Specific Association Between Alcohol Intake, High Grade of Di€Erentiation

Speci®c association between alcohol intake, high grade of dierentiation and 4q34-q35 deletions in hepatocellular carcinomas identi®ed by high resolution allelotyping

Olivier Bluteau1, Jean-Christophe Beaudoin1, Patricia Pasturaud1, Jacques Belghiti2, Dominique Franco3, Paulette Bioulac-Sage4, Pierre Laurent-Puig1 and Jessica Zucman-Rossi*,1

1INSERM U434, CEPH 27 Rue Juliette Dodu, 75010 Paris, France; 2HoÃpital Beaujon, 92110 Clichy, France; 3HoÃpital Antoine BeÂcleÁre, 92140 Clamart, France; 4HoÃpital Pellegrin, 33000 Bordeaux, France

One of the most frequent deletions in hepatocellular Introduction carcinoma (HCC) is that involving the long arm of chromosome 4 (30 to 70% of the cases). These Hepatocellular carcinoma (HCC) is one of the most chromosomal deletions are closely related to hepatitis common cancers worldwide, particularly in Asia and B virus (HBV) infection. A tumor suppressor gene (TSG) Africa. Recently, its incidence has substantially located on 4q has been proposed in liver carcinogenesis, increased in United Kingdom, France and United but has not been identi®ed as yet. Despite previous LOH States (Deuc et al., 1998; El-Serag and Mason, 1999; studies focused on 4q in HCC, a clear minimal common Taylor-Robinson et al., 1997). Etiological factors that region of deletion (MCRD) could not be delimited. To have been associated with the development of the further investigate the role of chromosome 4q LOH in disease are well known and include infection with the the pathogenesis of HCC, 85 microsatellite markers hepatitis B virus or hepatitis C virus, heavy alcohol spanning chromosome 4q were systematically analysed in intake, prolonged dietary exposure to A¯atoxin B1 and a series of 154 well-characterized primary liver tumors. primary hemochromatosis (Tsukuma et al., 1993). In 59 tumors (38%), LOHs were observed for at least Overall, HCC development is closely associated with two adjacent markers. Analysis of 31 tumors demon- cirrhosis and more than 80% of the tumors are found strating a partial or interstitial 4q deletion allowed to in a cirrhotic background. Nevertheless, the molecular de®ne three MCRDs of 15, 9 and 8 Mb at the 4q22, mechanisms of liver carcinogenesis are still poorly 4q34 and 4q35 regions, respectively. Seven putative elucidated. Although the spectrum of HCC risk factors candidate genes located in 4q22, DAPP1, BMPR1B, is very large, common genetic alterations occur in PKD2, HERC3, SMARCAD1, CEB1 and ENH were tumors. Among them, the most common are hyper- screened for mutations but no somatic alterations were ploidy, loss of heterozygosity (LOH), tumor suppressor identi®ed. Search for relationships between the speci®c gene (TSG) inactivation and oncogene activation regions of deletion and clinical parameters showed a (Buendia, 2000; Laurent-Puig et al., 2001). Cytogenetic, signi®cant association between loss of the 4q34-35 region allelotyping and comparative genomic hybridization with alcohol intake (P=0.005) and with high grade of (CGH) analyses have demonstrated frequent deletions dierentiation (P=0.02). These results are in contrast on chromosome 1p, 4q, 6q, 8p, 9p, 13q, 16 and 17p in with the close association between HBV infection and the HCC, suggesting existence of TSG on these chromo- whole 4q LOH and reveal heterogeneity of 4q LOH in some arms (Boige et al., 1997; Laurent-Puig et al., relation to dierent risk factors. In the light of these new 2001; Marchio et al., 1997; Nagai et al., 1997). TSG ®ndings, which link dierent 4q LOH regions to dierent like p53, RB1, M6P/IGF2R and most recently Axin1 etiologic factors, the molecular mechanisms underlying have been found altered as a consequence of 4q deletions in HCC and the targeted gene(s) remain to chromosomal losses of 17p, 13q, 6q and 16p, be identi®ed. respectively (Buetow et al., 1992; De Souza et al., Oncogene (2002) 21, 1225 ± 1232. DOI: 10.1038/sj/ 1995; Satoh et al., 2000; Zhang et al., 1994). For the onc/1205197 other chromosomal losses, TGS are not yet identi®ed and remain to be found. Keywords: hepatocellular carcinoma; loss of hetero- Previous cytogenetic and molecular genetic analyses zygosity; chromosome 4; mutation; alcohol intake have reported frequent deletion of the long arm of chromosome 4 in 30 to 74% of HCC tumors. We showed that chromosome 4q LOH occurs most frequently in a context of chromosome instability and is recurrently associated with LOH of 1p, 13q, 16p, 16q and Axin1 mutations (Laurent-Puig et al., 2001). *Correspondence: J Zucman-Rossi; E-mail: [email protected] Received 8 October 2001; revised 19 November 2001; accepted 26 Several allelotyping and CGH series revealed that November 2001 chromosome 4q LOH is more common in HBV Chromosome 4q deletions in hepatocellular carcinoma O Bluteau et al 1226 infected tumors, suggesting that TSG on 4q may overlapping deleted regions (Figure 1, group C). interact with this virus (Laurent-Puig et al., 2001; Genotyping adjacent SNP markers con®rmed inter- Marchio et al., 2000; Wong et al., 2000). Other stitial deletions in tumors CHC121 and CHC188. The associations of 4q LOH with large tumor size, telomeric MCRD had to be divided in two non- aggressive histological type and high serum alpha- contiguous subregions, de®ned by only one interstitial fetoprotein level have been described in HCC tumors deletion each. In 4q34 the MCRD is located between (Bando et al., 1999; Piao et al., 1998; Yeh et al., 1996), rs1564498 and D4S1584 markers de®ning a region of 9 but their independence from the HBV status remains Mb. The interstitial deletion in CHC121 de®ned a questionable. MCRD in 4q35 between D4S2943 and D4S426. A Despite previous allelotypes focused on chromosome precise physical map of this last region is not available, 4q in HCC, a de®nite minimal common region of as a lot of discrepancies exist between the dierent deletion (MCRD) still remains to be identi®ed. At least sequence databases, but its size can be estimated at ®ve non-overlapping regions have been reported, being approximately 8 Mb. the most frequent regions within 4q21-q25, 4q28 and 4q35 (Bando et al., 1999; Bluteau et al., 1999; Piao et Mutation detection in candidate genes al., 1998; Yeh et al., 1996). Since a stringent de®nition of the boundaries of the MCRD is the ®rst step In the common deleted region at 4q22 more than 30 necessary to localize and identify the 4q TSG, in this genes have been mapped on the UCSC genome draft study we systematically analysed 85 microsatellite map (Figure 2). Among them, we successively identi®ed markers spanning chromosome 4q to assess LOH in seven candidate genes on the basis of their known or a series of 154 well-characterized primary liver tumors. putative role in the control of the cell growth or in dierentiation. (1) Bone morphogenetic protein receptor 1B (BMPR1B or ALK6) is a transmembrane serine/threonine kinase member of the TGF-beta Results receptor's superfamily that plays a pivotal role in bone formation during embryogenesis and fracture repair. Deletion mapping of chromosome 4q Furthermore, germline mutations of BMPR1A located The whole series of primary liver tumors including 154 on chromosome 10q have been recently identi®ed in matched tumor and non-tumor DNA pairs was juvenile polyposis families (Howe et al., 2001). (2) The analysed with 85 microsatellite markers distributed on dual adaptor of phosphotyrosine and 3-phosphoinosi- chromosome 4q. Distance between any two genotyped tides (DAPP1) contains a src homology 2 (SH2) and a markers varies from 0 to 12.5 cM with an average of pleckstrin homology (PH) domains (Dowler et al., 2cM according to the Genethon genetic map (Figure 1999). This protein is likely to play an important role 1). Microsatellite instability was not observed in more in triggering signal transduction pathways that lie than 2% of the markers and all the tumors were downstream from receptor tyrosine kinases and PI3- informative for an average of 68% of the tested loci. kinase. Initially located within our 4q22 critical region, LOH was determined by quantitative ¯uorescent genotyping SNP in DAPP1 demonstrated a retention genotyping and all putative deletions de®ned by one of heterozygosity in tumor CHC241, pushing the gene marker only were con®rmed with a second micro- outside the MCRD (Figure 2). (3) Inactive mutations satellite or SNP genotype in the vicinity. In 59 cases of PKD2 are the cause of autosomal dominant (38%) a chromosome 4q deletion was observed. A polycystic kidney disease type II (ADPKD type II), a deletion was observed at all informative markers in 23 common autosomal dominant genetic disease charac- tumors consistent with a complete monosomy of 4q. In terized by progressive formation and enlargement of the other cases, deletions were partial, including the cysts in both kidneys and liver (Mochizuki et al., 1996). telomere (17 cases), interstitial (15 cases) or non- (4) The cyclin E binding protein 1 (CEB1/LOC51191) contiguous (four cases). Unique interstitial and partial belongs to the HECT family and also contains RCC1- deleted regions were used to de®ne the MCRDs, like repeats (Mitsui et al., 1999). (5) HERC3 is a whereas tumors showing multiple regions of deletion member of the HERC (domain homologous to E6 (Figure 1 group D), or including both the centromeric associated protein carboxy-terminus and RCC1 do- and the telomeric MCRDs (Figure 1 group A) were main protein) family. It may function both as guanine excluded from this analysis. The most proximal region, nucleotide exchange factor and E3 ubiquitin ligase and mapping on 4q22 between D4S395 and D4S2986, was may play a role in vesicular trac and ubiquitin- clearly de®ned by nine interstitial deletions (Figure 1, dependent processes (Cruz et al., 2001). (6) SMAR- group B). To further re®ne the extent of 4q22 MCRD, CAD1 is a DEAD/H box-containing molecule that we selected and sequenced ¯anking SNP markers in belongs to the snf2 helicase superfamily including critical tumors (CHC019 and CHC241) compared to proteins essential to genome replication, repair, and their matching non-tumor DNA. New 4q22 region expression (Adra et al., 2000). (7) ENH is a protein boundaries were de®ned by A/C-231 and D4S2986 with a LIM domain that belongs to the Enigma family markers delineating a 15 Mb critical region (Figure 2). proteins involved in cytoskeleton organization, cell The telomeric MCRD was more dicult to delineate lineage speci®cation, organ development, and oncogen- as CHC121, CHC210 and CHC188 demonstrated non- esis (Kuroda et al., 1996). Screening 28 HCC tumor

Oncogene Chromosome 4q deletions in hepatocellular carcinoma O Bluteau et al 1227

Figure 1 Schematic representation of the partial and interstitial 4q deletions in the series of 154 liver tumors. The order of the 85 microsatellite markers has been deduced from the UCSC sequence map (coordinates in Mega-bases indicated) and from the GeÂ neÂ thon genetic map (coordinates in centi-Morgan indicated). For each tumor, gray vertical boxes indicate the largest deleted region according to the status of the markers LOH (black circle), presenting a retention of heterozygosity (white circle) and homozygous (gray circle). Black bars to the right indicate location of the 4q22 and 4q34-35 minimal common regions of deletion. Tumors are classi®ed according to the extent of their deletion, including the two MCRDs (group A), the 4q22 MCRD (group B), the 4q34-35 MCRD (group C) and non-contiguous deleted region (group D)

Oncogene Chromosome 4q deletions in hepatocellular carcinoma O Bluteau et al 1228

Figure 2 Fine mapping of the 4q22 MCRD. Polymorphic markers are indicated in the left with a NCBI reference SNP ID when known. For tumors CHC019 and CHC241, the deleted regions are indicated as in Figure 1. Accession numbers of the BAC clones are indicated according to the golden path of the UCSC sequence map corresponding contigs in the NCBI map are indicated. Horizontal gray and black lines represent oriented and non-oriented gaps, respectively. Vertical black lines on the right indicate location of known genes in the region. Genes screened for mutations are mentioned by an * and bold characters

Oncogene Chromosome 4q deletions in hepatocellular carcinoma O Bluteau et al 1229 and cell line DNAs on the entire coding region of always inferior to 50% and (2) at least two adjacent BMPR1B, DAPP1, PKD2, SMARCAD1 and ENH for markers deleted to de®ne a region of LOH in each mutations revealed rare polymorphisms but no somatic tumor. The proportion of HCC demonstrating chro- alterations. To screen for alterations in CEB1 and mosome 4q loss reported here is consistent with that HERC3 genes, as they contain more than 20 exons, reported in a series of tumors presenting low rate of RT/PCR was performed, followed by direct sequencing HBV infection (30%) and previously allelotyped and on 10 RNAs extracted from HCC cell lines. No studied by CGH (Bando et al., 1999; Boige et al., 1997; somatic mutations, abnormal transcripts or clear Laurent-Puig et al., 2001; Nagai et al., 1997; Piao et downregulation of the transcription level were shown al., 1998; Yeh et al., 1996). using a semi-quantitative analysis of the RT ± PCR Tumors with complex LOH region have been products. discarded for MCRD analysis. Therefore, the pattern of allelic losses in the present study identi®ed three discrete regions of minimal deletions. Our de®nition of Correlations between clinical characteristics and specific the proximal 4q22 MCRD includes the two loci 4q MRCD D4S1534 and D4S2929 previously identi®ed by Bando The frequency of 4q LOH, whatever the region lost, et al., 1999 as hot spots of deletion. Even though these was similar in the three major histopathological two markers have been tested in the present allelotyp- subgroups: (a) HCC, 39% (53 of 133 tumors), (b) ing analysis, we have not found the same hot spot of FLC, 29% (two of seven tumors); and (c) mixed deletions in our series of tumors. To explain this hepato-cholangiocarcinoma 56% (®ve of nine tumors). discrepancy, dierences in the geographical origin of In contrast, all the ®ve benign liver tumors retained the patients and/or in the allelotyping techniques could both chromosomes 4q. Relationships between tumor be evoked. In particular, we eliminated all the putative histo-clinical parameters and the extent of 4q LOH interstitial deletions at a unique locus that could not be regions were further examined in the group of 133 con®rmed by a second polymorphic deleted marker to classical HCC. As described in Laurent-Puig et al., avoid genotyping artifacts. For the distal MCRDs in 2001, a close relation exists between 4q LOH, whatever 4q35, the region de®ned here is in agreement with that the region, and HBV infection (P50.0001), and described by Bando et al., 1999. absence of alcohol intake (P50.001). In contrast to We chose to focus our candidate genes screening these established relationships, the speci®c deletion of strategy on the 4q22 MCRD that was non-ambigu- the 4q34-35 region is associated with a high grade of ously de®ned and because tumors exhibiting a speci®c dierentiation of the tumors (Edmondson grade I or 4q22 deletion did not dier in their risk factors from II) (P=0.02) and a heavy alcohol intake (P=0.005) those that delete the 4q, whatever the subregion. The (Figure 3). Furthermore, the speci®c 4q22 deletions are 4q22 MCRD contains at least 30 known genes and we related to the small size of the tumors (P=0.008). To screened about a quarter of them for mutations. These test the relative in¯uence of the clinical parameters on screened genes have been selected on their putative the speci®c subregion of the deletions, we integrated function in cell growth and/or dierentiation. Since no the three variables (alcohol intake, dierentiation and alterations were found, we can exclude frequent and diameter) in a maximum-likelihood logistic regression recurrent nucleotide mutations of these genes in model. With this analysis, we demonstrated that heavy hepatocarcinogenesis. Finally, more than 50 known alcohol intake or high grade of dierentiation of the genes, located in dierent MCRDs, remain to be tested tumors are independently associated with the loss of in HCC tumors even if their possible involvement in 4q34-35 region. hepatocarcinogenesis could not be inferred by their amino acid analysis. One of the most intriguing results of this study was Discussion the identi®cation of the unexpected relationship between alcohol intake and 4q34-35 deletions. The speci®c Allelotype studies and CGH assays have identi®ed high deletion of the 4q34-35 region de®nes a particular group frequency of deletions on 4q in HCC, consistent with of tumors, all of which are well dierentiated and have the presence of one or more TGSs on this chromoso- been exposed to alcohol intake. This result is in mal arm (Bando et al., 1999; Boige et al., 1997; complete contrast to the established relationship Laurent-Puig et al., 2001; Nagai et al., 1997; Piao et between 4q LOH, whatever the subregion, and HBV al., 1998; Yeh et al., 1996). These studies, however, infection previously described (Bluteau et al., 1999; suer from the lack of extensive and systematic Laurent-Puig et al., 2001; Marchio et al., 2000; Wong et deletion mapping analysis in delineating the exact al., 2000). To explain this observation, a de®nite location of MRCDs. To further de®ne the critical mechanism of alcohol intake leading to generate regions of chromosome 4q deletions, we performed a deletions of the 4q34-35 region might be proposed. systematic high-density allelotyping. We identi®ed 59 Furthermore, since chromosome 4q deletions are rare tumors (38%) with a LOH of chromosome 4q events in tumors secondary to alcohol abuse, the corresponding to our criteria of selection, i.e. (1) a presence of a TGS located in 4q34-35 is questionable. high percentage of allelic height reduction on 4q when In this work we show heterogeneity in HCC tumors compared to the rest of the autosome with a value in relation to the speci®c 4q subregion deleted. This is

Oncogene Chromosome 4q deletions in hepatocellular carcinoma O Bluteau et al 1230 % of cases

Figure 3 Relationships between clinical parameters and 4q deletions strati®ed in speci®c MCRD. For each speci®c deleted region percentage of the patients presenting the clinical parameter indicated in x-axis is scored. Statistically signi®cant relationships are indicated with their P value obtained using a Fischer's exact test

an extremely important ®nding in the search for TGS on chromosome 4q, since the delimitation of the (Mediterranean area 15 cases, sub Saharan Africa 12 cases, the Antilles ®ve cases and Asia seven cases). Determination MCRDs is crucial. However, in the present series none of the dierent risk factors was performed using the methods of the tumors exhibiting an interstitial deletion of the described in Laurent-Puig et al., 2001, and alcohol abuse was proximal 4q22 had been associated to alcohol abuse. recorded as a risk factor when the average reported daily Subsequently, our delineation of the 4q22 MCRD alcohol intake was over 80 g during more than 10 years. The seems to be independent from the putative role of histological grade of tumor dierentiation was assigned alcohol intake as a risk factor. In conclusion, the LOH according to the Edmondson and Steiner grading system. data in this study support previous reports that at least After grading using the Edmondson classi®cation, tumors two regions are recurrently deleted on chromosome 4q were classi®ed in two groups (i.e. grade I + II and grade III in HCC. TGS(s) located on 4q remain to be identi®ed, + IV). but great heterogeneity in the risk factors associated to The HCC cell lines SNU-182, SNU-387, SNU-398, SNU- 423, SNU-449, SNU-475 and Hep 3B were purchased from HCC and underlying dierent deletions has to be taken American Type Culture Collection (Manassas, VA, USA) into consideration in the cloning approach. and cases PLC/PRF, Malhavu and HUH7 were kindly provided by Mehmet Ozturk (Bilkent University, Ankara, Turkey). Tumor tissue samples were dissected and non-tumor tissues Materials and methods (450 mg) were taken as far from the tumor as possible. Tissues were stored at 7808C until DNA extraction was Patients and samples performed using the Qiaquick extraction kit (Qiagen, From 1992 to 1998, primary liver carcinoma and matched Courtaboeuf, France). DNAs were quanti®ed by ¯uorometry non-tumor liver tissues were obtained from 154 patients from (Fluoroskan Thermo LabSystem, Cergy-pontoise, France). four French surgical departments. To the previously reported 133 classical HCC cases (Laurent-Puig et al., 2001), we added LOH analysis seven ®brolamellar carcinomas (FLC), nine tumors presenting mixed cholangio and hepato dierentiation and ®ve All the 154 tumor DNA and matching non-tumor DNA were benign tumors (two adenomas, three focal nodular hyperpla- genotyped using 85 microsatellite markers selected from the sias). The majority of the patients were born in France (115 GeÂ neÂ thon database. The markers used in this study are listed cases) while the others have dierent geographical origins with their localization on chromosome 4q in Figure 1,

Oncogene Chromosome 4q deletions in hepatocellular carcinoma O Bluteau et al 1231 according to the GeÂ neÂ thon genetic map and to the sequencing Les Ulis, France). For all samples demonstrating a draft genome map available at the UCSC web site (release of chromosome 4q22 LOH with an allelic height reduction in April 2001). Microsatellite markers were ampli®ed using PCR the tumor of more than 80%, DHPLC analyses were with one primer ¯uorescently labeled with FAM, TET, or performed both using tumor DNA pure and mixed with NED, using a touch down protocol and Amplitaq poly- 50% of the autologous non-tumor DNA sample to allow merase (Applied Biosystems, protocol available upon re- heteroduplex formation. Aberrant DHPLC fragments were quest). PCR products were analysed on an ABI 377 reampli®ed and sequenced directly on an ABI 377 automatic automatic sequencer (Applied Biosystems). Peak height sequencer (Applied Biosystems). Whenever an alteration was values and peak sizes were assigned using the Genotyper detected in tumor DNA, DNA from autologous normal software (Applied Biosystems) with an automatic data- tissue was also analysed. In case of frequent polymorphisms processing trace program developed in the laboratory and identi®ed in the GenBank database or experimentally (i.e. available on request. The threshold for LOH was de®ned as 15% of the exons), screening for mutations was performed by an allelic intensity reduction in the tumor of more than 50%, direct sequencing of the PCR products analysed with the after normalization on the second allele and when compared Factura and Autoassembler softwares (Applied Biosystems). with the non-tumor matching DNA. Since all tumors have In case of the HERC3, CEB1 and SMARCAD1, because been previously screened with 400 microsatellite markers of the large number of exons (HERC3 and CEB1) or because distributed on the whole genome, in order to assess the LOH 35% of the genomic sequence were not available (SMAR- results, the average percentage of allelic reduction obtained CAD1), reverse-transcriptase PCR was performed to search on chromosome 4q was compared, for each sample, with the genetic alterations in the 10 cell lines. In each case, the entire value obtained on the other autosomes. Chromosome 4q known coding sequence was ampli®ed in three (SMARCAD1 LOH was established if the mean value on 4q did not dier and HERC3) or ®ve (CEB1) overlapping fragments and more than 20% in respect to the percentage of allelic subsequently ampli®ed fragments were directly sequenced. reduction obtained for the other frequently lost chromosome PCR primers and sequencing primers used in this project are arms, i.e. 1p, 6q, 8p, 9p, 13q, 16 and 17p. available upon request. To re®ne the boundaries of MCRD regions, we PCR ampli®ed fragments of 0.8 kb randomly or centered on SNP Statistical analysis described in the Single nucleotide polymorphism consortium (http://www.ncbi.nlm.nih.gov/SNP). LOH status assignment To test for associations between chromosome 4q LOH and was determined by measurement of the relative height of the clinical parameters, comparisons were made using w2 or allelic peaks at the polymorphic position in the sequence of the Fisher exact test when appropriate. Three categories of 4q tumor PCR fragment compared to the matching non-tumor one. deletions were determined to assess the possible biological eect of a 4q deletion in patients: centromeric (4q22), telomeric (4q34-35) and deletions that include both regions. Physical map Logistic regression model was performed using the Stata All physical map informations used in this work were available software (Stata Corporation, College Station, TX, USA). in the Human Genome Project Working Draft at UCSC (http://genome.ucsc.edu/, (Lander et al., 2001)). In selective situations, to control the order of markers or to ®ll a gap, for example, MapViewer and Blast on GenBank databases at the Acknowledgments NCBI (http://www.ncbi.nlm.nih.gov) were used. We thank Dr Licia Selleri for helpful discussions and critical reading of the manuscript. We are very grateful to Catherine Giudicelli, Christelle Vaury, Hung Bui, Isabelle Search for gene alterations Legall and Christine BellanneÂ -Chantelot for help in the A panel of 21 tumors deleted on chromosome 4q22 and seven sequencing and genotyping. This work was supported by cell lines was tested for genetic alterations in candidate genes. grants from the Association pour la Recherche sur le Tumor DNA samples were assessed for mutations in seven Cancer (ARC no 4217), the Ligue deÂ partementale de Lutte identi®ed genes mainly using DHPLC with the Helix system Contre le cancer de la Dordogne, the MinisteÁ re de la according to the manufacturer's recommendations (Varian, Recherche and the Inserm.

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