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Clinical Cancer Research Vol. 10, 3013-3019, May 1, 2004
© 2004 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Comparative Study between DNA Copy Number Aberrations Determined by Quantitative Microsatellite Analysis and Clinical Outcome in Patients with Stomach Cancer

Seiji Suzuki1, Kaku Egami1, Koji Sasajima1, Mohammad Ghazizadeh4, Hajime Shimizu4, Hidehiro Watanabe1, Hirokazu Hasegawa1, Shinya Iida1, Takeshi Matsuda2, Yuji Okihama2, Masaru Hosone3, Kazuo Shimizu5, Oichi Kawanami4 and Takashi Tajiri6

Departments of 1 Surgery, 2 Gastroenterology, and 3 Pathology, Tama-Nagayama Hospital, Nippon Medical School, Tama-Shi, Tokyo; 4 Department of Molecular Pathology, Institute of Gerontology, Nippon Medical School, Nakahara-Ku, Kanagawa; and 5 Second Department of Surgery and 6 First Department of Surgery, Nippon Medical School, Bunkyo-Ku, Tokyo, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: We detected the relative DNA copy numbers (RCNs) at target loci in patients with stomach cancer with quantitative microsatellite analysis. We additionally clarified the relationship between DNA copy number aberrations and the clinical outcome of the patients.

Experimental Design: Fresh frozen samples were obtained from 30 patients who had undergone surgery for stomach cancer. Seven microsatellite loci in chromosomes 8q, 16q, and 20q and one gene-specific locus (ZNF217) were selected as the target loci. The DNA copy number was obtained relatively to a pooled reference consisting of six microsatellite primer sets selected from the regions where few aberrations have been reported in comparative genomic hybridization analysis. On the basis of the TaqMan PCR system, the internal probes used were carrying donor (6-carboxyfluorescein) and acceptor (6-carboxytetramethylrhodamine) fluorescent molecules complementary to CA repeats in the microsatellite markers and to one gene-specific oligomer in the gene-specific marker.

Results: Chromosome 8q gain, 20q gain, and 16q loss were detected in 18 (60.0%), 8 (26.7%), and 13 (43.3%) cases, respectively. Gains in the RCNs of D8S1801 and D8S1724 were most frequently found (36.7%). There was a significant correlation between the loss of D16S3026 and reduced survival duration (P = 0.0158), and the simultaneous aberrations of D8S1801 gain and D16S3026 loss (double marker positive) was significantly associated with reduced survival duration (P = 0.0008). According to Cox proportional hazards model, the double marker positive was a significant and independent factor indicating an unfavorable prognostic factor (relative risk, 17.176; 95% confidence interval, 2.782–106.026; P = 0.0022).

Conclusion: RCN aberrations in tumor tissues determined by quantitative microsatellite analysis enable identification of the prognostic factors that correlate with clinical outcome of the patients with stomach cancer.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Stomach cancer is the second common cause of all cancer deaths in males and the first in females in Japan (1) . Although the 5-year overall survival rate for stomach cancers detected and surgically or endoscopically treated in the early stage is surprisingly high, in stage IV tumors it unfortunately remains to be less than 20%. Stomach cancer detected in the late stage may not infrequently result in the patient’s death because of local recurrence, distant metastasis, or peritoneal dissemination, despite the performance of curative gastrectomy (1) . The patients suffering from super-advanced stomach cancer demonstrate a rapid decline and death when the diagnosis of stomach cancer is first established. Some of them, however, may at times survive for longer than expected. It is thought that individual differences in the pathogenesis and clinical outcome could be caused by variations of genetic change (2) .

