5-Aminolevulinic Acid Fluorescence in Detection of Peritoneal Metastases

Peritoneal involvement of gastrointestinal, gynecologic and mesothelial carcinomas are named as Peritoneal Surface Malignancies (PSM). Previous studies suggested that volume of peritoneal seeding has an impact on survival in PSM (Chu et al., 1989; Sadeghi et al., 2000; Jayne et al., 2002). The natural course of tumors at this stage is characterized by a short life expectancy (Sadeghi et al., 2000; Yan et al., 2007). Palliative systemic chemotherapy and best supportive care are widely used as treatment modalities. Surgery was performed in palliative intention to prevent tumor related complication as bleeding, obstruction or perforation. The options for management of PSM have been expanded over the past three decades. Sugarbaker introduced cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) as a new treatment modality for selected patients with


Introduction
Peritoneal involvement of gastrointestinal, gynecologic and mesothelial carcinomas are named as Peritoneal Surface Malignancies (PSM).Previous studies suggested that volume of peritoneal seeding has an impact on survival in PSM (Chu et al., 1989;Sadeghi et al., 2000;Jayne et al., 2002).The natural course of tumors at this stage is characterized by a short life expectancy (Sadeghi et al., 2000;Yan et al., 2007).Palliative systemic chemotherapy and best supportive care are widely used as treatment modalities.Surgery was performed in palliative intention to prevent tumor related complication as bleeding, obstruction or perforation.The options for management of PSM have been expanded over the past three decades.Sugarbaker introduced cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) as a new treatment modality for selected patients with RESEARCH ARTICLE

5-Aminolevulinic Acid Fluorescence in Detection of Peritoneal Metastases
Yutaka Yonemura 1,2,3,4 , Emel Canbay 1,2,3 , Haruaki Ishibashi 1,2 , Eisei Nishino 5 , Yoshio Endou 6 , Shouzou Sako 1 , Shun-Ichirou Ogura 7 PSM (Sugarbaker et al., 1989;Sugarbaker, 2006).This multimodal approach is directed to surgically eliminate all the visible nodules, followed by intraoperative direct instillation of heated chemotherapy in to the abdominal cavity to eradicate the remaining microscopic tumoral foci and free cancer cells.Studies from different centres have investigated the outcome and utility of this combined treatment modality in these patients.All these studies show that completeness of cytoreduction and extent of disease are the most relevant predictors of survival (Glehen et al., 2004;Verwaal et al., 2008;Yan et al., 2009;Glehen et al., 2010;Yang et al., 2011;Chua et al., 2012), suggesting the complete cytoreductive surgery is still crucial in management of patients with peritoneal surface malignancies.Therefore, determination of tumoral foci invisible with white light inspection prior to complete cytoreduction and HIPEC is mandatory to provide better survival outcomes in patients with PSM.
Diagnosis of PSM is challenging especially when the lesions are flat or small enough avoiding detection with preoperative investigations.The recent interest in this field has resulted new techniques that enable better identification of tumor extension into the abdominal cavity to improve diagnosis of peritoneal seeding in PSM.Initially, photodynamic diagnosis (PDD) has been proposed for PSM in animal models (Canis et al., 2001;Krammer and Plaetzer, 2008).The identification of peritoneal metastases was significantly increased by 5-aminolevulinic acid (ALA) administration followed by fluorescence detection in these studies.ALA is the natural precursor of the heme pathway.ALA induced protophorphyrin (Pp)IX accumulation from cancer cells has been applied as a red fluorescence detection marker of cancer using fluorescence blue light (Krammer and Plaetzer, 2008).More recently, PDD is used for detection of PSM originated from gastric cancer using laparoscopic method (Kishi et al., 2012).We previously reported PDD of peritoneal metastatic nodules in primary peritoneal papillary serous carcinoma as a case report after oral administration of 5-ALA (Canbay et al., 2014) and pivotal roles of peptide transporter PEPT1 (ALA influx transporter) and ATP-binding cassette (ABC) transporter ABCG2 (porphyrin efflux transporter) genes in accumulation of PpIX and 5-ALA induced photocytotoxicity of gastric cancer cells in vitro (Hagiya et al., 2012).Now, we present results of our prospective study.We aim to explore the role of 5-ALA in detection of peritoneal metastases and the mechanism underlying photosensitivity of metastatic nodules in vivo.

