Rapid genetic counseling and testing in newly diagnosed breast cancer: Patients' and health professionals' attitudes, experiences, and evaluation of effects on treatment decision making.

BACKGROUND: Rapid genetic counseling and testing (RGCT) in newly diagnosed high-risk breast cancer (BC) patients may influence surgical treatment decisions. To successfully integrate RGCT in practice, knowledge of professionals', and patients' attitudes toward RGCT is essential. METHODS: Between 2008 and 2010, we performed a randomized clinical trial evaluating the impact of RGCT. Attitudes toward and experience with RGCT were assessed in 265 patients (at diagnosis, 6- and 12-month follow-up) and 29 medical professionals (before and after the recruitment period). RESULTS: At 6-month follow-up, more patients who had been offered RGCT felt they had been actively involved in treatment decision-making than patients who had been offered usual care (67% vs 48%, P = 0.06). Patients who received DNA-test results before primary surgery reported more often that RGCT influenced treatment decisions than those who received results afterwards (P < 0.01). Eighty-seven percent felt that genetic counseling and testing (GCT) should preferably take place between diagnosis and surgery. Most professionals (72%) agreed that RGCT should be routinely offered to eligible patients. Most patients (74%) and professionals (85%) considered surgeons the most appropriate source for referral. CONCLUSIONS: RGCT is viewed as helpful for newly diagnosed high-risk BC patients in choosing their primary surgery and should be offered routinely by surgeons.

Does rapid genetic counseling and testing in newly diagnosed breast cancer patients cause additional psychosocial distress? results from a randomized clinical trial.

PURPOSE: Female breast cancer patients carrying a BRCA1/2 mutation have an increased risk of second primary breast cancer. Rapid genetic counseling and testing (RGCT) before surgery may influence choice of primary surgical treatment. In this article, we report on the psychosocial impact of RGCT. METHODS: Newly diagnosed breast cancer patients at risk for carrying a BRCA1/2 mutation were randomized to an intervention group (offer of RGCT) or a usual care control group (ratio 2:1). Psychosocial impact and quality of life were assessed with the Impact of Events Scale, Hospital Anxiety and Depression Scale, Cancer Worry Scale, and the EORTC QLQ-C30 and QLQ-BR23. Assessments took place at study entry and at 6- and 12-month follow-up visits. RESULTS: Between 2008 and 2010, 265 patients were recruited into the study. Completeness of follow-up data was more than 90%. Of the 178 women in the intervention group, 177 had genetic counseling, of whom 71 (40%) had rapid DNA testing and 59 (33%) received test results before surgery. Intention-to-treat and per-protocol analyses showed no statistically significant differences between groups over time in any of the psychosocial outcomes. CONCLUSIONS: In this study, RGCT in newly diagnosed breast cancer patients did not have any measurable adverse psychosocial effects.

Prognostic value of lymph node micrometastases in breast cancer: a multicenter cohort study.

BACKGROUND: To evaluate the prognostic meaning of lymph node micrometastases in breast cancer patients. METHODS: Between January 2000 and January 2003, 1411 patients with a cT(1-2)N(0) invasive breast carcinoma underwent surgery in 7 hospitals in the Netherlands. Sentinel lymph node biopsy was done in all patients. Based on lymph node status, patients were divided into 4 groups: (p)N(0) (n = 922), (p)N(1micro) (n = 103), (p)N(1a) (n = 285), and (p)N(≥1b) (n = 101). Median follow-up was 6.4 years. RESULTS: At the end of follow-up, 1121 women were still alive (79.4%), 184 had died (13.0%), and 106 were lost to follow-up (7.5%). Breast cancer recurred in 244 patients: distant metastasis (n = 165), locoregional relapse (n = 83), and contralateral breast cancer (n = 44). Following adjustment for possible confounding characteristics and for adjuvant systemic treatment, overall survival (OS) remained comparable for (p)N(0) and (p)N(1micro) and was significantly worse for (p)N(1a) and (p)N(≥1b) (hazard ratio [HR] 1.18; 95% confidence interval [95% CI] 0.58-2.39, HR 2.47; 95% CI 1.69-3.63, HR 4.36; 95% CI 2.70-7.04, respectively). Disease-free survival (DFS) was similar too in the (p)N(0) and (p)N(1micro) group, and worse for (p)N(1a) and (p)N(≥1b) (HR 0.96; 95% CI 0.56-1.67 vs HR 1.64; 95% CI 1.19-2.27, HR 2.95; CI 1.98-4.42). The distant metastases rate also did not differ significantly between the (p)N(0) and (p)N(1micro) group and was worse for (p)N(1a) and (p)N(≥1b) (HR 1.22; 95% CI 0.60-2.49, HR 2.26; 95% CI 1.49-3.40, HR 3.49; CI 2.12-5.77). CONCLUSIONS: In breast cancer patients survival is not affected by the presence of micrometastatic lymph node involvement.

