CYP2D6 genotype and adjuvant tamoxifen: meta-analysis of heterogeneous study populations.

The International Tamoxifen Pharmacogenomics Consortium was established to address the controversy regarding cytochrome P450 2D6 (CYP2D6) status and clinical outcomes in tamoxifen therapy. We performed a meta-analysis on data from 4,973 tamoxifen-treated patients (12 globally distributed sites). Using strict eligibility requirements (postmenopausal women with estrogen receptor-positive breast cancer, receiving 20 mg/day tamoxifen for 5 years, criterion 1); CYP2D6 poor metabolizer status was associated with poorer invasive disease-free survival (IDFS: hazard ratio = 1.25; 95% confidence interval = 1.06, 1.47; P = 0.009). However, CYP2D6 status was not statistically significant when tamoxifen duration, menopausal status, and annual follow-up were not specified (criterion 2, n = 2,443; P = 0.25) or when no exclusions were applied (criterion 3, n = 4,935; P = 0.38). Although CYP2D6 is a strong predictor of IDFS using strict inclusion criteria, because the results are not robust to inclusion criteria (these were not defined a priori), prospective studies are necessary to fully establish the value of CYP2D6 genotyping in tamoxifen therapy.

Rapid typing of neonatal Staphylococcus epidermidis isolates using polymerase chain reaction for repeat regions in surface protein genes.

Staphylococcus epidermidis is a significant pathogen in neonatal sepsis and other nosocomial infections. For further investigations of the colonisation patterns and invasive pathways, typing methods that are applicable on large populations of bacterial isolates are warranted. In the present study, a genotyping method based on polymerase chain reaction (PCR) for the repeat regions of four genes (sdrG, sdrF, aap and sesE) that encode for bacterial surface proteins was developed and applied to a sample of well-characterised neonatal blood isolates of S. epidermidis (n = 49). The PCR products were visualised on agarose gel (sdrG, sdrF and sesE) or by fragment analysis (aap). The discriminatory index (D-index) for genotyping of the different genes was compared to genotyping by pulsed-field gel electrophoresis (PFGE). The highest D-index for the PCR-based typing methods was found for the combination of sdrF, sdrG and aap (D-index 0.94), whereas the optimal two-gene combination (sdrF and aap) resulted in a D-index of 0.92. We conclude that the described method can be used for the genotyping of large populations of S. epidermidis isolates with a sufficient discriminatory capacity, and we suggest that the combination of sdrF and aap is the most suitable to use.

Incidence and prognosis in early onset breast cancer.

The aim of this study was to assess the incidence and prognosis in early onset breast cancer. Age-adjusted incidence and death rate for the 5394 Swedish women diagnosed with breast cancer under the age of 40 between 1960 and 1996 was studied using data from the Swedish Cancer Registry and Swedish Death Cause Registry. A total of 107 consecutive young patients with invasive breast cancer undergoing surgery during 1980-1993 in the Southeast Swedish health care region were retrospectively followed up and their cancers reviewed and graded blindly. The median follow-up time was 11.2 years. The applicability of the Nottingham Prognostic Index (NPI) as a prognostic tool was investigated. Grade, age, node status, tumour size, S-phase fraction and steroid receptor content were related to survival univariately and multivariately in a Cox proportional hazard analysis. The incidence of early onset breast cancer has increased moderately and the survival rate has not improved during the last 35 years. When young women are diagnosed with breast cancer their tumours are larger, their lymph nodes more often involved, and the median grade higher than in older with 64% having grade 3 tumours. Lymph node status was the strongest sole prognostic indicator but the use of NPI gave more accurate prognostic information than node status alone.

Candidate tumour suppressor genes at 11q23-q24 in breast cancer: evidence of alterations in PIG8, a gene involved in p53-induced apoptosis.

