Clinico-Pathological and Prognostic Significance of p53, Bcl-2 and Her-2/neu Protein Markers in Colorectal Cancer Using Tissue Microarray

Background: The prognostic role of her-2/neu has been established in breast cancer but remains controversial in colorectal cancer (CRC). Widespread genetic mutations in colorectal carcinogenesis exist on chromosome 17. Her- 2/neu gene and the tumor suppressor gene p53 are both located on this chromosome. Bcl-2 protein prolongs survival of a variety of cells by blocking apoptosis. The aim of this study is to evaluate the relationship between the overexpression of p53, bcl-2 and her-2/neu protein markers and the clinico-pathologic characteristics of CRC, and their influence on survival rates. Patients and Methods: One hundred and four cases of CRC had paraffin blocks with representative tissue, and sufficient follow-up data. They were arrayed and evaluated for protein marker expression using tissue microarray (TMA). Results: Ten (9.6%), 35 (33.7%) and 27 (26%) of the patients were her-2/neu, p53 and bcl-2 positive, respectively. None of the examined clinico-pathologic factors had a significant relation with her-2/neu overexpression. Patients with +ve bcl-2 had a significantly higher mean age (52.4-/+13.3years) compared to 45.4-/+14.4 years for bcl-2 negative patients, p=0.03. Positive p53 was overexpressed in 20/44 (45.5%), 6/17 (35%), 9/43 (21%) cases of the colon, recto-sigmoid, and rectal sites, respectively, p=0.05. For the whole population, p53 overexpression had a significantly lower disease-free survival (DFS). For patients with Dukes' stage B, overexpression of p53 protein had a significant reduced overall survival (OS) p=0.04, metastasis free survival (MFS) p=0.004, and DFS p=0.01 rates. Expression of bcl-2 had a significantly better MFS p=0.001, while her-2/neu overexpression worsened the OS rate significantly, p=0.04. Conclusion: This study recommends the application of TMA technique for its economic importance and reliable quick throughput. The results from this study also suggest that overexpression of p53, bcl-2, and her-2/neu protein markers appear to be useful in selecting a group of CRC patients with a worse prognosis and constitute potential candidates for adjuvant therapy. Key Words: Her-2/neu , p53 , Bcl-2 , Colorectal cancer , Tissue microarray , Prognostic factors.     

Localisation by in situ hybridisation of S100A4 (p9Ka) mRNA in primary human breast tumour specimens

Rodent S100A4 (p9Ka) induces a metastatic phenotype in benign rat mammary tumour cells and cooperates with the neu oncogene to produce metastatic tumours in a transgenic mouse model system. Human S100A4 possesses similar metastasis-inducing properties. S100A4 mRNA is now sought in human breast tumour-derived cell lines and tumour specimens. S100A4 mRNA is present in some cell lines derived from malignant breast cancers, but is not detectable in cells derived from benign breast tumours. In human tumour specimens, using in situ hybridisation, the mRNA for S100A4 is localised to the epithelial cells of carcinoma specimens, and in some normal breast specimens, to a stromal region surrounding the epithelial ducts. In carcinoma specimens, S100A4 mRNA is also found in the stromal region surrounding islands of cancer cells. For both the epithelial and stromal components, S100A4 mRNA is present at a higher level in carcinomas relative to benign breast tumour specimens. In general, there is a concordance between the S100A4 mRNA signal from the epithelial and stromal elements of the same carcinoma specimens. Using Northern blotting techniques, these results have been extended to a panel of 137 benign and malignant breast tumour specimens. The results show that S100A4 mRNA occurs in the more-malignant, rather than in the more-benign tumour specimens.     

Correlations among p53, Her-2/neu, and ras overexpression and aneuploidy by multiparameter flow cytometry in human breast cancer: evidence for a common phenotypic evolutionary pattern in infiltrating ductal carcinomas.

