© 2004 Foundation for Promotion of Cancer Research
Detection of Disseminated Cancer Cells in Linitis Plastica-type Gastric Carcinoma
1 Department of Surgery II, Nagoya University Graduate School of Medicine, 2 Department of Gastroenterological Surgery, Aichi Cancer Center Hospital and 3 Laboratory of Pathology, Aichi Cancer Center Research Institute, Nagoya, Japan
For reprints and all correspondence: Yasuhiro Kodera, Department of Surgery II, Nagoya University Graduate School of Medicine, 65, Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail: ykodera{at}med.nagoya-u.ac.jp
Received October 13, 2003; accepted July 5, 2004
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Abstract |
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 TOP Abstract INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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Background: Dissemination of cancer cells in the abdominal cavity may lead to peritoneal carcinomatosis, a common event in the linitis plastica type of gastric carcinoma. Reverse transcriptase polymerase chain reaction (RT-PCR) is a sensitive technique for detecting these cells.
Methods: Peritoneal washings were obtained during laparotomy from 47 consecutive linitis plastica patients who preoperatively were considered candidates for curative resection. Together with conventional cytological examination using Papanicolaou staining, real-time RT-PCR was performed to quantitate prospectively carcinoembryonic antigen (CEA) mRNA in these washings. Samples above a cutoff value for CEA mRNA were considered positive for molecular detection of disseminated cancer cells.
Results: Conventional cytological examination was positive in 43% (20/47). Positivity of CEA mRNA was much higher at 83% (39/47) and peritoneal carcinomatosis actually was observed in 77% (36/47), either at laparotomy or during postoperative follow-up. Although only two of eight patients who were negative for CEA mRNA suffered from peritoneal carcinomatosis, three more patients died of recurrences through other metastatic pathways. Multivariate analysis revealed that curability of the operation (knowledge of the CEA mRNA values excluded) and T categories were the only significant independent prognostic factors.
Conclusions: RT-PCR of the peritoneal washes had little value as a prognostic factor, but identified over 80% of the patients planned for curative surgery to have disseminated cancer cells in the peritoneal cavity. Almost the same proportion of patients actually suffered from peritoneal carcinomatosis. These facts indicate why treatment with surgery alone rarely cures patients with linitis plastica.
Key Words: carcinoembryonic antigen • reverse transcriptase polymerase chain reaction • peritoneal carcinomatosis • metastasis • cytology • stomach
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INTRODUCTION |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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Linitis plastica, a scirrhous carcinoma most commonly of gastric origin, is characterized by diffuse infiltration resulting in thickening and stiffening of the gastric wall. Despite improved treatment outcome for other types of gastric carcinoma in Japan, the prognosis in linitis plastica remains extremely poor (1). In this disease, death often results from peritoneal carcinomatosis, a consequence of dissemination of free cancer cells from the primary lesion. Gross findings indicating peritoneal seeding sometimes are evident at laparotomy. Even when this is not apparent, patients with this disease remain at high risk for peritoneal carcinomatosis (2,3). Since linitis plastica often involves the entire stomach, total gastrectomy is usually performed. Indications for radical resection therefore should be carefully weighed against potential complications of major surgery. Ideally, such resection should be performed only for patients with a chance of cure.
Cytological examination of peritoneal washings has been used to evaluate the risk of intraperitoneal recurrence in curatively resected cases of gastric carcinoma (1,4,5). This assessment recently has been added to the Japanese staging classification as a CY category. CY1 (i.e. positive cytological examination) immediately places the patient in Stage IV (6). Its prognostic value has also been reported in Western countries (7,8). Conventional examination of washings with Papanicolaou staining, however, may lack sensitivity (9) and improvement has been reported by investigators employing immunocytochemistry or reverse transcriptase polymerase chain reaction (RT-PCR). We established a new real-time RT-PCR protocol for rapid, quantitative detection of free cancer cells in peritoneal washings using carcinoembryonic antigen (CEA) mRNA as the cancer marker (10). In the present study, this technique was employed to evaluate more sensitively the risk for recurrence in patients with linitis plastica. Indications for radical surgical treatment of this disease might be reconsidered based on such RT-PCR data.
