Recommendations on qPCR/ddPCR assay validation by GCC.

Gene therapy, cell therapy and vaccine research have led to an increased use of qPCR/ddPCR in bioanalytical laboratories. CROs are progressively undertaking the development and validation of qPCR and ddPCR assays. Currently, however, there is limited regulatory guidance for the use of qPCR and a complete lack of any regulatory guidelines for the use of the newer ddPCR to support regulated bioanalysis. Hence, the Global CRO Council in Bioanalysis (GCC) has issued this White Paper to provide; 1) a consensus on the different validation parameters required to support qPCR/ddPCR assays; 2) a harmonized approach to their validation and 3) a consistent development of standard operating procedures (SOPs) for all the bioanalytical laboratories using these techniques.

Recommendations on ELISpot assay validation by the GCC.

Gene therapy, cell therapy and vaccine research have led to an increased need to perform cellular immunity testing in a regulated environment to ensure the safety and efficacy of these treatments. The most common method for the measurement of cellular immunity has been Enzyme-Linked Immunospot assays. However, there is a lack of regulatory guidance available discussing the recommendations for developing and validating these types of assays. Hence, the Global CRO Council has issued this white paper to provide a consensus on the different validation parameters required to support Enzyme-Linked Immunospot assays and a harmonized and consistent approach to Enzyme-Linked Immunospot validation among contract research organizations.

Prognostic impact of genetic variants of CYP19A1 and UGT2B17 in a randomized trial for endocrine-responsive postmenopausal breast cancer.

Polymorphisms of genes involved in estrogen synthesis have been linked to breast cancer risk, prognosis, and treatment response. We investigated the prognostic impact of a deletion spanning the entire UGT2B17 gene (UGT2B17*2) and genetic variants of the aromatase CYP19A1 and estrogen receptor α (ESR1) in 125 postmenopausal women with ER-positive breast cancer enrolled in a randomized pre-surgical trial. The UGT2B17*2 was estimated by copy number variation assays and the CYP19A1 rs10046/rs4646 and ESR1 rs2077647/rs2234693/rs9340799 by TaqMan allelic discrimination assays. Serum exemestane/17-hydroxy exemestane were determined by MS and estrone (E1)/estradiol (E2)/ by GC-MS/MS. The association of genetic polymorphisms with "any event" was assessed by the Cox proportional hazards models adjusted for confounders. The UGT2B17*2 was associated with higher levels of 17-hydroxy exemestane (P = 0.04) and better prognosis (HR = 0.45; 95% CI: 0.20-1.01; P = 0.05) compared with homozygote UGT2B17 wt. The CYP19A1 rs10046 A and rs4646 C alleles were associated with higher estrogen levels: rs10046 AA vs. AG/GG genotypes had median E1 of 35.9 vs. 27.4 pg/mL (P = 0.05) and E2 of 7.57 vs. 3.9 pg/mL (P < 0.004). After a median follow-up of 7 years, women carrying the "low estrogen" alleles rs10046 G and rs4646 A had a better prognosis compared with homozygote wt for both polymorphisms (HR = 0.40; 95% CI: 0.17-0.93; P = 0.03). Our analysis points to an impact of UGT2B17 and CYP19A1 in postmenopausal endocrine responsive breast cancer. Carriers of UGT2B17*2 and CYP19A1 low estrogen variants may have better prognosis, supporting studies addressing the role of these polymorphisms in optimizing endocrine therapy. Trial registration: http://www.isrctn.com/ISRCTN86894592.

SLCO1B1 polymorphisms and plasma estrone conjugates in postmenopausal women with ER+ breast cancer: genome-wide association studies of the estrone pathway.