Human solid tumors develop multiple genetic abnormalities that accumulate progressively in individual cells during the course of tumor evolution. We sought to determine whether there are specific sequences of occurrence of these progressive evolutionary changes in human breast cancers by performing correlated cell-by-cell measurements of cell DNA content, p53 protein, Her-2/neu protein, and ras protein by multiparameter flow cytometry in 56 primary tumor samples obtained at surgery. In addition, p53 allelic loss and Her-2/neu gene amplification were determined by fluorescence in situ hybridization in cells from the same samples. We reasoned that if there is a specific order in which genetic changes occur, the same early changes would be found consistently in the cells with the fewest abnormalities. We reasoned further that late-developing abnormalities would not occur alone in individual cells but would almost always be found together with the early changes inherited by the same cells. By these criteria, abnormalities involving p53 generally occurred early in the course of development of invasive breast cancers, whereas ras protein overexpression was found to be a late-occurring phenomenon. Within individual tumors, cellular p53 overexpression was often observed alone in individual cells, whereas ras protein overexpression was rarely observed in the absence of p53 overexpression and/or Her-2/neu overexpression in the same cells. Furthermore, the intracellular level of each abnormally expressed protein was found to increase progressively as new abnormalities were acquired. Infiltrating ductal carcinomas exhibited characteristic phenotypic patterns in which p53 allelic loss and/or p53 protein overexpression, Her-2/neu amplification and/or overexpression, aneuploidy, and ras overexpression accumulated within individual cells. However, this pattern was not a prominent feature of lobular breast cancers. All six lobular breast cancers studied were diploid. p53 allelic loss and/or early p53 overexpression, and late ras cooverexpression in the same cells were less common in lobular breast cancers than in infiltrating ductal carcinomas. Although Her-21neu overexpression was a common finding in lobular breast cancers, Her-2/neu amplification was not observed in these tumors.     

HER2/neu gene amplification and protein overexpression in G3 pT2 transitional cell carcinoma of the bladder: a role for anti-HER2 therapy?

HER2/neu is an oncogene encoding a type 1 tyrosine kinase growth factor receptor. Polysomy 17, gene amplification and HER2/neu protein overexpression are associated with a poor prognosis in transitional cell carcinomas (TCC) of the bladder. Due to the application of different laboratory techniques, the exact incidence of HER/neu abnormalities remains uncertain in TCC. Standardised laboratory techniques are therefore important in the determination of the HER2/neu status if an assessment of the potential value of anti-HER2/neu treatments in the clinical management of patients with TCC is to be made. In this study, 75 TCCs with evidence of detrusor muscle invasion at first clinical presentation were included. Gene amplification, polysomy 17 and HER2 copy number were assessed using fluorescence in situ hybridisation (FISH), with separate probes for chromosome 17 and HER2/neu. Protein overexpression was assessed using immunohistochemistry (IHC), with the CB11 antibody and a scoring system evaluating only membranous staining as positive. The mean patient age was 69.5 years (range 42-93 years) and the median survival was 15 months (range 1-156 months). Polysomy 17 occurred in 97%, increased HER/neu copy number in 92% and HER2/neu gene amplification in 7%. Protein overexpression occurred in 57% of cases. Polysomy 17 and HER2/neu protein overexpression are common in G3 pT2 TCCs of the bladder. However, gene amplification is uncommon. Mechanisms other than gene amplification may be responsible for protein overexpression in this tumour type. Evidence from breast cancer suggests that only tumours with HER2/neu gene amplification respond to the anti-HER2/neu therapy trastuzumab (Herceptin). If this were true for bladder cancer, only 4/75 (5%) of G3 pT2 TCCs would be suitable for treatment. The role of trastuzumab in these tumours remains untested at present.     

Loss of heterozygosity at selective sites on chromosomes 13 and 17 in human breast carcinoma.

