Overexpression of CDC25B and LAMC2 mRNA and Protein in Esophageal Squamous Cell Carcinomas and Premalignant Lesions in Subjects from a High-Risk Population in China

Esophageal cancer is a frequently fatal cancer that is common in some geographic regions of the world. Shanxi province in north-central China has one of the highest rates of esophageal cancer in the world. With a standardized incidence rate in excess of 100/100,000 person-years, esophageal cancer is the second leading cause of cancer death in this region (1, 2). Molecular events associated with the initiation and progression of esophageal squamous cell carcinoma (ESCC) remain poorly understood.

To better understand the role of genetics in the etiology of ESCC and to identify potential susceptibility genes, we previously compared tumor and matched normal tissues from ESCC patients from Shanxi Province using cDNA expression microarrays and identified 41 differentially expressed genes (28 underexpressed and 13 overexpressed). Two of the most prominently overexpressed genes, CDC25B and LAMC2, were chosen for further study here (3).

Whereas the importance of CDC25B (OMIM 116949; located on chromosome 20p13) in the etiology of ESCC is unknown, CDC25 phosphatases are critical components of the cellular regulatory machinery and work at the G₂-M checkpoint (4, 5). In 1995, Galaktionov et al. (6) found overexpression of CDC25B in breast cancer and suggested that the CDC25B phosphatases may contribute to the development of human cancer. Recent studies show that CDC25B is overexpressed in a variety of cancers, including esophageal cancer, and suggest that it may serve as an oncogene regulating G₂-M progression (7–20).

LAMC2 (laminin-5 γ2, OMIM 150292), located on chromosome 1q25-q31, encodes an extracellular epithelial basement membrane protein in normal tissue. LAMC2 represents a single isoform within the laminin family of proteins that contains three distinct polypeptides: the α3, β3, and γ2 chains (21, 22). LAMC2 is thought to play a crucial role in tumor cell adhesion, migration, and proliferation (23, 24). Expression of LAMC2 has been evaluated in a variety of cancers, including esophageal, colorectal, gastric, oral squamous cell, and prostate cancers (25–27), and has also been associated with invasiveness in cervical lesions (28).

In the present study, frozen tumor samples analyzed by real-time quantitative reverse transcription-PCR and immunohistochemistry applied to ethanol-fixed, paraffin-embedded tissue presented on a high-throughput tumor tissue microarray were used to evaluate CDC25B and LAMC2 mRNA and protein expression levels in ESCC patients from a high-risk area in China. These proteins were further evaluated in patients representing a morphologic spectrum of disease that included normal, dysplasia, and invasive ESCC.

This study was approved by the Institutional Review Boards of the Shanxi Cancer Hospital and the U.S. National Cancer Institute. Two different study populations were evaluated in the series of studies presented here.

The first study population consisted of patients who presented from 1996 to 2001 to the Shanxi Cancer Hospital in Taiyuan, Shanxi Province, People's Republic of China, who were diagnosed with ESCC and considered candidates for curative surgical resection. None of the patients had prior therapy and Shanxi was the ancestral home for all. After obtaining informed consent, patients were interviewed to obtain information on demographic and lifestyle cancer risk factors (e.g., smoking, alcohol drinking, and family history of cancer) and clinical data. Tumor tissue obtained during surgery was (a) snap frozen in liquid nitrogen, along with matching normal tissue, and stored at −130°C until used for RNA expression analysis or (b) fixed in ethanol and embedded in paraffin for histopathologic and protein expression analysis. In 2003, all patients (or their families) from this study population were recontacted to ascertain vital status. For those who had died, date and cause of death were determined. Additional information on treatment beyond surgery (i.e., radiotherapy and/or chemotherapy) was not obtained.

The 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 and gender information were available but no other covariate information was known. In addition to biopsies of any suspicious areas, two to three biopsies were obtained from normal-appearing mucosa in the mid-esophagus, one for local diagnostic purposes and the other(s) reserved for potential future analysis. All biopsies were ethanol fixed and paraffin embedded.

