The power of empirical data; lessons from the clinical registry initiatives in Scandinavian cancer care.

BACKGROUND: In Scandinavia, there is a strong tradition for research and quality monitoring based on registry data. In Denmark, Norway and Sweden, 63 clinical registries collect data on disease characteristics, treatment and outcome of various cancer diagnoses and groups based on process-related and outcome-related variables. AIM: We describe the cancer-related clinical registries, compare organizational structures and quality indicators and provide examples of how these registries have been used to monitor clinical performance, develop prediction models, assess outcome and provide quality benchmarks. Further, we define unmet needs such as inclusion of patient-reported outcome variables, harmonization of variables and barriers for data sharing. RESULTS AND CONCLUSIONS: The clinical registry framework provides an empirical basis for evidence-based development of high-quality and equitable cancer care. The registries can be used to follow implementation of new treatment principles and monitor patterns of care across geographical areas and patient groups. At the same time, the lessons learnt suggest that further developments and coordination are needed to utilize the full potential of the registry initiative in cancer care.

[Prostate cancer--new guidelines and the first care program]. / Prostatacancer--nya riktlinjer och första vårdprogrammet.

The Swedish National Board of Health and Welfare recently released an update of the national guidelines for the management of prostate cancer. These guidelines are produced using an evidence based method and the recommendations rely on cost-benefit assessment. They focus on controversial, new and expensive methods and the recipients are mainly healthcare administrators and commission groups. Based on these general guidelines, more detailed clinical guidelines have been published by the Confederation of Regional Cancer Centres in Sweden. The guidelines include a recommendation of evaluating systematic information on the pros and cons of PSA-testing to men aged 50 to 70 years with subsequent systematic testing for the men who choose to be tested. Active surveillance for low risk disease and radical treatment for high risk, non-metastatic disease are emphasised. Requirements for units managing prostate cancer patients are defined. The guidelines will be updated annually.

Heterozygosity for a coding SNP in COL1A2 confers a lower BMD and an increased stroke risk.

Genetic variation plays an important role in osteoporosis and a prime candidate gene is Collagen alpha2(I) (COL1A2). A coding polymorphism (rs42524) in COL1A2 has previously been associated with intracranial aneurysms. Here the effects of this polymorphism have been studied in relation to bone mineral density (BMD) and prevalences of stroke and myocardial infarction (MI). rs42524 was genotyped in elderly men (n = 2004) from the Swedish MrOS cohort. Genotypes were analysed for association to BMD and certain health parameters. Significant associations (overall P < 0.05), were observed between rs42524 genotype and BMD at several skeletal sites. Surprisingly, the heterozygote genotype class exhibited lower BMD than either homozygote group. When subjects were classified as heterozygotes or homozygotes, the heterozygous genotype was found to confer a lower BMD at total hip, femoral neck and trochanter Furthermore, the heterozygote genotype had an increased risk of stroke and MI, with population Attributable Risks being 0.12 and 0.08, respectively.

Expression of RANK-ligand in prostate cancer cell lines.

The molecular mediators of bone remodelling, receptor activator of nuclear factor-kappaB ligand (RANKL), receptor activator of nuclear factor-kappaB (RANK) and osteoprotegerine (OPG), are believed to be involved in the cellular mechanisms by which tumours metastasize to bone. RANKL is a potent stimulator of osteoclastic bone resorption and is expressed in a variety of tumour cells. We have investigated if the membrane bound form of RANKL is expressed in prostate cancer cell lines, and whether this expression might be regulated by the presence of human osteoblasts. Three prostate cancer cell lines were co-cultured with human osteoblast-like cells (hOB) and RANKL expression on cell surface was measured by FACS. We found basal expression of RANKL on the cell surface, and in co-culture with hOBs the number of cells expressing RANKL was increased between 2.5 and 4 times. These data suggest a signalling mechanism between bone cells and prostate cancer cells that might increase bone resorption and thereby promote bone metastases.

Association between repeat length of exon 1 CAG microsatellite in the androgen receptor and bone density in men is modulated by sex hormone levels.

