Review ArticleExperimental StudiesR

Body Fluid Biomarkers for Early Detection of Head and Neck Squamous Cell Carcinomas

KANG-DAE LEE, HYOUNG-SHIN LEE and CHANG-HO JEON
Anticancer Research April 2011, 31 (4) 1161-1167;

Abstract

Along with advancements in treatment, early detection of primary tumor, and relapse seems to remain a key factor for improving the survival rate of patients with head and neck squamous cell carcinoma (HNSCC), in which a high proportion of patients are diagnosed at an advanced stage. Recent advancements in basic research of molecular biology have improved the understanding of the molecular process of HNSCC progression and have led to identification and characterization of numerous biomarkers. Biomarkers of HNSCC are expected to facilitate the early detection of primary, and relapsed tumors. In the present article, we review the recent discoveries of potential biomarkers for the early detection of HNSCC. Most of the promising biomarkers have some limitations in clinical diagnosis. However, salivary interleukin-8 and melanoma-associated gene showed good sensitivity, specificity, convenience, and standardization. As HNSCC is a life-threatening disease, large-scale clinical validation is necessary for these two markers.

Head and neck squamous cell carcinoma (HNSCC) is the sixth common cause of cancer-related death and accounts for 6% of all cancer cases worldwide (1). Each year there are approximately 560,000 new cases and 300,000 deaths due to HNSCC (2). A slight decrease in the overall incidence of HNSCC has been detected in the past two decades. However, an increase in cancer in the base of the tongue and tonsillar cancer has been noted, which appears to be more pronounced in young adults in the USA and European countries (3). The most important risk factors in HNSCC are tobacco and alcohol consumption (4). Human papilloma virus has been reported to be associated with HNSCC in patients without a history of tobacco or alcohol consumption (5).

Successful treatment of the patients depends on early detection and correct therapy for each cancer phase. However, most cases of HNSCC are detected when the patient has become symptomatic from the effects of the primary disease or when lymphatic metastases are palpable. A patient's complaints of pain, bleeding, ulceration, mass, otalgia, and dysphagia will usually direct the clinician to the primary lesion, which is typically at least stage II, and often has associated cervical lymphadenopathy (6). It has been reported that only 30% of HNSCC cases in the USA are diagnosed at an early clinical stage and two-thirds of patients present with advanced stage III or IV tumors (7, 8).

Early Detection of HNSCC

Five-year survival rates of HNSCC have not improved despite improved locoregional control and reduced treatment-related morbidity (9). This can be attributed to a lack of suitable markers for screening, presentation of the disease at an advanced stage, and failure of advanced lesions to respond to treatment (10). The treatment of neoplasia is still most effective when the tumor burden is lowest at the primary site and when the lymphatic spread is the least. Thus, effective therapy for HNSCC will depend on early diagnosis and intervention. Despite the obvious advantage of earlier diagnosis of HNSCC, no strategy has yet proven to be a consistently effective means of diagnosing these malignancies at an early stage and none of the screening methods have been proven to decrease mortality from HNSCC (6, 7, 11).

Two pilot studies have shown that early genetic alterations in tumorigenesis can be successfully identified in body fluids that drain from the organ affected by the tumor (12), and in the serum or saliva (13, 14). Analysis of these markers in oral secretions and other accessible specimens may allow for early detection and screening of individuals at high risk for cancer. Depending on the location of the tumor, one may not be able to easily access and swab the tumor bed. Thus the use of the fluid phase of saliva may show unique advantages over the use of exfoliated cells. Although the use of salivary biomarkers did not identify the site from which the tumor originated, they were able to identify patients at risk (15). Salivary RNA, the most accessible and non-invasive biofluid of the body, harbors a wide spectrum of biological analytes informative for clinical diagnostic applications. Salivary RNA appears to enter the oral cavity from different sources, including the three major salivary glands, gingival crevice fluid, and desquamated oral epithelial cells. Park et al. (16) observed that the supernatant of saliva contains genuine human mRNAs. The amount of mRNA in saliva may be too low to be used for mRNA-specific RT-PCR technologies (17). However, many researchers have successfully amplified potential tumor markers by using salivary mRNA, and moreover, salivary mRNA can be effectively preserved by the RNAprotect Saliva reagent (18).

