Annals of Surgical Oncology Cite Track
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
10.1245/ASO.2004.12.043
Annals of Surgical Oncology 11:778-785 (2004)
© 2004 Society of Surgical Oncology
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ishii, T.
Right arrow Articles by Monden, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ishii, T.
Right arrow Articles by Monden, M.

ORIGINAL ARTICLES

Rapid Genetic Diagnosis With the Transcription—Reverse Transcription Concerted Reaction System for Cancer Micrometastasis

Takaaki Ishii, MD, Yoshiyuki Fujiwara, MD, Satoru Ohnaka, Toshinori Hayashi, Hirokazu Taniguchi, MD, Shuji Takiguchi, MD, Takushi Yasuda, MD, Masahiko Yano, MD and Morito Monden, MD

From the Department of Surgery and Clinical Oncology, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan (TI, YF, HT, ST, TY, MY, MM); and Scientific Instruments Division, Tosoh Corporation (SO, TH), Kanagawa, Japan.

Correspondence: Address correspondence and reprint requests to: Yoshiyuki Fujiwara, MD, PhD, Department of Surgery and Clinical Oncology, Graduate School of Medicine, Osaka University, 2-2 E2, Yamadaoka, Suita-City, Osaka 565-0871, Japan; Fax: 81-6-6879-3259; E-mail: fujiwara{at}surg2.med.osaka-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Detection of cancer micrometastases is required for improvement of cancer therapy. The aim of this study was to establish a rapid and practical genetic assay to detect micrometastasis in gastric cancer and to assess its clinical significance with respect to prognosis.

Methods: A novel RNA amplification system with transcription–reverse transcription concerted reaction (TRC) was introduced for quantitative detection of cancer-specific carcinoembryonic antigen messenger RNA. The sensitivity and quantitative aspects of the assay were assessed with the full-length carcinoembryonic antigen messenger RNA, a gastric cancer cell line (MKN-45), and metastatic lymph nodes obtained from patients with gastric cancer. Peritoneal lavage fluid specimens that were collected from gastric cancer surgery were subjected to the assay, and the clinical significance of the results was examined for prediction of recurrence and survival.

Results: The quantification, sensitivity, and reproducibility of the assay with the TRC reaction were equal to those of quantitative reverse transcriptase-polymerase chain reaction with LightCycler. The most important advantages of the assay were its simplicity and rapidity. Molecular diagnosis of peritoneal lavage fluid by the TRC reaction significantly correlated with depth of invasion, peritoneal metastasis, clinical stage, overall survival, and peritoneal recurrence-free survival.

Conclusions: Molecular diagnosis of peritoneal lavage fluid with the TRC reaction could be a useful prognostic indicator for peritoneal recurrence and survival. Because the TRC reaction is more rapid and simpler than reverse transcriptase-polymerase chain reaction as a format for detecting RNA sequences, it may enhance the genetic diagnosis of cancer micrometastasis and may improve cancer therapy.

Key Words: Gastric cancer • Peritoneal dissemination • CEA • Carcinoembryonic antigen


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Accurate diagnosis of cancer spread is one of the most important factors in the selection of appropriate surgical and nonsurgical treatment. Usually, clinicians depend on histopathologic examination (hematoxylin and eosin) of surgical specimens such as lymph nodes or peritoneal lavage fluid during and after surgical intervention to determine the clinical stage of cancer. However, the sensitivity of conventional microscopic diagnosis is insufficient for detecting micrometastasis, which is thought to cause recurrent disease after curative treatment. Recent developments in molecular biology have produced new diagnostic tools for detecting cancer micrometastasis, which is often missed by conventional hematoxylin and eosin.1–4 Furthermore, recent studies have confirmed the clinical significance of micrometastases detected by the new molecular techniques.5–7