Neuronal intranuclear inclusions, dysregulation of cytokine expression and cell death in spinocerebellar ataxia type 3.

OBJECTIVE: We analyzed the expression of the inflammatory mediators IL-1beta, IL-1ra, IL-6 and the transcription factors IRF-1 and C/EBPdelta (previously identified in a transgenic model of spinocerebellar ataxia type 3 (SCA3) by gene expression profiling) in the central nervous system of SCA3 patients in relation to neuronal cell loss and ataxin-3-positive neuronal intranuclear inclusions (NI), to identify a putative upregulation of cytokines or microglia in SCA3 brains and to investigate whether enhanced cytokine expression was a generalized event mediating neuronal dysfunction in SCA3. MATERIALS AND METHODS: Light- and electronmicroscopic immunohistochemistry was performed on SCA3 tissues derived from five patients from unrelated families with genetically confirmed diagnosis, and six individuals without a history of neurological or inflammatory disease. RESULTS: NI were found almost exclusively in brain regions that also showed neuronal cell loss, i.e. in pons and dentate nucleus neurons, rarely in putamen and thalamus, but not in cerebral or cerebellar cortex. NI displayed an irregular surface and were mostly attached to the nucleoli. Quantitative analysis of NI in the pons revealed an inverse relation of NI and cell loss, i.e. patients with more severe neuronal cell loss had a smaller proportion of neurons with NI. Thus, formation of NI is not necessarily an indicator of cell death but could exert a protective effect. We found increased expression of IL-1beta, IL-1ra, IL-6 and C/EBPdelta only in pons and dentate nucleus neurons and both in neurons with and without NI, suggesting that NI are not a prerequisite for transcriptional changes. CONCLUSIONS: Our data suggest that the selectively affected neuronal populations in SCA3 undergo a complex alteration of gene expression independent from the formation of NI.

Comprehensive analysis of the genetic factors determining expression and function of hepatic CYP2D6.

Variable expression and function of the cytochrome P4502D6 (CYP2D6) leads to distinct phenotypes termed ultrarapid (UM), extensive (EM), intermediate (IM) and poor metabolizer (PM). Whereas the PM phenotype is known to be caused by two null-alleles leading to absence of functional CYP2D6 protein, the large variability among individuals with functional alleles remained largely unexplained. In this study, we systematically investigated 76 liver biopsies from individuals with known sparteine metabolic ratios (MRS) for the relationships between CYP2D6 genotype, microsomal protein expression, bufuralol 1'-hydroxylase activity and in-vivo phenotype. Average CYP2D6 protein levels ranged from undetectable in PMs (MRS > 20) to 2.6 +/- 2.7 pmol/mg microsomal protein in IMs (1.2 < MRS< 20), 7.6 +/- 4.7 in EMs (0.2 < MRS < 1.2) and 23.8 +/- 7.7 in UMs (MRS < 0.2), respectively. Analysis with respect to genotype demonstrated gradually increased expression and function for individuals with no, one, two or three functional gene copies per genome. The recently discovered -1584 C/G promoter polymorphism was identified as another major factor for expression and function with the mutant [-1584G] promoter type being consistently associated with significantly higher expression than [-1584C]. To investigate functional differences between the detected variant protein forms CYP2D6.1, 2D6.2, 2D6.9 and 2D6.10, we expressed them recombinantly in insect cells. The most significant difference was a decrease in the relative P450 holoprotein content of all allelic forms, including the common functional variant 2D6.2, in comparison to 2D6.1, whereas only modest Km changes were observed. Taken together, these data provide further insight into the complex mechanisms that govern the highly variable expression and function of CYP2D6.

Inflammatory genes are upregulated in expanded ataxin-3-expressing cell lines and spinocerebellar ataxia type 3 brains.

Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine disorder caused by a CAG repeat expansion in the coding region of a gene encoding ataxin-3. To study putative alterations of gene expression induced by expanded ataxin-3, we performed PCR-based cDNA subtractive hybridization in a cell culture model of SCA3. In rat mesencephalic CSM14.1 cells stably expressing expanded ataxin-3, we found a significant upregulation of mRNAs encoding the endopeptidase matrix metalloproteinase 2 (MMP-2), the transmembrane protein amyloid precursor protein, the interleukin-1 receptor-related Fos-inducible transcript, and the cytokine stromal cell-derived factor 1alpha (SDF1alpha). Immunohistochemical studies of the corresponding or associated proteins in human SCA3 brain tissue confirmed these findings, showing increased expression of MMP-2 and amyloid beta-protein (Abeta) in pontine neurons containing nuclear inclusions. In addition, extracellular Abeta-immunoreactive deposits were detected in human SCA3 pons. Furthermore, pontine neurons of SCA3 brains strongly expressed the antiinflammatory interleukin-1 receptor antagonist, the proinflammatory cytokine interleukin-1beta, and the proinflammatory chemokine SDF1. Finally, increased numbers of reactive astrocytes and activated microglial cells were found in SCA3 pons. These results suggest that inflammatory processes are involved in the pathogenesis of SCA3.

Overexpression of bcl-2 results in reduction of cytochrome c content and inhibition of complex I activity.

Bcl-2 has been shown to exert its antiapoptotic activity predominantly at the level of mitochondria by preventing cytochrome c release. Whether Bcl-2 is involved in the regulation of mitochondrial function prior to an apoptotic stimulus remains elusive. Using functional and spectrophotometric measurements in an inducible PC12-Tet-on-bcl-2 cell line we demonstrate that induction of Bcl-2 overexpression rapidly reduced cytochrome b and c levels as well as complex I activity. To confirm that these changes were specific for Bcl-2 we generated a bcl-2 antisense construct under the control of the tetracycline responsive promotor. Transient transfection with this antisense plasmid prevented both the decrease of cytochrome b and c levels and the loss of complex I activity. The decrease of cytochrome b levels was paralleled by a decrease of cytochrome b mRNA levels while Northern blot analysis of cytochrome c mRNA expression did not reveal any overt changes in Bcl-2 cells. We propose that the antiapoptotic properties of Bcl-2 are related to the reduction of mitochondrial complex I activity and lowered mitochondrial cytochrome b and c levels.

Flupirtine and retigabine prevent L-glutamate toxicity in rat pheochromocytoma PC 12 cells.

Flupirtine is an analgesic drug thought to have NMDA receptor antagonistic and antiapoptotic effects. We investigated the effects of Ethyl-2-amino-6-(4-(4-fluorbenzyl)amino)-pyridine-3-carbamamic+ ++ acid, maleate (flupirtine) and the related compound N-(2-amino-4-(4-fluorobenzylamino)-phenyl)-carbamic acid, ethyl ester) (retigabine) (Desaza-flupirtine) on the toxicity of L-glutamate and L-3,4-dihydroxyphenylalanine (L-DOPA) in rat pheochromocytoma PC 12 cells in vitro. Both drugs (10 microM) markedly decreased nonreceptor-mediated necrotic cell death in PC 12 cultures treated with L-glutamate (10 mM) for 72 h. In contrast, apoptosis induced by L-DOPA (250 microM) after 48 h was not affected by either substance. While L-DOPA elicited massive generation of reactive oxygen intermediates, L-glutamate-induced cell death was accompanied by only slightly increased levels of reactive oxygen intermediates. Flupirtine and retigabine exerted anti-oxidative effects in PC 12 cultures independent of their ability to prevent cell death. Further examination of the protective action of flupirtine and retigabine against L-glutamate toxicity showed that it had no influence on monoamine oxidase (monoamine: oxygen oxidoreductase (deaminating), EC 1.4.3.4., MAO) activity. Thus, flupirtine and retigabine provided protection against cystine deprivation and L-glutamate toxicity but did not protect against L-glutamate under cystine-free conditions indicating that both compounds are sufficiently effective to compensate the oxidative stress elicited by cystine deprivation but not excessive activity of monoamine oxidase after L-glutamate treatment.

Cell death in polyglutamine diseases.

