBZ group,the TN+40 mg/kg CBZ group,and the TN+20 mg/kg CBZ group were lower than those in the TN group(all P<0.05).Spearman correlation analysis showed that the BDNF level in serum was negatively correlated with mechanical pain threshold(r=-0.650,P<0.01). Conclusion:CBZ treatment can inhibit the expression of BDNF and TrkB in the TGs of TN rats,reduce the level of BDNF in serum of TN rats and the phosphorylation of ERK signaling pathway,so as to inhibit TN.The serum level of BDNF can be considered as an indicator for the diagnosis and prognosis of TN.

The clinical data of one child with metanephric stromal tumor (MST) and BRAF V600E gene mutation admitted to the First Affiliated Hospital of Zhengzhou University in June 2022 was analyzed retrospectively.Literature was reviewed.The patient, a 2-year-old girl, was diagnosed with a tumor in the left abdomen.The maximum diameter of the tumor was 10.5 cm.A radical nephrectomy was performed on the left kidney, and postoperative pathology revealed MST.Microscopically, the tumor had no envelope and exhibited expansive growth.The tumor cells were fusiform or stellate, and nuclear division was visible in the cell-rich region.Dysplastic blood vessels were seen inside the tumor.The tumor cells around the blood vessels and invaginated renal tubules were arranged like onion skin.CD34 was detected positive by immunohistochemical staining, and BRAF V600E mutation was also detected positive by fluorescent polymerase chain reaction.A total of 21 relevant case reports were retrieved, including 16 in English and 5 in Chinese.Fifty-eight MST patients, including the one in this report were analyzed.These patients were aged 2 days to 15 years, with a median age of 2 years.Except for 2 patients with unknown sex, the ratio of male to female was about 1.4∶1.0.Most MST patients were asymptomatic, with an average tumor size of 5.3 cm.The tumor cell CD34 showed positive expression in different degrees.Eight patients received the BRAF V600E mutation detection, and the results were all positive.Fifty-eight patients underwent nephrectomy and were followed up for 0-156 months, of which 7 patients were assisted with radiotherapy and chemotherapy.During the follow-up, 1 patient died, and 1 patient had a relapse.MST is a rare benign renal stromal tumor. BRAF V600E mutations are detected in a variety of malignancies.This paper is the first to report MST with BRAF V600E mutation in China and points out the importance of molecular detection of BRAF mutation for accurate diagnosis of MST.

