We performed a genome-wide scan for loss of heterozygosity (LOH) in 22 intrahepatic cholangiocarcinoma (ICC) cases using 168 polymorphic microsatellite markers throughout all of the human chromosomes and 48 markers of which LOH is reportedly characteristic of hepatocellular carcinoma (HCC). Markers with LOH in more than 30% of informative cases were observed at 21 loci. Among these, eight markers on 6q (three loci), 4q (two loci), 9q, 16q, and 17p shared high frequencies of LOH with HCC in our previous study. As for gross appearance, mass-forming type tumors showed higher frequency of LOH (P < 0.001) compared with other types. Compared by tumor size (≤5 cm versus >5 cm), number (multiple versus solitary), and the International Union Against Cancer TNM classification (stage IVB versus II-IVA), LOH was observed more frequently in advanced stages (P < 0.01, respectively). However, LOH frequency does not differ regardless of lymph node status (pN0 versus pN1). Frequent LOH on 1p36 including the p73 locus was noted in large tumors without lymph node metastasis. These suggest that ICC shares some common carcinogenic steps with HCC such as LOH of 4q and 6q and that inactivation of tumor suppressor genes on chromosome 1p36 contributes to progression of ICC but not to metastatic traits.

ICC3 constitutes about 1 of 20 of primary liver cancers in Japan (1), and the number of ICCs treated surgically has been increasing according to the development of the imaging diagnosis. ICC shows quite different characteristics from HCC. The prognosis of ICC is generally poorer than that of HCC because the population at high risk such as subjects with viral infection and liver cirrhosis is not well characterized and ICC tends to metastasize to lymph nodes. However, ICC is still treated according to the same criteria as HCC. The Liver Cancer Group of Japan has proposed classification of ICC according to the gross appearances such as MF type, PI type, intraductal growth type, and combined type (2). Although the 5-year survival rate after surgery for the MF or intraductal growth type is higher than that for the MF + PI type (3), underlying molecular biological differences and clinicopathological features are not yet well understood.

Microsatellites are a class of short tandem repeats widely and evenly distributed throughout the whole genome (4), and these allow us to assess LOH (3) and microsatellite instability of various malignancies (5, 6, 7, 8). LOH in a chromosomal region is thought to be indicative of the presence of putative tumor suppressor genes at the locus (9, 10). Among primary liver cancers, genome-wide scan of LOH has been performed only for HCC (11, 12, 13, 14) but not for ICC.

To determine affected sites for putative tumor suppressor genes characteristic of ICC and to elucidate differences of genetic alterations between ICC and HCC, we performed a genome-wide scan of LOH analysis in 22 ICC cases using 168 polymorphic microsatellite markers at an interval of every 25 cM throughout all of the human chromosomes (screening markers) and 48 LOH markers reported to be associated with HCC (HCC hotspot markers). Additionally, we performed fine mapping of chromosome 1p36, one of the affected regions characteristic of ICC in this study, with 13 markers including seven screening and HCC hotspot markers. The relationship between the frequency and the location of LOH and clinicopathological features of ICC was also evaluated. This is the first large-scale study of genomic alterations in ICC using microsatellite markers.

Patients.

We investigated 21 patients with ICC who underwent surgical resection at the Department of Gastroenterological Surgery, Kyoto University (Kyoto, Japan) or its satellite hospitals and one autopsy case without surgical intervention (Table 1). In all of the cases, pathological diagnosis was confirmed independently by two different pathologists. The patients were 62.6 ± 12.0 years of age at diagnosis (mean ± SD; range, 29–79 years of age) and consisted of 11 males and 11 females. Clinical staging was determined according to the International Union Against Cancer TNM classification, and all of the cases were classified on the basis of gross appearance (2).

DNA Preparation.

Written informed consent was obtained from each patient according to the guidelines of the Ethical Committee of Kyoto University Faculty of Medicine. Tissue preparation and DNA extraction was performed by the method described previously (15).

Microsatellite Markers.

The microsatellite markers composed of 216 loci were the same as those used previously (15) for analysis of HCC. Of 216 markers, 168 screening markers (CHLC/Weber Human Screening Set 8A) were purchased from Research Genetics, Inc., and 48 HCC hotspot markers (10, 11) were synthesized. Fine mapping of allelic imbalance on chromosome 1p36 was performed in a total of 13 markers with an additional six markers other than screening markers and HCC hotspot markers.

PCR Amplification and Assessment of LOH.

PCR amplification was carried out with more than 10 ng of genomic DNA as a template, 200 mm of each deoxynucleotide triphosphate, 0.4 mm of each primer, 2.0 mm MgCl2, 0.25 units of TaKaRa Ex Taq polymerase, and 10 × Ex Taq Buffer (Takara Shuzo, Shiga, Japan) in a final volume of 10 μl. The PCR conditions were denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 55°C for 45 s, extension at 72°C for 1 min, and final extension at 72°C for 10 min in a thermal cycler (GeneAmp 9700 PCR System; Applied Biosystems, Chiba, Japan). Electrophoresis in a 377XL Automated Sequencer (Applied Biosystems) and LOH analysis was carried out as described previously (15). LOH was quantitatively assessed by calculating the LOH index, which was defined as the allele ratio in the normal tissue divided by the allele ratio in the tumor tissue. If the LOH index was less than 0.5 or more than 2.0, we defined the case as having LOH.

Statistical Analysis.

We correlated LOH frequency with several clinical variables, and the significance of association was determined using the χ2 test.

LOH Study.

