Clinical Significance of Cytokeratin 19 and Cytokeratin 20 in Predicting Recurrence of Bladder Transitional Cell Carcinoma

Objective： To investigate the expressions of cytokeratin 19 （CK19） and cytokeratin 20 （CK20） in bladder transitional cell carcinoma （TCC） and their clinical significance. Methods： The expression of CK19 and CK20 was detected in 54 cases of TCC by immunohistochemical methods and image processing techniques. Results： The expression of CK19 and CK20 was significantly stronger in the recurrent group than in the non-recurrent group （P〈0.01, P〈0.001, respectively）. Conclusion： The expression of CK19 and CK20 was obviously related with biological behaviors of TCC, suggesting that CK19 and CK20 could be used to predict the recurrence of TCC.

Myocardial metastasis from ZEB1-and TWIST-positive spindle cell carcinoma of the esophagus:A case report

BACKGROUND Metastatic cardiac tumors are known to occur more frequently than primary cardiac tumors,however,they often remain asymptomatic and are commonly dis-covered on autopsy.Malignant tumors with a relatively high frequency of cardiac metastasis include mesothelioma,melanoma,lung cancer,and breast cancer,whereas reports of esophageal cancer with cardiac metastasis are rare.CASE SUMMARY The case of a 60-year-old man who complained of dysphagia is presented.Upper gastrointestinal endoscopy showed a submucosal tumor-like elevated lesion in the esophagus causing stenosis.Contrast-enhanced computed tomography showed left atrial compression due to the esophageal tumor,multiple liver and lung metastases,and a left pleural effusion.Pathological examination of a biopsy speci-men from the esophageal tumor showed spindle-shaped cells,raising suspicion of esophageal sarcoma.The disease progressed rapidly,and systemic chemotherapy was deemed necessary,however,due to his poor general condition,adminis-tration of cytotoxic agents was considered difficult.Given his high Combined Positive Score,nivolumab was administered,however,the patient soon died from the disease.The autopsy confirmed spindle cell carcinoma(SCC)of the esophagus and cardiac metastasis with similar histological features.Cancer stem cell markers,ZEB1 and TWIST,were positive in both the primary tumor and the cardiac metastasis.CONCLUSION To the best of our knowledge,there have been no prior reports of cardiac metastasis of esophageal SCC.This case highlights our experience with a patient with esophageal SCC who progressed rapidly and died from the disease,with the autopsy examination showing cardiac metastasis.

Transitional cell carcinoma of the ovary – A case report with review of literature

Transitional cell carcinoma （TCC） of the ovary is a rare and recently recognized subtype of ovarian epithelial cancer. We presented the first case report from our Institute （Sheri Kashmir Instiute of Medical Sciences, Srinagar, India）, which was initially rnisdiagnosed as stromal cell carcinoma （granulosa cell tumour）, and on review of histopathology with immunohistochemistry, the diagnosis of TCC of the ovary was established. The aim of this article was to describe the typical case of primary TCC of the ovary and to review the literature for information on TCC management.

Expression and Significance of PTEN in Bladder Transitional Cell Carcinoma

Objective： To explore the expression of anti-oncogene PTEN in bladder transitional cell carcinoma and its clinical significance. Methods： Using immunohistochemical S-P methods, the expression of PTEN gene was detected in 62 specimens of bladder cancer and 18 specimens of normal bladder tissue. Results： In the 62 bladder cancers, the positive rate of PTEN was 53.2% （33/62）. All 18 normal bladder tissues were positive for the PTEN expression. The expression of PTEN was negatively correlated to tumor grades （P〈0.05）. Conclusion： The reduced expression of PETN might play an important role in carcinogenesis and progression of bladder cancer. Detection of PTEN might be useful for judgement of tumor development and prognosis.

Expression of KAI1/CD82 and MRP-1/CD9 in Transitional Cell Carcinoma of Bladder

The expression of KAI1/CD82 and MRP-1/CD9 in transitional cell carcinoma of bladder （TCCB） and its clinical significance were investigated. Immunohistochemistry was used to detect KAI1/CD82 and MRP-1/CD9 protein expression in 52 TCCB specimens. Correlation between the expression of KAI1/CD82 and MRP-1/CD9 to clinicopathologic factors was statistically analyzed. The results showed that the positive rate of KAI1/CD82 and MRP-1/CD9 in TCCB was 50% and 61.5%, respectively. The MRP-1/CD9 and KAI1/CD82 expression was significantly associated with grade of TCCB （P〈0.05）, but no correlation was found between MRP-1/CD9 or KAI1/CD82 expression and clinical stage of TCCB （P〉0.05）. The expression level of MRP-1/CD9 and KAI1/CD82 in recurrent TCCB samples was lower than that in non-recurrent samples （P〈0.05）. Meanwhile, the correlation between the KAI1/CD82 expression and MRP-1/CD9 expression was statistically significant （r=0.316, P〈0.05）. It was concluded that KAI1/CD82 and MRP-1/CD9 expression may be important prognostic indicators and potentially useful for assessing the biological behavior of TCCB.

