Polymorphisms in drug-metabolizing enzymes and steady-state exemestane concentration in postmenopausal patients with breast cancer.

Discovery of clinical and genetic predictors of exemestane pharmacokinetics was attempted in 246 postmenopausal patients with breast cancer enrolled on a prospective clinical study. A sample was collected 2 h after exemestane dosing at a 1- or 3-month study visit to measure drug concentration. The primary hypothesis was that patients carrying the low-activity CYP3A4*22 (rs35599367) single-nucleotide polymorphism (SNP) would have greater exemestane concentration. Additional SNPs in genes relevant to exemestane metabolism (CYP1A1/2, CYP1B1, CYP3A4, CYP4A11, AKR1C3/4, AKR7A2) were screened in secondary analyses and adjusted for clinical covariates. CYP3A4*22 was associated with a 54% greater exemestane concentration (P<0.01). Concentration was greater in patients who reported White race, had elevated aminotransferases, renal insufficiency, lower body mass index and had not received chemotherapy (all P<0.05), and CYP3A4*22 maintained significance after adjustment for covariates (P<0.01). These genetic and clinical predictors of exemestane concentration may be useful for treatment individualization in patients with breast cancer.

Aromatase inhibitor-induced modulation of breast density: clinical and genetic effects.

BACKGROUND: Change in breast density may predict outcome of women receiving adjuvant hormone therapy for breast cancer. We performed a prospective clinical trial to evaluate the impact of inherited variants in genes involved in oestrogen metabolism and signalling on change in mammographic percent density (MPD) with aromatase inhibitor (AI) therapy. METHODS: Postmenopausal women with breast cancer who were initiating adjuvant AI therapy were enrolled onto a multicentre, randomised clinical trial of exemestane vs letrozole, designed to identify associations between AI-induced change in MPD and single-nucleotide polymorphisms in candidate genes. Subjects underwent unilateral craniocaudal mammography before and following 24 months of treatment. RESULTS: Of the 503 enrolled subjects, 259 had both paired mammograms at baseline and following 24 months of treatment and evaluable DNA. We observed a statistically significant decrease in mean MPD from 17.1 to 15.1% (P<0.001), more pronounced in women with baseline MPD ≥20%. No AI-specific difference in change in MPD was identified. No significant associations between change in MPD and inherited genetic variants were observed. CONCLUSION: Subjects with higher baseline MPD had a greater average decrease in MPD with AI therapy. There does not appear to be a substantial effect of inherited variants in biologically selected candidate genes.

Predictors of recovery of ovarian function during aromatase inhibitor therapy.

BACKGROUND: Aromatase inhibitors (AIs) may cause a rise in estrogen levels due to ovarian function recovery in women with clinical chemotherapy-induced ovarian failure (CIOF). We carried out a prospective registry trial to identify predictors of ovarian function recovery during AI therapy. PATIENTS AND METHODS: Women with hormone receptor (HR)-positive breast cancer who remained amenorrheic and had hormonal levels consistent with ovarian failure after adjuvant chemotherapy were enrolled in a multi-institutional clinical trial of anastrozole. Subjects underwent frequent assessment using an ultrasensitive estradiol assay. Multivariable analysis was used to evaluate clinical and biochemical predictors of ovarian function recovery within 48 weeks. RESULTS: Recovery of ovarian function during AI therapy was observed in 13 of 45 (28.9%) assessable subjects after a median 2.1 months (range 0.6-11.9). Median age at chemotherapy initiation was statistically significantly different between those who regained ovarian function (43 years, range 40-51) and those who remained postmenopausal (49 years, range 44-52; P < 0.0001). CONCLUSIONS: A significant proportion of women with CIOF recover ovarian function during AI therapy, including a woman over age 50 at initiation of chemotherapy. Tamoxifen remains the standard of care for women with CIOF. If an AI is used, patients should be monitored frequently with high-quality estradiol assays. CLINICALTRIALS.GOV: NCT00555477.

