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Originally published as Ann Surg Oncol Early Release 10.1245/ASO.2004.02.016 on December 8, 2003

Annals of Surgical Oncology 11:14-20 (2004)
© 2004 Society of Surgical Oncology
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ORIGINAL ARTICLES

Application of Molecular Diagnosis for Detection of Peritoneal Micrometastasis and Evaluation of Preoperative Chemotherapy in Advanced Gastric Carcinoma

Takuji Mori, MD, Yoshiyuki Fujiwara, MD, PhD, Yurika Sugita, BA, Takashi Azama, MD, Takaaki Ishii, MD, Kazuhiro Taniguchi, MD, Keiji Yamazaki, MD, PhD, Shuji Takiguchi, MD, Takushi Yasuda, MD, PhD, Masahiko Yano, MD, PhD and Morito Monden, MD, PhD

From the Department of Surgery and Clinical Oncology, Graduate School of Medicine (TM, YF, YS, TA, TI, KT, ST, TY, MY, MM), Osaka University; and Department of Surgery, Shinsenri Hospital (KY), Osaka, 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: In advanced gastric cancer, peritoneal recurrence is the main cause of death after curative surgical resection. The aim of this report was to describe a novel approach for quantitative genetic diagnosis using peritoneal lavage for the identification of patients at high risk for peritoneal recurrence and for evaluation of the clinical response to intraperitoneal chemotherapy in advanced gastric cancer.

Methods: Nineteen patients with advanced gastric cancer who underwent staging laparoscopy and intraperitoneal chemotherapy before surgical resection or systemic chemotherapy between June 1999 and September 2001 were enrolled in this study. All peritoneal lavage specimens, collected at both staging laparoscopy and gastrectomy, were subjected to real-time quantitative genetic diagnosis.

Results: The reverse transcriptase polymerase chain reaction (RT-PCR) values decreased in 8 cases, stabilized as negative in 5, and increased in 6 during therapy. Patients whose RT-PCR values diminished and were ultimately negative survived except for one, and all but one patient whose values increased during treatment died of recurrence.

Conclusions: Quantitative evaluation of genetic changes can provide accurate, useful information on the effects of preoperative intra-abdominal chemotherapy and overall prognosis for patients with advanced gastric cancer.

Key Words: Gastric cancer • Molecular diagnosis • Peritoneal dissemination • Staging laparoscopy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recently, the prognosis for patients with gastric cancer has greatly improved, mainly because of increased detection of early gastric cancer due to advances in diagnostic methods. However, the prognosis of advanced gastric cancer, especially serosa-invading tumors, remains poor. In these advanced cases, peritoneal dissemination is the most common type of recurrence after surgery.1,2 Therefore, a new therapeutic strategy for these advanced gastric cancers is urgently needed.

To prevent peritoneal recurrence after surgery, early selection of high-risk patients for recurrence is very important. Preoperative staging laparoscopy is considered a necessity for detection of macroscopic dissemination and microscopic detection of free cancer cells in the abdominal cavity. However, peritoneal recurrence frequently occurs postoperatively, even with negative cytological findings. Therefore, the sensitivity of cytology and macroscopic observation is unsatisfactory for identification of high-risk patients, and new techniques are urgently needed.3,4 Furthermore, efficient preoperative therapy for these high-risk patients also is needed. We applied intraoperative chemotherapy, using two kinds of antitumor agents, for advanced gastric cancer with serosa-invading tumor.5–8

Recent advances in the understanding of the molecular biology of cancer have provided new methods for detection of cancer micrometastasis that are more sensitive than conventional cytological examination.9,10 Reverse transcriptase polymerase chain reaction (RT-PCR) analyses with cancer-specific RNA markers have been widely studied for the detection of cancer micrometastasis.11–13 However, because the conventional PCR amplification technique lacks reproducibility despite its high sensitivity, the reliability of the method must be improved for it to be clinically applicable. In this study, a real-time monitoring thermal cycler (LightCycler, Idaho Technology, Salt Lake City, UT) was used for the quantitative detection of cancer cells in peritoneal washes from patients with gastric cancer.