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PATIENTS AND METHODS |
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PATIENTS
Between 1995 and 2000, peritoneal lavage was carried out during laparotomy at the Aichi Cancer Center Hospital in 47 consecutive patients with histologically proven gastric adenocarcinoma of the linitis plastica type who underwent laparotomy. All patients were preoperatively staged with computed tomography and endoscopic ultrasonography. Patients with distant metastases, ascites, para-aortic lymphadenopathy and other signs suggestive of disseminated disease were excluded. Analyses of peritoneal washings by RT-PCR and also conventional cytological examination were performed prospectively. No patients were treated with neoadjuvant chemotherapy. Evaluation of peritoneal washings using RT-PCR was approved for all gastric carcinoma patients by the Institutional Review Board of the Aichi Cancer Center. Prior to surgery, informed consent was obtained from all patients for collecting and analyzing these samples. The abdominal cavity was examined thoroughly for metastatic lesions and for peritoneal deposits in particular. When potentially curative resection was deemed possible, total or subtotal gastrectomy with D2 lymphadenectomy was the treatment of choice. Palliative resection was performed and chemotherapy was given at the surgeon's discretion for patients with no possibility of an R0 resection. Gastrectomy was avoided for those with extensive invasion of the retroperitoneum and those with extensive grossly evident peritoneal dissemination. While chemotherapy was given later to patients diagnosed with a cancer recurrence during follow-up after curative resection, no chemotherapy was given in the adjuvant setting. For patients who underwent gastrectomy with D2 lymphadenectomy, the pathologically determined depth of cancer invasion (pT categories) and the number of involved lymph nodes (pN categories) were evaluated histologically. Staging was carried out according to the tumor–nodes–metastasis (TNM) classification (13). T categories for the patients who did not undergo resection were decided based on findings at laparotomy. Whenever peritoneal deposits were observed, samples were taken for histological confirmation of cancer dissemination. Patients who proved to have a positive cytological examination of peritoneal washings (CY+) were defined as having undergone R1 resection even in the absence of other metastatic lesions. CEA RT-PCR, on the other hand, was considered experimental at the time this study was conducted and the result was not reflected in the R classification.
POSTOPERATIVE SURVEILLANCE OF PATIENTS
The follow-up program consisted of interim history, physical examination and hematological and blood chemistry panels including tests for CEA and carbohydrate antigen 19-9, repeated every 3 months for the first postoperative year and every 6 months thereafter. Either abdominal ultrasonography or computed tomography was carried out every 6 months. Peritoneal recurrence, diagnosed primarily on the basis of clinical symptoms, digital examination, physical and radiological findings of bowel obstruction and ascites, was confirmed by paracentesis or laparotomy.
PERITONEAL WASHINGS
At the beginning of each operation, 100 ml of saline were introduced into the peritoneal cavity and aspirated after gentle agitation. These washings were centrifuged at 1800 r.p.m. for 5 min to collect intact cells, rinsed with phosphate-buffered saline (PBS), dissolved in ISOGEN RNA extraction buffer (Nippon Gene, Tokyo, Japan) and stored at –80°C until use. A portion of each peritoneal washing sample was examined cytopathologically using conventional Papanicolaou and Giemsa staining.
REAL-TIME RT-PCR
Real-time RT-PCR detection of CEA mRNA in the peritoneal washing samples was performed as described elsewhere (10). In brief, samples frozen in ISOGEN were thawed and total RNA was extracted using a guanidinium isothiocyanate–phenol–chloroform method. Extracted total RNA was preincubated with 50 ng of random hexanucleotide primer (Pharmacia Biotech, Uppsala, Sweden) in 9 µl of solution for 10 min at 70°C. After chilling on ice, 4 µl of 5x synthesis buffer (250 mM Tris–HCl, pH 8.3, 375 mM KCl and 15 mM MgCl2) plus 2 µl of 100 mM dithiothreitol, 4 µl of each dNTP at 2.5 mM and 1 µl of SuperScript II RNase H– reverse transcriptase (200 units/µl; I-gene CA) were added. The reaction mixture then was incubated for 40 min at 42°C and the resulting first-strand cDNA was immediately used for PCR amplification with a LightCycler (Roche Diagnostics, Mannheim, Germany). Real-time RT-PCR was performed by a single-step method using hybridization probes. The CEA-specific oligonucleotide primer pair consisted of sense primer, 5'-AACTTCTCCTGGTCTCTCAGCT-3', and an antisense primer, 5'-GCAAATGCTTTAAGGAAGAAG-3'. These were designed to recognize sequences in the M/3' and 3' exons of the CEA gene and to span large intron sequences. The donor probe (5'-TGAAATGAAGAAACTACACCAGG-FL-3') was labeled at the 3' end by fluorescence, whereas the acceptor probe (5'-LC-CTGCTATATCAGAGCAACCCCAA-P-3') was labeled at the 5' end with LightCycler-Red640 and