Prospective study to assess fluid accumulation and tenosynovial changes in the aromatase inhibitor-induced musculoskeletal syndrome: 2-year follow-up data.

BACKGROUND: Aromatase inhibitors (AIs) frequently lead to the AI-induced musculoskeletal syndrome (AIMSS). Looking into its pathophysiology, 6 months of AI therapy thickens the tendon sheath with intra-articular fluid (IAF) retention and loss of grip strength. We here report 24-month follow-up data. PATIENTS AND METHODS: A prospective cohort study of 33 postmenopausal breast cancer patients received adjuvant endocrine therapy; 27 received an AI and 6 received tamoxifen. At baseline, 6 and 24 months patients had a rheumatologic examination, including a grip strength test, and magnetic resonance imaging of both hands and wrists. The primary end point was tenosynovial changes; secondary end points were changes in morning stiffness, grip strength and IAF. RESULTS: Twenty-three AI and 5 tamoxifen patients completed all investigations. Between month 6 and 24, IAF further increased in AI users (P = 0.04) but not in tamoxifen users, and grip strength further decreased in both groups. The worsened tenosynovial changes were strongly correlated with a decrease in grip strength. At 24 months, morning stiffness continued to be present in over a third of AI users. CONCLUSION: AIMSS represents a substantial problem in breast cancer patients. It is associated with tenosynovial changes, IAF retention, joint stiffness and loss of grip strength that do not improve with prolonged use.

A SNP in steroid receptor coactivator-1 disrupts a GSK3ß phosphorylation site and is associated with altered tamoxifen response in bone.

The coregulator steroid receptor coactivator (SRC)-1 increases transcriptional activity of the estrogen receptor (ER) in a number of tissues including bone. Mice deficient in SRC-1 are osteopenic and display skeletal resistance to estrogen treatment. SRC-1 is also known to modulate effects of selective ER modulators like tamoxifen. We hypothesized that single nucleotide polymorphisms (SNP) in SRC-1 may impact estrogen and/or tamoxifen action. Because the only nonsynonymous SNP in SRC-1 (rs1804645; P1272S) is located in an activation domain, it was examined for effects on estrogen and tamoxifen action. SRC-1 P1272S showed a decreased ability to coactivate ER compared with wild-type SRC-1 in multiple cell lines. Paradoxically, SRC-1 P1272S had an increased protein half-life. The Pro to Ser change disrupts a putative glycogen synthase 3 (GSK3)ß phosphorylation site that was confirmed by in vitro kinase assays. Finally, knockdown of GSK3ß increased SRC-1 protein levels, mimicking the loss of phosphorylation at P1272S. These findings are similar to the GSK3ß-mediated phospho-ubiquitin clock previously described for the related coregulator SRC-3. To assess the potential clinical significance of this SNP, we examined whether there was an association between SRC-1 P1272S and selective ER modulators response in bone. SRC-1 P1272S was associated with a decrease in hip and lumbar bone mineral density in women receiving tamoxifen treatment, supporting our in vitro findings for decreased ER coactivation. In summary, we have identified a functional genetic variant of SRC-1 with decreased activity, resulting, at least in part, from the loss of a GSK3ß phosphorylation site, which was also associated with decreased bone mineral density in tamoxifen-treated women.

Plasma letrozole concentrations in postmenopausal women with breast cancer are associated with CYP2A6 genetic variants, body mass index, and age.

The associations between plasma letrozole concentrations and CYP2A6 and CYP3A5 genetic variants were tested in the Exemestane and Letrozole Pharmacogenomics (ELPH) trial. ELPH is a multicenter, open-label prospective clinical trial in women randomly assigned (n≈250 in each arm) to receive 2 years of treatment with either oral letrozole (2.5 mg/day) or oral exemestane (25 mg/day). CYP2A6 and CYP3A showed effects on letrozole metabolism in vitro. DNA samples were genotyped for variants in the CYP2A6 and CYP3A5 genes. Plasma letrozole concentrations showed high interpatient variability (>10-fold) and were associated significantly with CYP2A6 genotypes (P<0.0001), body mass index (BMI) (P<0.0001), and age (P=0.0035). However, CYP3A5 genotypes showed no association with plasma letrozole concentrations. These data suggest that CYP2A6 is the principal clearance mechanism for letrozole in vivo. CYP2A6 metabolic status, along with BMI and age, may serve as a biomarker of the efficacy of letrozole treatment or a predictor of adverse effects.

