Frequent Multiplication of the Long Arm of Chromosome 8 in Hepatocellular Carcinoma1

Home , Chromosome 8

[CANCER RESEARCH 53. 857-860. February 15. 1993] Frequent Multiplication of the Long Arm of Chromosome 8 in Hepatocellular Carcinoma1

Yoshiyuki Fujiwara, Monto Monden, Takesada Mori, Yusuke Nakamura,2 and Mitsuru Emi

Department of Biochemistry Â¡Y.F., Y. N.. M. E.l. Cancer Institute. 1-37-1 Kaini-lkebukuro. Tiishinia-ku. Tokyo 170. and the Second Department of Surgen' Â¡Y.F.. M. M., T. M.], Osaka University MÃ©diraiSellimi. 1-1-50 Fukushima. Fukushima, Osaka 53}, Japan

ABSTRACT normal and tumor DNAs enabled visualization of subtle changes that had occurred in tumor DNAs. Frequent allelic losses at loci on several chromosomes have been de We initiated a systematic RFLP analysis of paired DNAs from tected in human hepatocellular carcinomas, but other types of chromo HCCs and their corresponding normal tissues to examine whether somal abnormalities have not been characterized well. Using eight poly multiplication of chromosomal segments may take place during de morphic DNA markers on chromosome 8, we examined 120 primary hepatocellular carcinomas for abnormalities in the copy number of these velopment of HCC. In this paper, we present results of RFLP analysis loci in tumor cells. A 2- to 6-fold increase in intensities of bands repre at loci on chromosome 8, and we demonstrate that multiplication of senting single alÃeleswas observed in 32 of the 78 tumors that were single alÃeleson part or all of the long arm of chromosome 8 has informative for one or more of the markers, indicating an increase in copy occurred in a large proportion of HCCs. number ("multiplication") of alÃeleson8q. The regions that multiplied varied from almost the entire long arm to a distal segment of chromosome MATERIALS AND METHODS 8, but a terminal region distal to 8q24.11 was multiplied in all 32 cases. Similar multiplications on 8q were detected in nine of 56 colorÃ©ela!car Samples. Tumorsand theircorrespondingnoncanceroustissues were ob cinomas that were informative for one or more of the markers. Our study tained during surgery from 120 patients with HCC and 99 patients with CRC. demonstrated that polymorphic DNA markers can be used to detect nu All tissues were dissected in the operation room, fro/.en immediately, and merical abnormalities of chromosomal material in solid tumors in which stored at -80Â°Cuntil the DNA was isolated. cytogenetic analysis is technically difficult. DNA Extraction and Southern Blotting. Fro/en tissue samples were ground to a very fine powder in liquid nitrogen, suspended in lysis buffer, treated with proteinase K, and extracted by phenol-chloroform-isoamylalcohol INTRODUCTION as described elsewhere (11). Five ug of each DNA sample were digested Carcinogenesis is generally considered to be a multistep process overnight with 10times excess of restrictionen/.ymes (Boehringer Mannheim) that may involve cumulative mutations in oncogenes and tumor sup and fractionated by electrophoresis using a 0.8"7ragarose gel. The gel was pressor genes, as illustrated in a published genetic model for colorec- stained with ethidium bromide and photographed in order to visualize the tal tumorigenesis (1). Any one of several mechanisms, such as point actual amount of DNA present on each lane of the gel. The DNAs were then transferred to nylon membranes (Biodyne; Pall) in 0.1 NNaOH-0.1 MNaCl and mutation, amplification of genes, chromosomal translocation, and loss fixed by UV cross-linking (11). or gain of chromosomal material can activate oncogenes or inactivate tumor suppressor genes. In HCCs,1 LOHs described at loci on chro Probes and Hybridization. Of eight polymorphicmarkersused in this study, cosmid clones CI8-494 (D8S234), CI8-277 (D8SI94). CI8-512 mosomes 4q, 8p, 1Ip, 13q, 16q, and 17p (2-6) imply the presence of (D8S238). CI8-340 (D8S223). CI8-442 (D8S23I). CI8-212 (D8SI88). and putative tumor suppressor genes on these chromosomes and indicate CI8-134 (D8SI77) were described previously (12); they had been physically that HCC, like colon cancer, may arise from a complex process. Little locali/.ed on chromosome 8 by fluorescent in situ hybridization on R-banded is known, however, about other chromosomal mechanisms that may human prometaphase chromosomes. The chromosomal locations of these be involved in hepatocarcinogenesis. markers are shown in Tables 1 and 2. CI8-512 was recently reassigned to 8q11.21-q11.22.Â°*p380-8A,a polymorphic probe at the c-mvcgene that detects Cytogenetic analyses of primary cultured cells from solid tumors have often uncovered chromosomal abnormalities. For instance, kary- a RFLP with three alÃeles(13),was kindly provided by Dr. Tsujimoto; the c-mvc gene lies within band 8q24.12-q24.13 (14). A polymorphic restriction otypic observations have identified translocations, partial trisomies, or fragment of each cosmid was purified by agarose gel electrophoresis for use as losses of specific chromosomal regions as significant changes in renal a probe. Probes were labeled with [a-32P]dCTPby random-primer extension cell carcinomas (7). In HCC, however, cytogenetic data derived from (15). Prehybridization, hybridization, and autoradiography were carried out as primary cultured cells have been limited to a single case report (8), described elsewhere (11). The membranes were stripped in 0.4 NNaOH and because of technical difficulties in chromosome preparation. repeatedly hybridized. Recently the usefulness of polymorphic markers for detecting both Determination of Alleile Dosage. Only the cases showing constitutional LOH and "multiplications" (modest increases of chromosomal copy heterozygosity were used in the evaluation of allelic dosage. The signal inten number) in solid tumor tissues was proven by careful comparative sity of the polymorphic alÃeleswasquantified with a Hoefer GS-300 scanning studies of karyotypic and RFLP analyses; partial trisomy of chromo densitometer; the peak areas corresponding to each hybridizing signal were some 5q in renal cell carcinomas (9) and trisomy of chromosome 7 in calculated by electric integration using a GS 370 one-dimensional electro malignant mesotheliomas (10) were unambiguously demonstrated in phoresis data system (Hoefer Scientific Instruments). The difference in the amount of DNAs between paired normal and tumor autoradiographies of hybridization experiments. In these RFLP anal DNA may result in an increase or decrease in signal intensities of both alÃeles yses, comparison of the four bands that represented both alÃelesin in tumor DNA. Therefore, we measured the amount of DNA on each lane by ethidium bromide staining of the gel and compared that amount with the signals observed by control probes on other chromosomes. Information for the Received 9/1/92; accepted 12/1/92. amount of DNAs was taken into consideration when signal intensities for The costs of publication of this article were defrayed in part by the payment of page normal and tumor DNAs were compared. Some instances that required such charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. adjustments were displayed (see Fig. l, D to F). In these cases, overloading of 1The work was supported by a Grant-in-Aid for Cancer Research from the Ministry of tumor DNAwas detected by measurementof DNAamounts and was taken into Education. Science, and Culture of Japan. account in judging the multiplication of alÃeles.Ananalysis of allelic dosage - To whom requests for reprints should be addressed. ' The abbreviations used are: HCC. hepatocellular carcinoma: LOH, loss of heterozy- gosily; RFLP, restriction fragment length polymorphism; CRC, colorectal carcinoma. 4 M. Emi, Y. Fujiwara, and Y. Nakamura. unpublished data. 857

consisted of the comparison of signal intensities of four bands, two derived Table I Multiplication at loci on 8q in hepatoce/lular carcinoma from the alÃelesof the probed locus in constitutional DNA and the other two of patients derived from the same pair of alÃelesin tumor DNA. The extent of multipli ProbeC tested1109210785828810598Multiplication/informativecase1/51 cation of a given alÃelewas calculated by dividing the ratio of the intensity of 18^194C (2)"1/45 the multiplied alÃeleto that of the normal alÃelein tumor DNA by the corre 18-277C18-5I2C (2)4/34(12)5/37(14)3/30(10)4/24(17)12/27(44)26/73 sponding ratio measured in normal DNA; when the results exceeded 1.8, the 18-340CI8-442C18-212p380-8AC18-134LocusD8S234D8SI94D8S2J8D8S223D8S231D8S188MYCD8S177Location8pll.23-p.ll.228pll.23-pll.218qll.2l-qll.228qll.238q22.2q-22.38q24.ll-q24.128q24.l2-lq24.138q24.23-q24.3No. alÃelewas considered to be multiplied. Genotyping and Linkage Analysis. Genotyping and linkage analysis were carried out on 40 Centre d'Etude du Polymorphism Humain (CEPH) reference families according to a procedure previously described (16). (36) a Numbers in parentheses, percentage. RESULTS (Fig. 1C) or CI8-494, both located in a proximal portion of the short Multiplication of 8q in HCC. When paired normal and tumor arm. These data, which indicated that an entire long arm of one DNAs from 120 patients with HCC were analyzed for abnormalities homologue of chromosome 8 was multiplied in this tumor, provided in the copy number of chromosome 8, the signal intensity in one alÃele molecular evidence for partial polysomy. Fig. l, D and E, shows was increased in 26 of 73 tumor DNAs, that were informative with a examples for HCC 15, in which CI8-134 detected a 3.3-fold increase variable-number tandem repeat probe (CIS-134) at the telomeric band in intensity of the larger alÃeleafter variations in signal intensities of 8q. The same panel of samples was subsequently examined with caused by overloading of tumor DNA were adjusted; however, a more four additional polymorphic markers located on different segments of proximal probe (CI8-212) revealed the same intensity on both alÃeles, the long arm and with three markers on the proximal part of 8p. indicating that multiplication in this tumor is limited to a distal part of Representative autoradiographies and their densitometric profiles are the long arm. The frequencies of multiplication observed at each of the presented in Fig. 1. In HCC 10, for example, probe p380-8A revealed eight loci are shown in Table 1; 32 of 78 tumors informative for at an increased intensity of the larger alÃelein tumor DNA compared to least one locus revealed multiplication at one or more loci. Multipli its counterpart in normal DNA (Fig. \A ). Densitometoric quantifica cation was found almost exclusively on the long arm. tion showed that the larger alÃelewas multiplied 4.4-fold in the tumor DNA. Probe CI8-134 also detected multiplication in the same tumor; The multiplication data for each of the 32 tumors are summarized schematically in Fig. 2. Among the 26 cases informative for at least the intensity of the smaller alÃelewas increased 3.5-fold (Fig. IS). one of three probes at the telomeric region (CIS-134, p380-8A, and Three other probes on the long arm (CI8-212, CI8-340, and CI8-512) CI8-212) and for at least one of four probes near the centromere also detected multiplication of one alÃelein this tumor; however, no (CI8-340, CI8-512, CI8-277, and CI8-494), seven cases showed mul change in the intensity of alÃeleswas observed with probes CI8-277 tiplication at both telomeric and centromeric loci that suggested poly somy involving an almost entire long arm. Three tumors, HCC 15, 108, and 148, showed multiplication only in the distal loci of 8q; these p380-8A CIS-134 cases seemed to reflect partial polysomy limited to the distal 8q NT NT segment. The data presented in Fig. 2 indicate that a commonly multiplied region lies in a segment at 8q24.11-qter that includes the HCC loci defined by p380-8A and CIS-134; the genetic distance of these two loci was estimated to be 21 cM by two-point linkage analysis of genotypes of 40 CEPH reference families (the maximum lod score of 4.36 at the recombination frequency of 0.21). Multiplication of 8q in CRC. Ninety-nine CRC samples were analyzed for abnormalities in the dosage of chromosome 8 in the same CIS-134 CI8-212 manner. Examples of autoradiographies and densitometric profiles are NT NT presented in Fig. 1F. In Case 21, probe p380-8A detected increased

HCC intensity of a larger alÃelein tumor DNA compared to the same alÃele case IO HCC in normal DNA. Densitometric quantification showed that the alÃele case IS < was multiplied 4.9-fold. Frequencies of multiplication observed in CRCs at each of the eight marker loci on chromosome 8 are shown in Table 2. Multiplications were detected in 9 of 56 tumors informative with the probes p380-8A and CIS-134; these data are summarized schematically in Fig. 3. In two tumors, the probes detected multipli cation of an almost entire long arm, and in five, partial polysomy CI8-277 p380-8A distal toCI8-442orCI8-212. NT NT

