BRCA1 genomic deletions are major founder mutations in Dutch breast cancer patients.

To date, more than 300 distinct small deletions, insertions and point mutations, mostly leading to premature termination of translation, have been reported in the breast/ovarian-cancer susceptibility gene BRCA1. The elevated frequencies of some mutations in certain ethnic subpopulations are caused by founder effects, rather than by mutation hotspots. Here we report that the currently available mutation spectrum of BRCA1 has been biased by PCR-based mutation-screening methods, such as SSCP, the protein truncation test (PTT) and direct sequencing, using genomic DNA as template. Three large genomic deletions that are not detected by these approaches comprise 36% of all BRCA1 mutations found in Dutch breast-cancer families to date. A 510-bp Alu-mediated deletion comprising exon 22 was found in 8 of 170 breast-cancer families recruited for research purposes and in 6 of 49 probands referred to the Amsterdam Family Cancer Clinic for genetic counselling. In addition, a 3,835-bp Alu-mediated deletion encompassing exon 13 was detected in 4 of 170 research families, while an deletion of approximately 14 kb was detected in a single family [corrected]. Haplotype analyses indicated that each recurrent deletion had a single common ancestor.

High prevalence of founder mutations of the succinate dehydrogenase genes in the Netherlands.

Mutations in four genes encoding subunits or cofactors of succinate dehydrogenase (SDH) cause hereditary paraganglioma and pheochromocytoma syndromes. Mutations in SDHB and SDHD are generally the most common, whereas mutations in SDHC and SDHAF2 are far less frequently observed. A total of 1045 DNA samples from Dutch paraganglioma and pheochromocytoma patients and their relatives were analyzed for mutations of SDHB, SDHC, SDHD or SDHAF2. Mutations in these genes were identified in 690 cases, 239 of which were index cases. The vast majority of mutation carriers had a mutation in SDHD (87.1%). The second most commonly affected gene was SDHAF2 (6.7%). Mutations in SDHB were found in only 5.9% of samples, whereas SDHC mutations were found in 0.3% of samples. Remarkably, 69.1% of all carriers of a mutation in an SDH gene in the Netherlands can be attributed to a single founder mutation in SDHD, c.274G>T and p.Asp92Tyr. Moreover, 88.8% of all SDH mutation carriers carry one of just six Dutch founder mutations in SDHB, SDHD and SDHAF2. The dominance of SDHD mutations is unique to the Netherlands, contrasting with the higher prevalence of SDHB mutations found elsewhere. In addition, we found that most SDH mutation-related paragangliomas-pheochromocytomas in the Netherlands can be explained by only six founder mutations in SDHAF2, SDHB and SDHD. The findings underline the regional differences in the SDH mutation spectrum, differences that should be taken into account in the development of effective screening protocols. The results show the crucial role that demographic factors play in the frequency of gene mutations.

Mutations in SDHD are the major determinants of the clinical characteristics of Dutch head and neck paraganglioma patients.

OBJECTIVE: Head and neck paragangliomas (HNPGL) are associated with mutations in genes encoding subunits of succinate dehydrogenase (SDH). The aim of this study was to evaluate SDH mutations, family history and phenotypes of patients with HNPGL in the Netherlands. DESIGN: We evaluated the clinical data and the mutation status of 236 patients referred between 1950 and 2009 to Leiden University Medical Center. RESULTS: The large majority of the patients carried mutations in SDHD (83%), and the p.Asp92Tyr Dutch founder mutation in SDHD alone accounted for 72% of all patients with HNPGL. A mutation in SDHAF2 was found in 4%, mutations in SDHB in 3% and a mutation in SDHC was identified in a single patient (0·4%). Over 80% of patients presented with positive family history, of whom 99·5% carried a mutation in an SDH gene. SDH mutations were also found in 56% of isolated patients, chiefly in SDHD (46%), but also in SDHB (8%) and SDHC (2%). The clinical parameters of these different subgroups are discussed: including the age at diagnosis, associated pheochromocytomas, tumour multifocality and malignancy rate. CONCLUSION: The majority of Dutch patients with HNPGL present with a positive family history, in contrast to other European countries. The clinical characteristics of patients with HNPGL are chiefly determined by founder mutations in SDHD, the major causative gene in both familial and isolated patients with HNPGL. The high frequency of founder mutations in SDHD suggests a higher absolute prevalence of paraganglioma syndrome in the Netherlands.

Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma.

Hereditary paraganglioma (PGL) is characterized by the development of benign, vascularized tumors in the head and neck. The most common tumor site is the carotid body (CB), a chemoreceptive organ that senses oxygen levels in the blood. Analysis of families carrying the PGL1 gene, described here, revealed germ line mutations in the SDHD gene on chromosome 11q23. SDHD encodes a mitochondrial respiratory chain protein-the small subunit of cytochrome b in succinate-ubiquinone oxidoreductase (cybS). In contrast to expectations based on the inheritance pattern of PGL, the SDHD gene showed no evidence of imprinting. These findings indicate that mitochondria play an important role in the pathogenesis of certain tumors and that cybS plays a role in normal CB physiology.

Ever since Knudson.

Many publications have documented loss of heterozygosity (LOH) on many different chromosomes in a wide variety of tumours, implicating the existence of multiple tumour suppressor genes (TSGs). Knudson's two-hit hypothesis predicts that these LOH events are the second step in the inactivation of both alleles of a TSG. However, to date the number of TSGs identified that are inactivated mainly at the somatic level in cancers and are not inherited has remained disappointingly small. Here we postulate that the accurate mapping of LOH events in a series of tumours to define a common LOH region is greatly confounded by deficient LOH detection, genetic instability and intertumour heterogeneity. Finding the TSGs in chromosomal regions of frequent LOH might require 'brute-force' genomic approaches.

Genetic susceptibility for breast cancer: how many more genes to be found?

Today, breast cancer is the most commonly occurring cancer among women. It accounts for 22% of all female cancers and the estimated annual incidence of breast cancer worldwide is about one million cases. Many risk factors have been identified but a positive family history remains among the most important ones established for breast cancer, with first-degree relatives of patients having an approximately two-fold elevated risk. It is currently estimated that approximately 20-25% of this risk is explained by known breast cancer susceptibility genes, mostly those conferring high risks, such as BRCA1 and BRCA2. However, these genes explain less than 5% of the total breast cancer incidence, even though several studies have suggested that the proportion of breast cancer that can be attributed to a genetic factor may be as high as 30%. It is thus likely that there are still breast cancer susceptibility genes to be found. It is presently not known how many such genes there still are, nor how many will fall into the class of rare high-risk (e.g. BRCAx) or of common low-risk susceptibility genes, nor if and how these factors interact with each other to cause susceptibility (a polygenic model). In this review we will address this question and discuss the different undertaken approaches used in identifying new breast cancer susceptibility genes, such as (genome-wide) linkage analysis, CGH, LOH, association studies and global gene expression analysis.

Amplification of the neu (c-erbB-2) oncogene in human mammmary tumors is relatively frequent and is often accompanied by amplification of the linked c-erbA oncogene.

We investigated alterations in the structure and expression of oncogenes in mammary tumors and mammary tumor-derived cell lines. In 16 of 95 samples, we detected amplification of the human neu oncogene, also known as c-erB-2, accompanied by overexpression in the tumors from which intact RNA could be isolated. In 10 of these DNAs, the linked oncogene c-erbA was also amplified, whereas another gene on human chromosome 17, p53, was present in normal copy numbers. Overexpression of c-erbA could not be detected in the tumors analyzed. The relatively high frequency of neu amplification points to a functional role in human breast cancer. Coamplification of the c-erbA oncogene could contribute to this disease as well but is most likely fortuitous.

Multifactorial analysis of differences between sporadic breast cancers and cancers involving BRCA1 and BRCA2 mutations.