In this study we examined whether the androgen receptor (AR) gene CAG repeat polymorphism and serum androgen levels are associated with bone mineral density (BMD) and changes in BMD during 2-3 years in 229 healthy men 41-76 years old. Microsatellite analysis was performed on an automated sequencer. Indices of bioavailable testosterone (free testosterone [FT] and free androgen index) were calculated. BMD was measured using both dual-energy Xray absorptiometry and quantitative ultrasound. All participants completed a questionnaire regarding major possible osteoporosis risk factors. In linear regression analysis there was a modest positive association, which was independent of age and body mass index (BMI), between AR repeat length and BMD at all sites. Although this association was significant independent of BMI, analyses in the subgroup of obese men (BMI > 30) did not reach significance, while the effect was enhanced when analyzing only nonobese men (BMI < or = 30). There was no association between the AR gene polymorphism and rate of bone loss, FT, and BMD or testosterone and bone loss. Interestingly, the association between AR and BMD was modified by total testosterone. The lowest age- and BMI-adjusted average femoral neck BMD was found among men in the lowest tertile for both AR repeat length and FT, whereas men within the higher categories of these variables displayed the highest BMD. In conclusion, there is a positive association between the AR CAG repeat polymorphism and BMD, which is modified by androgen levels in healthy men.

Systematic assessment of the human osteoblast transcriptome in resting and induced primary cells.

Osteoblasts are key players in bone remodeling. The accessibility of human primary osteoblast-like cells (HObs) from bone explants makes them a lucrative model for studying molecular physiology of bone turnover, for discovering novel anabolic therapeutics, and for mesenchymal cell biology in general. Relatively little is known about resting and dynamic expression profiles of HObs, and to date no studies have been conducted to systematically assess the osteoblast transcriptome. The aim of this study was to characterize HObs and investigate signaling cascades and gene networks with genomewide expression profiling in resting and bone morphogenic protein (BMP)-2- and dexamethasone-induced cells. In addition, we compared HOb gene expression with publicly available samples from the Gene Expression Omnibus. Our data show a vast number of genes and networks expressed predominantly in HObs compared with closely related cells such as fibroblasts or chondrocytes. For instance, genes in the insulin-like growth factor (IGF) signaling pathway were enriched in HObs (P = 0.003) and included the binding proteins (IGFBP-1, -2, -5) and IGF-II and its receptor. Another HOb-specific expression pattern included leptin and its receptor (P < 10(-8)). Furthermore, after stimulation of HObs with BMP-2 or dexamethasone, the expression of several interesting genes and pathways was observed. For instance, our data support the role of peripheral leptin signaling in bone cell function. In conclusion, we provide the landscape of tissue-specific and dynamic gene expression in HObs. This resource will allow utilization of osteoblasts as a model to study specific gene networks and gene families related to human bone physiology and diseases.

The genetic architecture of a female sexual ornament.

Understanding the evolution of sexual ornaments, and particularly that of female sexual ornaments, is an enduring challenge in evolutionary biology. Key to this challenge are establishing the relationship between ornament expression and female reproductive investment, and determining the genetic basis underpinning such relationship. Advances in genomics provide unprecedented opportunities to study the genetic architecture of sexual ornaments in model species. Here, we present a quantitative trait locus (QTL) analysis of a female sexual ornament, the comb of the fowl, Gallus gallus, using a large-scale intercross between red junglefowl and a domestic line, selected for egg production. First, we demonstrate that female somatic investment in comb reflects female reproductive investment. Despite a trade-off between reproductive and skeletal investment mediated by the mobilization of skeletal minerals for egg production, females with proportionally large combs also had relatively high skeletal investment. Second, we identify a major QTL for bisexual expression of comb mass and several QTL specific to female comb mass. Importantly, QTL for comb mass were nonrandomly clustered with QTL for female reproductive and skeletal investment on chromosomes one and three. Together, these results shed light onto the physiological and genetic architecture of a female ornament.

The impact of estradiol on bone mineral density is modulated by the specific estrogen receptor-alpha cofactor retinoblastoma-interacting zinc finger protein-1 insertion/deletion polymorphism.