An increasing number of inherited neurodegenerative diseases are known to be caused by trinucleotide repeat expansions in the respective genes. At least nine disorders result from a CAG trinucleotide repeat expansion which is translated into a polyglutamine stretch in the respective proteins: Huntington's disease (HD), dentatorubral pallidolysian atrophy (DRPLA), spinal bulbar muscular atrophy (SBMA), and several of the spinocerebellar ataxias (SCA1, 2, 3, 6, 7 and 12). Although the molecular steps leading to the specific neuropathology of each disease are unknown and are still under intensive investigation, there is increasing evidence that some CAG repeat disorders involve the induction of apoptotic mechanisms. This review summarizes the clinical and genetic features of each CAG repeat disorder and focuses on the common mechanistic steps involved in the disease progression of these so-called polyglutamine diseases. Among the common molecular features the formation of intranuclear inclusions, the recruitment of interacting polyglutamine-containing proteins, the involvement of the proteasome and molecular chaperones, and the activation of caspases are discussed with regard to their potential implication for the induction of cell death.

The molecular biology of the autosomal-dominant cerebellar ataxias.

Autosomal-dominant cerebellar ataxias (ADCA) may present as progressive or paroxysmal disorders. While the progressive ataxias have been named spinocerebellar ataxias (SCA), the paroxysmal disorders are designated episodic ataxias (EA). Until now, three different mutational mechanisms resulting in distinctive pathogenesis have been identified. The first type of mutation present in SCA1, SCA2, SCA3, and SCA7 is an expanded CAG repeat in genes of unknown function that are translated into proteins with expanded polyglutamine tracts. A common ultrastructural feature of these disorders is the formation of neuronal intranuclear inclusions (NII) harboring the expanded disease proteins and a variety of other proteins. The pathogenic role of these inclusions has yet to be clarified. A second group of disorders is the result of mutations in genes that code for ion channels. In EA-1, a disorder characterized by episodes of ataxia provoked by movement and startle, missense mutations in a potassium channel gene, KCNA1, have been found. Patients with EA-2, another form of paroxysmal ataxia, carry nonsense mutations of the gene encoding the alpha1A voltage-dependent calcium channel subunit, CACNA1A, that are predicted to result in truncated channel proteins. In SCA6, a progressive ataxia, an expanded CAG repeat in the 3' translated region of the CACNA1A gene, has been found. The third type of mutation is an untranslated CTG expansion resembling the mutation found in myotonic dystrophy. It is associated with a progressive ataxia, SCA8.

High level expression of expanded full-length ataxin-3 in vitro causes cell death and formation of intranuclear inclusions in neuronal cells.

Spinocerebellar ataxia type 3 (SCA3) is caused by a CAG/polyglutamine repeat expansion in the SCA3 gene. To analyse the pathogenic mechanisms in SCA3, we have generated ataxin-3-expressing rat mesencephalic CSM14.1 cells. In these cells, a post-mitotic neuronal phenotype is induced by temperature shift. The isolated stable cell lines provided high level expression of non-expanded (Q23) or expanded (Q70) human full-length ataxin-3. CSM14.1 cells expressing the expanded full-length ataxin-3 developed nuclear inclusion bodies, strong indentations of the nuclear envelope and cytoplasmic vacuolation. These ultrastructural alterations were present prior to a significantly decreased viability of neuronally differentiated cells expressing expanded ataxin-3. The observed spontaneous cell death did not correlate with formation of intranuclear inclusions and was not apoptotic by ultrastructural analysis. No increased susceptibility to staurosporine-induced apoptosis was found for the expanded or non-expanded ataxin-3-expressing cell lines. These data show that high level expression of expanded full-length ataxin-3 in a neuron-like cell line generates ultrastructural alterations of SCA3 pathogenesis and results in increased spontaneous non-apoptotic cell death.

Genes involved in hereditary ataxias.

The hereditary ataxias are a group of inherited neurodegenerative disorders characterized by progressive ataxia that results from degeneration of the cerebellum and its afferent and efferent connections. Recent molecular research has led not only to the discovery of a number of causative mutations, but also shed light on the likely mechanisms by which these mutations cause the respective phenotypes. In Friedreich's ataxia (FRDA), the most common type of autosomal recessive ataxia, the loss of a mitochondrial protein, frataxin, results in overload of mitochondrial iron and oxidative stress. The autosomal dominant ataxias, spinocerebellar ataxia type I (SCAI), SCA2, SCA3 and SCA7, are caused by inheritance of an unstable, expanded CAG trinucleotide repeat. These disorders are assumed to be due to a novel deleterious function of the extended polyglutamine sequences within the proteins encoded by the respective genes. Recent observations in transgenic mice and in human post-mortem tissue suggest that the extended proteins are transported into the nucleus of neurons where they form intranuclear inclusions that disrupt normal nuclear function. In another group of dominant disorders, episodic ataxia type I and type 2 (EA-I, EA-2) and SCA6, the mutations affect genes that code for ion channels.