One of the most consistently deleted chromosomal regions in solid tumours is 11q23-q25, which consequently has been postulated to harbour one or more tumour suppressor loci. Despite large efforts to identify the responsible genes, the goal remains elusive, but as knowledge accumulates new candidates are emerging. The present study was undertaken in an attempt to assess the possible implication of four genes residing at 11q23-q24, in a population of early onset breast cancer (n=41). The coding sequence of PIG8, CHK1, LOH11CR2A and PPP2R1B were screened for mutations using the protein truncation test or single-strand conformational polymorphism, in combination with direct DNA sequencing. Varying proportions of alterations were detected, ranging from 6% in PPP2R1B to 39% in PIG8. Many of these changes were deletions, in some cases corresponding to complete exons, thus likely to represent splice variants, while others were presumed to arise from aberrant splicing, since they occurred at sites with resemblance to exon/intron borders. Considering only bona fide mutations, the highest alteration frequency (17%) was again found in PIG8. Most of these alterations were likely to have an adverse impact on the translated protein as they either altered the reading frame or affected phylogenetically conserved residues. Our data represent the first evidence of alterations in the PIG8 gene in human malignancies, a finding that substantiates its role as a potential tumour suppressor gene as suggested by its involvement in p53-induced apoptosis.

Catechol-O-Methyltransferase (COMT) gene polymorphism and breast cancer risk in young women.

Oestrogen exposure has long been considered to be a main risk factor of breast cancer. More recently, interest has also focused on the possible carcinogenic influence from oestrogen metabolites, such as catechol oestrogens. O-methylation, catalysed by Catechol-O-Methyltransferase (COMT), is one pathway by which the potentially carcinogenic catechol oestrogens can be inactivated. The gene coding for COMT protein contains a single-nucleotide polymorphism (SNP), resulting in an amino acid shift Val-->Met, which has been shown to determine high- and low-activity configuration of the enzyme. We hypothesized that the low-activity allele, COMT(Met), may be implicated in early onset breast cancer. In the present case-control study, including 126 young breast cancer patients (

Deletion mapping of chromosome segment 11q24-q25, exhibiting extensive allelic loss in early onset breast cancer.

Frequent allelic deletions at chromosome 11q24-q25 have been described in both early and late onset breast cancers, suggesting the existence of a gene locus implicated in the initiation and/or progression of the disease. In the present study we fine mapped this region further by loss of heterozygosity (LOH) analysis in a population of early onset breast cancer cases (n = 102, 22 to 36 years old). Loss of chromosomal material was assessed for possible association with patient survival as well as Nottingham histologic grade (NHG). Additionally, we investigated the involvement of the 11q24-q25 locus in a group of familial breast cancer cases with no detectable BRCA1 or BRCA2 gene alterations (n = 32, ages 28 to 40 years). Among the consecutive patients, extensive LOH was observed for all markers at 11q24-q25, with frequencies ranging from 42% to 54%. Deletion at the D11S4125 marker was found to be associated with reduced survival (p = 0.026), whereas the adjacent D11S387 marker correlated with higher histologic grade (p = 0.042). In the familial cases, the most telomeric markers showed substantially lower proportions of LOH, ranging from 10% to 21%. Comparison of the two patient groups demonstrated that this difference in LOH frequency was statistically significant for the D11S4098, D11S968, D11S387 and D11S4125 markers (p = 0.020, p = 0.029, p = 0.0070 and p = 0.0030, respectively). We conclude that 11q25 may harbor a gene implicated in early onset breast cancer. Our data suggest that the most probable position for this locus is defined by the markers D11S387 and D11S4125 and furthermore that it may play a less significant role in familial breast cancer cases not linked to either of the BRCA genes.

Protein accumulation and gene mutation of p53 in bilateral breast cancer. South-East Sweden Breast Cancer Group.