PCR was successfully performed in 4202 of 4884 microsatellite markers (86.0%). Of those, 2941 markers (70.0%) showed heterozygosity in the nontumorous part (informative loci), and LOH was found in 484 loci (16.5% of the informative loci). A representative electropherogram with LOH is shown in Fig. 1. Using screening markers, the frequency of LOH (number of loci with LOH/number of informative loci) in each case of ICC ranged from 1.6 to 66.4% (16.8 ± 17.7%; mean ± SD; Table 1). Five cases (22.7%) showed more than 34.5% (mean + 1 SD) of LOH frequency including two of three (66.7%) HCV antibody-positive cases.

LOH Frequency of Each Microsatellite Marker.

LOH frequency for each screening marker ranged from 0 to 44.4% (15.8 ± 10.4%; mean ± SD). As was defined previously (15), if LOH frequency at a locus was more than 30% of informative cases, we determined that LOH frequency of the locus was significantly high. Among 216 loci, we observed 21 loci with significantly high LOH frequency (Table 2). To compare ICC with HCC, LOH frequency of the loci in HCC from our previous study (15) is also presented in Table 2. The most frequent LOH in ICC was found at locus D6S474 (6q21; 44.4%), and LOH of chromosome 6q21-26 was commonly observed in ICC and HCC. Other common loci with high LOH were D16S2624, D6S1277, D4S1538, D6S290, D9S910, D4S1652, and D17S1303 in decreasing order. Among HCC hotspot markers, four loci, i.e., D1S170(9, 16), D4S1538, D6S305, and D6S290(11), showed a significantly high frequency of LOH.

LOH Frequency of Each Chromosome Arm.

Using screening markers, LOH frequency on each chromosome arm, calculated by (total number of markers with LOH)/(total number of informative markers on the same arm level), ranged from 3.7 to 30.8% (16.4 ± 6.8%; mean ± SD). To better understand these findings, LOH frequency at each chromosome arm of HCC was prepared using data from our previous study (15). As shown in Fig. 2, the total incidence of LOH did not differ between ICC and HCC. However, distribution of the LOH loci on chromosomes was quite different. In ICC, only 6q exceeded 30% of LOH, whereas six chromosome arms did in HCC. Chromosome arms with significantly high frequency of LOH in ICC were 6q (30.8%), 9p (29.8%), 17p (26.9%), Xp (26.7%), 1p (25.7%), 9q (25.5%), 6p (23.6%), 3q (23.4%), and 18q (23.2%). Although LOH frequencies of chromosome arms 6q and 9p were significantly high in HCC, they were not the highest. Chromosome arms 4q, 8p, and 16q showed a high frequency of LOH in HCC, whereas they did not in ICC. The LOH profile of chromosome arms with significant LOH frequency is summarized in Table 3. LOH frequency of TP53 locus in ICC was less than half of that in HCC (51.4%) of our previous study (15). LOH frequency was high through a wide range of the 1p36 region.

Fine Mapping of the Distal Region of Chromosome 1p.

Compared with HCC, a high incidence of LOH in chromosome 1p36 was characteristic of ICC. Therefore, we further performed fine mapping of this region to seek common allelic loss (Fig. 3). Thirteen cases (59.1%) showed LOH at one or more loci, and three distinctive loci with significantly high frequency of LOH were denoted. Those were the TP73, the 2.2-cM region between D1S214 and D1S1612 and the 13.3-cM region between D1S1597 and D1S507. LOH of TP73 was observed in 6 of 11 (54.5%) informative cases, and the incidence was the highest in this study.

Correlation between LOH Frequency and Clinicopathological Data.

Relationships between LOH frequencies with three different groups of microsatellite markers and clinical parameters are shown in Table 4. With regard to the gross appearance of the tumor, LOH was more frequent in the MF type than in other types, irrespective of the group of markers (P < 0.001). Using screening markers, LOH frequencies of chromosomes 1p, 6p, and 6q (P < 0.05) were significantly higher in the MF type than in other types (data not shown). Total LOH frequency of screening markers tended to be higher in more advanced tumors such as multiple tumors, tumors larger than 5 cm in diameter, and those in stage IVB. LOH frequency in female cases was significantly higher than in male cases and was especially observed on chromosomes 1q, 4p, 9q, and 12q (data not shown). Infection by HCV is associated with a general increase in LOH frequency. LOH of HCC hotspot markers did not show a clear relationship to clinical parameters except for gross appearance of ICC and tumor size. Interestingly, a high frequency of LOH at chromosome 1p36 was associated with tumor progression without metastasis.

Using highly polymorphic microsatellite markers, we carried out comprehensive analyses of LOH in ICC. A significantly high frequency of LOH was demonstrated in the distal part of chromosome 1p (1p36.12-23). Chromosome 1p35-36 is reportedly an affected region in HCC (9, 10), and its LOH is suggested to be involved in HCC development at early stages (12). However, our previous study (15) did not confirm significant LOH in Japanese HCC cases in this region except for D1S170. The present study suggests that at least three or more tumor suppressor genes including the p73 gene are localized in this region and that deletion in chromosome 1p36 is related to the progression of ICC without metastatic activity.

In the present study, chromosome 6q showed the highest LOH frequency among all of the chromosomes and especially in MF type ICC. In our previous study (15), LOH of the same region is suggested to be associated with the early phase of HCC development, especially in cases without hepatitis virus infection. These suggest that carcinogenic steps of MF type ICC and HCC share some fraction of genetic changes on chromosome 6q. LOH of 6q25-27, which harbors the mannose 6-phosphate/insulin-like growth factor ΙΙ receptor (M6P/IGFIIr) gene, is found in more than two-thirds of HCC cases (17, 18), indicating that inactivation of this gene might be one of common carcinogenic steps toward both ICC and HCC. Furthermore, the present study suggests the involvement of unknown tumor suppressor gene(s), located more centromeric from the M6P/IGFIIr locus in cholangiocarcinogenesis.