MTHFR C677T polymorphisms are associated with aberrant methylation of the IGF-2 gene in transitional cell carcinoma of the bladder

The purpose of this study was to determine the relationship between methylation status of the insulin-like growth factor 2 （IGF-2） gene and methylenetetrahydrofolate reductase （MTHFR） C677T gene polymorphisms in bladder transitional cell carcinoma tissues in a Chinese population. The polymorphisms of the folate metabolism enzyme gene MTHFR were studied by restrictive fragment length polymorphism （RFLP）, PCR-based methods of DNA methylation analysis were used to detect the CpG island methylation status of the IGF-2 gene. The association between the methylation status of the IGF-2 gene and clinical characteristics, as well as MTHFR C677T polymorphisms, was analyzed. Aberrant hypomethylation of the IGF-2 gene was found in 68.3% bladder cancer tissues and 12.4% normal bladder tissues, respectively, while hypomethylation was not detected in almost all normal bladder tissues. The hypomethylation rate of the IGF-2 gene in cancer tissues was significantly higher in patients with lymph node metastasis than in those without lymph node metastasis （46.3% vs 17.2%, P = 0.018）. No association was found between aberrant DNA methylation and selected factors including sex, age, tobacco smoking, alcohol consumption and green tea consumption. After adjusting for potential confounding variables the variant allele of MTHFR C677T was found to be associated with hypomethylation of the IGF-2 gene. Compared with wildtype CC, the odds ratio was 4.33 （95% CI=1.06-10.59） for CT and 4,95 （95% CI=1.18-12.74） for TT. MTHFR 677 CC and CT genotypes might be one of the reasons that cause abnormal hypomethylation of the IGF-2 gene, and the aberrant CpG island hypomethylation of the IGF-2 gene may contribute to the genesis and progression of bladder transitional cell carcinoma.

Plasmacytoid Transitional Cell Carcinoma of Bladder： A Clinico-pathological Study and Review of Literatures

Objective： To study the pathologic features of plasmacytoid transitional cell carcinoma of the bladder, and to analyze the diagnostic features, criteria for differential diagnosis and the clinical significance of the tumor. Methods： Two cases of bladder plasmacytoid transitional cell carcinoma were studied. Routine paraffin sections with HE staining, Pap smear and immunohistochemistry by S-P method were observed under a light microscope. Pathological and clinical data were analyzed by comparison with early reported cases in literatures. Results： A characteristic feature of this tumor was of deep invasion in the lamina propria and/or muscularis propria, in addition to the component of carcinoma in situ in the mucosa, when tumors were diagnosed. The histological pattern and cytological features showed similarity to a plasmacytoid tumor. The tumor cells were strongly positive for AE1/AE3, CEA and CK18. The prognosis appeared to be worse than ordinary transitional cell carcinoma. Conclusion： The plasmacytoid transitional cell carcinoma of bladder is rare but has typical pathological, immunohistological and clinical features. Pathologists should be aware of this kind of primary tumor of bladder.

RECURRENCE RISK FACTORS IN PATIENTS WITH TRANSITIONAL CELL CARCINOMA OF BLADDER

Objective： To study recurrence factors and set up a model to evaluate the prognosis of patients with bladder cancer. Methods： An analysis on recurrence-related factors was made by Cox＇s proportional hazards model analysis and logistic multiple linear regression model analysis in 212 patients with transitional cell carcinoma treated surgically from 1995-2001. These factors included clinical and pathologic figures. Results： The most important factor is metastasis to the regional lymph nodes, the Hazards ratio is 6.6 （P=0.0004）, followed by multiple tumors （Hr=2.255, P〈0.0001）, tumor in trigone and bladder neck （Hr=2.053, P〈0.0001）, stage （Hr=2.057, P〈0.0001）, grade （Hr=1.569, P=0.0081）, intravesical chemotherapeutic instillations （Hr-0.559, P=0.0011） and hematuria （Hr=0.762, P=0.0076）. A predicting equation was established, and the predicting values were calculated according to the individual features of patients. The predicting and actual values were compared, and the sensitivity, specificity and overall concordance were 83.5%, 67.6% and 80.1% respectively. Conelusion：The evaluation of prognosis could be made quite accurately based on these factors.

