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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Anticancer Res. Author manuscript; available in PMC Dec 12, 2011.
Published in final edited form as:
PMCID: PMC3236111
NIHMSID: NIHMS338797

Fascin and CK4 as Biomarkers for Esophageal Squamous Cell Carcinoma

Abstract

Background

Several studies have suggested that fascin, cytokeratin 14 and cytokeratin 4 may have significant roles as biomarkers for the progression and survival of esophageal squamous cell carcinoma (ESCC).

Methods

This study performed immunohistochemistry in tissue microarrays, profiling premalignant lesions and invasive tumors.

Results

Fascin increased across the following states as follows: normal epithelium (26%) to dysplasia (46%) to ESCC (68%), while CK4 was undetectable in ESCC (0%) compared to normal epithelium (45%) or dysplasia (41%). CK14 was elevated and invariant in expression. In regression analyses, compared to normal epithelium, higher fascin expression was associated with a 36% increased risk of dysplasia (odds ratio=1.36) and a 56% increased risk of invasive ESCC (odds ratio=1.56).

Conclusions

Expression of fascin is up-regulated in the transformation from normal epithelium, through dysplasia, into invasive carcinoma. Expression of CK4, CK14 and fascin did not correlate with patient survival. Fascin has a potential role as an early detection biomarker and CK4 as a tumor marker in ESCC.

Cancer of the esophagus is the fifth most common cause of cancer death in the world, with 562,000 deaths reported in 2004 (1). Because most patients present with late stage disease, esophageal cancer has a particularly poor prognosis, with only a 17% relative five-year survival (2). Two histological types of esophageal cancer occur, adenocarcinoma and squamous cell carcinoma, both with equally poor prognosis. Esophageal adenocarcinoma was relatively uncommon in the past, but its incidence has increased dramatically over the past 25 years, and it is now the predominant esophageal cancer in the U.S.A. (37). Esophageal squamous cell carcinoma (ESCC), however, still accounts for 80% of esophageal cancer cases worldwide and is extraordinarily common in several geographical regions of the world. China, with its large population and high rates of esophageal cancer, accounts for over half of the world’s esophageal cancers, and Shanxi Province, a region in north-central China, has among the highest rates in China. In populations with such high rates of ESCC, screening is an essential element of cancer control. Biomarkers associated with the transition from normal epithelium to pre-malignancy are optimal targets for early detection strategies. The goal, then, is to find new biomarkers that predict the development of pre-malignant lesions as well as invasive tumors and prognosis.

Previous studies have identified fascin, SPARC, CK4, and CK14 as potential biomarkers in ESCC (810). This study sought to validate and explore further the role of expression of these four biomarkers, by means of immunohistochemistry (IHC), in reference to progression from normal, epithelium through dysplasia to invasive carcinoma, as well as prognostic markers in invasive carcinoma.

Previous studies by this group compared tumor and matched normal tissues from ESCC patients from Shanxi Province in China using cDNA expression microarrays and 41 differentially-expressed genes were found (8). Additional validation at the RNA level was carried out by quantitative reverse transcription polymerase chain reaction (RT-PCR) and confirmed the initial expression array results (8, 11). A panel of these markers was previously studied by IHC in ESCC patients drawn from Beijing, China (9). Fascin was previously explored as a prognostic biomarker of survival in squamous cell carcinoma patients from Kyoto Japan (10). Recent studies demonstrated that the expression of cytokeratins may be related to the etiology of the tumors (12) and may offer new means of exploring the relationship between the etiology and the phenotype of cancer types.

In the present study, two sets of tissue microarrays (TMAs) were employed to investigate protein expression of four genes, fascin (actin-bundling protein, 55-KD, p55, FSCN1), cytokeratin 4 (CK4; Keratin 4, KRT 4), CK14 (Keratin 14, KRT 14), and SPARC (secreted protein acidic, rich in cysteine), in ESCC as potential biomarkers for early detection, tumor diagnosis and prognosis.

A tumor TMA, constructed of tumor specimens from patients with extensive clinical/pathological, exposure and outcome data was investigated. At the same time, a family of progression TMAs was constructed from biopsy specimens, obtained as part of a cancer screening effort. These progression TMAs enabled the investigation of the expression of the biomarkers in pre-invasive tumors. The patients assessed in the current validation study were from the same population previously studied for RNA expression (8); however, all subjects in the current study were independent of those evaluated for RNA expression.