In the current study, chromosome 9p showed frequent LOH (29.8%). Recently (19, 20, 21, 22, 23), frequent deletions on chromosome 9p21-22 have been observed in various human cancers, and putative tumor suppressor genes p16/MTS and p15/MTS have been mapped to 9p21-22 (24, 25). These genes have two different pathways of inactivation: hypermethylation of the promoter region or homozygous deletion (26, 27). To date, inactivation of the p15 gene (28) but not the p16 gene (29) is reported to play a role in tumorigenesis of a subset of human HCC. In contrast, the p16 gene rather than the p15 gene is involved in biliary tract carcinogenesis including ICC (30). It would be interesting to determine whether either of these genes is inactivated in ICC because D9S1118, mapped to 9p13.3-21.1, showed frequent LOH in the present study.

The E-cadherin gene, located on chromosome 16q22, plays an important role in a variety of human cancers as a suppressor of invasive growth (31, 32). Inactivation of the E-cadherin gene either by methylation of the CpG island in the promoter region (33, 34) or by LOH (35) contributes to dedifferentiation and invasive growth of HCC. Our study demonstrated a high frequency of LOH of D16S2624 (16q22.1-23.1) in both ICC and HCC, suggesting that inactivation of E-cadherin also plays an important role in ICC formation.

The tumor suppressor p53 gene, located on chromosome 17p13 (TP53), is frequently mutated in a wide variety of human cancers including HCC (36, 37, 38). We have found recently (39) that inactivation of p53 protein through MDM2 overexpression without p53 gene mutations is frequently observed in ICC. In the present study, LOH of TP53 was not frequently seen; however, we found frequent LOH of D17S1303 on 17p12, which is centromeric to the TP53 locus, suggesting that suppressor gene(s) other than p53 exist on the short arm of chromosome 17.

MF type tumors harbored significantly higher LOH frequency than others, indicating that carcinogenesis of MF type ICC would require more steps of genetic changes than other types of ICC. With regard to clinical data, the current study found that the frequency of LOH tended to be higher in more advanced tumors. This is consistent with the report that accumulation of genetic changes is associated with a progression and a higher histological grade of HCC (40, 41). In contrast, there was no significant difference in LOH frequency between pN0 and pN1. Furthermore, patients with MF type have a better 5-year survival rate than those with other types (42). These findings raise the hypothesis that inactivation of tumor suppressor genes are a crucial step in the progression of ICC but not in lymph node metastasis, which is an independent prognostic factor in ICC (3). In the present study, cases examined were composed of the same number; however, female cases demonstrated greater LOH frequency than male cases. We need further study to determine whether LOH of chromosome Xp could be attributed to this phenomenon.

Loci with LOH were distributed more widely in ICC than in HCC, indicating that ICC is composed of more heterogeneous subgroups of genetic changes than HCC. ICC arises anywhere in the liver from the intrahepatic large bile ducts (perihilar type) to small bile ducts at the lobular level (peripheral type). Because several precursor cell types are assumed to differentiate to hepatocytes and to duct cells (43), characteristic gene alterations that reflect cellular origin of different subtypes of ICC may exist.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

        
1

Partially supported by a Grant-in-Aid from the Ministry of Education, Sports and Culture and Health and Ministry of Labor, and Welfare of Japan, contracts with the Nuclear Safety Research Association of Japan, and Japan Space Forum.

                
3

The abbreviations used are: ICC, intrahepatic cholangiocarcinoma; HCC, hepatocellular carcinoma; MF, mass-forming; PI, periductal-infiltrating; LOH, loss of heterozygosity; HCV, hepatitis C virus.

Fig. 1.

Representative electropherogram for assessment of LOH. Size in bp is shown on the X axis at the top of the figure, and the peak heights in fluorescence are shown on the Y axis on the right. The top trace shows alleles of the normal tissue, and the bottom trace shows those of the corresponding tumor tissue. The LOH index is calculated as (T2 × N1)/(T1 × N2). A, an example of a normal case. The LOH index was 1.07, indicating retention of heterozygosity. B, an example of a LOH case. The LOH index was 0.19, indicating loss of the larger allele.

Fig. 1.

Representative electropherogram for assessment of LOH. Size in bp is shown on the X axis at the top of the figure, and the peak heights in fluorescence are shown on the Y axis on the right. The top trace shows alleles of the normal tissue, and the bottom trace shows those of the corresponding tumor tissue. The LOH index is calculated as (T2 × N1)/(T1 × N2). A, an example of a normal case. The LOH index was 1.07, indicating retention of heterozygosity. B, an example of a LOH case. The LOH index was 0.19, indicating loss of the larger allele.

Close modal
Fig. 2.

LOH frequency at each chromosome arm of ICC and HCC. Using 168 screening markers, LOH frequencies were calculated by (total number of markers with LOH)/(total number of informative markers on the same arm level). A, ICC; B, HCC.

Fig. 2.

LOH frequency at each chromosome arm of ICC and HCC. Using 168 screening markers, LOH frequencies were calculated by (total number of markers with LOH)/(total number of informative markers on the same arm level). A, ICC; B, HCC.

Close modal
Fig. 3.

Fine mapping at 1p36. The karyogram of chromosome 1p and positions of microsatellite markers are shown at the left of the figure. •, loss of heterozygosity; ○, retention of heterozygosity; −, not informative (hemizygosity); ×, PCR failure or LOH not detectable because of irregular allele bands.

Fig. 3.

Fine mapping at 1p36. The karyogram of chromosome 1p and positions of microsatellite markers are shown at the left of the figure. •, loss of heterozygosity; ○, retention of heterozygosity; −, not informative (hemizygosity); ×, PCR failure or LOH not detectable because of irregular allele bands.