CONTEXT: Estrogens regulate bone mass by binding to the estrogen receptor (ER)-alpha as well as ER-beta. The specific ERalpha cofactor retinoblastoma-interacting zinc finger protein (RIZ)-1 enhances ERalpha function in the presence of estrogen. OBJECTIVE: The objective of the study was to determine whether a RIZ P704 insertion (+)/deletion (-) (indel) polymorphism modulates the impact of estradiol on bone mineral density (BMD) and study the association between the polymorphism and BMD in elderly subjects. DESIGN: This was a population-based, prospective, and cross-sectional study, the Swedish MrOS Study, and the Malmö OPRA Study, respectively. SETTING: The study was conducted at three academic medical centers: Sahlgrenska Academy in Gothenburg, Malmö University Hospital, and Uppsala University Hospital. PARTICIPANTS: In total, 4058 men and women, aged 69-81 yr, were randomly selected from population registries. MAIN OUTCOME MEASURES: BMD (grams per square centimeter) was measured at femoral neck, trochanter, lumbar spine, and total body. RESULTS: The RIZ P704(+/+) genotype was associated with low BMD in both women (femoral neck, P < 0.001; trochanter, P < 0.01; lumbar spine, P < 0.05; total body, P < 0.01) and men (lumbar spine, P < 0.05). However, the association between the polymorphism and BMD was dependent on estradiol status. The positive correlation between serum estradiol and BMD was significantly modulated by the genotype with a stronger correlation in the P704(+/+) group than the P704(-/-) group (r = 0.19 vs. r = 0.08, P < 0.05). CONCLUSIONS: These large-scale studies of elderly men and women indicate that the ERalpha cofactor RIZ gene has a prominent effect on BMD, and the P704 genotype modulates the impact of estradiol on BMD. Further studies are required to determine whether this polymorphism modulates the estrogenic response to estradiol treatment.

Vitamin D receptor 3' haplotypes are unequally expressed in primary human bone cells and associated with increased fracture risk: the MrOS Study in Sweden and Hong Kong.

UNLABELLED: The VDR is a prime candidate gene for osteoporosis. Here, we studied three common VDR haplotypes in relation to bone phenotypes in 5014 participants of the global MrOS Study. We also studied the relative expression of the haplotypes in human bone cells. One haplotype was associated with increased fracture risk and differently expressed in primary human bone cells. INTRODUCTION: Vitamin D plays an essential role in skeletal metabolism by binding to its nuclear steroid receptor, the vitamin D receptor (VDR). The heritability of BMD is well established, and the VDR gene is considered a prime candidate suggested to partially account for genetically controlled BMD variance in the population. MATERIALS AND METHODS: Here, we reconstructed common haplotypes in the VDR 3' untranslated region (UTR) and studied the association to BMD and risk of vertebral fractures in elderly men from Sweden (n = 3014) and Hong Kong (n = 2000), all participants of the global MrOS Study. To assess any functional implications of the VDR polymorphisms, we studied allele-specific expressions of the different VDR 3' UTR haplotypes in the normal chromosomal context of 70 unrelated human trabecular bone samples. This was performed by quantitative genotyping of coding polymorphisms in RNA samples and in corresponding DNA samples isolated from the bone samples. RESULTS: Three major haplotypes were reconstructed and in agreement with the previously well-defined baT, BAt, and bAT haplotypes, herein denoted Hap1, Hap2, and Hap3. The Hap1 haplotype was independently associated with increased risk of vertebral fractures in Swedish men (OR, 1.655; 95% CI, 1.146-2.391; p < 0.01) and with lower lumbar spine BMD in elderly men from Sweden (p < 0.01) and Hong Kong (p < 0.05). The VDR gene was also shown to exhibit a 3' UTR haplotype dependent allelic imbalance, indicating that the VDR Hap1 allele was overexpressed in human trabecular bone samples. CONCLUSIONS: The results indicate that the relatively overexpressed VDR Hap1 haplotype could be considered a risk allele for osteoporosis regardless of ethnicity.

Quantitative trait loci for BMD and bone strength in an intercross between domestic and wildtype chickens.

