Christina Gersch

ESR1 mutations in circulating plasma tumor DNA from metastatic breast cancer patients

Purpose: Mutations in the estrogen receptor (ER)α gene, ESR1, have been identified in breast cancer metastases after progression on endocrine therapies. Because of limitations of metastatic biopsies, the reported frequency of ESR1 mutations may be underestimated. Here, we show a high frequency of ESR1 mutations using circulating plasma tumor DNA (ptDNA) from patients with metastatic breast cancer. Experimental Design: We retrospectively obtained plasma samples from eight patients with known ESR1 mutations and three patients with wild-type ESR1 identified by next-generation sequencing (NGS) of biopsied metastatic tissues. Three common ESR1 mutations were queried for using droplet digital PCR (ddPCR). In a prospective cohort, metastatic tissue and plasma were collected contemporaneously from eight ER-positive and four ER-negative patients. Tissue biopsies were sequenced by NGS, and ptDNA ESR1 mutations were analyzed by ddPCR. Results: In the retrospective cohort, all corresponding mutations were detected in ptDNA, with two patients harboring additional ESR1 mutations not present in their metastatic tissues. In the prospective cohort, three ER-positive patients did not have adequate tissue for NGS, and no ESR1 mutations were identified in tissue biopsies from the other nine patients. In contrast, ddPCR detected seven ptDNA ESR1 mutations in 6 of 12 patients (50%). Conclusions: We show that ESR1 mutations can occur at a high frequency and suggest that blood can be used to identify additional mutations not found by sequencing of a single metastatic lesion. Clin Cancer Res; 22(4); 993–9. ©2015 AACR.

Concordance Between CYP2D6 Genotypes Obtained From Tumor-Derived and Germline DNA

Formalin-fixed, paraffin-embedded tumors (FFPETs) are a valuable source of DNA for genotype association studies and are often the only germline DNA resource from cancer clinical trials. The anti-estrogen tamoxifen is metabolized into endoxifen by CYP2D6, leading to the hypothesis that patients with certain CYP2D6 genotypes may not receive benefit because of their inability to activate the drug. Studies testing this hypothesis using FFPETs have provided conflicting results. It has been postulated that CYP2D6 genotype determined using FFPET may not be accurate because of somatic tumor alterations. In this study, we determined the concordance between CYP2D6 genotypes generated using 3 tissue sources (FFPETs; formalin-fixed, paraffin-embedded unaffected lymph nodes [FFPELNs]; and whole blood cells [WBCs]) from 122 breast cancer patients. Compared with WBCs, FFPET and FFPELN genotypes were highly concordant (>94%), as were the predicted CYP2D6 metabolic phenotypes (>97%). We conclude that CYP2D6 genotypes obtained from FFPETs accurately represent the patients CYP2D6 metabolic phenotype.

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.

Genotyping concordance in DNA extracted from formalin‐fixed paraffin embedded (FFPE) breast tumor and whole blood for pharmacogenetic analyses

Cancer pharmacogenetic studies use archival tumor samples as a DNA source when germline DNA is unavailable. Genotyping DNA from formalin‐fixed paraffin embedded tumors (FFPE‐T) may be inaccurate due to FFPE storage, genetic aberrations, and/or insufficient DNA extraction. Our objective was to assess the extent and source of genotyping inaccuracy from FFPE‐T DNA and demonstrate analytical validity of FFPE‐T genotyping of candidate single nucleotide polymorphisms (SNPs) for pharmacogenetic analyses.

Comprehensive mutation and copy number profiling in archived circulating breast cancer tumor cells documents heterogeneous resistance mechanisms