Fascin is the first member of a family of proteins termed fascins to be cloned and is characterized by a highly conserved 55-KD actin-bundling protein (14). Fascin functions in the organization of two major forms of actin-based structures. One form is dynamic, cortical cell protrusions; the second form is cytoplasmic microfilament bundles such as filopodia, spikes, lamellipodial ribs, dendrites and microvilli (15). In normal epithelial cells, fascin expression is usually absent or very low, but is often up-regulated in several types of human cancer types, including cancer of the colon (16), breast (17), lung (18), bladder (19), stomach (20), and esophagus (9). Fascin overexpression is suggested to disrupt epithelial junctions and may increase the potential for tumor invasion and metastasis (19). Intermediate filaments are polymers that, together with actin and microtubules, form the cytoskeleton of cells that is critical in maintaining normal cell integrity. Epithelial cell intermediate filaments are keratins derived from a family of proteins that includes CK4 and CK14 (12). Cytokeratins are used extensively as diagnostic markers for various malignancies (21). SPARC, an extracellular matrix protein, also known as osteonectin or BM-40, plays important an role in development, tissue healing and remodeling and angiogenesis (22, 23). Increased mRNA expression of SPARC has been shown in a number of human cancers, including ESCC (8, 23, 24). SPARC protein is produced at high levels in many types of cancer, especially by cells associated with tumor stroma and vasculature (22). The SPARC protein has also been reported to be up-regulated in esophageal cancer (25, 26). In contrast, decreased expression of this protein has also been found in several cancers, with methylation of the SPARC promoter proposed as a possible mechanism (27).

In the present study, fascin, CK4, CK14, and SPARC protein expression levels were investigated by applying IHC assays to biopsy and tumor resection TMAs to enable the examination of the relationships between protein expression and risk factors, clinicopathological characteristics and survival in ESCC.

Materials and Methods

Patient selection, sample collection, and patient follow-up

This study was approved by the institutional review boards of the Shanxi Cancer Hospital in P.R. China and the National Cancer Institute in the U.S.A. Details of patient selection and TMA construction were previously described (13). The first study population consisted of patients seen between 1998 and 2001 at the Shanxi Cancer Hospital in Taiyuan, Shanxi Province, People’s Republic of China. Patients that were diagnosed with ESCC and considered candidates for curative surgical resection were identified and recruited to participate in the study. None of the patients had received prior therapy and Shanxi was the ancestral home for all of them. After obtaining informed consent, patients were interviewed to obtain information on demographic and lifestyle cancer risk factors (such as smoking, alcohol drinking and family history of cancer) and clinical data. Tumor tissue obtained during surgery was fixed in 70% ethanol and embedded in paraffin for protein expression analysis. In 2003, all patients (or their families) whose ethanol-fixed, paraffin-embedded resection tissues were examined for this study were re-contacted to ascertain vital status. For those who had died, the date and cause of death were determined.

A second study population included patients evaluated by the Yangcheng County Cancer Institute in Yancheng, Shanxi Province, People’s Republic of China between 2001 and 2002, and included both asymptomatic subjects invited for esophageal cancer endoscopic screening examinations and symptomatic subjects evaluated endoscopically for diagnostic purposes. Age (median: 42 years) and gender information (44% male) were available but no other covariate information was known. All biopsies were ethanol-fixed and paraffin-embedded.

TMA construction and validation

Details of the large tumor TMA construction were previously described (13). Tumor tissues were sampled from 313 ESCC cases with 0.6-mm-diameter tissue cores using an MTA-1 tissue arrayer (Beecher Instruments, Sun Prairie, WI, USA). Biopsied tissue samples from 95 subjects from Yangcheng were arrayed into four tissue microarray recipient blocks with 2.00 mm cores. After exclusion of cores with inadequate tissue following sectioning and staining, the final IHC analyses included cores from 265 ESCC patients in the large tumor TMA, of which 257 were available for analysis. Of 257 ESCC patients with data for at least one biomarker, survival information was available on 253; of these, 79 (31%) were alive at follow-up, while 174 (69%) were determined to have died.

Each of the patients contributed to one or more of the different biomarker analyses. Final numbers of patients for each of the biomarkers evaluated in this study are annotated in the tables.