UNLABELLED: With chicken used as a model species, we used QTL analysis to examine the genetic contribution to bone traits. We report the identification of four QTLs for femoral traits: one for bone strength, one for endosteal circumference, and two affecting mineral density of noncortical bone. INTRODUCTION: BMD is a highly heritable phenotype, governed by elements at numerous loci. In studies examining the genetic contribution to bone traits, many loci have been identified in humans and in other species. The goal of this study was to identify quantitative trait loci (QTLs) controlling BMD and bone strength in an intercross between wildtype and domestic chickens. MATERIALS AND METHODS: A set of 164 markers, covering 30 chromosomes (chr.), were used to genotype 337 F2-individuals from an intercross of domesticated white Leghorn and wildtype red junglefowl chicken. DXA and pQCT were used to measure BMD and bone structure. Three-point bending tests and torsional strength tests were performed to determine the biomechanical strength of the bone. QTLs were mapped using forward selection for loci with significant marginal effects. RESULTS: Four QTLs for femoral bone traits were identified in QTL analysis with body weight included as a covariate. A QTL on chr. 1 affected female noncortical BMD (LOD 4.6) and is syntenic to human 12q21-12q23. Also located on chr. 1, a locus with synteny to human 12q13-14 affected endosteal circumference (LOD 4.6). On chr. 2, a QTL corresponding to human 5p13-p15, 7p12, 18q12, 18q21, and 9q22-9q31 affected BMD in females; noncortical (LOD 4.0) and metaphyseal (LOD 7.0) BMD by pQCT and BMD by DXA (LOD 5.9). A QTL located on chr. 20 (LOD 5.2) affected bone biomechanical strength and had sex-dependent effects. In addition to the significant QTLs, 10 further loci with suggestive linkage to bone traits were identified. CONCLUSIONS: Four QTLs were identified: two for noncortical BMD, one for endosteal circumference, and one affecting bone biomechanical strength. The future identification of genes responsible for these QTLs will increase the understanding of vertebrate skeletal biology.

A TA-repeat polymorphism in the gene for the estrogen receptor alpha does not correlate with muscle strength or body composition in young adult Swedish women.

OBJECTIVES: There are conflicting data in the literature whether estrogens affect muscle strength. Prospective studies with hormone replacement therapy have not been able to convincingly demonstrate a muscular effect and the putative role of estrogen in the development of lean body mass is not established. Both lean mass and fat mass are known to be under strong genetic control and therefore we have investigated the relation between a TA-repeat in the gene for the estrogen receptor alpha (ERalpha) and muscle strength and body composition. METHODS: 175 healthy Swedish women, aged 20-39 were randomly selected from the population registry and included in the study. Body mass measurements (lean mass, fat mass, body weight and BMI) and muscle strength (quadriceps, hamstring and grip strength) were evaluated. The TA-repeat in the ERalpha gene was amplified by polymerase chain reaction. RESULTS: Alleles with a TA-repeat length of 16 repeats or shorter were denoted short (e), and repeat length of 17 repeats or longer were denoted long (E). Women homozygous for the short and long genotype were denoted ee (31%) and EE (21%), respectively, while heterozygous individuals were denoted Ee (48%). The frequencies were in Hardy-Weinberg equilibrium. No associations were found between ERalpha genotypes and muscle strength or body composition. CONCLUSION: The TA-repeat in the human ERalpha gene does not correlate with muscle strength or body mass measurements, indicating that body composition is not as sensitive to genetic variation in this receptor as other target organs for estrogen.

Interleukin-4 and interleukin-13 potentiate interleukin-1 induced secretion of interleukin-6 in human osteoblast-like cells.

Formation of interleukin-6 (IL-6) in osteoblasts and bone marrow stromal cells is believed to regulate osteoclast recruitment. The anti-inflammatory cytokines interleukin-4 and -13 (IL-4 and IL-13) stimulate IL-6 production in human osteoblasts. We investigated the relative potencies, and synergistic effects, between IL-4, IL-13 and interleukin-1 (IL-1) on IL-6 formation in human osteoblast-like cells. Isolated human osteoblast-like cells were incubated for 72 h in the presence of various concentrations of IL-4, IL-13 and IL-1, and IL-6 secretion was measured by ELISA. All cytokines stimulated the secretion of IL-6. The rank order of potency was IL-1>>IL-4>IL-13. There were no additive or synergistic effects between IL-4 and IL-13. However, co-stimulation with IL-1 and IL-4 resulted in a marked synergistic effect on IL-6 secretion. Co- stimulation with IL-1 and IL-13 gave a minor synergistic effect. In conclusion, IL-4/13 synergistically potentiates IL-1 induced secretion of IL-6 in human osteoblast-like cells.

Interleukin-6 promoter polymorphism is associated with bone quality assessed by calcaneus ultrasound and previous fractures in a cohort of 75-year-old women.