Objective:To investigate the evaluation efficacy and predictive prognostic value of alpha-fetoprotein (AFP) response in tyrosine kinase inhibitors (TKIs) in combination with PD-1 inhibitors (α-PD-1) for intermediate-to-advanced hepatocellular carcinoma (HCC).Methods:The retrospective cohort study was conducted. The clinicopathological data of 205 patients with intermediate-to-advanced HCC who were admitted to 9 medical centers, including Mengchao Hepatobiliary Hospital of Fujian Medical University et al, from March 2020 to July 2022 were collected. There were 178 males and 27 females, aged (52±12)years. Based on AFP response at 6-8 weeks after treatment, patients were divided into the AFP response group (AFP level decreased by ≥50% compared to baseline) and the AFP no response group (AFP level decreased by <50% compared to baseline). Observation indicators: (1) AFP response evaluation of anti-tumor efficacy; (2) comparison of patient prognosis; (3) analysis of factors affecting patient prognosis. Measurement data with normal distrubution were represented as Mean± SD, and measurement data with skewed distribution were represented as M(range) and M( Q1, Q3). Count data were described as absolute numbers, and comparison between groups was conducted using the chi-square test. The Kaplan-Meier method was used to draw survival curve and calculate survival rate, and the Log-Rank test was used for survival analysis. The COX proportional risk model was used for univariate analysis and the COX stepwise regression model was used for multivariate analysis. Results:(1) AFP response evaluation of anti-tumor efficacy. Before treatment, all 205 patients were positive of AFP, with a baseline AFP level of 1 560(219,3 400)μg/L. All 205 patients were treated with TKIs in combination with α-PD-1, and the AFP level was 776(66,2 000)μg/L after 6 to 8 weeks of treatment. Of the 205 patients, 88 cases were classified as AFP response and 117 cases were classified as AFP no response. According to the response evaluation criteria in solid tumors version 1.1, the objective response rate (ORR) and disease control rate (DCR) were 42.05%(37/88) and 94.32%(83/88) in patients of the AFP response group and 16.24% (19/117) and 64.10% (75/117) in patients of the AFP no response group, showing significant differences between them ( χ2=16.846, 25.950, P<0.05). According to the modified response evaluation criteria in solid tumors, the ORR and DCR were 69.32% (61/88) and 94.32% (83/88) in patients of the AFP response group and 33.33% (39/117) and 64.10% (75/117) in patients of the AFP no response group, showing significant differences between them ( χ2=26.030, 25.950, P<0.05). (2) Comparison of patient prognosis. All 205 patients were followed up for 12.4(range, 2.4-34.0)months after treatment. The median progression free survival time and total survival time were 5.5 months and 17.8 months, respectively. The 1-year, 2-year progression free survival rates were 20.8% and 7.2%, and the 1-year, 2-year overall survival rates were 68.7% and 31.5%, respectively. The median progression free survival time, 1-year and 2-year progression free survival rates were 9.7 months, 39.6% and 14.2% in patients of the AFP response group and 3.7 months, 7.8% and 2.0% in patients of the AFP no response group, showing a significant difference in progression free survival between them ( χ2=43.154, P<0.05). The median overall survival time, 1-year and 2-year overall survival rates were not reached, 85.2% and 56.3% in patients of the AFP response group and 14.6 months, 56.3% and 14.5% in patients of the AFP no response group, showing a significant difference in overall survival between them ( χ2=33.899, P<0.05). (3) Analysis of factors affecting patient prognosis. Results of multivariate analysis showed that invasion of large blood vessels, extrahepatic metastasis, combined hepatic artery intervention therapy, and AFP response were independent factors influencing progression free survival in patients with intermediate-to-advanced HCC who were treated with TKIs in combination with α-PD-1 ( hazard ratio=1.474, 1.584, 0.631, 0.367, 95% confidence interval as 1.069-2.033, 1.159-2.167, 0.446-0.893, 0.261-0.516, P<0.05), and Eastern Cooperative Oncology Group score, invasion of large blood vessels, extrahepatic metastasis, and AFP response were independent factors influencing overall survival in patients with intermediate-to-advanced HCC who were treated with TKIs in combination with α-PD-1 ( hazard ratio= 1.347, 1.914, 1.673, 0.312, 95% confidence interval as 1.041-1.742, 1.293-2.833, 1.141-2.454, 0.197-0.492, P<0.05). Conclusions:AFP response at 6-8 weeks after treatment can effectively evaluate anti-tumor efficacy of TKIs in combination with α-PD-1 for intermediate-to-advanced HCC. AFP response is the independent factor influencing progression free survival and overall survival in patients with intermediate-to-advanced HCC who were treated with TKIs in combination with α-PD-1.

Objective To investigate the expression of fibroblast growth factor (FGF)19 in neonatal hyperbilirubinemia and its correlation with intestinal flora characteristics and β-glucuronidase (β-GD) activity. Methods Seventy-five neonates with hyperbilirubinemia in Daqing Longnan Hospital from January 2022 to January 2023 were selected as the observation group and 45 neonates with non-hyperbilirubinemia during the same period were selected as the control group.Serum FGF19,total bilirubin and total bile acid levels,intesti-nal flora characteristics and β-GD activity were compared between the two groups.The correlation between FGF19,intestinal flora and β-GD activity with serum total bilirubin level and total bile acid (TBA) level in newborns with hyperbilirubinemia and the correlation between FGF19 with the intestinal flora characteristics and β-GD activity were analyzed by the Pearson method.Results The serum FGF19 level in the observation group was lower than that in the control group,the total bilirubin and TBA levels and β-GD activity were higher,the number of bifidobacterium and Lactobacillus were much less,the number of Escherichia coli was more,and the differences were statistically significant (P<0.05).The FGF19 level and number of bifidobac-terium and Lactobacillus in newborns with hyperbilirubinemia were negatively correlated with the levels of se-rum total bilirubin and TBA levels,while the number of Escherichia coli and β-GD activity were positively cor-related with the levels of serum total bilirubin and TBA (P<0.05).FGF19 in hyperbilirubinemia neonates was positively correlated with the number of bifidobacterium and Lactobacillus,and negatively correlated with Escherichia coli and β-GD activity (P<0.05).Conclusion The serum FGF19 in neonates with hyperbilirubinemia is abnormally low expressed,moreover which is related to intestinal flora disturbance and β-GD activity.