Materials and Methods
One hundred fifteen patients admitted to NPO Organization to Support Peritoneal Dissemination Treatment, Osaka, Japan with a clinical diagnosis of PSM included to this study.The local ethics committee in our hospital approved the current study (Ethical Committee of Kishiwada Tokushukai Hospital (No-24-05).A written informed consent was obtained from all patients.Exclusion criteria were extra abdominal and liver metastases.

Fluorescence Detection of Peritoneal Metastases
Each patient received 20mg/kg body weight of 5-ALA (Cosmo Bio Co., Ltd, Tokyo, Japan) dissolved in 50 ml water, then mixed with 50-100 ml orange juice.The mixture was given orally 4 hours before surgery.After oral administration, patients were kept away from direct sunlight or strong room light for 24 hours.Following standard evaluation under the standard white light, PDD was performed using a xenon lamp (300W) with blue light with a 375-445nm wavelength to detect fluorescence.Excision of tumor nodules from peritoneal surfaces was performed and subjected to histological examination and molecular analysis.Then cytoreductive surgery (CRS) with peritonectomy and hyperthermic intraperitoneal chemotherapy (HIPEC) were performed in all patients with PSM as described previously by Sugarbaker (1998).
Detection of PEPT1 and ABCG2 expression levels Total RNA was extracted from tissue samples, and first-strand cDNA was prepared from total RNA by RT reaction, as described previously (Hagiya et al., 2008).Thereafter, the first-strand cDNA preparations encoding PEPT1, ABCG2, and GAPDH were individually amplified by PCR in a Thermal Cycler Dice Mini (TaKaRa Bio, Otsu, Japan) with specific primer sets as previously reported (Hagiya et al., 2012).GAPDH is used as the internal control.The PCR reaction consisted of hot-start incubation at 95 o C for 5min and 35 cycles each of 95 o C for 30s and 60 o C for 1min.The resulting amplicons were separated by 2.5% agarose gel electrophoresis and detected with ethidium bromide under ultraviolet light.

Statistical analysis
Data were collected for all patients included in the study.Microsoft excel 10.0 was used to manage and analyse the data.For statistical analysis, the SPSS version

Diagnostic value of 5-ALA in PSM
A typical observation of metastatic nodules with sequence of white light and PDD is shown in Figure 1.About 0.5 mm tumor nodules were detected by PpIX fluorescence (Figure 2).Overall metastases detected 42 out of 118 patients with PSM (Table 1).Even though, there was no significant improvement in visualization of peritoneal tumor lesions, PDD was allowed enhanced detection of metastatic nodules of cholangiocarcinoma (100%), ovarian/extra ovarian primary peritoneal cancer (89%), and malign mesothelioma (67%).PDD detection rate of tumor nodules from colorectal adenocarcinoma was 53%, pancreas adenocarcinoma was 50%, from gastric and  small bowel adenocarcinoma were 33%, and appendiceal mucinous adenocarcinoma was 17%.Although very limited number of PSM patients with granulosa cell tumor (n=3) and GIST (n=4) investigated, tumor nodules were not visualized with PDD in both groups of patients.Nausea (n=1) and vomiting (n=1) as adverse effects following 5-ALA administration were observed in 2 patients (1,6%).Direct exposure of sunlight or strong room light was avoided for 24 hour after operation.Symptoms of porphyria were not observed in any of our patients group.
Characterization of porphyrin biosynthesis pathwayrelated genes PEPT1 and ABCG2 expression levels in 5-ALA-PDD positive specimens of patients with PSM We examined the expressions of PEPT1 and ABCG2 genes involved in the porphyrin biosynthesis pathway in tumor-specific PpIX accumulation following the administration of 5-ALA in vivo in clinical specimens NLR, negative likelihood ratio; PPV, positive predicting value; NPV, negative predicting value of patients with PSM.The mRNA levels of PEPT1 and ABCG2 were measured as shown in Figure 3 and expressions were evaluated relative to those of β-actin expression.PEPT1 mRNA levels were upregulated whereas ABCG2 mRNA levels were downregulated in tumor tissues of PSM patients detected with 5-ALA-PDD (p<0.05; Figure 3).These results suggest that high PEPT1 expression and low ABCG2 expression play pivotal role in tumor specific PpIX accumulation following 5-ALA administration in PDD of PSM patients.

PEPT1 and ABCG2 protein expressions in 5-ALA-PDD positive specimens of patients with PSM
We further determined the PEPT1 and ABCG2 protein expression levels in tumor tissue samples from PSM.We detected PEPT1 protein expression is upregulated and ABCG2 protein expression is downregulated in tumor tissues detected with 5-ALA-PDD (p<0.05; Figure 4).