Four chromosomal regions were tested for loss of constitutional heterozygosity in primary tumours from 85 Icelandic breast cancer patients. Loss of heterozygosity and other types of gene rearrangements were observed in 37% of informative cases at the retinoblastoma locus, RB1, on chromosome 13q. Allele losses on chromosome 17 were tested with two polymorphic probes on 17p and two on 17q. Loss of heterozygosity or other types of genetic rearrangement were detected in 43.5% of cases on 17p near the p53 gene and 40.5% on 17q. In our study abnormalities at the RB1 locus and on chromosome 17 frequently occurred together, indicating that the coincident inactivation of more than one tumour suppressor gene may, in some cases, play a part in tumour formation. No significant correlation was found between these losses and clinico-histological parameters. Family history of breast cancer was found to be more common among patients with RB1 deletions and this trend was strengthened in cases where there were deletions at both the RB1 locus and on chromosome 17.     

p53 allele losses, mutations and expression in breast cancer and their relationship to clinico-pathological parameters.

The p53 locus on the short arm of chromosome 17 at 17p 13.1 was examined for loss of heterozygosity, mutation, mRNA and protein expression in 60 primary breast cancers. Allele loss around the p53 locus was detected in 19/45 informative tumours (42%). p53 mutations in the evolutionarily conserved exons 5 to 9 were detected in 17/60 (28%) by amplification mismatch and confirmed by direct DNA sequencing. p53 mRNA expression was detected by Northern blot in 36/59 (61%) of tumours, and p53 protein expression using antibody 1801 on frozen-tissue sections in 13/44 of the tumours examined. p53 mutation was significantly associated with oestrogen-receptor-poor tumours (p less than 0.01) and hence with poor prognosis, but not with other clinical or pathological parameters. There was no statistical correlation between loss of heterozygosity around the p53 locus at 17p13.1 and p53 mutation. Furthermore, p53 mutation was not associated with p53 expression detected by immunohistochemical staining with antibody 1801 or as p53 mRNA. In addition, events on 17p (allele losses, p53 mutation, p53 expression) were independent of c-erbB-2 expression. In breast cancer, by contrast with colorectal, lung and ovarian cancer, there appears to be no clear association between p53 DNA abnormalities and p53 expression.     

Heparanase-1 gene expression in normal, hyperplastic and neoplastic prostatic tissue

Heparanase-1 (Hpa-1) has been implicated in tumour invasion and metastasis. In the present study, we evaluated the clinicopathological significance of Hpa-1 mRNA expression in prostate cancer and non-cancerous prostatic tissue by one-step polymerase chain reaction (PCR) of laser microdissected prostatic gland cells. In addition, cell type-specific expression of Hpa-1 mRNA in prostatic tissue was analysed by in situ hybridisation. Hpa-1 mRNA expression was found in 50% of normal and 40% of hyperplastic prostatic tissue. In situ hybridisation showed that Hpa-1 mRNA was strongly expressed in prostate gland cells. Of the 26 prostate carcinomas tested, 42% were positive for Hpa-1 mRNA. However, in non-cancerous prostatic tissue, Hpa-1 mRNA was significantly more often expressed than in less differentiated or more invasive prostate cancers (P<0.05). In situ hybridisation revealed only focal Hpa-1 mRNA expression in the neoplastic gland cells. Hpa-1 mRNA expression in the tumours significantly correlated with tumour differentiation and tumour stage (P<0.05). Our data indicate that Hpa-1 gene expression may be lost during dedifferentiation of prostatic gland cells.     

Overexpression of the p53 protein and allele loss at 17p13 in ovarian carcinoma.

