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Annals of Surgical Oncology 8:525-532 (2001)
© 2001 Society of Surgical Oncology

ORIGINAL ARTICLES

Evaluation of Metastatic Potential of Gastric Tumors by Staining for Proliferating Cell Nuclear Antigen and Chromosome 17 Numerical Aberrations

Ryusuke Terada, MD, Toru Yasutake, MD, Shirou Nakamura, MD, Takashi Hisamatsu, MD, Tohru Nakagoe, MD, Hiroyoshi Ayabe, MD and Yutaka Tagawa, MD

From the First Department of Surgery (RT, TY, SN, TH, TN, HA), School of Medicine; and the Department of Occupational Therapy (YT), School of Allied Medical Sciences; Nagasaki University, Nagasaki, Japan.

Correspondence: Address correspondence and reprint requests to: Ryusuke Terada, MD, Department of Surgery, Hokusho Central Hospital, 299 Akasaka-men, Emukae-cho, Kitamatsuura-gun, Nagasaki 859-6131, Japan; Fax: 81-956-65-2123; E-mail: TE0403{at}aol.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Aberrations in chromosome 17 are important in carcinogenesis. We recently reported that numerical aberrations in chromosome 17 were associated with tumor progression in gastric cancer. The aim of this study was to determine the biological characteristics of gastric tumor cells with chromosome 17 numerical aberrations.

Methods: Gastric tumor sections (n = 105) and metastatic lymph nodes (n = 16) were stained simultaneously for PCNA (proliferating cell nuclear antigen) and chromosome 17 centromere. Cancers were classified as follows: Group 1: PCNA(+) and numerical chromosomal aberration(+); Group 2: PCNA(-) and numerical chromosomal aberration(+); Group 3: PCNA(+) and numerical chromosomal aberration(-); and Group 4: PCNA(-) and numerical chromosomal aberration(-).

Results: The frequency of Group 1 cells correlated with lymphatic invasion (P < .0001), lymph node metastasis (P < .0001), and venous invasion (P < .01). The frequency of these cells in gastric lesions was lower than in metastatic lymph nodes (P < .01). Logistic regression analysis identified the depth of invasion followed by the frequency of Group 1 cells were two of the most significant independent factors that could predict lymph node metastasis and lymphatic invasion.

Conclusions: The frequency of gastric tumor cells positive for PCNA and chromosome 17 numerical aberrations may be an indicator of the metastatic potential of gastric cancers.

Key Words: PCNA • Chromosome 17 • Gastric cancer • Lymph node metastasis • Fluorescence in situ hybridization (FISH) • Two-color staining


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The study of human chromosomes is important in the investigation of malignancy. Aberrations in chromosome 17 are well characterized because they are important in tumor carcinogenesis or development.1–9 Numerical aberrations in chromosome 17 might be an early event in human solid tumor. For example, chromosome 17 loss has been reported in one case of breast hyperplasia6 (suggesting that some hyperplasias may be part of a progressive sequence to malignancy in breast cancer) and also in a tubulovillous adenoma of the colon.7 In gastric lesions, numerical aberrations in chromosome 17 have been also reported in gastric intestinal metaplasia and gastric cancers,8 suggesting that the alteration of chromosome 17 is significant in gastric carcinogenesis. We have recently demonstrated the presence of numerical aberrations in chromosome 17 at early stages during carcinogenesis and that such aberrations were associated with tumor progression in gastric cancer.9 Furthermore, our results showed a strong correlation between chromosome 17 numerical aberrations and lymph node metastasis in gastric cancer. Lymph node metastasis may be the main cause of treatment failure in gastric cancer patients, especially in minimally invasive operations. Therefore, the metastatic potential of tumors should be analyzed thoroughly by various molecular and biological techniques.

Recently, new techniques for immunophenotyping cells with chromosomal aberrations were described using cell lines.10–13 Moreover, a combination of fluorescent immunophenotyping and fluorescence in situ hybridization (FISH) procedures was introduced using satellite probes on cytological preparations.10 To date, these techniques have been applied on cytological preparations such as smears and frozen tissue specimens.10,11 These approaches may allow further characterization of gastric cancer cells obtained from biopsy specimens.

