Microarray Gene Expression Analysis of Tumorigenesis and Regional Lymph Node Metastasis in Laryngeal Squamous Cell Carcinoma

Background

Laryngeal squamous cell carcinoma (LSCC) is the most common type in head and neck squamous cell carcinoma (HNSCC), and the development and progression of LSCC are multistep processes accompanied by changes of molecular biology.

Objective

The purpose of this study was to investigate the molecular basis of tumorigenesis and regional lymph node metastasis in LSCC, and provide a set of genes that may be useful for the development of novel diagnostic markers and/or more effective therapeutic strategies.

Methods

A total number of 10 patients who underwent surgery for primary laryngeal squamous cell carcinoma were recruited for microarray analysis. LSCC tissues compared with corresponding adjacent non-neoplastic tissues were analysed by Illumina mRNA microarrays, and LSCC tissues with regional lymph node metastasis and LSCC tissues without regional lymph node metastasis were analyzed in the same manner. The most frequently differently expressed genes screened by microarrays were also validated by qRT-PCR in another 42 patients diagnosed for LSCC.

Results

Analysed by Illumina mRNA microarrays, there were 361 genes significantly related to tumorigenesis while 246 genes significantly related to regional lymph node metastasis in LSCC. We found that the six genes (CDK1, CDK2, CDK4, MCM2, MCM3, MCM4) were most frequently differently expressed functional genes related to tumorigenesis while eIF3a and RPN2 were most frequently differently expressed functional genes related to regional lymph node metastasis in LSCC. The expressions of these genes were also validated by qRT-PCR.

Conclusions

The research revealed a gene expression signature of tumorigenesis and regional lymph node metastasis in laryngeal squamous cell carcinoma. Of the total, the deregulation of several genes (CDK1, CDK2, CDK4, MCM2, MCM3, MCM4, EIF3a and RPN2) were potentially associated with disease development and progression. The result will contribute to the understanding of the molecular basis of LSCC and help to improve diagnosis and treatment.     

乳腺癌细胞hMSH2 蛋白、CD4、CD8的表达及其与淋巴结转移的关系

目的:探讨乳腺癌细胞hMSH2蛋白、CD4和CD8表达与淋巴结转移的关系及临床意义.方法:采用免疫组织化学方法检测49例乳腺癌(淋巴结转移25例,无淋巴结转移24例)患者癌细胞hMSH2蛋白、CD4和CD8的表达,并分析其表达与淋巴结转移的关系.结果:hMSH2蛋白在乳腺癌细胞细胞核内表达,CD4和CD8在乳腺癌细胞细胞浆和细胞膜表达.淋巴结转移组与无淋巴结转移组hMSH2蛋白、CD4和CD8的表达率分别为29%和68%(P＜0.05)、58%和44%(P＞0.05)、29%和68%(P＜0.05).结论:在有淋巴结转移的乳腺癌细胞hMSH2蛋白、CD4、CD8表达的改变可能与乳腺癌淋巴结转移有关.     

Clinical Features in Differentiated Thyroid Carcinoma Stratified By Lymph Node Status