Close modal
Table 1

Clinical data of ICC patients

CaseGenderAgeViral infectionHist. typeaDif.bGross appearanceTumor numbercTumor size (mm)StagedLOH percentagee
37 Mf 61  TA MF 75 II 21.8 (29/133) 
43 67  TA MF 55  37.9 (47/124) 
45 40  TMA MF + PI 42 IVA 3.1 (4/127) 
46 54  TA MF + PI 48 IVB 9.3 (12/129) 
47 65  TA PI 30 IIIB 1.6 (2/129) 
48 79  TA  70  4.2 (5/119) 
49 52  TA W-M MF + PI 47 IVB 4.1 (5/123) 
50 73 HCV(+) TA MF + PI 140 IVB 38.0 (54/142) 
51 58  TA MF + PI 46 IVB 5.2 (7/135) 
52 71  TA MF 56 IVB 42.6 (58/136) 
53 76  PTA MF + PI 30 IVB 15.9 (23/145) 
54 64  TA W-M MF + PI 50 IVB 11.8 (15/127) 
55 69    MF 80 II 16.9 (22/130) 
56 29 HBV(+) TA MF 75 II 26.1 (40/153) 
57 63  TA MF 68 IVB 39.2 (38/97) 
58 65  AS  MF + PI 40 IVA 3.8 (5/133) 
59 59  TA MF + PI 29 IIIA 2.2 (2/137) 
61 77 HCV(+) TA MF 44 IVB 7.3 (9/124) 
62 64  TA PI 40 IVA 4.4 (6/136) 
63 72  TA MF + PI 50 IIIB 2.9 (4/140) 
64 57 HCV(+) TA MF + PI 90 IVA 66.4 (71/107) 
65 65  TA     3.1 (4/127) 
CaseGenderAgeViral infectionHist. typeaDif.bGross appearanceTumor numbercTumor size (mm)StagedLOH percentagee
37 Mf 61  TA MF 75 II 21.8 (29/133) 
43 67  TA MF 55  37.9 (47/124) 
45 40  TMA MF + PI 42 IVA 3.1 (4/127) 
46 54  TA MF + PI 48 IVB 9.3 (12/129) 
47 65  TA PI 30 IIIB 1.6 (2/129) 
48 79  TA  70  4.2 (5/119) 
49 52  TA W-M MF + PI 47 IVB 4.1 (5/123) 
50 73 HCV(+) TA MF + PI 140 IVB 38.0 (54/142) 
51 58  TA MF + PI 46 IVB 5.2 (7/135) 
52 71  TA MF 56 IVB 42.6 (58/136) 
53 76  PTA MF + PI 30 IVB 15.9 (23/145) 
54 64  TA W-M MF + PI 50 IVB 11.8 (15/127) 
55 69    MF 80 II 16.9 (22/130) 
56 29 HBV(+) TA MF 75 II 26.1 (40/153) 
57 63  TA MF 68 IVB 39.2 (38/97) 
58 65  AS  MF + PI 40 IVA 3.8 (5/133) 
59 59  TA MF + PI 29 IIIA 2.2 (2/137) 
61 77 HCV(+) TA MF 44 IVB 7.3 (9/124) 
62 64  TA PI 40 IVA 4.4 (6/136) 
63 72  TA MF + PI 50 IIIB 2.9 (4/140) 
64 57 HCV(+) TA MF + PI 90 IVA 66.4 (71/107) 
65 65  TA     3.1 (4/127) 
a

Histological type: TA, tubular adenocarcinoma; TMA, tubular-mucinous adenocarcinoma; PTA, papillo-tubular adenocarcinoma; AS, adenosquamous carcinoma.

b

Histological differentiation: W, well differentiated; M, moderately differentiated; P, poorly differentiated.

c

S, solitary; M, multiple.

d

The International Union Against Cancer TNM classification.

e

(number of loci with LOH)/(total number of informative loci) at 168 screening markers.

f

M, male; F, female; HBV, hepatitis B virus.