BACKGROUND: Estrone (E1), the major circulating estrogen in postmenopausal women, promotes estrogen-receptor positive (ER+) breast tumor growth and proliferation. Two major reactions contribute to E1 plasma concentrations, aromatase (CYP19A1) catalyzed E1 synthesis from androstenedione and steroid sulfatase (STS) catalyzed hydrolysis of estrone conjugates (E1Cs). E1Cs have been associated with breast cancer risk and may contribute to tumor progression since STS is expressed in breast cancer where its activity exceeds that of aromatase. METHODS: We performed genome-wide association studies (GWAS) to identify SNPs associated with variation in plasma concentrations of E1Cs, E1, and androstenedione in 774 postmenopausal women with resected early-stage ER+ breast cancer. Hormone concentrations were measured prior to aromatase inhibitor therapy. RESULTS: Multiple SNPs in SLCO1B1, a gene encoding a hepatic influx transporter, displayed genome-wide significant associations with E1C plasma concentrations and with the E1C/E1 ratio. The top SNP for E1C concentrations, rs4149056 (p = 3.74E-11), was a missense variant that results in reduced transporter activity. Patients homozygous for the variant allele had significantly higher average E1C plasma concentrations than did other patients. Furthermore, three other SLCO1B1 SNPs, not in LD with rs4149056, were associated with both E1C concentrations and the E1C/E1 ratio and were cis-eQTLs for SLCO1B3. GWAS signals of suggestive significance were also observed for E1, androstenedione, and the E1/androstenedione ratio. CONCLUSION: These results suggest a mechanism for genetic variation in E1C plasma concentrations as well as possible SNP biomarkers to identify ER+ breast cancer patients for whom STS inhibitors might be of clinical value.

The 10th GCC Closed Forum: rejected data, GCP in bioanalysis, extract stability, BAV, processed batch acceptance, matrix stability, critical reagents, ELN and data integrity and counteracting fraud.

The 10th Global CRO Council (GCC) Closed Forum was held in Orlando, FL, USA on 18 April 2016. In attendance were decision makers from international CRO member companies offering bioanalytical services. The objective of this meeting was for GCC members to meet and discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at this closed forum included reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, biomarker assay validation, processed batch acceptance criteria, electronic laboratory notebooks and data integrity, Health Canada's Notice regarding replicates in matrix stability evaluations, critical reagents and regulatory approaches to counteract fraud. In order to obtain the pharma perspectives on some of these topics, the first joint CRO-Pharma Scientific Interchange Meeting was held on 12 November 2016, in Denver, Colorado, USA. The five topics discussed at this Interchange meeting were reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, processed batch acceptance criteria and electronic laboratory notebooks and data integrity. The conclusions from the discussions of these topics at both meetings are included in this report.

Bioanalytical method transfer considerations of chromatographic-based assays.

Bioanalysis is an important part of the modern drug development process. The business practice of outsourcing and transferring bioanalytical methods from laboratory to laboratory has increasingly become a crucial strategy for successful and efficient delivery of therapies to the market. This chapter discusses important considerations when transferring various types of chromatographic-based assays in today's pharmaceutical research and development environment.

The importance of chromatographic resolution when analyzing steroid biomarkers.

The analyses of endogenous substances as biomarkers presents challenges that are distinctly different from the analyses of drugs or other xenobiotic substances. This is particularly true for estrogens. When no matrix is available which does not contain some level of the biomarker of interest, specificity cannot be demonstrated. Therefore it cannot be known whether the analyte signal includes a response from another substance. This uncertainty is increased by the fact that biomarkers are often created as part of a complex biosynthetic process that also creates a large number of substances with very similar structures and sometimes the same mass. Because of this, the two most powerful selectivity tools in the analysis of drugs, mass selective detection and MS/MS, are often rendered ineffective. The only remaining selectivity tool is chromatography and as will be demonstrated these separations can be very challenging. Failure to achieve specificity is perhaps the leading cause for inaccuracy of biomarker data and inter-laboratory variability.

Estrogens and their precursors in postmenopausal women with early breast cancer receiving anastrozole.