Mechanisms of cell death in cerebellar disorders.

Recent advances in molecular genetic and cellular biology have provided new insights into the mechanisms leading to neuronal dysfunction and cell death in the disorders of the cerebellum. Trinucleotide repeat expansions have been identified as an important cause of inherited cerebellar ataxias and mechanisms involved in apoptotic cell death have become centerstage of scientific interest. In the present article, we review the recent findings in trinucleotide repeat disorders and address the possible link of glutamate neurotoxicity, impaired Ca2+ homeostasis and apoptosis.

Functional properties of CYP2D6 1 (wild-type) and CYP2D6 7 (His324Pro) expressed by recombinant baculovirus in insect cells.

The debrisoquine/sparteine or CYP2D6 genetic polymorphism of drug oxidation is a common cause for interindividual variability in drug response. We recently identified a mutant allele, designated CYP2D6-E or CYP2D6*7, which is associated with the poor metabolizer phenotype and occurs in Caucasian populations with a frequency of about 1%. In contrast to other loss-of-function alleles, a full length protein with a single amino acid substitution. His324Pro, is encoded by the CYP2D6*7 allele. To functionally analyze this mutant protein form of CYP2D6, recombinant baculoviruses were constructed to express the CYP2D6 cDNA. Up to 0.33 nmol of spectrally detected P450/mg of cell protein were produced in Spodoptera frugiperda cells, whereas Trichoplusia ni 5B1-4 cells reproducibly produced 0.8 nmol/mg (4% of total cell protein). Insect cell membranes were functionally characterized with cumene hydroperoxide or after reconstitution with purified rat NADPH:cytochrome P450 reductase. Km values for the substrates bufuralol and sparteine and other enzymatic properties were almost identical to those of human liver microsomes. The H324P mutation was introduced into the cDNA by site-directed mutagenesis and recombinant baculovirus was obtained. Expression under a variety of conditions demonstrated that mutant protein amounts comparable to the wild-type enzyme were produced. However, no spectrally detectable P450 was formed and no catalytic activity was detected. Furthermore, in contrast to the wild-type protein the mutant protein was almost exclusively located in a detergent-insoluble insect cell fraction. These results demonstrate that the H324P mutation is responsible for the in vivo poor metabolizer phenotype associated with the CYP2D6*7 allele by preventing normal protein folding and heme incorporation.

Influence of pharmacogenetics on drug disposition and response.

1. Pharmacogenetics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare defects or as polymorphisms. 2. Glucose-6-phosphate dehydrogenase deficiency (G6PD) is the most common inherited enzymopathy affecting approximately 400 million people. The main clinical complication associated with G6PD deficiency is haemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans. 3. The activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g. N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. The gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.

A missense mutation in exon 6 of the CYP2D6 gene leading to a histidine 324 to proline exchange is associated with the poor metabolizer phenotype of sparteine.

The sparteine/debrisoquine polymorphism is a clinically important genetic deficiency of cytochrome P4502D6-catalyzed oxidative drug metabolism. 5-10% of Caucasians designated as poor metabolizers have a severely impaired capacity to metabolize more than 30 therapeutically used drugs. Genotyping of a random Caucasian population for the known cytochrome P4502D6 mutations A, B and D which are associated with the poor metabolizer phenotype has revealed a substantial number of misclassified poor metabolizers indicating the existence of one or more unknown mutations which cannot be identified with the currently available genotyping assays. Therefore we have cloned and sequenced one nonfunctional cytochrome P4502D6 allele of a misclassified poor metabolizer and could identify a single missense mutation designated E mutation at position 3023(A-C) in exon 6. Direct sequencing analysis, FokI restriction analysis and a newly developed allele-specific polymerase chain reaction assay were applied to analyze for this mutation in a population study. Three out of 97 randomly selected Caucasians were carriers of this mutation and thus the E allele has a frequency of 1.5% (confidence interval95% = 0.33 - 4.54%). Since only 2 out of 4 misclassified poor metabolizers carried the E mutation, additional unknown mutant alleles must exist. Computer modelling suggests that the E mutation, which results in a histidine to proline exchange in position 324 of the protein, may cause an alteration of the 3D structure of CYP2D6 in close vicinity to the active site thereby leading to total loss of enzyme function.