Aromatase inhibitor-induced loss of grip strength is body mass index dependent: hypothesis-generating findings for its pathogenesis.

BACKGROUND: Our preliminary results showed that tenosynovial changes and decrease in grip strength are associated with the aromatase inhibitor-induced musculoskeletal syndrome (AIMSS). Here, we report the final results and assess the relationship between grip strength and body mass index (BMI). PATIENTS AND METHODS: We conducted a prospective study including postmenopausal early breast cancer patients receiving either an aromatase inhibitor (AI) or tamoxifen. Primary end point was change from baseline in tenosynovial abnormalities. Secondary end points were changes from baseline in morning stiffness, intra-articular fluid and grip strength and its association with BMI. RESULTS: After 6 months of therapy, 74% [95% confidence interval (CI) 51% to 89%] of AI-treated patients had worsened tenosynovial abnormalities, 56% (95% CI 34% to 75%) had increased intra-articular fluid, and 22% (95% CI 9% to 45%) had increased morning stiffness. Grip strength decreased 8% for the left hand (95% CI 2% to 21%) and 11% for the right (95% CI 4% to 17%). Regression analysis suggested that grip strength decreased more for subjects with high or with low BMI. CONCLUSIONS: AIMSS is characterized by tenosynovial changes, intra-articular fluid and morning stiffness. We hypothesize that the quadratic association between BMI and loss of grip strength reflects AI-induced changes on the endocrine control of the growth hormone insulin-like growth factor-I pathway.

Modeling hot flushes and quality of life in breast cancer survivors.

OBJECTIVES: To evaluate the relationships among measures of hot flushes, perceived hot flush interference, sleep disturbance, and measures of quality of life while controlling for potential covariates (patient and treatment variables). METHODS: Breast cancer survivors (n = 395) due to receive aromatase inhibitor therapy provided demographic information, physiological hot flush data via sternal skin conductance monitoring, hot flush frequency via written diary and electronic event marker, hot flush severity and bother via written diary, and questionnaire data via the Hot Flash Related Daily Interference Scale, Pittsburgh Sleep Quality Index, the EuroQOL, Hospital Anxiety and Depression Scale and the Center for Epidemiologic Studies Depression Scale. RESULTS: Confirmatory factor analysis supported a two-factor model for hot flush symptoms (frequency and severity). Although there was strong convergence among self-reported hot flush measures, there was a high degree of unexplained variance associated with physiological measures. This suggests that self-report and physiological measures do not overlap substantially. The structural model showed that greater hot flush frequency and severity were directly related to greater perceived interference with daily life activities. Greater perceived interference, in turn, directly predicted greater sleep disruption, which predicted lower perceived health state and more symptoms of anxiety and depression. CONCLUSIONS: Findings suggest hot flush interference may be the most appropriate single measure to include in clinical trials of vasomotor symptom therapies. Measuring and ameliorating patients' perceptions of hot flush interference with life activities and subjective sleep quality may be the most direct routes to improving quality of life.

Estrogen receptor genotypes, menopausal status, and the effects of tamoxifen on lipid levels: revised and updated results.

