A randomized trial exploring the biomarker effects of neoadjuvant sequential treatment with exemestane and anastrozole in post-menopausal women with hormone receptor-positive breast cancer.

Several adjuvant endocrine strategies exist for postmenopausal women with breast cancer. This study compared the effect of two sequences of aromatase inhibitor use [steroidal (exemestane) and non-steroidal (anastrozole)] on serological and pathological biomarkers when given in the neoadjuvant setting to postmenopausal women with breast cancer. Thirty women were assigned to receive exemestane 25 mg or anastrozole 1 mg each given for 8 weeks in a randomized sequence. The effect of this treatment on serum estrone sulfate and estradiol levels, as well as tumor changes in the proliferation biomarker Ki67 were evaluated at baseline, 8 weeks and 16 weeks. WHO clinical response criteria, patient preference, and quality of life were also assessed. Assessable data was available from 28 patients. There were no differences in concentration changes of serum estradiol or Ki67 between patients in the two arms. Overall clinical response rate was 68% (19/28 assessable patients) and clinical benefit was 93% (26/28 assessable patients). There was no significant difference in toxicity or quality of life scores. The majority of patients expressed a personal preference for anastrozole over exemestane. Results suggest that the order of steroidal and non-steroidal aromatase inhibitors has little effect on outcome. The majority of patients express clear preferences for drug treatments.

Vitamin D insufficiency in a large female SLE cohort.

The objective of this study was to determine the vitamin D status and its relationship with disease and therapy features and with bone mineral density in women with systemic lupus erythematosus. Non-pregnant systemic lupus erythematosus women with dual-energy X-ray absorptiometry and vitamin D measurements performed between May 1 2005 and August 31 2006 were studied. In each patient, the lowest T-score of the first dual-energy X-ray absorptiometry scan during the study period was used. In postmenopausal women, a T-score > or = 1.0 standard deviation was considered normal, between -1.0 and -2.5 standard deviations osteopenia and < or = 2.5 standard deviations osteoporosis; in premenopausal women a T-score > or = 2.5 standard deviations was normal and < or = 2.5 standard deviations defined as reduced bone density. 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D levels were determined at the time of dual-energy X-ray absorptiometry. A 25-hydroxyvitamin D level of <80 nmol/L was defined as sub-optimal and a level <40 nmol/L as deficient. Demographic and clinical variables were investigated for association with vitamin D levels by univariate and multivariate analyses. One-hundred and twenty-four systemic lupus erythematosus women had dual-energy X-ray absorptiometry scans and vitamin D assays performed during the study period. Sub-optimal 25-hydroxyvitamin D levels were found in 82 (66.7%) and deficient 25-hydroxyvitamin D levels in 22 (17.9%) patients. The disease-related features examined at the time of vitamin D assays or bone mineral density showed no correlation with vitamin D levels by univariate analyses. Neither 25-hydroxyvitamin D nor 1,25-dihydroxyvitamin D was associated with bone mineral density status among these patients. A multivariate logistic regression model identified season, cumulative glucocorticoid exposure, and serum creatinine as being associated with 25-hydroxyvitamin D levels, whereas ethnicity, glucocorticoid exposure, and serum creatinine were associated with 1,25-dihydroxyvitamin D levels. In conclusion, sub-optimal vitamin D status is common in women with systemic lupus erythematosus and is related to season, cumulative glucocorticoid dose, and serum creatinine.

Heterogeneous disease modeling for Hardy-Weinberg disequilibrium in case-control studies: application to renal stones and calcium-sensing receptor polymorphisms.

Renal stone formation due to hypercalciuria is a relatively common disorder with clear evidence for genetic predisposition, but cryptic phenotypic heterogeneity has hampered identification of candidate genes. The R990G single-nucleotide polymorphism (SNP) of the calcium sensing receptor (CASR) gene has been associated with hypercalciuria in stone formers and shows the appropriate functional phenotype in cell culture. In our preliminary association analysis of a case-control cohort, however, we observed significant Hardy-Weinberg disequilibrium (HWD) for the cases (n= 223), but not controls (n= 676) at the R990G locus, pointing us toward the general disease model incorporating HWD. Because there is an adjacent CASR SNP, A986S, which is in negative linkage disequilibrium with R990G, we extended the general disease model to enable testing of a two-site hypothesis. In our data set, there is no lack of fit (P= .345) for the single-locus model for the R990G genotype, and likelihood ratio testing favors a recessive effect with an eight-fold increase in risk (P < .001) for GG homozygotes, relative to wild-type, based on a population prevalence of 2%. Addition of the A986S genotype provides no additional information either by itself or when included in our two-site model.

