Serum hepatocyte growth factor as a marker of tumor activity in head and neck squamous cell carcinoma

Hepatocyte growth factor (HGF) has previously been reported to be elevated in the serum of patients with malignancy, including breast, colorectal and gastric cancers. Here, we evaluated the correlation between serum HGF and the progression of head and neck squamous cell carcinoma (HNSCC). The mean serum HGF levels in 71 healthy control subjects, 78 patients with primary HNSCC, and eight patients with recurrent HNSCC were 0.538 ± 0.163, 0.701 ± 0.252, and 0.925 ± 0.349 ng/ml, respectively. The increase in the HGF level was significantly correlated with tumor stage progression. The HGF level had decreased to normal at 1 month after curative resection of the tumors. During follow-up for several months, the HGF level significantly increased in recurrent HNSCC patients, whereas there was no increase in nonrecurrent patients. Our data suggest that serum HGF is significantly corrected with tumor progression and may be a strong predictor of recurrence in HNSCC.     

Macrophage colony-stimulating factor as a marker of ovarian carcinoma

Macrophage colony stimulating factor (M-CSF) is a cytokine which stimulates the proliferation and differentiation of phagocytic cells. We evaluated the usefulness of M-CSF as a serum tumor marker for ovarian malignancies and also assessed M-CSF production by tumor cells and the role of an autocrine system in such M-CSF production. The findings obtained were as follows: i) Serum M-CSF was a useful marker for malignant ovarian tumors. ii) M-CSF was a marker for both epithelial stromal tumors and for germ cell tumors. It was also a marker for dysgerminoma, for which no specific tumor marker is currently available. iii) The value of combined assays employing M-CSF was confirmed. iv) M-CSF production was demonstrated in various malignant ovarian tumor cell lines, but the presence of an autocrine system for M-CSF was not confirmed.     

Serum ferritin as a tumor marker in patients with squamous cell carcinoma of the head and neck

A double antibody enzyme immunoassay was used to measure serum ferritin levels in several different control and tumor-bearing populations collected from two institutions. The control groups consisted of normal volunteers, smokers, and Latter Day Saints. No statistically significant differences were noted in ferritin levels between pairs of these groups. Differences were noted among the normal groups when separated on the basis of age and sex, with higher ferritin levels in individuals older than 32 years of age and in men. By one-way analysis of variance, most control groups and subgroups were shown to have significantly lower levels (P less than 0.05) than the head and neck cancer group, with the exception of male smokers, who had levels comparable to male head and neck cancer patients. Serum ferritin levels were higher in head and neck cancer patients than in controls; however, there was no difference when compared with patients with other types of solid malignancies or when considering the anatomic site of the head and neck lesion. Ferritin levels were significantly (P less than 0.05) higher in patients with advanced (Stages III and IV) cancer than in those individuals with Stage I or II cancer. In patients with no evidence of clinical disease 5 years after treatment, the ferritin level had essentially returned to normal. In a group of head and neck cancer patients followed longitudinally, a significant decline in ferritin levels (P less than 0.05) was seen by 5 months after the completion of successful treatment. Furthermore, ferritin levels showed a tendency to increase or remain at high levels in patients with a poor prognosis and to decrease in those patients with a favorable prognosis, giving support to the contention that ferritin may prove to be a valuable tumor marker in head and neck cancer.     

