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From the Departments of Surgery (JM, YK, TK, MW, SO, YO, HH, YS, KK MK), Radiology (HF, AK), and Pathology (MM), School of Medicine, and Institute for Advanced Medical Research (KD), Keio University, Tokyo, Japan.
Correspondence: Address correspondence and reprint requests to: Yuko Kitagawa, MD, Department of Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160–8582, Japan; e-mail: kitagawa{at}sc.itc.keio.ac.jp
ABSTRACT
The clinical significance of micrometastasis in sentinel nodes (SNs) may differ in various organs. In particular, the prognostic value of SN micrometastases detected by reverse transcriptase-polymerase chain reaction (RT-PCR) is still controversial. We investigated the diagnostic and therapeutic significance of nodal molecular metastasis detected by nested RT-PCR for cytokeratin (CK) 19 mRNA in gastrointestinal cancer. In 51 cases with GI tract cancer treated by standard curative resection, SNs were identified by a radio-guided method. In 10 of 51 patients, 25 SNs and 3 non-SNs were histologically negative and RT-PCR positive. Three non-SNs with positive CK19 mRNA were randomly sampled from the same basin where histologically positive SNs were identified. Immunohistochemical analysis of six additional step sections obtained at 30-µm intervals with use of an anticytokeratin antibody showed clearly recognizable histological metastases in 4 of 25 histologically negative/RT-PCR-positive SNs (16%). In one case of esophageal squamous cell carcinoma with nodal micrometastasis identified by CK19 RT-PCR, extranodal local recurrence in the SN basin (left supraclavicular basin) was observed 6 months postoperatively. These findings suggest that nodal micrometastasis detected by nested RT-PCR has some clinical significance in GI cancer. Molecular assessment of the SN may be a valuable tool to complement routine histological examination for GI cancers.
Key Words: Colorectal cancer • Esophageal cancer • Gastric cancer • Sentinel node • Micrometastasis • RT-PCR
The validity of the sentinel node (SN) concept has been demonstrated with various solid tumors, including gastrointestinal (GI) cancers.1–5 Despite the multidirectional and variable lymphatic drainage from the GI tract, SN mapping and the detection of metastasis based on SN status seem to be feasible based on several recent reports. Accurate staging of early GI cancer by SN mapping could be useful for therapeutic decision-making. However, the clinical application of selective lymphadenectomy based on SN status is likely to be controversial because of the potential risk of false-negative results. A local recurrence following exclusion of lymph node dissection due to false-negative results of SN biopsy would present a serious problem in GI cancer. There have been several reports of the limitations of intraoperative histological examination to detect micrometastasis.6–8 Because the application of SN technology in GI surgery depends on the sensitivity of intraoperative assessment of SNs, we investigated the incidence and clinical significance of molecular metastasis in SNs detected by RT-PCR in GI tract cancer.
METHODS
Patient Population
We enrolled 51 consecutively treated patients with clinically localized, node-negative GI cancer (gastric cancer, n = 29; esophageal cancer, n = 11; rectal cancer, n = 11); all patients gave written informed consent. Clinical diagnosis was made by pathological examination of endoscopic biopsy specimens. The absence of visible lymph node involvement was confirmed by preoperative computed tomography and ultrasonography. The protocol was approved by an institutional review board and committee for radiation safety.
As previously described,9 2.0 mL of technetium–99m tin (Sn) colloid was endoscopically injected in four quadrants into the submucosal layer of the primary lesion before surgery. After standard resection (total or distal gastrectomy for gastric cancer; transthoracic esophagectomy for esophageal cancer; abdominoperineal resection or low anterior resection for rectal cancer) and level I/II lymph node dissection, a hand-held gamma probe (Navigator; Tyco Health Care Japan, Tokyo, Japan) was used to identify nodes that had 10 times more radioactivity than surrounding tissue. These nodes were defined as SNs.
