Polymorphisms associated with everolimus pharmacokinetics, toxicity and survival in metastatic breast cancer

<div><p>Purpose</p><p>Metastatic breast cancer (MBC) progressing after endocrine therapy frequently activates PI3K/AKT/mTOR pathway. The BOLERO-2 trial showed that everolimus-exemestane achieves increased progression free survival (PFS) compared with exemestane. However, there is great inter-patient variability in toxicity and response to exemestane-everolimus treatment. The objective of this study was to perform an exploratory study analyzing the implication of single nucleotide polymorphisms (SNPs) on outcomes from this treatment through a pharmacogenetic analysis.</p><p>Patients and methods</p><p>Blood was collected from 90 postmenopausal women with hormone receptor-positive, HER2-negative MBC treated with exemestane-everolimus following progression after prior treatment with a non-steroidal aromatase inhibitor. Everolimus pharmacokinetics was measured in 37 patients. Twelve SNPs in genes involved in everolimus pharmacokinetics and pharmacodynamics were genotyped and associations assessed with drug plasma levels, clinically relevant toxicities (non-infectious pneumonitis, mucositis, hyperglycemia and hematological toxicities), dose reductions or treatment suspensions due to toxicity, progression free survival (PFS) and overall survival.</p><p>Results</p><p>We found that <i>CYP3A4</i> rs35599367 variant (<i>CYP3A4*22</i> allele) carriers had higher everolimus blood concentration compared to wild type patients (P = 0.019). <i>ABCB1</i> rs1045642 was associated with risk of mucositis (P = 0.031), while <i>PIK3R1</i> rs10515074 and <i>RAPTOR</i> rs9906827 were associated with hyperglycemia and non-infectious pneumonitis (P = 0.016 and 0.024, respectively). Furthermore, <i>RAPTOR</i> rs9906827 was associated with PFS (P = 0.006).</p><p>Conclusions</p><p><i>CYP3A4*22</i> allele influenced plasma concentration of everolimus and several SNPs in PI3K/AKT/mTOR pathway genes were associated with treatment toxicities and prognosis. These results require replication, but suggest that germline variation could influence everolimus outcomes in MBC.</p></div

Baseline demographic and clinical characteristics.

<p>Baseline demographic and clinical characteristics.</p

Box plot representing everolimus blood concentration by <i>CYP3A4</i> rs35599367 (<i>CYP3A4*22</i>) genotype.

<p>“C/C” corresponds to <i>CYP3A4*22</i> wild type patients (n = 33), and “C/G” to <i>CYP3A4*22</i> heterozygous carriers (n = 4). Comparison between groups was performed using the Mann-Whitney-U test.</p

SNPs associated with toxicity.

<p>SNPs associated with toxicity.</p

SNPs included in the study and their genotype frequencies.

<p>SNPs included in the study and their genotype frequencies.</p

Kaplan-Meier curve for progression free survival by RAPTOR rs9906827 genotype.

<p>P-value corresponds to Cox regression analysis under a dominant genetic model including the number of previous chemotherapy lines as covariate. HR, hazard ratio; CI, confidence interval.</p

Gene-gene interaction results for susceptibility to classic Papillary Thyroid Carcinoma.

<p>SH- SNPHarvester; MSH- MegaSNPHunter; Light grey shading indicates interactions selected by each of the algorithms; dark grey green shading indicates the interaction fulfilling the established criteria to pass to stage 2 (replication) (SNP pair selected by at least three methods);</p>*<p>associated P-value; NA- not available (all genotypes combinations classified as neutral).</p

Association with risk of follicular cell-derived thyroid cancer for 9 candidate variants in the discovery and replication stages.

<p>Abbreviations: MAF = minor allele frequency; OR = odds ratio; CI = confidence interval; ESE = Exonic Splicing Enhancers; PTC = Papillary Thyroid Carcinoma; cPTC = classic PTC; fvPTC = follicular variant of PTC; FTC = Follicular Thyroid Carcinoma. The table is sorted by disease subtype and, within each group, by <i>P</i>-value.</p>a<p>Major/minor allele (in controls);</p>b<p>OR and CI were obtained using homozygotes for the most frequent allele in controls as the reference group;</p>c<p><i>P</i>-values are derived from Wald statistics;</p>d<p>Results adjusted for age and gender;</p>e<p>Results adjusted for age, gender and country.</p

Relative expression levels of STK17B in Pax8-silenced cells.

<p>PCCl3 cells were transiently (A) or stably (B) silenced for the transcription factor Pax8 (siPax8). As a control, wild type or siScramble transfected cells were used. The expression levels were assessed by means of qRT-PCR (A, upper panel) or western blot (A, lower panel, and B).</p

Epistatic model for SNPs in <i>PAX8</i> and <i>STK17B</i> and genotype frequencies for cPTC-cases and controls.

<p>Relative frequencies of the nine genotype combinations of the replicated interaction (<i>PAX8</i>-<i>STK17B</i>) are shown for cases and controls (red and blue columns, respectively). The cell containing the high-risk genotype combination is highlighted in light red, those with low-risk combinations in light blue, and those with neutral combinations are uncoloured. Figure1a - based on the discovery stage (series I); <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074765#pone-0074765-g001" target="_blank">Figure 1b</a> - based on the replication stage (series II and III); <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074765#pone-0074765-g001" target="_blank">Figure 1c</a> – based on both stages combined (series I, II and III).</p