日本産科婦人科学会香川地方部会雑誌 vol.8， No.，1pp.19 -21， 2006(平18.9月) + 者
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Yasuko Yamamoto1，
3)，
Kohkichi Hata2)，
Hiroyuki Ohsaki3)，
Tomoko T. Yamanushi3)，
Tadao Shiooka4)
，
Masayuki Nakano 4)，
Eiichiro Hirakawa3)u Department of Laboratory Science
，
School of Health Sciences，
Faculty of Medicines，
Kyoto University2) Department of Nursing
，
Kagawa Prefectural Col1ege of Hea1th Sciences3)Laboratory ofPathology
，
Department ofMedical Technology，
Kagawa Prefectural Col1ege ofHealth Sciences 4) Shikoku Cytopathologic InstituteAbstract
OBJECTIVE: The aim of this study was to evaluate whether podoplanin mRNA expression in body cavity fluids can be used as an adjunctive tool for cytological diagnosis.
METHODS : Twenty-two body cavity fluids
，
including 9 pleural fluids，
11 ascitic fluids，
1 peritoneal washig，
and 1 pericardial fluid were obtained from 22 patients. The podoplanin gene expression was assessed by real -time quantitative reverse transcription-polymerase chain reaction (RT同PCR).
RESULTS: The podoplanin expression level of negative samples of cytological diagnosis (meadian; 0.134
，
range; 0.002・0.542) was not significant from that of positive samples of cytological diagnosis (meadian;
0.048
，
range; 0.009・0.102).CONCLUSIONS: Although further examination is necessary
，
mRNA expression of podoplanin in body cavity fluids assessed by real-time quantitative RT-PCR will be useful in improving the accuracy of cytological diag -nosls.Key words: body cavity fluids
，
podoplanin，
mRNA expressionIntroduction In cytological preparations
，
reactive mesothelial ce11s in body cavity fluids are sometimes difficult to distin -guish from adenocarcinoma cells. Since Wang et al.1)in -dicated出atcarcino巴mbryonicantigen (CEA) could be se1'ved as an immunohistochemical ma1'ker in the diagnosis of mesothelioma. Although a number of other markers have been found to be help白1in cytological diagnosis， up to date， a good sensitive and specific marker fo1'mesothel -ial cells has not yet been1'ecognized. Acco'1dingly， the immunocytochemical distinction of mesothelial c巴11f1'om adenocarcinoma ce111argely d巴pendson the combined use of several markers， which are frequently expressed in mか sothelial ce11 (anti-human mesothelial ce11: HBME-l， Calretinin， thrombomodulin) and in ca1'cinomas (epithelial specific antigen: MOC同1，CEA， anti-human epithelial antigen: Ber-Ep4)，
respectively. Podoplanin， an approximately 38-kd membrane mucoprotein， is recently recognized as a ma1'ker ofmeso -thelioma. It was o1'iginally detected on the su1'face of rat glomemlar epithelial ce11s (podocyt巴s) and was found to be linked to the flattening ofthe foot processes in puromy -cin-induced nephrosis2. R)ecently，
Kimu1'a et al，3)1'eported that positive immunoreactivity for podoplanin were found in 5 of5 epithelioid mesotheliomas，
but in none of93 vari -ous adenocarcinomas， and suggested that this marker could assist in distinguishing between reactive mesothelial ce11 and adenoca1'cinoma. In the present sωdy，
we examined the mRNA ex閏 pression of podoplanin using the real-time quantitative1'e -verse t1'ansc1'iption-polymerase chain1'eaction(R'下PCR) in body cavity fluids， in order to assess whether the g巴n巴 巴xp1'巴ssionofpodoplanin can discriminat巴thereactive me-sothelial ce11 from adenocarcinoma cell. Materials and methods Cytology SpecimensFrom November 2005 to January 2006， body cavity