Interleukin 6 (IL-6) is a multifunctional cytokine and a potent stimulator of bone resorption and has been implicated in the pathogenesis of osteoporosis in postmenopausal women. The aim of this study was to investigate if a functional IL-6 promoter polymorphism (-174) was related to bone mass and fractures in a cohort consisting of 964 postmenopausal Caucasian women aged 75 years. Bone mineral density (BMD; g/cm2) of the femoral neck, lumbar spine and total body was measured using dual energy X-ray absorptiometry (DXA). Quantitative ultrasound (QUS) was also measured in the calcaneus and quantified as speed of sound (SOS; m/s), broadband ultrasound attenuation (BUA; dB/MHz), and stiffness index (SI). IL-6 genotypes was determined by restriction fragment length polymorphism (RFLP) using the restriction enzyme NlaIII. The frequencies of the different IL-6 genotypes were 27.5% (GG), 47.9% (GC), 24.6% (CC). The IL-6 polymorphism (presence of G) was independently related to a lower stiffness (beta=-0.07; P=0.03) and BUA (beta=-0.08; P=0.02), but not to BMD at any site measured by DXA. In the cohort, 420 subjects (44%) reported at least one fracture during their lifetime, and 349 (36%) reported at least one fracture after the age of 50. Using binary logistic regression, the IL-6 polymorphism (presence of G) was significantly related to an increased risk of a previous fracture during life (odds ratio 1.46, 95% CI 1.08-1.97) and to an increased risk of a fracture occurring after 50 years of age (odds ratio 1.37, 95% CI 1.004-1.88). The risk was further increased for fractures grouped as osteoporotic fractures (odds ratio 1.67, 95% CI 1.14-2.45), including forearm fractures (odds ratio 1.59, 95% CI 1.05-2.40). In conclusion, presence of G allele in the IL-6 promoter polymorphism at position -174 is independently related to previous fractures in postmenopausal women. This association may be related primarily to an altered bone quality identified by QUS and not a lower bone mass. This is also the first demonstration of association of IL-6 gene polymorphism to calcaneal QUS.

Genetic variation in the human vitamin D receptor is associated with muscle strength, fat mass and body weight in Swedish women.

OBJECTIVE: Bone mineral density (BMD) is under strong genetic control and a number of candidate genes have been associated with BMD. Both muscle strength and body weight are considered to be important predictors of BMD but far less is known about the genes affecting muscle strength and fat mass. The purpose of this study was to investigate the poly adenosine (A) repeat and the BsmI SNP in the vitamin D receptor (VDR) in relation to muscle strength and body composition in healthy women. DESIGN: A population-based study of 175 healthy women aged 20-39 years was used. METHODS: The polymorphic regions in the VDR gene (the poly A repeat and the BsmI SNP) were amplified by PCR. Body mass measurements (fat mass, lean mass, body weight and body mass index) and muscle strength (quadriceps, hamstring and grip strength) were evaluated. RESULTS: Individuals with shorter poly A repeat, ss and/or absence of the linked BsmI restriction site (BB) have higher hamstring strength (ss vs LL, P=0.02), body weight (ss vs LL, P=0.049) and fat mass (ss vs LL, P=0.04) compared with women with a longer poly A repeat (LL) and/or the presence of the linked BsmI restriction site (bb). CONCLUSIONS: Genetic variation in the VDR is correlated with muscle strength, fat mass and body weight in premenopausal women. Further functional studies on the poly A microsatellite are needed to elucidate whether this is the functionally relevant locus or if the polymorphism is in linkage disequilibrium with a functional variant in a closely situated gene further downstream of the VDR 3'UTR.

A survey of genetic and epigenetic variation affecting human gene expression.