Research on the prognosis of different types of microvessels in bladder transitional cell carcinoma

BACKGROUND At present,there is controversy on the role of microvessel density(MVD)in tumors as a prognostic indicator of bladder transitional cell carcinoma(BTCC).However,the MVD in tumors is simply classified based on the expression of several different vascular markers,which has not been related to analytical research on the prognosis of patients with BTCC.AIM To explore the classification of blood vessels in tumors and studied the relationship between MVD and the prognosis of patients with BTCC.METHODS The tissue mass was detected by tissue microarray and immunohistochemical analysis with monoclonal antibodies against CD31,CD34,CD105,and vascular smooth muscle actin to investigate the MVD in BTCC.The measurement data are expressed as the mean±SD.The difference between the groups was analyzed by the t-test,the counting data were analyzed byχ2 test.The Kaplan-Meier survival curve was estimated by the product-limit method.The log-rank time-series test was employed to compare the tumor-free survival curves.RESULTS The MVD was closely related to the pathological grade,invasive depth,and prognosis of BTCC.Significant differences were found between grade I and grade II,grade II and grade III,superficial and invasive type,and the tumor-free survival group and the recurrence or metastasis group(P<0.01).Multivariate analysis showed that undifferentiated MVD was an independent prognostic factor for patient survival time.An inverse correlation between undifferentiated tumor MVD and differentiated tumor MVD in BTCC was also shown.CONCLUSION The classification of blood vessels in BTCC could act as an important prognostic indicator and may also be of great significance in the treatment of cancer.

The Expression and Significance of KAl1 and Ki67 in Bladder Transitional Cell Carcinoma

OBJECTIVE To explore the expression and significance of the tumor metastatic suppressor gene the KAI1 and Ki67 antigen in bladder transitional cell carcinoma （BTCC）. METHODS Two-stepped immuno-histochemical staining was used to detect the expression of the KAI1 protein and Ki67 antigen. The reverse transcription polymerase chain reaction （RT-PCR） was used to detect the KAI1 mRNA in 54 BTCC specimens and 32 normal bladder counterparts. 13-actin was the internal control. RESULTS The KAI1 protein was mainly expressed on cell membranes at cell-to-cell borders, with uniform and continuous staining in normal bladder transitional epithelium. However, the number of positive-staining cells was greatly decreased in BTCC epithelium, and with an increase in the stage and Grade and appearance of lymph node metastasis the staining was non-uniform and discontinuous. The Ki67 antigen was expressed in the nucleus, and with an increase in the stage and Grade and appearance of lymph node metastasis, the Ki67 expression increased. The Ki67 antigen was negatively related to the expression of KAI1 （P〈0.01）.The expression level of KAI1 mRNA was much greater in normal bladder transitional epithelium compared to BTCC, moreover, with an increase in the Grade, infiltration depth and appearance of lymph node metastasis, the expression of KAI1 mRNA was reduced. CONCLUSION The expression of KAI1 protein may be used as a prognostic marker to indicate the degree of infiltration and metastasis. Measurement of KAI1 and Ki67 expression together may be helpful in evaluating the metastatic potential and prognosis of BTCC patients.

Simultaneous occurrence of transitional cell carcinoma and renal cell carcinoma in the same kidney:a casereport and review of the literature

This article reports a case of simultaneous occurrence of 2 primary renal tumors of different histology, a transitional cell carcinoma and a renal cell carcinoma. in the same kidney. The histological, immunohistochemical and ultrastructural changes of the tumors were described. A review of the literature to date revealed this case to be rare. only 24 other cases were reported previously.

