many patients with non - small cell lung cancer or gastrointestinal tract cancer have pleural effusion or ascites at diagnosis . generally ,
cytological examination is the most common method to detect malignant cells in pleural fluid or ascites ; however , the technique is known to miss up to more than 50% of tumors .
it is crucial to detect malignant cells in pleural fluid or ascites for staging work - up and for decision of treatment strategy , especially in non - small cell lung cancer .
pleural or peritoneal biopsy , which is the  gold standard  for diagnosis , is invasive for routine examination .
hence , it is important to develop a sensitive and less invasive diagnostic method to improve the detection of malignant cells in pleural fluid or ascites .
cd44 is a cell surface adhesion molecule implicated in diverse biologic processes , such as lymphocyte activation and homing , extracellular matrix adhesion and cellular migration .
cd44 is composed of at least 20 exons , of which 10 exons can be alternatively spliced into different combinations to make up various isoforms .
it has been reported that various human malignant tumors have been associated with over - expression of cd44 variant isoforms , and widespread attention has been focused on cd44 variant isoforms a candidate marker for detection of cancer .
however , the variant exons that have been associated with cancer have been detected in corresponding normal tissues in other studies .
recently , a novel molecular approach , cd44v8 - 10/cd44v10 competitive rt - pcr , has been developed for the detection of cancer cells over - expressing cd44v8 - 10 .
this method is based on the concept that the relative expression of cd44v8 - 10 transcripts to cd44v10 transcripts can be quantified by using the endogenous cd44v10 transcripts as a source of internal standard competitor rna .
we analyzed from benign and malignant pleural effusion and ascites by cd44 competitive rt - pcr for the detection of cancer cells over - expressing cd44v8 - 10 .
ags , a human gastric adenocarcinoma cell line(korean cell line bank , seoul , south korea ) was maintained in rpmi supplemented with 10% heat - inactivated fetal calf serum(fcs ) , 100 unit / ml penicillin and 0.1mg / ml streptomycin at 37c in an atmosphere containing 5% co2 .
the pleural fluid or ascites were obtained from 11 patients with malignant disease ( 5 non - small cell lung cancer , 5 gastric cancer , 1 pancreatic cancer ) and 24 patients with benign disease ( 14 tbc pleurisy , 10 liver cirrhosis ) , who were treated at hanyang medical center ( table 1 , 2 , 3 ) .
cellular components from pleural fluid or ascites were examined by both cytological and competitive rt - pcr analysis . for competitive rt - pcr analysis , 25 ml of pleural fluid or
ascites samples obtained from patients were centrifuged at 3,000 rpm for 10 min and the precipitated cells were washed with phosphate- buffered saline ( pbs ) and stored at 70c until use .
total rna was spectrophotometrically quantified at 260/280 nm and its quality was tested in 1% agarose gel to find intact 28s , and 18s rnas .
cdna was synthesized from 5 g of total rna in a 25 l reaction mixture containing 5 l of 5  reverse transcriptase reaction buffer , 200 m dntp , 100 m of random hexamer , 50 units of rnasin , 2 l of 0.1 m dithiothreitol , and 200 units of moloney leukemia virus reverse transcriptase .
the mixture was incubated at 37 c for 60 min , heated to 95 c for 10 min , and then chilled on ice .
the competitive rt - pcr was performed by using the sense primer , s2 ( 5-gacagaatccctgctaccaata-3 ) and the antisense primer , as2 ( 5-atgtgtcttggtctcctgataa-3 ) that were designed to amplify both cd44v8 - 10 and cd44v10 cdna , simultaneously , by utilizing sequence identity(17 ) .
five microliters of cdna were transferred into an eppendorf tube containing 200 m dntp , 1.5 mm mgcl2 , 2.5 u taq polymerase and 20 pmoles of primer s2 and as2 .
thirty - five cycles of amplification were performed in a thermocycler ( robocycler 40 : stratagene , la jolla , ca ) at 94 c for 1 min , 56 c for 1 min , and 72 c for 1 min , with a final extension step performed for 10 min at 72 c. to ensure the rna was of sufficient purity for rt - pcr , a pcr assay with primers specific for the gene -actin cdna was carried out in each case .
the primer sequences for -actin primers were as follows : 5-tgacggggtcacccacactgtgcccatcta-3 and 5-ctagaagcattgcggtggacgatggaggg-3. each series of rt - pcr reactions included mononuclear cell samples from peripheral blood as a negative control and ags cell samples as a positive control , respectively .
the pcr products were electrophoresed on a 1.5% agarose gel and visualized by ethidium bromide staining followed by uv transillumination .
the resulting images were density scanned and peak intensity of each band corresponding to the amplimers of cd44v8 - 10 and cd44v10 were then quantified by use of image analyzer , and cd44v8 - 10/cd44v10 ( v8- 10/v10 ) ratio was calculated ( biorad , fluor - s image analyzer , bms , usa ) .
all the samples were assessed in 2 independent pcr reactions , and the reported v8- 10/v10 ratios represent the mean values from these duplicate determinations .
