ANTICANCER RESEARCH 32: 5421-5428 (2012)

Staging Fluorescence Laparoscopy for Gastric Cancer by Using 5-Aminolevulinic Acid

YASUTOSHI MURAYAMA1, DAISUKE ICHIKAWA1, NORIAKI KOIZUMI1,2, SHUHEI KOMATSU1, ATSUSHI SHIOZAKI1, YOSHIAKI KURIU1, HISASHI IKOMA1, TAKESHI KUBOTA1, MASAYOSHI NAKANISHI1, YOSHINORI HARADA2, HITOSHI FUJIWARA1, KAZUMA OKAMOTO1, TOSHIYA OCHIAI1, YUKIHITO KOKUBA1, TETSURO TAKAMATSU2 and EIGO OTSUJI1

Departments of 1Surgery, Division of Digestive Surgery, 2Pathology and Cell Regulation, Kyoto Prefectural University of Medicine, Kyoto, Japan

Abstract. Background: Precise staging of gastric cancer is factors for patients with gastric cancer and common features essential when selecting for the appropriate treatment of metastasis, associated with advanced gastric cancer (2). approach. 5-Aminolevulinic acid (5-ALA) is metabolized and Accurate staging of gastric cancer is essential for planning accumulated as protoporphyrin IX (PpIX), which is a therapeutic strategies and evaluating the prognoses of photosensitizer. In this study, we evaluated the diagnostic patients. Enhanced computed tomography (CT) has been usefulness of 5-ALA for demonstrating peritoneal commonly used to assess the preoperative staging of gastric dissemination in patients with gastric cancer. Materials and cancer (3, 4). However, its diagnostic power is limited to Methods: Fluorescence laparoscopy (FL) was performed in detecting serosal invasion, tumor deposition as peritoneal 13 patients with gastric cancer. All patients received dissemination, and small superficial liver metastases. preoperative oral administration of 5-ALA. Fluorescence-light Diagnostic laparoscopy is a valuable method used in staging laparoscopies were sequentially performed intraoperatively. of gastric cancer. By performing accurate diagnostic Results: In four out of the 13 patients, primary tumors were laparoscopy, it is possible to avoid unnecessary laparotomy detected by FL and demonstrated serosal invasion on and to deliver neoadjuvant chemotherapy. In addition, we can histological examination. Five out of the 13 patients evaluate the effect of neoadjuvant chemotherapy by carrying demonstrated peritoneal metastases, and one patient out diagnostic laparoscopy (5-8). demonstrated superficial liver micrometastases, by FL. All of Photodynamic diagnosis (PDD) is being increasingly used. these lesions were diagnosed as metastatic lesions by PDD can discriminate between normal cellular organization hematoxylin and eosin staining. Conclusion: These findings and malignant lesions without surgical intervention. 5- show that FL diagnosis with 5-ALA is accurate and suitable Aminolevulinic acid (5-ALA) is an endogenous substance for the detection of peritoneal metastases and of superficial and a natural precursor of the heme pathway. 5-ALA is not a liver micrometastases in patients with gastric cancer. photosensitizer, but protoporphyrin IX (PpIX), to which 5- ALA is metabolized, is. 5-ALA is immediately metabolized Gastric cancer is the second most common cause of cancer- to heme in normal cells. On the other hand, since the activity related death worldwide (1). Peritoneal dissemination and of porphobilinogen deaminase is high in abnormal cells, and liver metastasis are among the most important prognostic the activity of ferrochelatase is low, PpIX accumulates in the mitochondria in cancer cells (9-13). When PpIX is excited by light of 405-nm, a fluorescence spectrum with a 635-nm peak is emitted. Various cancer diagnostic methods using the Correspondence to: Yasutoshi Murayama, Department of Surgery, properties of 5-ALA have been reported (14-20). In addition, Division of Digestive Surgery, Kyoto Prefectural University of we previously demonstrated the usefulness of administration Medicine Graduate School of Medical Science, 465 Kajii-cho of 5-ALA for the detection of lymph node micrometastases Hirokoji Kawaramachi, Kamigyo-ku, Kyoto 602-8566, Japan. Tel: in a mouse model (21). +81 752515527, Fax: +81 752515522, e-mail: murayama@ koto.kpu-m.ac.jp The purpose of this study was to assess the diagnostic capability of fluorescence laparoscopy using 5-ALA for Key Words: Staging laparoscopy, gastric cancer, peritoneal peritoneal dissemination and small superficial liver dissemination, liver metastasis, photodynamic diagnosis, 5- metastases that are difficult to identify by CT scanning in aminolevulinic acid (5-ALA), protoporphyrinIX (PpIX). patients with advanced gastric cancer.

