cytogenetic analysis plays an important role in the elucidation of the pathogenesis and prognosis of cancers . several techniques are now available for genome - wide screening of alterations in copy number . comparative genomic hybridization ( cgh ) is a molecular cytogenetic tool that allows the screening of copy number change , and has now become one of the most popular genome screening techniques ( 1 ) . the degenerate oligonucleotide primed polymerase chain reaction ( dop - pcr ) cgh technique permits genetic imbalance screening of the entire genome using only small amounts of tumor dna . it has also been successfully applied to microdissected archival paraffin - embedded tissue by several investigators ( 2 , 3 ) . intrahepatic cholangiocarcinoma ( icc ) is the second most common malignant tumor in the liver , arising from epithelial cells in the intrahepatic bile duct ( 4 ) . although the frequency of icc is low , its incidence in southeast asian is higher than that in europe and north america . icc is refractory to conventional chemotherapy and radiation treatment and curative surgical resection is not effective due to the high recurrence rate ( 5 ) . while risk factors including liver fluke infection , hepatolithiasis , congenital cysts , and sclerosing cholangitis have been reported ( 6 ) , the genetic mechanisms involved in the development of icc are not well understood and only a few cytogenetic studies of icc have been published . chromosomal gains of 5p , 7p , 8q , 13q , 15q , 17q , and 20q , and losses of 3p , 4q , 6q , 9p , 16q , 17p , and 18q were reported in icc ( 7 , 8) . the structural abnormalities of chromosomes 1 , 5 , 7 , and 12 were detected in icc cell lines ( 9 ) . our previous study demonstrated the chromosomal aberrations in chromosomes 3 , 6 - 8 , 12 , 14 , 17 , and 18 were analyzed by cross - species color banding ( 10 ) . loss of heterozygosity ( loh ) studies of icc has shown frequent allelic losses from various chromosomes including 1p , 1q , 3p , 6q , 8p , 9q , 14q , 17p , and 18q ( 11 ) . whereas specific genes responsible for the development of icc , genetic alterations of apc , c - erbb2 , k - ras , p53 , and p16 are proposed to be closely related to icc progression ( 12 - 14 ) . in this study , the dnas from 33 korean icc specimens were analyzed by dop - pcr cgh with the aim of identifying the non - random chromosomal aberrations of korean icc . a total 33 formalin - fixed , paraffin - embedded specimens of icc were collected . using a 4 m hematoxylin and eosin stained adjacent section as a guide , tumor - containing areas were microdissected from sectioned 10 m specimens with a sterile 25-gauge needle under a microscope . normal dna from human peripheral blood and tumor dna from microdissected cells were prepared by standard protocols . dop - pcr was performed in two separate steps according to the published protocol with some modifications ( 15 , 16 ) . briefly , in the preamplification step , the four initial cycles were performed at low stringency conditions ( denaturation at 95 for 3 min followed by 4 cycles of 94 for 1 min ; 25 for 1 min ; 56 for 3 min ; 74 for 2 min ) , followed by 35 cycles in high stringency conditions ( at 94 for 1 min ; 56 for 1 min ; 72 for 2 min ) . labellings of tumor dna with biotin-16dutp and control dna with digoxigenin-11dutp were performed by nick translation ( nick translation system , gibco / brl , rockville , md , u.s.a . ) . equal amounts of labeled normal control and tumor dna including 40 g of cot-1 dna ( gibco / brl ) and 1 g salmon sperm dna were precipitated with ethanol . labeled dna probes were mixed with hybridization buffer , denatured at 75 for 5 min and then applied onto the denatured metaphase slides . after incubation for 3 days , the slides were washed and the hybridization signal was detected using an avidin - fluorescein isothiocyanate ( fitc , 1:400 ) and mouse anti - digoxigenin ( 1:100 ) . metaphase slides were counterstained with dapi at a concentration of 0.1 g / ml in an antifade solution ( oncor , gaithersburg , md , u.s.a . ) for chromosomal identification . thresholds for gains and losses were defined as the theoretical value of 1.25 and 0.75 , respectively . the following regions were excluded for the analyses ; centromere , acrocentric short arms , telomere , and heterochromatin - rich region ( 17 ) . a total 33 formalin - fixed , paraffin - embedded specimens of icc were collected . using a 4 m hematoxylin and eosin stained adjacent section as a guide , tumor - containing areas were microdissected from sectioned 10 m specimens with a sterile 25-gauge needle under a microscope . normal dna from human peripheral blood and tumor dna from microdissected cells were prepared by standard protocols . dop - pcr was performed in two separate steps according to the published protocol with some modifications ( 15 , 16 ) . briefly , in the preamplification step , the four initial cycles were performed at low stringency conditions ( denaturation at 95 for 3 min followed by 4 cycles of 94 for 1 min ; 25 for 1 min ; 56 for 3 min ; 74 for 2 min ) , followed by 35 cycles in high stringency conditions ( at 94 for 1 min ; 56 for 1 min ; 72 for 2 min ) . labellings of tumor dna with biotin-16dutp and control dna with digoxigenin-11dutp were performed by nick translation ( nick translation system , gibco / brl , rockville , md , u.s.a . ) . equal amounts of labeled normal control and tumor dna including 40 g of cot-1 dna ( gibco / brl ) and 1 g salmon sperm dna were precipitated with ethanol . labeled dna probes were mixed with hybridization buffer , denatured at 75 for 5 min and then applied onto the denatured metaphase slides . after incubation for 3 days , the slides were washed and the hybridization signal was detected using an avidin - fluorescein isothiocyanate ( fitc , 1:400 ) and mouse anti - digoxigenin ( 1:100 ) . metaphase slides were counterstained with dapi at a concentration of 0.1 g / ml in an antifade solution ( oncor , gaithersburg , md , u.s.a . ) for chromosomal identification . thresholds for gains and losses were defined as the theoretical value of 1.25 and 0.75 , respectively . the following regions were excluded for the analyses ; centromere , acrocentric short arms , telomere , and heterochromatin - rich region ( 17 ) . all chromosomes were involved in imbalances and the mean number of imbalances per tumor specimen was 9 ( range , 2 - 18 ) . table 1 summarizes clinical and chromosomal findings in the 33 entire cases . a schematic summary of copy number changes as detected by cgh analysis in 33 abnormal cases is shown in fig . 1 . among the 33 cases , the common sites of copy number increases , in order of frequency , were , 20q ( 67% ) , 17 ( 61% ) , 11q11-q13 ( 42% ) , 8p12-qter ( 39% ) , 18p ( 39% ) , 15q22-qter ( 36% ) , 16p ( 36% ) , 3q25-qter ( 27% ) , 1q41-qter ( 24% ) , and 5p14-q11.2 ( 24% ) ( fig . sixteen tumors exhibited one or more sites of dna amplification ; the frequent sites of amplification were 20q13.2-qter ( 6 cases ) , 17p ( 3 cases ) , 17q23-qter ( 2 cases ) , and 7p ( 2 cases ) . the most frequent sites of copy number decreases were 1p32-pter ( 21% ) and 4q ( 21% ) . gains of 11q11-q13 , 17 , and 18p were more commonly detected in moderately and poorly differentiated tumors . the analysis of recurring chromosomal aberrations has become an integral part of the diagnostic and prognostic workup in many human cancers , and their molecular analyses have facilitated the identification of genes related to the pathogenesis of cancers . however , the characterization of complex chromosomal rearrangements was limited when conventional cytogenetic methods were used . in this study thirty - three icc were successfully analyzed using dop - pcr cgh . in consistent with our result , a few alteration such as copy number increases of 5p , 8q , 15q , 17q , and 20q the most common gain region was 20q , with eight tumors showing high amplification of 20q13-qter . gains of 17q and 20q are common finding in several solid tumors including hepatocellular carcinoma , pancreas , and breast tumors ( 18 - 20 ) . chromosome arm 20q contains many genes such as e2f1 ( 20q11 ) , aib1 ( 20q12 ) , btak / stk15 ( 20q13 ) , and nabc14 ( 20q13.2 ) , which are involved in several cancers ( 21 - 25 ) . gene amplification and protein expression of c - erbb-2 ( her-2/neu ) on 17q21 were observed in icc and breast cancer ( 26 , 27 ) . in particular , gains of 3q26-qter , 6p21 , 11q11.2-q13 , 16p and 18p are frequently observed in this study ( fig . chromosome region 3q26-qter harbors pik3ca and ect2 proto - oncogenes that are related to cell proliferation and carcinogenesis ( 28 ) . the frequent over - represented pik3ca has also been reported in ovarian , head and neck , and stomach cancers ( 29 - 31 ) . gain of 6p with a minimum overlapping region at 6p21 was frequently observed in this study . among the genes mapped to 6p21 , pim1 was known as a proto - oncogene related to the progression of thymic lymphomas in transgenic mice , although the role of pim1 in the other cancers is not elucidated ( 32 ) . in addition , ccnd3 on 6p21 shares considerable homology with proto - oncogene cyclin d1 and is involved in the cell cycle regulation at g1 to s phase transition ( 33 ) . gain of 11q13 is a common event in human cancers and is associated to poor prognosis ( 34 , 35 ) . chromosomal band 11q13 contained several cancer related genes such as fgf3 , fgf4 , and ems1 , and only ccnd1 and ems1 ( 36 , 37 ) . losses of 1p and 4q have been reported in icc ( 7 , 8) , in consistent with our current result ( fig . previous reports indicated that 1p loss is related to the early stage of hepatocarcinogenesis ( 38 ) and at least three or more tumor suppressor genes including the p73 gene are localized on 1p 36 . the deletion of 1p36 was reported to be related to the progression of icc without metastatic activity ( 39 ) . allelic loss from 4q appears to be a fundamental event in the development of hcc ( 40 ) . in conclusion , although chromosomal loss was found to be less frequent in this study , we were able to identify several genetic changes , which were previously reported . these findings suggest that the technical improvement in dop - pcr - cgh technique may reliably enhance the sensitivity and accuracy of the analysis . the recurrent gains and losses of chromosomal regions identified in this study provide candidate regions containing oncogenes or tumor suppressor genes involved in icc development and progression .
intrahepatic cholangiocarcinoma ( icc ) , a malignant neoplasm of the biliary epithelium , is usually fatal because of difficulty in early diagnosis and lack of availability of effective therapy . 
 the genetic mechanisms involved in the development of icc are not well understood and only a few cytogenetic studies of icc have been published . 
 recently , technique of degenerate oligonucleotide primed ( dop)-pcr comparative genomic hybridization ( cgh ) permits genetic imbalances screening of the entire genome using only small amounts of tumor dna . in this study 
 chromosomal aberrations in 33 korean icc were investigated by dop - pcr cgh . 
 the common sites of copy number increases were 20q ( 67% ) , 17 ( 61% ) , 11q11-q13 ( 42% ) , 8p12-qter ( 39% ) , 18p ( 39% ) , 15q22-qter ( 36% ) , 16p ( 36% ) , 6p21 ( 30% ) , 3q25-qter ( 27% ) , 1q41-qter ( 24% ) , and 5p14-q11.2 ( 24% ) . 
 dna amplification was identified in 16 carcinomas ( 48% ) . 
 the frequent sites of amplification were 20q , 17p , 17q23-qter , and 7p . 
 the most frequent sites of copy number decreases were 1p32-pter ( 21% ) and 4q ( 21% ) . 
 the recurrent chromosomal aberrations identified in this study provide candidate regions involved in the tumorigenesis and progression of icc .