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The incidence of esophageal squamous cell carcinoma (ESCC) is very high in northern China. This cancer has a very poor prognosis, mostly because it is usually diagnosed at a late stage. Detection an earlier stage can dramatically improve prognosis. Microscopic evaluation of esophageal balloon cytology (EBC) specimens has been the most common method for early detection of ESCC, but this technique is limited by low sensitivity and specificity. The use of molecular markers may improve these screening characteristics. This study evaluates whether measurement of gene methylation in EBC specimens may have utility for the detection of esophageal squamous dysplasia and early ESCC. We evaluated the presence of methylation in eight genes shown to be methylated in ESCC in previous studies in EBC specimens from 147 patients with endoscopic biopsy diagnoses ranging from normal mucosa through severe squamous dysplasia. Methylation status was determined using quantitative methylation-specific PCR techniques. The sensitivity and specificity of methylation of each individual gene and combinations of these genes to detect biopsy-proven high-grade (moderate or severe) squamous dysplasia was determined. For individual genes, the sensitivities ranged from 9–34% and the specificities ranged from 77–99%. Using a panel of four genes (AHRR, p16INK4a, MT1G, and CLDN3) resulted in sensitivity and specificity of 50% and 68%, respectively. This study suggests that evaluation of gene methylation in EBC samples may have utility for early detection of esophageal squamous dysplasia and early ESCC, however, identification of more sensitive methylation markers will be required for development of a clinically useful screening test.
Esophageal cancer is the eighth most commonly occurring cancer and the sixth most common cause of cancer death in the world (1). There is wide geographic variation in the incidence of esophageal cancer, with the highest rates occurring in China, Southcentral Asia, and Eastern and Southern Africa (1). In these high-risk areas, nearly all cases of esophageal cancer are esophageal squamous cell carcinoma (ESCC). The prognosis for esophageal cancer is poor, with five year relative survival rates of about 10% (2). The main reason for this poor prognosis is that most early cases are asymptomatic, so ESCC is usually diagnosed only at a late stage when the cancer has spread beyond the esophagus and is unresectable. Squamous dysplasia is the precursor lesion of ESCC, and subjects with moderate or severe dysplasia are at high risk (10-fold and 28-fold risk, respectively, compared to subjects with normal mucosa) for developing ESCC (3). When detected, squamous dysplasia and early ESCC are treatable by endoscopic techniques or esophagectomy, and survival can be improved dramatically (4). This highlights the necessity of developing of a clinically useful early detection test for squamous dysplasia and early ESCC.
Among the techniques currently used for ESCC screening in high-risk regions is esophageal balloon cytology (EBC) (5). In this technique, a deflated mesh-covered balloon is swallowed into the stomach, inflated, and then withdrawn, collecting esophageal mucosal cells which are stained and read visually like a Pap smear by a cytopathologist. The sensitivity of balloon cytology for detecting squamous dysplasia has been shown to be at about 50% (5–7), which is insufficient to be effective for population screening. Molecular markers may be able to improve the sensitivity and specificity of this method.
The main problems associated with cytological screening of the esophagus appear to be sampling error, in cell collection and in cell review, and insensitivity for finding rare abnormal cells among the cells that are reviewed. Blind balloon sampling may miss small mucosal lesions, and even if neoplastic cells from these lesions are successfully retrieved, they are still rare cells in a much larger population of normal cells, so they may not appear on the slides which are reviewed or they may be misinterpreted by the slide reader. The evaluation of molecular markers, such as gene methylation, cannot help cell collection, but it may be able to improve the other steps in this screening procedure. Gene methylation refers to abnormal methylation of CpG islands in the promoter regions of genes which are normally unmethylated, and this abnormal methylation results in the silencing of gene expression. DNA methylation assays have been shown to be highly sensitive (they can detect as few as 20 copies of methylated DNA among a large number of normal unmethylated copies) (8). In addition, hypermethylation of some genes has been shown to occur early in esophageal squamous dysplasia and is common in ESCC (observed in up to 80% of cases in some series) (9–11).
In this study we measured the prevalence of methylation in selected genes in balloon cytology specimens collected in a screening study conducted in Linxian, China, a county with some of the highest rates of ESCC in the world (12). The purpose of this study was to evaluate whether the presence of methylation in these genes in EBC samples could identify individuals with high-grade (moderate or severe) esophageal squamous dysplasia, who should be referred for endoscopic examination.