Replication of associations between LRP5 and ESRRA variants and bone density in premenopausal women.

UNLABELLED: Replication is a critical step to validate positive genetic associations. In this study, we tested two previously reported positive associations. The low density lipoprotein receptor-related protein 5 (LRP5) Val667Met and lumbar spine bone density are replicated. This result is in line with results from large consortiums such as Genomos. However, the estrogen-related receptor alpha (ESRRA) repeat in the promoter is not replicated although the polymorphism studied was functional and could have been a causative variant. INTRODUCTION: We sought to validate associations previously reported between LRP5 V667M polymorphism and lumbar spine (LS, p = 0.013) and femoral neck (FN, p = 0.0002) bone mineral density (BMD), and between ESRRA 23 base pair repeat polymorphism and LS BMD (p = 0.0036) in a sample of premenopausal Caucasian women using an independent sample. METHODS: For the replication sample, we recruited 673 premenopausal women from the Toronto metropolitan area. All women were Caucasian and had BMD measured. LRP5 V667M was genotyped by allele-specific PCR and ESRRA repeats by sizing of PCR products on agarose gels. RESULTS: We reproduced the same association as we reported previously between LRP5 V667M and LS BMD (p = 0.015) but not with FN BMD (p = 0.254). The combined data from the two populations indicate an effect size of 0.28SD for LS BMD (p = 0.00048) and an effect size of 0.26 SD for FN BMD (p = 0.00037). In contrast, the association we reported earlier between ESRRA repeats and LS BMD was not replicated in the sample from Toronto (p = 0.645). CONCLUSIONS: The association between LRP5 V667M and LS BMD is confirmed but not that between ESRRA repeats and LS BMD. This result indicates that it is imperative to validate any positive association in an independent sample.

Elevation of plasma homocysteine in natural menopause can not be explained by a lack of vitamin coenzyme availability: relevance to the risk of cardiovascular disease.

UNLABELLED: The risk of cardio vascular disease (CVD) doubles after menopause. Plasma homocysteine (hCy) is a risk factor which is influenced by vitamins B12,B6 and folate. The present study was conducted to examine the relationship of plasma hCy to the three vitamins and other contributing variables in early natural menopause. METHODS: Participants were healthy, non smoking Caucasian women 3 to 5 years postmenopausal (n = 26) or premenopausal between 30 and 45 y(n = 30). Anthropometric data, dietary records and plasma concentrations of hCy, vitamin B6, vitamin B12 and folate were obtained. RESULTS: The nutritional status of vitamins B6, B12 and folate as measured by dietary intake and blood concentrations was adequate in both groups. Mean fasting plasma total (t) hCy concentration of postmenopausal group was 2-fold higher than the value found for control group (P < 0.0001) without oral methionine loading. The difference between the two groups remained highly significant after adjustment for confounding variables by multivariate analysis, suggesting that the effect of estrogen deficiency was direct. CONCLUSION: In addition to the loss of the protective effects of estrogen on their cardiovascular physiology and lipid metabolism, postmenopausal women are exposed to higher plasma hCy concentrations and deleterious cardiovascular effects. The exact mechanism is not known but does not seem to be related to coenzyme deficiency.

Persistence of vitamin B12 insufficiency among elderly women after folic acid food fortification.