CD44 as a stem cell marker in head and neck squamous cell carcinoma

In the recent past, evidence is increasing indicating the existence of a subpopulation of resistant tumor cells in head and neck squamous cell carcinoma (HNSCC) that cannot be eradicated by established antineoplastic treatments. These cancer stem cells (CSCs) have features of somatic stem cells such as selfrenewal, proliferation and differentiation. CD44+ cells in tumors of the head and neck are referred to as CSCs of HNSCC. Expression profiling of CD44 in 29 HNSCC tumors was performed by fluorescence microscopy. ELISA analysis was performed to detect concentration of soluble CD44 in the peripheral blood of 29 HNSCC patients and 11 healthy controls. Expression of CD44 was determined in all HNSCC tissue samples (n=29). In all samples a surface staining pattern was found. The concentration of CD44 in the peripheral blood of HNSCC patients was significantly higher compared to a healthy control group (mHNSCC =13.5 ± 0.5 ng/ ml; mCont = 9.3 ± 0.6 ng/ml; P=0.6 x 10(-12)). The role of CD44 as a marker for CSCs in HNSCC remains to be ascertained. Further experiments might reveal its role as a diagnostic and prognostic factor, and possibly as a therapeutic target.     

The significance of Tenascin-C serum level as tumor marker in squamous cell carcinoma of the head and neck

BACKGROUND: Tenascin, an extracellular matrix glycoprotein, is transiently present in embryonic tissue, in benign granulation tissue, but also in several highly anaplastic tumors like fibrosarcoma, melanoma and squamous cell carcinoma of the skin. This study was performed to validate elevated Tenascin serum levels as a possible marker for head and neck squamous cell carcinomas (HNSCC). PATIENTS AND METHODS: Tenascin serum levels were evaluated in patients with primary (n = 92) and with recurrent (n = 28) HNSCC. Patients with benign, non inflammatory ear, nose and throat diseases (n = 16) served as the control. The Tenascin serum levels were measured by ELISA (Aventis). RESULTS: Serum Tenascin concentrations of patients with benign ENT diseases ranged between 0.37 and 2.19 micrograms/ml (n = 16, mean +/- SD: 1.23 +/- 0.59 micrograms/ml), of patients with HNSCC (primary diagnosis) between 0.05 and 8.75 micrograms/ml (n = 92, mean +/- SD: 1.81 (1.36 micrograms/ml) and of patients with recurrent HNSCC between 0.53 and 10.0 micrograms/ml (n = 28, mean +/- SD: 2.78 +/- 2.2 micrograms/ml). CONCLUSION: We found a significant elevation of Tenascin serum levels only in patients with higher tumor stages (T4/UICC4) (p < 0.01/p < 0.1) or recurrent disease compared to Tenascin serum levels in healthy controls. Thereby Tenascin serum levels cannot be used clinically as a routine serum marker for the control of head and neck cancer. Further investigations are necessary to evaluate whether the measurement of Tenascin levels as tumor markers could offer additional information to the clinical outcome of patients with HNSCC.     

Heat-Stable Alkaline Phosphatase A putative tumor marker of head and neck squamous cell carcinoma

Serum total alkaline phosphatase (AP) activity and heat-stable AP (HSAP) were investigated in patients with uncontrolled squamous cell carcinoma of the head and neck before and after treatment. No significant differences in AP activity were seen between normal subjects and cancer patients. However, the HSAP fraction of the total AP activity was significantly elevated prior to treatment and the level declined and remained low during successful treatment, while it increased with tumor progression or recurrences. Heat-stable AP was found to be a useful tumor marker of potential usefulness in the management of patients with squamous cell carcinoma of the head and neck.     

Circulating miRNA is a novel marker for head and neck squamous cell carcinoma

The aim of the study is to investigate the alteration of plasma miRNA in head and neck squamous cell carcinoma (HNSCC). Altered microRNAs (miRNAs) expression has been found in many cancers, including lung cancer, breast cancer, prostate cancer, bladder cancer and colorectal cancer. Many recent studies have demonstrated that aberrant plasma miRNAs were also found in various types of cancers. However the alteration of plasma expression in HNSCC remains unclear. In this present study, the expression profiles of ten miRNAs, let-7a, miR-21, miR26b, miR-34c, miR-99a, miR-133a, miR-137, miR-184, miR-194a, and miR-375, in plasma from 50 patients and 36 healthy subjects were evaluated using real-time quantitative polymerase chain reaction (PCR). Our results demonstrated that the expression level of miR-21 was significantly up-regulated in plasma samples obtained from HNSCC patients (p?<?0.01) than those from healthy subjects, which were in consistent with our finding in HNSCC tissues. A 7.7-fold increase of miR-21 in cancerous parts when compared to their non-cancerous counterparts (p?<?0.0001) was observed in HNSCC tissues. In addition, the expression levels of miR-21 and miR-26b were both reduced in post-operative HNSCC patients with good prognosis. In contrast, the concentration of plasma miR-21 and miR-26b stayed high after tumor removal in the expired cases. Our study suggests that detecting circulating miR-21 and miR-26b pre- and post-operatively might provide a novel tumor marker for HNSCC.     