The aim of this study was to investigate the frequency of p53 alterations in bilateral breast cancer and to evaluate a possible clonal relationship between the paired tumours regarding p53 alteration and other pathobiological variables. Tumours from 34 patients were investigated with immunohistochemistry, single strand conformation polymorphism analysis and DNA-sequence analysis applied to exons 5-8. Fifteen percent of the 68 tumours showed positive immunoreaction and/or presence of mutation. The occurrence of p53 accumulation was 9% and the prevalence of gene mutation 10%. No significant concordance was found between the tumours in the same patient for p53 alterations, progesterone receptor status or DNA ploidy. S-phase fraction showed a weak correlation, not statistically significant. Oestrogen receptor status was the only variable that exhibited a significant concordance. No convincing evidence was found for other associations between the paired tumours or for a high prevalence of p53 alterations in bilateral breast cancer.

C-erbB-2 overexpression and survival in early onset breast cancer.

Young breast cancer patients have a decreased survival rate and it has been demonstrated that young age is an independent predictor of adverse prognosis. Overexpression of c-erbB-2 protein (also known as HER-2/neu) has been shown to be a prognostic indicator in breast cancer in general and especially among patients with axillary nodal metastases. The present study was initiated to determine the prognostic significance of c-erbB-2 protein overexpression in early onset breast cancer. A population consisting of 110 young breast cancer patients, < or = 36-year-old at diagnosis, was analyzed with immunohistochemical staining for c-erbB-2 protein. Thirty patients (27%) were found to overexpress the c-erbB-2 protein. C-erbB-2 positivity was significantly associated with poor survival when all patients were included in the analysis (P = 0.002) and for patients with axillary nodal metastases (P = 0.0007). No such association was found for node-negative patients. Furthermore, the difference in prognosis in relation to c-erbB-2 among node-positive patients was maintained, when these were stratified in groups treated or not treated with adjuvant chemotherapy. The study indicates that overexpression of c-erbB-2 protein is a strong prognostic factor in young breast cancer patients with axillary nodal metastases. Moreover, the adverse prognosis associated with c-erbB-2 overexpression in node-positive patients was observed whether or not the patients had received adjuvant chemotherapy.

Effects of human platelet-derived growth factor-AB on sarcoma growth in vitro and in vivo.

Platelet-derived growth factor (PDGF) has been proposed to play an important role in the growth of tumors. In order to study the effects of PDGF-AB on tumor growth in vivo, sarcoma-bearing mice were treated with PDGF-AB. The tumors, a malignant fibrous histiocytoma and an osteosarcoma, had functional PDGF receptors in vitro, as demonstrated by stimulation of PDGF-AB using a [3H]thymidine incorporation assay. Immunohistochemistry also revealed that both sarcoma xenografts expressed PDGF receptors. The tumor-bearing mice were given human PDGF-AB for 14 days, either continuously by an intraperitoneally placed mini-osmotic pump, or by daily injections. No effects on tumor growth in vivo were observed, as measured by tumor volume, autoradiography or cell cycle distribution. The histological appearance and ploidy of the tumors remained unaltered. The results indicate that, although the tumor cells are stimulated by PDGF-AB in vitro, the in vivo milieu or tumor growth pattern may render the tumors less susceptible to exogenously administered PDGF-AB in vivo.

Association between CYP17 gene polymorphism and risk of breast cancer in young women.

Long-term exposure to oestrogens is a well-recognised risk factor for breast cancer, whereas little is known about the influence of polymorphisms of genes involved in oestrogen biosynthesis and metabolism. A candidate, containing a single bp polymorphism, T-->C, (designated, A2 allele), might be the CYP17 gene, which codes for an enzyme involved in oestrogen synthesis. This polymorphism creates an additional Sp1-type promoter site (CCACC box), which has been shown to be associated with increased serum oestrogen levels. We performed a case-control study, to evaluate association of the CYP17 gene polymorphism with risk of breast cancer in young women (younger than 37 years). We found a statistically significant increased risk in carriers of at least 1 A2 allele [odds ratio (OR), 2.0; 95% confidence interval (CI), 1.1-3.5, p = 0.027], and a trend toward a gene-dose effect illustrated by a slightly higher risk for A2-homozygous subjects (OR, 2.8) than for heterozygous women (OR, 1. 9). Furthermore, when we investigated the CYP17 genotype in relation to tumour characteristics, breast cancer patients with 1 or 2 A2 alleles tended to have lower oestrogen receptor levels (risk ratio, 0.70; CI, 0.41-1.2, p = 0.44). Our findings suggest that CYP17 gene polymorphism influences breast carcinogenesis in young women. Int. J. Cancer (Pred. Oncol.) 84:350-353, 1999.