PURPOSE: We determined hormone concentrations (estradiol [E2], estrone [E1], estrone conjugates [E1-C], androstenedione [A], testosterone [T]) before and on anastrozole therapy where we also determined plasma concentrations of anastrozole and its metabolites. EXPERIMENTAL: Postmenopausal women who were to receive adjuvant anastrozole for resected early breast cancer were studied. Pretreatment, blood samples were obtained for the acquisition of DNA and for plasma hormone measurements (E2, E1, E1-C, A, and T). A second blood draw was obtained at least 4 weeks after starting anastrozole for hormone, anastrozole and metabolite measurements. For hormone assays, a validated bioanalytical method using gas chromatography negative ionization tandem mass spectrometry was used. Anastrozole and metabolite assays involved extraction of plasma followed by LC/MS/MS assays. RESULTS: 649 patients were evaluable. Pretreatment and during anastrozole, there was large inter-individual variability in E2, E1, and E1-C as well as anastrozole and anastrozole metabolite concentrations. E2 and E1 concentrations were below the lower limits of quantitation in 79% and 70%, respectively, of patients on anastrozole therapy, but those with reliable concentrations had a broad range (0.627-234.0 pg/mL, 1.562-183.2 pg/mL, respectively). Considering E2, 8.9% had the same or higher concentration relative to baseline while on anastrozole, documented by the presence of drug. CONCLUSIONS: We demonstrated large inter-individual variability in anastrozole and anastrozole metabolite concentrations as well as E1, E2, E1-C, A, and T concentrations before and while on anastrozole. These findings suggest that the standard 1mg daily dose of anastrozole is not optimal for a substantial proportion of women with breast cancer.

Validation of a method for quantifying enzalutamide and its major metabolites in human plasma by LC-MS/MS.

BACKGROUND: Enzalutamide is an androgen receptor inhibitor that targets multiple steps in the androgen receptor signaling pathway. Oral enzalutamide was recently approved by the US FDA and health authorities in other regions for the treatment of patients with metastatic castration-resistant prostate cancer who previously received docetaxel. The objective of this study was to validate a method for quantification of enzalutamide and its two major metabolites in human plasma. RESULTS: The analytes were extracted from plasma by an LLE procedure, separated by reversed phase HPLC and detected by MS/MS in positive mode ESI. The quantitation range was 0.0200-50.0 µg/ml. CONCLUSION: The method proved to be rapid and simple, and met FDA validation criteria.

TSPYL5 SNPs: association with plasma estradiol concentrations and aromatase expression.

We performed a discovery genome-wide association study to identify genetic factors associated with variation in plasma estradiol (E2) concentrations using DNA from 772 postmenopausal women with estrogen receptor (ER)-positive breast cancer prior to the initiation of aromatase inhibitor therapy. Association analyses showed that the single nucleotide polymorphisms (SNP) (rs1864729) with the lowest P value (P = 3.49E-08), mapped to chromosome 8 near TSPYL5. We also identified 17 imputed SNPs in or near TSPYL5 with P values < 5E-08, one of which, rs2583506, created a functional estrogen response element. We then used a panel of lymphoblastoid cell lines (LCLs) stably transfected with ERα with known genome-wide SNP genotypes to demonstrate that TSPYL5 expression increased after E2 exposure of cells heterozygous for variant TSPYL5 SNP genotypes, but not in those homozygous for wild-type alleles. TSPYL5 knockdown decreased, and overexpression increased aromatase (CYP19A1) expression in MCF-7 cells, LCLs, and adipocytes through the skin/adipose (I.4) promoter. Chromatin immunoprecipitation assay showed that TSPYL5 bound to the CYP19A1 I.4 promoter. A putative TSPYL5 binding motif was identified in 43 genes, and TSPYL5 appeared to function as a transcription factor for most of those genes. In summary, genome-wide significant SNPs in TSPYL5 were associated with elevated plasma E2 in postmenopausal breast cancer patients. SNP rs2583506 created a functional estrogen response element, and LCLs with variant SNP genotypes displayed increased E2-dependent TSPYL5 expression. TSPYL5 induced CYP19A1 expression and that of many other genes. These studies have revealed a novel mechanism for regulating aromatase expression and plasma E2 concentrations in postmenopausal women with ER(+) breast cancer.

Variation in anastrozole metabolism and pharmacodynamics in women with early breast cancer.

