High expression of ALDH1 and SOX2 diffuse staining pattern of oral squamous cell carcinomas correlates to lymph node metastasis

One of the major factors involved in the prognosis of oral squamous cell carcinoma (OSCC) patients is metastasis. Recent progress in cancer stem‐like cell/cancer‐initiating cell (CSC/CIC) research indicates that CSCs are related to metastasis. Aldehyde dehydrogenase 1 – (ALDH1) and SRY‐related HMG‐box gene 2 (SOX2) have recently been shown to be putative CSC markers for several human malignancies. The aim of this study was to determine the association of ALDH1 and SOX2 expression in oral squamous cell carcinoma (OSCC) with lymph node metastasis. Immunohistochemical staining of ALDH1, SOX2 and Ki67 was performed in 80 OSCC tissues. High expression rates of ALDH1 (2%–40%) were found to be related to lymph node metastasis (P = 0.0017). Interestingly, we found that SOX2 staining could be classified into two patterns: (i) peripheral staining pattern; and (ii) diffuse staining pattern. The diffuse staining pattern showed a significant correlation with lymph node metastasis (P < 0.001). No correlation was found between Ki67 staining and lymph node metastasis (P = 0.4724). The ALDH1 positive staining rates in metastatic lymph nodes were higher than that in corresponding primary OSCC tissues. These results indicate that high expression rates of ALDH1 and SOX2 diffuse staining patterns might be novel prediction markers for OSCC lymph node metastasis.     

Histopathological study of lymphatic invasion in squamous cell carcinoma (O-1N) with high potential of lymph node metastasis

The process of lymph node metastasis was studied in an animal model (termed O-1N) that was successfully established using a metastatic tumor to the submandibular lymph node from a chemically induced squamous cell carcinoma of the hamster tongue. The model has been maintained by serial transplantation of metastatic tumors into the buccal pouch. Lymphovascular invasion of transplanted O-1N in the tongue was examined in serial histologic sections. Lymphatic vessels were distinguished from blood vessels by Masson's trichrome stain for vascular smooth muscle, BSA-I lectin binding for vascular endothelium, and laminin and type IV collagen immunostaining for the vascular basement membrane. Transplanted tumors enlarged progressively with invasion of surrounding tissues of the tongue and resulted in lymph node metastasis in all animals with successful takes. Local growth of the tumors in the tongue was accompanied by stromal proliferation with abundant dilated lymphatic vessels which contained clusters of tumor cells. On serial sections, the carcinoma cell clusters in lymphatics in the close proximity of tumor nests were in continuity with adjacent tumor nests, whereas such continuity was not recognized in those occurring apart from tumor nests. The formation of isolated carcinoma cell clusters resulting from disintegration of elongated processes of tumor nests with invasion of lymphatics and subsequent transport in lymphatics and deposition in lymph nodes in clusters were well demonstrated in other serial sections. The key step of lymph node metastasis therefore appears to be direct invasion of lymphatic vessels by tumor cells, similar to their invasion of adjacent tissues but different from the way that blood cells escape through vessel walls. Proliferation of lymphatics around tumor nests and transport of tumor cells in clusters would also contribute to the production of metastatic deposits in lymph nodes.     

A nomogram for predicting lymph node metastasis in superficial esophageal squamous cell carcinoma

Superficial esophageal squamous cell carcinoma (SESCC) is defined as carcinoma with mucosal or submucosal invasion, regardless of regional lymph node metastasis (LNM). The lymph node status is not only a key factor to determine the training strategy, but also the most important prognostic factor in esophageal cancer. In this study, we establish a clinical nomogram for predicting LNM in patients with SESCC. A predictive model was established based on the training cohort composed of 711 patients who underwent esophagectomy for SESCC from December 2009 to June 2018. A prospective cohort of 203 patients from June 2018 to January 2019 was used for validation. Favorable calibration and well-fitted decision curve analysis were conducted and good discrimination was observed (concordance index [C-index], 0.860; 95% confidence interval [CI], 0.825–0.894) through internal validation. The external validation cohort presented good discrimination (C-index, 0.916; 95% CI, 0.860–0.971). This model may facilitate the prediction of LNM in patients with SESCCs.     