Bcl-2 as prognostic factor in head and neck squamous cell carcinoma

A series of 66 cases of oral squamous cell carcinoma (OSCC) was retrospectively analyzed by immunohisto-chemistry for bcl-2 expression to verify its predictive value for clinical outcome in patients with OSCC. After grouping for bcl-2 expression, OSCCs were statistically analyzed for the variables age, gender, histological grading (G), TNM, staging, recurrence, and overall survival rate. Univariate and multivariate (Cox regression) analyses were performed. Thirty-six OSCC (54.5%) showed expression for bcl-2, whereas 30 (44.5%) were negative. No statistical association was found between bcl-2 expression and any variables considered at baseline. Overall disease-specific survival rate at 72 months was 51%, independently from the extent of the tumor. In terms of prognostic significance, the bcl-2-positive group showed more than 60% survival at 72 months whereas the bcl-2-negative group showed none. An independent association of bcl-2 expression was found with an improved overall survival rate (p = 0.048), although grading and staging were established to be the best baseline markers of prognosis. On the basis of these results, it is possible to suggest bcl-2 as an early marker of prognosis: lack of bcl-2 expression could constitute a hallmark of aggressive biological behavior in OSCC.     

Pituitary tumor-transforming gene expression is a prognostic marker for tumor recurrence in squamous cell carcinoma of the head and neck

Background  

The proto-oncogene pituitary tumor-transforming gene (PTTG) has been shown to be abundantly overexpressed in a large variety of neoplasms likely promoting neo-vascularization and tumor invasiveness. In this study, we investigated a potential role for PTTG mRNA expression as a marker to evaluate the future clinical outcome of patients diagnosed with primary cancer of the head and neck.     

Identification of osteopontin as a prognostic plasma marker for head and neck squamous cell carcinomas.

PURPOSE: Tumor hypoxia modifies treatment efficacy and promotes tumor progression. Here, we investigated the relationship between osteopontin (OPN), tumor pO(2), and prognosis in patients with head and neck squamous cell carcinomas (HNSCC). EXPERIMENTAL DESIGN: We performed linear discriminant analysis, a machine learning algorithm, on the NCI-60 cancer cell line microarray expression database to identify a gene profile that best distinguish cell lines with high Von-Hippel Lindau (VHL) gene expression, an important regulator of hypoxia-related genes, from those with low expression. Plasma OPN levels in 15 volunteers, 31 VHL patients, and 54 HNSCC patients were quantitatively measured by ELISA. The relationships between plasma OPN levels, tumor pO(2) as measured by the Eppendorf microelectrode, freedom from relapse (FFR), and survival in HNSCC patients were evaluated. RESULTS: Microarray analysis indicated that OPN gene expression inversely correlated with that of VHL. These findings were confirmed by Northern blot analysis. ELISA studies and Western blot in a HNSCC cell line demonstrated that hypoxia exposure resulted in increased OPN secretion. Patients with VHL syndrome had significantly higher plasma OPN levels than healthy volunteers. Plasma OPN level inversely correlated with tumor pO(2) (P = 0.003, r = -0.42). OPN levels correlated with clinical outcomes. The 1-year FFR and survival rates were 80 and 100%, respectively, for patients with OPN levels 450 ng/ml (P = 0.002 and 0.0005). Multivariate analysis revealed that OPN was an independent predictor for FFR and survival. CONCLUSIONS: Plasma OPN levels appeared to correlate with tumor hypoxia in HNSCC patients and may serve as noninvasive tests to identify patients at high risk for tumor recurrence.     