OBJECTIVE To observe the effect of Qiai(Artemisia argyi Levl.et Van.var.argyi cv.Qiai)essential oil on the healing of infected skin wounds and explore its mechanism.METHODS 30 Kunming mice were randomly divided into five equal groups:blank control group,substrate group,positive control group(3 mL·kg-1 Mupirocin ointment),low-dose group(each patch containing 7.3 mg of Qiai essential oil),and high-dose group(each patch containing 57.6 mg of Qiai essential oil).After anesthe-sia,a 10 mm full-thickness circular skin wound was created on the back of each mouse.Following hemostasis,100 μL of Staphylococ-cus aureus bacterial solution(1.5×108 CFU·mL-1)was applied to the wound.The wounds in each group were treated in the afore-mentioned manner,the dressing was changed daily,and the healing of the skin wound was observed.On the 12th day post-surgery,the mice were euthanized and the skin around the wounds was collected for HE and Masson staining to observe pathological features;qPCR and Western blotting were used to detect the expression of genes related to wound healing(Col1a1,Col3a1,Fn1,VEGFA),in-flammatory factors(IL-1β,IL-6,TNF-α),and the PI3K/Akt signaling pathway in the wound skin tissue.Further,in NIH-3T3 mouse embryonic fibroblasts,the potential impact of Artemisia argyi volatile oil on PI3K/Akt signaling pathway activity was investigated using the PI3K inhibitor PI-103.RESULTS Animal experiments have shown that compared to the blank control group,the relative wound area in both the low-dose and high-dose groups significantly decreased;the infiltration of inflammatory cells in the wound tis-sues reduced,while the expression of collagen fibers increased;qPCR showed that,compared to the blank control group,the expres-sion of VEGFA,Col3a1,and Col1a1 in the low-dose and high-dose groups significantly increased,while the expression of inflamma-tory factors IL-1β and TNF-α decreased;further Western blotting revealed that compared to the blank control group,both the phos-phorylated PI3K/total PI3K ratio and the phosphorylated Akt/total Akt ratio in the low-dose and high-dose groups significantly in-creased in a dose-dependent manner.Cellular experimental results also confirmed that the volatile oil from Artemisia argyi exerts a reg-ulatory effect on the PI3K/Akt signaling pathway in NIH-3T3 cells.CONCLUSION The essential oil of Qiai can promote wound healing,with its mechanism possibly involving the activation of the PI3K/Akt signaling pathway,suppression of inflammatory factor ex-pression,and promotion of collagen expression.

Objective:To explore the clinical features and genetic etiology of a child with Central core disease (CCD).Methods:A child with CCD who was treated at the Children′s Hematology Department of the First Affiliated Hospital of Zhengzhou University in February 2022 was selected as the study subject. Muscle biopsy was performed. Peripheral blood samples were collected from the child and his parents for the extraction of genomic DNA. The child was subjected to whole exome sequencing (WES), and candidate variant was verified by Sanger sequencing.Results:The child, a 12-year-old boy, had manifested motor retardation, facial weakness, ptosis, pectus carinatum, scoliosis, etc. Muscle biopsy showed that the central nucleus muscle fibers and atrophic muscle fibers were mainly type I. WES revealed that the child has harbored c. 10561G>A (p.G3521S) and c. 3448T>C (p.C1150R) compound heterozygous variants of the RYR1 gene. Sanger sequencing confirmed that they were inherited from his mother and father, respectively. Based on the guidelines from the American College of Medical Genetics and Genomics, both variants were considered as likely pathogenic (PS4+ PM1+ PM2_Supporting+ PP3; PM1+ PM2_Supporting+ PM3+ PP3). Conclusion:By combining his clinical manifestation and results of muscle pathology and genetic testing, the child was diagnosed with CCD, which may be attributed to the c. 10561G>A (p.G3521S) and c.3448T>C (p.C1150R) compound heterozygous variants of the RYR1 gene.