Amplification and detection of molecular markers specific to cancer cells or their organs of origin has generally been performed by reverse transcriptase (RT)-polymerase chain reaction (PCR).1 However, such procedures are complicated and have multiple steps, including the RT reaction and thermal cycling, and thus take several hours. Therefore, further refinements are required for the clinical application of molecular diagnostic techniques. In this study, we report the application of a novel method of quantitative genetic diagnosis with the transcription-RT concerted (TRC) reaction system8 for detection of cancer micrometastasis and prediction of cancer recurrence in patients with gastric cancer. The method amplifies and measures a cancer-specific messenger RNA (mRNA), carcinoembryonic antigen (CEA), in a single tube at a constant temperature without thermal cycling in three steps: denaturing, annealing, and extension for PCR. The reaction at a single temperature may produce a stable result and more accurate quantification. Another advantage of this method is that it amplifies RNA directly and thus avoids the need for RT to convert RNA to complementary DNA (cDNA) before amplification. This advantage should facilitate rapid diagnosis, which could be applied for intraoperative diagnosis of micrometastasis. These advantages may allow more reliable and practical genetic diagnosis of cancer micrometastasis.

The prognosis of advanced gastric cancer invading the gastric serosa is very poor, even after curative resection, mainly because of the high incidence of peritoneal recurrence.9–11 Peritoneal recurrence develops from micrometastasis, which originates from free cancer cells seeded from a primary gastric tumor.12–16 However, morphological diagnosis by cytological examination of peritoneal lavage fluid has a low sensitivity for detection of free cancer cells in the peritoneal cavity.17–20 Therefore, there is an urgent need for more sensitive methods to detect micrometastasis in the peritoneal cavity.

In this study, the TRC amplification format targeted for cancer-specific CEA mRNA was introduced as a novel rapid and quantitative genetic diagnosis for the detection of occult cancer cells in the peritoneal cavity of patients with advanced gastric cancer.8 The clinical significance of the molecular diagnosis was assessed by the peritoneal recurrence rate and overall survival.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Peritoneal Lavage Specimens
Between October 1999 and August 2002, 60 patients with histopathologically confirmed gastric cancer were enrolled onto this study. They represented all patients who underwent laparotomy without preoperative therapy at the Department of Surgery and Clinical Oncology, Osaka University. The peritoneal lavage fluid specimens were collected at surgery from the pouch of Douglas and the cavity under the left hemidiaphragm. Peritoneal lavage fluids from 15 patients who had laparotomy for benign diseases such as cholelithiasis, achalasia, and gastroesophageal reflux disease were also collected at the Department of Surgery and Clinical Oncology, Osaka University, and served as the control samples. All specimens were collected after written, informed consent was obtained from the patients. The experimental procedures were approved by the ethics review committee of our institution.

At the beginning of the operation, 100 mL of saline was instilled into the pouch of Douglas, and another 100 mL of the same solution was injected into the cavity under the left hemidiaphragm. Subsequently, 50 mL of peritoneal lavage fluid was collected from each cavity. Half of the peritoneal lavage fluid was subjected to conventional cytology, and the remaining half was used for molecular diagnosis. The cytological examination was performed after Papanicolaou staining. For molecular diagnosis, after centrifugation at 300 x g for 5 minutes, the cell pellet was dissolved with 1 mL of TRIzol reagent (Invitrogen, Carlsbad, CA). The mixture was stored at –80°C until RNA isolation. If at least one peritoneal lavage of the pouch of Douglas and the cavity under the left hemidiaphragm showed positive results, the patient was diagnosed as positive for cytology or molecular diagnosis.

Metastatic Lymph Nodes
Between June and August 2003, 18 metastatic lymph nodes were collected from 5 patients with gastric cancer. All patients underwent gastrectomy with lymphadenectomy at the Department of Surgery and Clinical Oncology, Osaka University. All specimens were collected after written, informed consent was obtained from the patients.