In this study, we used the above system to identify patients with gastric cancer who had a high risk for postoperative peritoneal recurrence and assessed the utility of these quantitative analyses of free cancer cells in peritoneal lavages for evaluation of intraperitoneal chemotherapy performed before surgery.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Studies
Animal experimental procedures
We recently established a peritoneal metastasis model in nude mice, using the gastric cancer cell MKN-45, which had been transduced with an enhanced green fluorescent protein (EGFP)–expressing plasmid vector.14 This model facilitates evaluation of the status of peritoneal seeding in an accurate way because of the sensitive detection of micrometastasis in the peritoneal cavity of mice. MKN-45-EGFP (1 x 106/mice) was injected intraperitoneally into 4-week-old female BALBc nu/nu mice (Japan Clea, Tokyo) on day 0. Fourteen mice were allocated to each group. They were killed 14 or 21 days after tumor cell injection. The status of peritoneal seeding was assessed by the number of fluorescent microfoci under fluorescent stereomicroscopy. Peritoneal lavage specimens were collected after washing with 5 mL of saline, and the amount of carcinoembryonic antigen (CEA) mRNA was measured with the LightCycler. The relationship between the number of fluorescent foci and the quantitative genetic analysis in peritoneal lavages was assessed. Prior to this experiment, we confirmed that PCR primers for human CEA mRNA did not amplify any products from peritoneal washes from mice.

Human Studies
Patient eligibility
Between June 1999 and September 2001, 19 patients with histologically confirmed adenocarcinoma of the stomach were enrolled in this study. The tumor stages were diagnosed as T3 (serosa penetration) or T4 (invasion of adjacent organs) at preoperative staging laparoscopy, according to the Japanese Classification of Gastric Carcinoma.15 (There are no differences between the Japanese Classification of Gastric Cancer and the International Union Against Cancer [UICC] T3 and T4 stages of gastric cancer.)

The follow-up period ranged from 8 months to 39 months (mean ± SD, 16.00 ± 8.98).

Study Design
Staging laparoscopy was initially performed in all cases with use of general anesthesia. After laparoscopic intraperitoneal inspection, peritoneal lavage specimens were collected from the pouch of Douglas and left subphrenic cavity. After the staging laparoscopy, patients were allocated to either the intraperitoneal chemotherapy group (cytologically negative cases) or the sequential chemotherapy group, involving both intraperitoneal and systemic chemotherapy (cytologically positive cases and cases with macroscopic peritoneal dissemination).

At the second surgery (gastrectomy or staging laparoscopy) after the chemotherapy, peritoneal lavage specimens were also collected (Fig. 1) and were subjected to quantitative genetic diagnosis with the LightCycler, with use of CEA as the target gene.



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  		FIG. 1. Schematic representation of study design. P(0), no evidence of peritoneal dissemination; P(1), peritoneal dissemination; CY(0), cytologically negative; CY(1), cytologically positive.

 
Intraperitoneal and Systemic Chemotherapy
The protocol for intraperitoneal chemotherapy was as follows: mitomycin (MMC, 20 mg on day 1) and cisplatin (CDDP, 20 mg on days 1–5) in 1000 mL of saline were administered into the abdominal cavity through a drainage tube held at the staging laparoscopy. The catheter was clamped for 2 hours and the intraperitoneal solution was drained through the catheter (Fig. 2).



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  		FIG. 2. Intraperitoneal chemotherapy regimen and position of drainage tube. The protocol for intraperitoneal chemotherapy was as follows: MMC (Mitomycin C, 20 mg on day 1) and CDDP (cisplatin, 20 mg on days 1–5) were administered into the abdominal cavity through the drainage tube introduced at staging laparoscopy.

 
The protocol for systemic chemotherapy was as follows: docetaxel (60 mg/m2 on day 1), CDDP (10 mg/m2 on days 1–5), and 5-fluorouracil (5-FU, 350 mg/m2 on days 1–5) were combined for patients with positive cytologic results and/or with macroscopic peritoneal dissemination. One course consisted of 4 weeks of therapy.