Cloning of Human Uroplakin Ⅱ Gene from Chinese Transitional Cell Carcinoma of Bladder and Construction of Its Eukaryotic Expression Vector

Summary: To clone Uroplakin Ⅱ gene from Chinese transitional cell carcinoma (TCC) of bladder and construct its eukaryotic expression vector, the molecular cloning method was used to extract total RNA from a GⅢ/ T 3N 0M 0 tissue sample of the bladder TCC patients. The primers were designed by Primer 5.0 software. Full length cDNA of Uroplakin Ⅱ gene was amplified by reverse transcription polymerase chain reaction (RT-PCR), assayed by nucleic acid sequencing and then inserted between XbaⅠ and HindⅢ restrictive sites of eukaryotic expression vector pcDNA3.0. The recombinant was assayed by restricted enzyme digestion. Under the induction of Lipofectamine 2000, the recombinant was transfected into Uroplakin Ⅱ negative bladder cancer cell line EJ. Cellular expression levels of Uroplakin Ⅱ were detected by RT-PCR. The nucleic acid sequencing results indicated that Chinese Uroplakin Ⅱ cDNA (555 bp) was successfully cloned. The BLAST analysis demonstrated that the cloned sequence is 100 % homologous with sequences reported overseas. The GenBank accession number AY455312 was also registered. The results of restricted enzyme digestion indicated that eukaryotic vector pcDNA-UPⅡ for Uroplakin Ⅱ was successfully constructed. After being transferred with pcDNA-UPⅡ for 72 h, cellular Uroplakin Ⅱ mRNA levels were significantly improved (P<0.01). It is concluded that human Uroplakin Ⅱ gene was successfully cloned from Chinese TCC tissues, which provided a basis for further exploration of the roles of Uroplakin Ⅱ gene in TCC biological behaviors and potential strategies for targeted biological therapy of TCC.

Expression of connexin43 and its correlation with the expressions of Bcl-2 and Bax proteins in transitional cell carcinoma of the bladder

Objective: To study the expression of connexin43 (Cx43) gene and its correlation with the apoptosis related genes Bcl-2 and Bax in transitional cell carcinoma of the bladder (BTCC), and to investigate the role of Cx43 in the BTCC. Methods: Reverse transcription polymerase chain reaction was used to detect the expression of Cx43 mRNA and immunocytochemistry technique was used to detect the expressions of Bcl-2 and Bax proteins in 60 cases of BTCC tissues, and compared with that of 15 cases of pericancerous tissues and 15 cases of normal bladder tissues. Results: The positive expression rate of Cx43 mRNA in 60 cases of BTCC tissues was 43.33% which was significantly lower than that in pericancerous tissues (73.33%) and normal tissues (100%) (χ2 = 17.58, P < 0.01). The expression of Cx43 had significant negative correlation with the pathological degree and lymph node metastasis of BTCC (χ2 = 9.33 and 9.74, respectively, P < 0.01). However, no correlation was found between the expression and patient sex, age, clinical staging and diameter and growth pattern of BTCC (P > 0.05). Expression of Cx43 negatively correlated with Bcl-2 protein (r = -0.63, P < 0.01) and positively correlated with Bax protein (r = 0.52, P < 0.01). Conclusion: The down-regulated expression of Cx43 gene was closely associated with the development, invasion and metastasis of BTCC. It could be used as a prognostic indicator for BTCC. Cx43 gene may have antagonistic effects with Bcl-2 gene and synergic with Bax gene in the initiation and progression of BTCC.

Differential expression of microRNA clusters in bladder transitional cell carcinoma

Objective： The aim of the study was to investigate the differential expression of microRNAs （miRNAs） in bladder transitional cell carcinoma （BTC）. Methods： Fresh tissues were obtained from patients with BTC （9 cases; 3 cases with grade Ⅰ, 3 cases with grade Ⅱ, 3 cases with grade III） and those with normal bladder mucosa （3 cases） and stored in liquid nitrogen. Total RNA was extracted using TRizol reagent and RNA was quantified and quality control was performed, miRNA probes were labeled with Hy3TM fluorescence, then hybridized with a miRCUR^M array labeling kit. miRNAarrays were scanned and analyzed and the scanned result was validated using reverse transcription-polymerase chain reaction （RT-PCR）. Results： In four groups of differentially expressed genes obtained from grade Ⅰ, grade Ⅱ, grade Ⅲ, and grade Ⅰ ＋ grade Ⅱ ＋ grade Ⅲ BTC tissues compared with normal bladder mucosa, hsa-miR-29b-1＊ was upregulated, and hsa-miR-923 and hsa-miR-300 were downregulated. The hsa-miR-29b-1＊, hsa-miR-300, and hsa-miR-923 findings were confirmed by real-time RT-PCR. Conclusion： Genes that were differentially expressed between BTC and normal bladder mucosa may be involved in the pathogenesis and development of BTC, and may be useful for further studies of BTC-related genes.