Mouse monoclonal antibodies PAb 240 and PAb 1801 which specifically immunoprecipitate p53 protein, were used to examine 27 fresh ovarian tumours (16 serous adenocarcinomas, six endometrioid carcinomas, one mucinous adenocarcinoma, one mucinous borderline tumour and three benign adenomas). Eleven out of 16 (69%) serous adenocarcinomas and one endometrioid tumour showed positive staining with one or both antibodies and none of the mucinous or benign tumours stained with either antibody. DNA from tumour and peripheral blood leukocytes was used to identify allelic deletions on chromosome 17p in tumours. 11/12 positively staining tumours showed less of heterozygosity (LOH) on 17p at the nearest informative locus to the p53 gene. In this series of ovarian tumours, LOH on 17p correlates closely with the aberrant expression of the p53 protein in a high proportion of advanced stage serous adenocarcinomas. This observation suggests that the p53 tumour suppressor gene is involved in the evolution of epithelial ovarian cancer (EOC) and may have prognostic significance.     

Her-2/neu-triggered intracellular tyrosine kinase activation: in vivo relevance of ligand-independent activation mechanisms and impact upon the efficacy of trastuzumab-based treatment

Proteolytic cleavage of the Her-2/neu extracellular domain (ECD) has been shown to initiate receptor phosphorylation representing Her-2/neu activation in vitro. The present investigation was performed to evaluate the clinical relevance of ECD cleavage for Her-2/neu activation and the consequences of active intracellular Her-2/neu signalling reflected by tyrosine kinase phosphorylation in patients treated with the anti-Her-2/neu antibody trastuzumab. Sera from 62 patients receiving trastuzumab-based treatment for Her-2/neu overexpressing metastatic breast cancer were assessed for pretreatment ECD levels using an enzyme-linked immunosorbent assay. In parallel, Her-2/neu activation status of tumour specimens was assessed by immunohistochemistry using a Her-2/neu phosphorylation state specific antibody (PN2A) and correlated with the patients' ECD levels and clinical course of disease. Serum ECD levels were significantly higher in 15 (24%) patients with tumours exhibiting activated Her-2/neu as compared to those without detectable Her-2/neu phosphorylation (median 148.2 vs 28.5 ng ml(-1), P=0.010). Whereas response rate only showed a trend to be higher in patients with Her-2/neu-phosphorylated breast cancer (47 vs 34%, P=0.197), both uni- and multivariate analyses revealed that the median progression-free survival under trastuzumab-based treatment was significantly longer in patients with Her-2/neu-phosphorylated breast cancer-11.7 (95% CI 5.2-18.3) months-when compared to the progression-free survival of 4.5 (95% CI 3.4-5.6) months observed in patients with tumours lacking phosphorylated Her-2/neu (P=0.001). Proteolytic cleavage of the ECD represents a biologically relevant ligand-independent mechanism of Her-2/neu activation in vivo. The influence of Her-2/neu activation status upon the outcome of trastuzumab-based therapies merits further investigation in larger prospective trials.     

HER2/neu overexpression in the development of muscle-invasive transitional cell carcinoma of the bladder

The mortality from transitional cell carcinoma (TCC) of the urinary bladder increases significantly with the progression of superficial or locally invasive disease (pTa/pT1) to detrusor muscle-invasive disease (pT2+). The most common prognostic markers in clinical use are tumour stage and grade, which are subject to considerable intra- and interobserver variation. Polysomy 17 and HER2/neu gene amplification and protein overexpression have been associated with more advanced disease. Standardised techniques of fluorescence in situ hybridisation and immunohistochemistry, which are currently applied to other cancers with a view to offering anti-HER2/neu therapies, were applied to tumour pairs comprising pre- and postinvasive disease from 25 patients undergoing treatment for bladder cancer. In the preinvasive tumours, increased HER2/neu copy number was observed in 76% of cases and increased chromosome 17 copy number in 88% of cases, and in the postinvasive group these values were 92 and 96%, respectively (not significantly different P=0.09 and 0.07, respectively). HER2 gene amplification rates were 8% in both groups. Protein overexpression rates were 76 and 52%, respectively, in the pre- and postinvasive groups (P=0.06). These results suggest that HER2/neu abnormalities occur prior to and persist with the onset of muscle-invasive disease. Gene amplification is uncommon and other molecular mechanisms must account for the high rates of protein overexpression. Anti-HER2/neu therapy might be of use in the treatment of TCC.     