Surgical Specimens
Peritoneal lavages were collected from patients undergoing staging laparoscopy or gastrectomy at Osaka University and Shinsenri Hospital between September 1999 and August 2001. The pouch of Douglas and the left subphrenic cavity were washed with 100 mL of saline, and 50 mL of lavage fluid was collected from each site. Half of the specimen was centrifuged at 1500 rpm for 15 minutes, and the pellet was suspended in 0.5 mL of reagent (TRIzol, Molecular Research Center, Cincinnati, OH) and transferred to a 1.5-mL tube. The mixture was stored at -80°C until RNA extraction. The remaining half was subjected to cytological examination after Papanicolaou staining.

RNA Extraction
Total cellular RNA was extracted from cell pellets of peritoneal lavage specimens with use of TRIzol reagent, according to the protocol supplied by the manufacturer. RNA extraction was carried out with TRIzol reagent in a single-step method, as described previously.16 The mixture was minced and homogenized with a disposable homogenizer (IEDA; Tokyo, Japan) at room temperature, mixed with 0.2 mL of chloroform, and centrifuged at 12,000 rpm for 10 minutes. The supernatant was then transferred to a fresh tube and 0.5 mL of 100% 2-propanol was added. RNA was precipitated after centrifugation at 12,000 rpm, washed with 75% ethanol, and diluted in diethyl pyrocarbonate–treated water. Purified RNA was quantified and assessed for purity with ultraviolet spectrophotometry.

Reverse Transcription
Complementary DNA (cDNA) was generated with avian myeloblastosis virus reverse transcriptase (RT, Promega, Madison, WI), according to the protocol recommended by the manufacturer. In brief, 1 µg of RNA was incubated at 70°C for 5 minutes and then placed on ice before the addition of RT reaction reagents with oligo-(dT)15 primer. The RT reaction was performed at 42°C for 90 minutes, followed by heating at 95°C for 5 minutes.

Serial Dilutions as a Standard
The cancer cell line MKN-45, which highly expressed the target gene, was obtained from the Japanese cell bank, was cultured according to the instructions provided by the manufacturer, and was subjected to RNA isolation. For constructing a standard curve for CEA, cDNA was synthesized from 1 µg of RNA extracted from 102–106 cells of MKN-45.

Real-Time PCR
A real-time monitoring thermal cycler, LightCycler, and detection system (Roche Diagnostics, Mannheim, Germany) were used for amplification and online quantification, respectively. PCR was performed in glass capillaries, which ensured rapid equilibration between the air and the reaction components because of the high surface-to-volume ratio of the capillaries. For detection of the CEA amplification product, LightCycler-DNA Master SYBR Green I (Boehringer Mannheim, Mannheim, Germany) was used. Real-time PCR was performed in a 20-µL PCR reaction containing 0.2 µM of each primer, 1x LC-DNA Master SYBR Green, 4 mM of MgCl2, and 2 µL of cDNA as a template. Forward and reverse primers for CEA were 5'-TCTGGAACTTCTCCTGGTCTCTCAGCTGG and 5'-TGTAGCTGTTGCAAATGCTTTAAGGAAGAAGC, respectively, and the estimated product size was 160 bp. The PCR conditions were set up as follows: one cycle of denaturing at 95°C for 2 minutes, followed by 45 cycles of 95°C for 15 seconds, 62°C for 5 seconds, and 72°C for 18 seconds. Fluorescence was acquired at the end of every 72°C extension phase.

Detection and Quantification Analysis with the LightCycler
Quantification data were analyzed with LightCycler analysis software (Roche Diagnostics), as described by the manufacturer. In this analysis, the background fluorescence was removed by setting a noise band. The log-linear portion of the standard amplification curve was identified, and the crossing point was the intersection of the best-fit line through the log-linear region and the noise band. The standard curve was a plot of the crossing points versus the amount of initial total RNA extracted from the MKN-45 cell line.17

Statistical Analysis
Spearman’s correlation coefficient by rank (Spearman’s rank correlation) was introduced to assess the significance of group associations. Values of P < .05 were considered to be significant.

Ethical Considerations
Both the human and animal study protocols were approved by the Human and Animal Ethics Review Committees of Osaka University School of Medicine. A signed consent form was obtained from each subject participating in the study.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Animal Studies
Relationships between number of fluorescent foci in abdominal cavity and CEA mRNA in peritoneal lavage fluid
There was a positive correlation between the number of fluorescent foci in the abdominal cavity and the quantitative molecular values of CEA mRNA (r = .05, P = .0012; Fig. 3).