Evaluation of soluble CD44v6 as a potential serum marker for head and neck squamous cell carcinoma.

In recent years, the measurement of soluble CD44 levels in the circulation of patients with malignant diseases has been introduced as a new and simple diagnostic tool for the detection of human cancer. The high CD44v6 expression in head and neck squamous cell carcinoma (HNSCC) would enable the use of soluble CD44v6 proteins present in the circulation of HNSCC patients as a marker of disease. In the present study, we determined CD44v6 plasma levels using a domain-specific ELISA in healthy volunteers, non-cancer patients, and HNSCC patients before and after surgical removal of the tumor. A difference between the CD44v6 plasma levels of HNSCC patients and controls could not be observed. Moreover, surgical removal of the tumor did not result in a reduction of the CD44v6 plasma level in the HNSCC patients. In addition, the spectrum of soluble v6-containing CD44 proteins present in the plasma of HNSCC patients and controls was determined by immunoprecipitation experiments, but again, tumor-related isoforms could not be distinguished in patient samples. Additional experiments to unravel the biological source of these circulating proteins indicated surprisingly that the v6-containing proteins present in the circulation of healthy individuals are only released in part, if at all, by activated lymphocytes or other nucleated blood cells. Most circulating CD44v6 proteins seem to be derived from the normal epithelial cell compartments, including breast cells, colon cells, and squamous cells. Taken together, these data do not support the use of soluble CD44v6 as a tumor marker in HNSCC or any other tumor type that has developed from tissues producing soluble isoforms.     

Re-irradiation for head and neck squamous cell carcinoma

Introduction

Local recurrences after curative treatment have a potential for cure with salvage surgery or with re-irradiation.

Staging of head and neck squamous cell carcinoma using the MET oncogene product as marker of tumor cells in lymph node metastases

In head and neck squamous cell carcinomas (HNSCC), metastasis to cervical lymph nodes is a major determinant of patient outcome. To detect metastases, we used the MET oncogene as marker, which encodes the receptor for hepatocyte growth factor/scatter factor, mediating epithelial cell motility and invasiveness. The MET gene is expressed in epithelia and over-expressed in carcinomas of specific histotypes, but not in lymphatic tissue. A total of 151 lymph nodes from 20 squamous cell carcinomas were studied with both in-depth histology and end-point and real-time quantitative RT-PCR. MET-encoded sequences were found in 61 of 151 nodes (40%), of which 24 (16%) were found metastatic by in-depth histopathology. Parallel routine histopathologic analysis of 654 lymph nodes from the same cases identified 36 metastases (5%). Real-time quantitative RT-PCR was used to measure MET gene-specific mRNA in normal tissues, primary tumors and lymphatic metastases and showed a 2-8-fold increased expression in tumor cells which metastasize. RT-PCR for 3 cytokeratins expressed in HNSCC (K4, K10 and K13) proved to be less sensitive in detecting occult lymphatic metastases. Western blot analysis demonstrated the presence of the full-size MET receptor in primary tumors and lymph node metastases; immunohistochemistry showed receptor localization in tumor cells. Altogether, these data demonstrate that the MET gene product is a valuable marker with which to detect occult tumor cells in lymph nodes, thanks to its high expression in metastatic cells. After RT-PCR analysis we were able to attribute a more advanced stage to 10 out of 20 HNSCC cases, including 5 cases classified as tumor-free after routine histopathology.     

