Effect of CYP2D6 polymorphisms on breast cancer recurrence.

BACKGROUND: Previous studies evaluating the effect of cytochrome P450 2D6 (CYP2D6) polymorphisms on outcomes of adjuvant tamoxifen therapy have been conflicting due to differences in study design, concomitant medications that alter CYP2D6 metabolism, and tamoxifen adherence. METHODS: The authors performed CYP2D6 genotyping from whole blood and fresh frozen tumor samples (n 106) in patients at The University of Texas MD Anderson Cancer Center who were receiving, or had received, tamoxifen as adjuvant therapy for early breast cancer (EBC), using the AmpliChip CYP450 Test. Each patient's medical history was assessed for drugs that affected CYP2D6. Fifty-five patients who had experienced breast cancer recurrence were matched (by date of diagnosis, menopausal status, clinical stage [TNM Staging System], and race) to patients without recurrence. RESULTS: Unadjusted for other patient characteristics, the odds ratio for disease recurrence associated with CYP2D6 functional status was 1.0 (95% confidence interval, 0.35-2.85). After adjustment for stage, CYP2D6 inhibitors (moderate or strong vs none), and follow-up time, no significant association was found between CYP2D6 genotype and breast cancer recurrence in patients who were treated with adjuvant tamoxifen for EBC. CONCLUSIONS: This case-control study demonstrated no significant effect of CYP2D6 genotype on risk of recurrence in breast cancer patients who received adjuvant tamoxifen therapy.

Comprehensive CYP2D6 genotype and adherence affect outcome in breast cancer patients treated with tamoxifen monotherapy.

The association between CYP2D6 genotype and outcome in breast cancer patients treated with adjuvant tamoxifen remains controversial. We assessed the influence of comprehensive versus limited CYP2D6 genotype in the context of tamoxifen adherence and co-medication in a large cohort of 618 patients. Genotyping of 33 CYP2D6 alleles used two archival cohorts from tamoxifen-treated women with invasive breast cancer (Dundee, n = 391; Manchester, n = 227). Estimates for recurrence-free survival (RFS) were calculated based on inferred CYP2D6 phenotypes using Kaplan-Meier and Cox proportional hazard models, adjusted for nodal status and tumour size. Patients with at least one reduced function CYP2D6 allele (60%) or no functional alleles (6%) had a non-significant trend for worse RFS: hazard ratio (HR) 1.52 (CI 0.98-2.36, P = 0.06). For post-menopausal women on tamoxifen monotherapy, the HR for recurrence in patients with reduced functional alleles was 1.96 (CI 1.05-3.66, P = 0.036). However, RFS analysis limited to four common CYP2D6 allelic variants was no longer significant (P = 0.39). The effect of CYP2D6 genotype was increased by adjusting for adherence to tamoxifen therapy, but not significantly changed when adjusted for co-administration of potent inhibitors of CYP2D6. Comprehensive genotyping of CYP2D6 and adherence to tamoxifen therapy may be useful to identify breast cancer patients most likely to benefit from adjuvant tamoxifen.

Author Correction: Ultra-high throughput single-cell analysis of proteins and RNAs by split-pool synthesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Ultra-high throughput single-cell analysis of proteins and RNAs by split-pool synthesis.

Single-cell omics provide insight into cellular heterogeneity and function. Recent technological advances have accelerated single-cell analyses, but workflows remain expensive and complex. We present a method enabling simultaneous, ultra-high throughput single-cell barcoding of millions of cells for targeted analysis of proteins and RNAs. Quantum barcoding (QBC) avoids isolation of single cells by building cell-specific oligo barcodes dynamically within each cell. With minimal instrumentation (four 96-well plates and a multichannel pipette), cell-specific codes are added to each tagged molecule within cells through sequential rounds of classical split-pool synthesis. Here we show the utility of this technology in mouse and human model systems for as many as 50 antibodies to targeted proteins and, separately, >70 targeted RNA regions. We demonstrate that this method can be applied to multi-modal protein and RNA analyses. It can be scaled by expansion of the split-pool process and effectively renders sequencing instruments as versatile multi-parameter flow cytometers.

DNA microarray technology in the clinical environment: the AmpliChip CYP450 test for CYP2D6 and CYP2C19 genotyping.

