Linkage analysis and allelic imbalance in human breast cancer kindreds using microsatellite markers from the short arm of chromosome 3

Eight Icelandic breast cancer kindreds were subjected to linkage analyses with respect to 28 microsatellite loci dispersed along the short arm of chromosome 3. Breast tumors derived from these kindreds were concurrently scored for allelic imbalance with ten of the markers. Linkage to most markers could be excluded on the basis of negative LOD scores and haplotype analyses, although some moderately positive LOD scores resulted. A high frequency of imbalance in the familial tumors was seen with two of the markers in comparison with results obtained from sporadic material. The highest frequency (68%) of imbalance was detected with the marker D3S1217, which is located on 3p14.2-p14.1. Imbalance at the D3S1211 locus, which is more telomeric (3p24.2-p22), was not significantly elevated in the familial tumors. We suggest that the genetic defect responsible for breast cancer susceptibility in these families either promotes instability in the 3p14.2-p14.1 region or enhances the selective advantage of such changes.The Nordic primer bank that provided the microsatellite markers is supported by the Nordic Council of Ministers. This work was supported by the Nordic Cancer Union, Icelandic Cancer Society, the Science Fund of Iceland, the University of Iceland Research Fund the Science Fund of the University Hospital of Iceland and the Memorial Fund of Bergthora Magnusdottir and Jakob B. Bjarnason

Loss of heterozygosity at chromosome 1p in human breast cancer: Association with high S-phase, reduced patient survival and deletions at other chromosome regions

232 human primary invasive breast tumors were analyzed with 13 polymorphic microsatellite markers specific to chromosome 1p. Loss of heterozygosity (LOH) was observed in 126 cases or 54% of the tumors. One marker, D1S496, at the 1p35 region showed the highest LOH, 28%. High frequencies of LOH were also detected by the markers, D1S488, D1S167 and D1S435, at the 1p31 region, 25%, 24% and 26% LOH, respectively. This suggests the presence of tumor suppressor genes at these two regions. Tumors with and without LOH at 1p were tested for association with clinico-pathological features of the tumors such as estrogen- and progesterone-receptor content (ER and PgR), age at diagnosis, tumor size, node status, histological type, S-phase fraction, ploidy, survival and LOH at chromosomes 3p, 6q, 9p, 11p, 11q, 13q, 16q, 17p and 17q. A significant association was found between LOH at chromosome 1p and high S-phase fraction and lower survival rate. Association was also found between LOH at 1p and chromosome regions 3p, 6q, 9p and 17q. A multivariate model including prognostic variables, showed that LOH at 1p is an independent prognostic variable and patients who have breast tumors with LOH at 1p have approximately a two-fold increase in relative risk of death. We conclude that screening for 1p deletions gives additional prognostic information that might be useful in breast cancer treatment.This work was financially supported by the University of Iceland Graduate Research fund, the Icelandic Research Council and the Nordic Council of Ministers

Loss of heterozygosity at chromosome 11 in breast cancer: Association of prognostic factors with genetic alterations

We examined DNA from 116 female and four male breast cancer patients for loss of heterozygosity (LOH). DNA was analysed by polymerase chain reaction using ten microsatellite markers on chromosome 11. Three distinct regions of LOH were identified: 11p15.5, 11q13 and 11q22-qter with a LOH frequency of 19, 23 and 37-43% respectively. The marker D11S969 showing the highest frequency of LOH (43%) is located at the 11q24.1-q25 region. No previous molecular genetic studies have shown frequent LOH at the region telomeric to q23 on chromosome 11. Southern analysis revealed that LOH at 11q13 was due to amplification, whereas LOH at 11q22-qter was due to deletion. LOH at 11p15.5 was associated with paucity of hormone receptor proteins, high S-phase and positive node status. An association was found between LOH at 11q13 and positive node status. LOH at the 11q22-qter region correlated with a high S-phase fraction. A significant association was found between LOH at 11p15 and chromosome regions 17q21 (the BRCA1 region) and 3p.Nordic Council of Ministers, University of Iceland Graduate Research Fund, the Nordic Cancer Union, the Icelandic Cancer Society, the Memorial Fund of Bergthora Magnusdottir and Jakob B Bjarnason, the Science Fund of Iceland and the Science Fund of the University Hospital of Iceland