Objective: Cervical lymph node (CLN) metastases (mets) often occur in differentiated thyroid cancer (DTC), especially in the central compartment, and are a major predictor of local recurrence. We examined clinical endpoints in three groups of patients based on status of lymph node involvement: those with definite lymph node involvement (N1), negative lymph nodes (N0), and no lymph nodes resected (Nx). We correlated these endpoints with clinical and pathologic features of these patients.Methods: Medical records of 261 patients with DTC who underwent thyroidectomy between 2006 and 2018 at our center were reviewed. Lymph node status of patients was categorized based on American Joint Committee on Cancer (AJCC) 8th edition criteria as N1, N0, and Nx. We performed statistical analysis to assess the differences among these groups, using one-way analysis of variance. When significant differences were found, pairwise comparisons were conducted among the three groups. Statistical significance was defined as 2-tailed P<.05 for all tests.Results: There were significant differences among the groups in tumor multicentricity, tumor category/size, AJCC stage, and the presence of thyroglobulin auto-antibodies (TgAbs). There were no difference in age, gender, or histopathology. N1 patients had a higher incidence of multicentricity, larger tumor sizes, and were more likely to have elevated TgAbs. There were no significant differences between the N0 and Nx groups.Conclusion: This study shows that larger and multi-centric tumors are associated with increased likelihood of CLN mets in DTC. We suggest increased vigilance for CLN mets in tumors >2 cm, multicentric tumors, and patients with elevated TgAbs.Abbreviations: AJCC = American Joint Committee on Cancer; CLN = cervical lymph node; DTC = differentiated thyroid cancer; FTC = follicular thyroid cancer; mets = metastases; N0 = no cancer in any lymph nodes; N1 = cancer in a lymph node; N1a = cancer in a central compartment lymph node; N1b = cancer in a lateral neck lymph node; Nx = lymph nodes not resected or examined; PTC = papillary thyroid cancer; TgAb = thyroglobulin auto-antibody     

胃癌组织中溶菌酶的免疫组织化学及其与浸润转移的关系

应用微波－ＳＰ法免疫组化技术,检测了９７例胃癌和３０例手术胃切端正常胃粘膜组织中溶菌酶（ＬＺＭ）表达状况。其阳性率分别为８５．６％和６６．７％,前者显著高于后者（Ｐ＜０．０５）。ＬＺＭ在胃癌中的表达阳性率比较,差分化癌和粘液细胞癌均显著高于分化型腺癌（Ｐ值均＜０．０５）,有淋巴结转移性胃癌显著高于无淋巴结转移者（Ｐ＜０．００５）。在一组原发部位和淋巴结转移性胃癌配对标本中,ＬＺＭ表达无明显差异性,提示胃癌ＬＺＭ活性增高发生在淋巴结转移之前。结果表明,ＬＺＭ表达与胃癌细胞的分化程度及生物学行为密切相关     

Overexpression of phosphatase regenerating liver 3 in oesophageal squamous cell carcinoma associated with metastasis and its comparison with phosphatase regenerating liver 1

Expression of PRL3 (phosphatase of regenerating liver 3) protein was examined with immunohistochemistry in 60 cases of ESCC (oesophageal squamous cell carcinoma) with matched lymph node metastasis (n = 40) and 6 cases of oesophageal adenocarcinoma. Its associations with PRL1 and clinicopathological parameters were analysed. The results showed the frequency of PRL3 protein expression was significantly higher in ESCC (39/60, 65%) than in normal oesophageal mucosa (0/20, P < 0.001); higher in ESCC with lymph node metastasis (30/40, 75%) than in ESCC without lymph node metastasis (9/20, P = 0.022), as well as higher in metastatic ESCC in lymph node (38/40, 95%) than in the primary ESCC (39/60, 65%, P < 0.001). PRL3 was expressed in 1 out of 6 oesophageal adenocarcinomas, but showed no nuclear staining of PRL1. Expression of PRL3 protein was positively associated with the grade and partially with the stage of ESCC. These results suggest that expression of PRL3 protein may be involved in the metastasis of ESCC and serve as a biomarker for prediction of ESCC metastasis.     