RNA Extraction
Total cellular RNA was extracted from cell pellets of peritoneal lavage fluid samples and cancer cell lines by using TRIzol reagent according to the protocol provided by the manufacturer. In brief, the mixture was minced with disposable homogenizers (IEDA, Tokyo, Japan), mixed with .2 mL of chloroform, and centrifuged at 12,000 x g for 15 minutes. The supernatant was transferred to a fresh tube and mixed with .5 mL of 100% isopropyl alcohol. After incubation for 10 minutes at room temperature, RNA was precipitated by centrifugation, washed with 75% ethanol, and diluted with diethyl pyrocarbonate—treated water.

RNA was extracted from metastatic lymph nodes with the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the protocol provided by the manufacturer, after homogenization with disposable homogenizers. The entire procedure takes approximately 40 minutes (Fig. 1A). The quality and quantity of isolated RNA was checked by ultraviolet spectrophotometer.



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 1. (A) The outline of transcription–reverse transcription concerted reaction (TRC) and reverse transcriptase (RT)-polymerase chain reaction (PCR) experiments. (B) Elementary steps of TRC reaction for detection of CEA messenger RNA (mRNA). The TRC reaction performs RNA sequence amplification isothermally in the presence of the carcinoembryonic antigen (CEA)-specific intercalation activating fluorescence (INAF) probe. The progress of the reaction is monitored by measuring the fluorescence intensity of the reaction mixture. cDNA, complementary DNA; AMV, avian myeloblastosis virus; dsDNA, double-stranded DNA.

 
Sequences of Primers and Probes for TRC
Synthetic oligonucleotide sequences of a pair of primers, a scissors probe for TRC amplification, and an intercalation activating fluorescence (INAF) probe for detection of CEA mRNA are listed in Table 1. Numbers in parentheses indicate the corresponding position of the target genome sequences (GenBank Accession No. M29540). Sequences of the promoter primers indicated by the letters in italics are the T7 RNA polymerase-binding sequences. The sequences of the primers, a scissors probe, and the INAF probe were designed to bind to the secondary-structure-free sites of CEA mRNA. The INAF probe is a DNA oligonucleotide linked with an intercalating fluorescence dye, oxazole yellow. The 3'-OH of the scissors probe and the INAF probe was capped with an amino group and glycolic acid, respectively, to avoid undesired enzymatic elongation by the avian myeloblastosis virus (AMV) RT reaction. Synthetic oligonucleotides of primers and a scissors probe were provided by Sawady Technology Co. (Tokyo, Japan). Synthesis of the INAF probe for CEA amplicons was performed as described previously.8


View this table:
[in this window]
[in a new window]
  		TABLE 1. Synthetic oligonucleotide sequences of a pair of primers, a scissors probe for amplification, and an INAF probe for detection of CEA mRNA in the TRC reaction
 
Preparation of Standard RNA for TRC Quantification
Standard RNA containing the full-length CEA mRNA was prepared by in vitro transcription of promoter-bearing double-stranded DNA as a template with T7 RNA polymerase. The DNA template for CEA was synthesized from RNA extracts of a gastric cancer cell line, MKN-45, by RT-PCR with a pair of synthetic oligonucleotide primers with sequences derived from CEA mRNA. The sense primer has the T7 RNA polymerase-binding sequences in its 5' end to provide the preferred transcription initiation site. The resultant RNA fragment (2312 nucleotides containing CEA mRNA) was purified by gel filtration with CHROMA-SPIN 30 columns (Clontech, Palo Alto, CA). The concentration of the RNA obtained was estimated by high-performance liquid chromatography (gel permeation chromatography; G4000SW column; Tosoh Corp., Tokyo, Japan). The calibrator set was prepared by diluting the transcribed RNA to the appropriate concentration with TE buffer.