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  		FIG. 3. Relationship between the number of fluorescent foci in the abdominal cavity and carcinoembryonic antigen (CEA) mRNA in peritoneal lavage in the nude mice model. There was a positive correlation between the number of fluorescent foci in the abdominal cavity and the molecular quantification values (r = .05, P = .0012).

 
Human Studies
Patient characteristics
Characteristics of the patients are listed in Table 1. There were 12 men and 7 women, and the mean (± SD) age was 57.9 (± 9.6) years (range, 34–70). Type 3 and 4 tumors accounted for more than 90% of the cases. Histopathologically, undifferentiated tumors (poorly differentiated adenocarcinoma, signet-ring cell carcinoma, mucinous adenocarcinoma) were predominant (14 of 19 cases; 73%). At the staging laparoscopy, the clinical staging revealed 5 patients in stage IIIA, 3 in stage IIIB, and 11 in stage IV disease, according to the Japanese Classification of Gastric Cancer.


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  		TABLE 1. Clinicopathological characteristics of patients
 
Change of quantitative genetic values
The results of quantitative analysis of peritoneal lavage specimens were compared for 19 patients who underwent chemotherapy. Over the course of treatment, including intraperitoneal chemotherapy, the quantitative molecular (PCR) values changed from positive to negative in 6 cases, stabilized as negative throughout the treatment in 5 cases, and were positive both before and after chemotherapy in 8 cases.

Survival and outcomes
Differences in genetic diagnosis and clinical outcomes are illustrated in Figs. 4 and 5Go. Over the course of intraperitoneal chemotherapy, the quantitative molecular values of 11 patients changed from positive to negative or stabilized as negative throughout treatment (Fig. 4). All but one of these patients survived, and that patient died of peritoneal recurrence. On the other hand, the PCR values of eight patients did not show any transformation to negative in spite of chemotherapy, and all died of recurrence except for one patient, who survived without any recurrence (Fig. 4). Overall, 10 (53%) of the 19 patients had recurrence after treatment.



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  		FIG. 4. Results of quantitative genetic diagnosis of peritoneal lavages and clinical outcomes. Eleven patients changed from positive to negative or stabilized as negative throughout treatment (POM, postoperative periods in months; Pre and Post, genetic quantification of peritoneal lavages before and after chemotherapy; NED, no evidence of disease; AWD, alive with disease; P, peritoneal recurrence; Ly, lymph node metastasis; CY(0), cytologically negative.

 


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  		FIG. 5. Results of quantitative genetic diagnosis of peritoneal lavages and clinical outcomes. Eight patients did not show any transformation to negative (POM, postoperative periods in months; Pre and Post, genetic quantification of peritoneal lavages before and after chemotherapy; NED, no evidence of disease; AWD, alive with disease; P, peritoneal recurrence; Ly, lymph node metastasis; B, bone metastasis; CY(0), cytologically negative; CY(1), cytologically positive; 7,8, RT-PCR analysis before chemotherapy was not done.

 
Pattern of recurrence and causes of death
We also assessed the recurrence types in all patients enrolled in this study. Ten (53%) of 19 patients had recurrence after treatment. Of these 10 patients, 7 developed peritoneal recurrence, 2 had local recurrence, and 1 had bone metastasis with peritoneal metastasis. Eight patients died of recurrence and two patients are still alive with peritoneal recurrence. Nine patients (47%) are alive and have no signs of recurrence.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In advanced gastric cancer, peritoneal dissemination is the main cause of death after surgical resection (the most frequent type of recurrence after curative surgery).18,19 The prognosis for patients with gastric cancer with peritoneal dissemination is extremely poor.

It is generally accepted that peritoneal dissemination originates from peritoneal cancer cells that have exfoliated from the serosa of primary tumors. Despite the increasing number of reports on detection of free cancer cells in peritoneal lavage specimens with use of RT-PCR analysis,20–22 molecular diagnosis with peritoneal lavage specimens has not been applied as a clinical strategy in cancer management. The aim of our study was to develop quantitative genetic diagnosis and to apply it for the identification of patients at high risk for peritoneal dissemination and for the evaluation of efficacy of chemotherapy.

