Micrometastasis of Gastric Cancer S

Micrometastasis of Gastric Cancer

Shoji Natsugoe, Masataka Matsumoto, Hiroshi Okumura, Akihiro Nakajo, Koki Tokuda, Futoshi Miyazono, Sumiya Ishigami, Shuichi Hokita,

Sonshin Takao, and Takashi Aikou

Introduction

Even patients with gastric cancer who undergo complete resection and have no his- tological evidence of lymph node metastasis sometimes experience recurrence after operation [1–3]. Why do cancers recur despite the performance of macroscopically and histologically radical resections? This recurrence is probably caused by micrometastasis to the lymph nodes, circulating blood, and abdominal cavity [4,5]. It is known that occult lymph node micrometastases have been identified by detailed histological examination in additional sections [6,7]. In recent years, the development of sensitive immunohistochemical technique and reverse transcription-polymerase chain reaction (RT-PCR) has led to the detection of micrometastases [4,5]. The concept of isolated tumor cells (ITC) has been introduced in the TNM classification [8]. In this issue, ITC was defined as single tumor cells or small clusters of cells not more than 0.2 mm in greatest dimension that are usually detected by immunohisto- chemistry or molecular methods, but which may be verified with hematoxylin and eosin (H&E) stains. The clinical significance of ITC or micrometastasis is still unknown in gastric cancer patients.

Since 1996, our laboratory has pursued gene diagnosis to detect lymph node micrometastasis,free cancer cells in the circulating blood,and disseminated cancer cells in the abdominal cavity in patients with gastric cancer. In the present chapter, we demonstrate the current results of micrometastasis,including ours,and discuss the role, significance, and problem of perioperative gene diagnosis in gastric cancer surgery.

Lymph Node Micrometastasis

Immunohistochemical Detection of Micrometastasis

We examined lymph node micrometastasis by immunohistochemistry using AE1/AE3 (Dako, Carpinteria, CA, USA) in 1761 lymph nodes obtained from 67 gastric cancer patients who were diagnosed as free of lymph node metastasis by routine histologi-

329 Department of Surgical Oncology and Digestive Surgery, Kagoshima University School of Medicine, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan

e-mail: [email protected]

cal examination [9]. Tumor involvement of lymph nodes was divided into two cate- gories: tumor cell microinvolvement (TCM), individual tumor cells without a change in the stroma and micrometastasis; and cluster formation of tumor cells with stromal reaction (Fig. 1) [5,10]. Thirty (1.5%) of the 1761 lymph nodes showed micrometas- tasis and/or TCM. Micrometastasis with or without TCM was found in 10 patients, and TCM alone was found in 4 patients; 6 (18.2%) of the 33 patients with T1 tumor and 8 (23.5%) of the 34 patients with T2 tumor had occult lymph node metastasis.

The 5-year survival rate was worse among those with micrometastasis with or without TCM than among those without micrometastasis (Fig. 2) [10–19]. Some authors have reported the detection rate and clinical outcome in patients with micrometastasis (Table 1). The rate of micrometastasis ranged from 8% to 90%. There were some prob- lems regarding the difference of antibody used, identification between micrometasta- sis and immune cells, and estimation of micrometastasis, especially in single cells. It is important to apply such methods in the clinical field. Recently, we have introduced

400

X

Fig. 1. a Micrometastasis in a lymph node as detected by immunohistochemical staining with anticytokeratin AE1/AE3. Micrometastasis was defined as metastasis consisting of tumor cells or a small cluster of carcinoma cells with surrounding stromal change. b Tumor cell microin- volvement (TCM) in a lymph node. Microinvolvement was defined as carcinoma cells without surrounding stromal change

0 50%

100%

0 1 2 3 4 5

MM (-) (n=57)

MM and / or TCM (+) (n=10) P<0.05

year

Fig. 2. Survival curve of patients who did or did not have micrometastasis in one or more lymph nodes. MM, micrometastasis; TCM, tumor cell microinvolvement

a b

rapid immunohistochemical detection of lymph node micrometastasis during oper- ation, which took 30 min to complete [20]. Such a method is useful for determining distant lymphadenectomy by examination of regional nodes for advanced cancer and examining the presence or absence of nodal involvement in sentinel node navigation surgery for early cancer.

Micrometastasis Detected by RT-PCR

We examined 312 lymph nodes obtained from 50 patients (pT1, 41; pT2, 5; pT3, 3;

and pT4, 1) with node-negative gastric cancer [21]. The clinical characteristics of micrometastasis were investigated after RT-PCR using carcinoembryonic antigen (CEA) as a primer and immunohistochemistry using anticytokeratin antibody (AE1/AE3) were performed. The number of patients and micrometastases detected by RT-PCR was 14 and 17 and by immunohistochemistry was 7 and 8, respectively. RT- PCR was a more sensitive method than immunohistochemistry. Micrometastasis by RT-PCR correlated with depth of tumor invasion and lymphatic invasion. Regarding pT1 tumor, 9 patients with micrometastasis had tumors that were of the macroscop- ically depressed type and 2 cm or more in diameter. These results supported the indi- cation of endoscopic mucosal resection.