The allelic imbalance of 1p, 1q, 3p, 5q, 8p, 12q, 16q, 17p, 17q, and 18q was reported previously in stomach cancer (3, 4, 5, 6) and specific genetic changes including amplification and/or overexpression of c-MYC (7) , ERBB2 (8) , c-MET (8) , KSAM (9) , AIB-1(10) , BTAK (11) , DcR-3(12) have also been found. Additionally, mutation or down-regulation of TP53(13) , K-RAS (14) , APC (15) , DCC (15 , 16) , FHIT (17) , CDH1(18) , RUNX3 (19) , and NM23 (20) has been reported. Recently, comparative genomic hybridization (CGH) enables detection of the recurrent abnormal regions in the DNA copy number encoding new genes that contribute to carcinogenesis and the progression of stomach cancer. Several reports dealing with CGH analysis on stomach cancer disclosed gains of 1q, 7q, 8q, 17q, and 20q and losses of 1p, 4q, 5q, 16q, and 17p (21, 22, 23, 24, 25) . However, the resolution of CGH is not high enough to enable the precise detection of copy number aberrations limited to a pinpoint region within a chromosomal arm. To overcome this drawback of CGH, Ginzinger et al. (26) introduced quantitative microsatellite analysis (QuMA), and they selected microsatellite loci and gene-specific loci for minute target areas on the basis of the detection by CGH of the recurrent abnormal regions in DNA copy numbers and those associated with a poor clinical outcome. QuMA is characterized by the relative quantification of the DNA copy number using a pooled reference consisting of six or seven microsatellite markers from the regions where few copy number aberrations have been shown in CGH analysis. Ginzinger et al. (26) emphasized that the use of a pooled reference reduced the possibility that loss/gain of a single reference marker might mislead the assignment of copy number at the test locus. Three papers dealing with the identification of DNA copy number aberrations in clinical samples by QuMA have been published previously (26, 27, 28) . In the present study, we selected seven microsatellite loci and a gene-specific locus in 8q, 16q, and 20q that had been identified previously as recurrent abnormal regions by CGH. Accordingly, we detected the relative DNA copy numbers (RCNs) in each of the test loci using QuMA and clarified the relationship between the RCNs and the clinical outcome in patients with stomach cancer.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Specimens.
Fresh frozen samples were obtained from each of a series of 30 patients who had undergone gastrectomy for stomach cancer at Tama-Nagayama Hospital, Nippon Medical School. There were 9 female and 21 male patients with an average age of 66 years (range, 42–85). The final stage and histopathological findings of those 30 patients, according to the Japanese Classification of Gastric Carcinoma (29) , are listed in Table 1Citation . None of the patients was preoperatively treated with neoadjuvant chemotherapy. The surgically obtained samples had been stored at –80°C until we began to extract the genomic DNA. Only the samples of which H-E preparation revealed >70% tumor tissue were used in DNA extraction.


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  		Table 1 Clinicopathological findings in a series of thirty operated patients with stomach cancer

 
The genomic DNA was extracted from tumor specimens by proteinase K/SDS digestion followed by phenol/chloroform/isoamyl alcohol extraction. Normal DNA was prepared from peripheral blood lymphocytes from 16 healthy volunteers.

QuMA.
We assessed the DNA copy number at eight loci using a quantitative real-time PCR assay described by Ginzinger et al. (26) . As the target areas, we selected seven microsatellite loci (D8S530, D8S1724, D8S1801, D16S3140, D16S3026, D20S911, and D20S185) in three chromosomal arms (8q, 16q, and 20q) and one gene-specific locus (ZNF217). Those selected loci had previously been frequently reported to be abnormal in DNA copy numbers by CGH analysis (22, 23, 24, 25, 26) . On this basis of the TaqMan PCR system performed on the ABI 7700 instrument (Applied Biosystems, Foster City, CA), we used internal probes carrying donor (6-carboxyfluorescein) and acceptor (6-carboxytetramethylrhodamine) fluorescence molecules complementary to CA repeats in the micosatellite markers and to a gene-specific oligomer in the gene-specific marker (Table 2)Citation . Copy numbers were detected relative to a pooled reference consisting of six primer sets (D2S2242, D6S1559, D10S1686, D11S1312, D14S1011, and D15S197) selected from regions where few aberrations have been reported in CGH analysis (22, 23, 24, 25, 26) . During the PCR cycle, fluorescence intensity of donor was determined by exonuclease degradation of TaqMan probe, and then the amplified material and PCR cycle were plotted. If the PCR efficiency for the combination of primer sets and a probe were >0.95, the relative DNA copy number at test locus was defined as RCN = 2 x 2{Delta}Ct(normal) – {Delta}Ct(sample), where {Delta}Ct = Ct (test marker) – Ct (reference marker). Ct was the cycle number at a constant threshold during the linearly amplified phase. {Delta}Ct (normal) was the mean of measurement on 16 normal DNAs at each test locus. PCR efficiency (E) can be detected from the slope of a four points curve where the log of the input of four kinds of DNA mass is plotted against the Ct. The equation of the curve is derived from XCt = X0(1 + E)Ct, where XCt = DNA quantity at the constant threshold and X0 = quantity of input DNA. The slope of this four points curve, S, is equal to 1/log(1 + E) or E = 10(–1/S) – 1. We excluded the primer sets at test loci where the SD of {Delta}Ct (normal) for 16 normal DNAs was >0.25.