BACKGROUND: We have previously demonstrated that breast cancers associated with inherited BRCA1 and BRCA2 gene mutations differ from each other in their histopathologic appearances and that each of these types differs from breast cancers in patients unselected for family history (i.e., sporadic cancers). We have now conducted a more detailed examination of cytologic and architectural features of these tumors. METHODS: Specimens of tumor tissue (5-microm-thick sections) were examined independently by two pathologists, who were unaware of the case or control subject status, for the presence of cell mitosis, lymphocytic infiltration, continuous pushing margins, and solid sheets of cancer cells; cell nuclei, cell nucleoli, cell necrosis, and cell borders were also evaluated. The resulting data were combined with previously available information on tumor type and tumor grade and further evaluated by multifactorial analysis. All statistical tests are two-sided. RESULTS: Cancers associated with BRCA1 mutations exhibited higher mitotic counts (P = .001), a greater proportion of the tumor with a continuous pushing margin (P<.0001), and more lymphocytic infiltration (P = .002) than sporadic (i.e., control) cancers. Cancers associated with BRCA2 mutations exhibited a higher score for tubule formation (fewer tubules) (P = .0002), a higher proportion of the tumor perimeter with a continuous pushing margin (P<.0001), and a lower mitotic count (P = .003) than control cancers. CONCLUSIONS: Our study has identified key features of the histologic phenotypes of breast cancers in carriers of mutant BRCA1 and BRCA2 genes. This information may improve the classification of breast cancers in individuals with a family history of the disease and may ultimately aid in the clinical management of patients.

Thermographic study of in vivo modulation of vascular responses to phenylephrine and endothelin-1 by dexamethasone in the horse.

REASONS FOR PERFORMING STUDY: In vitro, glucocorticoids potentiate vasoconstriction of equine digital vessels to catecholamines and this has been implicated as a mechanism of glucocorticoid-induced laminitis. This observation has never been confirmed in vivo. OBJECTIVES: To study the effects of glucocorticoid therapy on vasoconstrictor responsiveness in the horse in vivo. METHODS: In a blinded, randomised cross-over experiment, 9 horses were treated with either dexamethasone (0.1 mg/kg bwt i.v. q. 24 h) or saline i.v. for 6 days. The changes in local average skin temperature before (baseline) and after intradermal injections of the alpha1-adrenoceptor agonist phenylephrine (PHE; 10(-4), 10(-5), 10(-6), 10(-7) and 10(-8) mol/l), endothelin-1 (ET-1; 10(-5), 10(-6), 10(-7), 10(-8) and 10(-9) mol/l) or ET-1 plus a blocker (BQ-123 10(-6) mol/l; RES-701 10(-6) mol/l; and L-NAME 10(-4) mol/l) were investigated with a thermograph. RESULTS: Dexamethasone (DEX) decreased baseline skin temperatures, suggesting reduced blood flow as a consequence of an increase in vasomotor tone. This was accompanied by potentiation of the response to PHE as demonstrated by a left shift in the dose-response curve and a decrease in the EC50. Dexamethasone did not potentiate ET-1, but the interplay with the lower baseline temperature resulted in a significantly lower skin temperature for this vasoconstrictor after DEX. The different ET-1 blockers had no effect on ET-1 modulated skin temperatures. CONCLUSIONS: Dexamethasone decreases skin perfusion. This is accompanied by a potentiated alpha1-adrenoceptor agonist response and a greater response to ET-1. POTENTIAL RELEVANCE: Glucocorticoid therapy probably decreases perfusion of the equine hoof. During disease states that already are characterised by hypoperfusion and/or increased levels of circulating catecholamines, glucocorticoid therapy could, according to the vascular model of laminitis, tilt the balance in favour of laminitis.

Biochemical and quantitative histochemical study of reduced pyridine nucleotide dehydrogenation by human colonic carcinomas.

This study shows a marked increase in the activity of the soluble enzyme DT-diaphorase and of the histochemical activity of the reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate nitroblue tetrazolium menadione-mediated reductases in human colonic carcinomas when compared with the enzymatic activities of portions of the colon uninvolved by the carcinomatous process. The activity of the reductases in histological sections was quantitated with a microphotometer. It is believed that the increase in histochemical nitroblue-tetrazolium reductase activity in the histochemical reactions in colonic carcinomas is a real reflection of the activity of the DT-diaphorase, because the increase in the dehydrogenation of reduced nicotinamide adenine dinucleotide equals the dehydrogenation of reduced nicotinamide adenine dinucleotide phosphate when measured biochemically in the soluble fraction, or histochemically, by microspectophotometry in tissue sections; meanwhile, the biochemical dehydrogenation of NAD(P)H by the particulate fractions shows that the enzymatic activities are not altered by the neoplastic process. The biological significance of these changes is discussed in the text.