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Table I. Comparison of 5-ALA-mediated fluorescence laparoscopic imaging and pathological examination.

Examination by FL Pathology

Case Gender Age Depth of Peritoneal Liver Depth of Peritoneal Liver Histology Operation (years) invasion dissemination metastasis invasion dissemintion metastasis

1 Male 66 SE – + SE – + tub2>por2 Distal gastrectomy 2 Male 67 ~SS – SS – sig>por2 Total gastrectomy 3 Male 76 ~SS + + sig Bypass 4 Male 76 SE + SE + tub1>tub2 Total gastrectomy 5 Female 65 ~SS – SS – sig>por2 Distal gastrectomy 6 Male 68 SE + SE + por2>por1 Total gastrectomy 7 Male 69 SE + + laparotomy 8 Male 61 ~SS – SS – sig>por2 Distal gastrectomy 9 Female 65 ~SS – SS – tub2>por1 Total gastrectomy 10 Male 58 ~SS – SS – tub2>tub1 Distal gastrectomy 11 Female 75 SE – SE – por2>sig Total gastrectomy 12 Male 81 ~SS – SS – por1>por2 Total gastrectomy 13 Female 51 SE + + laparotomy tub1: Well-differentiated adenocarcinoma, tub2: moderately-differentiated adenocarcinoma, por1: poorly-differentiated adenocarcinoma solid type, por2: poorly-differentiated adenocarcinoma non-solid type, sig: signet ring cell carcinoma, SS: tumor invades subserosa, SE: tumor perforates serosa, FL: fluorescence laparoscopy.

Materials and Methods Suspicious lesions detected under the fluorescence mode were sampled. In patients requiring gastrectomy, the procedure was Patients. This study was performed at the University Hospital of performed under laparotomy. Kyoto Prefectural University of Medicine, Kyoto, Japan, and approved by the Ethics Committee of Kyoto Prefectural University Fluorescence images and fluorescence spectrum. The system used for of Medicine, Kyoto, Japan. A total of 13 patients underwent surgery stereomicroscopic analyses of fluorescence and the spectral analytic for advanced gastric cancer between April 2010 and February 2012 system were as previously described (21). We applied the excitation at after written informed consent. Exclusion criteria included the 405±20 nm and observed the fluorescence at wavelengths of >430 nm. presence of porphyria and pyloric stenosis. We compared the fluorescence spectra of peritoneal disseminated lesions with that of pure PpIX (Sigma-Aldrich, St. Louis, MO, USA) Fluorescence laparoscopic analysis system. The system used for dissolved in dimethyl sulfoxide (DMSO). fluorescence laparoscopic analyses is composed of a laparoscopic videoscope (OTV-Y0007; Olympus, Tokyo, Japan) equipped with a Fluorescence analysis of primary tumors. The resected primary video system center (EVIS EXCERAII CV-180; Olympus) and a lesions were subjected to formalin fixation overnight and were xenon light source (EVIS EXCERAII CLV-180; Olympus). 5-ALA- processed for pathological examination. The specimens were induced fluorescence images (>450 nm) (MAJ-Y0048; Olympus) observed using fluorescence stereomicroscopy. were taken after excitation at 380-430 nm. We performed the observation in white-light mode using the same conditions as for Histopathogical examination. Formalin-fixed, paraffin-embedded standard laparoscopy, and for the fluorescence observation, we specimens were prepared from surgically-removed lesions for changed to fluorescence mode. histopathological diagnosis.