MDR1/P-Glycoprotein Overexpression in Bladder Transitional Cell Carcinoma and its Correlation with Expression of Survivin and Fas

OBJECTIVE To explore the expression of the MDR1/P-glycoprotein, Fas and survivin and to examine their correlation with the biologic behavior of bladder transitional cell carcinoma (BTCC). METHODS Immunohistochemistry was used to examine the expression of P-gp, survivin and Fas in BTCC (n=64) and normal bladder mucosa (n=12). RESULTS The expression level of P-gp and survivin in BTCC was higher compared to normal bladder mucosa (P<0.01) and their expression was strongly correlated with clinical grading (P<0.01). In BTCC and normal bladder mucosa Fas expression was 50% and 100%, respectively (P< 0.01). Recurrent BTCC showed higher expression than primary BTCC (P< 0.01) and the expression of P-gp in BTCC had a reverse correlation with Fas expression but no correlation with survivin expression. CONCLUSUON The MDR of BTCC was strongly correlated with the ex- pression of P-gp and Fas, but was not correlated with survivin expres- sion. Thus, enhancing cancer sensitivity to chemotherapy by reversing multidrug resistance with reversal agents or up-regulating Fas expres- sion by apoptotic enhancing agents, might be a potential therapy to pre- vent tumor recurrence and invasiveness.

Neoadjuvant Chemotherapy for Transitional Cell Carcinoma of the Bladder： A Single Centre Experience

Urothelial cancers usually recur distantly rather than loco-regionally. In patients with pT2 and pT3/pT4 tumors, local recurrence has been observed in 3-4% and 11-16%, respectively, whereas distant failure has occurred in 10-27% and 19-35%, respectively. Despite local therapy most patients with muscle invasive transitional cell carcinoma （TCC） of the bladder die of systemic relapse, indicating a need for effective adjunctive systemic treatment. We determined whether neoadjuvant chemotherapy improved overall survival. This study evaluated the role of neoadjuvant combination chemotherapy with gemcitabine/cisplatin （GC） in improving the outcome of this group of patients. A total of 44 patients （84% Male, 16% Female） with newly diagnosed bladder cancer （T3-4, N0-2, M0） were subjected to initial 3 cycles of GC, then managed according to response. Patients were assessed clinically after each cycle and by Interim CT scan after 3 cycles of chemotherapy and those who achieved complete or partial response underwent radical cystectomy. We enrolled 63 patients, 19 of whom were found to be ineligible; thus, 44 were assigned to receive neoadjuvant chemotherapy followed by surgery. Average size of the largest tumor was greater that 30mm in 77% patients. According to Computed Tomographic findings 70% patients belonged to Stage T4A. The overall response rate to GC was 50%, and incomplete response was achieved in 25% whereas 25% patients were lost to follow up. Twenty two patients who had complete response, underwent cystectomy and diversion. It was observed that those patients who underwent radical cystectomy with ureterosigmoidostomy had an increased serum creatinine in comparison to patients who had ileal conduit. The size of the effect is modest and combination chemotherapy can be administered safely without adverse outcomes resulting in delayed local therapy. Further efforts to identify the patients most likely to benefit from neoadjuvant therapy are necessary to optimize its use.

Expressions and clinical significances of MMP-2 and TIMP-2 mRNA in bladder transitional cell carcinomas

Objective:The aim of the study was to investigate the expressions of matrix metalloproteinase-2(MMP-2) and tissue inhibitor of MMP-2(TIMP-2) mRNA in transitional cell carcinomas of bladder and discuss their clinical significances.Methods:Using RT-PCR and real time quantitative PCR(RQ-PCR) technique,the expressions of MMP-2 and TIMP-2 mRNA of 45 cases of bladder carcinoma(tumor group) and 10 cases of normal bladder tissue(control group) were analyzed.Results:MMP-2 and TIMP-2 were not expressed in control group.MMP-2 was expressed in 30 cases tumor samples and TIMP-2 was expressed in 26 cases.The expression of MMP-2 mRNA in non-muscle invasive bladder cancers and grade I cancers was lower than that in muscle invasive bladder cancers and grades II-III cancers respectively.The differences were statistically significant(P<0.05).The expression of MMP-2 in recurrent patients was higher than that in incipient patients.TIMP-2 mRNA expression decreased with grades and stage.The expression of TIMP-2 in non-muscle invasive bladder cancers and grade I cancers was higher than that in muscle invasive bladder cancers and grades II-III cancers respectively.There was statistical difference between two groups(P < 0.05).TIMP-2 expression in incipient patients was higher than that in recurrent patients,but the difference was not significant(P>0.05).Conclusion:These results suggest that MMP-2 and TIMP-2 play an important role in the invasion step of transitional cell carcinoma of bladder.MMP-2 may become a new approach to the diagnosis of bladder carcinoma.