Objective:To explore the clinical features, treatment and prognosis of refractory hyponatremia caused by secondary adrenal cortical insufficiency (SACI) after chemotherapy in children with tumors of the lymphoid hematopoietic system.Methods:Clinical data including age, sex, serum sodium level, time of onset of hyponatremia, duration of hyponatremia, cortisol level, 24 h urinary sodium level, plasma/urine osmotic pressure, treatment and prognosis of 3 acute lymphoblastic leukemia (ALL) children and 1 lymphoblastic lymphoma (LBL) child with SACI-caused refractory hyponatremia treated at the First Affiliated Hospital of Zhengzhou University from January 2020 to June 2023 were analyzed retrospectively.Results:The 4 patients were all boys, and they received Dexamethasone chemotherapy.The primary clinical manifestations were fatigue, moderate hyponatremia in 3 cases, and mild hyponatremia in 1 case.Hyponatremia occurred after 1 week of glucocorticoid therapy and persisted for 10-20 days (mean 14.25 days).The 24 h urinary sodium level increased, plasma osmotic pressure slightly decreased or was at the low limit of the normal range, and urine osmotic pressure was greater than plasma osmotic pressure.The serum cortisol level at 8∶00 AM.decreased in 1 LBL case, increased in 1 ALL case, and remained normal in 2 ALL cases.After treatment with Hydrocortisone [10 mg/(m 2·d), taken orally in 2 doses], hyponatremia was quickly corrected within 3-5 days. Conclusions:When receiving glucocorticoid therapy, children with tumors of the lymphoid hematopoietic system may have SACI, and the hyponatremia caused by it generally appears 1 week after glucocorticoid therapy and persists for a long time.Laboratory tests are similar to the syndrome of inappropriate antidiuretic hormone secretion, but the response to intravenous/oral supplementation of concentrated sodium and water restriction treatment is poor.The addition of Hydrocortisone can quickly correct hyponatremia, and the prognosis is excellent.

Objective:To study the expression level of peripheral blood microRNA(miRNA)-21 and miRNA-192 in patients with papillary thyroid carcinoma (PTC) and their relationship with pathological features and prognosis.Methods:A total 107 PTC patients admitted to Huaian Cancer Hospital in Jiangsu Province from June 2018 to June 2021 were selected as the PTC group, another 76 healthy individuals who underwent physical examination during the same period were selected as the control group. Real-time fluorescence quantitative PCR (qRT-PCR) was used to measure the expression levels of miRNA-21 and miRNA-192 in the peripheral blood of two populations, and the expression of miRNA-21 and miRNA-192 in the peripheral blood of the two populations and patients with different characteristics was compared and analyzed. Kaplan-Meier (K-M) method was used to analyze the relationship between the expression levels of miRNA-21, miRNA-192 in peripheral blood of PTC patients and prognosis.Results:The expression level of miRNA-21 in the peripheral blood of patients in the PTC group (2.21 ± 0.64) was higher than that in the control group (1.01 ± 0.02), while miRNA-192 (0.42 ± 0.14) was lower than that in the control group (0.99 ± 0.03, t = 16.33, 34.90, P < 0.001). In different clinical stages, the expression level of miRNA-21 in peripheral blood of patients in stages Ⅲ - Ⅳ was higher than that in stages Ⅰ - Ⅱ, and the level of miRNA-192 was lower than that in stages Ⅰ - Ⅱ ( t = 9.98, 11.75, P < 0.001). In the case of lymph node metastasis, the expression level of miRNA-21 in peripheral blood of patients with lymph node metastasis was higher than that of the group without lymph node metastasis, and the level of miRNA-192 was lower than that of the group without lymph node metastasis ( t = 12.81, 18.75, P < 0.001). There was no statistically significant difference in the expression levels of miRNA-21 and miRNA-192 in peripheral blood of patients with different genders, ages, body mass index, and tumor sizes ( t = 0.57, 0.42, 0.54, 0.62, 1.15, 1.47, 0.74, 1.13, P > 0.05). The K-M survival curve results showed that the progression free survival time (39.02 months) with high miRNA-21 expression was lower than that with low miRNA-21 expression (55.97 months, P = 0.026); the progression free survival time (34.97 months) with low miRNA-192 expression was lower than that with high miRNA-192 expression (57.04 months, P = 0.008). Conclusion:The high expression of miRNA-21 and low expression of miRNA-192 in peripheral blood of PTC patients, and they are closely related to the clinical stage, lymph node metastasis, and prognosis of patients.