OBJECTIVE: To estimate the associated risk of folate and vitamin B12 (B12) insufficiency, as well as vitamin repletion, following folic acid food fortification. DESIGN: Retrospective cross-sectional study over a 5-year period. SETTING: Two large laboratory databases in the provinces of Ontario and British Columbia, Canada. PARTICIPANTS: Canadian women aged 65 years and over who underwent concomitant clinical testing of serum folate and B12 during the pre-fortification period of January 1996 to December 1997 in Ontario (n = 733) and British Columbia (n = 3839), and in the near-complete post-fortification period of January 1998 to December 2000 in Ontario (n = 4415) and British Columbia (n = 6677). MEASUREMENTS: Geometric mean concentrations of serum folate and B12 before and after folate fortification. Prevalence ratios (PR) were used to separately compare the post- and pre-fortification period rates of folate deficiency (below 6.0 nmol/L); B12 insufficiency (below 150 pmol/L); and B12 insufficiency in combination with supraphysiological concentrations of serum folate (above 45 nmol/L). RESULTS: The mean baseline folate and B12 concentrations were similar between provinces. Using the combined provincial data, the mean serum folate concentration increased by 64% after fortification, from 14.8 to 24.2 nmol/L (p < 0.001). The average B12 concentration increased from 280 to 300 pmol/L, which was more pronounced in BC (p < 0.001) than in Ontario (p = 0.16). The prevalence of folate deficiency declined from 6.3% to 0.88% after fortification (PR 0.14, 95% confidence interval [CI] 0.11-0.18), while the decline in B12 deficiency was less pronounced (PR 0.78, 95% CI 0.71-0.86). CONCLUSIONS: The prevalence of combined B12 insufficiency with supraphysiological concentrations of serum folate increased from 0.09% pre-fortification to 0.61% post (PR 7.0, 95% CI 2.6-19.2). The introduction of folic acid food fortification was associated with a substantial improvement in the folate status of Canadian women aged 65 years and older, paralleled by a large decline in the rate of folate deficiency. Improvement in the B12 status of these women was far less pronounced. Because the prevalence of combined B12 insufficiency and supraphysiological concentrations of serum folate may have increased with folic acid food fortification, consideration should be given to confirming this finding, and possibly, to the addition of B12 to folate fortified foods.

N-acetylcysteine neither lowers plasma homocysteine concentrations nor improves brachial artery endothelial function in cardiac transplant recipients.

BACKGROUND: N-acetylcysteine is a novel antioxidant that has been reported to reduce plasma homocysteine concentrations and improve endothelial function. Cardiac transplant recipients have a high incidence of coronary endothelial dysfunction and hyperhomocysteinemia, both of which may lead to the development of transplantation coronary artery disease. It was hypothesized that N-acetylcysteine would reduce plasma homocysteine concentrations and improve brachial endothelial function in cardiac transplant recipients. PATIENTS AND METHODS: A cohort of stable cardiac transplant recipients was recruited from the outpatient clinic at the Toronto General Hospital, Toronto, Ontario. Brachial artery endothelial functions were studied according to standard techniques to determine flow-mediated dilation of the brachial artery. Plasma homocysteine concentrations were assayed using high performance liquid chromatography with electrochemical detection and pulsed integrated amperometry. After baseline testing, patients were treated in an unblinded fashion with N-acetylcysteine 500 mg/day. After 10 weeks of therapy, patients returned for follow-up endothelial function and homocysteine testing. RESULTS: Thirty-one patients were initially enrolled. Two patients withdrew due to excessive gastrointestinal upset. Two patients did not return for follow-up testing. The remaining 27 patients tolerated the treatment well. At baseline, 85% of the patients had hyperhomocysteinemia (greater than 15 mol/L) with a mean plasma concentration of 18.6 4.7 mol/L. No changes in homocysteine concentrations were seen at follow-up. At baseline, the average flow-mediated dilation was only 4.7 6.3%. No changes were seen at follow-up. CONCLUSIONS: Hyperhomocysteinemia and brachial endothelial dysfunction are common in stable cardiac transplant recipients and are unaffected by supplementation with N-acetylcysteine.

Declining rate of folate insufficiency among adults following increased folic acid food fortification in Canada.

