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Clinical Cancer Research Vol. 9, 5137-5141, November 1, 2003
© 2003 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Prediction of Nodal Metastasis by Comparative Genomic Hybridization in Biopsy Specimens from Patients with Superficial Esophageal Squamous Cell Carcinoma

Takashi Ueno, Akira Tangoku, Shigefumi Yoshino, Toshihiro Abe, Hideto Hayashi, Hiroaki Toshimitsu, Kiichiro Hashimoto, Tomomitsu Satoh, Atsunori Oga, Tomoko Furuya, Masaaki Oka and Kohsuke Sasaki1

Departments of Pathology [T. U., H. T., K. H., A. O., T. F., K. S.] and Surgery II [T. U., A. T., S. Y., T. A., H. H., H. T., K. H., T. S., M. O.], Yamaguchi University School of Medicine, Ube 755-8505, Yamaguchi, Japan

ABSTRACT

Purpose: Selection of appropriate protocols for treatment of superficial esophageal squamous cell carcinoma (SESCC) is dependent on lymph node metastasis status. Therefore, it is important to know whether lymph node metastasis is present before treatment.

Experimental Design: In this study, we examined the relation between DNA sequence copy number aberrations detected by comparative genomic hybridization and lymph node metastasis in 26 surgically resected SESCCs (training samples). We then assessed whether the genetic information is predictive for nodal status in biopsy specimens from eight newly enrolled patients with SESCC (blinded samples).

Results: Pathological examination revealed that 17 of 26 training samples (65.4%) did not have associated lymph node metastasis. Gains of 8q24 and/or 20q12-qter were observed in 12, including all (nine of nine) with nodal metastasis. Fourteen training samples did not have gain of either 8q24 or 20q12-qter. Of the blinded samples, two showed no gain of 8q24 or 20q12-qter, and as anticipated the postoperative pathological examination revealed no nodal metastasis. The remaining six blinded samples had gains of 8q24 and/or 20q12-qter, and lymph node metastasis was detected by postoperative examination in four of these tumors.

Conclusions: Absence of gains of 8q24 and/or 20q12-qter appears to be associated with absence of lymph node metastasis in patients with SESCC; therefore, less invasive surgery can be chosen.

Introduction

ESCC2 is one tumor of the digestive tract that has poor prognosis (1) . The prognosis can be improved when treatment is optimized for each patient according to the biological characteristics of the tumor. In SESCCs, it is important to determine whether lymph node metastasis is present, because the therapeutic strategies differ for cases with and without metastasis (2) . If the presence of lymph node metastasis could be assessed with a reliable diagnostic method before treatment, less invasive treatments could be used to treat patients with SESCC without metastasis.

In general, multiple genetic changes are present in malignant solid tumors including ESCC (3, 4, 5, 6, 7, 8, 9) , and these genetic alterations affect the biological characteristics of a tumor. The relationship between genetic alterations and biological characteristics should be elucidated to estimate the biological behavior in each tumor for optimal treatment of patients. This relation has not been clarified in SESCC. A comprehensive method for analysis of genetic alterations related to biological behaviors of tumors is needed.

CGH provides a genome-wide assessment of chromosomal gains and losses (10) . The number of DSCNAs detected by CGH is correlated with the disease stage and biological behavior of a tumor (11, 12, 13, 14) . Some genetic changes are linked to patient prognosis, lymph node metastasis, and distant organ metastasis in ESCC (8) . Such data allow us to estimate the biological characteristics of ESCC from biopsy specimens with the goal of optimizing treatment for each patient.

Here, we report that the status of 8q24 and 20q12-qter as detected by CGH allows prediction of nodal status in patients with SESCC.

Materials and Methods

Patients
We studied 34 cases of SESCC comprising 26 training samples and 8 blinded samples as described below. In this study, SESCC is defined as cancers with tumor invasion limited to the mucosal and/or submucosal layer (15) . All patients were admitted to our department between 1997 and 2002 (Table 1)Citation . The patients were 30 men and 4 women with an average age of 62.7 years (range, 46 to 82 years). All patients were diagnosed as having SESCC by preoperative examination with endoscopy and endoscopic ultrasonography. All underwent transthoracic esophagectomy with lymphadenectomy without preoperative radiotherapy and/or chemotherapy per the routine manner. Tumor stage and grade were determined based on the surgical staging system of the International Union Against Cancer (16) . Tumor depth was subclassified histologically into m1, m2, m3, sm1, sm2, and sm3 based on the guidelines from the Japanese Society for Esophageal Diseases (15) . Namely, tumors limited to the epithelial layer, proper mucosal layer, and muscularis mucosa are designated as m1, m2, and m3, respectively. In addition, tumors extending to the upper third, middle third, and lower third of the submucosa are defined as sm1, sm2, and sm3, respectively. The study protocol was approved by the Institutional Review Board for Human Use at the Yamaguchi University School of Medicine in May 1995, and informed consent for this study was obtained from all patients.