Human papillomavirus as a marker of the natural history and response to therapy of head and neck squamous cell carcinoma

There has been a gradual change in the demographics of head and neck carcinoma. Although relatively uncommon, the incidence of oropharyngeal carcinoma has been increasing despite declining tobacco consumption and contrary to a diminishing incidence of cancers at other head and neck sites. It is now clear that the incidence of human papillomavirus (HPV)-associated oropharyngeal cancers is rising, likely as a consequence of changing life styles and sexual behaviors. Many studies have contributed to understanding the characteristics of HPV-related oropharyngeal carcinoma, which usually presents as nonkeratinizing squamous cell carcinoma of low to intermediate T-category and affects middle-aged white men, having higher socioeconomic status and no or brief history of tobacco consumption. The diagnosis of this distinct neoplastic entity can be firmly established by a combination of p16 immunohistochemical and in situ hybridization assays. Compared with the traditional smoking-associated head and neck squamous cell carcinoma, HPV-related oropharyngeal carcinoma has a favorable natural history and responds better to treatment. Consequently, patients with this cancer have better long-term survival than those with HPV-unrelated head and neck squamous cell carcinoma (eg, 5-year overall survival rate of >80% versus ～40% for patients with stage III-IV tumors), and hence they are more likely to experience chronic therapy-induced morbidity. Therefore, changes in evaluation, staging, and treatment are needed for this patient group. However, attempts to change the treatment for HPV-associated oropharyngeal carcinoma should take place in a closely monitored clinical trial setting. In this article, we summarize the epidemiology, diagnosis, and clinical behavior of HPV-associated oropharyngeal carcinoma, with emphasis on prognostic and biomarker discovery aspects, and discuss briefly the current thoughts on changing the treatment paradigms aimed at reducing morbidity while preserving the high tumor control probability through well-coordinated prospective trials.     

miR-1 as a tumor suppressive microRNA targeting TAGLN2 in head and neck squamous cell carcinoma

Based on the microRNA (miRNA) expression signatures of hypopharyngeal and esophageal squamous cell carcinoma, we found that miR-1 was significantly down-regulated in cancer cells. In this study, we investigated the functional significance of miR-1 in head and neck squamous cell carcinoma (HNSCC) cells and identified miR-1-regulated novel cancer pathways. Gain-of-function studies using miR-1 revealed significant decreases in HNSCC cell proliferation, invasion, and migration. In addition, the promotion of cell apoptosis and cell cycle arrest was demonstrated following miR-1e transfection of cancer cells. A search for the targets of miR-1 revealed that transgelin 2 (TAGLN2) was directly regulated by miR-1. Silencing of TAGLN2 significantly inhibited cell proliferation and invasion in HNSCC cells. Down-regulation of miR-1 and up-regulation of TAGLN2 were confirmed in HNSCC clinical specimens. Our data indicate that TAGLN2 may have an oncogenic function and may be regulated by miR-1, a tumor suppressive miRNA in HNSCC. The identification of novel miR-1-regulated cancer pathways could provide new insights into potential molecular mechanisms of HNSCC carcinogenesis.     

Serum matrix metalloproteinase-9 in head and neck squamous cell carcinoma is a prognostic marker

The aim of this study was to determine whether serum matrix metalloproteinase-9 (MMP-9) could predict cause-specific and relapse-free survival in patients with squamous cell carcinoma of head and neck. Furthermore, this study was designed to investigate whether there is an association between MMP-9 immunohistochemical staining and serum MMP-9 levels. Pretreatment serum levels of MMP-9 were quantitatively measured by ELISA assay in 67 patients presenting with a primary head and neck squamous cell carcinoma. The results were compared with the corresponding immunohistochemical staining results, clinical data and the patients' outcome. The follow-up time for all of the patients was at least 5 years. There was a statistically significant correlation between circulating MMP-9 and MMP-9 immunohistochemical staining in the corresponding tumors (p = 0.028). The cause-specific and relapse-free survival rates were clearly lower among patients with high MMP-9 serum levels (> 73 ng/ml). The 5-year cause-specific survival-rate was 40% in a patient group with high serum MMP-9, and 69% for patients with a low MMP-9 level (p = 0.027). In the same follow-up period, the cumulative relapse-free survival rate was 36% in patients presenting with a high serum MMP-9 and 66% in those with a low MMP-9 level. No correlation was found between MMP-9 serum levels and the traditional clinical or histopathologic factors. The results suggest for the first time that pretreatment serum MMP-9 level could serve as a prognostic factor in head and neck squamous cell carcinoma.     