Frequent allelic losses at 11q24.1-q25 in young women with breast cancer: association with poor survival.

Previous studies have demonstrated that the pathological features of breast cancer are more aggressive in younger women than in their older counterparts, and that young age may be an independent marker for adverse prognosis. These findings have raised the question whether these differences are also present at the molecular level. In order to characterize the genetic alterations associated with early-onset breast cancer, 102 cases selected for age under 37 at diagnosis were examined for loss of heterozygosity (LOH) at nine different loci on chromosomes 11, 13 and 17. Ninety cases (88%), exhibited LOH for at least one marker. The D17S855 marker, intragenic in the BRCA1 gene, showed a high proportion of LOH (63%), whereas the intragenic marker for the TP53 gene, HP53, exhibited LOH in 43% of the cases. On chromosome 11, frequencies of LOH peaked at the D11S969 and D11S387 markers, which expressed LOH in 53% and 48% of the informative cases, whereas D11S1818, which is proximate to the ATM gene, exhibited an LOH frequency of 24%. A statistically significant correlation was found between LOH at the D11S387 marker and poor survival (P = 0.028). No such correlation was found for the adjacent D11S969 marker, located approximately 500 kb centromeric to D11S387. We conclude that one or more as yet unidentified genes, situated in chromosome bands 11q24.1-q25, could be involved in the initiation and/or progression of breast cancer in younger women.

Association of breast cancer progression with a vitamin D receptor gene polymorphism. South-East Sweden Breast Cancer Group.

The vitamin D3 receptor gene (VDR) contains a TaqI RFLP that is associated with increased VDR mRNA stability, increased serum levels of 1alpha,25-dihydroxyvitamin D3 (1,25-D3), and decreased risk for prostate cancer. Determination of the TaqI genotype, in a group of young women with breast cancer (n = 111; age, <37 years) and a control population (n = 130), revealed no overall association to risk for breast cancer. However, patients without TaqI site (TT genotype) showed a significantly increased risk for lymph node metastasis (relative risk, 1.8, 95% confidence interval, 1.3-2.6). Furthermore, a tendency toward an increased survival was found among estrogen receptor-positive, tamoxifen-treated patients who were homozygous for the TaqI site (P = 0.075). We conclude that polymorphism in the VDR gene may influence tumor progression and tamoxifen treatment response in early-onset breast carcinomas.

p53 and survival in early onset breast cancer: analysis of gene mutations, loss of heterozygosity and protein accumulation.

The p53 protein has proven to be central in tumorigenesis by its cell cycle regulatory properties and both gene mutations and protein accumulation have been associated with poor prognosis in breast cancer. The present study was undertaken to investigate the prognostic significance of gene mutations, p53 protein accumulation and of loss of heterozygosity (LOH) at the TP53 locus in young (age < 37 years) breast cancer patients. In total, gene mutations were found in 21 of the 123 patients (17%), LOH in 20 of the 47 informative cases (43%) and protein accumulation in 47 of the 102 available cases (46%). Log rank analysis revealed no significant association between survival and TP53 mutations (in general), p53 protein accumulation or LOH. However, missense mutations localised to the zinc binding domain were significantly (P = 0.0007) associated with poorer prognosis. As indicated in this as well as other studies, p53 protein accumulation is frequently found in young breast cancer patients, but this protein overexpression appears to be of minor significance for survival. Nevertheless, the present report also suggests that specific mutations contribute substantially to tumour aggressiveness.