TRC Reaction
The protocol of the TRC reaction was described previously8 (Fig. 1B). In brief, 20 µL of the TRC buffer was added to 5 µL of the RNA extract in a thin-walled PCR tube, followed by the addition of 5 µL of the enzyme mix. The tube containing the mixture was closed and set in a dedicated instrument, the TRC monitor, to measure the fluorescence intensity of the reaction mixture incubated at 44°C (excitation wavelength, 470 nm; emission wavelength, 520 nm). The TRC buffer contained 90 mM Tris-HCl (pH 8.6), 165 mM KCl, 26 mM MgCl2, 1.5 mM dithiothreitol, .38 mM deoxynucleoside triphosphate, 4.5 mM nucleoside triphosphate, 5.4 mM inosine triphosphate, .3 U/µL ribonuclease inhibitor, 1.5 µM forward primer, 1.5 µM reverse primer, .24 µM scissors probe, 23 nM INAF probe, and 16% dimethyl sulfoxide. The enzyme mixture consisted of .72 mg/mL bovine serum albumin, 12% sorbitol, 1.3 U/µL AMV RT (TaKaRa, Kyoto, Japan), and 34 U/µL T7 RNA polymerase (Life Technologies, Gaithersburg, MD).

Real-Time Monitoring of TRC Reaction
The experimental apparatus, the TRC monitor, was constructed on a round incubator block and rotating fluorescence scanning unit.8 The temperature of the incubator block was controlled at optimal TRC conditions (44°C), and 32 thin-walled PCR tubes were installed and set in a circle. These were assembled into one unit to enable synchronous scanning of the fluorescence while the tube was irradiated. The light-emitting diode turns like a beacon to irradiate the excitation light (470 nm) into a tube from outside. The fluorescence (520 nm) is transferred from the bottom of the tube to a photomultiplier through a light guide.

RT Reaction
Complementary DNA was generated with AMV RT (Promega, Madison, WI) by using the protocol recommended by the manufacturer. Briefly, 1 µg of RNA mixed with RT reaction reagents, including oligo-(dT)15 primer, was incubated at 42°C for 15 minutes, followed by heating at 95°C for 5 minutes for enzyme inactivation.

Real-Time Quantitative PCR With the LightCycler
Quantitative PCR was performed by using real-time PCR with a LightCycler (Idaho Technology, Salt Lake City, UT). PCR reagents contained 1x LightCycler DNA Master SYBR Green I (Roche Diagnostics, Mannheim, Germany), .2 µM of each primer, 3 mM MgCl2, and 2 µL of cDNA template. PCR conditions for CEA were as follows: one cycle of denaturing at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, 62°C for 5 seconds, and 72°C for 10 seconds. The intensity of fluorescence was calculated at each cycle, and the standard curve was constructed with 3-fold serial dilutions of cDNA obtained from cancer cell lines. The primer sequences for PCR amplification were as follows: CEA antisense, 5'-TGTAGCTGTTGCAAATGCTTTAAGGAAGAAGC-3'; and CEA sense, 5'-TCCTGGAACTCAAGCTCTTC-3'. The amplified product size was 160 base pairs for CEA mRNA.

Statistical Analysis
Spearman’s correlation coefficient by rank (Spearman’s rank correlation) was introduced to assess the significance of group associations. The Kaplan-Meier survival model was used to estimate overall and relapse-free survival. The log-rank test and generalized Wilcoxon’s test were used to determine statistical differences between groups. P < .05 was considered significant. The entire statistical analysis was performed with StatView software (SAS Institute Inc., Cary, NC).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Profile of CEA mRNA Amplification by TRC
The profile of TRC amplification with diluted standard CEA mRNAs is shown in Fig. 2A. The ordinate represents the ratio of fluorescence intensities, and the abscissa shows the reaction time. Production of CEA amplicons was detected from 102 copies of standard CEA mRNA in the tube. The reaction time to reach the cutoff value (1.2 times the starting fluorescence intensity) showed an excellent linear correlation with the logarithm of the starting copy numbers, and the standard curve could be constructed for quantification of a starting copy number in a sample (Fig. 2B).