IHC analysis

Slides were stained according to manufacturer’s protocols for fascin (Dako, Carpenteria CA, USA; 1:50 dilution), CK4 (Novocastra Lab, Newcastle, UK; 1:40 dilution), CK14 (Lab Vision, Fremont, CA, USA; 1:50 dilution) and SPARC (Novacastra Lab; 1:40 dilution). In brief, 5-μm-thick deparaffinized sections were pretreated with 3% H2O2 in methanol for 10 min. Antigen retrieval included pressure cooker treatment for 5 or 25 min, blocking with 10% goat serum for 1 h, followed by incubation with primary antibodies at an appreciated dilution of 1:40 or 1:50 overnight at 4°C. Secondary antibody (anti-mouse IgG (H+L), Vector Laboratories, Burlingame, CA, USA; 1:500 dilution) was applied for 1 h at room temperature, followed by the ABC (Vector Laboratories) solution for 1 h at room temperature. Slides were developed with 0.02% 3′, 3′-diaminobenzidine (DAB) solution (Sigma, St. Louis MO, USA), counterstained with hematoxylin, dehydrated in ethanol, and cleared in xylene.

Immunohistochemistry assessment and scoring

For assessment of fascin, CK4 and CK14 proteins, two scores were assigned to each core: (i) the cytoplasmic staining intensity [categorized as 0 (absent), 1 (weak), 2 (moderate), or 3 (strong)]; and (ii) the percentage of positively stained epithelial cells [scored as 0 (0% positive), 1 (1–25%), 2 (26–50%), 3 (51–75%), or 4 (>75%)]. An overall protein expression score was calculated by multiplying the intensity and positivity scores (overall score range, 0–12). This overall score for each patient was further simplified by dichotomizing it to negative (overall score of ≤ 3) or positive (score of ≥ 4).

Statistical analysis

All statistical analyses were performed using SAS software (SAS Corp., Cary, NC, USA). Chi-square tests were used for statistical analyses of the relations between expression levels of proteins of target genes and lifestyle risk factors, as well as clinicopathological characteristics (Table I) (13). Survival was calculated from the date of surgery to the date last known alive or date of death. Overall survival was examined visually with Kaplan-Meier curves and analyzed by log-rank tests. Spearman non-parametric correlation coefficients were used to assess correlations among biomarkers, risk factors, and clinicopathological features. Polytomous logistic regression was used to evaluate the relations of protein expression scores (the exposures of interest) to the outcomes of dysplasia and ESCC cases with normal tissue (the reference outcome) in subjects in the biopsy TMA while adjusting for age and gender; odds ratios (OR) and 95% confidence intervals (95% CI) were computed. All p-values were two-sided and considered statistically significant if p<0.05.

Table I
Patient characteristics in biomarker protein expression tumor TMA study of four selected genes, with unadjusted and adjusted hazard ratios for death.

Results

Patient characteristics and survival analysis

Clinical features of the patients evaluated in the tumor TMA with available clinicopathological information are presented in Table I. Males and females were comparable for all characteristics evaluated except that smoking and alcohol consumption were more common in males. Hazard ratios (HR) for death by risk factors and clinicopathological features in ESCC cases represented on the tumor TMA are also shown in Table I. Higher tumor stage and the presence of lymph node metastasis both were associated with reduced survival, even after adjustment for risk factors and other clinicopathological features.

Tumor TMA protein expression and relation to survival

Fascin was positive in 56% of ESCC cases, while CK4 positive staining was detected in 84% of ESCCs, and CK14 was positive in 33% of ESCCs (Table II and Figure 1C). SPARC staining was present in the desmoplastic stroma but negative in the tumor epithelium (Figure 1). Only a small fraction of TMA cores contained desmoplastic stroma and these numbers were insufficient for analysis. Analysis of protein expression of these four genes in relation to survival showed no associations, whether evaluated as continuous overall scores or as binary positive/negative variables.

Figure 1
Examples of IHC for the biomarkers examined in this study. The photomicrographs are representative images of IHC staining patterns. Scale bar is 100 μm. (A) Cytokeratin 4, (B) Cytokeratin 14, (C) Fascin, demonstrating diffuse cytoplasmic staining ...
Table II
Distribution of protein expression for four selected genes, Cox proportional hazard ratio (HR) and 95% CI for overall survival biomarker status for ESCC patients in tumor TMA.

Correlations among biomarkers, risk factors, and clinicopathological features

Among the biomarkers, the tumor TMA revealed that only fascin and CK14 were highly correlated in a positive manner (r = 0.28) (Table III). Fascin expression was also positively correlated with male gender and tobacco use. Protein expressions for the other three biomarkers examined were not associated with the risk factors studied. Among the clinicopathological features examined, higher expression of fascin and CK14 were both associated with lower tumor grade and well-differentiated tumors.

Table III
Spearman correlations between four selected biomarkers and five risk factors and four clinicopathological features in tumor TMA.