Using patients from the first study population, total RNA was extracted from each patient's matched frozen tumor and normal surgical resection tissues using Trizol reagent (Life Technologies) in accordance with the manufacturer's instructions. RNA quality and quantity were determined using the RNA 6000 LabChip/Agilent 2100 Bioanalyzer (Agilent Technologies) or electrophoresis on 1.2% denaturing agarose gel/spectrophotometer. RNA purification was done according to the manufacturer's instructions for the RNeasy Mini kit (Qiagen, Inc.) and RNase-Free DNase Set digestion (Qiagen). Reverse transcription of RNA was done by adding 5 μg total RNA, 1 μL of oligo(dT)_12-18 (500 μg/mL), 1 μL (200 units) of SuperScript II reverse transcriptase, 1 μL (2 units) of Escherichia coli RNase, and 1 μL of 10 mmol/L deoxynucleotide triphosphate (Invitrogen).

All real-time PCRs were done using an ABI Prism 7000 Sequence Detection System (Perkin-Elmer Applied Biosystems). Primers and probes for target genes (CDC25B and LAMC2) and an internal control gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were designed by Perkin-Elmer Applied Biosystems. A singleplex reaction mix was prepared according to the manufacturer's protocol of “Assays-on-Demand Gene Expression Products,” including 10 μL Taqman Universal PCR Master Mix, No AmpErase UNG (2×), 1 μL of 20× Assays-on-Demand Gene Expression Assay Mix (all Gene Expression assays have a FAM reporter dye at the 5′ end of the Taqman MGB probe and a nonfluorescent quencher at the 3′ end of the probe), and 9 μL of cDNA (90 ng) diluted in RNase-free water to a total volume of 20 μL. Each sample for each gene was run in triplicate. The thermal cycling conditions included an initial denaturation step at 95°C for 10 min, 40 cycles at 95°C for 15 s, and 60°C for 1 min.

Using these quantitative methods requires that the PCR efficiencies of all genes be similar and, preferably, ≥90%. Efficiency was measured using a standard curve generated by serial dilutions of the RNA. Consequently, the initial RNA concentration of 100 ng/μL was serially diluted 10-fold for the real-time PCR assay according to the standard protocol of Applied Biosystems. The relative standard curve quantitation method was previously described (29).⁸

Details of the 2^-ΔΔCT method have been previously described (29, 31).⁸ Briefly, the mean target gene mRNA expression level for the three mRNA measurements was calculated. The 2^-ΔΔCT method was used to calculate relative changes in gene expression determined from real-time quantitative PCR experiments. In the present study, the data are presented as the fold change in target genes CDC25B and LAMC2 expression in tumors normalized to the internal control gene (GAPDH) and relative to the normal control (matched normal as calibrator). Results of the real-time PCR data were represented as C_T values, where C_T was defined as the threshold cycle number of PCR at which amplified product was first detected. There is an inverse correlation between C_T and amount of target: lower amounts of target correspond to a higher C_T value and higher amounts of target have lower C_T values. The average C_T was calculated for both the target genes and GAPDH and the ΔC_T was determined as (the mean of the triplicate C_T values for the target gene) minus (the mean of the triplicate C_T values for GAPDH). The ΔΔC_T represented the difference between the paired tissue samples, as calculated by the formula ΔΔC_T = (ΔC_T of tumor − ΔC_T of normal). The N-fold differential expression in the target gene of a tumor sample compared with the normal counterpart was expressed as 2^-ΔΔCT (29, 31).⁸ In the present study, the range of mRNA expression was defined by the N-fold change as follows: overexpressed (N-fold change ≥2.0), normal (N-fold range from 0.5001 to 1.9999), or underexpressed (N-fold change ≤0.5).

Tumor tissue samples from 313 ESCC cases from the first study population were collected, fixed in ethanol, and embedded in paraffin. H&E-stained sections from a single random block from each patient were reviewed to define representative tumor regions (M.J.R.). A targeted core sample of each region was obtained using a manual tissue arrayer MTA-1 (Beecher Instruments) as previously described (32). Briefly, tissue cylinders with a diameter of 0.6 mm were punched and arrayed on a recipient paraffin block. Sections (5 μm) of the tissue array (“recipient”) block were cut and placed on glass slides using the tape transfer system (adhesive-coated slides PSA-CS4x, Instrumedics, Inc.) to support the adhesion of 0.6-mm array elements. To evaluate potential changes in tissue morphology between serial sections, the 1st and the 50th sections of the tissue microarray block were stained with H&E and reviewed (M.J.R. and S.M.H.).