modified at the 3' end by phosphorylation to block extension. To prove the integrity of the isolated RNA, real-time RT-PCR analysis for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was carried out using specific primers (sense, 5'-TGAACGGGAAGCTCACTGG-3'; antisense, 5'-TCCACCACCCTGTTGCTGTA-3'). Nucleotide sequences of the GAPDH hybridization probes were 5'-TCAACAGCACACCCACTCCT-FL-3' for the donor and 5'-LC-CACCTTTGACTCTGGGGCT-P-3' for the acceptor. All primers and probes were synthesized and purified by reversed-phase high-performance liquid chromatography (HPLC) by Nihon Gene Research Laboratories (Sendai, Japan). PCR amplification of CEA mRNA using the LightCycler was carried out within a LightCycler capillary in 10 µl of reaction mixture consisting of a master mixture containing Taq DNA polymerase, a dNTP mixture and buffer (LightCycler DNA master hybridization probes, Roche Diagnostics); 4.0 mM MgCl2; sense and antisense primers at 0.25 µM; each probe at 0.4 µM; and 1 µl of template cDNA. Prior to amplification, 0.16 µl of anti-Taq DNA polymerase antibody (TaqStart antibody, Clontech Laboratories, Palo Alto, CA) was added to the reaction mixture with incubation at room temperature for 5 min to block primer elongation. For CEA amplification, this inactivation step (95°C, 90 s) was followed by 50 rounds of amplification in the LightCycler at 95°C (0 s) for denaturation, 50°C (10 s) for annealing and 72°C (10 s) for extension, with temperature change at 20°C/s. Real-time PCR monitoring was achieved by measuring the fluorescent signal at the end of the annealing phase for each cycle. For GAPDH amplification, the same temperature profile was used except for the extension step (72°C for 20 s). GAPDH was quantified only to ensure that mRNA was successfully extracted.
A moderately differentiated, CEA-producing adenocarcinoma cell line derived from a liver metastasis of a colon cancer (COLM-2) was used for preparing external standards for CEA mRNA. Serial 10-fold dilutions of a suspension containing 106 COLM-2 cells was made to which 107 peripheral blood leukocytes were added. RNAs were then extracted and converted into cDNAs. Each run consisted of six external standards, a negative control without a template and patient samples with unknown mRNA concentrations. Quantitation of mRNA in each sample was performed automatically with reference to a standard curve constructed for each run by the LightCycler software. A cutoff value of 0.1 for CEA mRNA (equivalent to one-tenth of CEA mRNA contained in a single COLM-2 cell) was established with the aid of a receiver operating characteristics (ROC) curve which was constructed with data concerning CEA mRNA values and the presence or absence of free intra-abdominal cancer cells (presence of cancer cells in this analysis was defined as either having peritoneal metastasis at surgery or suffering from relapse as peritoneal carcinomatosis within 2 years of surgery) from 189 gastric carcinoma patients, as reported previously (14). At this cutoff value, both the sensitivity and specificity of this detection system were 84%.
STATISTICAL ANALYSIS
Survival analysis was carried out using Kaplan–Meier curves. No patient in the current series died of causes other than cancer progression. The generalized Wilcoxon method was used to evaluate the difference between the curves. Correlation between the results of cytology or RT-PCR and the presence of peritoneal carcinomatosis at surgery or during the follow-up were evaluated by chi-squared tests. The Mann–Whitney test was used to compare the CEA mRNA values between the patients who were negative and positive for the conventional cytological examination. Prognostic factors for linitis plastica first were identified through univariate analysis. Factors thus identified were evaluated as covariates to determine independent prognostic factors, using the Cox regression hazards model for multivariate analysis.
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RESULTS |
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PATIENT DEMOGRAPHICS (TABLE 1)
The mean age of the patients was 56.5 years (range, 30–81 years). On average, patients were younger than those previously reported with other types of gastric carcinoma. The male to female ratio was 25:22. Of the 47 patients, only 17 were treated with potentially curative resection. Twenty-two patients underwent palliative resection, while gastrectomy was not performed in eight other patients. The primary lesion invaded the gastric serosa (T3 or T4 status) in most patients (46 of 47), with 12 patients showing invasion into adjacent structures (T4). Lymph node metastasis was also observed in almost all patients evaluated (38 of 39 patients). Of these 38 patients, 21 (45%) were classified as pN3 (i.e. >15 involved lymph nodes). The mean number of histologically involved lymph nodes for all 39 patients was 19.4 (range, 0–55). Macroscopically, 20 patients had peritoneal deposits (P+) and 20 patients had a positive conventional cytological examination of peritoneal washings (CY+). Cytological examination was positive in six of 27 patients who had no macroscopic peritoneal seeding (P–) and in 14 of the 20 P+ patients.