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 2. Fluorescence monitoring of the carcinoembryonic antigen (CEA) messenger RNA (mRNA) calibrators with the transcription–reverse transcription concerted reaction (TRC) monitor. (A) The profile of TRC amplification with diluted standards of CEA mRNAs. Numbers on each curve indicate the starting copies of the calibrator RNA (0, 102, 103, 104, 105, and 107 copies). The ordinate represents fluorescence intensities, and the abscissa shows the reaction time. (B) The number of starting copies of CEA mRNA calibrators on a logarithmic scale is plotted versus the time needed for the fluorescence enhancement to reach a cutoff level of 1.2. CEA mRNA in a sample can be quantified on the basis of this linear relationship as the standard curve.

 
Comparative Analysis Between TRC and RT-PCR in Detecting Cancer Cells
Serially diluted MKN-45 cells (0, 1, 10, 102, and 103 cells) were mixed with normal leukocytes (103 cells) obtained from healthy volunteers. These specimens were subjected both to TRC with the TRC monitor and to RT-PCR with the LightCycler. Fluorescence monitoring of the TRC reaction is shown in Fig. 3A. As shown in Fig. 3B, one cancer cell can be detected by both methods, and quantitative results showed a significant association. Furthermore, we examined the reproducibility of TRC and RT-PCR experiments with 5 aliquots of MKN-45 cells (10 cells) mixed with leukocytes (103 cells).



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 3. (A) The profile of transcription–reverse transcription concerted reaction (TRC) amplification with MKN-45 cells (0, 1, 10, 102, and 103 cells) mixed with normal leukocytes (103 white blood cells; WBC) and calibrators (103 and 107 copies). The quantitative values from TRC and reverse transcriptase-polymerase chain reaction are plotted versus the number of MKN-45 cells and their correlation. (B) These specimens were subjected to both TRC and RT-PCR with the LightCycler.

 
The index conversion value of the number of CEA mRNA copies was 7.4% in TRC and 9.4% in RT-PCR (data not shown). The TRC reaction was equivalent to RT-PCR in terms of reproducibility. These results showed that the sensitivity, quantification, and reproducibility were almost equal between the TRC and RT-PCR methods. The entire reaction from tissue sampling to diagnosis can be shortened to approximately 1 hour with TRC, instead of the 3 hours required with the LightCycler.

Quantitative Analysis With TRC and RT-PCR by Using Dissected Metastatic Lymph Nodes
Metastatic lymph nodes obtained from gastric cancer surgery were subjected to both TRC and RT-PCR analysis. As shown in Fig. 4, the quantitative values of TRC correlated significantly with those of RT-PCR of all histopathologically confirmed metastatic lymph nodes that were scored positive for metastasis.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 4. Quantitative analysis with transcription–reverse transcription concerted reaction (TRC) and reverse transcriptase-polymerase chain reaction by using dissected metastatic lymph nodes.

 
Correlation Between TRC-Based Diagnosis of Peritoneal Lavage Fluid Samples and Clinicopathologic Characteristics of Patients With Gastric Cancer
The clinicopathologic findings of the 60 participating patients are listed in Table 2. The male/female ratio was 46:14, and age was 66.8 ± 9.3 years (mean ± SD; range, 44–85 years). Molecular diagnosis with TRC reaction correlated significantly with depth of invasion (T), peritoneal metastasis (P), clinical stage, and cytology, and all patients with positive cytology or with simultaneous peritoneal metastasis scored positive for metastasis by TRC (Table 2). However, all 23 peritoneal lavage fluid samples from 15 patients who had laparotomy because of benign disease were negative for CEA by TRC.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Comparison of clinicopathologic characteristics of patients with gastric cancer based on TRC diagnosis of peritoneal lavage fluid samples
 