All SNs were examined for cytokeratin (CK) 19 mRNA. As control samples to exclude nonspecific contamination, non-SNs from the same lymphatic basin (sentinel basin) and a non-SN from a different lymphatic basin were randomly sampled (Fig. 1). Bivalved lymph nodes were analyzed by nested RT-PCR for the expression of CK19. Formalin-fixed paraffin-embedded specimens of the other half of the bivalved nodes were examined by routine hematoxylin and eosin staining. Lymph nodes examined by nested RT-PCR were reevaluated in serial sections (six sections at 30-µm intervals) by immunohistochemistry with anticytokeratin antibody (AE1/AE3).
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Reverse transcription for 2 µg of total RNA was performed in a 25-µL reaction mixture according to the manufacturer’s instructions (M-MLV Reverse Transcriptase RNase H minus; Promega, Madison, WI). Two µM of oligo-d(T)18 was used as the RT primer.
The first PCR for CK19 mRNA was performed in 20 µL of reaction mixture, containing 2 µL of cDNA solution, PCR reaction buffer mix (Takara, Tokyo, Japan), 0.2 mM dNTP, 0.5 unit Taq HS (Takara), and 1 µM of each outer PCR primer set (5'-AGAGCTGGCCTACCTGAAGAAG-3'and 5'-AAGGACAGCAGAAGCCCCAGAG-3'). Then, a 2-µL aliquot of the first PCR product was used as the template for the nested PCR. The conditions for the nested PCR were the same as for the first PCR except for the PCR primer set (5'-AGGTGGATTCCGCTCCGGGCA-3' and 5'-ATCTTCCTGTCCCTCGAGCA-3'). The thermal conditions of the first PCR and the nested PCR on GeneAmp 9600 (ABI, Foster City, CA) were as follows: initial heating at 94°C for 5 minutes and 35 cycles of PCR (95°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute). The detection and evaluation of the nested PCR products of CK19 mRNA (461 bp) were performed by 2% agarose gel electrophoresis with ethidium bromide staining.
The quality of purified total RNA and cDNA synthesis was ascertained by amplification of glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA. The PCR for GAPDH mRNA was performed in a 20-µL reaction mixture containing 2 µL of cDNA solution, PCR reaction buffer mix (Takara), 0.2 mM dNTP, 0.5 unit Taq HS (Takara), and 1 µM of each PCR primer set (5'-TGAACGGGAAGCTCACTGG-3'and 5'-TCCACCACCCTGTTGCTGTA-3'). A 307-bp fragment was generated by the PCR.
The results of nested RT-PCR were compared with those of pathological examination in a blinded fashion.
Positive RNA Control
In order to generate a PCR-positive control, the PCR product of CK19 mRNA was cloned into pGEM-T Easy vector (Promega). Then, the RNA fragment of the inserted sequence was synthesized by T7 RNA polymerase (Promega). After removal of vector DNA by DNase I treatment, RNA was purified according to the manufacturer’s instructions. Finally, the yield of the RNA fragment was measured spectrophotometrically at 260 nm. The positive control, diluted at 108 copies/µL, was stored at -80°C.
RESULTS
Sentinel Node Mapping
In all 51 patients, at least one SN was identified by the gamma probe (detection rate, 100%). The mean number of SNs was 3.5 (esophagus, 3.1; stomach, 4.5; rectum, 3.4). Lymph node metastases were detected in 13 cases by routine histopathological examination. In all of these cases, histologically detected metastasis was present in the SN. The performance of SN mapping in this series was compatible to our previous experience.3,5,9
Sensitivity of CK19 Nested RT-PCR
We examined 331 nodes (176 SNs and 155 non-SNs) by nested RT-PCR for CK19. The sensitivity of nested RT-PCR was determined by performing serial dilutions of RNA-positive control. When 0 to 1000 copies of the positive controls were amplified by the nested RT-PCR, the amplification product of CK19 was clearly detected at over 10 copies/PCR (Fig. 2). To see the positive rate at the low target concentration, we amplified diluted positive controls (0, 1, 3, 10 copies, n = 9). The positive rates were 0% (0 of 9), 22% (2 of 9), 67% (6 of 9), and 100% (9 of 9), respectively.
