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Ann Thorac Surg 1998;66:1715-1718
© 1998 The Society of Thoracic Surgeons

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Original articles: general thoracic

Detection of micrometastases in histologically negative lymph nodes in esophageal cancer

^a Department of Surgery, University of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania, USA
^b Department of Pharmacology, University of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania, USA
^c Department of Pathology, University of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania, USA

Address reprint requests to Dr Luketich, 200 Lothrop St, C800, PUH, Pittsburgh, PA 15213
e-mail: (luketich{at}pittsurg.nb.upmc.edu)

Presented at the Thirty-fourth Annual Meeting of The Society of Thoracic Surgeons, New Orleans, LA, Jan 26–28, 1998.

	Abstract

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Background. New molecular techniques may identify micrometastases in histologically negative lymph nodes and have an impact on the staging of esophageal cancer. We investigated the role of the reverse transcriptase–polymerase chain reaction (RT–PCR) assay to identify micrometastases in esophageal cancer.

Methods. The RT–PCR assay to detect carcinoembryonic antigen (CEA) messenger ribonucleic acid (mRNA) was performed on lymph nodes from patients with esophageal cancer and benign esophageal disorders. The presence of CEA mRNA in lymph nodes was considered evidence of metastases.

Results. Histopathologic study revealed metastases in 50 (41%) of 123 lymph nodes from 30 patients with esophageal cancer. All histologically positive lymph nodes contained CEA mRNA by RT–PCR. Of 73 histologically negative lymph nodes, 36 (49%) contained CEA mRNA, a significant increase compared with the histopathologic diagnosis (p < 0.001). Lymph nodes in patients with benign disease contained no CEA mRNA. In 10 patients, histologic stage was N0. Five of them were also negative by RT–PCR, and all are alive with only one recurrence. In the remaining 5 patients, RT–PCR was positive for occult lymph node metastases; 2 have died of disease, and 1 is alive with recurrent disease.

Conclusions. In patients with esophageal cancer, RT–PCR detects more lymph node metastases than does histopathology. Initial follow-up suggests a positive RT–PCR with negative histologic findings may have poor prognostic implications. Further studies will be needed to confirm any clinical implications.

	Introduction

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
The presence of lymph node metastases is the most important prognostic feature in patients with esophageal cancer. The application of molecular biologic techniques may allow the recognition of micrometastases in lymph nodes that are histologically negative and could have important clinical implications for staging and treatment options. A new technique that uses a carcinoembryonic antigen (CEA)–specific nested reverse transcriptase–polymerase chain reaction (RT–PCR) assay to detect CEA–producing metastatic cells from bone marrow aspirates has been described [1]. In a recent pilot study, this technique was used to identify the messenger ribonucleic acid (mRNA) for CEA in histologically negative lymph nodes of patients with esophageal, gastric, colorectal, and breast carcinoma [2]. The objective of our study was to further investigate the application of CEA–specific RT–PCR assay to detect micrometastases in lymph nodes from patients with potentially resectable esophageal cancer.

	Material and methods

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Informed consent was obtained from all patients as specified in University of Pittsburgh Cancer Institute protocol 94-121.

Tissue samples
One hundred twenty-three lymph nodes were obtained from 30 patients with carcinoma of the esophagus. The lymph nodes were taken during combined laparoscopic and videothoracoscopic staging or esophagectomy. As a control, 26 lymph nodes were obtained from 13 patients undergoing surgical treatment of a benign esophageal disease, such as gastroesophageal reflux or achalasia. Five lymph nodes were evaluated from a patient with a history of long-standing reflux and Barrett’s metaplasia. In addition to lymph nodes, 16 samples of esophageal carcinoma (14 adenocarcinoma, 2 squamous cell carcinoma) and 11 samples of normal esophagus were studied. Each sample was carefully dissected from perinodal fat and soft tissue and sectioned into two pieces. One piece was fixed and embedded in paraffin and underwent routine histologic analysis. The other piece was immediately placed in liquid nitrogen and stored at -70°C until RNA extraction.

After the RT–PCR assay, the results of histologic examination and RT–PCR analysis were compared. The ² test was used to determine significant differences in positivity between histologic analysis and RT–PCR assay.