Table 2

Microsatellite marker loci demonstrating significant frequency of LOHa

LocusaLocalizationRepeat typeLOHb in ICC (%)LOH in HCCc (%)
D1S552 1p 36.12-13 tetra- 2/6 (33.3) 14.8 
D1S170 1p 36.13 di- 3/9 (33.3) 25.0 
D1S1597 1p 36.13-23 tetra- 5/16 (31.3) 20.7 
D2S1356 2p21 tri- 3/10 (30.0) 17.9 
D2S2976 2p 25.3-p 37.3 tetra- 3/9 (33.3) 18.2 
D4S1538 4q 13.3 di- 5/15 (33.3) 35.7 
D4S1652 4q 35.1 tetra- 4/13 (30.8) 42.9 
F13A1 6p 25.1-p 24.3 tetra- 5/13 (38.5) 26.7 
D6S474 6q 21 tetra- 8/18 (44.4) 36.4 
D6S290 6q 25.1 di- 5/15 (33.3) 32.3 
D6S1277 6q 26 tetra- 5/13 (38.5) 32.3 
D6S305 6q 25.2-q 27 di- 6/18 (33.3) 23.5 
D9S1118 9p 13.3-p 21.1 tetra- 7/19 (36.8) 22.5 
D9S910 9q 22.2 tri- 4/12 (33.3) 30.0 
D12S391 12p 12.3 tetra- 5/16 (31.3) 21.1 
D14S1280 14q 11.2 tetra- 4/12 (33.3) 3.4 
D15S655 15q 25.3 tri- 5/15 (33.3) 8.8 
D16S2624 16q 22.1-q 23.1 tetra- 5/12 (41.7) 43.3 
D17S1303 17p 12 tetra- 4/13 (30.8) 54.5 
D18S844 18q 23 tri- 3/10 (30.0) 8.6 
DXS6810 Xp 21.1 tetra- 4/10 (40.0) 12.5 
LocusaLocalizationRepeat typeLOHb in ICC (%)LOH in HCCc (%)
D1S552 1p 36.12-13 tetra- 2/6 (33.3) 14.8 
D1S170 1p 36.13 di- 3/9 (33.3) 25.0 
D1S1597 1p 36.13-23 tetra- 5/16 (31.3) 20.7 
D2S1356 2p21 tri- 3/10 (30.0) 17.9 
D2S2976 2p 25.3-p 37.3 tetra- 3/9 (33.3) 18.2 
D4S1538 4q 13.3 di- 5/15 (33.3) 35.7 
D4S1652 4q 35.1 tetra- 4/13 (30.8) 42.9 
F13A1 6p 25.1-p 24.3 tetra- 5/13 (38.5) 26.7 
D6S474 6q 21 tetra- 8/18 (44.4) 36.4 
D6S290 6q 25.1 di- 5/15 (33.3) 32.3 
D6S1277 6q 26 tetra- 5/13 (38.5) 32.3 
D6S305 6q 25.2-q 27 di- 6/18 (33.3) 23.5 
D9S1118 9p 13.3-p 21.1 tetra- 7/19 (36.8) 22.5 
D9S910 9q 22.2 tri- 4/12 (33.3) 30.0 
D12S391 12p 12.3 tetra- 5/16 (31.3) 21.1 
D14S1280 14q 11.2 tetra- 4/12 (33.3) 3.4 
D15S655 15q 25.3 tri- 5/15 (33.3) 8.8 
D16S2624 16q 22.1-q 23.1 tetra- 5/12 (41.7) 43.3 
D17S1303 17p 12 tetra- 4/13 (30.8) 54.5 
D18S844 18q 23 tri- 3/10 (30.0) 8.6 
DXS6810 Xp 21.1 tetra- 4/10 (40.0) 12.5 
a

Bold, HCC hotspot markers; italic, commonly affected markers in ICC of this study and in HCC from our previous study.

b

Cases with LOH/informative cases.

c

Okabe et al., Ref. 15.

Table 3

Summary of the results of LOH analysis on chromosome arms with significant LOH frequencya