Comparison Between TRC Diagnosis of Peritoneal Lavage Fluid and Prognosis
Examination of all 60 patients with advanced gastric cancer showed that patients with TRC-positive lavages had a significantly worse prognosis than TRC-negative cases (Fig. 5A). The peritoneal recurrence-free survival curves and overall survival curves of 51 cured patients (9 patients who had simultaneous peritoneal dissemination at surgery or staging laparoscopy were excluded) are shown in Fig. 5B and 5C, respectively. Even in cured cases, TRC-positive patients showed a poorer prognosis in peritoneal recurrence-free survival or overall survival than TRC-positive patients. These results suggest that molecular diagnosis of peritoneal lavage fluid by TRC could be a useful prognostic indicator for peritoneal recurrence and survival of patients with gastric cancer.



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 5. Survival analysis based on transcription–reverse transcription concerted reaction (TRC) diagnosis of peritoneal lavage fluid. (A) Overall survival of all 60 patients. (B) Peritoneal recurrence-free survival of 51 curative patients (9 patients who had simultaneous peritoneal dissemination at surgery or staging laparoscopy were excluded). (C) Overall survival of the 51 cured patients.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we developed a novel diagnostic technique for detecting micrometastasis in cancer patients by using a TRC amplification system, which enabled us to detect cancer-specific mRNA sequences in a completely homogeneous way and to perform rapid quantitative detection of cancer micrometastasis with high sensitivity. This method does not require heat-denaturing annealing steps or postamplification analysis. The method is based on performing isothermal RNA sequence amplification, a TRC reaction, in the presence of the INAF DNA probe and measuring the fluorescence intensity of the reaction mixture during the reaction. The INAF DNA probe binds to specific sequences of produced RNA amplicons and enhances the fluorescence. The progress of producing amplicons can be monitored in real time by measuring the fluorescence intensity of the reaction mixture in a closed vessel.

Micrometastasis, defined as invisible metastasis missed by conventional microscopic examination, is considered to be of great clinical significance in the field of oncology. The 6th edition of the International Union Against Cancer tumor-node-metastasis classification of malignancies introduced the new concept of micrometastasis detected by molecular analysis and immunohistochemistry.21 For molecular diagnosis, PCR is a powerful and commonly used tool for detecting cancer-related sequences with high sensitivity, and it has been widely studied for clinical application in various types of malignancies. However, PCR-based methods have several engineering obstacles that must be overcome before they are applied for high-throughput automation in the clinical setting. PCR-based detection of cancer-specific sequences requires an RT step after RNA extraction. Furthermore, PCR requires more than 30 cycles of 3 steps with different incubation temperatures. Therefore, the method is time consuming. The LightCycler format can quantify cancer-specific mRNAs by using real-time monitoring of PCR products, and the results are more reproducible and reliable. We previously developed a rapid RT-PCR–based diagnostic technique for cancer micrometastasis with the LightCycler, which facilitated a rapid (intraoperative) assay for detection of lymph node micrometastasis, and we applied this method to esophageal cancer surgery.22 The entire procedure takes 3 hours from the tissue sampling to completion of the test, and this duration limits the introduction of our method to long operations such as esophageal cancer surgery. Further developments in molecular biology should shorten the assay time, thus allowing the PCR assay to be widely used during cancer surgery.

In this study, we showed that the TRC technique is superior to the RT-PCR format for detecting RNA sequences because of its rapidity and simplicity. The entire reaction from tissue sampling to diagnosis can be shortened to approximately 1 hour with TRC, instead of the 3 hours required with the LightCycler.

With this system, intraoperative genetic diagnosis can be performed in short operations such as gastric surgery. Recently, we showed that intraperitoneal chemotherapy could improve the prognosis of gastric cancer patients with peritoneal lavage fluid diagnosed as micrometastasis-positive by RT-PCR.23 Therefore, the intraoperative TRC diagnosis of peritoneal lavage fluid might be able to select patients who should have intraperitoneal chemotherapy at the time of surgery. Further clinical studies are needed to address the significance of intraoperative TRC diagnosis.