Involvement of ZEB1 and E-cadherin in the invasion of lung squamous cell carcinoma

This study intended to investigate the expression of the ZEB1 and E-cadherin proteins in lung squamous cell carcinoma (LSCC) tissues and to examine the clinicopathological correlation between protein levels and LSCC. RT-PCR and Western blot were used to examine the expression of ZEB1 and E-cadherin mRNAs and proteins in LSCC tissues as well as in adjacent normal tissues, and then analyze the relationship between the clinicopathological characteristics and the expression changes of ZEB1 and E-cadherin mRNAs in LSCC. In addition, RNAi was used to knockdown the expression of the ZEB1 gene in Human HCC827 cells; subsequently, changes in the invasive ability of the resultant cells were studied. The positive rates of ZEB1 and E-cadherin mRNAs in LSCC tissues were 69.2 and 38.5 %, respectively. They differed significantly from the corresponding positive rates in the adjacent normal lung tissues (15.4 and 80.8 %, p < 0.05). There was a negative correlation between the protein levels of ZEB1 and E-cadherin in LSCC tissues (r = -0.714, p < 0.001); in addition, it was found that ZEB1 protein expression in LSCC tissues was significantly higher than that in the neighboring normal lung tissues (p < 0.05), and its expression was also significantly higher in patients with lymph node metastases and distant metastases compared to those patients without metastatic disease (p < 0.05). On the contrary, E-cadherin expression was significantly lower in LSCC tissues than that in the neighboring normal tissue (p < 0.05). It was lower in patients with lymph node metastasis and distant metastasis compared to patients without metastatic disease (p < 0.05). However, the expression of ZEB1 and E-cadherin was independent of gender, age, tumor size, or tumor differentiation level (p > 0.05). Transfection of ZEB1 siRNA into HCC827 cells significantly reduced the ZEB1 protein level (p < 0.01) and significantly elevated E-cadherin levels (p < 0.01). Moreover, significantly less ZEB1 siRNA-transfected cells migrated through Transwell chambers in the LSCC tissue than that in the control groups (untransfected or transfected with control siRNA, p < 0.01). The expression of the ZEB1 gene in LSCC tissues is downregulated with the expression of E-cadherin. On the other hand, the expression of siRNA against ZEB1 promotes E-cadherin expression and suppresses the invasive ability conferred by E-cadherin. In conclusion, our data suggested that overexpression of the ZEB1 gene is possibly associated with the occurrence, development, invasion of LSCC.     

The Expression of SIRT1 and DBC1 in Laryngeal and Hypopharyngeal Carcinomas

Rationale and Objective

Sirtuin 1 (SIRT1) plays an important role in tumorigenesis and is increased in many human tumors. DBC1 is a negative regulator of SIRT1 via promotion of p53-mediated apoptosis. It is necessary to investigate the expression of SIRT1 and DBC1 in laryngeal and hypopharyngeal squamous cell carcinomas (LSCC and HSCC) and its correlation with available clinical parameters.

Methods

The mRNA levels of SIRT1 and DBC1 were measured in 54 paired LSCC or HSCC tumors and corresponding adjacent noncancerous mucosae using quantitative RT-PCR (qRT-PCR). The protein levels of SIRT1 and DBC1 were also evaluated in 120 cases of patients with LSCC or HSCC using immunohistochemical staining. The correlation between SIRT1 and DBC1 expression and clinical parameters was analyzed with Pearson chi-square test.

Results

qRT-PCR assay showed that, compared with the paired adjacent noncancerous mucosae, SIRT1 mRNA was significantly decreased in tumors. The immunohistochemical results indicated that the SIRT1 protein was also downregulated in tumors compared with noncancerous mucosae. Moreover, decreased SIRT1 was significantly correlated with the tumor clinical stage and lymph node metastasis. Additionally, DBC1 mRNA was significantly increased in tumors compared with noncancerous mucosae. The immunohistochemical results indicated that the DBC1 protein was downregulated in tumors, which is inconsistent with the results obtained by qRT-PCR. Finally, decreased DBC1 protein was significantly correlated with tumor differentiation, lymph node metastasis, and p53 expression.

Conclusions

SIRT1 and DBC1 might be involved in the pathophysiology of laryngeal and hypopharyngeal squamous cell carcinomas and are associated with lymph node metastasis and p53 positive staining in LSCCs and HSCCs.     