Inhibition of cervical lymph node metastasis by marimastat (BB-2516) in an orthotopic oral squamous cell carcinoma implantation model

Activation of matrix metalloproteinase-2 (MMP-2) is a common event in head and neck squamous cell carcinoma. An OSC-19 cell line, derived from human oral squamous cell carcinoma and known to metastasize to cervical lymph nodes, was implanted into the lingual margin of mice. The effect of marimastat (BB-2516), a broad MMP inhibitor, on the suppression of regional cervical lymph node metastasis was evaluated with an orthotopic implantation nude mice model. Marimastat was given immediately after OSC-19 implantation and continuously administered by an osmotic pump. The mice were divided into three groups by marimastat dose; Group A; 0 mg/kg/day, Group B; 30 mg/kg/day, and Group C; 150 mg/kg/day. Twenty-one days after implantation, primary oral tumors and cervical lymph nodes were resected. Cervical lymph node status was microscopically examined. Activation of MMP-2 in primary oral tumor was examined by gelatin zymography. Both cervical lymph node metastasis and activation of MMP-2 were significantly suppressed in Group C (P<0.05). Moreover, the Group C mice had a significantly better survival than group A (P=0.0026). There was a significant difference between Group A and Group C in terms of proliferation of tumor cells by proliferating cell nuclear antigen immunostaining (P=0.0120). These results suggest a positive role for marimastat in the inhibition of MMP-2 activation and prevention of cervical lymph node metastasis in oral squamous cell carcinoma (OSCC). Improvement of survival in patients with OSCC could be expected using adjuvant therapy with marimastat. This revised version was published online in July 2006 with corrections to the Cover Date.     

Vascular invasion of O-1N, hamster squamous cell carcinoma with high potential of lymph node metastasis: Ultrastructural comparison between lymphatics and blood vessels

The ultrastructural modes of lymphatic and blood vessel invasions were studied comparatively In hamsters with squamous cell carcinoma (O-1N) that had a high potential for lymph node metastasis. The endothellal injury, which was caused by mechanical stretching with the growth of O-1N, was the lnltial and characteristic feature common to both vascular invasions. Tumor cell nests penetrating the lymphatic lumen through disrupted endothellal cells still maintained their volume and continuity to the underlying tumor cell nests. In contrast, pronounced microthrombotic end neutrophllic reactions occurred at the site of blood vessel penetration. Within the lymphatic lumen, large clusters of O-1N cells were kept longer In spite of lymphocytic and macrophagic reactions. In blood vessels, clusters of tumor cells that had passed through dense fibrin layers were reduced In size and further disintegrated into smaller pieces by neutrophils. In conclusion, lymphatic invasion is a mechanical process, and smooth and direct invasion of large tumor cell nests into lymphatic vessels Is responsible for causing more prompt and frequent lymph node metastasis in O-1N than a hematogenous type.     

Exposure to chewing tobacco promotes primary oral squamous cell carcinoma and regional lymph node metastasis by alterations of SDF1α/CXCR4 axis

A high incidence of oral squamous cell carcinoma (OSCC) is observed in South‐East Asian countries due to addictions such as chewing tobacco. Local invasion and distant metastases are primary causes of poor prognosis in OSCC. This study aimed to understand the alterations in metastasis biomarkers, such as stromal cell–derived factor‐1α (SDF‐1 or SDF1α) and its receptor C‐X‐C chemokine receptor type 4 (CXCR4), in OSCC patient samples that were stratified based on the history of addiction to chewing tobacco. Targeted immunohistochemical staining and Western blotting were performed on primary tumour and metastatic lymph node (LN) tissues in parallel. Overexpression of hepatocyte growth factor (HGF), activated form of its cognate receptor tyrosine kinase, c‐Met (p‐Met), GRB2‐associated‐binding protein 1 (Gab1), phospho‐protein kinase B (pAkt), nuclear factor kappa B (NF‐κB) and cyclooxygenase‐2 (COX‐2) were observed in primary tumour and metastatic lymph nodes in both chewer and non‐chewer cohorts. Variance analysis showed significant positive correlation between them (P < .0001) indicating upregulation of these biomarkers upon ligand‐induced activation of c‐Met in both tobacco chewers and non‐chewers. Significantly higher expressions of SDF1α and CXCR4 were observed in both primary tumours and metastatic lymph nodes of tobacco chewers (P < .0001) and coincided with overexpressed HGF. In contrast, no significant correlation was observed between expression of HGF and that of SDF1α and CXCR4 in non‐chewers. Together, our findings provide important insights into the association of HGF/c‐Met and the SDF1α/CXCR4 axis in lymph node metastasis and to an aetiological link with the habit of chewing tobacco.     