Biomarkers may be compromised by their insufficient diagnostic sensitivity and specificity and are mostly utilized as an auxiliary approach to assist in clinical decision-making (19). However, recent reports have introduced several promising biomarkers for early detection of HNSCC, which still require continuous studies to validate their clinical feasibility for diagnosis. In the present article, we review the recent discoveries of potential biomarkers for the early detection of HNSCC. These have been summarized in Table I.

Microsatellite instability (MSI). In Nunes et al.'s study (20), tumor cell-derived DNA was detected in 16 (84%) of the 19 examined cases using mouth washing (MW) and lesion brushing (LB). They were able to detect loss of heterozygosity (LOH) in four patients with T2N0M0 tumors and one with a T1N0M0 tumor. The detection of tumor DNA in MW or LB samples could also have value in the analysis of pre-malignant lesions, such as erythroplakia and leukoplakia. A few studies attempting to analyze MSI in HNSCC studies using different sets of microsatellite markers have reported similar microsatellite alteration rates (75-95%) with lower rate of MSI, ranging from 12.5 to 35.0% (21-23).

MSI studies showed lack of uniformity of the study methods, choice and number of the markers (24). To date, no standard approach for the study of MSI has been developed for the early detection of HNSCC.

Methylation. Hypermethylation of cytosine-phosphate-guanine (CpG) dinucleotide-rich promoter regions leads to gene inactivation, which seems to occur early in the head and neck carcinogenesis (25). Rosas et al. (26) reported methylation-specific PCR (MSP) to be a highly specific tool (96%) for HNSCC detection by using saliva specimens. In 2008, Carvalho et al. (27) studied a large sample size of 527 controls and 211 HNSCC patients using multiple panels of methylated promoter regions with quantitative-MSP in serum and salivary rinses. The sensitivity and specificity for detection of HNSCC in the serum and salivary rinses were 65% and 72%, and 35% and 90% respectively.

The promoter hypermethylation rate was low in salivary rinses despite the presence of methylation in primary tumors due to the dilution effect of normal, non-methylated genomes present in salivary rinses from normal mucosa. Normal control tissue showed substantial rates of methylation in a subset of promoters (27, 28). Carvalho et al. suggested that prior studies had missed the phenomenon of promoter methylation in tissues from individuals without a cancer diagnosis due to small sample size. Promoter hypermethylation can be associated with age, race, or tobacco and alcohol exposure (29-31).

Thus, methylation markers still have some limitations to their use in the clinical laboratory such as sensitivity, specificity, robustness and convenience for HNSCC detection using body fluids.

Metalloproteinases (MMPs). MMP are proteolytic enzymes that cause degradation of extracellular matrix (ECM) allowing for tumor cell migration (11). They comprise a large and ever-growing family of Zn2+ -and Ca2+-dependent endopeptidases (32, 33). Several MMPs, including MMP-1, the gelatinases MMP-2 and MMP-9, and the stromelysins MMP-3 and MMP-10, have been implicated in cancer cell invasion and metastasis (34, 35). Although a major function of MMPs in metastasis is to facilitate the breakdown of the ECM, including the basement membrane, they play substantial roles in the maintenance of a microenvironment that facilitates growth and angiogenesis of tumors at primary and metastatic sites. MMPs have been reported to play direct roles in other cellular activities such as differentiation, proliferation, and apoptosis (32). High levels of MMP-2 or MMP-9 were detected in plasma of patients with many kinds of cancer, including HNSCC (36), breast and colorectal carcinoma (37), lung (38) and ovarian (39) tumors.

In the cases of HNSCC, Kuropkat et al. (40) showed there to be significant increase of MMP-3, -8 and -9 in serum, and Yen et al. (41) reported that MMP-1 and MMP-10 were suitable markers for oral cancer detection, with gingiva and margin as controls. Ranuncolo et al. (42) detected enhanced MMP-9 activity using euglobulin plasma fraction demonstrating 80% positive rates with 10 cases of stage I disease. The acetic acid precipitation of euglobulin may increase MMP-9 activity through the concentration of the samples (43). The acid treatment converts latent MMPs to catalytically active forms, without proteolytic cleavage of the N-terminal inhibitory sequence (44). However, Somiari et al. (45) reported that MMP-9 activity was unable to discriminate between a breast cancer group and benign disease group. To utilize MMP-9 as a tumor marker, more study would be necessary to evaluate its specificity against the benign disease group.