A 5’-tiRNA fragment that inhibits proliferation and migration of laryngeal squamous cell carcinoma by targeting PIK3CD

tRNA-derived small RNAs (tsRNAs) participate in several biological processes, including carcinogenesis. The correlations between tsRNAs and human cancers are attracting substantial attention. Nevertheless, the involvement of tsRNAs in laryngeal squamous cell carcinoma (LSCC) progression remains unclear. We constructed tsRNAs expression profiles in LSCC and adjacent normal tissues by next-generation sequencing. Interestingly, we identified a specific 5′-tiRNA fragment (tRF-33-Q1Q89P9L842205) that was significantly downregulated and was closely associated with lymph node metastasis and advanced stages of LSCC. Importantly, we found that tRF-33-Q1Q89P9L842205 suppressed cell growth, proliferation, migration, invasion and induced apoptosis in LSCC by directly silencing phosphoinositide 3-kinase catalytic subunit (PIK3CD). We speculated that tRF-33-Q1Q89P9L842205 is a potential diagnostic biomarker for LSCC and acts as a tumor suppressor by directly targeting PIK3CD.     

Clinical predictors of right upper paraesophageal lymph node metastasis from papillary thyroid carcinoma

ABSTRACT: BACKGROUND: Central and lateral lymph node metastases are quite common in patients with papillary thyroid carcinoma, and the predictors for those metastases have been well studied. Right upper paraesophageal lymph node metastasis has rarely been studied. The aim of this study was to identify the clinicopathological characteristics that may be risk factors for right upper paraesophageal lymph node metastasis in patients with papillary thyroid carcinoma. METHODS: This was a prospective observational study of 243 patients with papillary thyroid carcinoma (PTC) who underwent total thyroidectomy and comprehensive central lymph node dissection with or without lateral lymph node dissection between April 2008 and January 2010. The clinicopathologic findings from these patients were investigated and the patterns of lymph node metastasis were analyzed in the patients who had right upper paraesophageal lymph node disease. RESULTS: Of the 243 patients undergoing lymph node dissection, 14 had right upper paraesophageal lymph node metastases. Two of these patients had right upper paraesophageal lymph node metastasis only, without central compartment metastasis. Univariate analysis of clinicopathologic findings showed that right upper paraesophageal lymph node metastasis had significant association with larger primary tumors, multifocal tumors, extrathyroid extension, and lymphatic invasion (p <0.05 for each factor). CONCLUSIONS: Although there were no independent predictors of right upper paraesophageal lymph node metastasis, it can be the only site of metastasis without other compartmental metastasis. Therefore, during surgery for patients with central or lateral lymph node metastases from PTC, it may be helpful to examine the right upper paraesophageal lymph nodes.     

抑癌基因MTUS1在结直肠癌的表达与其淋巴结转移呈负相关

微管相关肿瘤抑制基因1(MTUS1)是一个新的肿瘤抑制基因,编码血管紧张素II AT受体结合蛋白（ATIPs）. MTUS1基因表达下调可促进肿瘤细胞增殖,但MTUS1基因表达在结直肠癌转移中的作用尚不知晓.本文以接受根治性手术、经病理检查证实的50例结直肠癌患者新鲜癌组织、癌旁组织和远癌组织为材料,探索MTUS1基因表达与结直肠癌淋巴结转移的关系. 荧光定量PCR及蛋白质免疫印迹揭示,有淋巴结转移的结直肠癌患者的癌及癌旁组织MTUS1 基因表达的mRNA及蛋白质水平明显低于无淋巴结转移的患者. MTUS1 蛋白质在淋巴结转移的结直肠癌患者的癌及癌旁组织表达下调,并进一步得到免疫组织化学的验证.实验结果提示,MTUS1的表达量与肿瘤的侵润深度有明显的负相关性.结合临床资料,推断抑癌基因MTUS1在肿瘤的发生、发展及淋巴转移中发挥重要作用.     