p53 mutations in childhood thyroid tumors from Belarus and in theyroid tumors without radiation history

Mutations in the p53 tumour-suppressor gene (exons 5–8) were investigated in 31 Belarussian childhood thyroid tumours (24 cases of papillary thyroid carcinoma, 3 benign tumours and 2 cases each of thyroiditis and goiter); 33 thyroid tumours from juveniles and adults without radiation exposures (25 carcinomas of various histological types, including 11 papillary carcinomas and 8 adenomas) and 6 tumours from adults (4 papillary carcinomas, 1 adenoma, 1 goiter) served as controls. The mutational spectrum of p53 differed greatly between the childhood thyroid carcinomas from Belarus and the control groups. In the control groups of 29 malignant thyroid tumours, 7 different mutations were detected on exons 5–8, none of which occurred among the 15 papillary carcinomas in this group. Five mutations were found in tissue samples of the 24 childhood papillary carcinomas, and they were all the same p53 point mutation (CGA |iO CGG) on codon 213 of exon 6. To determine whether this mutation is simply a polymorphism or whether it is specific to the tumour cells, laser-assisted microdissection was applied to collect various areas of tumorous and non-tumorous cells (10–20 cells per sample) from each paraffin-embedded tissue section of 8 of the papillary thyroid carcinomas. Using PCR-SSCP and sequence analysis on these cells, the very same p53 mutation on codon 213 was detected in various microdissected tumour samples of 2 cases, but it was not found in any microdissected non-tumorous sample. The exclusive occurrence of this p53 mutation in selective microdissected samples of tumour cells, even as homozygous mutation in 1 case, reflects a distinct tumour heterogeneity within papillary childhood thyroid carcinomas. Int. J. Cancer 73:802–807, 1997. © 1997 Wiley-Liss, Inc.     

Allele loss of tumour suppressor genes on chromosome 17 in human testicular germ cell tumours

The molecular genetics of testicular germ cell tumours (TGCT) are still largely unknown. We investigated 20 TGCT tumours for allelic losses (LOH) of tumour supressor genes BRCA1, TP53 and of THRA1 on chromosome 17. We observed an overall loss of 50% for the whole chromosome. Detailed deletion mapping revealed no losses for the BRCA1 gene, 42% LOH for THRA1 and 11% allelic loss for the region telomeric to BRCA1. We observed 11% LOH for TP53. Our results suggest that allelic losses of BRCA1 and TP53 genes do not play a pivotal role in TGCT but that dysfunction of THRA1 or tumour suppressor gene(s) in this region may have an impact in the development of this cancer.     

Evidence that p53 behaves as a tumour suppressor gene in sporadic breast tumours

Using the polymerase chain reaction, we have amplified exons 5 and 6 of the human p53 gene from paired samples of tumour and blood DNA of 60 patients with sporadic breast tumours and from placental or tonsil DNA of 30 normal controls. The patients' DNAs were previously examined for loss of heterozygosity of markers distal to the p53 gene on chromosome 17p and tumour samples were assayed for p53 mRNA levels. Hydroxylamine modification of mismatched base pairs was used to identify mutations in the amplified product. We have directly sequenced many of the samples including all those with mutations and have identified the particular mutation in each case. Exons 5 and 6 code for amino acids 126 through to 224 and constitute approximately 25% of the total coding region of the gene. They encompass part of the SV40 binding region as well as 2 of 5 areas known to be highly conserved in evolution and mutations in this region have been shown to be associated with tumorigenicity. We have found mutations in 13% of the primary tumours (8/60), all of which show allele loss of markers in the 17p region. No mutations were found in exons 5 and 6 of 30 normal controls. Our results are the first to identify mutations in p53 in primary breast tumours. Since our analysis has so far been confined to only part of the coding region of the gene, it is likely that further studies will reveal a mutation frequency in excess of 13%.     