20 The Quantitative mRNA Detection of Podoplanin in Body Cavity Fluids 産婦香川会誌8巻 1号 Table 1 Clinicopathologic data in body cavity fluids Case Specimen source Cytologic diagnosis Histological diagnosis Podoplanin expression 1巴V巴I 1 Pleural fluid Negative， inflammatory cell N/A 0.004 2 Ascitic fluid Negative， inflammatory cell N/A 0.091 3 Ascitic fluid Negative， inflammatory cell N/A 0.133 4 Ascitic fluid Negative， inflammatory c巴II N/A 0.419 5 Pleural fluid Negative， inflammatory cell N/A 0.134 6 Pleural fluid Negative， inflammatory cell N/A 0.542 7 Pleural fluid Negative， inflammatory cell N/A 0.300 8 Ascitic fluid Negative， inflammatory cell N/A 0.013 9 Pleural fluid Negative， inflammatory cell N/A 0.008 10 Ascitic fluid Negative， inflammatory cell N/A 0.002 11 Pleural fluid Negativ， i巴 nflammatory cell N/A 0.145 12 Pleural fluid Negative， inflammatory cell N/A 0.003 13 Peritoneal washing Negative N/A 0.145 14 Ascitic fluid N巴gative，reactive mesothelial cell N/A 0.459 15 Ascitic fluid Positive， adenocarcinoma 16 Ascitic fluid Positive， adenocarcinoma 17 Pericardial fluid Positive， adenocarcinoma 18 Ascitic fluid Positive， adenocarcinoma 19 Ascitic fluid Positive， adenocarcinoma 20 Ascitic fluid Positive， adenocarcinoma 21 Pleural fluid Positive， adenocarcinoma 22 Pleural fluid Positive， ad巴nocarcinoma fluids submitted for cytological examination were collec -ted at the Shikoku CytopathologicInsti旬te
，
Kagawa，
Ja開 pan. We evaluated 22 cytological specimens of serous eι 白sions (9 pleural，
11 peritoneal，
1 peritoneal washing and 1 pericardial). These specimens w巴recollected from metastatic adenocarcinoma (n=8)，
inflammatory cells and reactive mesothelial cell (n= 14)，
respectively. The primary sites of metastatic adenocarcinoma were the lung cancer (n=3)， gastric cancer (n=2)， pancr巴a，ticcancer (nニ1，) and ovarian cancer (n=2) (Table 1). In all cases，
the diagnosis was later confrrmed by clinical or pathological findings. Specimens for remnant cytological examination were stored at必Ooc. RNA preparation and quantitative real-time PCR procedureTotal RNA was isolated企ombody cavity fluids using TRIZOL reagent (Invitrogen Lifi巴Technology
，
Car-lsbad
，
CA). The 5μg of total RNA was used for the sy任thesis of single-strand complementary DNA (cDNA) with random primer and reverse transcriptase (RT; promega Corp.， Madison， WI) in total volume of20'&， and th巴n 3
，
&
was used as a template for real-time PCR. We performed real幽timequantitative PCR using the TaqMan system (Applied Biosystems，
Foster City，
CAふ
The expression levels of podoplanin and intemal reference GAPDH were measured by multiplex PCR using TaqMan probes labeled with 6-carboxyfluorescein (FAM) or VIC，
respectively. The podoplanin and GAPDH primer/probe set were purchased合nmApplied Biosystems. Real幽time PCR amplification and product detection were performed using an ABI PRIS乱17300sequence system (Applied Bio -Pancreatic cancer 0.102 Ovarian cancer 0.009 Lung cancer 0.071 Ovarian cancer 0.009 Gastric cancer 0.035 Gastric cancer 0.023 Lung cancer 0.066 Lung cancer 0.061 N/A= not available systems)
，
following the recommended protocol企 ommanu-facture. The simultaneous measurement of podoplanin gene-FAM and GAPD日開VICpermitted normalization of the amount of cDNA added per sample. Th巳quantityofcDNA for podoplanin gene was normalized to the quantity of GAPDH cDNA in each sample. Relative expression was detetmined by using the
L
J
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method according to the manufacturer's protocol (User Bulletin #2). Each assay included a standard curve sample in duplicate，