Previous studies have identified a significant correlation between the proliferative activity of various malignant neoplasms on one hand and metastatic potential, recurrence, and overall prognosis on the other.14 In this regard, the rate of tumor growth is partly dependent on the proliferative activity, and a gain in the growth rate of proliferating cells has been observed in a number of conditions associated with increased risk of gastric cancer, such as pernicious anemia,15 chronic gastritis,16 and in the postantrectomy stomach.17 In these conditions, further characterization of the proliferating cells needs to be examined for a full assessment of their malignant potential.

In this study, we used a new system for simultaneous detection of proliferating cell nuclear antigen (PCNA) and centromere-specific DNA probe for human chromosome 17 in freshly obtained gastric cancer tissue specimens.10 Our aim was to further clarify the biological and clinical significance of proliferating cells with chromosome aberrations in gastric tumors. PCNA, a marker of cell proliferation, is a well-characterized auxiliary protein of DNA polymerase delta.18–22 PCNA is an intranuclear 36-kD polypeptide whose expression and synthesis is linked with cell proliferation.18,19 PCNA staining of nuclei is a sensitive and reliable index of the S phase of cell proliferation and is now routinely used in the study of human pathology specimens.22 Therefore, we postulated that simultaneous detection of cells positive for PCNA and chromosome 17 copy numbers in the same sample should enhance the prediction of the aggressiveness of gastric cancer.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Surgical Specimens
The study involved 105 patients with gastric cancer (76 men, 29 women) with a mean age of 64.5 years. None had received chemotherapy or radiotherapy before surgical treatment, and none had double cancer or synchronous multiple gastric cancers. The clinicopathological features of participating patients are listed in Table 1. The study protocol was approved by the Human Ethics Review Committee of Nagasaki University School of Medicine, and a signed consent form was obtained from each subject. A total of 105 fresh tissues of primary gastric tumors and metastatic nodes from 16 of these patients were collected immediately after surgical resection. A biopsy gun was used when gastric tissue was difficult to collect, particularly in patients with early gastric cancer. The edge of the tissue was snap frozen in liquid nitrogen and stored at -80°C. Lymph node status was determined by strict quality-controlled node dissection and pathological examination. All tumors were fixed with 10% buffered formalin, and paraffin blocks were prepared for histological analysis. The specimens were evaluated for histopathological diagnosis and classified separately by two pathologists according to Japanese Research Society for Gastric Cancer guidelines.23


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  		TABLE 1. Characteristics of gastric cancer patients (n = 105)
 
Simultaneous Staining of PCNA and Chromosome 17 Centromere
The methods of Weber-Matthiesen et al.10 were used with slight modifications. Frozen sections were washed twice for 5 minutes in cold phosphate-buffered saline (PBS). The slide for PCNA staining was fixed in acetone at -20°C for 15 minutes, followed by washing in cold PBS, and further fixation in methanol at -20°C for 15 minutes. The slide was then air dried and treated with 10% normal goat serum at room temperature for 30 minutes, followed by incubation with fluorescein isothiocyanate (FITC)-conjugated PC-10 (1:20 in PBS; DAKO, Glostrup, Denmark) for 60 minutes at room temperature. As a negative control for PCNA, PC-10 was replaced with FITC-conjugated mouse IgG2a (DAKO). After the slides were washed twice in 4 x SSC/0.05% Tween 20 for 5 minutes each, the slide was fixed in 4% paraformaldehyde in PBS for 3 minutes at 4°C, followed by a brief wash in distilled water. Before FISH, the slide was dehydrated in a series of ethanol solutions (70%, 90%, and 100%). The hybridization proceeded according to Pinkel et al.24 with minor modifications. The DNA probe (D17Z1, biotin-labeled alpha-satellite centromere probes) was purchased from Oncor (Gaithersburg, MD). The hybridization mixture (total 10 µl), containing 1-µl human placental DNA (Sigma Co., St. Louis, MO); 10% dextran sulfate; 50% formamide/2 x SSC; and 1-µl DNA probe was denatured in 70% formamide/2 x SSC, pH 7.0, at 70°C. The sample was denatured for 2.5 minutes in 70% formamide/2 x SSC, pH 7.0, at 70°C. The hybridization mixture (10 µl) was added to a slide under a coverslip (18x18 mm) at 37°C. After hybridization for 60 minutes at 37°C, slides were washed three times for 10 minutes in 50% formamide/2 x SSC, pH 7.0, at 45°C.; in 2 x SSC, pH 7.0, at 45°C; and in 2 x SSC, pH 7.0, at room temperature.

