Selection of Patients by Membrane Transporter Expressions for Aminolevulinic Acid (ALA)- Guided Photodynamic Detection of Peritoneal Metastases

Background. Photodynamic diagnosis (PDD) with 5-aminolevulinic acid (ALA) was used to detect peritoneal metastasis (PM). This study was done to verify the roles of the expressions of the peptide transporter PEPT1 and ATP-binding cassette transporter ABCG2 genes on the selection of patients in ALA PDD. Methods. The study group comprised 138 patients with PM. After oral administration of 5-ALA, PM was evaluated by PDD. Tissue protoporphyrin IX (PpIX) levels, and PEPT1/ABCG2 mRNA expressions were determined. Results. The tumor detection rate on PDD was 45.6% (63/138). PDD is a safe technique for the detection of PM from ovarian cancer, Selection of Patients by Membrane Transporter Expressions for Aminolevulinic Acid (ALA)-Guided Photodynamic Detection of Peritoneal Metastases http://www.ijSciences.com Volume 4 – September 2015 (09) 67 mesothelioma, and colorectal cancer. PpIX levels of ALA-PDD-positive PM were significantly higher than those of ALA-PDD-negative PM. The PpIX levels in PM that simultaneously expressed PEPT1 and ABCG2 mRNA were significantly higher than those in PM that expressed either PEPT1 or ABCG2 mRNA, as well as PM that expressed neither PEPT1 nor ABCG2 mRNA. PM with PEPT1 mRNA up-regulation showed simultaneous up-regulation of ABCG2 mRNA. PpIX accumulates in PM with PEPT1 up-regulation. At the same time, PpIX may be excreted into stromal tissue through ABCG2 transporter, resulting in the accumulation of excess PpIX in the stromal tissue near cancer cells. Conclusions. Preoperative evaluations of the expressions of the PEPT1 and ABCG2 genes by RT-PCR methods might facilitate the selection of patients most likely to benefit from ALA-PDD.


INTRODUCTION
Peritoneal metastasis (PM) has been considered a terminal disease, and palliative chemotherapy and/or best supportive care wass widely used for treatment. In  6 . Recently, PDD is used to detect PM from gastric cancer and serous carcinoma 7,8 .
We previously reported that the peptide transporter PEPT1 (SLC15A1, ALA influx transporter) and ATPbinding cassette (ABC) transporter ABCG2 (porphyrin efflux transporter) genes play pivotal roles in the accumulation of PpIX in gastric cancer cell lines 9 .
We now present the results of a prospective study evaluating PDD for the detection of PM from various primary sites. A major aim was to clarify the key molecules in PpIX accumulation after oral administration of ALA.

PATINTS AND METHODS
The study group comprised 138 patients with PM from 48 appendiceal mucinous neoplasms, 20 ovarian cancers, 7 mesotheliomas, 34 colorectal cancers, 5 pancreas cancers, and 24 gastric cancers (52 men and 86 women; mean age, 56.9 ± 11.9 years). The local ethics committee in our hospitals approved the study protocol, and written informed consent was obtained from all patients.
Patients were informed about the adverse effects of ALA-PDD, such as nausea, vomiting and skin photosensitivity in accordance with the Common Terminology Criteria for Adverse Events, version 4.0.

Fluorescence detection of peritoneal metastases, tumor sampling, and CRS
Each patient received 20 mg/kg body weight of 5-ALA (Cosmo Bio Co., Ltd, Tokyo, Japan) dissolved in 50-100 ml of orange juice. The mixture was given orally 2 hours before surgery. After treatment with 5-ALA, patients were kept away from direct sunlight for 24 hours.
After laparotomy, standard evaluations of the distribution and size of PM were done under white light.
Tumor tissues and normal peritoneum were discriminated under white light. Then, all lights in the operation room were turned off, and PDD was performed using a xenon lamp (300 W) emitting violet light with a wavelength of 375-445 nm for fluorescence excitation, and the abdominal cavity was examined 5 .
Two specimens were taken from red fluorescentpositive peritoneal nodules or from fluorescent-negative peritoneal nodules, and two specimens were collected from red fluorescent-negative normal peritoneum. The red fluorescence intensity of each specimen was evaluated and categorized, and the results were recorded.
Half of each sampled specimen was examined histopathologically to confirm the presence of cancer cells. The obtained specimens were immediately stored at -80℃ in the dark for further analysis. All patients with PM received cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy 8 .

High-performance liquid chromatographic (HPLC) analysis of protoporphyrin IX (PpIX)
Samples were treated as described previously, with some modifications 9 . Porphyrins were then separated using an HPLC system (Type Prominence, Shimadzu, Kyoto, Japan) equipped with a reversed-phase C18 column (CAP- 250 mm; Shiseido, Tokyo, Japan) 9 . Porphyrins were continuously detected with a fluorospectrometer porphyrin concentrations in samples were estimated from calibration curves obtained with standard porphyrins 9 .

ABCG2, and ferrochelatase in tumors and normal peritoneum
Total RNA was extracted from tissue samples using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer's protocols, and first-strand cDNA was prepared from total RNA by reverse transcriptase reaction, as described previously 9 . Then, the first-strand

Immunohistochemical analyses
The expressions of PEPT1, ABCG2, and ferrochelatase in paraffin-embedded tumor or normal tissue sections were detected by immunohistochemical staining with the DAKO LSAB+system-HRP as described previously 13  Santa Cruz Biotechnology) were used.

Statistical Analysis
Data were collected for all patients included in the study.
The primary outcome variable of statistical analysis was the metastatic lesions detected by PDD. For statistical analysis, the SPSS version 17.0 statistical software package (SPSS, Chicago, Ill) was used.

Diagnostic value of 5-ALA for PM and side effects
Typical examples of metastatic nodules on PDD are shown in Figures 1 and 2. Figure 1 shows PM from ovarian cancer. Figure

PpIX concentrations in peritoneal tissue and PM
The PpIX level was significantly higher in PM nm/mg-protein, respectively.

PEPT1, ABCG2, and ferrochelatase mRNA expressions and PpIX levels in PM
The PpIX level was significantly higher in PEPT1

DISCUSSION
Experimental and clinical studies have demonstrated that ALA PDD can detect PM from different cancers 5,6,8 . In gastric cancer, the accuracy of fluorescence imaging by laparoscopy was higher than that of white light imaging 7  ABCG2 is a known ALA efflux transporter 15, 16 .
Accordingly, PpIX accumulation in bladder cancer strongly correlates with the PEPT1 up-regulation and ABCG2 down-regulation 13 . In contrast to bladder cancer 13 and brain tumor 12  The present study confirmed that the oral administration of ALA is associated with very few adverse reactions because ALA is an intrinsic molecule and is rapidly metabolized to PpIX through the porphyrin/heme pathway.
In conclusion, ALA-PDD-guided surgery may improve