RegionLocusTypebMFcOthersc?cLOH percentagee
37435255565761454647495051535458596263644865
1q31.3-1p36.31 D1S1612 tetra- • − − • ○ − • ○ ○ ○ ○ ○ ○ • ○ ○ ○ ○ × − − ○ 25.0 
1p36.31 D1S160 di- ○ − − ○ • • × ○ ○ − × ○ ○ × • × − ○ ○ ○ − ○ 23.1 
1p36.31 D1S214 di- • − • • ○ ○ ○ − − − − − − ○ ○ ○ ○ ○ ○ − ○ ○ 21.4 
1p36.13-23 D1S1597 tetra- − ○ • • − ○ • ○ − ○ ○ • ○ − ○ ○ − ○ − • ○ ○ 31.3 
1p36.13 D1S170 di- ○ − ○ • ○ − − • • − − − − − − − − ○ − ○ − ○ 33.3 
1p36.12-13 D1S552 tetra- − − • • − × − − ○ − − − − − ○ − − ○ ○ − − × 33.3 
1p36.11-21 D1S199 di- ○ • − ○ ○ ○ × − − × × × ○ • × × × − ○ − × ○ 22.2 
1p33 D1S255 di- − − − ○ − ○ − − ○ − − − ○ − − − − − − − ○ − 0.0 
1p33 D1S186 di- ○ − • ○ ○ ○ × ○ ○ ○ − ○ ○ • ○ × ○ ○ ○ − ○ ○ 11.8 
1p32.3 D1S2134 tetra- ○ × • − ○ − ○ • ○ ○ − ○ ○ ○ ○ ○ − − ○ • • ○ 25.0 
1q22-1p31.2 D1S1665 tetra- ○ • • ○ ○ − − − ○ ○ ○ − ○ ○ − ○ ○ • ○ × ○ ○ 18.8 
1p31.1 D1S159 di- × × • − ○ × × × × ○ − × ○ ○ × − × × − × × − 20.0 
1p22.2 D1S167 di- ○ ○ − ○ ○ ○ ○ ○ • − − ○ − ○ − ○ − ○ ○ ○ − ○ 6.7 
1p21.1 D1S248 di- ○ ○ • ○ ○ ○ ○ ○ • ○ ○ − ○ ○ − ○ ○ ○ ○ ○ ○ ○ 10.0 
1p13.1 D1S187 di- ○ − − × ○ ○ − × ○ − ○ − − ○ × ○ ○ ○ ○ − − ○ 0.0 
3q21.1-q13.31 D3S2460 tetra- − ○ • − ○ − ○ ○ • ○ • ○ ○ − ○ ○ ○ ○ − • ○ − 25.0 
3q21.3-q25.2 D3S1744 tetra- ○ − − • ○ − ○ • ○ − ○ − ○ ○ − ○ ○ ○ ○ • • ○ 25.0 
3q26.2-q27 D3S2427 tetra- − • ○ • ○ • ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ − ○ ○ • ○ ○ 20.0 
3q27 D3S2418 tri- − • − • ○ ○ − ○ − − − − ○ − ○ ○ − ○ ○ • − ○ 25.0 
6p25.1-p24.3 F13A1 tetra- • • ○ − − • ○ ○ − − • ○ − − − − ○ ○ ○ • − ○ 38.5 
6p22.1 D6S2439 tetra- • • • ○ ○ − ○ − × ○ − • × ○ ○ ○ ○ − ○ − × ○ 28.6 
6p21.1 D6S1017 tetra- − • ○ ○ ○ − − ○ − ○ ○ − ○ • ○ × ○ − ○ − ○ ○ 14.3 
6q11.2-6p12.1 D6S1053 tetra- − ○ ○ ○ ○ − ○ ○ − ○ ○ • ○ − − − ○ − ○ • ○ − 14.3 
6q16.3 D6S1056 tetra- • ○ ○ − • − • ○ × − ○ • ○ − ○ ○ ○ ○ ○ − ○ ○ 25.0 
6q21 D6S474 tetra- • ○ • • − • • − ○ ○ ○ • ○ • ○ ○ ○ × − • ○ ○ 44.4 
6q22-q27 GATA184A08 tetra- • − ○ ○ • ○ − ○ ○ ○ ○ • ○ ○ ○ ○ − ○ ○ • ○ ○ 21.1 
6q25.1 D6S290 di- − • • ○ • ○ ○ − ○ ○ − × ○ • − ○ ○ ○ − • ○ − 33.3 
6q25.3 D6S305 di- • ○ • • • ○ ○ ○ ○ ○ − • ○ • ○ − ○ ○ ○ ○ − − 33.3 
6q26 D6S1277 tetra- • − − • − − − − ○ ○ ○ • ○ • ○ ○ − ○ ○ • − − 38.5 
6q26-q27 D6S1027 tri- × • × − • − × ○ ○ − − − − • ○ ○ ○ ○ ○ − ○ ○ 25.0 
9q24.1 D9S2169 tetra- ○ − • ○ ○ • − − ○ ○ − • ○ ○ ○ − − ○ ○ • − ○ 26.7 
9p23 D9S925 tetra- ○ • • − ○ − ○ ○ ○ ○ − • ○ ○ ○ × ○ − × − − − 23.1 
9p13.3-21.1 D9S1118 tetra- − • • ○ ○ • • − ○ ○ ○ • ○ ○ • ○ ○ ○ ○ • − ○ 36.8 
9q12-9q21.13 D9S273 di- • − − ○ ○ − ○ − − − − • − ○ − − − − ○ ○ × − 25.0 
9q21.31-32 D9S922 tetra- ○ • ○ ○ ○ • − ○ ○ ○ ○ • − − • − ○ ○ ○ − − − 26.7 
9p22.2 D9S910 tri- − ○ ○ − − • • − − ○ ○ • ○ ○ • − ○ − ○ − − − 33.3 
9q33.2-3 D9S934 tetra- ○ • ○ − ○ • − ○ ○ ○ ○ • ○ ○ • ○ ○ ○ − • ○ ○ 26.3 
9q34.3 D9S158 di- − − − − ○ × ○ ○ − × ○ • ○ − ○ × ○ ○ − − × × 11.1 
17p13.3 D17S1308 tetra- ○ ○ − ○ • − − ○ − − ○ • − − ○ ○ ○ ○ − • ○ × 23.1 
17p13.1 TP53 di- • ○ ○ ○ − ○ ○ ○ ○ ○ ○ • • ○ ○ ○ • ○ ○ • ○ ○ 23.8 
17p13.1 D17S786 di- − ○ ○ ○ ○ − • ○ ○ ○ − ○ − ○ ○ ○ ○ ○ ○ − ○ ○ 5.9 
17p12 D17S1303 tetra- − − − ○ • − − ○ ○ ○ ○ • • − ○ ○ ○ • − × − ○ 30.8 
17p12 D17S953 di- ○ − ○ ○ ○ ○ ○ ○ ○ ○ − × ○ ○ ○ − ○ ○ ○ − ○ ○ 0.0 
18q11.2-18p11. D18S877 tetra- − ○ − − ○ • − ○ • ○ ○ • − ○ − • ○ − ○ − ○ ○ 28.6 
18q21.1 D18S858 tri- − − ○ − − − ○ ○ ○ ○ ○ • • ○ − ○ − − ○ • − − 25.0 
18q22.1 D18S878 tetra- • ○ ○ ○ ○ ○ ○ − ○ ○ ○ • ○ ○ ○ ○ ○ ○ − • ○ ○ 15.0 
18q23 D18S844 tri- • × − − ○ − × × ○ − ○ • ○ × × ○ ○ − ○ • × − 30.0 
Xq28-Xp22.33 DXS6814 tetra- − ○ ○ ○ • − ○ ○ ○ ○ ○ • − ○ • − − ○ − − ○ − 21.4 
Xp21.3 DXS9896 tetra- − ○ − × × − ○ − ○ ○ − − − ○ × − × • − − − − 16.7 
Xp21.1 DXS6810 tetra- • ○ • − ○ • − − − − − ○ − ○ ○ ○ − − − − − • 40.0 