View this table:
Table I.

Summary of biomarkers for detection of head and neck cancer.

Interleukin (IL)-6 and IL-8. In two studies (13, 15) encompassing 32 patients with primary T1 or T2 squamous cell carcinoma of the oral cavity or oropharynx (OSCC), IL-8 was detected at higher concentrations in the saliva of patients with OSCC and IL-6 was detected at higher concentrations in the serum of patients with OSCC, indicating that IL-8 in saliva and IL-6 in serum hold promise as biomarkers for OSCC. These cytokines have also been linked with increased tumor growth and metastasis, and could contribute to carcinogenesis while their expression is silenced in normal keratinocytes. Others have also detected elevated concentrations of IL-6 and IL-8 in cell line supernatants, tumor specimens, and the serum of patients with HNSCC (36, 46). Zimmermann et al. (18) reported that four salivary mRNAs (OAZ, SAT, IL-8, and IL-1β) collectively had a discriminatory power of 91% sensitivity and specificity for oral cancer detection. However, in the study of SahebJamee et al. (47), conducted with 9 patients with OSCC, the concentration of salivary IL-8 in the tumor group was higher than that of 9 age- and sex-matched controls, but this was not statistically significant. From the literature review, it would appear to be an attractive marker for HNSCC screening, however a large number of cases would be necessary to validate the sensitivity and specificity of salivary IL-8 as a biomarker for HNSCC.

Cytokeratin 17 (CK-17). Cytokeratins are other candidates for diagnostic markers of OSCC, as they are overexpressed in OSCC compared to normal mucosa (48). Cytokeratins are among the major components of intracellular filament networks, while they cannot be found in lymph nodes, bone marrow, and peripheral blood. Constitutive expression of CK-17 in the lung occurs only in normal basal cells (49). Overexpression of cytokeratins is often associated with tumor progression and prognosis (50). CK-17 has been identified as a specific immunohistochemical marker in SCC of the larynx (51). Toyoshima et al. (48) demonstrated CK-17 mRNA overexpression in patients with T1 and T2 OSCC. In a subsequent study (52), overexpression of CK-17 mRNA was detectable in OSCC cells of 40 patients (76.9%, n=52). Wei et al. (53) also reported overexpression of CK-17 protein in oral SCC under both in vivo and in vitro conditions. However, most studies were performed using the tissue samples of cancer not saliva or serum. Moreover in the study of Domachowske et al. (50), expression of an mRNA fragment encoding CK-17 in respiratory syncytial virus-infected epithelial cells was increased 12-fold by 96 hours after infection. Thus more studies using body fluids of HNSCC patients and data for specificity are inevitable.

MicroRNA (miRNA). miRNAs are small noncoding RNA sequences with potent regulatory potential for normal biological processes ranging from cell growth, and differentiation to death (54). It has been demonstrated that genes for miRNAs genes are dysregulated in various types of cancer and that miRNAs themselves can act as both oncogene and as tumor suppressors (55). Many research groups have shown that miRNAs are differentially expressed in various cancer cells compared with normal cells, and it seems that miRNAs can more accurately classify different types of solid tumors than can mRNA, suggesting that miRNAs can be used to detect cancer (56). In addition, the fold change in mRNA between cancer cells and normal cells is relatively small, whereas the expression level of many miRNAs exhibits fold changes of tens to hundreds (57).

In HNSCC, Park et al. (58) found miR-125a and miR-200a were present at significantly lower levels (P<0.05) in the saliva of OSCC patients than in controls. However, their sensitivity and specificity were not satisfactory. Hui et al. (59) reported that one-third of the miRNAs were dysregulated in formalin-fixed samples of HNSCC. They concluded that miR-106b-25 cluster and miR-375 likely mediate HNSCC development and progression, and that miR-451 could be a potential prognostic marker for relapse of the disease.

Although miRNAs are considered as promising markers for early detection of HNSCC, it is too early for their application as a screening tool in the clinical field.