IHC analyses of biopsy TMAs

SPARC protein was not detected by IHC within ESCC tumor cells, but rather in the desmoplastic stroma. As such, the biopsy TMAs were not stained for SPARC, since they contained only superficial epithelium and lacked the necessary underlying stromal structures.

Fascin staining was cytoplasmic in pattern and only apparent in the basal layer of the normal esophageal epithelium. However, dysplasias exhibited positive staining of fascin in all cell layers from the basal layer to the granular layer of the epithelium. In ESCC, immunoreactivity of fascin was homogenously present in the tumor cells. The frequency of positive staining of this protein increased monotonically across the transition from normal tissue (26%) to dysplasia (45%) to ESCC (69%) (Table IVa).

Table IV
Prevalence of overall protein expression level scores by pathologic diagnosis in subjects with healthy, dysplasia, or invasive ESCC on biopsy TMAa.

IHC revealed that CK4 protein was diffusely stained in the epithelium of normal esophageal tissue and was considered positive in 45% of subjects with normal histology (Table IVb). The remainder of the samples from patients with normal tissue cores showed weak focal staining that was below the threshold for positivity. CK4 expression was also positive in 41% of samples from patients with dysplasia, but none of the invasive ESCCs in the biopsy sample series.

IHC expression of CK14 was limited to the cytoplasm and was detected in the basal cells of the epithelium in normal esophageal tissue and dysplastic samples and diffusely expressed in all epithelial cells in tumors. The prevalence of CK14 staining was high and invariable across morphologic categories: 89% in normal tissue, 86% in dysplasias, and 82% in ESCCs (Table IVc).

Biopsy TMA polytomous regression analyses

Age- and gender-adjusted ORs for increasing protein expression of CK4, CK14 and fascin are shown in Table V for subjects represented on the biopsy TMA. Higher fascin scores were associated with higher risks for both dysplasia (OR, 1.33; 95% CI, 1.07–1.67) and ESCC (OR, 1.56; 95% CI, 1.24–1.97). Compared to normal tissue samples, higher CK4 scores did not affect risk of dysplasia, but were associated with markedly decreased risk of ESCC (OR, 0.04; 95% CI, 0.01–0.24). CK14 score was unassociated with risk of either dysplasia or ESCC.

Table V
Age-, gender-adjusted OR and 95% CIs for increasing protein expression by histology from biopsy TMA (polytomous regression).

Discussion

Previous studies from this group showed differential expression of the four genes, fascin, CK4, CK14 and SPARC at the RNA level in ESCC by both cDNA array as well as by confirmatory real-time RT-PCR methods (8, 11). Xue et al., using a different cohort of patients, examined the expression of these biomarkers by IHC in both tumors and dysplastic lesions (9), while Hashimoto et al. examined the expression of fascin as a prognostic biomarker in tumors only (10). The data of the present study are a validation at the protein level of the same cohort of patients previously evaluated at the level of RNA expression (11).

The present study demonstrated that fascin is overexpressed at the protein level in ESCC, which is concordant with prior RNA expression findings (8, 11). Increased fascin expression also correlated with the presence of dysplasia and invasive cancer of the esophagus. These results are largely concordant with the findings of Hashimoto et al., although their data were limited to comparisons of normal tissue and tumor epithelium (10). The present findings are similar to those of Xue et al., both in premalignant and tumor lesions (9). In vitro studies by Xie et al. suggested that fascin expression correlates with cell proliferation and invasive properties (28). The observation that fascin is associated with cellular proliferation and invasion is congruent with the observation that fascin is up-regulated during progression from normal epithelium to invasive tumor. The same molecular signals are anticipated to be involved in cell proliferation and migration, before and after invasion.