Flow cytometric detection of aneuploidy in colorectal adenomas.

Flow cytometry has been used to study the incidence of aneuploidy in a series of 55 colorectal adenomas (29 tubular adenomas, 22 tubulovillous adenomas, and 4 villous adenomas). For comparison, 5 nonadenomatous polyps, 4 normal mucosa samples from colectomy specimens and 16 colorectal cancers were measured. Fifteen (27%) adenomas were aneuploid, 33 (63%) were diploid, and 7 (11%) were peridiploid. The aneuploidy incidence increased with the size of the adenomas (less than 1 cm, 0%; 1 to 2 cm, 30%; greater than 2 cm, 50% aneuploid cases, respectively) but was less dependent on the histological type or degree of dysplasia. However, the degree of aneuploidy [mean DNA index of aneuploid stem lines] was significantly higher in tubulovillous adenomas [1.26 +/- 0.33 (SD)] than in tubular adenomas [1.09 +/- 0.04] and only slightly lower than in carcinomas [1.59 +/- 0.26]. The progressive increase in ploidy abnormality with size and histological type strongly supports the evidence for the adenoma-carcinoma sequence in the development of colorectal cancer.

The role of DNA damage and inhibition of poly(ADP-ribosyl)ation in loss of clonogenicity of murine L929 fibroblasts, caused by photodynamically induced oxidative stress.

Reactive oxygen species are used to eradicate malignant cells in photodynamic therapy as well as in other cancer therapies. Despite many efforts, the pathways leading to cellular damage and cell killing due to the action of these species are poorly understood. In previous studies with hematoporphyrin derivative-sensitized L929 murine fibroblasts, the only parameter for which a relation with photodynamically induced reproductive cell death could not be excluded was inhibition of DNA excision repair. The present results show that loss of clonogenicity of these cells in fact is related to a series of effects, including the development of slight, irreperable DNA damage, a virtually complete inhibition of poly(ADP-ribosyl)ation activation, a transient elevation of the intracellular calcium concentration and, after a lag time of about 8 h, DNA fragmentation caused by endonuclease activity. This conclusion is supported by the observation that photodynamic treatment inhibited the repair of X-ray-induced DNA strand breaks and suppressed X-ray- and methyl methanesulfonate-induced enhancement of poly(ADP-ribosyl)ation. Our experimental results further suggest that in this cell line the photodynamically induced inhibition of enhanced poly(ADP-ribosyl)ation could well be involved in inhibition of repair of DNA strand breaks and in activation of endonuclease activity.

Flow cytometric analysis of DNA ploidy in canine mammary tumors.

DNA ploidy has been determined using flow cytometry in 23 nonmalignant and 34 malignant (primary and metastatic) mammary tumors from 46 dogs. This parameter was compared with clinical stage, histology, and estrogen and progesterone receptor analysis. Twenty-one of 34 cancers (61.8%) from 32 dogs were DNA aneuploid. Aneuploidy was also found in 4 of 23 nonmalignant tumors (17.4%) from 20 dogs. Regional lymph nodes were involved in 6 of 10 diploid and 3 of 9 aneuploid cancers of dogs with operable disease. The aneuploidy incidence was higher in dogs that had distant metastasis at initial diagnosis (8 of 11) than in those presented with local or locoregional disease (9 of 19), although this difference was not statistically significant. DNA aneuploidy incidence was not found to be related to histological tumor type, histological malignancy grade, nuclear grade, or steroid receptor presence. Heterogeneity in DNA content was found in 4 of 32 cancers (30 dogs) in samples from primary or locally recurrent lesions. In 3 of 16 cancers that were analyzed both at the primary and at secondary sites of growth, a significant variation in DNA content was observed. The degree of aneuploidy in the dog cancers was much lower than seen for human breast carcinomas with a relatively high frequency of hypoploid stemlines (7 of 34 cancers, 20.6%). The frequency distribution of DNA indices in dog mammary cancers indicates that aneuploidy evolution probably differs from that of human breast cancer.

