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Ann Thorac Surg 1996;62:835-838
© 1996 The Society of Thoracic Surgeons

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Original Articles: General Thoracic

Meanings of c-erbB and int-2 Amplification in Superficial Esophageal Squamous Cell Carcinomas

Department of Surgery, Keio University School of Medicine, Tokyo, Japan

Accepted for publication April 22, 1996.

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Background. Accumulation of genetic abnormalities is linked to the development and progression of cancer. We therefore analyzed the correlation between the clinical characteristics of superficial esophageal squamous cell carcinoma patients and oncogene amplifications.

Methods. Between 1980 and 1991, there were 63 cases of superficial esophageal carcinoma (Tis and T1 cancer) at Keio University Hospital. The T1 cases were divided into two groups: T1a cases, in which the tumor had invaded the lamina propria, and T1b cases, in which the tumor had invaded the submucosa. DNA was isolated from paraffin-embedded blocks. Oncogene amplification was determined by slot-blot hybridization.

Results. Amplification of int-2 and c-erbB was detected in 14 and 5, respectively, of the 54 cases. Three of 12 T1b patients with int-2 amplification died of distant organ metastasis. The survival rate for the group with int-2 amplification was significantly lower than that without int-2 amplification. All 4 T1b patients with c-erbB amplification had lymph node metastasis at operation.

Conclusions. These findings mean that genetic abnormalities are a useful marker for treating patients with superficial esophageal squamous cell carcinomas.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
The survival rate of patients with esophageal carcinoma is generally low [1]. To improve treatment outcome, it is necessary to detect the disease in the early stage and treat it more appropriately. In Japan, endoscopic examination with iodine staining is commonly performed, and many superficial esophageal carcinomas (Tis and T1 cancer) have been detected recently [2].

The resected cases of Tis and T1 carcinoma increased from 398 cases in 18 years between 1966 and 1983 to 1,584 cases in 6 years between 1984 and 1989 in Japan [3]. By histopathologic analysis, simple removal of lesions, such as by transhiatal esophagectomy or endoscopic mucosal resection, is indicated in Tis and T1a cases because lymph node metastasis was found in only 3.5% of cases [3]. Curative operation with lymph node dissection is indicated in T1b cases because lymph node metastasis was found often (35%) [3]. Therefore precise diagnosis in terms of depth of invasion must be performed and appropriate treatment must be selected. However, it is not easy to accurately distinguish between T1a or T1b lesions preoperatively. Assessment of the malignant potential of each case on the basis of special biological indicators, such as oncogene abnormalities, in addition to depth of invasion, would be useful in improving treatment selection for Tis and T1 carcinomas.

Amplification of the c-erbB and int-2 oncogenes has been observed in carcinoma of the esophagus and the breast [4–6]. These abnormalities are closely correlated with survival rate and pattern of recurrence after curative operation. In esophageal squamous cell carcinomas, it has been reported that c-erbB amplification is significantly correlated with lymph node metastasis at operation and that int-2 amplification is correlated with a high incidence of eventual metastasis to distant organs [4, 5].

As the genetic abnormalities and clinical characteristics in early carcinomas of the esophagus may differ from those in advanced cases and this genetic information could serve as indicators for treatment of superficial esophageal squamous cell carcinoma, we examined the relation between oncogene amplification and clinicopathological characteristics in superficial esophageal squamous cell carcinoma.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Patients
A total of 63 patients with superficial esophageal carcinomas (Tis and T1 cancer) who had undergone esophagectomy at Keio University Hospital between 1980 and 1991 were examined. According to clinical stage based on the TNM Classification of the Oesophagus [7] before resection, 4 patients had stage 0, 52 patients had stage I, 3 patients had stage II A, and 4 patients had stage II B disease. Forty-five patients had undergone esophagectomy with thoracotomy and 18 patients had undergone esophagectomy without thoracotomy. The T1 cases were divided into two groups: T1a cases, in which the tumor had invaded the lamina propria, and T1b cases, in which the tumor had invaded the submucosa. Tis, T1a, and T1b lesions were found in 6, 12, and 45 cases, respectively. DNA was obtained from formalin-fixed, paraffin-embedded blocks of the primary tumors and normal tissues adjacent to the tumors as controls. The histopathologic findings of cancer tissues of the patients at operation were investigated by hematoxylin and eosin staining of sections from the blocks. Patients were followed up every 3 months in the outpatient clinic for more than 3 years. Recurrence was diagnosed based on the results of diagnostic imaging, including routine chest roentgenograms, computed tomographic scans, and ultrasonography. Clinical and pathologic classification was based on the TNM Classification of the Oesophagus [7].