Detection of hybridization signals from the DNA probe was performed with Texas Red avidin (EY Laboratories, Inc., San Mateo, CA) in 4 x SSC/1% BSA for 60 minutes at room temperature. Finally, nuclei were counterstained with antifade solution containing 0.2 µg/ml diamidinophenylindole (DAPI). Microscopy was performed with a Nikon FXA epifluorescence microscope (Nikon, Tokyo, Japan). Hybridization signals (Texas Red) and PCNA expression (FITC) were simultaneously visualized through dual bandpass filters for red and green. Cells were examined for the simultaneous presence of PCNA expression and chromosome 17 signal numbers, using 200 nuclei per slide. Numerical chromosomal aberrations represented the sum of chromosomal loss and gain. Accordingly, tumor cells were classified into the following four groups based on two parameters: PCNA (+ or -) and chromosomal aberration (+ or -). The classification of cancer cells was defined as follows: Group 1, PCNA(+) and numerical chromosomal aberration(+); Group 2, PCNA(-) and numerical chromosomal aberration(+); Group 3, PCNA(+) and numerical chromosomal aberration(-); and Group 4, PCNA(-) and numerical chromosomal aberration(-). As described above, 200 nuclei per slide were examined and divided into four groups, and each sample was evaluated by determining the percentage of cells in each of the above groups.

The FISH signal was evaluated as described previously.25,26 Briefly, nonoverlapping intact nuclei were counted, and split centromere signals were counted as one. Small lymphocytes and granulocytes were excluded from analysis. Nuclei with no signals were regarded as not reactive with the probe and were not counted. When the frequency of nuclei without signals was >15% relative to the total number of counted nuclei, or when it was difficult to evaluate PCNA expression and signals due to background noise, the sample was excluded from further analysis. Chromosomal numerical aberrations were diagnosed as chromosomal loss (single signal) or gain (triple or more signals) of each cell.

Statistical Analysis
The StatView J-5.0 statistical program package (Abacus Concepts, Inc., Berkeley, CA) for the Macintosh computer was used for all analyses. Data were expressed as the mean ± standard deviation (SD). The Mann-Whitney U-test (two groups) and Kruskal-Wallis test (more than three groups) were used to examine the statistical significance of difference between group data. Wilcoxon’s signed-rank test was used to compare the frequency of cells between primary lesions and metastatic lymph nodes. Logistic regression analysis was used for analysis of factors that determined lymph node metastasis, lymphatic invasion, venous invasion, and depth of invasion. A probability value < 0.01 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Simultaneous Detection of PCNA And Centromere-Specific DNA Probe for Chromosome 17 in Primary Lesions of Gastric Cancers
Figure 1(a, b, and c) shows representative examples of simultaneous expression of PCNA and chromosome 17 signal numbers in primary lesions of gastric cancer. These findings confirmed that the PC-10 and DNA probe reacted with their true targets and that counting the resultant fluorescent signals provided valid information with respect to PCNA and copy numbers of chromosome 17. The frequencies of tumors containing Group-1, Group-2, Group-3, and Group-4 cells were 13.5% ± 7.9% (range, 0%–44%; median, 12.0%); 19.3% ± 9.2% (range, 4%–45.5%; median, 17.0%); 17.5% ± 8.4% (range, 4%–53%; median, 16.5%); and 49.7% ± 12.8% (range, 16.5%–77%; median, 49.0%), respectively, among 105 tumors.



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  		FIG. 1. Simultaneous two-color staining of nuclei by PCNA (green) and FISH (red) using chromosome 17 specific probe.

Cells of Group 1 (long yellow arrows), Group 2 (short yellow arrows), Group 3 (long white arrows), and Group 4 (short white arrows). (a) Cells in well-differentiated adenocarcinoma, (b) cells in moderately differentiated adenocarcinoma, (c) cells in poorly differentiated adenocarcinoma, (d) cells in metastatic lymph node. Original magnification, x1000.

 
The frequency of PCNA-positive cells with chromosome 17 numerical aberrations (Group 1) correlated significantly with lymph node metastasis (P < .0001), lymphatic invasion (P < .0001), and venous invasion (P < .01). There was a significant correlation between the frequency of PCNA-positive cells with chromosome 17 disomy (Group 3) and lymphatic invasion (P < .01) (Table 2). The frequency of cells in each group was not associated with histologic type, tumor size, and depth of invasion.