Nuclear distribution of the Ki-67 antigen during the cell cycle: comparison with growth fraction in human breast cancer cells.

It has been claimed that the commercially available Ki-67 monoclonal antibody recognizes a nuclear antigen which is solely expressed in cycling cells. Therefore, at present, Ki-67 is increasingly used as a tool in evaluating growth fractions (GFs) of human tumors. Here we describe specific patterns in the expression and topological distribution of this antigen during the cell cycle in MCF-7 human breast cancer cells. Our results support earlier findings that the antigen belongs to a class of antigens associated with the structural organization of meta- and anaphase chromosomes, and proteins located near the cortical regions of prenucleolar bodies and nucleoli. Using 5'-bromodeoxyuridine-labeling technology, we show that the expression may be undetectably low at the onset of DNA replication. Comparison of Ki-67 fractions (KFs) and GFs as estimated from continuous 5'-bromodeoxyuridine-labeling curves revealed that KF was invariably higher than GF: in exponentially growing cov362.c14 human ovary cancer cells, KF was only 3.5% higher than GF; in MCF-7 cells, 11.3 +/- 4.6%. In MCF-7 cultures either growing under suboptimal conditions or treated with 10(-6) M tamoxifen, the difference was more pronounced. Furthermore, we evaluated the decrease of Ki-67-positive cells in nutritionally deprived and cell cycle-specific, drug-treated cultures. Since the results indicate that nonproliferating cells may retain the antigen for a considerable period of time, KF may not always be a reliable indicator of GF.

Detection of chromosome aneuploidy in interphase nuclei from human primary breast tumors using chromosome-specific repetitive DNA probes.

We have used in situ hybridization with chromosome specific repetitive DNA sequences as a probe to reveal particular chromosomes as distinct spots or clusters of signal within interphase nuclei. Using karyotypically defined cells and cell lines, we show that the number of signals obtained per nucleus correlates with the number of particular chromosomes present in that nucleus. Further, admixtures of karyotypically different cell lines could be detected. In situ hybridization of nuclei and metaphase spreads derived from the breast cancer cell line MCF-7 shows that a deviant number of spots/nucleus indicates a numerical and/or structural chromosomal aberration. In seven primary breast tumors studied, we detected numerical aberrations of the target sites of chromosomes 1 and/or 18. Although all had a single peak in DNA flow measurements, six of the cases appeared to be heterogeneous with respect to their spots/nucleus content.

Evidence for limited molecular genetic heterogeneity as defined by allelotyping and clonal analysis in nine metastatic breast carcinomas.

To investigate genetic intratumor heterogeneity, 42 samples of nine primary breast carcinomas and 29 related lymph node metastases were examined for DNA ploidy status, allelotype, and X chromosome inactivation pattern. Two primary breast carcinomas showed DNA index heterogeneity and five contained a single DNA aneuploid tumor stemline, whereas the two remaining primary tumors were solely DNA diploid. Most primary DNA tumor stemlines recurred in lymph node metastases (9 of 11). The allelotype, constructed with 31 different probes mapping to 23 different chromosome arms showed allelic imbalances on nearly all chromosome arms investigated. All tumors contained multiple allelic imbalances (range, 3-12). An allelic imbalance present in a primary tumor was consistently present in all DNA samples of that primary tumor and also in all DNA samples of related lymph node metastases, irrespective of DNA index heterogeneity. X chromosome inactivation pattern analysis with probe M27 beta (DXS255) confirmed the presence of clonal tumor cell populations in these tumors at the time of diagnosis. Densitometry of autoradiograms, which by eye showed retention of heterozygosity, revealed a narrow clustering of allelic imbalance factors between 1.0 and 1.4. In contrast, autoradiograms visually showing an allelic imbalance exhibited a marked interprobe, intertumor and intratumor variation in allelic imbalance factors. No relation between densitometry results and DNA ploidy status was found. Thus, at the time of diagnosis, an advanced primary breast carcinoma consists of a clonal tumor cell population with an established complement of allelic imbalances in all parts of the primary tumor and in the related lymph node metastases. Secondary to the establishment of allelic imbalances, intratumor heterogeneity for the copy number of involved alleles may develop, which in turn probably precedes metastasis.