A microdissection approach to detect molecular markers during progression of prostate cancer.

To investigate the underlying mechanisms of carcinogenesis, we have developed a technique to determine the frequency of genetic changes in prostatic carcinoma tissue. We have demonstrated that at a ratio of between 1:4 and 1:9 mutant-normal alleles, the signal from a mutant TP53 allele is not apparent after polymerase chain reaction (PCR) amplification and further direct sequencing or single-strand conformation polymorphism (SSCP) analysis. To bypass this problem, which is inherent in the heterogeneity of the prostate tissue and of the tumour, we selected areas of graded prostate tumours (Gleason score) from cryosectioned preparations and microdissected these cells (20-100 cells). After anionic resin removal of proteins, PCR amplification of TP53 gene exons 5/6 and SSCP analysis, an abnormal SSCP band shift was observed in suspected tumour cells, compared with microdissected stromal cells used as an internal control, while (1) a crude preparation of tissue DNA carrying the tumour did not show any abnormality and (2) immunostaining by a set of monoclonal antibodies against TP53 protein remained negative. Nucleotide sequence analysis of the different bands confirmed the presence of a mutation in the TP53 gene exon 6 position 13,336 in an abnormal band for one specimen, while no mutation was detected in the normal SSCP band. By targeting recognised tumour cells we can find DNA mutations which are undetectable using the standard technique of whole-tissue DNA extraction, particularly in a heterogeneous tumour such as carcinoma of the prostate.     

Loss of heterozygosity for defined regions on chromosomes 3, 11 and 17 in carcinomas of the uterine cervix.

Loss of heterozygosity (LOH) frequently occurs in squamous cell carcinomas of the uterine cervix and indicates the probable sites of tumour-suppressor genes that play a role in the development of this tumour. To define the localization of these tumour-suppressor genes, we studied loss of heterozygosity in 64 invasive cervical carcinomas (stage IB and IIA) using the polymerase chain reaction with 24 primers for polymorphic repeats of known chromosomal localization. Chromosomes 3, 11, 13, 16 and 17, in particular, were studied. LOH was frequently found on chromosome 11, in particular at 11q22 (46%) and 11q23.3 (43%). LOH on chromosome 11p was not frequent. On chromosome 17p13.3, a marker (D17S513) distal to p53 showed 38% LOH, whereas p53 itself showed only 20% LOH. On the short arm of chromosome 3, LOH was frequently found (41%) at 3p21.1. The beta-catenin gene is located in this chromosomal region. Therefore, expression of beta-catenin protein was studied in 39 cases using immunohistochemistry. Staining of beta-catenin at the plasma membrane of tumour cells was present in 38 cases and completely absent in only one case. The tumour-suppressor gene on chromosome 3p21.1 may be beta-catenin in this one case, but (an)other tumour-suppressor gene(s) must also be present in this region. For the other chromosomes studied, 13q (BRCA-2) and 16q (E-cadherin), only sporadic losses (< 15% of cases) were found. Expression of E-cadherin was found in all of 37 cases but in six cases the staining was very weak. No correlation was found between clinical and histological parameters and losses on chromosome 3p, 11q and 17p. In addition to LOH, microsatellite instability was found in one tumour for almost all loci and in eight tumours for one to three loci. In conclusion, we have identified three loci with frequent LOH, which may harbour new tumour-suppressor genes, and found microsatellite instability in 14% of cervical carcinomas.     