A retrospective analysis of histological prognostic factors for the development of lymph node metastases from auricular squamous cell carcinoma

Clark R R, Soutar D S & Hunter K D (2010) Histopathology 57 , 138–146
A retrospective analysis of histological prognostic factors for the development of lymph node metastases from auricular squamous cell carcinoma Aims: Squamous cell carcinoma (SCC) of the auricle has a high risk of metastatic spread, which is associated with high mortality. Identification of patients with a high risk of lymph node metastases would allow prophylactic treatment to the draining lymph nodes, but there are no established clinical or histopathological criteria to predict which tumours have a high risk of metastasis. The aim was to determine such criteria. Methods and results: The study was a retrospective analysis of the clinical and histological features of 229 cases of SCC of the auricle, with a minimum of 2 years’ clinical follow‐up. Overall, lymph node metastases were present in 24 cases (10.5%). Of the patients with metastatic disease 66.7% died, despite multi‐modality treatment. Tumours with a depth of invasion >8 mm or a depth of invasion between 2 and 8 mm in conjunction with evidence of destructive cartilage invasion, lymphovascular invasion or a non‐cohesive invasive front had a high risk of metastasis (56% and 24%, respectively). Conclusions: Patients with high‐risk tumours, as assessed histopathologically, should be considered for prophylactic therapy to or staging of the regional lymph nodes.     

Tenascin-C expression in Merkel cell carcinoma lymph node metastasis

Expression of tenascin-C (Tn-C) has been shown to correlate with invasion and metastasis in Merkel cell carcinoma (MCC). Cytokeratin-20 (CK-20) is used in differential diagnostics of the primary tumour. The aim of this study was to demonstrate the expression of Tn-C in MCC lymph node metastases. Immunohistochemical staining was performed for five metastatic lymph nodes using a monoclonal antibody against Tn-C and CK-20. All five metastatic lymph nodes expressed Tn-C. The expression concentrated around the vascular structures, invasion borders and fibrotic septae. One of the metastatic lymph nodes was strongly positive for CK-20 while the others showed a focal or negative pattern. The normal lymphoid tissue was negative for Tn-C. Tn-C detected metastatic MCC tissue within the lymph nodes undisputedly. There was a clear distinction between the metastatic and normal lymphatic tissue. Furthermore, invasion to the surrounding tissue was easily demonstrated. Contrary to previous studies, CK-20 expression seemed to fluctuate.     

Histo-blood group ABO antigen in oral potentially malignant lesions and squamous cell carcinoma--genotypic and phenotypic characterization

Loss of histo-blood group A/B antigens is frequent in oral cancer. It is unclear whether this alteration is due to loss of the chromosomal region encoding the genes. The aim was to investigate genotypic alterations in the ABO locus in oral potentially malignant lesions and carcinomas. Seventy-three cases which expressed A/B antigen in normal epithelium by immunohistochemical (IHC) staining were investigated. Both tumour and normal cells were collected from paraffin-embedded tissue by laser microdissection. DNA was extracted and analysed by PCR coupled with restricted digestion analysis in order to establish the ABO genotype. Total and patchy loss of A/B antigen expression was found in 24/32 carcinomas, 6/7 leukoplakias with severe dysplasia, 12/17 leukoplakias with mild and moderate dysplasia, and 6/17 leukoplakias without dysplasia. Specific A/B allele loss was found in 8/24 cases with carcinoma and 3/24 cases with mild and moderate dysplasia by genotyping analysis. O allele loss was found in 10 cases involving all four groups. In patients with heterozygous genotypes, A/B allelic loss by genotyping analysis was always followed by loss of A/B antigen expression by IHC staining. Loss of A/B antigen expression in tissues which had intact ABO alleles was, however, found and may be explained by other genetic and epigenetic changes.     

Intratumoral c-Met expression is associated with vascular endothelial growth factor C expression, lymphangiogenesis, and lymph node metastasis in oral squamous cell carcinoma: implications for use as a prognostic marker

Hepatocyte growth factor has been identified as a lymphangiogenic factor in experimental animal models. However, the correlation between hepatocyte growth factor or c-Met expression and lymphangiogenesis in human spontaneous tumors has been rarely reported, and the distribution pattern of c-Met on tumor-related lymphatic vessels remains to be further investigated. Lymphatic vessel density, lymphatic invasion, the expression of hepatocyte growth factor, c-Met, and vascular endothelial growth factor C proteins were evaluated by immunohistochemistry in 76 cases of oral squamous cell carcinoma. The distribution of c-Met on lymphatic endothelium was examined. High expression of c-Met in tumor cells was significantly associated with advanced clinical stage (P = .045), high expression of vascular endothelial growth factor-C (P < .001), higher peritumoral lymphatic vessel density (P = .003), higher incidence of peritumoral lymphatic invasion (P = .032), and positive lymph node status (P = .005), in spite of its negative expression on most lymphatic vessels. Patients with high–c-Met expression tumors exhibited shorter overall survival and disease-free survival (P < .001 and P = .010, respectively). Taken together, our results provide indirect evidence for an association and possible regulatory link of c-Met with the lymphangiogenic factor, vascular endothelial growth factor C, and, by extension, with lymphangiogenesis and lymph node metastasis, suggesting important prognostic significance of c-Met for patients with oral squamous cell carcinoma.     