In the spring of 2002, 740 asymptomatic Linxian adults, aged 50–64, were recruited for a primary screening study of two EBC samplers (7). Each participant underwent an EBC exam and a subsequent upper endoscopy exam with Lugol’s iodine staining biopsies of all unstained lesions and standard biopsies of normal-appearing mucosa (13). All biopsies were read by two experienced pathologists, using previously developed criteria (14). The current study used a subset of these subjects who had worst biopsy diagnoses of normal (n=50), esophagitis (n=25), mild dysplasia (n=25), moderate dysplasia (n=26), severe dysplasia (n=20) and early invasive ESCC (n=1). Approximately half of the subjects with a worst biopsy diagnosis of either severe dysplasia (n=11 (52%)) or moderate dysplasia (n=11 (42%)) had an additional endoscopic biopsy showing a dysplastic lesion of less severity. All participants gave informed consent for the screening procedures, and the protocol was approved by the Institutional Review Board of the U.S. National Cancer Institute and the Cancer Institute of the Chinese Academy of Medical Sciences. This research was supported by the Intramural Research Program of the NIH, National Cancer Institute.
Balloon cytology specimens were collected in 1 ml of phosphate buffered saline (PBS) and 50% of this sample was frozen. DNA was extracted from 50 µl of each frozen balloon cytology balloon specimen using Qiagen Tissue and Blood extraction kits (Qiagen, Valencia, CA), following the manufacturer’s instructions. Bisulfite modification was performed using the EZ DNA Methylation Gold Kit (Zymo Research, Orange, CA), following the manufacturer’s instructions. Gene methylation analysis was conducted using quantitative methylation-specific PCR (QMSP), based on the Taqman chemistry (Applied Biosystems, Foster City, CA). The genes for this analysis were selected because they were shown to be methylated in ESCC in previous studies (6, 10, 11, 15), and this methylation was confirmed in our laboratory.
Primers and hybridization probes were designed to bind specifically to bisulfite-converted sequences in the CpG islands in the promoters of each of the genes. The primers and probes for each of the assays are listed in Table 1. The assays were carried out using the Applied Biosystems 7900HT Fast Real Time PCR System (Applied Biosystems, Foster City, CA). The reactions were carried out in a reaction volume of 10 µL using Taqman Fast Universal PCR Master Mix, No AmpErase UNG. Each PCR mixture contained 300nM of each primer, 100nM probe, and 1X Taqman Buffer. Amplification and detection were carried out using the following profile: one step at 95°C for 20 sec, 50 cycles at 95°C for 1 sec, and 60°C for 20 sec. Standards consisting of serial dilutions with known amounts of methylated DNA (25 ng–0.025 ng) and unmethylated DNA were run on every plate. All samples were within the assay’s range of sensitivity and reproducibility based on an internal reference standard (ACTB Ct value <36). All samples were run in duplicate, with standards and controls on every plate. All samples with Ct value less than 42 were considered positive for methylation.
All statistical analyses were conducted using the STATA 8.0 software package (Stata Corporation, College Station, TX). The relative methylation level for each gene in each sample was calculated as the percent of that gene’s DNA that was methylated. The formula used was as follows: ng of the gene DNA methylated derived from the standard curve of the positive control/ ng of ACTB methylated derived from the standard curve of the positive control × 100. All samples had at least 0.1% methylation for each gene (background). The relative methylation level was used to create a dichotomous methylation status variable based on methylation > 0.1% (yes/no). The single patient with early ESCC was combined with the severe dysplasia patients for analysis. The sensitivity and specificity for methylation in discriminating patients with high-grade dysplasia (including moderate and severe dysplasia) from all other patients was calculated. Methylation of individual genes and combinations of genes were analyzed to determine an optimal gene panel. All statistical tests were two-sided and considered significant if p<0.05.
This study included 147 participants who had a worst squamous histologic diagnosis ranging from normal to severe dysplasia. There were 83 men and 64 women aged 40 to 67 years (mean=55). There were no significant differences in patient characteristics such as age, gender, family history of cancer, smoking or alcohol consumption across diagnostic category (data not shown).
The average DNA yield after extraction was 8.67 ng/ul, and ranged from 0.419 – 69.27 ng/ul. Promoter methylation was present in the EBC samples from patients across the disease spectrum, and most genes show an increasing prevalence of methylation with increasing severity of the patients’ worst disease (Table 2). For example, AHRR methylation was present in 6% of normal patients but in 20% of patients with severe dysplasia. The prevalence of methylation in individual genes ranged from 0–12% in patients with normal mucosa, 0–32% in patients with mild dysplasia, 4–35% in patients with moderate dysplasia, and 10–34% in patients with severe dysplasia.