Relationship of A,B,H blood group antigens with pathomorphological grading and prognosis of transitional-cell carcinoma of the urinary bladder

After being labelled with monoclonal antibodies against A,B, H blood group antigens,100 specimens of transitional-cell carcinoma of the urinary bladder were studied with ABC immunohistochemical technique and the cases were followed up.It was found that the overall positive rate of A,B,H antigens was 63%. The mortality rate and recurrence were significantly lower in the positive group than in the negative group(P<0.01)and 5-years survival rate was higher in the positive than in the negative(P<0.01).The findings suggest that the expression of blood group antigens is more effective for the prognosis of transitional-cell bladder carcinoma than the pathomorphological grading.

Long-term follow-up of Ta transitional cell carcinoma of bladder after treatment of TURBt plus intravesical therapy

To stduy the association between the prognosis of Ta transitional cell carcinoma (TCC) of the bladder and risk-related factors.Methods A total of 88 cases (62 males and 26 females;mean age,61 years;age range,41-81 years) of initial T.TCC of the bladder treated with transurethral resection of bladder tumor (TURBt) plus intravesical chemotherapy or immunotherapy were enrolled.Among them,there were 26 cases of G1,61 cases of G2 and 1 case of G3.For tumor site,62 cases (16 cases of G1,45 of G2,1 of G3) had single tumor and 26 cases (10 cases of G1,16 of G2) had multi-site tumors.The mean follow-up was 113 months (range,56-168 months).The tumor grade,original tumor number and their association with the recurrence and progression of this type of TCC were retrospectively analyzed.Results The overall recurrence rate (RR) was 60% (53/88).In single tumor group,RR of G1 cases was 25% (4/16);RR of G2 cases was 62% (28/45) and the total RR was 52% (32/62).In multi-site tumor group,RR of G1 cases was 80% (8/10),RR of G2 cases was 75% (12/16) and the total RR was 77% (20/26).The RR of multi-site tumor group was significantly higher than that of single tumor group (P<0.01).In single tumor group,RR of G2 cases was significantly higher than that of G1 cases (P<0.001).In multi-site tumor group,there was no association of RR with tumor grade.There was no progression in G1 tumor cases.The progression rate was 42.5% (17/40) in G2 tumor cases;among them,30% (12/40) progressed to T1G2 tumors and 12.5% (5/40) progressed to T2G2 tumors.The RR of cases who received thiotepa,mitomycin and BCG were 75% (12/16),68% (30/44) and 40% (11/27),respectively.Tumor specific mortality was 1.14% (1/88,a T2G3 case).Conclusion The multi-site Ta TCC of the bladder has relatively higher RR and greater chance of progression after the treatment of TURBt plus intravesical chemotherapy or immunotherapy,especially in the poor differentiated tumors,thus active treatment and close follow-up are essential in clinical practice.9 refs.

Application of single-cell RNA sequencing in head and neck squamous cell carcinoma

Single-cell RNA sequencing has been broadly applied to head and neck squamous cell carcinoma(HNSCC) for characterizing the heterogeneity and genomic mutations of HNSCC benefiting from the advantage of single-cell resolution. We summarized most of the current studies and aimed to explore their research methods and ideas, as well as how to transform them into clinical applications. Through single-cell RNA sequencing, we found the differences in tumor cells’ expression programs and differentiation tracks. The studies of immune microenvironment allowed us to distinguish immune cell subpopulations, the extensive expression of immune checkpoints, and the complex crosstalk network between immune cells and non-immune cells. For cancerassociated fibroblasts(CAFs), single-cell RNA sequencing had made an irreplaceable contribution to the exploration of their differentiation status, specific CAFs markers, and the interaction with tumor cells and immune cells. In addition, we demonstrated in detail how single-cell RNA sequencing explored the HNSCC epithelial-tomesenchymal transition(EMT) model and the mechanism of drug resistance, as well as its clinical value.