A recent advance in surgery has been the introduction of sentinel node navigation surgery to determine the extent of surgery—primarily the need for lymphadenectomy. The sentinel node concept was introduced for melanoma and breast cancers and has since been assessed in other tumors, including gastrointestinal cancers.24–26 However, the clinical application of this technique requires accurate intraoperative detection of metastasis to lymph nodes. Our genetic diagnostic system may enhance the spread of sentinel node navigation surgery and provide cancer patients with more effective surgical treatment in the future.


   ACKNOWLEDGMENTS
 
Supported by a grant-in-aid from the Ministry of Education, Culture, and Science of Japan.

The acknowledgments are available online in the fulltext version at www.annalssurgicaloncology.org. They are not available in the PDF version.


   FOOTNOTES
 
A novel molecular diagnosis with transcription–reverse transcription concerted reaction was introduced for detection of cancer micrometastasis and could be a prognostic indicator in gastric cancer patients.

Received for publication December 29, 2003. Accepted for publication May 4, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Raj GV, Moreno JG, Gomella LG. Utilization of polymerase chain reaction technology in the detection of solid tumors. Cancer 1998; 82: 1419–42.[CrossRef][Medline]
  2. Datta YH, Adams PT, Drobyski WR, Ethier SP, Terry VH, Roth MS. Sensitive detection of occult breast cancer by the reverse-transcriptase polymerase chain reaction. J Clin Oncol 1994; 12: 475–82.[Abstract]
  3. Mori M, Mimori K, Inoue H, Barnard GF, Tsuji K, Nanbara S. Detection of cancer micrometastases in lymph nodes by reverse transcriptase-polymerase chain reaction. Cancer Res 1995; 55: 3417–20.[Abstract/Free Full Text]
  4. Noguchi S, Aihara T, Motomura K, Inaji H, Imaoka S, Koyama H. Detection of breast cancer micrometastasis in axillary lymph nodes by means of reverse-transcriptase polymerase chain reaction. Am J Pathol 1996; 148: 649–56.[Abstract]
  5. Hayashi N, Ito I, Yanagisawa A, et al. Genetic diagnosis of lymph-node metastasis in colorectal cancer. Lancet 1995; 345: 1257–9.[CrossRef][Medline]
  6. Liefers GJ, Jansen AMC, Velde CJH, et al. Micrometastasis and survival in stage II colorectal cancer. N Engl J Med 1998; 339: 223–8.[Abstract/Free Full Text]
  7. Miyake Y, Fujiwara Y, Ohue M, et al. Quantification of micrometastasis in lymph nodes of colorectal cancer using real-time fluorescence polymerase chain reaction. Int J Oncol 2000; 16: 289–93.[Medline]
  8. Ishiguro T, Saitoh J, Horie R, et al. Intercalation activating fluorescence DNA probe and its application to homogeneous quantification of a target sequence by isothermal sequence amplification in a closed vessel. Anal Biochem 2003; 314: 77–86.[CrossRef][Medline]
  9. Baba H, Korenaga D, Okamura T, Saito A, Sugimachi K. Prognostic factors in gastric cancer with serosal invasion. Arch Surg 1989; 124: 1061–4.[Abstract]
  10. Hiratsuka M, Iwanaga T, Furukawa H, et al. Important prognostic factors in surgically treated gastric cancer patients. Gan To Kagaku Ryoho 1995; 22: 703–8.[Medline]
  11. Yoo CH, Noh SH, Shin DW, Choi SH, Min JS. Recurrence following curative resection for gastric carcinoma. Br J Surg 2000; 87: 236–42.[CrossRef][Medline]
  12. Koga S, Kaibara N, Iitsuka Y, Kudo H, Kimura A, Hiraoka H. Prognostic significance of intraperitoneal free cancer cells in gastric patients. J Cancer Res Clin Oncol 1984; 108: 236–8.[CrossRef][Medline]