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  		Table 1 Clinical data

 
Tumor Tissue Specimens and DNA Extraction
Training Samples.
We examined surgically resected human SESCCs from 26 patients. Of these patients, 17 did not have lymph node metastasis, and the remaining 9 did. Tumor tissue specimens were frozen and stored at -80°C until use. Microdissection was performed as described previously to reduce contamination by normal tissues (17 , 18) . In brief, 30-µm frozen tissue sections, numbering between 20 and 30 sections per specimen, were prepared and stained with H&E. Under microscopic observation, tumor tissues were microdissected manually from surrounding stromal tissue with a disposable fine needle, and tissue fragments were collected and transferred into a microtube. High-molecular-weight genomic DNAs were extracted from tumor tissues with a DNA extraction kit (SepaGene; Sankojunyaku Co., Ltd., Tokyo, Japan) according to the manufacturer’s instructions. Control DNA was extracted from peripheral blood lymphocytes.

Blinded Samples.
To evaluate the predictive value of DSCNAs with respect to status of lymph node metastasis, we used fresh preoperative biopsy specimens from eight newly enrolled SESCC patients. Usually, a single biopsy specimen was subjected to CGH analysis. After collection of tumor tissues by microdissection, tumor DNA was extracted, and to amplify DNA, degenerate oligonucleotide primed-PCR was performed with universal primer 6-MW (5'-CCGACTCGAGNNNNNNATGTGG-3') as described previously (18 , 19) . Control DNA extracted from peripheral blood lymphocytes provided by healthy volunteers was also amplified by degenerate oligonucleotide primed-PCR. Postoperative histopathological examination revealed that four patients had lymph node metastasis, and four did not.

CGH and Digital Image Analysis
CGH and digital image analyses were conducted as described previously (11 , 17, 18, 19, 20, 21) . Briefly, DNAs extracted from tumors and lymphocytes were labeled by nick-translation with SpectrumGreen (Vysis, Inc., Downers Grove, IL) and SpectrumRed (Vysis), respectively. Each labeled DNA sample (200 ng) and 10 µg of Cot-1 DNA (Life Technologies, Inc., Gaithersburg, MD) were mixed in 10 µl of hybridization buffer and cohybridized onto normal denatured metaphase chromosomes for 72 h at 37°C. The slides were mounted in antifade solution containing 0.15 mg/ml 4,6-diamino-2-phenylindole as counterstain. Images were captured with an Olympus BX60 fluorescence microscope equipped with a 100x UplanApo objective lens and a cooled charge-coupled device camera (SenSys 1400; Photometrics, Ltd., Tucson, AZ). The digital image analysis system (QUIP STM XL; Vysis) was used for image analysis in this experiment. Approximately 20, at least 10, representative images were analyzed. Increases and decreases in DNA sequence copy number were defined by green-to-red ratios of 1.2 and 0.8, respectively, as described previously (11 , 18) . High-level copy number increases in subregions (amplifications) in contrast to whole arm gains were defined as a tumor:control ratio of 1.4.

Statistical Analysis
Statistical analysis was performed with StatView software (SAS Institute, Inc., Cary, NC). Fisher’s exact test was performed to test differences between DSCNAs and clinicopathological features. Differences in the total number of DSCNAs in the patients were tested by the nonparametric Mann-Whitney U test. For all statistical tests, P < 0.05 was considered significant.

Results

CGH Analysis of Training Samples.
In the training samples, the number of DSCNAs ranged from 2 to 19, with an average of 9.3 per tumor that included 5.0 gains and 4.3 losses. A gain of 3q26-qter was the most frequent aberration (69.2%) observed. Recurrent losses of DNA copy number were found at 3p and 18q22-qter (61.5 and 53.8%, respectively). The cytogenetic aberrations are summarized in Fig. 1Citation .



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  		Fig. 1. Summary of DNA copy number aberrations detected by CGH in training samples (n = 26) of SESCC. Gains are shown to the right of the chromosome ideograms. Thick lines, amplified regions. Losses are shown to the left of the chromosome ideograms. Green and pink lines, gains and losses in tumors without lymph node metastasis (n = 17), respectively; blue and red lines, gains and losses in tumors with lymph node metastasis (n = 9), respectively.