Fluorescence laparoscopy using 5-ALA. Patients received a dose of Results 10 to 15 mg per kg body weight of 5-ALA (Cosmo Bio Co., Ltd., Tokyo, Japan) dissolved in 20 ml of 50% glucose solution 3 h prior Diagnosis using fluorescence laparoscopy. Details of the to surgery. All patients were protected from direct light for 24 h patients’ characteristics are summarized in Table I. The mean age after administration of 5-ALA, to avoid sunburn. was 68 years (range 51 to 81 years), and the male/female ratio The patient was placed in the supine position under general was 2.3. Two patients underwent distal gastrectomy, three anesthesia. A small subumbilical incision was made and a 12-mm patients total gastrectomy, one patient gastrojejunostomy, and disposable trocar was inserted for the camera, and two or three two patients explorative laparotomy. In six out of the 13 patients, 5-mm disposable trocars for retracting or dissecting organs were inserted into the upper abdominal wall. Under 10-mmHg abdominal the lesions of serosal invasion were observed as primary tumors air pressure, we performed a careful examination of the abdominal and exhibited a specific speckled pattern of PpIX fluorescence cavity, liver surface, and omentum. Laparoscopic examination was signal by fluorescence laparoscopy. Tumor in four patients performed under alternate white light mode and fluorescence mode. demonstrated serosal invasion on histological examination

5422 Murayama et al: Staging Laparoscoy for Gastric Cancer with 5-ALA

Figure 1. Imaging of serosal invasion of gastric cancer (A), peritoneal dissemination (B), and superficial liver micrometastasis (C) with 5- aminolevulinic acid (5-ALA) using fluorescence laparoscopy. White-light (left) and fluorescence (right) images are shown. On the fluorescence laparoscopic image, metastatic lesions were positive for 5-ALA-induced protoporphyrinIX (PpIX).

(Figure 1A); two patients were not diagnosed histologically. Five and C). All of these lesions were diagnosed as metastatic lesions out of the 13 patients demonstrated peritoneal disseminations, by hematoxylin and eosin staining (data not shown). Table II and one patient demonstrated superficial liver micrometastases shows the comparative diagnostic accuracy of fluorescence as red signals of PpIX on fluorescence laparoscopy (Figure 1B laparoscopic imaging and white light imaging for the lesions of

5423 ANTICANCER RESEARCH 32: 5421-5428 (2012)

Figure 2. Imaging of an excised specimen of a primary lesion after formalin fixation. White-light (left) and fluorescence (center) images and hematoxylin and eosin (H&E)-stained sections are shown. The red signal of protoporphyrinIX (PpIX) and the cancerous lesion of the gastric wall were identical. Scale bar=1 mm.

peritoneal metastasis after 5-ALA administration. The white light Table II. The number of peritoneal disseminations as determined by imaging diagnosis of a white nodule was peritoneal fluorescence laparoscopy and white light imaging. dissemination. On the other hand, we diagnosed a nodule with White light Fluorescence red fluorescence as peritoneal metastasis by using fluorescence imaging (no.) laparoscopy (no.) laparoscopy. The accuracy of the fluorescence imaging was greater than that of the white light imaging. The side-effects of H&E analysis (no.) Positive Negative Positive Negative elevation of liver enzyme levels, nasal swelling, edema, and photosensitivity reaction are known to occur following ALA Positive 12 0 12 0 Negative 2 0 0 2 administration at 60 mg per kg of body weight. We used a concentration of 10 to 15 mg per kg of body weight. None of the patients developed notable side-effects. The results showed that laparoscopic fluorescence imaging by administration of 5- ALA, detected more metastatic lesions compared to white light laparoscopy and can provide qualitative diagnoses. years after surgery (22). Moreover, most cases of death are associated with recurrence. It is thought that this is due to Visualization of primary tumor in excised specimens by 5- micrometastases that could not be diagnosed at the time of ALA administration. We analyzed excised specimens after surgery. Therefore development of diagnostic technology that overnight formalin fixation but before paraffin embedding. provides accurate staging diagnosis is urgently needed. We could not distinguish cancer lesions from normal tissue Although significant improvements in preoperative tumor under white light (Figure 2, left), but detected red staging have been developed due to diagnostic modalities fluorescence by fluorescence microscopy observation (Figure such as CT scanning, Magnetic Resonance Imaging (MRI), 2, center). Furthermore, the portion with red fluorescence by and positron emission tomography-CT, peritoneal fluorescence observation was confirmed by hematoxylin and dissemination and small superficial liver metastases are only eosin (H&E) staining (Figure 2, right) to be cancerous tissue. detected during surgery in many cases (23, 24). Several The fluorescence spectra of both the resected peritoneal reports have indicated that preoperative staging of peritoneal dissemination and the pure PpIX were determined by using a dissemination and superficial metastasis aids in avoiding spectrophotometer. The result of each measurement is shown unnecessary laparotomy (5-8). However, standard staging in Figure 3. It was confirmed that the result for the emission laparoscopy cannot provide an accurate diagnosis without spectrum of peritoneal dissemination was in accordance with biopsy. Many patients with advanced gastric cancer have that for the pure PpIX spectrum, and it was confirmed that peritoneal dissemination, and peritoneal dissemination is a PpIX had accumulated at the cancer site. significant prognostic factor. Effective therapy has been reported (25, 26), and it is thought that discovery at an Discussion earlier stage is important for a positive prognosis. Furthermore, accurate prognostic judgment and planning of Despite improvements in therapeutic resection techniques, postoperative chemotherapy are strongly associated with 60-70% patients with gastric cancer who have undergone a peritoneal dissemination (27). The most common route of radical curative resection, experience recurrence within two peritoneal dissemination is tumor invasion as far as the