First, we examined the correlation between the CEA mRNA values in peritoneal lavage specimens and the quantity of disseminated tumor in the peritoneal cavity. Recently, we established a peritoneal dissemination model with nude mice, using MKN-45 cells transduced with an EGFP-expressing plasmid vector.14 Using this model, we could detect cancer cells that were invisible macroscopically. We used this model for accurate assessment of peritoneal dissemination. Using real-time PCR, we also calculated the relative number of CEA-expressing cells in peritoneal lavage specimens, in comparison with the CEA-expressing MKN-45 cell line as a standard control. We analyzed the relationship between CEA mRNA values in peritoneal lavage specimens and the number of fluorescent foci and confirmed a significant association between quantitative molecular values of peritoneal washes and the number of fluorescent foci (P < .05; Fig. 2). These results showed that quantitative genetic analysis of peritoneal lavage specimens with the real-time PCR format could reflect the amount of tumor in the abdominal cavity and potentially could assess the effect of antitumor drugs on peritoneal dissemination, which could not be evaluated by conventional methods. Therefore, we applied quantitative molecular diagnosis of peritoneal lavage specimens in combination with staging laparoscopy as a therapeutic strategy for advanced gastric cancer.

In our clinical study, 8 cases showed reduction of CEA RT-PCR values, 5 cases showed stabilization of the negative values, and 6 cases showed increased values throughout the treatment. Patients whose RT-PCR values decreased and finally became negative (under the range of detection) survived except for one, whereas patients whose values increased died after recurrences, except for one patient who survived for 22 months. Accordingly, the serial quantitative genetic analysis of peritoneal lavage specimens has possibilities as a useful diagnostic tool for evaluation of therapeutic efficacy and clinical outcomes. Moreover, our results indicated that a diagnosis of cytologically negative (CY0) and genetically positive (RT-PCR+) was a good indication for intra-abdominal chemotherapy, and when the RT-PCR values of peritoneal lavage specimens changed from positive to negative with treatment, the prognosis might improve. In contrast, a diagnosis of cytologically positive (CY1) and molecularly positive (RT-PCR+) rarely reduced RT-PCR values, despite intra-abdominal and systemic chemotherapy, and thus a poor prognosis would be expected. In addition, future patients similar to the five negative stabilizers who were negative at the beginning and end of the study should not necessarily be treated with peritoneal chemotherapy, even in the presence of T3,T4 gastric cancer, because no patients who were originally negative became positive.

To date, most chemotherapy regimens have failed to improve the survival of patients with peritoneal dissemination.23–30 This may be partly because of the difficulty of early detection of peritoneal dissemination and of the evaluation of therapeutic effect in cases of peritoneal dissemination. If precise evaluation of chemotherapeutic efficacy on peritoneal dissemination were possible, it would then be possible to determine whether chemotherapy should be continued or whether another treatment for peritoneal dissemination should be offered. For these reasons, a new diagnostic modality for early identification of high-risk patients and for evaluation of chemotherapy is needed. Yano et al.31 reported that staging laparoscopy was necessary to determine the therapeutic strategy for patients with advanced gastric cancer. The combination of molecular diagnosis of peritoneal lavage specimens and staging laparoscopy would permit surgical oncologists to accurately stage the disease and to select appropriate treatments on the basis of evidence.

In conclusion, this molecular genetic quantitative system using peritoneal lavage fluid samples enabled us to detect free cancer cells in the peritoneal cavity with higher sensitivity and to identify patients at high risk for peritoneal recurrence. Furthermore, the therapeutic efficacy of chemotherapy for these patients can be predicted with serial quantitative genetic analyses. We recommend that serial molecular quantification of peritoneal lavage specimens should be performed for patients undergoing staging laparoscopy for advanced gastric cancer to enable identification of those who require preoperative chemotherapy.


   ACKNOWLEDGMENTS
 
The acknowledgments are available online in the full-text version at www.annalssurgicaloncology.org. They are not available in the PDF version.

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


   FOOTNOTES
 
Quantitative evaluation of genetic changes in peritoneal lavage fluid can provide accurate, useful information on the effects of preoperative intra-abdominal chemotherapy and overall prognosis for patients with advanced gastric cancer.

Received for publication February 21, 2003. Accepted for publication September 17, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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