DNA Extraction
The blocks were cut into 5 or more sections 20 µm thick with a microtome. One section was examined by hematoxylin and eosin staining to identify the neoplastic and normal tissue areas. Samples of neoplastic and normal tissue were then taken from the unstained section. DNA was extracted following the method described by Dubeau and associates [8]. After deparaffinization with 100% xylene and dehydration with 100% ethanol, the final pellet was resuspended in 50 mmol/L Tris-HCl (pH 8.0), 10 mmol/L EDTA, and 150 mmol/L NaCl containing 1% sodium dodecyl sulfate (SDS) and proteinase K (200 µg/mL) and incubated at 37°C for 7 days. After incubation, DNA was extracted with a mixture of 24 parts chloroform and one part isoamyl alcohol after the incubation. The suspension was transferred to a 37°C water bath, and RNase digestion was performed immediately. Sodium acetate was added to each sample, and the DNA was precipitated with 2.5 volumes of cold ethanol. The DNA was then incubated at -70°C for at least 1 hour and centrifuged for 15 minutes at 19,500 g. The pellet was washed with 70% ethanol, dried thoroughly, and resuspended in TE solution.

Probes
The probes used were pE7, a 2.4-kilobase pair Cla I-Cla I fragment of the c-erbB gene [9]; 7C22, a 5.1-kilobase pair EcoR I-EcoR I fragment of the pSP64 gene [10]; SS6, a 0.9-kilobase pair Sac I-Sac I fragment of the int-2 gene [11]; and DRD2, a 1.6-kilobase pair BamH I fragment of the dopamine receptor D2 gene [12]. The c-erbB gene, pSP64 gene, and int-2 gene probes were provided by the Japanese Cancer Research Bank. The dopamine receptor D2 gene probe was obtained from American Type Culture Collection. The 7C22 and DRD2 were used as internal control probes of chromosome 7 and chromosome 11.

Slot-Blot Analysis
DNA concentrations were estimated spectrophotometrically. DNA (10 µg) was dissolved in an equal volume of 0.8 N NaOH, and 200 µL of 0.4 N NaOH was added. The samples were applied to a nylon membrane (Hybond-N; Amersham Life Science, Buckinghamshire, England) and incubated for 30 minutes at room temperature. The filters were then rinsed in 5 x SSPE and dried at room temperature. Probes were prepared by multiprime radioactive labeling with [³²P]d-CTP using an oligolabeling kit (Pharmacia, Uppsala, Sweden). Filters were prehybridized in 5 x Denhardt's solution, 50% formamide, 5 x SSPE, 0.5% SDS, and 100 µg of denatured salmon sperm DNA at 42°C for 2 hours and hybridized to the labeled probe at 42°C for 12 hours in the same solution. In the case of probes pE7 and 7C22, the filters were washed twice at 42°C for 10 minutes in 2 x SSPE and 0.5% SDS, once for 15 minutes in 1 x SSPE and 0.5% SDS and once for 10 minutes in 0.1 x SSPE and 0.5% SDS; in the case of probes SS6 and DRD2, the filters were washed twice at 65°C for 10 minutes, once for 15 minutes and once for 10 minutes. Autoradiograms were evaluated quantitatively using a bioimage analyzer (BAS 2000, Fujix, Tokyo, Japan). Cases in which more than threefold gene copy numbers were detected were estimated for gene amplification. The probes were removed from the filters by pouring boiled 0.5% SDS solution onto them, and the filters were rehybridized with other probes.

Statistical Analysis
Patient groups with and without oncogene amplification were compared by the ² test. The cumulative survival rates of patient groups excluding deaths from other causes were calculated by the Kaplan-Meier method, and the survival curves were compared with each other using the generalized Wilcoxon test.

	Results

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Histopathologic Analysis
The relations between depth of invasion of superficial esophageal squamous cell carcinoma and both vessel invasion and lymph node metastasis are shown in Figure 1. In Tis carcinoma, there was neither vessel invasion nor lymph node metastasis. Vessel invasion was found in 25% of patients with T1a carcinoma, but there was no lymph node metastasis, whereas vessel invasion and lymph node metastasis were found in 51% and 43%, respectively, of patients with T1b carcinoma.

View larger version (13K): [in this window] [in a new window]   Fig 1. . (A) Relation between the depth of invasion and vessel invasion of superficial esophageal squamous cell carcinoma and (B) relation between the depth of invasion and lymph node metastasis of superficial esophageal squamous cell carcinoma with lymphodectomy through right thoracotomy. The black portion of the bars represents positive cases. (Tis = invasion of epithelium; T1a = invasion of lamina propria; T1b = invasion of submucosa.)

Amplification of c-erbB and int-2 Genes
DNA was extracted from 54 of 63 cases: 4 of Tis carcinoma, 8 of T1a carcinoma, and 42 of T1b carcinoma. Amplification of the c-erbB gene was detected in 1 of the 8 T1a cases (12%) and 4 of the 42 T1b cases (10%). Amplification of the int-2 gene was detected in 1 of the 4 Tis cases (25%), 2 of the 8 T1a cases (25%), and 12 of the 42 T1b cases (29%). Coamplification of c-erbB and int-2 genes was detected in 2 cases of T1b carcinoma.