The proportion of patients with one or more methylated genes also increased with the degree of dyplasia (Fig 1). No methylation was identified in any of the eight genes in 70% of normal patients, 52% of patients with esophagitis or mild dysplasia, 48% of patients with moderate dysplasia, and 43% of patients with severe dysplasia. The percent of patients with 3 or more genes methylated in their EBC samples increased from 6% in normal, 4% in esophagitis, and 4% in mild dysplasia to 15% in moderate and 29% in severe dysplasia.
The sensitivities and specificities of finding methylation in the eight single genes or in several combinations of genes for identifying patients with high-grade (moderate or severe) dysplasia are shown in Table 3. For each of the eight individual genes, the sensitivities ranged from 9–34% and specificities ranged from 77–99%. Using combinations of genes improved sensitivity but reduced specificity. For example, finding methylation in AHRR, p16INK4a, or MT1G had a sensitivity and specificity of 30% and 86%, respectively. Adding CLDN3 to these three genes markedly improved the sensitivity, to 50%, but reduced the specificity, to 68%.
Esophageal squamous cell carcinoma is a common disease in many countries, and, because of late diagnosis, it has a very poor prognosis. Early detection of ESCC and its precursor lesion, squamous dysplasia, should improve cure rates and reduce mortality, but there is currently no accurate and affordable way to screen the large numbers of asymptomatic people who are at high risk for this disease. The most common current screening technique in high-risk populations, esophageal balloon cytology (EBC), has only a 50% sensitivity for detecting squamous dysplasia or early ESCC, and only a 5% sensitivity for detecting high-grade dysplasia or early ESCC (7). The current study is part of an effort to identify molecular markers that may improve this sensitivity.
There are three main problems with EBC for the detection of early esophageal neoplasia. Blind balloon sampling may miss small mucosal lesions, so the sample that is collected may not include diagnostic abnormal cells (sampling error #1). Also, because visual examination of cell preparations is time-consuming, current methods for reading EBC samples evaluate only a minority of the collected cells, so rare abnormal cells may have been collected but may not be evaluated (sampling error #2). Finally, rare abnormal cells that are evaluated on the cytology preparations may be missed or misinterpreted by those reading the slides (reading error). Molecular markers, including gene methylation, cannot help incomplete cell sampling (sampling error #1), but may be able to reduce the other two sources of screening error.
In this study we evaluated promoter methylation in 8 genes in EBC specimens from 147 asymptomatic high-risk Chinese adults, and we tested the ability of this methylation to detect individuals with high-grade (moderate or severe) esophageal squamous dysplasia. For most genes, methylation was more common in the EBC samples of patients with worse disease, consistent with previous findings in esophageal tissue specimens (9, 10, 16). Methylation of individual genes had sensitivities and specificities ranging from 9–34% and 77–99%, respectively, for identifying patients with high-grade squamous dysplasia. A panel of four genes (AHRR, p16INK4a, CLDN3, and MTIG) had a sensitivity to 50% and a specificity of 65%. While these figures are no better than the sensitivity and specificity of traditional visual evaluation of EBC specimens (6, 7), this is only the first evaluation of balloon samples for methylation in a few candidate genes, so these figures probably represent minimum values for this technique.
Our findings of gene methylation in balloon cytology samples from subjects believed to have a normal esophagus after endoscopy with Lugol’s iodine staining is not surprising in light of previous studies that found methylation in normal esophageal mucosa from patients without cancer (16). In addition, Guo et al (10) evaluated methylation of eight genes in tissues with a spectrum of histologic diagnoses and found that most examples of squamous dysplasia, including low-grade dysplasia, had at least one methylated gene. This suggests that promoter methylation may be an early event in carcinogenesis. Thus, given the cross-sectional nature of our study, it is unclear if the methylation-positive samples in subjects with normal endoscopic and histologic findings identified field effects associated with occult early neoplasia or if they were non-specific findings unrelated to carcinogenesis. A prospective analysis would be required to distinguish these two possibilities.
Our study had several strengths, including using the gold-standard exam, endoscopy with Lugol’s iodine staining and biopsy, to define the disease status in all subjects. Also, we used a very sensitive and validated method to detect gene methylation in the balloon cytology samples. Our study also had some limitations, including testing only a moderate number of EBC samples and examining only a limited panel of genes. Future studies should use high-throughput methods to test larger numbers of clinical samples and genes.
In summary, this study suggests that measuring gene methylation in balloon cytology specimens may have promise as a primary screening technique for squamous dysplasia and early ESCC in high-risk regions. However, identification of more sensitive panels of methylation markers will be required, and sampling error in EBC cell collection will need to be minimized. Prospective studies evaluating multiple genes that have a high prevalence of methylation in ESCC should also shed light on the clinical usefulness of methylation markers in the early detection of ESCC.