Evaluation of the Association of Polymorphisms With Palbociclib-Induced Neutropenia: Pharmacogenetic Analysis of PALOMA-2/-3.

BACKGROUND: The most frequently reported treatment-related adverse event in clinical trials with the cyclin-dependent kinase 4/6 (CDK4/6) inhibitor palbociclib is neutropenia. Allelic variants in ABCB1 and ERCC1 might be associated with early occurrence (i.e., end of week 2 treatment) of grade 3/4 neutropenia. Pharmacogenetic analyses were performed to uncover associations between single nucleotide polymorphisms (SNPs) in these genes, patient baseline characteristics, and early occurrence of grade 3/4 neutropenia. MATERIALS AND METHODS: ABCB1 (rs1045642, rs1128503) and ERCC1 (rs3212986, rs11615) were analyzed in germline DNA from palbociclib-treated patients from PALOMA-2 (n = 584) and PALOMA-3 (n = 442). SNP, race, and cycle 1 day 15 (C1D15) absolute neutrophil count (ANC) data were available for 652 patients. Univariate and multivariable analyses evaluated associations between SNPs, patient baseline characteristics, and early occurrence of grade 3/4 neutropenia. Analyses were stratified by Asian (n = 122) and non-Asian (n = 530) ethnicity. Median progression-free survival (mPFS) was estimated using the Kaplan-Meier method. The effect of genetic variants on palbociclib pharmacokinetics was analyzed. RESULTS: ABCB1 and ERCC1_rs11615 SNP frequencies differed between Asian and non-Asian patients. Multivariable analysis showed that low baseline ANC was a strong independent risk factor for C1D15 grade 3/4 neutropenia regardless of race (Asians: odds ratio [OR], 6.033, 95% confidence interval [CI], 2.615-13.922, p < .0001; Non-Asians: OR, 6.884, 95% CI, 4.138-11.451, p < .0001). ABCB1_rs1128503 (C/C vs. T/T: OR, 0.57, 95% CI, 0.311-1.047, p = .070) and ERCC1_rs11615 (A/A vs. G/G: OR, 1.75, 95% CI, 0.901-3.397, p = .098) were potential independent risk factors for C1D15 grade 3/4 neutropenia in non-Asian patients. Palbociclib mPFS was consistent across genetic variants; exposure was not associated with ABCB1 genotype. CONCLUSION: This is the first comprehensive assessment of pharmacogenetic data in relationship to exposure to a CDK4/6 inhibitor. Pharmacogenetic testing may inform about potentially increased likelihood of patients developing severe neutropenia (NCT01740427, NCT01942135). IMPLICATIONS FOR PRACTICE: Palbociclib plus endocrine therapy improves hormone receptor-positive/human epidermal growth factor receptor 2-negative advanced breast cancer outcomes, but is commonly associated with neutropenia. Genetic variants in ABCB1 may influence palbociclib exposure, and in ERCC1 are associated with chemotherapy-induced severe neutropenia. Here, the associations of single nucleotide polymorphisms in these genes and baseline characteristics with neutropenia were assessed. Low baseline absolute neutrophil count was a strong risk factor (p < .0001) for grade 3/4 neutropenia. There was a trend indicating that ABCB1_rs1128503 and ERCC1_rs11615 were potential risk factors (p < .10) for grade 3/4 neutropenia in non-Asian patients. Pharmacogenetic testing could inform clinicians about the likelihood of severe neutropenia with palbociclib.

Exploring the Use of Serum-Derived Small Extracellular Vesicles as Liquid Biopsy to Study the Induction of Hepatic Cytochromes P450 and Organic Anion Transporting Polypeptides.

Liver-derived small extracellular vesicles (sEVs), prepared from small sets of banked serum samples using a novel two-step protocol, were deployed as liquid biopsy to study the induction of cytochromes P450 (CYP3A4, CYP3A5, and CYP2D6) and organic anion transporting polypeptides (OATP1B1 and OATP1B3) during pregnancy (nonpregnant (T0), first, second, and third (T3) trimester women; N = 3 each) and after administration of rifampicin (RIF) to healthy male subjects. Proteomic analysis revealed induction (mean fold-increase, 90% confidence interval) of sEV CYP3A4 after RIF 300 mg × 7 days (3.5, 95% CI = 2.5-4.5, N = 4, P = 0.029) and 600 mg × 14 days (3.7, 95% CI = 2.1-6.0, N = 5, P = 0.018) consistent with the mean oral midazolam area under the plasma concentration time curve (AUC) ratio in the same subjects (0.28, 95% CI = 0.22-0.34, P < 0.0001; and 0.17, 95% CI = 0.13-0.22, P < 0.0001). Compared with CYP3A4, liver sEV CYP3A5 protein (subjects genotyped CYP3A5*1/*3) was weakly induced (≤ 1.5-fold). It was also possible to measure liver sEV-catalyzed dextromethorphan (DEX) O-demethylation to dextrorphan (DXO), correlated with sEV CYP2D6 expression (r = 0.917, P = 0.0001; N = 10) and 3-hour plasma DXO-to-DEX concentration ratio (r = 0.843, P = 0.002, N = 10), and show that CYP2D6 was not induced by RIF. Nonparametric analysis of liver sEV revealed significantly higher CYP3A4 (3.2-fold, P = 0.003) and CYP2D6 (3.7-fold, P = 0.03) protein expression in T3 vs. T0 women. In contrast, expression of both OATPs in liver sEV was unaltered by RIF administration and pregnancy.