Fascin-1, Ezrin and Paxillin Contribute to the Malignant Progression and Are Predictors of Clinical Prognosis in Laryngeal Squamous Cell Carcinoma

Aims

Fascin-1, ezrin and paxillin, cytoskeleton-associated proteins, have been implicated in several human cancers, but their role in laryngeal squamous cell carcinoma (LSCC) is unknown. We investigated the association of their expression and clinicopathologic factors and their prognostic value in LSCC.

Materials and Methods

Quantitative RT-PCR and western blot analyses were used to examine mRNA and protein levels in 10 fresh LSCC specimens and 10 corresponding adjacent normal margin (ANM) tissues from patients undergoing surgery in 2012. We used immunohistochemistry to retrospectively study 216 paraffin blocks of LSCC samples from patients (193 men) who had undergone surgery between 2000 and 2006 and had not received special treatment before the diagnosis. Univariate analysis of patient survival involved the Kaplan–Meier method. Multivariate analyses involved the Cox proportional hazards model.

Results

The relative mRNA and protein levels of fascin-1, ezrin and paxillin were significantly greater in LSCC than ANM tissue (P<0.05). The high expression of fascin-1, ezrin or paxillin was positively correlated with poor tumor differentiation, cervical lymph node metastasis (N+), and advanced clinical stage (III+IV) (P<0.05) but not sex or metastasis. In addition, a high expression of fascin-1 (P = 0.007) or ezrin (P = 0.047) was associated with advanced tumor stage (T3+T4). The expression of fascin-1 was higher in smokers than non-smokers (P = 0.019). A high expression of fascin-1, ezrin or paxillin was associated with poor prognosis.

Conclusions

Fascin-1, ezrin and paxillin may be prognostic of poor outcome with LSCC after surgery. Our study may lead to establishing new molecular therapeutic targets and/or prognostic biomarkers in LSCC.     

Mean nuclear volume in squamous cell carcinoma of the vulva

OBJECTIVE: To compare mean nuclear volume (MNV) estimated by the stereologic intercept method in lymph node-positive and -negative cases of squamous cell carcinoma of the vulva. STUDY DESIGN: This retrospective study consisted of 53 cases (lymph node metastasis, n = 19; cases without lymph node metastasis, n = 34) of squamous cell carcinoma of the vulva. MNV was estimated with the help of an image cytometer. The nuclear point intersection method was used to measure MNV. The mean nuclear volumes of both lymph node-positive and -negative cases were compared. RESULT: MNV in the lymph node-negative and -positive cases was 717.0 +/- 533.1 and 1,961.4 +/- 1,369.6 microns 3, respectively (P < .000, Mann-Whitney U test). There was a significant difference in MNV between the 2 groups of tumors. CONCLUSION: The observations from the present study suggest that estimation of MNV of malignant squamous cells from the vulva on conventional histopathology sections may provide an objective and useful diagnostic tool in predicting lymph node metastasis.     

Primary Tumor-Secreted Lymphangiogenic Factors Induce Pre-Metastatic Lymphvascular Niche Formation at Sentinel Lymph Nodes in Oral Squamous Cell Carcinoma

Objectives

The objectives of this study were to evaluate the formation of lymphvascular niches in lymph nodes of patients with oral squamous cell carcinoma (OSCC), and investigate the roles of lymphangiogenic and angiogenic factors, such as vascular endothelial growth factor (VEGF)-A, VEGF-C, and VEGF-D, expressed in the primary tumors.

Materials and Methods

Forty-four patients with previously untreated clinically late T2 or T3 OSCC of cN0 were evaluated for primary tumors and 166 sentinel lymph nodes (SLNs). Primary tumors were immunohistochemically analyzed for expressions of VEGFs. Densities of lymphatic vessels (LVD_podoplanin) and high endothelial venules (HEVD) in the SLNs were also calculated using antibodies for each marker, podoplanin and MECA-79, respectively.