CRC DISCUSSION case 2I Although losses of heterozygosity have been detected on several chromosomes in hepatocellular carcinomas, no information about other numerical abnormalities of chromosomes has been available for this type of tumor. In the present study, we demonstrated by means of JL1L RFLP analysis that multiplication of an entire arm or parts of chro mosome 8q had existed in 32 of 78 HCCs. Fig. l. Autoradiograms from Southern hybridizations and densitometric quantifications of paired normal (N) and tumor (T) DNAs. Case numbers and probe names are shown, LOH studies in human primary tumors have become more effi respectively, on the left and at the lap of autoradiograms. See text for explanations. cient because of an increase in available RFLP markers that enable 858

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,1i4 4fiC5Â«)) 404)l 4v)4i!S1nÂ» H:|(t iV 4(I) 4[1)4Â«5II44>Ã•*i;11lii 5) (i 6(i;â€¢ /) t"CIS9)1449;Â»â€¢ Â»))tu) 1Â¡i13IIS H;j) 14)1 Fig. 2. Schematic representation of multiplications at loci on chromosome 8 in hepatocellular carcinomas. Case numbers are shown below, and the physical location of each marker on chromosome 8 (12) is indicated to the right of the idiogram. Closed circles, multiplication ( + ); open circles, multiplication (-); shaded circles, LOH.

Table 2 Multiplication at loci on 8q in colorectal cancer toxin-induced rat hepatoma (23), no amplification of c-mvr in human of patients primary HCCs has been demonstrated to date. Considering the rather ProbeC tested9586939974787981Multiplication/informativecase2/45 modest increase of copy number detected in the present study, mul 18-494C (4)"1/40 tiplication of this gene in HCCs may well have escaped detection in 18-277C18-512C (3)0/30 previous studies where nonpolymorphic DNA fragments were used as (0)1/25 18-340C (4)0/23 probes. Although Kinzler el al. (24) reported that the amplification 18^442C18-212p380-8AC18-I34LocusD8S234D8SI94D8S238D8S223D8S231D8S188MYCD8SI77Location8pll.23-p.ll.228pll.23-pll.218qll.2l-ll.228qll.238q22.2-22.38q24.11-24.128q24.(4)3/25(12)5/31 unit containing the c-myc gene was 90 to 300 kilobases in their

12-24.138q24.23-q24.3No. (16)8/50(16) analysis of five cell lines derived from human cancers of the lung, brain, colon, and hematopoietic tissue, it is difficult to directly com " Numbers in parentheses, percentage. pare their results to those found here in primary HCCs, because these authors used cultured cell lines which had been passed repeatedly in reliable detection of allelic loss, even when tumor DNAs are con taminated with DNA from normal cells. Multiplication of chromo somes is also examined in tumors contaminated with DNA from 23 normal cells. It is obvious that the use of polymorphic DNA markers 22 will greatly improve the sensitivity and accuracy of estimations of copy numbers, because the dosage of each alÃelecan be compared 21 with its counterpart. 12 In all 32 HCCs showing gains of chromosomal material on 8q, only one of any pair of homologous alÃeleswas multiplied: the magnitude 11 CI8-494 O of multiplication of an alÃeleranged from 2- to 6-fold. The multiplied T regions appeared to vary in size, but all were relatively large; multi 11 CI8-277 plication involving almost the entire long arm was detected in seven tumors, and three others revealed multiplication of the distal part of 12 8q. The degrees of multiplication and the sizes of the regions involved CI8-512 0 indicate that the chromosomal changes in these tumors represent 13 trisomy, tetrasomy, or polysomy of all or parts of 8q, rather than the "gene amplification" phenomenon represented by double-minutes or CI8-340 heterogeneously staining regions in which a minute segment of a 21 chromosome containing a drug resistance gene or an oncogene, for CI8-442 O example, is amplified up to several hundredfold. 22 Cytogenetic analyses have frequently detected trisomy or tetrasomy C18-212 of 8q in acute nonlymphocytic leukemia, myelodysplastic syndrome, gastric cancer, and uveal melanoma (17-20). Isochromosomes of 8q 23 have been observed in colon cancers and in lung cancers (21, 22). p380-8A Frequent observation of gain of 8q in these hematopoietic malignan cies and cancers, together with our findings in HCC and CRC, em 24 C18-134 phasizes the likelihood of etiological significance for this type of .. chromosomal aberration in carcinogenesis. Plasmid 380-8A, a polymorphic marker for the c-myc gene located 8 18 20 21 27 39 53 125 146 153 at 8q24, was used in the present study. One of the alÃelesof the c-myc Fig. 3. Schematic representation of multiplication at loci on chromosome 8 in colorec- gene was multiplied 2- to 6-fold in 44% of the informative HCCs tal carcinomas. Case numbers are shown below, and the physical location of each marker on chromosome 8 is indicated to the right of the Â¡diagram.Closed circles, multiplication tested. Although amplification of c-myc has been reported in an afla- ( + ); open circles, multiplication (-). 859