The presence of well-differentiated or poorly differentiated foci in each tumor was also determined (M.J.R. or S.M.H.). Well-differentiated foci generally consisted of cells with low nuclear-to-cytoplasmic ratios, approximating that seen in histologically normal-appearing cells, and “hard-appearing” or “dense-appearing” cytoplasm consistent with squamous differentiation. Squamous “pearls,” or mature-appearing cells forming concentric rings, were focally identified in association with well-differentiated areas. Poorly differentiated regions were generally composed of cells with high nuclear-to-cytoplasmic ratios and less mature-appearing cytoplasm.

After exclusion of cores with inadequate tissue following sectioning and tissue transfer, the final immunohistochemical analysis included cores from 275 ESCC cases.

Biopsied tissue samples from 95 subjects from Yangcheng were collected, fixed in ethanol, and embedded in paraffin. The initial diagnostic group for biopsies was assigned based on worst reported histology among all diagnostic biopsies obtained at endoscopy. Subjects were assigned to diagnostic groups (approximately one third were normal epithelium, approximately one third were dysplasia, and approximately one third were invasive ESCC) and arrayed into four tissue microarray recipient blocks with 2.00-mm cores, essentially transplanting the entire biopsy from the donor block to the recipient tissue microarray. Dysplastic biopsies were all diagnosed as mild or moderate dysplasia, with shown abnormal features that extended between one third and two thirds of the depth of the epithelial thickness, with concurrence of two pathologist (M.T. and S.M.H.). The recipient blocks were sectioned as described above, the 1st and 25th sections were stained with H&E, and a definitive array-specific pathologic diagnosis was assigned. Only subjects whose initial pathologic diagnosis and array-specific pathologic diagnosis were in agreement were included in the results presented here. Subjects were also excluded when tissue or staining was inadequate on a specific section.

Slides from both the tumor and biopsy tissue microarrays were stained according to the manufacturer's protocols for CDC25B and LAMC2. In brief, 5-μm-thick deparaffinized sections were pretreated with 3% H₂O₂ in methanol for 10 min. Antigen retrieval included pressure cooker treatment for 10 min (for CDC25B) or protease XXIV (Biogenex) treatment for 10 min (for LAMC2) and 10% normal goat serum for 1 h to block endogenous peroxidase activity, followed by incubation with primary antibodies [CDC25B rabbit polyclonal (Cell Signaling Technology) or mouse anti-laminin-5 monoclonal p3E4 (Chemicon)] at a dilution of 1:50 for overnight at 4°C. The next day, slides were treated by the secondary antibody [biotinylated anti-rabbit IgG (H+L) for CDC25B and anti-mouse IgG (H+L) for LAMC2, 1:500 dilution; Vector Laboratories] for 1 h at room temperature followed by the avidin-biotin complex method (Vector Laboratories) solution for 1 h at room temperature. Slides were developed with 0.02% 3,3′-diaminobenzidine solution (Sigma), counterstained with hematoxylin, dehydrated in ethanol, and cleared in xylene. Concurrent positive and negative control sections were stained for all antibodies.

Stains were reviewed by three pathologists (M.T., M.J.R., and S.M.H.) and discussed to determine an appropriate analytic approach. Following the establishment of criteria, all cores on both arrays were read by a single pathologist (M.T.) using the described criteria.

CDC25B assessment considered both cytoplasmic and nuclear staining. Two scores were assigned to each core: (a) the cytoplasmic staining intensity [categorized as 0 (absent), 1 (weak), 2 (moderate), or 3 (strong)] and (b) 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). The rationale for the choice of cutoff for CDC25B protein expression was arbitrary but based on simple criteria that considered visually discernible differences in both intensity and percent cells positive. To be clearly positive for intensity, we required moderate or strong intensity (a score of 2 or 3). Similarly, we considered that over 25% of cells should be stained for a clear-cut positive in this category (a score of ≥2). Thus, the overall score, the product of these two scores, had to be ≥4 to be declared positive.

LAMC2 is normally found in the basement membrane of the epithelial-connective tissue interface and is expressed and secreted by the basal epithelial cells. When expressed in tumor cells, LAMC2 protein can be found in the cytosol, as well as secreted extracellularly. In an effort to accurately categorize the pattern of expression of LAMC2, six categories of LAMC2 expression were used and each specimen was categorized as having only one pattern (M.T.). Categories, as modified from Kuratomi et al. (33), included (a) continuous (continuous staining in the basement membrane only as seen in normal epithelium), (b) cytoplasmic (cytoplasmic staining in cancer cells), (c) diffuse (expressed diffusely in infiltrating cancer cells), (d) peripheral (expressed in peripheral cancer cells of tumor nests), (e) mixed (peripheral plus cytoplasmic or diffuse plus cytoplasmic), or (f) negative.