Discussion
This study provided promising results in detection of metastatic nodules with 5-ALA induced photodynamic method in selected patients with PSM.Even though overall detection rate was 35.6%, PDD was allowed enhanced detection of metastatic nodules in all cholangiocarcinoma patients, in majority of ovarian/extra ovarian primary peritoneal cancer patients (89%) and malign mesothelioma patients (67%).In addition to this, metastatic tumor nodules could be able to detect in PSM patients originated from colorectal adenocarcinoma (53%), from gastric adenocarcinoma (33%), and appendiceal mucinous adenocarcinoma (17%).On the other hand, PDD was not effective in diagnosis of metastatic peritoneal nodules in PSM patients with granulosa cell tumor and GIST.Although different determination ratio was obtained in PSM patients, sensitivity of this method was 100% if metastases could be able to detect with PDD.
Furthermore, upregulation of PEPT1 (ALA influx transporter) and downregulation of ABCG2 (porphyrin efflux transporter) genes could play pivotal role as an underlying mechanism in 5-ALA induced tumor specific PpIX accumulation for photodynamic diagnosis of peritoneal metastatic nodules in patients with PSM.
5-ALA has approved as a photosensitizer of PDD (Ladner et al., 2001).ALA-PDD guided surgery has improved surgical results and recurrence-free survival rates in patients with malignant glioma (Stummer et al., 2006).Moreover, the introduction of 5-ALA with cystoscopy for early bladder cancer (Daniltchenko et al., 2005;Denzinger et al., 2007) and urinary tract transitional cell carcinoma (Ahmad S et al., 2012) has increased the number of recurrence-free patients and improved recurrence-free survival after five years compared to white-light cystoscopy.
For peritoneal metastasis, intraperitoneal application of 5-ALA improved the sensitivity of peritoneal metastasis identification in ovarian cancer (Loning et al., 2004).Oral 5-ALA administration has improved the detection of peritoneal metastases in patients with pancreatic adenocarcinoma (Orth et al., 2000).Our results from ovarian carcinoma, pancreas adenocarcinoma and cholangiocarcinoma are similar with these previous studies.
5-ALA-PDD improved the detection sensitivity over white-light examination during staging laparoscopy in patients with peritoneal carcinomatosis originated from gastrointestinal cancers (Zopf et al., 2005) and gastric cancer (Hagiya et al., 2012;Zopf et al., 2005).Finally, they conclude that 5-ALA-PDD yielded more lesions detected than the conventional white-light examination.In our study, 5-ALA-PDD also improved the determination of small metastatic nodules less than 0.5mm in patients with PSM.A recent systematic review analyzed 12 human and 18 animal studies (Almerie et al., 2015).They have found that PDD was safe with no significant adverse effects.Detection of malignant peritoneal nodules was increased by 21%-34% in comparison with white light alone.The sensitivity and specificity of PDD were reported at 83%-100% and 95%-100%, respectively.These findings were also supported by multiple animal studies, which have shown an increase in the sensitivity of tumor detection when using PDD (72%-91%) in comparison with white light alone (39%).
In this study, we investigated the roles of PEPT1 and ABCG2 in 5-ALA induced PpIX accumulation in vivo.Our results strongly suggest that PSM share a common mechanism with previously reported in vitro results for tumor-specific PpIX accumulation following the administration of 5-ALA.
There are some limitations of this study; first of all, we had very limited sample size in some subgroups of peritoneal metastases.Specially, sample size for peritoneal metastases of cholangiocarcinoma, granulosa cell tumor and GIST were very limited due to natural occurrence of these disease are rare even metastases are the most rare to complete significant sample size.Another limitation, we only detect the samples in patients with peritoneal metastases.Comparison of diagnostic value of 5-ALA between diseases and their peritoneal metastases as well as other diseases needs to be performed to clarify exact role of 5-ALA based fluorescence production in detection of metastases.
In conclusion, 5-ALA guided surgery can be performed safely with high detection rate of peritoneal implants around 5mm, in subgroups of PSM patients originated from common bile duct and ovarian/extra ovarian primary peritoneal carcinoma compared to those of originated from colorectal, stomach, primary peritoneal malign mesothelial origin and appendiceal mucinous neoplasms to achieve complete cytoreduction.Continued research will clarify whether this diagnostic intervention will be altered the prognosis and therapeutic approaches of PSM.

Figure 1 .
Figure 1.White Light and Photodynamic Detection of Peritoneal Metastatic Nodules in Patients with PSM