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  		TABLE 2. The relationship between four cell groups and metastatic parameters
 
To identify other factors that correlate with lymph node metastasis, we performed logistic regression analysis. Such analysis showed that the two most significant factors that correlated with lymph node metastasis were depth of invasion and the frequency of PCNA-positive cells with chromosome 17 numerical aberrations (Group 1) (Table 3). Using the same analysis, we identified the frequency of cells in Groups 1 and 3 as independent significant determinants of lymphatic invasion (Table 4). Logistic regression analysis could not identify significant factors that correlated to venous invasion and depth of invasion (data not shown).


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  		TABLE 3. The logistic regression analysis of variables in relation to positive lymph node metastasis
 

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  		TABLE 4. The logistic regression analysis of variables in relation to positive lymphatic invasion
 
Simultaneous Detection of PCNA and Centromere-Specific DNA Probe for Chromosome 17 in Metastatic Lymph Nodes of Gastric Cancer
In 16 primary lesions, the frequencies of tumors containing Group-1, Group-2, Group-3, and Group-4 cells were 19.1% ± 8.9% (range, 9%–44%); 20.2% ± 6.5% (range, 11.5%–32.5%); 14.3% ± 4.7% (range, 6.5%–21.5%); and 46.4% ± 12.8% (range, 16.5%–69.5%), respectively.

In 16 metastatic lymph nodes, the frequencies of tumors containing Group-1, Group-2, Group-3, and Group-4 cells were 32.2% ± 13.6% (range, 10.5%-60%); 22.3% ± 13.3% (range, 3.5%–44%); 20.4% ± 12.7% (range, 7%–50%); and 27.6% ± 12.3% (range, 6%–53%), respectively.

Figure 1d shows an example of simultaneously detected PCNA expression and chromosome 17 signal numbers in a metastatic lymph node from gastric cancer. In the same group of patients, comparison of the frequency of Group 1 in 16 paired samples of gastric tumors and metastatic lymph nodes showed a higher percentage of Group 1 in metastatic lymph nodes than in the tumor itself (Fig. 2). On the other hand, there were no significant differences in the frequencies of Groups 2, 3, and 4 between primary cancer lesions and metastatic lymph nodes (data not shown).



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  		FIG. 2. Comparison of frequency of cells positive for PCNA with chromosome 17 aberrations between primary gastric lesions and metastatic lymph nodes.

The frequency of Group 1 was significantly greater in metastatic lymph nodes compared with primary lesions (P = .0023).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The increased number of genetic aberrations in certain tumors may be associated with a growth advantage27 and may contribute to malignant progression.28,29 Chromosomal anomalies may reflect oncogenic changes,28–31 and aberrations in chromosome copy number may reflect karyotypic instability and relate to tumor aggressiveness.29,30 Previous studies reported that elimination of chromosome 17 aberrations might be important in the tumor aggressiveness of several carcinomas.1–9 We have also shown that alterations of chromosome 17 might be linked with tumorigenesis and tumor progression.9 The main purpose of this study was to further characterize cells with alterations of chromosome 17. Our results successfully demonstrated simultaneous two-color staining with PC-10 (anti-PCNA antibody) and DNA probe (D17Z1) of frozen human gastric cancer material. We were able to classify cells obtained from each tumor and successfully used this method to classify the tumors in the four groups.

Previous studies reported that rapid simultaneous immunophenotyping of chromosomally aberrant cells might be useful in determining the biological characteristics of individual cancer cells.10–13 In the first part of our study, we confirmed that simultaneous two-color staining with PC-10 and D17Z1 was possible methodologically by using several cell lines. In addition, there were no significant differences in PCNA positivity and chromosome 17 copy numbers between those tumor tissues stained simultaneously and those stained separately for the protein and gene (data not shown).

PCNA is involved in DNA replication and is necessary for adequate leading strand synthesis, acting as an auxiliary protein of DNA polymerase {delta}.18,19 This protein is present in nuclei throughout the cell cycle and is synthesized in the late G1 and S phase. Hence, its level correlates with the cell proliferative state.19 However, there are at least two different forms of PCNA.20 The first, a detergent-extractable form, is present in significant amounts in proliferating cells but is almost undetectable in resting cells. Another form of PCNA is detergent resistant and closely related to DNA synthesis. Because previous studies indicated that PCNA used with acetone/methanol fixation is similar to BrdU as a marker for the S phase,21 specimens were fixed using these methods in our study. Cancer cells were fixed again with 4% paraformaldehyde and rapid FISH maintained the antigenicity of PCNA.