All statistical analyses were done using Statistical Analysis Systems (SAS Corp.). Spearman correlation coefficients and linear regression analyses were used to examine the effects of patient characteristics on the N-fold differences for both CDC25B and LAMC2 RNA expression levels for patients in the RNA expression study. For all tissue microarray analyses presented here (including lifestyle risk factors, clinical/pathologic characteristics, and survival), the patient was the unit of analysis. Although multiple tissue cores existed for approximately one third of the cases in the tumor tissue microarray, only a single core per patient (the one with the worst histologic diagnosis) was evaluated. Survival time was calculated from the date of surgery to the date of death or the date last known alive. Overall survival was examined by CDC25B overall protein expression score and by LAMC2 protein expression patterns graphically with Kaplan-Meier curves and analyzed statistically with proportional hazards regression models (SAS PHREG procedure) adjusted for lifestyle [gender, age, tobacco use, alcohol use, and family history of upper gastrointestinal (UGI) cancer] and tumor characteristics (grade, stage, metastasis, and degree of differentiation) as covariates. CDC25B protein expression scores were compared across morphologic categories (normal, dysplasia, and ESCC) using polytomous regression (SAS CATMOD procedure) to adjust for age and gender. All P values were two sided and considered statistically significant if P < 0.05.

Table 1 shows patient characteristics for the three analytic groups in the study: (a) the mRNA expression group, (b) the tumor tissue microarray protein expression group, and (c) the biopsy tissue microarray protein expression group.

Quantitative reverse transcription-PCR analyses to determine CDC25B and LAMC2 mRNA levels were done using paired normal-tumor mRNA extracts and the 2^-ΔΔCT method. PCR efficiency (E) was 92.4% for CDC25B (Y = 3.5173X + 36.88) and 91.7% for LAMC2 (Y = 3.5399X + 30.90), and correlation coefficients between RNA concentrations and C_T values were 0.99 for both CDC25B and LAMC2. CDC25B mRNA expression values (data not shown) ranged from 0.1- to 177.3-fold change in tumor relative to normal tissues (median, 3.3). RNA expression was increased more than 2-fold in 64% (47 of 73), unchanged (between 2-fold underexpressed and overexpressed) in 23% (17 of 73), and decreased more than 2-fold in 12% (9 of 73) of cases. Similarly, the range of LAMC2 differential mRNA expression values (data not shown) was 0.4- to 401.7-fold in tumor tissues relative to normal tissue in the ESCC patients tested, with a median of 16.3. Overexpression of LAMC2 mRNA was increased more than 2-fold in 89% (65 of 73), unchanged (between 2-fold underexpressed and overexpressed) in 8% (6 of 73), and decreased more than 2-fold in 3% (2 of 73) of the cases.

CDC25B mRNA expression was unrelated to any of the patient or tumor characteristics examined (i.e., gender, age, tobacco use, alcohol use, family history of UGI tract cancer, or tumor stage or grade) in univariate or multivariate linear regression models (all P > 0.10). LAMC2 mRNA expression was positively associated with age (multiple linear regression coefficient, 0.047; P = 0.02) and negatively associated with tumor grade (multiple linear regression coefficient, −0.808; P = 0.02).

Forty-four of 73 (60%) patients showed mRNA overexpression on both genes, 13 patients (18%) showed LAMC2 mRNA overexpression and normal CDC25B mRNA expression, and 8 patients (11%) showed LAMC2 mRNA overexpression but underexpression of CDC25B mRNA. A modest positive correlation was observed between CDC25B and LAMC2 mRNA expression (Spearman r = 0.25; P = 0.04).

Table 2 shows CDC25B protein intensity, percent positive cells, and overall scores for the 243 subjects with evaluable tissue for this marker. Figure 1 shows a low power image of a H&E stain of the entire tumor tissue microarray. The CDC25B protein was randomly distributed in the epithelia and the intensity of staining was generally moderate (77%) or strong (19%; see Fig. 2A-D for examples of absent, mild, moderate, and strong intensity staining). With an overall score cutoff of ≤3 as negative, 59% of cases were positive.