INTRODUCTION: An important technological advance in genetic testing is the DNA microarray, which allows for the simultaneous testing of thousands of DNA sequences. The AmpliChip CYP450 Test employs this microarray technology for cytochrome P450 (CYP) 2D6 and CYP2C19 genotyping. Isoenzymes encoded by these genes are responsible for the metabolism of many widely prescribed drugs. The objectives of this study were to identify CYP2D6 and CYP2C19 alleles and phenotypes in a psychiatric patient population in Kentucky, and to describe practical issues associated with DNA microarray technology. METHODS: A total of 4,532 psychiatric patients were recruited from three state hospitals in Kentucky. Whole blood, buccal swabs, or saliva samples were genotyped with the AmpliChip CYP450 Test to derive a predicted phenotype. RESULTS: In this cohort, the overall prevalence of CYP2D6 poor metabolizers was 7.6% (95% CI 7%, 8.3%), 8.2% in the Caucasians (95% CI 7.4%, 9.%) and 1.8% in the African Americans (95% CI 0.9%, 3.5%). The overall prevalence of CYP2D6 ultrarapid metabolizers was 1.5% (95% CI 1.2%, 1.9%), 1.5% in the Caucasians (95% CI 1.1%, 1.9%) and 2.0% in the African Americans (95% CI 1.1%, 3.7%). The overall prevalence of CYP2C19 poor metabolizers was 2.0% (95% CI 1.8%, 2.7%), 2.2% in Caucasians (95% CI 1.6%, 2.5%) and 4.0% in African Americans (95% CI 2.6%, 6.1%). CONCLUSION: We also propose a numeric system for expression of CYP2D6 and CYP2C19 enzyme activity to aid clinicians in determining treatment strategy for patients receiving therapeutics that are metabolized by the CYP2D6 or CYP2C19 gene products.

First identification of a recombinant form of hepatitis C virus in Austrian patients by full-genome next generation sequencing.

Hepatitis C virus (HCV) intergenotypic recombinant forms have been reported for various HCV genotypes/subtypes in several countries worldwide. In a recent study, four patients living in Austria had been identified to be possibly infected with a recombinant HCV strain. To clarify results and determine the point of recombination, full-genome next-generation sequencing using the Illumina MiSeq v2 300 cycle kit (Illumina, San Diego, CA, USA) was performed in the present study. Samples of all of the patients contained the recombinant HCV strain 2k/1b. The point of recombination was found to be within the HCV NS2 gene between nucleotide positions 3189-3200 based on H77 numbering. While three of four patients were male and had migration background from Chechnya (n = 2) and Azerbaijan (n = 1), the forth patient was a female born in Austria. Three of the four patients including the female had intravenous drug abuse as a risk factor for HCV transmission. While sequencing techniques are limited to a few specialized laboratories, a genotyping assay that uses both ends of the HCV genome should be employed to identify patients infected with a recombinant HCV strain. The correct identification of recombinant strains also has an impact considering the tailored choice of anti-HCV treatment.

Analytical performance of a real-time PCR-based assay for V600 mutations in the BRAF gene, used as the companion diagnostic test for the novel BRAF inhibitor vemurafenib in metastatic melanoma.

Melanomas frequently harbor BRAFV600 mutations. Vemurafenib (RG7204/PLX4032), a small-molecule inhibitor of mutant BRAF, has shown striking clinical efficacy in BRAFV600 mutant melanoma, creating the need for a well-validated companion diagnostic to select patients for treatment. We describe analytic performance characteristics of the cobas 4800 BRAF V600 Mutation Test, the test used to select patients for the pivotal vemurafenib trials. This real-time polymerase chain reaction assay was designed to detect the V600E (1799T>A) mutation DNA from formalin-fixed paraffin-embedded tissue samples. Sensitivity was assessed using blends of cell lines or tumor DNA, and tumor specimens with low levels of mutant alleles, as determined by 454 sequencing (a quantitative next-generation pyrosequencing method). A >96% hit rate was obtained across all specimen types with 5% mutant alleles at a DNA input of 125 ng, an amount readily obtained from one 5-µm section. The cobas test showed a higher sensitivity and specificity than direct bidirectional sequencing in a panel of 219 melanoma specimens. Cross reactivity with V600K and V600D was observed. Repeated testing of 5 specimens by 2 operators, using different instruments and reagent lots, yielded correct calls in 158/160 tests (98.8%). A set of 26 highly pigmented samples were identified that gave invalid test results. A simple 1:2 dilution resulted in a valid test result of 76% in such cases. The cobas test is a reproducible assay that detects some non-V600E mutations and is more accurate than direct sequencing in detecting BRAFV600E.