Detection of Genetic Alterations in Esophageal Squamous Cell Carcinomas and Adjacent Normal Epithelia by Comparative DNA Fingerprinting Using Inter-Simple Sequence Repeat PCR¹

Tumors are composed of populations of abnormal cells capable of altering their genomes in response to changes in their milieu (1, 2). The abnormal biological properties of tumor cells are acquired by a microevolutionary process that can enhance their overall growth, survival, and metastatic potential (1). Tumor progression is driven by clonal evolution in which a few neoplastic clones are selected from a variety of randomly generated cell variants. Rather than a few specific mutations leading to genetic instability, mechanisms of somatic mutation generate an evolutionary diversity, resulting in tumor heterogeneity, and eventually clonal predominance can be observed (1, 2). Thus, the critical genetic alterations are those that facilitate tumor heterogeneity, and these are necessary for tumor progression and development (3). Subsequently, or concurrently, a set of mutations necessary for malignancy is generated and expanded by clonal selection. For gastrointestinal cancers, tumor progression has been proposed to follow a stepwise acquisition of multiple changes that involve multiple oncogenes, tumor suppressor genes, and cell cycle-regulating genes (4, 5).

Different methods have been used to examine the alterations of cancer genomes. Typical examples are the observation of clonal cytogenetic abnormalities (6), detection of amplifications and/or deletions at the gene or transcript level (7), localization of point mutations for cancer-related genes (4), and examination of allelic losses or replication error phenotypes at microsatellite loci that are relevant to carcinogenesis (8). Another approach to detect the alterations of cancer genomes is the generation of DNA fingerprint profiles that can be produced by PCR-based methods. Examples include the detection of genetic alterations in colorectal, lung, and ovarian cancers by arbitrarily primed PCR genomic fingerprinting (9, 10, 11) and the application of ISSR-PCR³ analysis on sporadic colorectal cancer (12).

ISSR-PCR has also been described as microsatellite-primed PCR in the literature (13, 14) because it specifically amplifies regions of the genome between microsatellites. The method of comparing inter-repeat profiles from the whole genome was first used in the identification and differentiation of different eukaryotic species because of its accuracy and reproducibility (13, 14). The significance of the changes in the inter-repeat sequences for carcinogenesis was subsequently suggested by Basik et al. (12). In the present study, ISSR-PCR was applied to ESCC and premalignant lesions. By observing the fingerprinting profiles generated from the malignant and premalignant populations compared with the normal genomes, we hoped to follow the process of tumor progression in ESCC and compare the observable alterations at the genome level with the clinicopathological features of the disease.

For the initial ISSR-PCR DNA fingerprint profiling, ESCC samples with gross tumor appearance were collected prospectively from 19 surgically resected specimens after esophagectomy between June 1996 and March 1997 in Queen Mary Hospital, Hong Kong. All of the selected ESCC samples had more than 80% viable tumor cells as shown by histological assessment. Morphologically normal epithelial tissues at least 10 cm away from the tumors were also sampled from each patient. Apparently normal marginal epithelia that were adjacent to the tumors by 1 cm were collected from 13 patients. Sterile equipment was used for every sampling of tumor, normal, and marginal epithelia. One-half of each sample was fixed in 10% formalin for histological assessment as described previously (15), and the other half was snap-frozen in liquid nitrogen and stored at −85°C for later DNA extraction using the method described previously (16). For every five cryostat sections cut from the frozen blocks for DNA extraction, an additional H&E-stained section was prepared for histological assessment to confirm the presence or absence of tumor cells. To determine the frequency of occurrence of the specific gene alterations by direct genomic PCR analysis for the ISSR-PCR profile bands in ESCC, an additional batch of 41 archival ESCCs collected between 1989 and 1993 in Queen Mary Hospital that were not pretreated with chemoradiotherapy were also included in the present study. All these ESCC samples had more than 80% viable tumor cells as shown by histological assessment.

For ISSR-PCR DNA fingerprint profiling, 50 ng of genomic DNA were used in each 10-μl reaction as the template. The sequences of the three primers are as follows: (a) (GTG)₅, 5′-GTGGTGGTGGTGGTG-3′; (b) (GACA)₄, 5′-GACAGACAGACAGACA-3′; and (c) (GATA)₄, 5′-GATAGATAGATAGATA-3′. In each reaction, 0.2 μm each primer was end-labeled with [γ-³³P]ATP (Amersham, Aylesbury, United Kingdom) using T4 polynucleotide kinase (Promega, Madison, WI). Each PCR reaction contained the same amount of labeled and unlabeled primer. The PCR protocol (with an annealing temperature of 56°C) and the procedures for electrophoresis and autoradiography were as described previously (12). ³³P-end-labeled molecular weight markers (HaeIII-cut φ174 DNA) were also run on the side lanes of the gels for the size comparison.