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  		Table 2 Primers and probes at the target loci used for QuMAa to detect RCN

 
PCR.
PCR was performed in triplicates with 50 µl of reaction volume of 1x PCR buffer A (PE Biosystem), 2.5 mM MgCl2, 0.4 µM each primer sets, 200 µM each deoxynucleotide triphosphate, 60 nM GT probe or 100 nM gene-specific probe, and 0.025 unit/µl Amp TaqGold (PE Biosystem) with 5 ng of genomic DNA. Cycling parameters were as follows: 95°C for 12 min x1 cycle and (95°C for 12 s, 55°C for 20 s, and 72°C for 45 s) x40 cycles.

Statistical Analysis.
To determine whether the obtained DNA copy number might be significantly different from normal DNA, we calculated a tolerance interval (T.I.) with a 95% confidence interval by using the pooled SD of {Delta}Cts at each test locus and two additional microsatellite loci (D8S1775 and D16S3104) on 16 unrelated normal DNAs as described before (26 , 27) . T.I. = 2 x 2 to the power ±{2.271 x the square root of [{Sigma}i (ni-1) x SDi2/{{Sigma}i (ni-1)]} where ni = number of normal DNAs analyzed per microsatellite or gene-specific locus, and 2.271 was the two-sided tolerance limit factor for the total degrees of freedom ([{Sigma}i (ni-1)] = 150). The obtained RCNs over T.I. were scored as significantly increased RCNs, and those below T.I. were scored as significantly reduced RCN. To clarify whether the DNA copy number aberrations determined by QuMA might be prognostic factors, we assessed the relationship between the DNA copy number aberrations and the clinical outcome of the patients using Kaplan-Meier survival curves and log-rank statistics. Tumor-specific survival from the date of operation was used. Furthermore, the parameters that were significant in the univariate analysis with the log-rank test were tested with the Cox proportional hazards model to assess the parameters that might be significantly and independently influential in survival duration.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PCR Analysis.
{Delta}Ct and SD of {Delta}Ct at seven microsatellite loci, one gene-specific locus (ZNF217), and two other micosatellite loci for unrelated normal 16 DNAs are listed in Table 3Citation . The T.I. was calculated as 1.45–2.76 at each locus using the pooled SD of {Delta}Ct. The RCNs obtained from the tumor samples were abnormal at each test locus if they were beyond the T.I. of 1.45–2.76. We judged DNA copy numbers >2.76 as being significantly increased RCNs and those <1.45 as being significantly reduced RCNs. The RCNs determined by QuMA in the 30 patients are listed, and the DNA copy number aberrations are shown in Fig. 1Citation . Chromosome 8q gain, 20q gain, and 16q loss were found in 18 (60.0%), 8 (26.7%), and 13 (43.3%) cases, respectively. The most frequently aberrant regions in the gained RCNs were D8S1801 and D8S1724 (36.7%). Although there were seven cases with simultaneous gains at more than one loci in 8q, losses at more than one loci in 16q were found in four, and gains in 20q were found in three cases. Distribution of the DNA copy number aberrations was varied. There were eight cases (26.7%) in whom chromosomal aberrations at >3 loci were found in at least one of three chromosomal arms (8q, 16q, and 20q). In stage IB tumor and higher, several copy number aberrations were observed at each of the test loci.


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  		Table 3 {Delta}Ct (normal) and standard deviation of {Delta}Ct (normal) at each of test loci and two additional microsatellite loci (D8S1775 and D16S3104)

 


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  		Fig. 1. Relative DNA copy numbers at each of target loci determined by introduced quantitative microsatellite analysis in a series of 30 operative patients with stomach cancer. White boxes indicate normal in relative DNA copy numbers.