There were some reports of micrometastasis detected by RT-PCR (Table 2) [21–24].

The detection rate of micrometastasis was different because of various stages of the patients in each report. The RT-PCR method is actually more sensitive than the other methods. However, there are some problems for RT-PCR technique as follow: (1) some complicated procedures for management of specimens, (2) contamination of other specimens, (3) selection of primer, (4) presence of pseudogene, and (5) suitable setup of sensitivity for amplification. The relationship between micrometastasis detected by RT-PCR and clinical outcome is still unknown. In the near future, we should analyze a large number of patients with micrometastasis under certain conditions such as the same primer and the same stage.

The question arises whether occult micrometastasis implant and proliferate in the lymph node. Izbicki and Hosch reported that implantation and proliferation of

Author [Reference] Antibody Case Patients Positivity Prognosis Siewert et al. 1996 [10] AE1/AE3,Ber-EP4 100 T1–T3 90 (+)

Ishida et al. 1997 [11] CK,CEA 109 St.I–IV 18 (+)

Harrison et al. 2000 [12] CAM5.2 25 T1–T4N0 36 (+)

Nakajo et al. 2001 [9] AE1/AE3 67 T1,T2,N0 21 (+)

Yasuda et al. 2002 [13] CAM5.2 64 T2T3N0 32 (+)

Lee et al. 2002 [14] AE1/AE3 153 T1–T4 49 (+)

Stachura et al. 1998 [15] CK18 40 T1 8 (-)

Kikuchi et al. 1999 [16] AE1/AE3 51 T2T3N0 43 (-)

Saragoni et al. 2000 [17] CK 139 T1N0 17 (-)

Choi et al. 2002 [19] CK8 88 T1(SM) 32 (-)

Fukagawa et al. 2001 [18] CK 107 T2N0 36 (-)

(+), significant; (-), not significant

micrometastasized cancer cells in the lymph nodes were confirmed by an experiment of transplantation metastasis using nude mice [25,26]. Further study should be required in the relationship between proliferation of micrometastasis and autoim- mune system.

Free Cancer Cells in the Circulating Blood

Circulating cancer cells recently have been detectable with RT-PCR. We examined the relationship between molecular detection of circulation cancer cells according to the time course during surgical procedure and blood-borne metastases [27]. Blood samples from 57 patients with gastric cancer were obtained from the portal vein, peripheral artery and superior vena cava before and after tumor dissection. After total RNA was extracted from each blood sample, CEA-specific RT-PCR was performed (Fig. 3). CEA mRNA was detected in the blood of 21 (36.8%) of 57 patients. CEA mRNA expression was not detected in the blood obtained from 15 healthy volunteers and 15 patients with benign disease. The positive rate increased in proportion to the depth of tumor. The incidence of positive CEA mRNA did not differ among the various sites of blood sampling. The appearance of circulating cancer cells was related to the sur- gical maneuver. A significant relationship was found between the detection of CEA mRNA and blood-borne metastases (Table 3). These results suggested that surgical

Author [Reference] Primer LN Patients Positivity Prognosis

Noguchi et al. 1996 [22] CK19 100 12 15 Unknown

Mori et al. 1995 [23] CEA 87 13 54 Unknown

Okada et al. 2001 [24] CEA,CK20,MAGE3 414 28 12 Unknown

Matsumoto et al. 2002 [21] CEA 312 50 5 Unknown

LN, lymph node

Portal Artery Vein

B A B A B A marker

CEA

GAPDH

Fig. 3. Example of expression of carcinoembryonic antigen (CEA) mRNA. CEA mRNA was positive in the portal vein after resection. GAPDH, glyceraldehyde phosphate dehydrogenase. B, before resection; A, after resection

maneuvers were a possible cause of hematogeneous metastasis, and patients with positive CEA mRNA had a high risk of blood-borne metastasis even after curative resection.

The frequency of free cancer cells in blood detected by RT-PCR reportedly ranged from 10% to 37% (Table 4) [27–32]. Epithelial markers such as cytokeratin and CEA were generally used as primers. To date, as the clinical significance is still unclear because of the small number of patients, we should clarify clinical outcome by long- term follow-up in patients with disseminated tumor cells in blood in a large number of patients.

Although the frequency of hematogeneous metastasis differed depending on the animal species or cancer cell lines, an animal experiment revealed that cancer metas- tasis developed only when more than a certain number of cancer cells was injected intravenously [33–35]. However, the mechanism of cancer metastasis still remains unclear as to whether most free cancer cells die or are unable to adhere to the vascu- lar endothelium. Basically, it is necessary to clarify the characteristics of the cancer cells that are considered to establish a metastatic lesion. These cells possess the ability to adhere and infiltrate to the vascular endothelium, proliferate outside the blood vessel, and subsequently induce vascularization [36,37].