Results

In 25 patients, all lymph nodes were metastasis-negative, whereas, in 19 patients, metastasis was positive for at least one lymph node (either at SLN, non-SLN, or nodal recurrence). From the analyses of 140 SLNs without metastasis, LVD_podoplanin in 50 SLNs of metastasis-positive cases was significantly higher than that in 90 SLNs of metastasis-negative cases (p = 0.0025). HEVD was not associated with lymph node metastasis. The patients with VEGF-A-High or VEGF-D-High tumors had significantly higher LVD_podoplanin than patients with their Low counterparts (p = 0.0233 and p = 0.0209, respectively). In cases with lymph node metastasis, the VEGF-D-expression score was significantly higher than in those without lymph node metastasis (p = 0.0006).

Conclusions

These results suggest that lymph node lymphangiogenesis occurs before metastasis in OSCC. VEGF-A and VEGF-D play critical roles in this process. VEGF-D is a potential predictive marker of positive lymph node metastasis in cN0 patients.     

Genome-wide 5-Hydroxymethylcytosine Profiling Analysis Identifies MAP7D1 as A Novel Regulator of Lymph Node Metastasis in Breast Cancer

Although DNA 5-hydroxymethylcytosine(5 hmC) is recognized as an important epigenetic mark in cancer, its precise role in lymph node metastasis remains elusive. In this study, we investigated how 5 hmC associates with lymph node metastasis in breast cancer. Accompanying with high expression of TET1 and TET2 proteins, large numbers of genes in the metastasis-positive primary tumors exhibit higher 5 hmC levels than those in the metastasis-negative primary tumors. In contrast, the TET protein expression and DNA 5 hmC decrease significantly within the metastatic lesions in the lymph nodes compared to those in their matched primary tumors. Through genomewide analysis of 8 sets of primary tumors, we identified 100 high-confidence metastasis-associated5 hmC signatures, and it is found that increased levels of DNA 5 hmC and gene expression of MAP7 D1 associate with high risk of lymph node metastasis. Furthermore, we demonstrate that MAP7 D1, regulated by TET1, promotes tumor growth and metastasis. In conclusion, the dynamic5 hmC profiles during lymph node metastasis suggest a link between DNA 5 hmC and lymph node metastasis. Meanwhile, the role of MAP7 D1 in breast cancer progression suggests that the metastasis-associated 5 hmC signatures are potential biomarkers to predict the risk for lymph node metastasis, which may serve as diagnostic and therapeutic targets for metastatic breast cancer.     

Systematic analysis of genes involved in oral cancer metastasis to lymph nodes

Oral cancer remains a deadly disease worldwide. Lymph node metastasis and invasion is one of the causes of death from oral cancer. Elucidating the mechanism of oral cancer lymph node metastasis and identifying critical regulatory genes are important for the treatment of this disease. This study aimed to identify differentially expressed genes (gene signature) and pathways that contribute to oral cancer metastasis to lymph nodes. The GSE70604-associated study compared gene profiles in lymph nodes with metastasis of oral cancer to those of normal lymph nodes. The GSE2280-associated study compared gene profiles in primary tumor of oral cancer with lymph node metastasis to those in tumors without lymph node metastasis. There are 28 common differentially expressed genes (DEGs) showing consistent changes in both datasets in overlapping analysis. GO biological process and KEGG pathway analysis of these 28 DEGs identified the gene signature CCND1, JUN and SPP1, which are categorized as key regulatory genes involved in the focal adhesion pathway. Silencing expression of CCND1, JUN and SPP1 in the human oral cancer cell line OECM-1 confirmed that those genes play essential roles in oral cancer cell invasion. Analysis of clinical samples of oral cancer found a strong correlation of these genes with short survival, especially JUN expression associated with metastasis. Our study identified a unique gene signature – CCND1, JUN and SPP1 – which may be involved in oral cancer lymph node metastasis.