 
Relation of DSCNAs to Lymph Node Metastasis in Training Samples.
The average number of DSCNAs was smaller in tumors without lymph node metastasis (stage I tumors; n = 17) than in those with lymph node metastasis (stage IIb tumors; n = 9; 7.5 versus 12.9, P = 0.004). Gain of 8q24 or 20q12-qter was associated with lymph node metastasis (P = 0.005 and 0.02, respectively; Table 2Citation ). Nodal metastasis was detected in 9 of 12 patients with 8q24 and/or 20q12-qter gains. In contrast, tumors with neither 8q24 nor 20q12-qter gain showed no nodal metastasis. There was a distinct difference in the frequency of nodal metastasis between them (P = 0.00007; Table 2Citation ).


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  		Table 2 Relationship of DSCNAs to lymph node metastasis in training samples

 
CGH Analysis of Blinded Samples.
There were no differences in chromosomal aberrations between biopsy and surgical specimens; the DNA copy number aberrations at 8q and 20q in the eight blinded samples are summarized in Fig. 2Citation . Two of eight tumors showed no gain of 8q24 or 20q12-qter, and postoperative histopathological examinations revealed no nodal metastasis for these two tumors. Gains of 8q24 and/or 20q12-qter were detected in the remaining six tumors, and postoperative pathological examination revealed lymph node metastasis in four.



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  		Fig. 2. Summary of DNA copy number gains of chromosomes 8 and 20 detected by CGH in blinded samples (n = 8). Green lines, aberrations in tumors without lymph node metastasis (n = 4); blue lines, aberrations in tumors with lymph node metastasis (n = 4).

 
Discussion

Flexible endoscopic procedures allow easy detection of SESCC (22) , and therapeutic modalities for SESCC have improved (23, 24, 25) . However, patients with SESCC still have poor prognosis in comparison with early cancers of other parts of the digestive tract. This is because of anatomical and biological characteristics, such as early spreading to the surrounding tissue and early metastasis to the lymph nodes, inherent to ESCC.

It is important to provide treatment that is optimized to the individual patient to improve prognosis. Selection of treatment protocols for SESCC depends greatly on lymph node status. Unfortunately, there are no reliable markers available to distinguish cases without lymph node metastasis from those with it before treatment.

Detection of specific DSCNAs in biopsy specimens facilitates prediction of the presence or absence of nodal metastasis at the time of histological diagnosis of SESCC. Information concerning nodal status permits selection of the best treatment protocol for each patient with SESCC. Less invasive methods can be used to treat patients without 8q24 or 20q12-qter gains. Because lymph node metastasis was always accompanied by gains of 8q24 and/or 20q12-qter, transthoracic esophagectomy with lymphadenectomy would be recommended for treatment of patients with SESCC having these abnormalities. There was a difference in the frequency of 8q24 and 20q12-qter gains between training and blinded samples. This is probably due to the difference in the frequency in nodal metastasis between two samples, and this is evidence for the correlation between these genetic changes and nodal status.

Although gains of 8q24 and 20q12-qter are closely linked to lymph node metastasis in SESCC, genes involved in esophageal carcinogenesis have not been localized to these regions. Our previous study indicated that c-MYC, which is located at 8q24.1, was rarely involved in ESCC (26) . We have initiated studies to identify genes associated with lymph node metastasis of ESCC.

The present data indicate that CGH analysis of biopsy specimens provides valuable information regarding nodal status in SESCC and allows selection of an appropriate treatment protocol for each patient. To verify the results, however, large-scale studies at the molecular genetic level are necessary.

FOOTNOTES

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.

This work was supported by Grant-in-Aid for Science Research 14370071 from the Ministry of Education, Science, Sports and Culture of Japan.

1 To whom requests for reprints should be addressed, at Department of Pathology,Yamaguchi University School of Medicine, 1-1-1 Minami-kogushi, Ube 755-8505, Yamaguchi, Japan. Phone: 81-836-22-2221; Fax: 81-836-22-2223; E-mail: kohsuke{at}yamaguchi-u.ac.jp Back

2 The abbreviations used are: SESCC, superficial esophageal squamous cell carcinoma; ESCC, esophageal squamous cell carcinoma; CGH, comparative genomic hybridization; DSCNA, DNA sequence copy number aberration. Back

Received 2/24/03; revised 6/ 4/03; accepted 6/ 5/03.

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Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
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