Eleven selected bands from the ISSR-PCR profiles were cut out of the polyacrylamide gel based on their discrete and nonoverlapping appearance with other bands in the autoradiographs. DNA was extracted by boiling the gel slices in 30 μl of water for 10 min. Eight μl of each DNA fragment were then cloned into 1 μg of pGEM-T Easy vector (Promega). Cycle sequencing was done with the Big-Dye sequencing kit (Perkin-Elmer) using the forward and reverse primers as suggested by the supplier of the vector, and the sequences were then analyzed by an ABI 310 Genetic Analyzer (Perkin-Elmer). The sequences were matched against the genomic sequence data in the European Molecular Biology Laboratory and GenBank databases.

Two specific primers were designed for the direct genomic PCR analysis of the 11 ISSR-PCR profile bands in 60 ESCCs. The primer sequences for the amplification of selected profile bands by PCR are summarized in Table 2. Patient DNA (30 ng) was used for each 20-μl PCR reaction with 10 pmol of each primer, 1 unit of Taq polymerase (Promega), 0.2 mm each deoxynucleotide triphosphate, and 2 mm MgCl₂. The reaction was carried out for 35 cycles after an initial denaturation at 95°C for 4 min under the following conditions: 95°C for 30 s; 52°C for 30 s; and 72°C for 30 s. In addition, the β-globin gene sequences (17) were also amplified simultaneously in the same reaction tube as the internal PCR controls to normalize the amount of DNA of each sample analyzed. The products of the multiplex PCR amplification were run in a 3% agarose gel and visualized under UV light after staining with ethidium bromide (Fig. 2). Molecular weight markers (HaeIII-cut φ174 DNA) were run on the side lanes of the gels for the size comparison.

First, to ensure the reproducibility of ISSR-PCR DNA fingerprint profiling in our laboratory, the method was performed on two independent DNA preparations from the normal esophageal epithelia of the same patient. Identical profiles were generated from the two independent DNA preparations from the same patient (Fig. 1). Tissue samples collected from 19 patients with ESCC were then analyzed by ISSR-PCR. The clinicopathological features of these patients are summarized in Table 1. In the 13 apparently normal marginal epithelia collected, extremely rare tumor cells were found in 2 samples (patients 2 and 7) by histological assessment, dysplasia was noted in 2 other samples (patients 5 and 17), and the rest were histologically normal. The profiles generated by ISSR-PCR for each individual patient, using each of the three repeating primers, were aligned together with those generated from the corresponding normal epithelium. Evidence of amplification and/or deletion of chromosomal sequences was observed as shown by changes in intensities or gains and/or losses of profile bands of tumors compared with the corresponding normal epithelia. All of the 19 ESCCs showed evidence of genetic alteration compared with their normal epithelia controls using at least one of the repeating primers (Table 1). Six of the 19 patients (patients 2, 5, 6, 17, 18, and 19; 32%) showed abnormal profile bands for all three primers. Representative examples of autoradiographs are shown in Fig. 2 I.

Ten profile bands showing gains and one profile band showing loss in tumors compared with the corresponding normal epithelia were cloned, and their origins were determined by sequencing (Table 2). Profile band A from patient 6 shows 98% homology to the GenBank sequence that can be matched against a nonexonic genomic sequence (97% homology) with accession number AL050325 isolated from chromosome 20. Profile band C showing gain in the tumor specimen from patient 19 shows 98% homology to an expressed sequence tag sequence with accession number AA078562 that has been mapped to the chromosome 7q22 region. The other profile bands were anonymous and could not be matched significantly with any known sequences in the European Molecular Biology Laboratory and GenBank databases.

To show that the genetic alterations observed by ISSR-PCR are true somatic mutations occurring in the tumors and represent true amplifications in the genome, specific primers were designed for 11 ISSR-PCR profile bands based on the sequencing results (Table 2). Using the primers specific for these gene alterations, direct genomic PCR amplification was performed on the original patient DNA samples and confirmed the bona fide nature of the gains or losses of 11 profile bands in the original cases. Selected examples are shown in Fig. 2 II.

To determine the frequency of occurrence of these specific gene alterations in ESCC, DNA from the tumor and the corresponding normal epithelium from 60 ESCCs, including 41 additional cases of ESCC, was analyzed by direct genomic PCR using the specific PCR primers for ISSR-PCR profile bands (Table 2). For normalization of the DNA quantity of the tumor and the corresponding normal epithelium samples for the PCR analysis, β-globin gene amplification was used as the internal control (17). The frequencies of the occurrence of these specific gene alterations are summarized in Table 3. Significantly, 26 of 60 (43%) tumors showed DNA amplification for the expressed sequence tag sequence of chromosome 7 (the band intensity was at least two times stronger than that of the corresponding normal control), whereas the frequency of other individual gene alterations ranged from 7% to 15%.