In vitro BPDE-induced DNA adducts in peripheral lymphocytes as a risk factor for squamous cell carcinoma of the head and neck.

The level of DNA adducts under the same conditions of carcinogen exposure and cell proliferation reflects an integrated measure of carcinogen metabolism and DNA repair. Therefore, such DNA adduct levels have the potential to be a biomarker for susceptibility to chemical carcinogenesis. In a pilot study of 91 patients with squamous cell carcinomas of the head and neck and 115 controls who were frequency matched by age, sex, ethnicity, and smoking status, we applied a newly developed in vitro assay of benzo[a]pyrene diol epoxide (BPDE)-induced DNA adducts in short-term peripheral lymphocytes cultures. Levels of BPDE-DNA adducts were found to be significantly higher in cases than in controls (mean +/- SD, 76.8 +/- 77.4/10(7) and 47.1 +/- 48.0/10(7) nucleotides, respectively; p < 0.001). Using the median level of control values (35/10(7)) as the cut-off point, about 66% of cases were distributed above this level. Logistic regression analysis revealed that the level of BPDE-induced DNA adducts was an independent risk factor (odds ratio = 2.22; 95% confidence interval = 1.22--4.04) after adjustment for age, sex and smoking status. Further stratified analyses showed that levels of the induced adducts between cases and controls were significantly higher in both age groups, that is, younger or older than 60, as well as in both men and women. Smoking had a positive effect on the induced adducts. The highest level of induced adducts was seen in current smokers, then former smokers and non-smokers. There was a statistically significant dose--response relationship between the quartile levels of BPDE-induced DNA adducts and the risk of head and neck cancer (trend test, p = 0.003). Despite the relatively small sample size, the association of BPDE-induced DNA adducts and cancer risk suggests that this assay has the potential to complement with other biomarkers in identifying individuals at increased risk of developing tobacco-related cancers.     

Frequently methylated tumor suppressor genes in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is a very aggressive cancer. In advanced stages, the patient has poor chances of receiving effective treatment, and survival rates are low. To facilitate timely diagnosis and improve treatment, elucidation of early detection markers is crucial. DNA methylation markers are particularly advantageous because DNA methylation is an early event in tumorigenesis, and the epigenetic modification, 5-methylcytosine, is a stable mark. A genome-wide screen using Restriction Landmark Genomic Scanning found a set of genes that are most commonly methylated in head and neck cancers. Five candidate genes: septin 9 (SEPT9), sodium-coupled monocarboxylate transporter 1 (SLC5A8), functional smad-suppressing element on chromosome 18 (FUSSEL18), early B-cell factor 3 (EBF3), and iroquois homeobox 1 (IRX1) were methylated in 27% to 67% of the HNSCC patient samples tested. Furthermore, approximately 50% of the methylated tumor samples shared methylation between two of the five genes (most commonly between EBF3 and IRX1), and 15% shared methylation between three of the five genes. Expression analysis revealed candidate gene down-regulation in 25% to 93% of the HNSCC samples, and 5-aza-2'-deoxycytidine treatment was able to restore expression in at least 2 of 5 HNSCC cell lines for all of the genes tested. Overexpression of the three most frequently down-regulated candidates, SLC5A8, IRX1, and EBF3, validated their tumor suppressor potential by growth curve analysis and colony formation assay. Interestingly, all of the candidates identified may be involved in the transforming growth factor beta signaling pathway, which is often disrupted in HNSCC.