Flow cytometric DNA ploidy analysis of feline mammary tumors.

Flow cytometric DNA analysis was performed on biopsies from 9 nonmalignant and 111 malignant (primary and metastatic) feline mammary lesions. In our series, 46.3% of the primary mammary carcinomas appeared to be aneuploid, whereas all but one benign breast lesion were diploid. The degree of aneuploidy in carcinomas was low, with a relatively high number of primary tumors (12 of 82) displaying hypodiploidy. Aneuploidy was not found to be correlated with any specific histological tumor type, vascular invasion, tumor size, or histological malignancy grade or with the separate components thereof. Comparison of the ploidy in primary and metastatic tumors from the same cases revealed a remarkable stability, both in time and location of appearance of the metastases. It is concluded that with respect to DNA ploidy feline mammary carcinoma has more in common with canine mammary carcinoma than with human mammary carcinoma. Further prospective studies are necessary to clarify the implications of aneuploidy in feline mammary carcinoma for tumor behavior and prognosis.

Frequent somatic imbalance of marker alleles for chromosome 1 in human primary breast carcinoma.

Loss of heterozygosity at particular chromosomal loci in the tumor cell, as evidenced by restriction fragment length polymorphism analysis, has been taken as a hallmark of the presence of tumor suppressor genes. Recent studies of breast carcinoma have suggested that such genes might be located on the short as well as on the long arm of chromosome 1. We report here that comparison of constitutional and tumor genotypes of 84 breast tumors at 7 polymorphic chromosome 1 loci indicates a frequent imbalance of alleles on both 1p (12 of 61 informative patients) and 1q (37 of 71 informative patients). In about one-half of these cases, however, this imbalance was consistent with a gain in copy number of one allele in tumor DNA relative to normal DNA, rather than loss of the other. In 10 tumors we performed chromosome 1 enumeration in the interphase nucleus using in situ hybridization with a probe detecting the heterochromatin region at 1q12. These experiments confirmed the supernumerary presence of region 1q12 in those tumors showing an allelic copy number gain of 1q. We suggest that there are several genes on chromosome 1 serving as targets for these changes, some of them associated with breast cancer development through their deletion and others through an increase in copy number.

Isolation of two distinct epithelial cell lines from a single feline mammary carcinoma with different tumorigenic potential in nude mice and expressing different levels of epidermal growth factor receptors.

From a single spontaneous feline mammary carcinoma, two subpopulations of epithelial tumor cells have been isolated. The variant cells were established as cell lines designated K248C and K248P. DNA ploidy analysis showed that the two cell lines represented cell populations already present in the original tumor. Chromosome analysis confirmed the feline origin of K248C and K248P and demonstrated that in addition to unique marker chromosomes characteristic for each cell line, both cell lines had several marker chromosomes in common. These data suggest that the two cell populations arose from a hypothetical single ancestor which diverged during tumor progression. The K248C and K248P cell lines differed from one another with respect to their tumorigenicity in athymic mice and epidermal growth factor (EGF) receptor content. The K248C cells were highly tumorigenic as indicated by a short latency period and high take rate. The K248P cells were poorly tumorigenic. Southern blot analysis revealed that the K248C cells contained an amplified EGF receptor gene that was accompanied by elevated levels of EGF receptor RNA and protein. The K248C cells were growth inhibited in vitro at EGF concentrations that stimulated growth of K248P cells. The amplification of the EGF receptor gene could be detected only in DNA derived from K248C cells at high passage numbers and not in DNA derived from the original tumor and K248C cells at low passage numbers. These data suggest that amplification of the EGF receptor gene occurred during establishment of the K248C cell line.