We previously reported that the ESR1 XbaI genotypes were associated with baseline and tamoxifen-induced serum lipid profiles. The analysis in that study was carried out by PCR followed by restriction-enzyme digestion. After reanalysis using more robust TaqMan assays, the findings related to ~10% of the genotypes for the ESR1 XbaI single-nucleotide polymorphism (SNP) were revised. For the other genotypes (i.e., ESR1 PvuII, ESR2, and CYP2D6), the results were nearly identical to those in the previous study. Upon reanalysis, previously reported associations between the ESR1 Xba1 genotypes and baseline triglyceride and low-density lipoprotein (LDL) cholesterol levels were no longer observed. Previously reported associations between the ESR1 XbaI genotypes and tamoxifen-induced changes in levels of total cholesterol, triglycerides, and high-density lipoprotein (HDL) cholesterol were also no longer observed. However, the following observations from the original report did not change: (i) the levels of circulating lipids are lower in women taking tamoxifen; (ii) there is an association between the ESR2-02 genotypes and changes in triglyceride levels; and (iii) neither ESR1 PvuII nor CYP2D6 is associated with any changes in serum lipid concentrations in patients receiving treatment with tamoxifen.

Inflammatory cytokines and aromatase inhibitor-associated musculoskeletal syndrome: a case-control study.

BACKGROUND: The aromatase inhibitor (AI)-associated musculoskeletal syndrome (AIMSS) occurs in approximately 50% of AI-treated patients. Inflammatory mediators are associated with oestrogen signalling and may change with oestrogen depletion. We hypothesised that AIMSS may be associated with changes in circulating inflammatory markers. METHODS: Patients with breast cancer were enrolled in a trial of adjuvant AI therapy. Changes in pain and function during therapy were assessed prospectively. We selected 30 cases with AIMSS and 22 controls without AIMSS, matched for demographics and prior therapy. Serum samples collected at baseline and during treatment were assayed for multiple inflammatory cytokines and lipid mediators using multiplex assays. RESULTS: Before AI therapy, mean serum concentrations of 6 of 36 assayed factors were statistically significantly lower in cases than controls (all P<0.003). No statistically significant changes during AI therapy relative to pre-treatment were observed between cases and controls for any of the inflammatory markers tested. CONCLUSION: AIMSS is probably not associated with a systemic inflammatory response. Pre-treatment cytokine levels may predict for development of AIMSS.

Lack of association between oestrogen receptor polymorphisms and change in bone mineral density with tamoxifen therapy.

BACKGROUND: Tamoxifen, a selective oestrogen receptor (ER) modulator, increases bone mineral density (BMD) in postmenopausal women and decreases BMD in premenopausal women. We hypothesised that inherited variants in candidate genes involved in oestrogen signalling and tamoxifen metabolism might be associated with tamoxifen effects in bone. METHODS: A total of 297 women who were initiating tamoxifen therapy were enrolled in a prospective multicentre clinical trial. Lumbar spine and total hip BMD values were measured using dual-energy X-ray absorptiometry (DXA) at baseline and after 12 months of tamoxifen therapy. Single-nucleotide polymorphisms (SNPs) in ESR1, ESR2, and CYP2D6 were tested for associations in the context of menopausal status and previous chemotherapy, with a mean percentage change in BMD over 12 months. RESULTS: The percentage increase in BMD was greater in postmenopausal women and in those patients who had been treated with chemotherapy. No significant associations between tested SNPs and either baseline BMD or change in BMD with 1 year of tamoxifen therapy were detected. CONCLUSION: The evaluated SNPs in ESR and CYP2D6 do not seem to influence BMD in tamoxifen-treated subjects.

Cytochrome P450 2D6 activity predicts discontinuation of tamoxifen therapy in breast cancer patients.

The selective estrogen receptor modulator tamoxifen is routinely used for treatment and prevention of estrogen-receptor-positive breast cancer. Studies of tamoxifen adherence suggest that over half of patients discontinue treatment before the recommended 5 years. We hypothesized that polymorphisms in CYP2D6, the enzyme responsible for tamoxifen activation, predict for tamoxifen discontinuation. Tamoxifen-treated women (n=297) were genotyped for CYP2D6 variants and assigned a 'score' based on predicted allele activities from 0 (no activity) to 2 (high activity). Correlation between CYP2D6 score and discontinuation rates at 4 months was tested. We observed a strong nonlinear correlation between higher CYP2D6 score and increased rates of discontinuation (r(2)=0.935, P=0.018). These data suggest that presence of active CYP2D6 alleles may predict for higher likelihood of tamoxifen discontinuation. Therefore, patients who may be most likely to benefit from tamoxifen may paradoxically be most likely to discontinue treatment prematurely.