High frequency of LOH at chromosome 18q in human breast cancer: Association with high S-phase fraction and low progesterone receptor content

Human primary breast cancers were analysed for somatic loss of heterozygosity (LOH) at chromosome 18 with 15 polymorphic microsatellite markers. LOH was observed in 148 of the 228 cases analyzed (65%). Three smallest common deletion regions (SCDR) were detected on the long arm of chromosome 18. The marker D18S51 at the region 18q22 showed the highest LOH (42%). Tumors with and without LOH at 18q were tested for association with clinico-pathological features of the tumors, such as estrogen and progesterone receptor content, age at diagnosis, tumor size, node status, histological type, S-phase fraction DNA ploidy and LOH at other chromosomal regions. A significant association was found between LOH at 18q and high S-phase fraction (99.9% confidence interval) and low progesterone receptor. content (99%, confidence interval). Furthermore, an association was found between LOH at 18q and LOH at 1p, 7q, 9p, 13q and 17q. We conclude that there are three separate LOH target regions at chromosome 18q and that inactivation of one or. mole genes at these regions might be important for human breast carcinogenesis.The Icelandic Research Council and the Icelandic Cancer Societ

Different tumor types from BRCA2 carriers show wild-type chromosome deletions on 13q12-q13

In this study we examined loss of heterozygosity (LOH) on chromosome 13q12-13 in 50 tumors from BRCA2 carriers in five families showing strong evidence of linkage to BRCA2. In addition to high frequency of LOH in female breast cancer, LOH was observed in tumors of the prostate, ovary, cervix, colon, male breast, and ureter. All detected losses involved the wild-type chromosome. These results suggest that BRCA2 is a tumor suppressor gene and may be involved in the tumorigenesis of several cancer types in addition to breast cancer.This work was supported by the Nordic Cancer Union, the Science Fund of Iceland,the University of Iceland Graduate Research Fund, the Science Fund of the University Hospital of Iceland, and Ihc Memorial Fund of Bergthora Magnusdottir and Jakob B. Bjarnason

Loss of heterozygosity at chromosome 6q correlates with tumor progression and patient survival

Several chromosome regions exhibit loss of heterozygosity (LOH) in human breast carcinoma and are thought to carry tumor suppressor genes. We have analysed human breast tumors with 9 polymorphic microsatellite markers that are specific to chromosome 6q. The mapping of smallest region of overlap (SRO) indicated location of candidate suppressor genes at 6q23 and 6q27. Variations in estrogen receptor (ER) expression were independent of the number of copies of the corresponding gene. Tumors with and without LOH on chromosome 6q were tested for association with clinicopathological factors. A significant association was found between LOH at 6q and the following: high S-phase, aneuploidy, deletions at chromosomes 3p and 9p and lower survival rate. In a multivariate model LOH at 6q is an independent prognostic variable and patients having tumors with LOH have approximately twofold increase in relative risk of death. It can be concluded that the 6q deletions give additional prognostic information that might be useful in breast cancer treatment.The Nordic primer bank that provided the microsatellite markers is supported by the Nordic Council of Ministers. This work was supported by the Students' innovation fund, Nordic Cancer Union, Icelandic Cancer Society, the Science Fund of Iceland, the Science Fund of the University Hospital of Iceland and the Memorial Fund of Bergthora Magnusdottir and Jakob B. Bjarnason

High incidence of loss of heterozygosity at chromosome 17p13 in breast tumours from BRCA2 mutation carriers