Human malignant fibrous histiocytomas in vitro: growth characteristics and their association with expression of mRNA for platelet-derived growth factor, transforming growth factor-alpha and their receptors.

Eight human malignant fibrous histiocytomas were examined in vitro, in order to relate their growth properties to mRNA expression for platelet-derived growth factor (PDGF), PDGF receptor (PDGF-R), transforming growth factor-alpha (TGF-alpha) and the epidermal growth factor receptor (EGF-R). Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that all cell lines expressed mRNA for PDGF-R alpha and/or PDGF-R beta; six cell lines expressed mRNA for the PDGF-A chain, with one cell line coexpressing PDGF-B chain mRNA; seven cell lines expressed mRNA for TGF-alpha whereas six cell lines expressed EGF-R mRNA. Conditioned medium from three cell lines contained PDGF; none of the cell lines released TGF-alpha. Two cell lines grew without serum requirements; whereas both expressed mRNA for PDGF, PDGF-R, TGF-alpha and EGF-R, other cell lines, unable to grow without serum, showed the same combination of growth factor/growth factor receptor expression. The two cell lines able to grow without serum were also shown to be stimulated by the addition of PDGF-BB. These findings show that simultaneous expression of mRNA for a growth factor and its receptor does not necessarily imply an autocrine or paracrine loop. However, two of our cell lines fulfil the requirements of possible PDGF-related autocrine and paracrine regulation.

Flow cytometric DNA-heterogeneity in paraffin-embedded endometrial cancer.

To evaluate the degree of intratumoral DNA ploidy heterogeneity in endometrial carcinoma, the authors examined curettage specimens from 30 patients with clinical stage I and II endometrial carcinoma. The curettage material was obtained before the onset of treatment. A representative sample from each tumour was chosen and a 50 microm section was cut. The paraffin block of tumor was then divided into 4 equal parts and a 50 microm section was cut from each part. All tumour samples were analysed separately by flow cytometry. DNA ploidy heterogeneity was noted in 5/29 cases (17%). In three cases DNA-aneuploid stem cell lines were found only among the 4-part sections and were not detected when the whole tumor section was analysed.

Frequent allelic losses on chromosome 13q in human male breast carcinomas.

Loss of genetic material on chromosomes 13q and 17 has been suggested to be of importance in the initiation and progression of female breast cancer, but their involvement is less well illustrated in male breast carcinomas. The present study was designed to investigate the incidence of allelic loss and microsatellite instability for chromosomes 13q, 17p and 17q in 13 sporadic male breast carcinomas using matched normal-tumour DNA samples and seven polymorphic microsatellite markers. Genetic imbalance was found in one or more informative markers in 85% of the patients, with more frequent loss of heterozygosity and microsatellite instability at loci on chromosome 13q. Thus, a high incidence of allelic losses was observed at the retinoblastoma gene (4/6) and likewise at the D13S263 locus (7/12), which also exhibited the highest frequency of microsatellite instability. The intragenic microsatellite in intron 1 of the TP53 gene on chromosome 17p revealed loss of heterozygosity in 3 of 8 informative patients. The investigated proximal region of chromosome 13q is postulated to harbour several potential tumour suppressor genes associated with female breast cancer. The high incidence of allelic losses at the D13S263 microsatellite, located distal to both the BRCA2 and the Brush-1 loci but proximal to the retinoblastoma gene, possibly indicates the presence of an additional tumour suppressor gene which may be involved in male breast carcinomas. However, this hypothesis needs verification in an extended study of male breast carcinomas.

Contraction of collagen lattices by cells from Dupuytren's nodules.