RNA extraction
Total cellular RNA was extracted from all samples by a modified guanidinium thiocyanate extraction method [3] using an RNA isolation kit (RNeasy; Qiagen, Chatsworth, CA). Tissue lysis and homogenization were performed with an automated tissue homogenizer (PowerGen 35 homogenizer; Fisher Scientific). Disposable generator tips were used to prevent RNA cross-contamination.

Reverse transcription
Complementary deoxyribonucleic acid (cDNA) was synthesized from 1 to 5 µg of total RNA using a complementary DNA synthesis kit (superscript preamplification system; GibcoBRL, Gaithersburg, MD). The oligo-dT priming method was used.

PCR
Three CEA–specific oligonucleotide primers for a nested PCR were designed from previously published sequences [1, 2, 4]. The primer sequences for CEA were as follows: primer A, 5'-TCTGGAACTTCTCCTGGTCTCTCAGCTGG-3'; primer B, 5'-TGTAGCTGTTGCAAATGCTTTAAGGAAGAAGC-3'; and primer C, 5'-GGGCCACTGTCGGCATCATGATTGG-3'. The nested PCR was done in two successive steps. Step 1 was performed in a 20-µL reaction volume containing 1x PCR buffer (Boehringer Mannheim), 250 µmol/L dNTPs, 1 µmol/L primer A and primer B, and 0.5 U Taq DNA polymerase (Boehringer Mannheim). Twenty rounds of amplification were performed in a thermocycler (DNA Thermocycler 480; Perkin Elmer) under the following cycling conditions: 95°C (1 minute) denaturation, 72°C (1 minute) extension, and a final extension at 72°C (10 minutes). Two microliters of step 1 reaction product was transferred to another tube containing primers B and C, PCR buffer, dNTPs, and Taq DNA polymerase in the same concentrations as in step 1. Step 2 was also performed in a 20-µL reaction volume. The second PCR reaction was carried out under the same conditions as step 1. Step 1 yields a 160-base pair (bp) PCR product, and step 2 produces a 131-bp product.

As a positive control for RNA quality, a PCR assay for ß-actin was performed for each sample. The primer sequences were as follows: 5'-CCTGGCACCCAGCACAATGA-3' and 5'-ACGAAGGCTCATCATTCAAA-3'. The actin PCR was performed with 30 rounds of amplification under the following cycling conditions: 94°C (30 seconds) denaturation, 68°C (45 seconds) annealing, 72°C (1 minute) extension, and a 7-minute final extension at 72°C.

To check for possible contamination with genomic DNA, RT-PCR reactions were also performed without a reverse transcription step.

PCR product analysis
The PCR products were analyzed on a 1.5% agarose gel containing 0.5 µg/mL of ethidium bromide. A 100-bp ladder (Boehringer Mannheim) served as a molecular weight marker. The PCR products were separated by electrophoresis at 80 V for 2 to 3 hours.

	Results

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Normal esophagus and carcinoma samples
The RT–PCR amplification of all 16 samples of esophageal carcinoma and 11 samples of normal esophagus demonstrated the 131-bp CEA band. No product was observed in control PCR reactions performed without reverse transcription, thus demonstrating that there was no contamination with genomic DNA.

Lymph node samples
Histologic examination detected metastases in 50 (41%) of the 123 lymph node specimens from the 30 patients with esophageal cancer. All histologically positive lymph node samples demonstrated a positive CEA signal by RT–PCR. Of the 73 histologically negative lymph nodes, 36 (49%) contained CEA mRNA by RT–PCR (Fig 1). Overall, 86 (70%) of the 123 lymph nodes were positive by RT–PCR compared with only 50 (41%) with evidence of metastases by histopathologic study (p < 0.001). In 20 (67%) of the 30 patients, RT–PCR indicated an increased number of metastatic lymph nodes compared with histologic review.