Onchocerciasis in a nonendemic population: clinical and immunologic assessment before treatment and at the time of presumed cure.

Although suppressive therapy for onchocerciasis with intermittent ivermectin prevents the development of pathology in endemic populations, the clinical and immunologic effects of therapy in the absence of continued exposure are unknown. To address this question, 14 patients treated with ivermectin for onchocerciasis acquired >10 years ago during temporary residence in Africa were reevaluated. None had evidence of continued infection or pathology at follow-up. Although eosinophilia, serum IgE, and antifilarial antibody levels decreased after ivermectin therapy, none of these parameters was useful in predicting the resolution of symptoms in infected patients. Peripheral blood mononuclear cells isolated from patients at follow-up were more responsive to parasite antigen in vitro, which is as assessed by proliferation and production of interferon-gamma and interleukin (IL)-5. In contrast, antigen-induced levels of IL-10 were significantly decreased at follow-up, consistent with diminished down-regulatory factors rather than a switch from type 2 to type 1 immune responses.

Genes for Tfb2, Tfb3, and Tfb4 subunits of yeast transcription/repair factor IIH. Homology to human cyclin-dependent kinase activating kinase and IIH subunits.

Genes for the Tfb2, Tfb3, and Tfb4 subunits of yeast RNA polymerase transcription factor IIH (TFIIH) are described. All three genes are essential for cell viability, and antibodies against Tfb3 specifically inhibit transcription in vitro. A C-terminal deletion of Tfb2 caused a defect in nucleotide excision repair, as shown by UV sensitivity of the mutant strain and loss of nucleotide excision repair activity in cell extracts (restored by the addition of purified TFIIH). An interaction between Tfb3 and the Kin28 subunit of TFIIH was detected by the two-hybrid approach, consistent with a role for Tfb3 in linking kinase and core domains of the factor. The deduced amino acid sequence of Tfb2 is similar to that of the 52-kDa subunit of human TFIIH, while Tfb3 is identified as a RING finger protein homologous to the 36-kDa subunit of murine CAK (cyclin-dependent kinase activating kinase) and to the 32-kDa subunit of human TFIIH. Tfb4 is homologous to p34 of human TFIIH and is identified as the weakly associated 37-kDa subunit of the yeast factor. These and other findings reveal a one-to-one correspondence and high degree of sequence similarity between the entire set of yeast and human TFIIH polypeptides.

A yeast transcriptional stimulatory protein similar to human PC4.

A yeast protein has been identified that stimulates basal transcription by RNA polymerase II, binds both single- and double-stranded DNA, and interacts with both a general transcription factor and a transcriptional activator. Phosphorylation appears to regulate these interactions. The gene for the transcriptional stimulatory protein, termed TSP1, was cloned and found to be dispensable for yeast cell viability. The deduced amino acid sequence is similar to that of mammalian coactivator protein PC4.

TFG/TAF30/ANC1, a component of the yeast SWI/SNF complex that is similar to the leukemogenic proteins ENL and AF-9.

The SWI1/ADR6, SWI2/SNF2, SWI3, SNF5, and SNF6 gene products are all required for proper transcriptional control of many genes in the yeast Saccharomyces cerevisiae. Genetic studies indicated that these gene products might form a multiprotein SWI/SNF complex important for chromatin transitions preceding transcription from RNA polymerase II promoters. Biochemical studies identified a SWI/SNF complex containing these and at least six additional polypeptides. Here we show that the 29-kDa component of the SWI/SNF complex is identical to TFG3/TAF30/ANC1. Thus, a component of the SWI/SNF complex is also a member of the TFIIF and TFIID transcription complexes. TFG3 interacted with the SNF5 component of the SWI/SNF complex in protein interaction blots. TFG3 is significantly similar to ENL and AF-9, two proteins implicated in human acute leukemia. These results suggest that ENL and AF-9 proteins interact with the SNF5 component of the human SWI/SNF complex and raise the possibility that the SWI/SNF complex is involved in acute leukemia.