  13. Abe S, Yoshimura H, Tabara H, et al. Curative resection of gastric cancer: limitation of peritoneal lavage cytology in predicting the outcome. J Surg Oncol 1995; 59: 226–9.[Medline]
  14. Ribeiro U Jr, Gama-Rodrigues JJ, Safatle-Ribeiro AV, et al. Prognostic significance of intraperitoneal free cancer cells obtained by laparoscopic peritoneal lavage in patients with gastric cancer. J Gastrointest Surg 1998; 2: 244–9.[CrossRef][Medline]
  15. Bando E, Yonemura Y, Takeshita Y, et al. Intraoperative lavage for cytological examination in 1,297 patients with gastric carcinoma. Am J Surg 1999; 178: 256–62.[CrossRef][Medline]
  16. Nakanishi H, Kodera Y, Yamamura Y, et al. Rapid quantitative detection of carcinoembryonic antigen-expressing free tumor cells in the peritoneal cavity of gastric-cancer patients with real-time RT-PCR on the LightCycler. Int J Cancer 2000; 89: 411–7.[CrossRef][Medline]
  17. Nakanishi H, Kodera Y, Torii A. Detection of carcinoembryonic antigen-expressing free tumor cells in peritoneal washes from patients with gastric carcinoma by polymerase chain reaction. Jpn J Cancer Res 1997; 88: 687–92.[Medline]
  18. Kodera Y, Nakanishi H, Yamamura Y, et al. Prognostic value and clinical implications of disseminated cancer cells in the peritoneal cavity detected by reverse transcriptase-polymerase chain reaction and cytology. Int J Cancer 1998; 79: 429–33.[CrossRef][Medline]
  19. Kodera Y, Nakanishi H, Ito S, et al. Quantitative detection of disseminated free cancer cells in peritoneal washes with real-time reverse transcriptase-polymerase chain reaction: a sensitive predictor of outcome for patients with gastric carcinoma. Ann Surg 2002; 235: 499–506.[CrossRef][Medline]
  20. Ueno H, Yoshida K, Hirai T, Kono F, Kambe M, Toge T. Quantitative detection of carcinoembryonic antigen messenger RNA in the peritoneal cavity of gastric cancer patients by real-time quantitative reverse transcription polymerase chain reaction. Anticancer Res 2003; 23: 1701–8.[Medline]
  21. Sobin LH. TNM, sixth edition: new developments in general concepts and rules. Semin Surg Oncol 2003; 21: 19–22.[CrossRef][Medline]
  22. Yoshioka S, Fujiwara Y, Sugita Y, et al. Real-time rapid reverse transcriptase-polymerase chain reaction for intraoperative diagnosis of lymph node micrometastasis: clinical application for cervical lymph node dissection in esophageal cancers. Surgery 2002; 132: 34–40.[CrossRef][Medline]
  23. Mori T, Fujiwara Y, Sugita Y, et al. Application of molecular diagnosis for detection of peritoneal micrometastasis and evaluation of preoperative chemotherapy in advanced gastric carcinoma. Ann Surg Oncol 2004; 11: 14–20.[Abstract/Free Full Text]
  24. Morton DL, Wen DR, Wong JH, et al. Technical details of intraoperative lymphatic mapping for early stage melanoma. Arch Surg 1992; 127: 392–9.[Abstract]
  25. Giuliano AE, Kirgan DM, Guenther JM, Morton DL. Lymphatic mapping and sentinel lymphadenectomy for breast cancer. Ann Surg 1994; 220: 391–401.[Medline]
  26. Kitagawa Y, Fujii H, Mukai M, et al. The role of the sentinel lymph node in gastrointestinal cancer. Surg Clin North Am 2000; 80: 1799–809.[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ishii, T.
Right arrow Articles by Monden, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ishii, T.
Right arrow Articles by Monden, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS