ANTICANCER RESEARCH 24: 2681-2688 (2004) Deletions on Chromosome 4 in Sporadic and BRCA Mutated Tumors and Association with Pathological Variables HREFNA K. JOHANNSDOTTIR1, GUDRUN JOHANNESDOTTIR1, BJARNI A. AGNARSSON1, HANNALEENA EEROLA2,3, ADALGEIR ARASON1, OSKAR TH. JOHANNSSON4,5, PÄIVI HEIKKILÄ6, VALGARDUR EGILSSON1, HAKAN OLSSON5, ÅKE BORG5, HELI NEVANLINNA2 and ROSA B. BARKARDOTTIR1 1Department of Pathology and 4Department of Oncology, Landspitali-University Hospital by Hringbraut, IS-121, Reykjavik, Iceland; 2Department of Obstetrics and Gynecology, Biomedicum Helsinki, P.O. Box 700, 00029 HUS, Helsinki; 3Department of Oncology, P.O. Box 180, 00029 HUS, Helsinki; 5Department of Oncology, Klinikgatan 7, University Hospital of Lund, SE-22185; 6Department of Pathology, P.O. Box 400, 00029 HUS, Helsinki, Finland Abstract. Background: Chromosomal aberrations in breast 4p16.3-p16.1, 4q27-q32.1 and 4q35.1-4qter, have not been tumors from BRCA1 and BRCA2 germ-line mutation carriers reported in breast cancer previously. Conclusion: The results are considerably more frequent than what is seen in sporadic manifest the frequent alterations of chromosome 4 in BRCA1- breast tumors. According to Comparative Genomic associated breast tumors and indicate the location of several Hybridisation analysis (CGH), deletions on chromosome 4 are genes of potential importance in breast cancer development. one of the most frequent events in BRCA1-associated tumors, suggesting inactivation of specific tumor suppressor genes. Breast cancer is the most frequently diagnosed malignancy Materials and Methods: In the present study, 16 microsatellite in women in the Western countries (1). Of the many risk markers covering chromosome 4 were used to map loss of factors that have been identified for breast cancer, family heterozygosity (LOH) in tumors from BRCA1 (n=41) as well history is the best established one (2). Approximately half of as in tumors from BRCA2 (n=66) mutation carriers and in high risk familial breast cancer cases can be explained by tumors from unselected cases of breast cancer (n=68). Results: inactivation of mutation in one of the two major breast The frequency of LOH in these groups ranged from 16-73% in cancer susceptibility genes, BRCA1 or BRCA2, although the BRCA1-associated tumors, 13-42% in BRCA2-associated proportion varies in different populations (3-5). Tirkkonen tumors and 8-33% in unselected tumors. LOH was significantly et al. have used comparative genomic hybridisation (CGH) more frequent in BRCA1-associated tumors as compared to to define genetic alterations in breast tumors from BRCA1 BRCA2-associated tumors and unselected tumors, and and BRCA2 mutation carriers (6). The study revealed a high particularly high (over 70%) at 4q35.2. Pathological variables frequency of overall chromosomal copy number changes in that were found significantly associated (p≤0.05) with LOH at BRCA1 and BRCA2 mutation carriers in comparison with specific markers were: high percentage of cells in S-phase, tumors from unselected breast cancer patients. This elevated negative estrogen receptor status, young age at diagnosis and frequency of genetic aberrations could possibly reflect the large tumors. Deletion mapping indicates the existence of seven important roles of BRCA1 and BRCA2 in maintaining non-overlapping regions at chromosome 4, which were identified genomic integrity and stability (reviewed in (7)). in all three groups of tumors. Three of these seven regions, Furthermore, the pattern of genetic changes observed suggest that BRCA1 and BRCA2 tumors have a different spectrum of genetic changes, indicating distinct progression pathways depending on the genetic background. Correspondence to: Rosa B. Barkardottir, Department of Pathology, In the CGH analysis by Tirkkonen et al., deletions on House 14 by Eiriksgata, Landspitali-University Hospital by chromosome 4q were among the most frequently observed Hringbraut, IS-121, Reykjavik. Tel: 354-5438033, Fax: 354-5434828, e-mail: [email protected] events in BRCA1 tumors (6). As the resolution of CGH is limited, a more refined approach is needed to define the Key Words: Breast cancer, BRCA1, BRCA2, loss of heterozygosity. regions involved. In the present study, mapping of LOH 0250-7005/2004 $2.00+.40 2681 ANTICANCER RESEARCH 24: 2681-2688 (2004) Table I. Frequency of loss of heterozygosity at chromosome 4 in BRCA1 and BRCA2 mutated tumors and unselected tumors and the association between loss of heterozygosity at respective markers and pathological variables. Marker Cytogenetic location Loss of heterozygosity/informative samples (%) Association with (Mb)* BRCA1 BRCA2 Unselected Total pathological variables** (p value) D4S2936 4p16.3 (0.7) 11/26 (42) 14/33 (42) 4/32 (13) 29/91 (32) S-phase (0.01) D4S43 4p16.3 (2.3) 6/14 (43) 9/26 (35) 3/31 (10) 18/71 (25) ER (0.001) D4S1614 4p16.3 (2.7) 9/15 (60) 6/22 (27) 4/27 (15) 19/64 (30) D4S394 4p16.1 (7.0) 10/18 (56) 15/42 (36) 7/54 (13) 32/114 (28) S-phase (0.03) D4S1546 4p15.31 (20.5) 8/22 (36) 14/38 (37) 6/42 (14) 28/102 (27) D4S2971 4q12 (53.6) 3/19 (16) 6/45 (13) 6/29 (21) 15/93 (16) S-phase (0.02) D4S3000 4q12 (57.2) 7/19 (37) 8/47 (17) 4/42 (10) 19/108 (18) D4S2964 4q21.21 (81.2) 14/27 (52) 12/38 (32) 6/31 (19) 32/96 (33) S-phase (0.005) D4S414 4q22.1 (92.9) 7/20 (35) 12/40 (30) 3/40 (8) 22/100 (22) D4S2917 4q25 (109.2) 8/18 (44) 14/43 (33) 12/36 (33) 34/97 (35) D4S427 4q27 (121.9) 9/22 (41) 12/38 (32) 4/29 (14) 25/89 (28) age (0.02) D4S424 4q31.21 (142.8) 11/24 (46) 13/48 (27) 4/33 (12) 28/105 (27) S-phase (0.004), size (0.02) D4S413 4q32.1 (158.9) 14/23 (61) 12/48 (25) 10/42 (24) 36/113 (32) S-phase (0.03) D4S1595 4q34.1 (175.0) 12/19 (63) 12/44 (27) 9/30 (30) 33/93 (35) S-phase (0.003) D4S1554 4q35.1 (185.4) 11/24 (46) 15/47 (32) 7/44 (16) 33/115 (29) age (0.05) D4S426 4q35.2 (189.8) 11/15 (73) 14/38 (37) 7/36 (19) 32/89 (36) ER (0.02) * localisation of markers in megabases according to UCSC Genome Bioinformatics (http://genome.uscs.edu/) ** the pathological variables tested were estrogen receptor status (ER), progesterone receptor status (PgR), histological type of tumor, tumor size, age at diagnosis, percentage of cells in S-phase, ploidy and tumor grade with microsatellite markers covering chromosome 4 in al. (8). DNA was extracted from blood samples according to Miller tumors from BRCA1 as well as in BRCA2 carriers and et al. (9). Mapping of chromosome 4 was performed using 16 unselected breast cancer patients was performed. The microsatellite markers (MWG-Biotech AG, Ebersberg, Germany) (Table I). PCR was performed in 15 Ìl reactions with 25 ng DNA, deletion frequency was found to be very high at specific loci 2 mM MgCl2, 0.16 mM nucleotides (Amersham Pharmacia Biotech in BRCA1-associated tumors and, although in most cases at Inc, Buckinghamshire, England), 0.32 ÌM each primer and 0.3 u a lower frequency, the same loci are also affected in the Taq DNA polymerase together with the enclosed 10xbuffer (MBI other subgroups of breast tumors. Fermentas, St. Leon-Rot, Germany). Reactions were as follows: 30 sec at 94ÆC, 45 sec at 55ÆC and 45 sec at 72ÆC for 35 cycles. The Materials and Methods reactions were preceded with denaturation at 94ÆC for 3 min and followed by 10 min at 72ÆC. Samples. DNA from 175 paired blood and breast tumor samples were subjected to analysis of LOH on chromosome 4. The sample LOH analysis. The PCR products were separated on a 6.5% set included tumors from BRCA1 (n=41) and BRCA2 (n=66) polyacrylamide gel (8M urea) in 1 x TBE buffer, and blotted to mutation carriers as well as tumors from unselected cases of breast Hybond-N+ nylon transfer membrane (Amersham Pharmacia cancer (n=68). The Swedish samples were collected at the Biotech Inc, Buckinghamshire, England). The DNA bands were Department of Oncology/Oncogenetic clinic at the Lund University visualised using ECL (Amersham Pharmacia Biotech Inc, Hospital (18 BRCA1 mutated and 22 unselected breast tumors), Buckinghamshire, England) according to the manufacturer’s the Finnish samples at the Department of Obstetrics and protocol with the modification of employing HEPES buffer (40 Gynecology and Oncology, Helsinki University Hospital (15 mM K-HEPES, pH 7.2 with 1mM CoCl2), instead of cacodylate, BRCA1 and 17 BRCA2 mutated tumors), and the Icelandic for tailing oligomers with terminal transferase for probe samples at the Department of Pathology, Landspitali-University preparation. Absence of or significant decrease in the intensity Hospital (8 BRCA1, 49 BRCA2 mutated tumors and 46 unselected of one allele relative to the other was considered LOH. tumors). The study was approved by the respective Ethical and Data Protection Committees in each country. Statistical analysis. A Chi-square test with Yates correction was used to compare LOH frequencies between the three groups of DNA extraction and PCR. DNA extraction from paraffin-embedded breast tumors as well as to evaluate the relationship between tissue and fresh tumor tissue was performed according to Smith et LOH and different pathological variables. Fisher’s exact test was 2682 Johannsdottir et al: LOH at Chromosome 4 in Breast Tumors Figure 1. The frequency of loss of heterozygosity on chromosome 4 in tumors from patients with germ-line mutation in BRCA1 (■), BRCA2 ( ) and unselected tumors (■). a = significant difference between the frequency of LOH in BRCA1 mutated versus unselected tumors. b = significant difference between the frequency of LOH in BRCA1 versus BRCA2 mutated tumors.