TFIIF-TAF-RNA polymerase II connection.

RNA polymerase transcription factor IIF (TFIIF) is required for initiation at most, if not all, polymerase II promoters. We report here the cloning and sequencing of genes for a yeast protein that is the homolog of mammalian TFIIF. This yeast protein, previously designated factor g, contains two subunits, Tfg1 and Tfg2, both of which are required for transcription, essential for yeast cell viability, and whose sequences exhibit significant similarity to those of the mammalian factor. The yeast protein also contains a third subunit, Tfg3, which is less tightly associated and at most stimulatory to transcription, dispensable for cell viability, and has no known counterpart in mammalian TFIIF. Remarkably, the TFG3 gene encodes yeast TAF30, and furthermore, is identical to ANC1, a gene implicated in actin cytoskeletal function in vivo (Welch and Drubin 1994). Tfg3 is also a component of the recently described mediator complex (Kim et al. 1994), whose interaction with the carboxy-terminal repeat domain of RNA polymerase II enables transcriptional activation. Deletion of TFG3 results in diminished transcription in vivo.

Relationship of CDK-activating kinase and RNA polymerase II CTD kinase TFIIH/TFIIK.

KIN28, a member of the p34cdc2/CDC28 family of protein kinases, is identified as a subunit of yeast RNA polymerase transcription factor IIH (TFIIH) on the basis of sequence determination, immunological reactivity, and copurification. KIN28 is, moreover, one of three subunits of TFIIK, a subassembly of TFIIH with protein kinase activity directed toward the C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II. Itself a phosphoprotein, KIN28 interacts specifically with the two largest subunits of RNA polymerase II. Previous work of others points to two further associations: KIN28 interacts in vivo with the cyclin CCL1, and KIN28 and CCL1 are homologous to human MO15 and cyclin H, which form the cyclin-dependent kinase-activating kinase (CAK). We show that human CAK possesses the CTD kinase activity characteristic of TFIIH.

Yeast TFIIE. Cloning, expression, and homology to vertebrate proteins.

Genes encoding both the 66- and the 43-kDa subunits of yeast RNA polymerase II initiation factor a, designated TFA1 and TFA2, have been isolated. Both genes are essential for cell viability. The bacterially expressed gene products could replace factor a in transcription in vitro, and both recombinant subunits were required for activity. The deduced amino acid sequences of the TFA1 and TFA2 gene products were homologous to those of the large and small subunits of human TFIIE, respectively, identifying factor a as the yeast homolog of TFIIE.

A versatile synthetic peptide-based ELISA for identifying antibody epitopes.

A simple, versatile and very inexpensive procedure for cross-linking synthetic peptides to the polystyrene surfaces of micro-well assay plates for use in ELISA was developed. The method is based on the use of poly-L-lysine (PLL) as the anchor protein for synthetic peptides which were then easily and covalently linked to the PLL using glutaraldehyde. The synthetic peptides used for the study were based on the amino acid sequence of the equine infectious anemia virus (EIAV) envelope sequence and evaluated as antigens in an ELISA designed to detect antibodies in serum of EIAV-infected horses and ponies. The ELISA using cross-linked peptides proved to be significantly more sensitive when compared to assays where passively coated peptides were used. In one instance, a peptide was identified that was not recognized by any of our antisera and appeared not to bind to the assay plates. However, once this peptide was cross-linked to the assay plate it proved to be very useful for detecting EIAV-specific antibodies. This cross-linking approach functioned equally well with peptides of various charges and sizes and did not appear to alter epitopes contained in the peptides.