 
DNA Copy Number Aberrations and Clinical Outcome.
The clinicopathological and genetic variables analyzed by the Kaplan-Meier method with log-rank statistics are shown in Table 4Citation . There was a significant correlation between the D16S3026 (16q24.2) loss detected by QuMA and reduced survival duration (P = 0.0158). In addition to the D16S3026 loss, final stage and depth of invasion were significantly associated with reduced survival duration (P = 0.0150, 0.0276). The gain of D8S1801 (8q24.1–24.2) copy number seemed to be related to reduced survival duration (P = 0.1903), although the ostensible relation was not confirmed by statistical significance. On the other hand, the association between the simultaneous aberration of D8S1801 gain and D16S3026 loss (double marker positive) and reduced survival duration was proven to be statistically significant (P = 0.0008; Fig. 2Citation ). In a multivariate analysis by the Cox proportional hazards model, the D16S3026 loss was a significant and independent unfavorable prognostic factor, as was advanced final stage (relative risk, 5.929; 95% CI, 1.327–26.489; P = 0.0198; Table 5Citation , Model A). Furthermore, in the other model, the double marker positive was a significant and independent poor prognostic factor (relative risk, 17.176; 95% CI, 2.782–106.026; P = 0.0022; Table 5Citation , Model B).


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  		Table 4 Ps determined by log-rank statistics indicate the relationship between cumulative survival and each of clinicopathological or molecular parameters

Double marker positive, D8S1801 gain and D16S3026 loss.

 


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  		Fig. 2. Kaplan-Meier survival curves demonstrate a significant difference between simultaneous aberrations of D8S1801 gain and D16S3026 loss (double marker positive) determined by introduced quantitative microsatellite analysis and reduced survival duration (P = 0.0008).

 

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  		Table 5 Summaries of Cox proportional hazards model

Double marker positive, D8S1801 gain and D16S3026 loss.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The purpose of this paper was to present some evidence of the clinical usefulness of QuMA especially in evaluation of the prognosis for patients with stomach cancer. QuMA has the advantages of allelic imbalance studies in which all target loci are potentially informative and neither paired normal tissue nor locus-specific probes are required. The DNA copy numbers at gene-specific loci, in addition to microsatellite loci, can be obtained by QuMA using gene-specific primer sets and gene-specific internal probes (26) .

Ginzinger et al. (26) pointed out that the 20q gains detected by QuMA were well in accord with those seen with fluorescence in situ hybridization in various breast cancer cell lines. Furthermore, Nigro et al. (28) demonstrated the concordance of the DNA copy number between QuMA and fluorescence in situ hybridization in the detection of the loss at 1p and 19q in patients with oligodendroglioma. These results indicate that QuMA can determine the copy numbers at the target loci associated with tumorigenesis, progression, and clinical outcome for many clinical samples if the target area and pooled reference for QuMA were selected for some limited samples by CGH.

The distribution of the DNA copy number aberrations in each of 8q, 16q, and 20q varied among individuals. There were 12 cases whose copy number aberrations were both gains (8q or 20q) and losses (16q). Although the proportion of genetic aberration depends on the selection of the target chromosome, we think that these findings reflect the variety of genetic instabilities in the tumor tissue. Although copy number aberrations were observed from stage IB tumors in our study, the number of cases at the stage IA was only two and should be increased to achieve a substantial result in the study on early genetic change, including analysis of a precancerous lesion.