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RESULTS OF REAL-TIME RT-PCR AND RELATION TO OTHER VARIABLES INDICATING THE PRESENCE OF INTRAPERITONEAL CANCER CELLS
GAPDH mRNA was detected in all samples tested, indicating that RNA extraction was successful for all samples. CEA mRNA exceeded the cutoff value (CEA+) in 39 samples (83%), including 14 of 17 patients who underwent R0 resection, 18 of 22 patients who underwent R1/R2 resection and seven of eight patients who underwent exploratory laparotomy (Table 2). Individual CEA mRNA levels quantified by the LightCycler system are shown in Fig. 1, stratified according to the T categories. Of the 34 patients with T3 cancer, 13 (38%) were CY+ and 27 (79%) were CEA+. Of the 12 patients with T4 cancer, seven (58%) were CY+ and 11 (92%) were CEA+. As for correlations between positivities of RT-PCR, cytological examination and peritoneal deposits at surgery, 19 of 20 P+ patients and all 20 CY+ patients were CEA+ (Table 3). Even among 21 CY–/P– patients, 14 patients (67%) were CEA+ (Table 4). The median CEA mRNA value for CY– patients was 0.267 and was significantly lower (U = 74, P < 0.0001) than that of CY+ patients (median: 265).
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OUTCOME FOR PATIENTS WITH LINITIS PLASTICA-TYPE GASTRIC CARCINOMA
The patients were followed postoperatively for a median interval of 1355 days or until death. Overall survival of the 47 patients was extremely poor, with a 5-year survival rate of 10% and a 50% survival time of 419 days. Only five patients are disease free to date, after follow-up intervals ranging from 782 to 2215 days. Peritoneal carcinomatosis was observed during the course of the disease in 35 of 47 patients overall (75%) and in 30 of 38 patients (79%) who died of the disease. Although both cytology and RT-PCR correlated significantly with the incidence of peritoneal carcinomatosis and were good predictors of this pattern of disease failure (Table 5), RT-PCR was superior in terms of sensitivity. Of 27 CY– patients, six had peritoneal deposits at surgery and 11 other patients suffered from peritoneal carcinomatosis postoperatively, whereas only two of eight CEA– patients have had this pattern of recurrence to date. Nevertheless, RT-PCR was found not to be a significant indicator of prognosis, given the lack of difference in survival between CEA– and CEA+ patients (Fig. 2). Three of the remaining six CEA– patients died of bone metastasis and only three patients have survived to date. On the other hand, the outcome for CY+ patients was extremely poor with no long-term survivor and the difference in survival between CY– and CY+ was statistically significant (Fig. 2).
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CEA mRNA values ranged from 0 to 18 460 and were considered to correlate with the number of cancer cells and, ultimately, the risk for peritoneal carcinomatosis. Patients were stratified according to the CEA mRNA values into three groups [CEA mRNA <0.1 (n = 8), 0.1
CEA mRNA <10 (n = 13) and CEA mRNA 
10 (n = 26)] and the survival was compared between the groups (Fig. 3). Prognosis of the patients with CEA mRNA values of 10 was found to be extremely poor, approaching that of CY+ patients.
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PROGNOSTIC FACTORS FOR LINITIS PLASTICA-TYPE GASTRIC CARCINOMA
Univariate analysis with all 47 patients showed that T category (T4 vs T2 and T3: hazard ratio 4.93, P < 0.0001), cytological findings (CY+ vs CY–: hazard ratio 4.55, P < 0.0001), curability (palliative resection and no resection vs curative resection: hazard ratio 5.59, P < 0.0001) and a gross finding of peritoneal deposits (P+ vs P–: hazard ratio 2.00, P = 0.0379) were significant prognostic factors, whereas RT-PCR was not (hazard ratio 1.41, P = 0.4783). Nodal metastasis was not evaluated as a variable, since patients who did not receive systemic lymphadenectomy could not be assessed pathologically with respect to pN category. Of the four prognostic factors, only T category (T4 vs other classes) and curative resection (R0 vs other procedures) were found to be significant independent prognostic factors by multivariate analysis (Table 6).
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DISCUSSION |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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Linitis plastica is a category of gastric carcinoma with a propensity towards diffuse infiltration, massive lymph node metastasis and peritoneal seeding. With regard to treatment outcomes, this study only adds to growing numbers of patients reported to have poor survival (2,3). Whether linitis plastica is a surgical disease still remains rather problematic (1), but results of this study regarding the detection rate of free cancer cells in the peritoneal washes illustrate the reason why efforts to confront the disease surgically often turn out to be futile.