a no-tenト plate control and a cDNA sample for fluid specimen in trip開 licate. Samples with a coefficient ofvariance higher than 10 % were retested. Statistical analysis Mann-Whitney U test was us巴das appropriate for the evaluation of significant differences betw巴enend-poin -ts.Pく0.05was considered significant.R
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Each podoplanin expression level in each specimen was shown in Table1.The podoplanin expression level of negative samples of cytological diagnosis (meadian; 0.134，
rang巴
;
0.002 -0.542) was not significant from that ofposi司 tive samples of cytological diagnosis (meadian; 0.048，
range; 0.009 -0.102).D
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The distinction of reactive mesothelial cell from metastatic adenocarcinomas to the serosal membranes is sometimes very difficult in cytological diagnosis. VariousOLIVE 香川大学学術情報リポジトリ
2006年9月
markers such as HBMι1， calretinin， MOC-31， Ber・"
EP4
，
and BG8 have been used to distinguish them. How-eve耳 cyotochemicalstains are relatively insensitive and lack of specificity4，5).Politi et a.l6) assessed the useぬlnessofthe叩ti
-bodies HBME-l， calretinin， MOC-31， Ber-EP4， and BG8. Statistical significance was found with HB恥fE-l
，
calr・'etinin
，
MOC-31 and Ber-EP4 in the differential diag-nosis ofreactive m巴sothelialcell proliferations
，
malignantmesothelioma
，
and metastatic adenocarcinoma from 134 serous effusions. The sensitivity ofHBME-l and calretinin for mesothelial cells was 98% and 100%，
and th巴specifi回city was 71 % and 80%
，
r巴，spectively. Both antibodies sta-in巴dreactive m巴sothelialcells as well as malignant
meso-thelioma cells
，
with calr'etinin showing a stronger intensity of imrnunostaining. The sensitivity of the stain for aderトocarcinoma was 86.3% for MOC-31
，
77.5% for B巴r-EP4，
and 67.5% for BG8
，
and when those stains w巴recombined，
the sensitivity was 100%. These data suggest that immun-ocytochemical studies performed on Papanicolaou-stained cytological smears with HBME-l， calretinin， MOC-31，
Ber-EP4
，
and BG8 proved to be useful in the differentiation between metastatic adenocarcinoma and mesothe1ial pro -liferation6). Politi et a.l6)concluded that no single diag圃 nostic technique alone can establish th巴diagnosisin all cases ofbody cavity fluid， 巴ithermalignant or benign. In difficult cases，
which imrnunocytochemistry is necessary，
we believe that the combination of various markers may significantly improve the diagnostic accuracy.Recent1y
，
Kimura et al.3)showed the podoplaninis a usefull maker of mesothelial cell， They perf01med im -munohistochemical staining of podoplanin in 5 malignant mesotheliomas and 118 other tumors
，
including 93 aden同 ocarcinomas，
4 squamous cell carcinomas，
6 gastrointest -inal stromal tumors and 5 endocrine tumors. Imrnunoreac -tivity for podoplanin was demonstrated positive on the membrane of tumor cells in all mesotheliomas. All the other tumors were negative for podoplanin. Among many markers used for differential diagnosis of malignant meso-thelioma，
podoplanin has the potential to be an excellent tumor marker in both specificity and sensitivity.We examined the podoplanin mRNA expression in body cavity fluids using quantitative real-time RT・.PCR
，
and noted that the n
山本他
tumor markers such as M OC司1
，
CEA，
and Ber-Ep4 mightsignificant1y improv巴thediagnostic accuracy. Th巴mRNA expression of podoplanin assessed by real同timequantitative RT-PCR paves the way for白rtherinvestigation to improve the accuracy ofthe cytological diagnosis.