ags , a human gastric adenocarcinoma cell line(korean cell line bank , seoul , south korea ) was maintained in rpmi supplemented with 10% heat - inactivated fetal calf serum(fcs ) , 100 unit / ml penicillin and 0.1mg / ml streptomycin at 37c in an atmosphere containing 5% co2 .
the pleural fluid or ascites were obtained from 11 patients with malignant disease ( 5 non - small cell lung cancer , 5 gastric cancer , 1 pancreatic cancer ) and 24 patients with benign disease ( 14 tbc pleurisy , 10 liver cirrhosis ) , who were treated at hanyang medical center ( table 1 , 2 , 3 ) . cellular components from pleural fluid or ascites were examined by both cytological and competitive rt - pcr analysis . for competitive rt - pcr analysis , 25 ml of pleural fluid or
ascites samples obtained from patients were centrifuged at 3,000 rpm for 10 min and the precipitated cells were washed with phosphate- buffered saline ( pbs ) and stored at 70c until use .
total rna was spectrophotometrically quantified at 260/280 nm and its quality was tested in 1% agarose gel to find intact 28s , and 18s rnas .
cdna was synthesized from 5 g of total rna in a 25 l reaction mixture containing 5 l of 5  reverse transcriptase reaction buffer , 200 m dntp , 100 m of random hexamer , 50 units of rnasin , 2 l of 0.1 m dithiothreitol , and 200 units of moloney leukemia virus reverse transcriptase .
the mixture was incubated at 37 c for 60 min , heated to 95 c for 10 min , and then chilled on ice .
the competitive rt - pcr was performed by using the sense primer , s2 ( 5-gacagaatccctgctaccaata-3 ) and the antisense primer , as2 ( 5-atgtgtcttggtctcctgataa-3 ) that were designed to amplify both cd44v8 - 10 and cd44v10 cdna , simultaneously , by utilizing sequence identity(17 ) .
five microliters of cdna were transferred into an eppendorf tube containing 200 m dntp , 1.5 mm mgcl2 , 2.5 u taq polymerase and 20 pmoles of primer s2 and as2 .
thirty - five cycles of amplification were performed in a thermocycler ( robocycler 40 : stratagene , la jolla , ca ) at 94 c for 1 min , 56 c for 1 min , and 72 c for 1 min , with a final extension step performed for 10 min at 72 c. to ensure the rna was of sufficient purity for rt - pcr , a pcr assay with primers specific for the gene -actin cdna was carried out in each case .
the primer sequences for -actin primers were as follows : 5-tgacggggtcacccacactgtgcccatcta-3 and 5-ctagaagcattgcggtggacgatggaggg-3. each series of rt - pcr reactions included mononuclear cell samples from peripheral blood as a negative control and ags cell samples as a positive control , respectively .
the pcr products were electrophoresed on a 1.5% agarose gel and visualized by ethidium bromide staining followed by uv transillumination .
the resulting images were density scanned and peak intensity of each band corresponding to the amplimers of cd44v8 - 10 and cd44v10 were then quantified by use of image analyzer , and cd44v8 - 10/cd44v10 ( v8- 10/v10 ) ratio was calculated ( biorad , fluor - s image analyzer , bms , usa ) .
all the samples were assessed in 2 independent pcr reactions , and the reported v8- 10/v10 ratios represent the mean values from these duplicate determinations .
to examine the validity of the rt - pcr method as a quantitative assay , 210 normal mononuclear cells were mixed in separate tests with an increasing number of ags gastric cancer cells expressing cd44v8 - 10 .
the resultant mixtures were subjected to competitive rt - pcr . as shown in fig . 1 when the v8- 10/v10 ratios were plotted against the number of added ags cells , linear relationships were observed .
twenty one samples , 14 from patients with benign pleural fluid and 7 from patients with malignant effusion were analyzed by competitive rt - pcr by using the set of primers , s2 and as2 , that can co - amplify cd44v8 - 10 and cd44v10 .
the cd44v10 transcript was predominantly expressed in comparison with the cd44v8 - 10 transcript in all 14 of the pleural effusion samples from patients with benign disease ( v8 - 10/v10 ratio : 0.2190.948 ) , whereas 6 of 7 samples from patients with malignant cancers ( 5 non - small cell lung cancer , 2 gastric cancer ) presented a predominant expression of cd44v8 - 10 transcript ( table 1,3 ) .
the representative data are shown in figure 2 and 3 . when we place the diagnostic cutoff value for the v8 - 10/v10 ratio at 1.0 , the sensitivity and specificity of competitive rt - pcr were 85.7% and 100% , respectively .
fourteen samples , 10 from patients with benign ascites ( 10 liver cirrhosis ) and 4 from patients with malignant ascites ( 3 gastric cancer , 1 pancreatic cancer ) were analyzed as previously described .