CK4 protein is expressed in normal, non-keratinizing stratified squamous epithelia of the upper digestive tract (21). It is also expressed in several tumors of the upper digestive tract, including esophageal adenocarcinoma (29, 30); however, few studies have characterized the CK4 protein expression in ESCC (19, 20, 30). Chung et al. reported that CK4 protein expression is decreased in dysplasia and ESCC by 80% and 85%, respectively, compared to normal epithelial cells (29). Xue et al. reported that CK4 is underexpressed in ESCC and dysplasia (9). In the current study, CK4 was negative in all invasive ESCCs in the biopsy TMA, but positive in 84% of tumors in the tumor TMA. A precise explanation for these differences in CK4 expression is unknown, however, several explanations are possible. Recently, Singha et al. studied the CK4 expression in populations at low- and high-risk for ESCC in India (12). Neither the low- nor the high-risk population showed positive CK4 expression by their dichotomized scoring system. Differences in CK4 expression in ESCC observed in different study populations may be due to unrecognized factors that differentiate these tumors at the molecular level; alternatively, these differences may also have been influenced by technical issues in the collection and preparation of samples. In the current study, the subjects whose samples were used to create the biopsy and tumor TMAs came from different geographical locations within Shanxi Province, which may have affected the CK4 expression pattern. Furthermore, the samples were not procured or processed using a uniform protocol. Biopsy tissues were sampled from superficial esophageal tissues, whereas the tissues in the tumor TMA were obtained from deep portions of surgically resected invasive tumors without relation to normal epithelium. As CK4 expression decreased in the transition from normal tissue to invasive tumor in the report by Chung et al. (29), a difference in the expression throughout the tumor exhibited by the weak and punctate staining observed on the tumor TMA may have resulted in the different CK4 expression patterns observed in the present study. In an mRNA expression study, Vianne et al. reported that well-differentiated ESCC shows focal CK4 expression, whereas CK4 expression was not detected in poorly-differentiated ESCC (30), suggesting that the degree of histological differentiation may also influence the CK4 expression in ESCC.

CK14 expression results in ESCC are conflicting in the published literature. The previous cDNA array (11) and real-time RT- PCR studies (unpublished data) by this group showed overexpression of CK14 at the mRNA level. However, overexpression of CK14 protein likely does not occur in all ESCC cases. Real-time quantitative RT-PCR of 70 ESCC patients previously showed that only 66% of patients overexpressed CK14 (≥ 2-fold change), while 23% expressed CK14 RNA in the normal tissue range (1.999–0.50) and 11% underexpressed CK14 (unpublished data). Kan et al. reported that CK14 is under-expressed in a cDNA microarray experiment using an ESCC cell line (31). One of the previous reports from this group revealed that CK14 expression level is decreased in ESCC compared with normal tissue using multiplex tissue immunoblotting, although sample size was small (29). The study by Xue et al. showed that CK14 is overexpressed in both ESCCs and its precursor lesions (9). In the current study, CK14 protein expression was not different across the spectrum of normal tissue, dysplasia, or invasive tumor. Discrepancies between mRNA and protein expression data are not unusual (32). Taken together, these data suggest that CK14 is not a viable biomarker, at least not using IHC methods.

SPARC is a non-structural component of extracellular matrix-associated matricellular glycoprotein. It is known that SPARC protein is present within focal adhesions and cytoskeletal structures and that it influences levels of cell adherence and migration capacity and may play a role in the invasion and metastasis of tumors. In the present study, we did not identify overexpression of SPARC protein in ESCC by IHC, which is inconsistent with several other studies of esophageal cancer (26, 27, 33). Porte et al. reported overexpression of SPARC mRNA in ESCC, but did not evaluate protein levels (27). Yamashita et al. reported high expression of SPARC by both mRNA and Western blotting (26). They performed IHC for SPARC expression to confirm the localization of SPARC in ESCC specimens; however, the prevalence of SPARC by IHC was not reported. Che et al. found up-regulated SPARC protein expression in 30 of 36 (83%) ESCC cases studied (33). Using tissue immunoblotting, Chung et al. demonstrated elevated SPARC protein expression in the stroma underlying dysplasia and the desmoplastic stroma invaded by tumor cells, but did not observe SPARC expression in epithelial cells (29). One possible factor contributing to the reported differences in studies of SPARC protein expression in ESCC is the use of different antibodies. Yamashita et al. (26) and Che et al. (33) used a different IHC antibody from the present study or the Chung et al. immunoblotting study (29). Xue et al. also used the same antibody as in the present study and reported SPARC expression by IHC in just 6% of stage II-IV ESCCs, and none of the stage I ESCC cases evaluated (9); results similar to our own.

In conclusion, this study showed that fascin is overexpressed at the protein level in ESCC, a result concordant with prior RNA expression findings, as well as previous IHC studies (9). Fascin expression progressively increased across the transition from normal tissue to dysplasia to ESCC and increased fascin expression was associated with increased risk of both dysplasia and invasive cancer of the esophagus. CK4 expression was decreased in ESCC compared to either normal tissue or dysplasia and increased CK4 was associated with decreased risk of ESCC. These finding are discordant with other cohorts and suggest epigenetic factors that may play a role in CK4 protein expression. These results affirm a potential role for fascin as an early detection marker in ESCC.

Acknowledgments

This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Division of Cancer Epidemiology and Prevention, and Center for Cancer Research. The authors wish to thank Kimberly Parker, Langston Lim and Kris Ylaya for technical assistance.

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