5424 Murayama et al: Staging Laparoscoy for Gastric Cancer with 5-ALA

Figure 3. Typical normalized fluorescence spectrum of peritoneal dissemination with 5-aminolevulinic acid (5-ALA) administration in advanced gastric cancer patients (left) and typical normalized fluorescence spectrum of protoporphyrinIX (PpIX) dissolved in dimethyl sulfoxide (DMSO) (right).

gastric serosal layer (28). Conventional cytology has low seems not only to make it easy to look for metastatic sensitivity (29). Therefore, a means of diagnosis of serosal nodules, but also to allow diagnosis of whether they are invasion is necessary. metastases without H&E staining. Because neoadjuvant Various fluorescent probes which are cancer-specific have chemotherapy has been developed for peritoneal been developed and applied (30-33). The advantages of the dissemination (25, 26), a more accurate diagnosis of fluorescence diagnosis are that it is a minimally invasive peritoneal metastasis is necessary. In addition, it is not procedure, the diagnostic device is compact, and it makes possible to change chemotherapy regimens upon shifting to real-time diagnosis possible. However, only a few fluorescent surgery when an accurate diagnosis is not possible at second probes can be applied in a clinical setting, for reasons that staging laparoscopy after neoadjuvant chemotherapy has include safety considerations. Fluorescence diagnosis using been initiated. Therefore, a fluorescence laparoscopic method 5-ALA has been applied for diagnosis of such conditions as using 5-ALA will be useful. However, there is a limitation bladder cancer and brain tumor in a clinical setting (17-20). of this study in terms of the object of clinical application. A Because 5-ALA is an endogenous substance and is included weak point of this technique is that deep observations are not in many food types, there is no report of any serious side- possible because the penetration of light is low. This method effect associated with administering it (14, 33). As for our can be used to diagnose large lymph node metastases without results, it was confirmed that fluorescence laparoscopic excision, but the micrometastases are difficult to detect diagnosis using 5-ALA had sufficient sensitivity for without resection by observation in the abdominal cavity detecting peritoneal disseminations and superficial liver (data not shown). If deep observation were possible, micrometastases. This technique can accurately diagnose diagnosis of lymph node metastasis would be possible and it metastases without the need for biopsy. No acute or major could be possible to select individual lymph nodes for complications were observed, although patients should avoid extraction. Therefore a cancer-specific probe, sensitive to direct sun exposure for 24 h after 5-ALA administration. near-infrared light excitation should be developed. Because Our study indicates that PDD using fluorescence this study was a pilot study, it includes only a few patients laparoscopy after 5-ALA administration offers the potential and was a comparison between sampled metastatic lesions for the detection and accurate diagnosis of peritoneal and pathological examination results. However, the number dissemination and superficial liver micrometastases in of cases will be increased in the future, and it is necessary patients with advanced gastric cancer. Fluorescence diagnosis to further examine the diagnostic capability. In addition, it is

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