Dose-Dependent Inhibition of OATP1B by Rifampicin in Healthy Volunteers: Comprehensive Evaluation of Candidate Biomarkers and OATP1B Probe Drugs.

To address the most appropriate endogenous biomarker for drug-drug interaction risk assessment, eight healthy subjects received an organic anion transporting polypeptide 1B (OATP1B) inhibitor (rifampicin, 150, 300, and 600 mg), and a probe drug cocktail (atorvastatin, pitavastatin, rosuvastatin, and valsartan). In addition to coproporphyrin I, a widely studied OATP1B biomarker, we identified at least 4 out of 28 compounds (direct bilirubin, glycochenodeoxycholate-3-glucuronide, glycochenodeoxycholate-3-sulfate, and hexadecanedioate) that presented good sensitivity and dynamic range in terms of the rifampicin dose-dependent change in area under the plasma concentration-time curve ratio (AUCR). Their suitability as OATP1B biomarkers was also supported by the good correlation of AUC0-24h between the endogenous compounds and the probe drugs, and by nonlinear regression analysis (AUCR-1 vs. rifampicin plasma Cmax (maximum total concentration in plasma)) to yield an estimate of the inhibition constant of rifampicin. These endogenous substrates can complement existing OATP1B-mediated drug-drug interaction risk assessment approaches based on agency guidelines in early clinical trials.

Caspase-like activity during aging and cell death in the toxic dinoflagellate Karenia brevis.

The observation of caspase-like activity during cell death has provided a new framework for understanding the evolutionary and ecological contexts of programmed cell death in phytoplankton. However, additional roles for this caspase-like activity, the enzymes responsible, and the targets of this enzyme activity in phytoplankton remain largely undefined. In the present study, the role of caspase-like activity in aging and ROS-mediated cell death were investigated and death programs both dependent on and independent of caspase-like activity were observed in the toxic dinoflagellate, Karenia brevis. The dual use of in situ caspase 3/7 and TUNEL staining identified previously undescribed death-associated morphotypes in K. brevis. In silico motif analysis identified several enzymes with predicted caspase-like activity in the K. brevis transcriptome, although bona fide caspases are absent. Lastly, computational prediction of downstream caspase substrates, using sequence context and predicted secondary structure, identified proteins involved in a wide range of biological processes including regulation of protein turnover, cell cycle progression, lipid metabolism, coenzyme metabolism, apoptotic and autophagic death. To confirm the computational predictions, a short peptide was designed around the predicated caspase cleavage site in a predicted novel K. brevis caspase 3/7-like target, S-adenosylmethionine synthetase (KbAdoMetS). Cleavage of the peptide substrate with recombinant caspase 3 enzyme was determined by MALDI-TOF MS, confirming that KbAdoMetS is indeed a bona fide caspase substrate. These data identify the involvement of caspase-like activity in both aging and cell death in K. brevis and identify novel executioner enzymes and downstream targets that may be important for bloom termination.

Subcellular localization of dinoflagellate polyketide synthases and fatty acid synthase activity.

Dinoflagellates are prolific producers of polyketide secondary metabolites. Dinoflagellate polyketide synthases (PKSs) have sequence similarity to Type I PKSs, megasynthases that encode all catalytic domains on a single polypeptide. However, in dinoflagellate PKSs identified to date, each catalytic domain resides on a separate transcript, suggesting multiprotein complexes similar to Type II PKSs. Here, we provide evidence through coimmunoprecipitation that single-domain ketosynthase and ketoreductase proteins interact, suggesting a predicted multiprotein complex. In Karenia brevis (C.C. Davis) Gert Hansen & Ø. Moestrup, previously observed chloroplast localization of PKSs suggested that brevetoxin biosynthesis may take place in the chloroplast. Here, we report that PKSs are present in both cytosol and chloroplast. Furthermore, brevetoxin is not present in isolated chloroplasts, raising the question of what chloroplast-localized PKS enzymes might be doing. Antibodies to K. brevis PKSs recognize cytosolic and chloroplast proteins in Ostreopsis cf. ovata Fukuyo, and Coolia monotis Meunier, which produce different suites of polyketide toxins, suggesting that these PKSs may share common pathways. Since PKSs are closely related to fatty acid synthases (FAS), we sought to determine if fatty acid biosynthesis colocalizes with either chloroplast or cytosolic PKSs. [(3) H]acetate labeling showed fatty acids are synthesized in the cytosol, with little incorporation in chloroplasts, consistent with a Type I FAS system. However, although 29 sequences in a K. brevis expressed sequence tag database have similarity (BLASTx e-value <10(-10) ) to PKSs, no transcripts for either Type I (cytosolic) or Type II (chloroplast) FAS are present. Further characterization of the FAS complexes may help to elucidate the functions of the PKS enzymes identified in dinoflagellates.