OBJECTIVE: Canada introduced a mandatory folic acid food fortification program in November 1998. We investigated whether the rate of folate and vitamin B12 insufficiency among adults has changed since this mandatory fortification program was implemented. METHODS: We conducted a retrospective cross-sectional study using a large Ontario laboratory database. We included all individuals who underwent evaluation of their serum folate, red cell folate and serum vitamin B12 between April 1, 1997 to July 31, 1998 (Period A), August 1, 1998 to January 30, 1999 (Period B) and February 1, 1999 to March 31, 2000 (Period C). RESULTS: A total of 8,884 consecutive samples were analyzed during the period of study. Mean age was 57.4 years (SD 21.1), and 63.2% were female. The prevalence of serum folate insufficiency (below 3.4 nmol/L) fell from 0.52% in Period A to 0.22% in Period C [prevalence ratio (RR) 0.41, 95% confidence interval (CI) 0.18-0.93)]. The prevalence of red cell folate insufficiency (below 215 nmol/L) declined from 1.78% during Period A to 0.41% in Period C (RR 0.23, 95% CI 0.14-0.40). No significant difference was observed between periods in the prevalence of B12 insufficiency below 120 pmol/L (3.93% versus 3.11%, respectively; RR 0.79, 95% CI 0.62-1.01). CONCLUSIONS: There has been a significant decline in the prevalence of folate, but not vitamin B12 insufficiency, following Canadian folic acid food fortification. These changes may have important implications for the prevention and detection of folate and vitamin B12 insufficiency, including identifying the benefits of folic acid food fortification and the need to further consider fortification or supplementation with vitamin B12.

Sun exposure questionnaire predicts circulating 25-hydroxyvitamin D concentrations in Caucasian hospital workers in southern Italy.

INTRODUCTION: Recent sun exposure should correlate with circulating 25-hydroxyvitamin D [25(OH)D] due to ultraviolet B (UVB)-catalyzed cutaneous synthesis of vitamin D. METHODS: A Sun Exposure Score was calculated for healthy adults using a recall questionnaire assessing daily Time in Sun (<5 min, 5-30 min, >30 min) and Skin Exposure (face/hands; face/hands and arms; face/hands and legs; and "bathing suit") for 1 week in each of the winter and summer (n=47 and 23, respectively; n=18 participated in both). Concentrations of 25(OH)D were measured by DiaSorin RIA on end-of-week sera. RESULTS: Mean serum 25(OH)D was higher in summer than winter (58.6+/-16.5 nmol/L vs. 38.8+/-29.0 nmol/L, respectively, P=0.003 unpaired). The calculated Sun Exposure Score correlated strongly with serum 25(OH)D during summer (Spearman's rho=0.59, P=0.003); based on the Pearson coefficient of determination, summer Sun Exposure Score explained 38% of the variability in summer serum 25(OH)D. The Sun Exposure Score did not correlate with 25(OH)D in the winter (rho=0.19, P=0.210). The summer correlation was largely explained by the Time in Sun (rho=0.58, P=0.004) rather than area of Skin Exposed (rho=0.10, P=0.660). Although there was a correlation between winter and summer Sun Exposure Scores (rho=0.63, P=0.005), there was no summer vs. winter correlation in serum 25(OH)D (rho=0.08, P=0.76). CONCLUSION: This simple 1-week sun exposure recall questionnaire predicted summer serum 25(OH)D concentrations, accounting for 38% of the variability in 25(OH)D among healthy Italian adults.

Testing for equality of standardized composite measures of linkage disequilibrium.

We described the approximate variance for a composite measure of linkage disequilibrium (LD), which depends only on genotype frequencies and allows for departure from Hardy-Weinberg disequilibrium (Hamilton et al. 2006). Here we extend this work to allow for inferences about differences in composite LD coefficients, as we illustrate with two examples. Simulations demonstrate consistency of type I error rates under most conditions. Calculations for power and sample size are also given.

Azathioprine and diffuse alveolar haemorrhage: the pharmacogenetics of thiopurine methyltransferase.

Current guidelines support the use of corticosteroids and azathioprine as one possible treatment strategy for idiopathic pulmonary fibrosis (IPF). However, some patients with genetic polymorphisms of thiopurine methyltransferase (TPMT) are at risk of severe azathioprine myelotoxicity. The current authors present the case of an 85-yr-old Caucasian male with IPF who developed diffuse alveolar haemorrhage as a complication of azathioprine-induced myelosuppression. Leukocyte genetic TPMT testing revealed that the patient had homozygous polymorphisms associated with the absence of TPMT activity and severe azathioprine-induced myelotoxicity. Thiopurine methyltransferase deficiency should be considered in patients who develop leukopenia early in treatment with azathiopurine, or who present with severe marrow suppression at usual doses. For centres with equipped laboratories, a dosing suggestion is provided based on thiopurine methyltransferase testing. Even with screening strategies, frequent monitoring of complete blood count and liver biochemistry should remain the mainstay of surveillance for azathioprine toxicity.