The three repeating primers [(GTG)₅, (GACA)₄, and (GATA)₄] were chosen for ISSR-PCR profiling because these repeating sequences are somatically stable in normal human genomes (18, 19), and they are diverse because they can be found in both autosomes and sex chromosomes (20, 21). ISSR-PCR amplifies the inter-repeat sequences (13, 14), and the observed alterations of the profiles from tumors in the present study and in that of Basik et al. (12) include changes in intensities, gains and/or losses, which, as shown here, correlate with deletions and amplifications of genomic fragments. The profile bands with increased or decreased intensities in tumor specimens must reflect spontaneous somatic mutations consisting of gains and losses of chromosomal sequences occurring in tumors that became detectable by tumor clonality. These genetic alterations can be regarded as markers for the clonal evolution of tumor progression, showing the emergence and/or deletion of tumor clones (1, 2, 16).

Eight of 13 patients (patients 5, 7, 8, 13, 14, 17, 18, and 19; 62%) were found to have matching gains or losses of profiling bands in the ESCC and the corresponding apparently normal marginal epithelia adjacent to the tumors for at least one primer used. For the marginal epithelia of these eight patients, two patients (patients 5 and 17) had dysplastic epithelia, one patient (patient 7) had extremely rare tumor cells, and the other five patients were histologically normal. This observation suggests that the generation of widespread genetic alterations can be an early event in tumor progression and that these alterations can be detected by ISSR-PCR in premalignant stages. However, it should be pointed out that although all of the necessary precautions were taken to avoid any contamination of histologically normal marginal epithelia with tumor, one could never be sure that absolutely no tumor cells were present in the nontumorous sample. PCR reactions are very sensitive, almost certainly more sensitive than histopathological assessment. Nevertheless, our findings are consistent with previous reports showing the presence of genomic abnormalities in premalignant lesions before they are morphologically recognized (22, 23) and the concept of field cancerization, by which early genetic events are shared by cells in a local anatomical area before they appear to be morphologically neoplastic (24).

Of the 13 patients providing marginal epithelia, spectra of increasing intensities were noted in 3 patients (patients 1, 2, and 5; 23%). The intensities of the amplified bands in a fingerprinting profile are semiquantitative, and the intensity of an amplified band is proportional to the concentration of its corresponding template sequence (12). For patients 1 and 5, the extra bands were detected in their marginal epithelia, and the intensities of the bands were further increased in their tumor samples (Table 1), indicating further genomic amplification in tumors. It is worth noting that the marginal epithelia of patients 1 and 5 were histologically normal and dysplastic, respectively. Thus, the observed genetic alterations preceded even the appearance of premalignant lesions in patient 1. For patient 2, the spectrum of increasing intensity was observed in the normal, marginal, and tumor samples. Because squamous cell carcinoma was also present in the marginal epithelium of patient 2 (Table 1), such an increased band intensity in the tumor profile might be related to either an increased amount of tumor genomes in the tumor sample or an increase in the copy number of certain genomic sequences.

The finding of genetic alterations in all of the ESCC tumors and 62% of epithelia surrounding ESCC derives from the ability of the ISSR-PCR method to detect abnormalities in one reaction, throughout the genome, and in the absence of any information about loci-specific sequences. Moreover, the comparative DNA profiling analysis appears to be more sensitive than histological assessments in showing early genetic alterations in preneoplastic epithelia because five of eight (63%) samples of the marginal epithelia that matched tumor abnormalities are histologically normal. The sensitivity and reproducibility of the method suggest the application of ISSR-PCR for early detection of ESCC and also for the detection of early tumor recurrence. Another interesting means to test the usefulness of ISSR-PCR for early detection of cancer would be to investigate Barrett’s esophageal tissues, Barrett’s dysplasia, and adenocarcinomas (25).

As shown in the present study, the ISSR-PCR method also offers an opportunity to identify the genomic fragments that show amplifications or deletions in ESCC. Several amplified chromosomal regions and genes in ESCC have been implicated in the development and progression of ESCC, such as chromosomal regions 11q13 (5) and 13q34 (26), c-myc oncogene, and epidermal growth factor receptor genes (27). The overexpressions of these genes have also been suggested to be involved in tumorigenesis (4). In the present study, the detections of amplified exonic fragments of 7q22 from patient 19 with a high frequency (43%) are novel to ESCC. The chromosomal overrepresentation of 7q22 has also been shown by comparative genomic hybridization to be involved in pancreatic carcinomas (28) and nonpapillary renal cell carcinomas (29). The involvement of exonic amplification in carcinogenesis of ESCC is currently being investigated and will be reported later.

In summary, here we describe our results of the application of ISSR-PCR to detect somatic mutations in different tissues from ESCC patients including tumor and marginal epithelia in comparison with the corresponding normal epithelium, and we show that ISSR-PCR profiling offers a powerful approach to identify novel genomic fragments that may be involved in the carcinogenesis of esophageal cancers. Analysis of 19 ESCCs by this technique detected frequent changes in the intensities of the profile bands not only in tumor samples but also in apparently normal marginal epithelia. From these data, it is concluded that (a) widespread genetic alterations can be an early event in tumor progression in ESCC, and these alterations can be detected by ISSR-PCR in tumors and premalignant stages; and (b) the observed genetic alterations can be detected before the appearance of premalignant lesions and may provide the basis for a general screening method for predisposition to the development of patent disease.