RegionLocusTypebMFcOthersc?cLOH percentagee
37435255565761454647495051535458596263644865
1q31.3-1p36.31 D1S1612 tetra- • − − • ○ − • ○ ○ ○ ○ ○ ○ • ○ ○ ○ ○ × − − ○ 25.0 
1p36.31 D1S160 di- ○ − − ○ • • × ○ ○ − × ○ ○ × • × − ○ ○ ○ − ○ 23.1 
1p36.31 D1S214 di- • − • • ○ ○ ○ − − − − − − ○ ○ ○ ○ ○ ○ − ○ ○ 21.4 
1p36.13-23 D1S1597 tetra- − ○ • • − ○ • ○ − ○ ○ • ○ − ○ ○ − ○ − • ○ ○ 31.3 
1p36.13 D1S170 di- ○ − ○ • ○ − − • • − − − − − − − − ○ − ○ − ○ 33.3 
1p36.12-13 D1S552 tetra- − − • • − × − − ○ − − − − − ○ − − ○ ○ − − × 33.3 
1p36.11-21 D1S199 di- ○ • − ○ ○ ○ × − − × × × ○ • × × × − ○ − × ○ 22.2 
1p33 D1S255 di- − − − ○ − ○ − − ○ − − − ○ − − − − − − − ○ − 0.0 
1p33 D1S186 di- ○ − • ○ ○ ○ × ○ ○ ○ − ○ ○ • ○ × ○ ○ ○ − ○ ○ 11.8 
1p32.3 D1S2134 tetra- ○ × • − ○ − ○ • ○ ○ − ○ ○ ○ ○ ○ − − ○ • • ○ 25.0 
1q22-1p31.2 D1S1665 tetra- ○ • • ○ ○ − − − ○ ○ ○ − ○ ○ − ○ ○ • ○ × ○ ○ 18.8 
1p31.1 D1S159 di- × × • − ○ × × × × ○ − × ○ ○ × − × × − × × − 20.0 
1p22.2 D1S167 di- ○ ○ − ○ ○ ○ ○ ○ • − − ○ − ○ − ○ − ○ ○ ○ − ○ 6.7 
1p21.1 D1S248 di- ○ ○ • ○ ○ ○ ○ ○ • ○ ○ − ○ ○ − ○ ○ ○ ○ ○ ○ ○ 10.0 
1p13.1 D1S187 di- ○ − − × ○ ○ − × ○ − ○ − − ○ × ○ ○ ○ ○ − − ○ 0.0 
3q21.1-q13.31 D3S2460 tetra- − ○ • − ○ − ○ ○ • ○ • ○ ○ − ○ ○ ○ ○ − • ○ − 25.0 
3q21.3-q25.2 D3S1744 tetra- ○ − − • ○ − ○ • ○ − ○ − ○ ○ − ○ ○ ○ ○ • • ○ 25.0 
3q26.2-q27 D3S2427 tetra- − • ○ • ○ • ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ − ○ ○ • ○ ○ 20.0 
3q27 D3S2418 tri- − • − • ○ ○ − ○ − − − − ○ − ○ ○ − ○ ○ • − ○ 25.0 
6p25.1-p24.3 F13A1 tetra- • • ○ − − • ○ ○ − − • ○ − − − − ○ ○ ○ • − ○ 38.5 
6p22.1 D6S2439 tetra- • • • ○ ○ − ○ − × ○ − • × ○ ○ ○ ○ − ○ − × ○ 28.6 
6p21.1 D6S1017 tetra- − • ○ ○ ○ − − ○ − ○ ○ − ○ • ○ × ○ − ○ − ○ ○ 14.3 
6q11.2-6p12.1 D6S1053 tetra- − ○ ○ ○ ○ − ○ ○ − ○ ○ • ○ − − − ○ − ○ • ○ − 14.3 
6q16.3 D6S1056 tetra- • ○ ○ − • − • ○ × − ○ • ○ − ○ ○ ○ ○ ○ − ○ ○ 25.0 
6q21 D6S474 tetra- • ○ • • − • • − ○ ○ ○ • ○ • ○ ○ ○ × − • ○ ○ 44.4 
6q22-q27 GATA184A08 tetra- • − ○ ○ • ○ − ○ ○ ○ ○ • ○ ○ ○ ○ − ○ ○ • ○ ○ 21.1 
6q25.1 D6S290 di- − • • ○ • ○ ○ − ○ ○ − × ○ • − ○ ○ ○ − • ○ − 33.3 
6q25.3 D6S305 di- • ○ • • • ○ ○ ○ ○ ○ − • ○ • ○ − ○ ○ ○ ○ − − 33.3 
6q26 D6S1277 tetra- • − − • − − − − ○ ○ ○ • ○ • ○ ○ − ○ ○ • − − 38.5 
6q26-q27 D6S1027 tri- × • × − • − × ○ ○ − − − − • ○ ○ ○ ○ ○ − ○ ○ 25.0 
9q24.1 D9S2169 tetra- ○ − • ○ ○ • − − ○ ○ − • ○ ○ ○ − − ○ ○ • − ○ 26.7 
9p23 D9S925 tetra- ○ • • − ○ − ○ ○ ○ ○ − • ○ ○ ○ × ○ − × − − − 23.1 
9p13.3-21.1 D9S1118 tetra- − • • ○ ○ • • − ○ ○ ○ • ○ ○ • ○ ○ ○ ○ • − ○ 36.8 
9q12-9q21.13 D9S273 di- • − − ○ ○ − ○ − − − − • − ○ − − − − ○ ○ × − 25.0 
9q21.31-32 D9S922 tetra- ○ • ○ ○ ○ • − ○ ○ ○ ○ • − − • − ○ ○ ○ − − − 26.7 
9p22.2 D9S910 tri- − ○ ○ − − • • − − ○ ○ • ○ ○ • − ○ − ○ − − − 33.3 
9q33.2-3 D9S934 tetra- ○ • ○ − ○ • − ○ ○ ○ ○ • ○ ○ • ○ ○ ○ − • ○ ○ 26.3 
9q34.3 D9S158 di- − − − − ○ × ○ ○ − × ○ • ○ − ○ × ○ ○ − − × × 11.1 
17p13.3 D17S1308 tetra- ○ ○ − ○ • − − ○ − − ○ • − − ○ ○ ○ ○ − • ○ × 23.1 
17p13.1 TP53 di- • ○ ○ ○ − ○ ○ ○ ○ ○ ○ • • ○ ○ ○ • ○ ○ • ○ ○ 23.8 
17p13.1 D17S786 di- − ○ ○ ○ ○ − • ○ ○ ○ − ○ − ○ ○ ○ ○ ○ ○ − ○ ○ 5.9 
17p12 D17S1303 tetra- − − − ○ • − − ○ ○ ○ ○ • • − ○ ○ ○ • − × − ○ 30.8 
17p12 D17S953 di- ○ − ○ ○ ○ ○ ○ ○ ○ ○ − × ○ ○ ○ − ○ ○ ○ − ○ ○ 0.0 
18q11.2-18p11. D18S877 tetra- − ○ − − ○ • − ○ • ○ ○ • − ○ − • ○ − ○ − ○ ○ 28.6 
18q21.1 D18S858 tri- − − ○ − − − ○ ○ ○ ○ ○ • • ○ − ○ − − ○ • − − 25.0 
18q22.1 D18S878 tetra- • ○ ○ ○ ○ ○ ○ − ○ ○ ○ • ○ ○ ○ ○ ○ ○ − • ○ ○ 15.0 
18q23 D18S844 tri- • × − − ○ − × × ○ − ○ • ○ × × ○ ○ − ○ • × − 30.0 
Xq28-Xp22.33 DXS6814 tetra- − ○ ○ ○ • − ○ ○ ○ ○ ○ • − ○ • − − ○ − − ○ − 21.4 
Xp21.3 DXS9896 tetra- − ○ − × × − ○ − ○ ○ − − − ○ × − × • − − − − 16.7 
Xp21.1 DXS6810 tetra- • ○ • − ○ • − − − − − ○ − ○ ○ ○ − − − − − • 40.0 
a