Free Cancer Cells in the Peritoneal Cavity

Peritoneal dissemination is one of the most common modes of gastric cancer recur- rence, even after curative resection [38]. Cytological examination of peritoneal lavage is a useful means of detecting free cancer cells in the peritoneal cavity and predict- ing recurrence [39–41]. However, some patients with negative cytological findings

Expression of CEA mRNA

Negative Positive P value

No recurrence 36 17 0.029

Recurrence 0 4

Number of patients 36 21

Table 4. Reports of free cancer cells in blood detected by RT-PCR

Author [Reference] Primer Case Positivity Recurrence

Yeh et al. 1998 [28] CK19 34 21 (+)

Majima et al. 2000 [29] CEA,CK20 52 10, 10 Unknown

Nishida et al. 2000 [30] CAM5.2 36 22 Unknown

Piva et al. 2000 [31] CEA 30 37 Unknown

Miyazono et al. 2001 [27] CEA 57 37 (+)

Ikeguchi et al. 2003 [32] CK20 55 27 Unknown

(+), significant

have been diagnosed later with peritoneal metastasis. We investigated free cancer cells in the peritoneal lavage fluid by both conventional cytological examination (Papani- colaou and Giemsa staining) and CEA-specific RT-PCR. Peritoneal lavage was per- formed in 136 patients who underwent curative gastrectomy [42]. After laparotomy, the left subphrenic and Douglas cavities were filled with 200 ml isotonic sodium chlo- ride and peritoneal lavage fluid was collected. Among 136 patients, 5 patients (3.6%) were positive for free cancer cells by cytological examination and 30 (22.1%) were positive by RT-PCR. The frequency of RT-PCR results increased according to lymph node metastases, lymphatic invasion, tumor depth, and stage grouping. The incidence of peritoneal recurrence was significantly higher in patients with positivity than those with negativity by RT-PCR. Among cytologically negative patients, survival was sig- nificantly shorter in patients with positive than in those with negative CEA mRNA expression (see Fig. 3). Therefore, the technique of RT-PCR was more sensitive than cytological examination in the detection of free cancer cells and prediction of peritoneal recurrence.

There have been several reports regarding the detection of free cancer cells in the peritoneal cavity by the immunohistochemical or RT-PCR method (Table 5) [42–47].

According to these reports, the detection rate was higher in immunostaining than in RT-PCR. It is necessary to compare the sensitivity of both methods using peritoneal fluid obtained from same patients. In all reports, interestingly, the presence of free cancer cells detected by such methods correlated well with peritoneal recurrence.

Therefore, it should be considered that patients with positive finding by molecular diagnosis are at a risk of developing peritoneal recurrence.

It is difficult to make a decision of intraperitoneal chemotherapy for these cases when disseminated metastasis is not macroscopically observed. Early detection and eradication of free cancer cells before the development of metastases could help to improve the outcome of patients after tumor resection. The development of useful

CY:negative. CEA-mRNA:positive (n=25)

CY:negative. CEAmRNA:negative (n=106)

0 6 12 18 24 30 36 42 p=0.072

p<0.0001 100

(%)

50

Months after surgery

CY:positive (n=5)

Fig. 4. Cumulative rate of peritoneal recurrence according to cytology results and CEA mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR). CY, cytology

tactics for disseminated micrometastasis in the peritoneal cavity is expected in the near future.

Conclusions

Owing the development of biological and genetic techniques, the presence of micrometastasis in the lymph node, circulating blood, and abdominal cavity has been confirmed. The mechanism of implantation and proliferation of micrometastasis should be basically clarified. Furthermore, we should also investigate the relationship between micrometastasis and the autoimmune system. Clinically, the significance of micrometastasis should be surveyed in a large number of patients with the same stage and surgery. Because it may take a long time for occult micrometatsasis to form recur- rent disease, long-term follow-up data are required to elucidate the clinical significance.

Although surgery is performed to primarily treat many gastric cancer patients, it is inversely the final chance for survival of cancer cells. Therefore, the perioperative prevention of micrometastasis is important when curative surgery is performed.

Patients with micrometastasis seem to have a high risk of cancer recurrence. This concern may also allow the selection of therapeutic tactics to prevent metastasis. We must fight these invisible enemies simultaneously with surgery. In recent years, drugs that inhibit cancer cell infiltration and vascularization of the primary lesion have been used clinically as antimetastatic agents [48–50]. It is advantageous that these agents cause only slight side effects and may be useful for preventing cancer metastasis peri- operatively. The antitumor effects of these agents, in combination with gene diagno- sis, should be evaluated by randomized controlled studies in the near future.

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Table 5. Reports of free cancer cells in peritoneal lavage fluid detected by immunostaining and RT-PCR

Author [Reference] Antibody/Primer Case Positivity Prognosis Immunostaining

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