Breast tumours from BRCA1 and BRCA2 mutation carriers are genetically instable and display specific patterns of chromosomal aberrations, suggestive of distinct genetic pathways in tumour progression. The frequency of abnormalities affecting chromosome 17p and the TP53 gene was determined in 27 breast tumours from 26 female patients carrying the Icelandic BRCA2 founder mutation (999del5). Loss of heterozygosity (LOH) was detected in 23 of the 27 tumours (85%). The majority of tumours manifesting LOH had lost a large region on 17p, although a more restricted loss, including the TP53 locus was seen in a few tumours. Positive p53 immunostaining was observed in 18 of 26 tumours (69%). However, mutations in the TP53 gene were detected in only three tumours (11%), including a missense (codon 139) and a nonsense mutation (codon 306) in two tumours with moderate p53 expression and a frameshift deletion (codon 182) in a tumour with no detectable p53 expression. Positive p53 immunostaining, mainly weak, was observed in 16 of the 24 tumours (66%) without TP53 mutation. The high frequency of LOH at chromosome 17p13 suggests that one or more genes from this region are involved in the development of BRCA2-induced breast cancer. The frequent finding of weak overexpression of, presumably wild type p53 protein, suggests an alternative mechanism of TP53 involvement specific to these tumours.This work was financially supported by the Nordic Cancer Union, The Icelandic Cancer Society, The Memorial Fund of Bergthora Magnusdottir and Jakob B Bjarnason, the Icelandic Research Council, the Science Fund of the University Hospital of Iceland and the Research Fund of the University of Iceland

Chromosome imbalance at the 3p14 region in human breast tumours: High frequency in patients with inherited predisposition due to BRCA2

Our previous studies have indicated that genetic aberrations in the 3p14 region are more frequent in malignant tumours from hereditary breast cancer patients than sporadic breast cancers. The main purpose of this study was to test if BRCA2 susceptibility alleles contribute to imbalance in the 3p14 region. We mapped allelic imbalance at 3p14 in tumours from Icelandic sisters affected with breast cancer using a set of 10 microsatellite markers (tel-D3S1295-D3S1234-D3S1300-D3S1600-D3S1233-D3S1217-D3S1261-D3S1296-D3S1210-D3S1284-cen). The patients were of known carrier status with respect to the 999del5 mutation in BRC42 which is the most common cause of hereditary breast cancer in Iceland. Of 103 patients, 32 in the group were mutation carriers. A high degree of imbalance was observed in tumours from BRCA2 mutation carriers, ranging from 44 to 88% for individual markers. This was significantly higher than the percentage of imbalance in tumours from non-carriers, where the frequency ranged from 25 to 43%. In both groups, we noted elevated 3p14 imbalance in patients with bilateral disease. Allelic imbalance was most commonly observed near the marker D3S1210 (3p14.1-p12) and the FHIT gene (3p21.1-p14.2) for both groups. We conclude that genomic aberrations in 3p14 are especially frequent in tumours with BRCA2 gene defects, and suggest that this is caused by regional loss of chromosome stability rather than selection.Icelandic Research Council and the Science Fund of the University Hospital of Iceland

Mapping of chromosome 3 alterations in human breast cancer using microsatellite PCR markers: Correlation with clinical variables

Human breast tumours were analyzed with polymorphic microsatellite markers specific to chromosome 3p. Allelic imbalance (AI) was observed in 34% of the tumours. Microsatellite markers from two regions showed higher percentage of imbalance suggesting the presence of tumour suppressor genes or genes important for malignancy. Microsatellite instability was also found, implying errors in DNA replication. No significant correlation was found between AI and conventional prognostic variables. However, a striking correlation was found between AI and tumour S-phase fraction; AI was also significantly correlated with low steroid receptor content. A multivariate model including prognostic variables, showed that AI was without exception a significant prognostic variable; patients having tumours with AI had approximately a four-fold increase in relative risk of death. We conclude that screening for 3p deletions gives prognostic information and further investigations should reveal whether this finding could assist in treatment of the disease.This work was financially supported by the Nordic Cancer Union, Icelandic Cancer Society, The University of Iceland Research Fund, the Memorial Fund of Bergthora Magnusdottir and Jakob B. Bjarnason, the Science Fund of Iceland and the Science Fund of the University Hospital of Iceland. The Nordic primer bank in Uppsala, Sweden, is supported by the Nordic Council of Ministers

Identification of a novel splice-site mutation of the BRCA1 gene in two breast cancer families: Screening reveals low frequency in Icelandic breast cancer patients.

The purpose of this study was to characterise the Icelandic BRCAl allele segregating in these breast cancer families and to estimate its significance in a population-based screening effort.Icelandic Research Council, Science Fund of the University Hospital of Iceland, Nordic Cancer Union and Memorial Fund of Bergthora Magnusdottir and Jakob B. Bjarnason