Table 3 shows the outcome of 275 ESCC cases whose tissue was evaluable for LAMC2. None of these tumors showed the continuous pattern (Fig. 3A) seen in normal epithelium. The majority of cases showed the mixed pattern (52%; Fig. 3E) followed by peripheral (18%; Fig. 3D) and negative (18%; Fig. 3F) patterns. Cytoplasmic (Fig. 3B) and diffuse (Fig. 3C) patterns were seen in <10% of cases each.

The 238 ESCC patients analyzed here for protein expression and survival were successfully followed for up to 6 years after diagnosis to ascertain vital status. Overall survival rates were 71% (1 year), 47% (2 years), 29% (3 years), 11% (4 years), and 5% (5 years), with a median survival of 22 months.

Relations of protein expression to survival adjusted for lifestyle and tumor characteristics are shown in Table 4 for CDC25B and in Table 5 for LAMC2. Although both higher tumor stage [hazard ratio (HR), 1.96; 95% confidence interval (95% CI), 1.12-3.42] and presence of metastasis (HR, 2.06; 95% CI, 1.49-2.84) were significantly associated with death, our CDC25B protein overall expression score did not predict survival either alone or adjusted for other explanatory variables in our multivariate Cox proportional hazards models (multivariate HR, 1.00; 95% CI, 0.96-1.05; P = 0.90).

As with the multivariate Cox model for CDC25B, tumor stage (HR, 2.09; 95% CI, 1.19-3.69) and metastasis (HR, 1.97; 95% CI, 1.42-2.73) were also significant predictors of survival in the multivariate LAMC2 protein expression model. Unlike CDC25B, however, three of the four LAMC protein expression patterns were associated with markedly shorter survival than the cytoplasmic pattern (the pattern with the longest survival time; see Fig. 4 for Kaplan-Meier survival curves for all five LAMC2 patterns). Cases with the diffuse pattern had the shortest survival time (HR, 3.54; 95% CI, 1.41-8.87) followed by cases with no expression (the negative pattern, HR, 2.69; 95% CI, 1.29-5.63) and cases with the peripheral pattern (HR, 2.30; 95% CI, 1.14-4.63). Although not statistically significant, cases with the most common pattern, the mixed pattern, also seemed to have shortened survival (HR, 1.75; 95% CI, 0.91-3.35). Median survival times varied more than 2-fold across the five LAMC2 protein expression patterns. In decreasing order of survival time, these patterns (and survival times) were cytoplasmic (34.9 months), mixed (25.9 months), peripheral (19.4 months), diffuse (17.5 months), and negative (14.1 months).

The design and construction of the biopsy tissue microarray are shown in Fig. 5A and B. The prevalence of CDC25B protein expression intensity, percent cells positive, and overall score by pathologic diagnosis are shown in Table 6. No differences were seen in intensity of staining by pathology diagnoses, but the percent of cells staining positive did vary significantly by pathologic diagnosis, as did the overall protein expression score. Dichotomized into negative (overall score of 1 or 2) versus positive (overall score of ≥3), no normal subjects were positive, whereas 26% of dysplasia subjects and 50% of invasive cancer cases were positive.

The prevalence of LAMC2 protein expression patterns by pathologic diagnosis is shown in Table 7. Although overall there was a statistically significant association between expression patterns and pathologic diagnoses, distributional differences were found only among invasive cancer cases. All subjects with either a normal or dysplastic biopsy showed the continuous pattern, and this pattern is not seen in tumor samples. The most common expression patterns observed in cases with invasive cancer were the peripheral pattern (39%) and the mixed pattern (39%).

Age- and gender-adjusted polytomous regression models showed that, compared with ESCC cases, increased overall CDC25B protein expression scores were associated with reduced odds of having dysplasia (odds ratio, 0.55 for each unit increase in CDC25B score; 95% CI, 0.32-0.96; P = 0.03) or normal (odds ratio, 0.23; 95% CI, 0.09-0.60; P = 0.003) morphology on biopsy. The uniform distribution of LAMC2 expression pattern (i.e., all continuous) in normal and dysplasia cases did not permit multivariate model-based analyses.