Other investigators examined chromosomal aberrations using various cutoff levels in FISH. However, in our study, we used the frequency (%) of cells in each section and classified tumors into four groups. Unfortunately, we were unable to evaluate the parameters that correlate with gastric tumor metastasis to the liver due to the small sample size (only six cases). However, the frequency of PCNA-positive cells with chromosome 17 numerical aberrations correlated with lymph node metastasis, lymphatic invasion, and venous invasion. Therefore, the median ratio (12.0%) of Group 1 is useful as a cutoff value. When the median ratio (12.0%) of Group 1 was used as the cutoff value, logistic regression analysis identified the frequency of PCNA-positive cells with chromosome 17 numerical aberrations as independent determinants of lymph node metastasis and lymphatic invasion. The cell fraction of PCNA-positive and chromosomally aberration-negative (Group 3) was also identified as the independent determinant with respect to lymphatic invasion. Furthermore, the frequency of PCNA-positive cells with chromosome 17 numerical aberrations (Group 1) was higher in metastatic lymph nodes than in primary lesions (Fig. 2). However, the frequencies of cells in other groups (Groups 2, 3, and 4) were not elevated in metastatic lymph nodes compared with primary lesions. Interestingly, the frequency of cells negative for PCNA but positive for chromosome 17 aberrations (Group 2) did not correlate with lymph node metastasis, lymphatic invasion, and venous invasion. These results suggest that proliferating cells (Group 1, Group 3) easily invade lymphatic vessels and that the growth of proliferating cells (Group 1) with chromosome 17 numerical aberrations may be enhanced after invasion of lymph nodes.

Tumor suppressor genes, wild-type p5332 or nm2333 (considered as a metastasis suppressor gene), are located on chromosome 17. Loss of heterozygosity at the p53 locus has been found frequently in human gastric cancer.34–36 Immunostaining has revealed reduced nm23 immunoreactivity in lymph nodes and liver metastases compared to primary tumors in surgically resected and in autopsy cases of human gastric cancer.37 Cells positive for PCNA with chromosome 17 numerical aberrations may represent damage of some tumor suppressor genes, such as wild-type p53 or nm23. Induction of wild-type p53 activates p21WAF1/CIP1, a universal cyclin-CDK38 inhibitor and PCNA-interacting protein, which arrests cell growth.38,39 Therefore, our data suggest that the cell fraction (Group 1) positive for PCNA and chromosome 17 numerical aberrations could result in increased growth by a breakdown of the cell cycle.

Solid tumors are heterogeneous and contain cells with–as well as without–metastatic potential. The prognosis of cancer patients is influenced mainly by tumor growth, local infiltration, and metastasis. The ability of malignant tumor cells to invade the extracellular matrix and to metastasize is believed to be partially due to the expression of cellular adhesion receptors that interact with components of the extracellular matrix and the basement membrane. Therefore, cells positive for PCNA and chromosome 17 aberrations (Group 1) may represent those with alterations in the expression of adhesion molecules.

Preoperative staging of gastric cancer is suboptimal, especially with regard to lymph node metastasis. Therefore, there is still a need for additional and reliable information, preferably obtained by the least invasive techniques, such as examination of biopsy specimens. In this study, we performed simultaneous two-color staining of gastric biopsy material, including 12 early gastric cancer specimens obtained by biopsy gun. Our method of two-color staining can be performed with material obtained from primary gastric cancer using endoscopic biopsy before surgery. Our results also indicate that simultaneous two-color staining of PCNA and chromosome 17 centromere is a practical and useful method for the preoperative diagnosis of gastric cancer.

In conclusion, we have demonstrated in the present study that gastric tumors containing PCNA-positive cells with chromosome 17 aberrations have a high metastatic potential (especially lymph node metastasis). Our results also indicate that simultaneous staining for PCNA and chromosome 17 centromere is a useful diagnostic tool for clinical evaluation of patients with gastric cancer.


   Acknowledgments
 
The authors thank Dr. F.G. Issa (Word-Medex, Sydney, Australia) for careful and critical editing of the manuscript.

Received for publication August 14, 2000. Accepted for publication December 14, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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