The identification of human sequence polymorphisms that regulate gene expression is key to understanding human genetic diseases. We report a survey of human genes that demonstrate allelic differences in gene expression, reflecting the presence of putative allele-specific cis-acting factors of either genetic or epigenetic nature. The expression of allelic transcripts in heterozygous samples is assessed directly by relative quantitation of intragenic marker alleles in messenger or heteronuclear RNA derived from cells or tissues. This survey used 193 single-nucleotide polymorphisms (SNPs) from 129 genes expressed in lymphoblastoid cell lines, to identify 23 genes (18%) with common allele-specific transcripts whose expression deviated from the expected equimolar ratio. A subset of these deviations, or "allelic imbalances," can be observed in multiple samples derived from reference CEPH ("Centre d'Etude du Polymorphisme Humain") pedigrees and demonstrate a spectrum of patterns of transmission, including cosegregation of allelic skewing across generations compatible with Mendelian inheritance as well as random monoallelic expression for three genes (IL1A, HTR2A, and FGB). Additional studies for BTN3A2 provide evidence of SNPs and haplotypes in complete linkage disequilibrium with high- and low-expressing transcripts. The pipeline described herein offers tools for efficient identification and characterization of allelic expression allowing identification of regulatory sequence variants as well as epigenetic variation affecting human gene expression.

Genetic variation in the ATP-binding cassette transporter gene ABCG2 (BCRP) in a Swedish population.

The ATP-binding cassette transporter ABCG2 (also named breast cancer resistance protein, BCRP) functions as a drug efflux transporter and is expressed at high levels in the human small intestine. The aim of this study was to screen the human ABCG2 gene for genetic variation. The regions of the gene most likely to affect function, namely the coding parts, exon/intron boundaries, 5' untranslated region and 3' untranslated region and the proposed promoter region, were included in the screening. DNA was obtained from 60 Swedish individuals. The screening was performed using a polymerase chain reaction-denaturing high-performance liquid chromatography approach followed by sequence analysis. Eight sites of genetic variation were identified. The sequence variations considered to be most likely to affect transcription level or transport function were a CTCA deletion in the 5' flanking region, a single nucleotide polymorphism (SNP) in a 5' flanking CpG island, two non-synonymous SNPs, changing valine at amino acid position 12 to methionine and glutamine at position 141 to lysine, respectively. Genotyping of these sequence variations revealed linkage between the CTCA deletion and the SNP changing glutamine 141 for lysine. This information forms the basis for future association studies to investigate the genetic basis of differences of drug disposition due to sequence variation in the ABCG2 gene.

Osteoprotegerin secretion from prostate cancer is stimulated by cytokines, in vitro.

Osteoprotegerin (OPG), a member of the tumor necrosis receptor family, is produced by various tissues and inhibits osteoclast differentiation and activity. Since the metastasis of prostate cancer to bone often induces osteosclerosis, the possibility that these tumor cells secrete OPG is of interest. We have investigated whether the prostate cancer cell lines LNCaP, PC-3, and DU-145 produce and secrete OPG in vitro and if the production might be regulated by cytokines involved in remodeling of bone. OPG transcripts were detected by RT-PCR in all cell lines. OPG in culture media was analyzed by ELISA. In all three lineages, treatment with tumor necrosis factor-alpha and interleukin-1 beta dose dependently (5-5000 pM) stimulated the OPG secretion. Treatment with tumor necrosis factor-beta in increasing concentrations (1-1000 pM) stimulated OPG secretion in PC-3 but had no effect on the DU-145 and LNCaP cells. Dexamethasone (100 pM) had a small, but not significant, inhibitory effect on OPG secretion from DU-145 and LNCaP. In human non-malignant prostate cells, used as controls, there was no effect of IL-1 or TNFs on the secretion rate of OPG.

A single nucleotide polymorphism in the promoter region of the human gene for osteoprotegerin is related to vascular morphology and function.

Osteoprotegerin (OPG) is a secreted member of the tumor necrosis factor receptor family, and has previously been shown to regulate bone mass by inhibiting osteoclast differentiation and activation. Recent evidence indicates that OPG also plays a role in the vascular system, since ablation of the OPG gene in mice results in calcification of the aorta and renal arteries, and association has been found between serum levels of OPG and cardiovascular mortality. This study presents a novel single nucleotide polymorphism, a T/C transition located 129 bp upstream the TATA-box of the human OPG gene, detected by sequence analysis. The OPG genotype was determined by restriction fragment length polymorphism in a cohort consisting of 59 healthy subjects. The intima-media thickness (IMT) in the common carotid artery and maximal post-ischemic forearm blood flow (FBF) were investigated. Subjects with the CC genotype showed a significantly increased IMT (p<0.05) and a concommitantly reduced maximal FBF (p<0.01) as compared to those with the T allele. Thus, our results show that the polymorphism in the promoter region of OPG is associated with both vascular morphology and function in apparently healthy subjects.