Actin and myosin. De Jong et al. (60) used advanced mass spectrometry-based quantitative proteomics analysis of the pooled soluble fraction of whole saliva from 12 individuals with pre-malignant lesions and 4 with malignant lesions. Actin and myosin proteins displayed increases in their abundance levels in soluble saliva from those with malignant lesions, compared to those with pre-malignant lesions. The observed increases in protein abundance were due to increased actin and myosin expression within the exfoliated cells with the onset of invasive oral malignancy, and not simply an increase in the number of cells in saliva. Actin and myosin are key cytoskeletal proteins enabling cell motility and invasion, behavior central to epithelial tumorigenesis (61, 62). Actin isoforms have been observed to be increased in abundance in in vitro tumor cell migration studies (63), invasive basal cell carcinoma (64), cervical squamous cell carcinoma (65), esophageal squamous cell carcinoma (66), as well as invasive OSCC (67). Likewise, increases in myosin abundance have been reported in proteomic studies of OSCC tissue (68). However other studies seem to contradict these findings (69); Turhani et al. showed a decrease of myosin light chain expression in the HNSCC tissues. More rigorous determination in a larger number of samples of sensitivity and specificity for both proteins is necessary to better understand their potential as HNSCC biomarkers.

Melanoma-associated gene (MAGE). MAGE suppress apoptosis and plays an important role in carcinogenesis (70). Several tumor-specific shared antigen families, such as MAGE, GAGE (G antigen), BAGE (B melanoma antigen), and LAGE (L antigen family 3)/NY-ESO-1 (New York esophageal carcinoma 1), which are recognized by autologous cytotoxic T lymphocytes (CTL), have been characterized at the molecular level (71-73). These antigens usually consist of peptides derived from intracellular proteins and are presented to CTL by HLA class I molecules. The antigens are of particular interest in tumor immunology because their expression seems to be restricted to tumor cells such as melanoma, HNSCC (74), ovary cancer (75), bladder cancer (76), and lung cancer (77) as well as gastrointestinal carcinomas including colorectal cancer (78). Muller-Richter et al. (79) detected MAGE-A expression in all of five oral cancer cell lines and Ries et al. (80) reported that 85.5% of tumor specimens expressed MAGE-A gene. Other studies (80, 81) demonstrated more than 90% expression rates in HNSCC tissue. Metzler et al. (82) reported a case of a persistent suspicious-looking leukoplakia in which histopathologic examination revealed no malignancy with the brush biopsy and an incisional biopsy. However, expression analysis of MAGE-A indicated significant MAGE-A3 and A4 expression pattern in the biopsy specimen; the lesion was excised completely and was identified as early invasive carcinoma. MAGE expression was also detected in the sputa of HNSCC patients (83). Thus it could be accepted as a useful marker for early detection of HNSCC. Since the expression of MAGE has not been recognized in normal tissues, except for the testis (84). Expression of MAGE products was clinically tested as a potential new target for active specific immunotherapy for oral cancer (85), then 15 out of 21 oral carcinoma expressed one of MAGE-A1 to -A6. Moreover, Pastorcic-Grgic et al. reported that the 5-year survival rate in pharyngeal cancer was poorer in cases with MAGE-A expression, demonstrating the application of MAGE as a prognostic factor (86).

Conclusion

Along with advancement in treatments, early detection of the primary tumor, and relapse, seems to remain a key factor for improving the survival rate of patients with HNSCC, in which a high proportion of patients are diagnosed at an advanced stage. Recent advancements in basic research of molecular biology have improved the understandings of the molecular process of HNSCC progression and have led to identification and characterization of numerous biomarkers. Biomarkers of HNSCC are expected to facilitate the early detection of primary, and relapsed tumor. Moreover, they could be introduced as predictive and prognostic markers, and eventually as therapeutic targets for the treatment of HNSCC. To be utilized as a biomarker for HNSCC in the clinical laboratory, the analysis for the biomarker should be accurate, standardized and simple to perform.

We reviewed eight promising biomarkers for early detection of HNSCC and most had some limitations to their use in clinical diagnosis. However, salivary IL-8 and MAGE showed good sensitivity, specificity, convenience, and standardization. As HNSCC remains a life-threatening disease, large-scale clinical validation is necessary for these two markers to play a routine role in detection of HNSCC.

Footnotes

  • This article is freely accessible online.

  • Received December 21, 2010.
  • Revision received March 2, 2011.
  • Accepted March 3, 2011.

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Body Fluid Biomarkers for Early Detection of Head and Neck Squamous Cell Carcinomas
KANG-DAE LEE, HYOUNG-SHIN LEE, CHANG-HO JEON
Anticancer Research Apr 2011, 31 (4) 1161-1167;
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