•, LOH; ○, retention of heterozygosity; −, not informative; ×, PCR failed or LOH not detectable because of irregular allele pattern.

b

Type of nucleotide repeats.

c

Gross appearance.

d Case number.

e

LOH percentage, cases with LOH/informative cases.

Table 4

Correlation between frequency of LOH and clinical parameters

Clinical parametersnScreening markersHCC hotspot markers13 markers on 1p36
LOHaP                  bLOHaP                  bLOHaP                  b
Gender        
 Male 11 161/1199 (13.4) <0.001 36/262 (13.7) NSc 21/88 (23.9) NS 
 Female 11 225/11198 (20.1)  42/275 (15.3)  20/84 (23.8)  
Gross appearance        
 MF 199/708 (28.1) <0.001 44/188 (23.4) <0.001 28/60 (46.7) <0.001 
 MF + PI or PI 13 178/1408 (12.6)  34/268 (12.7)  13/96 (13.5)  
Tumor number        
 Multiple 93/398 (23.4) 0.004 9/83 (10.8) NS 2/25 (8.0) 0.030 
 Solitary 17 289/1817 (15.9)  69/427 (16.2)  39/137 (28.5)  
Tumor size        
 5 cm < 11 328/1123 (29.2) <0.001 56/284 (19.7) 0.002 31/93 (33.3) 0.006 
 ≤5 cm 10 54/1092 (4.9)  22/226 (9.7)  10/69 (14.5)  
LN metastasis        
 pN1 11 188/1168 (16.1) NS 36/271 (13.3) NS 15/84 (17.9) 0.006 
 pN0 150/846 (17.7)  34/197 (18.3)  25/66 (37.9)  
Stage (UICC)d        
 IVB 184/948 (19.4) 0.003 37/210 (17.6) NS 19/50 (27.5) NS 
 II–IVA 10 154/1066 (14.4)  33/258 (12.8)  21/81 (25.9)  
Viral infection        
 HCV (+) 91/291 (31.3) <0.001 10/72 (13.9) NS 6/23 (26.1) NS 
 Nonviral infection 17 266/1904 (14.0)  57/433 (13.2)  33/139 (23.7)  
Clinical parametersnScreening markersHCC hotspot markers13 markers on 1p36
LOHaP                  bLOHaP                  bLOHaP                  b
Gender        
 Male 11 161/1199 (13.4) <0.001 36/262 (13.7) NSc 21/88 (23.9) NS 
 Female 11 225/11198 (20.1)  42/275 (15.3)  20/84 (23.8)  
Gross appearance        
 MF 199/708 (28.1) <0.001 44/188 (23.4) <0.001 28/60 (46.7) <0.001 
 MF + PI or PI 13 178/1408 (12.6)  34/268 (12.7)  13/96 (13.5)  
Tumor number        
 Multiple 93/398 (23.4) 0.004 9/83 (10.8) NS 2/25 (8.0) 0.030 
 Solitary 17 289/1817 (15.9)  69/427 (16.2)  39/137 (28.5)  
Tumor size        
 5 cm < 11 328/1123 (29.2) <0.001 56/284 (19.7) 0.002 31/93 (33.3) 0.006 
 ≤5 cm 10 54/1092 (4.9)  22/226 (9.7)  10/69 (14.5)  
LN metastasis        
 pN1 11 188/1168 (16.1) NS 36/271 (13.3) NS 15/84 (17.9) 0.006 
 pN0 150/846 (17.7)  34/197 (18.3)  25/66 (37.9)  
Stage (UICC)d        
 IVB 184/948 (19.4) 0.003 37/210 (17.6) NS 19/50 (27.5) NS 
 II–IVA 10 154/1066 (14.4)  33/258 (12.8)  21/81 (25.9)  
Viral infection        
 HCV (+) 91/291 (31.3) <0.001 10/72 (13.9) NS 6/23 (26.1) NS 
 Nonviral infection 17 266/1904 (14.0)  57/433 (13.2)  33/139 (23.7)  
a

LOH frequency is calculated as follows, number of loci with LOH/total number of informative loci (%).

b

Statistical differences were evaluated using the χ2 test.

c

NS, not significant; LN, lymph node; HCV, hepatitis C virus.

d

The International Union Against Cancer (UICC) TNM classification.

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