Her-2/neu gene amplification compared with HER-2/neu protein overexpression on ultrasound guided core-needle biopsy specimens of breast carcinoma

Genomic amplification and oncoprotein overexpression of Her-2/neu was studied on ultrasound core needle biopsy specimens of the infiltrative ductal carcinomas of the breast. We performed two colour fluorescence in situ hybridization (FISH) for Her-2/neu and chromosome 17 and compared the FISH results with the immunohistochemical overexpression of Her-2/neu protein by 2 antibodies (DAKO HercepTest and the BioGenex monoclonal antibody AM 134-5M). Furthermore, following radical mastectomy with axillary dissection, Her-2/neu status of the patients were compared with the well known histopathological prognostic factors such as histologic grade, tumor stage, lympho/ vascular invasion, surgical margin status and Paget s disease. Amplification was demonstrated 27% of the cases. Her-2/neu protein overexpression was detected in 47% and 80% of the cases with CB11 and HercepTest respectively. We revealed statistically significant association between the tumor, oncoprotein expression and oncogene amplification (p<0.05). The results of our study showed that combination of IHC and FISH methods enhances the evaluation of tumor genetics at both gene and protein level for the analysis of Her-2/neu in breast carcinoma.     

Allele loss from large regions of chromosome 17 is common only in certain histological subtypes of ovarian carcinomas.

Using a panel of ten polymorphic markers, we examined the frequency of loss of heterozygosity (LOH) on chromosome 17 in 55 sporadic ovarian tumours. LOH on 17p and 17q was observed to be 50% and 62% respectively. LOH at D17S5 was detected in 24/36 (67%) of malignant cases and in 19/43 (44%) at TP53; the marker D17S855 intragenic to the BRCA1 gene showed allele loss in 50% (20/40) cases. The data presented here suggest that loss of the whole chromosome 17 is a relatively frequent event (30%) in ovarian carcinomas and this observation is especially frequent for serous, transitional cell and anaplastic histological subtypes. Mucinous and endometrioid ovarian tumours showed only short interstitial deletions (4/11, 36%). The overall frequency of the short deletions was relatively low (7/43, 16%) in our panel of carcinomas. Amplification of c-erbB-2/neu oncogene was detected in 32% (11/34) of the carcinomas tested; the gene was amplified only in those histological subtypes in which high incidence of LOH on chromosome 17 was observed, and was associated with advanced stages of the disease. We conclude that different histological types of tumour may have different aetiological mechanisms, and tumour-suppressor genes on chromosome 17 might be associated specifically with serous and transitional cell ovarian carcinomas.     

Amplification and protein over-expression of the neu/HER-2/c-erbB-2 protooncogene in human breast carcinomas: relationship to loss of gene sequences on chromosome 17, family history and prognosis

c-erbB-2 gene amplification and protein over-expression were investigated in 89 primary tumours and 24 metastases from Norwegian breast cancer patients. Amplification occurred in 22.5% of the primary tumours and 50% of the metastases. The amplification was negatively correlated to the oestrogen receptor (ER) content in both the primary tumours and the metastases. No significant differences between amplified and non-amplified tumours were observed with regard to node status, clinical stage, tumour size or menopausal status, although correlations of borderline significance were found between node status, clinical stage and high degree of gene amplification. All the amplified tumours were of the invasive ductal type. Follow-up data of patients observed for more than 1 year showed a significantly higher recurrence rate in the c-erbB-2 amplified group. Allele loss of chromosome 17p and of 7q was seen in 55% and 48% of the tumours respectively. No significant correlation was found between these losses and clinico-histological parameters. More than 50% of the tumours with a loss of 17q sequences had an amplification of c-erbB-2 which is located on 17q12-21, indicating that only one of the chromosomes may be involved in the amplification of the c-erbB-2. A trend towards a correlation between loss of 17q and high degree of amplification were found. No correlation was found between positive family history of breast cancer and c-erbB-2 gene amplification, nor loss of 17p or 17q sequences. Our data support the hypothesis that amplification correlates with aggressive tumour behaviour, and thus may be used as a prognostic factor in breast carcinomas. The allele losses on 17p and 17q points to tumour suppressor gene or genes on this chromosome, although not as predisposing genes in families.