In a previous 8K cDNA array study, we found that CDC25B and LAMC2 were both overexpressed in ESCC patients from Shanxi Province, a high-risk area in China (3). In the present study, we evaluated mRNA expression of these two genes in 73 new ESCC patients from the same high-risk area using a more quantitative method, real-time quantitative reverse transcription-PCR, and confirmed our previous results: CDC25B mRNA was overexpressed in nearly two thirds of cases and LAMC2 mRNA was overexpressed in 89%. Although CDC25B overexpression in ESCC has been previously reported (8, 34), to our knowledge, our two studies represent the first reports of overexpression of LAMC2 mRNA in ESCC.

In addition to confirming CDC25B and LAMC2 mRNA overexpression, using high-throughput tissue microarray platforms, we also showed overexpression of both CDC25B and LAMC2 proteins in roughly similar proportions of ESCC cases as those with overexpression of mRNA.

By following up the ESCC cases examined on our tumor tissue microarray to determine their vital status, we were also able to evaluate the relation of protein expression to survival. None of the metrics we evaluated for CDC25B protein expression showed a relation to survival. Four previous studies also reported on the relation of CDC25B protein expression to survival in ESCC cases (15, 16, 34, 35), but none reported a significant association.

The biopsy tissue microarray used here contained normal and premalignant tissue samples in addition to invasive cancers and permitted examination of the concordance between molecular and morphologic progression, an important approach in the evaluation of potential early detection markers. CDC25B protein expression increased progressively as morphology worsened: expression was positive in none of the normal subjects, one fourth of the dysplasia subjects, and one half of the invasive cancer cases. Only one previous publication has reported on CDC25B protein expression in ESCC precursor lesions (35), with results substantially different than we reported here. Xue et al. found much higher CDC25B positivity in normal tissue (47%) and observed little difference across the morphologic spectrum of mild/moderate dysplasia (79% positive), severe dysplasia/carcinoma in situ (61% positive), and invasive ESCC (68% positive). These results likely reflect a field effect as all of their comparison tissues were adjacent to tumors taken from ESCC cases. Although more definitive results on the predictive value of CDC25B will require characterization of CDC25B status in patients with no or only premalignant disease who are subsequently followed for the development of cancer, based on our findings, CDC25B seems to at least partially distinguish normal from abnormal tissue, and this is an important criterion in the development of a classifier or classification schema in an early detection strategy. Although its sensitivity is too low to be a satisfactory stand-alone predictor, CDC25B might be useful as part of a panel of predictors.

Although it is known that DNA damage results in cell cycle arrest at the G₂-M transition through inactivation of CDC25 (13), the precise mechanism by which CDC25B up-regulation participates in tumor progression remains unclear. One possibility is that up-regulation reduces the duration of G₂-M arrest, which alters mitotic spindle formation and provides insufficient time for DNA repair, and consequently leads to increased apoptosis (36–38). In support of this hypothesis, CDC25B transgenic animals have increased susceptibility to tumorigenesis induced by DNA-damaging agents (39). The lack of correlation of CDC25B with outcome in the present study may be due to the inability to detect differences in posttranslational modifications of CDC25B and/or the effect of different (and unknown) therapeutic interventions on the patients beyond surgical resection.

Dichotomizing CDC25B protein expression into positive versus negative is highly dependent on the selection of a cutoff criterion. Using an overall score of ≥4 (equivalent to at least moderate intensity in more than one fourth of the cells), 59% of ESCC cases in our study were called positive. This 59% positivity is consistent with reports from previous ESCC studies [68% for Xue et al. (35), 50% for Kishi et al. (16), 48% for Nishioka et al. (15), and 32% for Nakamura et al. (34)].

It is unclear at this time whether CDC25B protein expression in invasive tumors is clinically or therapeutically important. Although a correlation between CDC25B expression and tumor stage or grade has been observed in non–small cell lung carcinoma (40), ovarian cancer (41, 42), breast cancer (43, 44), and colorectal cancer (9), we did not find such a correlation for ESCC in the present study, nor did we find an association between CDC25B protein expression and survival. However, there is evidence in other studies of ESCC that CDC25B-positive tumors may be more responsive to chemo-radiotherapy. One report from Japan found that CDC25B protein was strongly expressed in 46% of ESCC patients who were sensitive to radiation, but only in 6% who were resistant (8). These same investigators also reported that CDC25B overexpression in an ESCC cell line resulted in suppressed G₂-M arrest and enhanced apoptosis, offering a potential mechanistic explanation in support of the observation about radiotherapy sensitivity (13). It remains for future ESCC studies to further elucidate the relations among CDC25B overexpression, tumor differentiation, and therapy (particularly radiation and chemotherapy) to better understand this phenomenon and to determine if such biomarkers will be clinically useful to predict therapy-specific responses.