The enhanced sensitivity achievable with the RT-PCR technique in detecting various types of micrometastasis and minimal residual disease has been well documented (15–17). Additionally, application to the detection of free cancer cells in peritoneal washings has been reported by the present authors and other investigators (10,11,14). Using conventional cytological examination with Papanicolaou staining, the detection rate of cancer cells in peritoneal washings ranged from 18 to 24% among patients with serosally invading (T3) gastric carcinoma (5,9,12,18). The detection rate in a similar population exceeded 30% using immunocytochemical techniques (12,19), while a remarkably high positivity rate of 67% was achieved with real-time RT-PCR technique (10). In the present study, positivity for CEA mRNA was 85% overall and 82% for T3 patients, both with no signs of metastatic disease after preoperative imaging studies. This percentage is much higher than reported percentages obtained by conventional cytological examination, but may seem expected to those familiar with the behavior of this disease. The incidence of peritoneal metastases and recurrences actually were high and were observed in 75% of the patients to date. The true incidence of peritoneal metastasis may have been even higher, because early diagnosis of this pattern of failure is difficult and patients who died of other types of metastatic disease may also have harbored peritoneal deposits without being noticed. Hence the incidence of peritoneal metastasis observed in the current series can be considered as almost equivalent to the positivity for CEA RT-PCR, implicating that the high positivity should not be attributed solely to false-positive results.
7Results of conventional cytological examination reflected survival, but were not an independent prognostic factor over and above curability of the operation and presence or absence of invasion to the adjacent organs. Since curability is decided after combined analysis of several known prognostic factors including the results of cytological examination, it is not surprising that its prognostic value exceeded that of the cytological examination alone. At the same time, the patients treated with R2 resection could have been excluded from the multivariate analysis to assess more adequately the prognostic value of cytological examination. Unfortunately, the small number of patients available in this study precluded the use of more refined statistical analyses.
To our disappointment, enhancement of the sensitivity by the use of molecular biology techniques further reduced the prognostic impact of exploring peritoneal washing samples. This is possibly because the presence of intraperitoneal cancer cells does eventually prevent the patients from being cured, but may not always be the direct cause of death, especially when the patient has other patterns of recurrent disease. Indeed, a small number of patients who did not have detectable intraperitoneal cancer cells still died of cancer that may have spread through lymphatic or hematogenous pathways. It is apparent from the mean CEA mRNA values that CY+ denotes presence of a large quantity of cancer cells and it is perhaps only in such a situation that free cancer cells directly affect survival time. This led to the idea that altering the cutoff value of RT-PCR may improve its prognostic value and elevating the cutoff value from 0.1 to 10 actually raised the accuracy of RT-PCR as a tool for selecting patients with a dismal prognosis. Given that this is a prospective study testing the CEA RT-PCR performed with a predetermined cutoff value, however, the fact that the current RT-PCR analysis was found not to be a significant prognostic factor remains our conclusion.
On the other hand, CEA RT-PCR could identify patients at risk for peritoneal carcinomatosis in a consecutive case series with linitis plastica-type gastric carcinoma. This knowledge might be useful in the future selection of treatment strategy for linitis plastica. Based on the lack of benefit from radical surgery for linitis plastica patients with CY+ status, the authors recently proposed that this subset of patients should be treated primarily with chemotherapy even in the absence of other unfavorable factors (1). The eligibility of this stomach-conserving strategy might be extended to include patients with CEA+ status. Peritoneal washings in this case can be collected at the time of preoperative staging laparoscopy. On the other hand, some investigators might propose the opposite, using more radical multimodality treatment for this subset of patients such as employing adjuvant systemic and/or intraperitoneal chemotherapy (20–23) or chemohyperthermia (24,25) in combination with radical surgery.
To summarize, over 80% of patients with linitis plastica who had no evidence of metastatic disease preoperatively were found in this study to present with free cancer cells in the abdominal cavity and almost the same percentage of the patients eventually developed peritoneal carcinomatosis. This suggests that a majority of surgery for patients with linitis plastica may turn out to be non-curative. Despite these findings, the CEA RT-PCR as performed in the current study was found not to be a significant prognostic determinant in patients with this disease.
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Acknowledgments |
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This study was supported in part by a grant from the Ministry of Education, Science and Culture, Japan. The authors thank Miss N. Yamada and S. Kato for expert technical assistance.
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References |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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