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1993 American Association for Cancer Research. MULTIPLICATION OF Kq IN HEPATOCELLULAR CARCINOMAS vitro and were preselected for the presence of a high degree of c-myc Blanquct. V., Brechot, C., Redecker, A., and Govindarajah, S. Loss of heterozygosity amplification (>50-fold). suggests tumor suppressor gene responsible for primary hepaioccllular carcinoma. Proc. Nati. Acad. Sci. USA. 86: 8852-8855. 1989. In the tumors presenting multiplications of 8q in this study, c-myc 3. Emi, M., Fujiwara, Y, Nakajima, T.. Tuchiya, E., Tsuda, H.. Hirohashi, S.. Miyaki. might be the gene playing an important role during carcinogenesis. M.. and Nakamura, Y. Frequent loss of heterozygosity for loci on chromosome 8p in hepatocellular carcinoma, colorÃ©ela!cancer, and lung cancer. Cancer Res.. 52; 5368- However, our findings indicated that the common region of multipli 5372, 1992. cation spans a segment of 8q24.11 to Sqter, which is at least 21 cM 4. Wang. H. P.. and Rogler, C. E. Deletions in human chromosome arms I Ip and I3q in long and probably contains several hundreds of unknown genes. primary hepatocellular carcinomas. Cytogenet. Cell Genet., 48: 72-78, 1988. 5. Zhang. W.. Hirohashi, S., Tsuda, H., Shimasato, Y. Yokota, J., Terada, M.. and Hence, we assume that the increase in the copy number of one or Sugimura, T. Frequent loss of heterozygosity on chromosome 16 and 4 in human more of these genes confers a growth advantage during develop hepatocellular carcinoma. Jpn. J. Cancer Res., 81: 108-111, 199(). ment and/or progression of tumors. Identification of such gene(s) and 6. Fujimori. M.. Tokino, T.. HiÃ±o,O., Kitagawu, T., Imamura, T., Okamoto, E., Mit- sunobu. M., Ishikawa. T., Nakagama, H.. Harada. H.. Yagura. 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USA, 88: 194-198. 1991. We examined the presence of 8q multiplication in CRCs as this type 10. Tiainen. M.. Kere, J., Tammilehto, L.. Mattson. K., and Knuutila. S. Abnormalities of chromosome 7 and 22 in human malignant pleural mesothelioma. Genes Chromo of tumor has also shown frequent LOH on chromosome 8p (13). The somes Cancer, 4: 176-182, 1992. 8q multiplication was observed at a lower frequency in CRCs com 11. Sato, T., Tanigami. A., Yamakawa. K.. Akiyama. F., Kasumi. F., Sakamoto, G., and pared to that in HCCs. Although we observed no multiplications with Nakamura, Y Allelotype of breast cancer: cumulative alÃelelosses promote tumor probes on chromosome 2q, 9q, 10p, and 20p,5 the significance of progression in primary breast cancer. Cancer Res.. 50: 7184-7189, 1990. 12. Emi, M.. Takahashi, E., Koyama. K.. Okui. K.. Oshimura, M.. and Nakamura. 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Yoshiyuki Fujiwara, Morito Monden, Takesada Mori, et al.

Cancer Res 1993;53:857-860.

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