Aromatase inhibitors play a prominent role in the management of postmenopausal women with endocrine-sensitive breast cancer, but there is large variability in both efficacy and tolerability. The purpose of our study was to define interindividual variation in anastrozole metabolism and pharmacodynamics among patients treated with the approved daily dose of 1 mg in a standard practice setting as adjuvant therapy for resected early breast cancer. This study was performed in 191 women in whom pretreatment and during anastrozole plasma concentrations of estrone (E1), estradiol (E2), estrone conjugates, androstenedione, and testosterone were determined and correlated with plasma concentrations of anastrozole and anastrozole metabolites. There were large interindividual variations in plasma anastrozole and anastrozole metabolite concentrations, as well as pretreatment and postdrug plasma E1, E2, and E1 conjugate and estrogen precursor (androstenedione and testosterone) concentrations. E1 and E2 concentrations were below the lower limit of quantitation (LLQ) in most patients after anastrozole therapy (83% for both), but those with detectable concentrations had a broad range (1.58-45.2 and 0.635-97.0 pg/mL, respectively). E1 conjugates after anastrozole therapy were above the LLQ in most patients (93%), with wide interpatient variability (3.50-2,990 pg/mL). Two patients seemed to extensively metabolize anastrozole and failed to display substantial decreases in estrogens. Acknowledging the potential factor of variable compliance, our results showed large interindividual variation in anastrozole metabolism and its effect on circulating estrogens in postmenopausal patients. These findings may have implications with regard to efficacy and adverse events and may indicate the need to "individualize" therapy with this drug.

Safety and pharmacokinetics of sirolimus-eluting stents in the canine cerebral vasculature: 180 day assessment.

OBJECTIVE: We evaluated local and systemic pharmacokinetics and pharmacodynamics of sirolimus-eluting stents (SES) in canine cerebral vessels. METHODS: SES (1.5 x 8 mm, 79 microg/479 microg sirolimus) and control stents (1.5 x 8 mm stainless steel with or without polymer) were implanted in canine basilar and ventral spinal arteries. Animals were sacrificed for local pharmacokinetic (36 animals at 1, 3, 8, 30, 90, 180 days) and pharmacodynamic (60 animals at 3, 30, 90, 180 days) assessment. RESULTS: Postrecovery adverse clinical events were not serious, requiring no unscheduled treatment. Histologically, brain and spinal cord sections revealed scattered microinfarcts and minimal gliosis consistent with postprocedure changes in all four stent-treatment groups. All stented vessels at all time points demonstrated good luminal patency with low injury and inflammation scores and no thrombosis of either stented or branch arteries. Endothelialization was complete in all stent groups by 30 days. Intimal smooth muscle cell scores were reduced in both SES groups at 30, 90, and 180 days. Systemic sirolimus levels peaked between 1 and 7 hours postimplant (maximum concentration, 1.2 +/- 1.47, 79 microg; 4.5 +/- 1.23 ng/ml, 479 microg), then declined rapidly to 1 ng/ml or less by 96 hours. Peak local tissue sirolimus levels were 41.5 ng/mg (79 microg) and 65 ng/mg (479 microg). CONCLUSION: SES in canine cerebral vessels were associated with good luminal patency to 180 days, with complete endothelialization and no evidence of acute thrombosis. This model has shown that SES deployed within the brain do not cause neurotoxicity during a 180-day time course, even when exaggerated doses are used. The findings support the contention that SES are safe to use and maintain patency in cerebral vessels.

Sirolimus PK trial: a pharmacokinetic study of the sirolimus-eluting Bx velocity stent in patients with de novo coronary lesions.

This study was conducted to assess the systemic drug release and distribution of sirolimus-eluting stents. Early results with sirolimus-eluting stents have demonstrated a favorable outcome for reducing restenosis post coronary intervention. However, the clinical systemic pharmacokinetics of sirolimus released from these stents has not been investigated. Sirolimus-eluting stents (150-178 mcg/18 mm stent) were implanted in 19 patients with coronary artery disease using standard techniques. Blood samples were obtained at multiple times to determine the kinetics of sirolimus release and elimination. Non-compartmental analysis showed that the maximum blood concentration of sirolimus occurred between 3 and 4 hr after implantation, with a peak concentration of 0.57 +/- 0.12 ng/mL (mean +/- SD) and 1.05 +/- 0.39 ng/mL in patients receiving one or two stents, respectively. Terminal-phase elimination half-life was independent of the number of stents and averaged at 213 hr, a value longer than that seen in patients following oral dosing. The apparent clearance was 1.46 +/- 0.45 L/hr with an apparent volume of distribution in the terminal phase of 407 +/- 111 L (data for both stent doses pooled). Minimal measurable blood levels were detectable at 7 days. Peak whole blood level following sirolimus stent implantation in humans is proportional to the number of stents implanted. The prolonged terminal half-life may reflect kinetics of blood clearance combined with continued drug elution and secondary local tissue release.