The aim of this study was to see if nodular cells in Dupuytren's disease differed from dermal cells in their contractile capacity and motility. Ten surgical specimens from patients with Dupuytren's disease and contracture of the finger of more than 45 degrees were harvested and the nodular cells were explanted and cultured. Dermal fibroblasts from the forearm were used as control cells. Both types of cell had the same growth pattern. The morphology on confocal laser scanning microscopy was also similar in both types of cell. Dermal control cells caused significantly more contraction of collagen lattices compared with fibroblasts from nodules of Dupuytren's contracture. The F-actin content was equal in both groups. Platelet derived growth factor, PDGF-BB (but not PDGF-AA), increased the chemotactic activity of both cell types, but there were no differences between them. The results indicate that at a late state of the disease cells from Dupuytren's nodules lose their contractile capacity and regain a phenotype resembling that of dermal fibroblasts.

Correlations of Ki-67 and PCNA to DNA ploidy, S-phase fraction and survival in uveal melanoma.

In 79 patients with uveal melanoma, the tumours were investigated by DNA flow cytometry and immunohistochemical staining of PCNA and Ki-67. S-phase as a continuous variable was significantly correlated with Ki-67 (P = 0.033), but not with PCNA. DNA ploidy was not correlated with either of the two antigens. Ki-67 was significantly correlated with histopathological type (P < 0.001) and tumour size (P < 0.001). Large tumours and epithelioid cell type were associated with a high frequency of Ki-67 positive cells. A high level of Ki-67 positivity (> or = 6.5%) was also associated with a shorter survival (P = 0.0037), and when adjusted for DNA ploidy, histopathological type and tumour size, Ki-67 in the multivariate analysis remained an important prognostic factor (P = 0.017).

Correlation of DNA ploidy and S-phase fraction with chemotherapeutic response and survival in a randomized study of disseminated malignant melanoma.

DNA ploidy and S-phase fraction were measured by flow cytometry in the tumour tissue of 87 patients with disseminated malignant melanoma, who had been classified either as responders or with progressive disease in a study of the effects of 2 chemotherapeutic regimens. The patients had been randomized to receive treatment with dacarbazine (DTIC) and vindesine (Eldesine) with or without addition of cisplatin (Platinol). Tumour tissue was obtained from both the primary tumours and the last histologically verified metastases, but in some cases only the primary tumours or the last metastases could be evaluated. There was a significantly higher mean S-phase value in melanoma metastases from patients with complete or partial responses compared with patients with progressive disease. Neither the S-phase fraction of the primary tumour, nor the DNA ploidy of the primary tumour or of the last histologically verified metastases taken before inclusion into the study were associated with therapeutic response. In the multivariate analysis, both the anatomical location of the metastases and the S-phase fraction measured on the last metastases remained significant prognostic factors of response. In the univariate survival analysis, there was an association between high S-phase fractions of the metastases and longer survival. In the multivariate survival analysis, the S-phase fraction, the number of involved metastatic sites and the treatment response were independent predictive factors. We conclude that, in disseminated melanoma treated with chemotherapy, a high S-phase fraction measured in the last histologically verified metastases is associated with a higher response rate and a longer survival. Our results clearly support the role of S-phase measurement as a potential tool for selecting patients for treatment.

c-erbB-2 expression and benefit from adjuvant chemotherapy and radiotherapy of breast cancer.

Frozen tissue from primary tumours of 152 premenopausal breast cancer patients, who participated in a trial comparing radiotherapy with adjuvant chemotherapy (cyclophosphamide, methotrexate, 5-fluorouracil, CMF), was analysed for c-erbB-2 protein expression, measured by flow cytometry. The relative risk of distant recurrence or death in the chemotherapy group as compared with the radiotherapy group was 3.0 (95% confidence interval (CI) 1.1-7.8) for patients whose tumours showed high c-erbB-2 levels and 0.87 (95% CI 0.43-1.7) for those with tumours with low levels of c-erbB-2 protein. Patients with highly proliferative tumours that did not overexpress c-erbB-2 benefited most, in terms of survival, from CMF. In addition, we found an increased risk of locoregional recurrence for tumours overexpressing c-erbB-2 when radiotherapy was replaced by chemotherapy.