D8S1801 is located in the vicinity of the c-Myc gene on 8q24.1. Several reports (7 , 30, 31) on c-Myc amplification and/or overexpression had been published in stomach cancer. In breast cancer, Yokota et al. (32) reported that the copy number gains of several genes at 8q24.1, in addition to c-Myc, were frequently observed with the differential-PCR method using microsatellite markers. Although a significant difference was not found between D8S1801 gain and reduced survival duration (P = 0.1903), the copy number gain of tumor-promoting genes at 8q.24.1, besides c-Myc, may contribute to tumorigenesis and/or progression in stomach cancer. Although no reports on tumor-promoting genes at 8q13 and 8q21 have been previously presented in stomach cancer, Fejzo et al. (33) reported DNA copy number gains at 8q12-q22 in breast cancer cell lines and samples by fluorescence in situ hybridization analysis. The high frequency of the D8S530 and D8S1724 copy number gains suggests that tumor-promoting genes in stomach cancer might be located at these regions. In breast cancer, ZNF217is considered an oncogene as described by Colins et al. (34) . Ginzinger et al. (26) stated that there was a significant correlation between the ZNF217gain detected by QuMA and the reduced survival duration in patients with ovarian cancer. Rosenberg et al. (35) reported ZNF 217gene amplification seen with a CGH array in cell lines of gastroesophageal cancer, and our report is apparently the first report on ZNF217copy number gain in patients with stomach cancer. In our study, however, no significant correlation between ZNF217 gain by QuMA and reduced survival duration could be elicited. As for the 16q loss, several reports on allelic imbalance studies have been published in stomach cancer (5 , 18 , 36, 37, 38) . Although we selected only two test loci (16q21, 16q24.2) in our study, a high frequency (43.3%) of loss was observed at either of two loci in 16q. Simultaneous loss at both loci, however, was found in only three cases. Association of loss of CDH1with tumorigenesis and progression was reported previously in allelic imbalance studies in stomach cancer (38 , 39) . Our present study revealed that a significant difference was found between the copy number loss of D16S3026 at 16q24.2 and reduced survival duration. Mori et al. (34) reported that a high frequency of allelic imbalance was observed at 16q23.3-q24 in stomach cancer, and tumor suppressor genes might be located in these areas. The above-stated results suggest that genes other than CDH1located distal to 16q22 contribute to the biological actions associated with the progression of stomach cancer. In ovarian cancer, an association between a poor clinical outcome and the copy number loss of D16S3026 detected by QuMA has been reported (27) . Furthermore, a significant association between an allelic imbalance at 16q24.1-q24.2 and reduced survival duration in prostate cancer has been described by Elo et al. (40) . Bandoh et al. (41) reported that an allelic imbalance at 16q24.1–24.2 was frequently observed in hepatocellular carcinoma. These results imply that regions distal to 16q23 may code the candidate tumor suppressor genes in various malignant tumors. Although the Cox proportional hazards model in our study showed that the simultaneous aberrations of D8S1801 gain and D16S3026 loss might be a significant and independent unfavorable prognostic factor in patients with stomach cancer, the interpretation of statistical analysis is to be limited in making a definite conclusion because of a small number of the patients. On the other hand, the study on a large number of target loci for many clinical samples is cost-expensive and time-consuming. Therefore, we focused on the limited number of candidate microsatellite loci that might be strongly related to tumor progression and clinical outcome. The interaction that may contribute to acquisition of an aggressive phenotype in malignant tumors among different chromosomal regions can be easily clarified by QuMA. Nowadays, the biologically malignant behavior of the tumors may be assessed, and the clinical outcome may be anticipated by final stage and pathological findings including histological typing and grading. If the DNA copy numbers of target loci associated with clinical outcome and tumor progression were taken into consideration in addition to those clinicopathological findings, the biological malignancy might be more comprehensively evaluated, and a new system of diagnosis and treatment for malignant tumors could be established.

In summary, we found a significant correlation between simultaneous aberrations of D8S1801 gain and of D16S3026 loss, detected by QuMA, and reduced survival duration in patients with stomach cancer. By using the QuMA technique with many patients, we can rapidly detect the DNA copy numbers of prognosis-predictive markers and apply the results to the diagnosis and treatment for malignant tumors.


   FOOTNOTES
 
Grant support: Grants-in-Aid for Scientific Research No.13671351 from the Japanese Society for the Promotion of Science.

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.

Requests for reprints: Seiji Suzuki, Department of Surgery, Tama-Nagayama Hospital, Nippon Medical School, 1-5-1, Nagayama, Tama-Shi, Tokyo 206-8512, Japan. Phone: 81-42-371-2111; Fax: 81-42-372-3784; E-mail: seiji{at}nms.ac.jp

Received 9/17/03; revised 12/18/03; accepted 1/ 8/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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