Approximate variance for a standardized composite measure of linkage disequilibrium.

The approximate variance for the standardized measure of gametic linkage disequilibrium has been described. However, this approach assumes knowledge of the phase of double heterozygotes or Hardy-Weinberg equilibrium. Here we give the approximate variance for a composite measure of linkage disequilibrium which depends only on genotype frequencies. We show by simulation that this variance approximation is valid over a range of allele probabilities and departures from Hardy-Weinberg equilibrium.

Standardizing a composite measure of linkage disequilibrium.

The maximum and minimum are obtained for a composite measure of linkage disequilibrium used with genotypic data when the phase of double heterozygotes cannot be determined. These bounds are used to standardize the composite measure in the same way used for D', the standardized gametic measure of linkage disequilibrium. Standardization produces a measure which lies between -1 and 1, and allows comparison of linkage disequilibrium between populations. The method is illustrated using two loci in the CASR gene.

Use of CA-125 and ultrasound in high-risk women.

Our objective was a retrospective study reporting on ovarian cancer screening in a high-risk female population using both CA-125 and ultrasound over a 7-year period. We used risk estimates of carrying a BRCA mutation that were based on family history. Subjects were screened with CA-125 and ultrasound every 6 months. Each of 311 high-risk subjects had between 1 and 17 screening visits. Overall, 33 of 1209 (2.7%) CA-125 results were abnormal (>35 U/ml); 226 of 1342 (17%) ultrasounds were abnormal, with abnormalities ranging from benign appearing cystic changes to more ominous patterns. Since entry into the program, 29 subjects (9%) have undergone surgery. In 20 of these, the preoperative screening was normal; in six, only the ultrasound was abnormal, and in two, only the CA-125 was abnormal (46-91 U/ml). In only one subject undergoing surgery were both serial CA-125 levels (52-91 U/ml) and ultrasound abnormal. In 7 years of screening, one patient (0.3%) has been diagnosed with ovarian cancer (stage IA, grade 1 endometrioid adenocarcinoma). Overall, 31 (10%) subjects have completed BRCA testing. We conclude that despite screening results comparable to other studies, the detection of only one ovarian cancer over 7 years is lower than expected. Explanations for this observation are discussed. Despite the limitations of CA-125 and ultrasound, we continue to recommend these screening modalities for high-risk women. At the present time, they offer the best opportunity to detect ovarian cancers early. With increasing knowledge of BRCA testing, more women may benefit from this testing in assessing their personal risk.

Is serum ferritin an additional cardiovascular risk factor for all postmenopausal women?

BACKGROUND: Most of the studies on cardiovascular disease (CVD) risk factors in menopause have focused on serum lipid(lipoprotein) abnormalities and were conducted in populations which were not well controlled for several important influential factors. METHODS: Two homogenous groups of 30 apparently healthy Caucasian premenopausal women and 3-5 years postmenopausal women who were nonobese, nonsmoking and not using estrogen were compared in a well-controlled cross-sectional design. Fasting serum ferritin and plasma total homocysteine (tHcy) were evaluated concomitantly to classical serum lipid(lipoprotein) risk factors. Relationships between risk factors and the influence of other contributing variables such as diet and body weight were also examined. RESULTS: Serum total cholesterol (p < 0.01), low-density lipoproteins (LDL; p < 0.05) and triglycerides (p < 0.05) of postmenopausal women were greater than that of their menstruating counterparts, even though they ate a CVD-preventive diet, had similar body weight and body fat distribution. Their serum ferritin was almost 3-fold greater (p < 0.0001) but was still within normal limits, except for the 38.5% of postmenopausal women who exhibited values above the 80 mug/l limit that has been associated with sharp increases in the rate of heart disease in either gender. Serum ferritin was low in one third of the postmenopausal group (as low as in the premenopausal control group, whose dietary iron intake was slightly below the nutritional recommendation). The mean plasma tHcy of the postmenopausal group was almost twice as elevated (p < 0.0001). Both ferritin and tHcy were found to be linked to serum cholesterol. The correlation between tHcy and triglycerides was also significant. CONCLUSION: Early menopause is not associated with blood iron overload and CVD risk factor in an important proportion of women.