< 1.0 ( range : 0.1260.756 ) . however , only one of four malignant ascites ( gastric cancer ) revealed > 1.0 ( table 2,3 ) .
to examine the validity of the rt - pcr method as a quantitative assay , 210 normal mononuclear cells were mixed in separate tests with an increasing number of ags gastric cancer cells expressing cd44v8 - 10 .
the resultant mixtures were subjected to competitive rt - pcr . as shown in fig . 1 when the v8- 10/v10 ratios were plotted against the number of added ags cells , linear relationships were observed .
twenty one samples , 14 from patients with benign pleural fluid and 7 from patients with malignant effusion were analyzed by competitive rt - pcr by using the set of primers , s2 and as2 , that can co - amplify cd44v8 - 10 and cd44v10 .
the cd44v10 transcript was predominantly expressed in comparison with the cd44v8 - 10 transcript in all 14 of the pleural effusion samples from patients with benign disease ( v8 - 10/v10 ratio : 0.2190.948 ) , whereas 6 of 7 samples from patients with malignant cancers ( 5 non - small cell lung cancer , 2 gastric cancer ) presented a predominant expression of cd44v8 - 10 transcript ( table 1,3 ) .
when we place the diagnostic cutoff value for the v8 - 10/v10 ratio at 1.0 , the sensitivity and specificity of competitive rt - pcr were 85.7% and 100% , respectively .
fourteen samples , 10 from patients with benign ascites ( 10 liver cirrhosis ) and 4 from patients with malignant ascites ( 3 gastric cancer , 1 pancreatic cancer ) were analyzed as previously described .
< 1.0 ( range : 0.1260.756 ) . however , only one of four malignant ascites ( gastric cancer ) revealed > 1.0 ( table 2,3 ) .
it has been known that conventional cytological analysis does not have high sensitivity to detect malignant cells in pleural fluid or ascites . at present , there is no reliable biological marker that is used for routine clinical examinations .
recently , molecular biological techniques make it possible to detect cancer cells in those samples . since it has been reported that a cd44 splice variant containing variant exon 6 could confer metastatic potential on rat pancreatic adenocarcinoma cells , a great deal of interest
many investigators reported that the expression of specific cd44 variant exons was detected in human carcinomas , such as bladder cancer , non - small cell lung cancer and breast cancer , indicating that molecular approaches of cd44 splice variant might be a promising tool for cancer diagnosis .
other investigators demonstrated that among the cd44 splice variants , cd44v8 - 10 was predominantly expressed in human colon cancer and non - small cell lung cancer by rt - pcr using the set of primers that are able to amplify all cd44 variant isoforms and dna sequence analysis .
however , cd44v10 was also expressed in normal leukocytes , so the discrimination method should be needed .
recently , okamoto et al developed noble competitive rt - pcr with which to quantify the relative expression of cd44v8 - 10 transcripts to cd44v10 transcripts .
the endogenous cd44v10 transcripts , which were identified as the predominant cd44 splice variants in leukocytes , were used as internal standard , competitor rnas for measuring the expression level of cd44v8 - 10 transcripts .
when we applied the competitive rt - pcr assay to pleural fluid and ascites , our results showed that the v8 - 10/v10 ratios in all the samples associated with benign disease were less than < 1.0 , whereas malignant diseases revealed significantly higher than those associated with benign diseases .
although the v8 - 10/v10 ratio displayed large variation between the benign diseases , the ratios were consistently low in various benign diseases suggesting that the v8- 10/v10 ratio might be a useful molecular marker for cancer diagnosis .
these findings indicate that the cd44v8 - 10/v10 ratio may be an important molecular marker for cancer diagnosis , especially in exfoliated cells from pleural fluid .
cd44 competitive rt - pcr assay is a useful and adjunct to cytological examination in cancer diagnosis , especially in detecting exfoliated cancer cells in pleural effusion . to determine the exact role of cd44 variant in the diagnosis of exfoliated cells from pleural
 background : cd44 is a cell surface adhesion molecule which has been implicated in various biologic functions as lymphocyte homing and activation , cellular migration and extracellular matrix adhesion . over - expression of cd44v8 - 10 has been found in several cancers and is considered to be associated with tumor progression and metastasis . 
 recently , a novel molecular method , cd44v8 - 10/cd44v10 competitive reverse transcription - polymerase chain reaction ( rt - pcr ) has been developed for detecting cancer cells over - expressing cd44v8-10.methods : we analyzed from benign and malignant pleural effusion and ascites by cd44 competitive rt - pcr and compared to the conventional cytology.results : the cd44 competitive rt - pcr analysis showed that all the 24 samples associated with benign disease presented a predominant expression of the cd44v10 transcript ( v8 - 10/v10 ratio : 0.1260.948 ) , whereas 6 of 7 malignant pleural samples associated with cytology positive cancer expressed the cd44v8 - 10 transcript ( v8 - 10/v10 ratio > 1.00).conclusion : these results indicate that cd44 competitive rt - pcr assay is a useful and adjunct to cytological examination in cancer diagnosis , especially in detecting exfoliated cancer cells in pleural effusion . 
Introduction
MATERIALS AND METHODS
Cancer cell lines
Pleural fluid or Ascites
RNA Preparation and Competitive RT-PCR
RESULTS
Quantitation of CD44v8-10 variant by CD44v8-10/CD44v10 competitive RT-PCR
Competitive RT-PCR for pleural samples
Competitive RT-PCR for ascites samples
DISCUSSION
CONCLUSION