KiSS‐1 expression in oral squamous cell carcinoma and its prognostic significance

Downregulated expression of KiSS‐1 has been correlated with tumor progression, metastasis, and patient prognosis in various human malignancies. However, there is no information regarding the expression of KiSS‐1 in oral squamous cell carcinoma (OSCC). Our aims were to examine KiSS‐1 expression in OSCC tissue samples and cell lines and to determine its prognostic significance. KiSS‐1 expression was significantly lower in lymph node (LN) metastases than in primary tumor tissues. Five of six OSCC cell lines showed absence or relatively low expression of KiSS‐1. Correlations between KiSS‐1 expression and clinicopathological parameters were statistically assessed. There were significant correlations between KiSS‐1 expression and LN metastasis (p = 0.007), TNM stage (p = 0.024), and local recurrence (p = 0.012). In the Kaplan–Meier survival analysis, negative KiSS‐1 expression significantly correlated with poorer overall survival (OS) and disease‐free survival (DFS) (p = 0.000 and 0.000, respectively). Multivariate analysis using Cox regression modeling revealed that KiSS‐1 expression was an independent prognostic factor for both OS and DFS (p = 0.001 and 0.000, respectively). Our findings suggested that KiSS‐1 downregulation may play a role in tumor progression and metastasis of OSCC and may be a reliable biomarker for predicting clinical outcome in OSCC.     

Overexpression of CyclinD1 and underexpression of p16 correlate with lymph node metastases in laryngeal squamous cell carcinoma in Chinese patients

Background CyclinD1 and p16 are involved in the regulation of G1 checkpoint and may play an important role in the tumorgenesis of laryngeal squamous cell carcinoma (LSCC). Previous studies have examined the level of expression of cyclinD1 or p16 in LSCC but no such information is available for their relation and their correlation with lymph node metastasis in Chinese patients. Patients and methods A total of 58 patients underwent surgical resection of laryngeal tumours in the Department of Otolaryngology, Qilu Hospital Shandong University between January 2001 and December 2002. All pathologic specimens were available for immunohistochemical study using antibodies against cyclinD1 and p16. Results Compared with normal epithelium, expression of CyclinD1 in the LSCC was significantly higher (62.1% vs. 10.0%, P < 0.05), expression of p16 was significantly lower (48.3% vs. 90.0%, P < 0.05); CyclinD1 expression in LSCC was up-regulated in TNM classification (r _s = 0.409, P < 0.05) as well as with cervical lymph node metastases (r _s = 0.294, P < 0.05); p16 expression in LSCC was down-regulated with cervical lymph node metastases (r _s = −0.275, P < 0.05); negative significant correlation between p16 immunostaining and CyclinD1 was observed in LSCC (r _s = −0.331, P < 0.05). Conclusion There was a negative correlation between CyclinD1 expression and p16 expression in LSCC. The over-expression of CyclinD1 and the under-expression of p16 may play a significant role in the incidence and development of LSCC and may be important indicators for cervical lymph node metastases in Chinese patients of LSCC. Zhi-jie Fu and Zhi-yong Ma are contributed to this work equally.     

Overexpression of ROCK1 and ROCK2 inhibits human laryngeal squamous cell carcinoma

Rho-associated coiled-coil containing protein kinase (ROCK) over-expression has been implicated in the progression of many tumor types. The aim of this study was to explore the roles of ROCK1 and ROCK2 in human laryngeal squamous cell carcinoma (LSCC). ROCK1 and ROCK2 expression levels were examined in 50 cases of human LSCC samples by immunohistochemistry. Effects of ROCK1 and ROCK2 on LSCC cell proliferation and motility were investigated in the presence of the ROCK inhibitor Y-27632. The results showed that ROCK1 expression was positively correlated with tumor size and lymph node metastasis (P < 0.05); ROCK2 positively correlated with tumor size (P < 0.05). Inhibition of ROCK1 and ROCK2 by Y-27632 significantly inhibits proliferation, migration, and invasion of LSCC cells. Our data indicate that expression of ROCK1 and ROCK2 are closely associated with tumor growth and lymph node metastasis of LSCC. Thus, these two ROCK isoforms may be useful as molecular makers for LSCC diagnosis and may be useful therapeutic targets as well.