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Although several technical aspects remain to be clarified, the SN concept appears valid in GI cancer. In addition, SNs may possess utility for ultrastaging. Nested RT-PCR to detect CK19 mRNA was a more sensitive method than routine histopathological examination of resected lymph nodes. The results of CK19 nested RT-PCR may have clinical significance because 16% of histologically negative/PCR-positive SNs showed histological metastases on additional step sections. The clinical utility of histologically negative/PCR-positive nodes is still unclear. Nevertheless, the high incidence of CK19 mRNA positivity in SNs vs. non-SNs and the absence of CK19 mRNA expression in nodes randomly sampled from distant nonsentinel lymphatic basins suggest the correlation of CK19 mRNA expression with the metastatic presence of cancer cells, rather than nonspecific contamination.
The fact that all of the CK19 mRNA-positive non-SNs were harvested from basins with histologically positive SNs supports our contention and suggests the clinical significance of an SN basin in GI cancer. The case of esophageal squamous cell carcinoma diagnosed as pN0 by routine histopathological examination but found to be node-positive by nested RT-PCR emphasizes the possible clinical impact of recognizing molecular metastasis. A randomized controlled trial has shown that postoperative adjuvant chemotherapy with 5-fluorouracil and cisplatin can prevent recurrence in node-positive cases.10 This patient with pN0 carcinoma did not receive adjuvant chemotherapy. Subsequently, extranodal recurrence adjacent to the SN basin with CK19 mRNA expression was recognized. These findings suggest that molecular metastasis in GI cancer detected by nested RT-PCR may have some clinical significance.
The concept of the SN basin as a diagnostic and therapeutic target is also clinically important. We are currently developing minimally invasive surgical approaches using SN navigation.11 An SN basin contains metastatic non-SNs in addition to metastatic SNs in cN0 cases with nodal micrometastasis. SN basin dissection is recommended rather than pinpoint sampling of SNs to reduce the risk of false-negative results.12
Molecular staging may be useful to determine indications for adjuvant therapy in GI cancer. In addition, molecular staging of SNs may alter the surgical approach for GI cancers. For example, laparoscopic local resection is theoretically feasible for curative treatment of SN-negative early gastric cancer based on intraoperative SN biopsy. We contend that the limitations of intraoperative histological examination should be offset by intraoperative molecular information. Although the sensitivity of nested RT-PCR in this study was extremely high, it is not suitable for intraoperative use because of time constraints. We are currently developing an intraoperative real-time RT-PCR system to evaluate the SN status. Furthermore, the quantitative evaluation of molecular metastasis using multiple markers is necessary to exclude the possibility of nonspecific contamination. A reliable system to assess molecular metastasis is essential for the safe introduction of individualized modified surgery based on sentinel node navigation.
ACKNOWLEDGMENTS
The authors are indebted to Prof. J. Patrick Barron, of the International Medical Communications Center of Tokyo Medical University, for his review of the manuscript.
The acknowledgments are available online in the fulltext version at www.annalssurgicaloncology.org. They are not available in the PDF version.
FOOTNOTES
Findings suggest that nodal micrometastasis detected by nested reverse transcriptase polymerase chain reaction has some clinical significance in gastrointestinal cancer. Molecular assessment of the sentinal node may be a valuable tool to complement routine histological examination for gastrointestinal cancers.
Received for publication November 21, 2003. Accepted for publication December 10, 2003.
REFERENCES
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  S. Gretschel, A. Bembenek, M. Hunerbein, S. Dresel, W. Schneider, and P. M. Schlag Efficacy of Different Technical Procedures for Sentinel Lymph Node Biopsy in Gastric Cancer Staging Ann. Surg. Oncol., July 1, 2007; 14(7): 2028 - 2035. [Abstract] [Full Text] [PDF]
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 M. Ito, Y. Minamiya, H. Kawai, S. Saito, H. Saito, K. Imai, and J.-i. Ogawa Intraoperative detection of lymph node micrometastasis with flow cytometry in non-small cell lung cancer J. Thorac. Cardiovasc. Surg., September 1, 2005; 130(3): 753 - 758. [Abstract] [Full Text] [PDF]
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