LAMC2 protein expression is highly variable, but overexpression is seen in most invasive carcinomas (26–28) except for breast (45) and prostate (46) cancer, where expression typically is lost. The altered protein expression patterns for LAMC2 identified in the present study are largely consistent with studies of different cancers (26–28) and other studies of ESCC (25, 35, 47, 48). The earliest (and smallest) of these published ESCC studies (47) described four laminin staining patterns by basement membrane continuity (thick and continuous, thin and continuous, thin and discontinuous or fragmentary, and unrecognizable) in 33 surgically treated cases of ESCC but saw no differences in survival by pattern. Yamamoto et al. (25) and Fukai et al. (48) both found strong associations for LAMC2 expression (defined as immunostaining in >30% of cells at the invasive front) and several unfavorable clinicopathologic features in ESCC cases, including poor survival. Xue et al. (35) also observed increased LAMC2 positivity (defined as clusters of positive cells >30%) in ESCC cases with higher-stage disease and shorter survival. Yamamoto et al. (25) used multivariate analysis and showed that LAMC2 expression predicted poor survival even after adjustment for clinicopathologic variables.

This study did not examine the pattern of expression but rather categorized expression based on positive expression at the “invasive front” or interface between tumor and underlying normal tissue. However, Fukai et al. (48) found that LAMC2 expression predicted survival in univariate but not in multivariate models, suggesting that LAMC2 was not an independent predictor of survival. Xue et al. (35) presented only univariate results, so it cannot be determined if the association observed in this study for LAMC2 with survival was independent of other factors or not. In the present study, LAMC2 protein patterns were highly associated with survival, even after adjustment for important clinicopathologic factors. In our study, we were limited to a small core of tissue, obtained from a representative tissue block, and we could not address issues of relationship to the invasive front of the tumor. Although several molecular events have been characterized at the invasive front, determining the exact location of this front in a surgical specimen can be challenging and may be hard to reproduce in clinical practice. Further evidence for the prognostic importance of LAMC2 protein patterns comes from a small series of tongue cancer cases, which showed that the 3-year survival rate for 7 cases with a diffuse pattern was worse than for 13 patients with a peripheral pattern (14% versus 68%, respectively; P = 0.02; ref. 33). Although the LAMC2 protein expression patterns we reported are not directly comparable with the positivity scores recorded by Yamamoto et al. (25), Fukai et al. (48), or Xue et al. (35), all four of these studies found an association between LAMC2 protein expression and survival, strongly suggesting that LAMC2 is an important prognostic factor in ESCC.

Little is known about the mechanism or function of cytoplasmic accumulation of LAMC2 in tumor cells. It is thought that LAMC2 protein expression in carcinoma cells at the invasive front contributes to a more aggressive phenotype in malignant cells, resulting in tumor progression (26). LAMC2 is a pivotal hemidesmosomal protein involved in cell stability and filament formation anchorage. Reduced LAMC2 expression can result in insufficiencies of these two elements, which may in turn result in less stable epithelial-stromal junctions. This instability may help to increase the invasive and migration potential of malignant cells (46). This scenario is consistent with the clinicopathologic associations observed by Yamamoto et al. (25) noted above and is not inconsistent with our results, particularly for cases with the negative pattern of LAMC2 protein expression, who had the shortest survival time. Future studies with more ESCC cases and longer follow-up time will be required to sort out this particularly important and therapeutically relevant issue for LAMC2.

In summary, the CDC25B and LAMC2 mRNA overexpression found in the overwhelming majority of ESCC tumors in the current study confirmed the results of our previous 8K cDNA array study. Immunohistochemical evaluation of protein expression largely paralleled RNA findings, and LAMC2 protein expression patterns strongly predicted survival. CDC25B protein expression scores increased with morphologic progression across the continuum of normal to dysplasia to invasive ESCC. The strong relation of LAMC2 pattern of protein expression to survival suggests a role in prognosis, whereas the association of CDC25B with morphologic progression indicates a potential role as an early detection marker.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Paul Albert for thoughtful input in the design of our tissue array and analysis of these data, Binbing Yu for running selected analyses, and Kimberly Tuttle for assistance in tissue microarray construction.