Relation Between c-erbB Amplification and Lymph Node Metastasis of T1b Carcinoma at Operation
Lymph node metastasis was observed in all 4 cases with c-erbB amplification. Only 1 of the 4 cases in which there were six metastatic regional lymph nodes was diagnosed as node-positive preoperatively. The other 3 cases with lymph node metastasis could not be diagnosed preoperatively. Lymph node metastasis was detected in 10 of the 32 cases without amplification (31%). The incidence of lymph node metastasis at operation in the patients with c-erbB amplification was significantly greater than in those without amplification (p < 0.05 by ² test). That sensitivity was 29% and specificity was 100% (Table 1). On the other hand, there was no significant relation between the incidence of lymph node metastasis at operation and int-2 amplification.

Outcome of Patients With int-2 Amplification
Int-2 amplification was detected in 15 cases: 1 Tis case, 2 T1a cases, and 12 T1b cases. The Tis and T1a patients are alive. Seven of the T1b patients are alive, and 5 have died: 3 of distant organ recurrence, 1 of regional lymph node recurrence, and 1 of other disease. There was no amplification in 39 patients: 35 are alive and 4 have died, 1 of regional lymph node recurrence and 3 of other disease. There was int-2 amplification in 15 patients and 27% subsequently died of esophageal cancer, whereas 1 of 39 (3%) with no int-2 amplification died of esophageal carcinoma. Coamplification of c-erbB and int-2 was detected in 2 patients: 1 died of distant organ recurrence and 1 of other disease.

Prognostic Value of T1b Cases With int-2 Amplification
The cumulative survival curves for the groups of T1b carcinoma with and without int-2 amplification were calculated by the Kaplan-Meier method. The two groups with and without int-2 amplification were not significantly different in regard to age, sex, histology, or pattern of lymph node metastasis. The 5-year survival rate was 55% in the group with int-2 amplification and 97% in the group without amplification. The survival rate in the group with amplification was significantly less than in the group without amplification (p < 0.05) (Fig 2).

View larger version (15K): [in this window] [in a new window]   Fig 2. . Cumulative survival curves of T1b carcinoma patients with int-2 amplification (n = 12; solid line) and patients without int-2 amplification (n = 29; broken line). (a = p < 0.05.)

	Comment

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

The incidence of c-erbB and int-2 amplification in early esophageal cancer is no different than in advanced cases (ie, 10% to 12% for c-erbB and 25% to 29% for int-2) [4, 5]. Although the accumulation of gene abnormalities was observed in adenoma-carcinoma sequence of colon, these gene amplifications in esophageal cancer had already occurred in the early stage of carcinoma.

Patients with Tis or T1a carcinoma did not have lymph node metastasis at operation or recurrence. In T1b lesions, however, lymph node metastasis was found in 43% of the patients at operation and 7 patients died, 3 of distant organ recurrence and 4 of regional lymph node recurrence. T1b cancers have a high rate of lymph node metastasis at operation and death due to disease. These findings suggested the following two conclusions. First, simple removal of the lesion such as by transhiatal esophagectomy or endoscopic mucosal resection should be recommended to treat Tis and T1a cases, and postoperative adjuvant chemotherapy or radiotherapy is unnecessary. Second, a more radical treatment approach to T1b cancers should be recommended. However, there is another problem. It is not easy to distinguish between T1a and T1b lesions preoperatively, making it difficult to select the treatment modality in borderline cases and difficult to choose postoperative adjuvant therapy for T1b carcinoma.

Information about gene abnormalities seems to provide the means of overcoming these problems. All patients with c-erbB amplification of T1b carcinoma had lymph node metastasis at operation. Moreover, 3 of the 4 cases of lymph node metastasis were not detected by computed tomography or ultrasonography preoperatively. Thus, c-erbB amplification may help identify some patients not otherwise staged (by computed tomography or ultrasonography) who would be candidates for this more aggressive approach.

A high incidence of metastasis (3 of 12) to distant organs was observed in T1b patients with int-2 amplification but none of the 30 T1b patients without int-2 amplification. Therefore, occult distant metastasis may be detected before resection; in such cases, it would be better to combine effective systemic therapy with operation.

Thus, additional preoperative information on gene amplification is used in the treatment of superficial esophageal squamous cell carcinoma in our department of surgery. If precise diagnosis of depth of invasion can be performed in Tis and T1a cases, simple removal of lesions, such as by transhiatal esophagectomy or endoscopic mucosal resection, is indicated. In T1b cases with int-2 amplification, chemotherapy after operation is indicated. If precise diagnosis of depth of invasion between T1a and T1b cases cannot be performed in borderline cases with c-erbB amplification, a radical operation with lymph node dissection is indicated. In borderline cases with int-2 amplification, chemotherapy after operation is indicated. The above strategy has been adopted in our department of surgery for several years, and we have been collecting data.

	Acknowledgments

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
We thank Miss Sachiko Matsuda for her expert technical assistance. This work was supported by grants-in-aid from the Ministry of Education, Science, and Culture and the Ministry of Health and Welfare, Japan.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Address reprint requests to Dr Ueda, Department of Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku Tokyo 160, Japan.

	References

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 

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