View larger version (24K): [in this window] [in a new window]   Fig 1. Ethidium bromide gel demonstrating complementary deoxyribonucleic acid (cDNA) products of reverse transcriptase–polymerase chain reaction (RT-PCR) assay. The known size of the RT–PCR cDNA fragment of carcinoembryonic antigen (CEA) is 131 base pair (131 bp fragment). Lane 1 is the 100 base pair ladder; lane 2 is empty; lane 3 is a CEA–positive control (pc); lane 4 is a negative control (nc); and lanes 5 through 15 are lymph node samples. The results of histologic study are shown below each lane. (+ = histologically positive lymph node; - = histologically negative lymph node.)

	Comment

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Lymph node staging in patients with esophageal cancer is an important determinant of survival [5–7]. Some studies have shown that the precise location and number of lymph nodes involved allows stratification of survival [8, 9]. However, accurate staging of esophageal cancer by means other than pathologic stage at the time of radical resection has been elusive. Conventional imaging modalities used to assess lymph node and distant metastases are inaccurate in more than 40% of patients compared with minimally invasive surgical staging [10, 11]. Positron emission tomography offers a substantial improvement over computed tomography in detecting distant metastatic disease but is only 45% sensitive in detecting metastases to lymph nodes smaller than 1 cm in diameter [11]. Endoscopic ultrasound is accurate in assessing the depth of tumor invasion, but its utility in detecting small lymph node metastases has been questioned [12–14].

Currently, the most accurate way to stage patients with esophageal cancer other than surgical resection is by minimally invasive surgical biopsy [14, 15]. This requires an extensive surgical dissection to avoid sampling errors and understaging. Once the biopsies have been done, further errors can take place in the histologic evaluation; up to 20% of histologically negative lymph nodes are positive on reexamination [16]. Immunohistochemical staining of lymph nodes to detect tumor markers may result in a higher detection rate of micrometastases compared with histologic examination, but this technique is time-consuming and labor intensive and is not in widespread clinical use [14–20].

A CEA–specific RT–PCR assay has been developed to detect carcinoma cells in bone marrow samples from patients with colorectal, pancreatic, or gastric carcinoma [1]. In a recent pilot study [2], this method was modified to detect lymph nodes metastases in patients with esophageal, gastric, colorectal, and breast cancer. Our preliminary experience with RT–PCR to detect mRNA for CEA confirms that this technique has the potential to more accurately reveal lymph node metastases in esophageal cancer compared with histologic evaluation. As expected, CEA was present in all samples of esophageal carcinoma and normal esophagus and thus appears to be a marker specific for epithelial cells. Importantly, CEA was not present in any of the lymph nodes from patients without cancer. Thus, in the absence of cancer, there is no migration of epithelial cells from the esophagus to the lymph nodes. Histologic assessment of samples from patients with esophageal cancer detected metastases in 50 of the 123 lymph nodes studied. All histologically positive lymph nodes were also positive by RT–PCR for CEA, a finding indicating that there were no false-negative results with this method. The application of RT–PCR to these samples was positive in 86 of the 123 lymph nodes, a significant increase compared with the histopathologic findings (p < 0.001).

In limited clinical follow-up, RT–PCR results were important prognostically. For example, in 10 patients, all nodes were negative by histopathologic study. Of the 5 patients whose results were also negative by RT–PCR, all are currently alive with a mean follow-up of 10.8 months, and 4 have no evidence of recurrence. In contrast, of the 5 patients with positive lymph nodes by RT–PCR in the setting of negative histology, 3 have recurrence and 2 have died of metastatic disease.

The CEA–specific RT–PCR assay is an extremely sensitive technique that facilitates the detection of histologically occult lymph node metastases in patients with esophageal cancer. Our preliminary results demonstrate a significant increase in detecting lymph node metastases in esophageal cancer compared with histopathologic results. Limited clinical follow-up suggests that lymph nodes positive by RT–PCR but negative histopathologically may have a similarly poor prognosis as histologically positive lymph nodes. Additional studies with clinical follow-up will be required to further define the role of RT–PCR in the diagnosis and treatment of patients with esophageal cancer.

	Acknowledgments

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Supported by ACS grant IRG-58-36.

	References

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): James D. Luketich Tracey L. Weigel Jill M. Siegfried Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Luketich, J. D. Articles by Siegfried, J. M. Search for Related Content PubMed PubMed Citation Articles by Luketich, J. D. Articles by Siegfried, J. M.