Addressing drug resistance is a core challenge in cancer research, but the degree of heterogeneity in resistance mechanisms in cancer is unclear. In this study, we conducted next-generation sequencing (NGS) of circulating tumor cells (CTC) from patients with advanced cancer to assess mechanisms of resistance to targeted therapy and reveal opportunities for precision medicine. Comparison of the genomic landscapes of CTCs and tissue metastases is complicated by challenges in comprehensive CTC genomic profiling and paired tissue acquisition, particularly in patients who progress after targeted therapy. Thus, we assessed by NGS somatic mutations and copy number alterations (CNA) in archived CTCs isolated from patients with metastatic breast cancer who were enrolled in concurrent clinical trials that collected and analyzed CTCs and metastatic tissues. In 76 individual and pooled informative CTCs from 12 patients, we observed 85% concordance in at least one or more prioritized somatic mutations and CNA between paired CTCs and tissue metastases. Potentially actionable genomic alterations were identified in tissue but not CTCs, and vice versa. CTC profiling identified diverse intra- and interpatient molecular mechanisms of endocrine therapy resistance, including loss of heterozygosity in individual CTCs. For example, in one patient, we observed CTCs that were either wild type for ESR1 (n = 5/32), harbored the known activating ESR1 p.Y537S mutation (n = 26/32), or harbored a novel ESR1 p.A569S (n = 1/32). ESR1 p.A569S was modestly activating in vitro, consistent with its presence as a minority circulating subclone. Our results demonstrate the feasibility and potential clinical utility of comprehensive profiling of archived fixed CTCs. Tissue and CTC genomic assessment are complementary, and precise combination therapies will likely be required for effective targeting in advanced breast cancer patients.Significance: These findings demonstrate the complementary nature of genomic profiling from paired tissue metastasis and circulating tumor cells from patients with metastatic breast cancer. Cancer Res; 78(4); 1110-22. ©2017 AACR.

Abstract P6-09-11: Genetic variation in CYP3A affects steady-state exemestane concentrations but does not explain inter-race difference

Background: Exemestane is a third generation steroidal aromatase inhibitor (AI) used for the treatment of estrogen receptor (ER) positive breast cancer in postmenopausal women. Differences in AI treatment efficacy and side effects may be due, in part, to variability in drug exposure. We previously reported that patients who self-report as white and those who carry the low-activity CYP3A4*22 single nucleotide polymorphism (SNP) have increased exemestane steady-state concentrations. Additional SNPs in CYP3A may contribute to pharmacokinetic variability and explain this inter-race difference. CYP3A5*3 (rs776746) is a non-expresser genotype that is far more common in European (minor allele frequency (MAF)∼0.94) than African (MAF∼0.18) individuals. CYP3A7*1C (rs45446698) is believed to tag adult expression of the fetal CYP3A7 enzyme and is relatively uncommon in tested cohorts (European MAF=0.04, African MAF Methods: 500 patients were randomly assigned to either drug on the Exemestane and Letrozole Pharmacogenetics (ELPh) Study. Clinical data and DNA were collected at baseline and blood samples were collected after 1 or 3 months of treatment to measure steady-state exemestane concentration via HPLC/MS. Genotyping for CYP3A5*3 and CYP3A7*1C was performed via Taqman Allelic Discrimination. Pharmacogenetic association with log-transformed concentrations were tested for each variant by inclusion in a multivariable model with CYP3A4*22 and self-reported race, assuming additive genetic effect, using Tobit regression to censor concentrations below the lower limit of quantification. SNPs with suggestive p-values 40, body mass index (BMI), and prior chemotherapy) to assess their independent contribution. Results: In 231 evaluable patients there was a suggestive trend toward lower steady-state exemestane concentrations for CYP3A7*1C carriers (6.3 vs. 8.0 ng/mL) in the model including CYP3A4*22 and race (p=0.083). In the final multivariable model each CYP3A7*1C allele decreased exemestane concentration 31.5% (p=0.035, Table 1). CYP3A5*3 was not associated with exemestane concentration (p>0.2). Conclusions: Patients with breast cancer who carry CYP3A7*1C have lower steady-state exemestane concentrations but this association does not explain the greater concentrations in self-reported white patients. Ongoing analyses will determine whether exemestane concentration predicts treatment efficacy or toxicity, and if so, whether genetic and clinical factors can be useful for individualizing dosing to optimize outcomes. CYP3A7*1C should be prioritized for analyses of pharmacokinetic variability of other CYP3A substrates. Citation Format: Hertz DL, Kidwell KM, Gersch CL, Desta Z, Storniolo AM, Stearns V, Skaar TC, Hayes DF, Henry NL, Rae JM. Genetic variation in CYP3A affects steady-state exemestane concentrations but does not explain inter-race difference [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-09-11.

Abstract P2-02-19: Somatic genetic profiling of circulating tumor cells (CTC) in metastatic breast cancer (MBC) patients

Introduction: Somatic mutations, including those in TP53 , PIK3CA , and estrogen receptor alpha ( ESR1 ), are key to the biology of cancer and response to therapy. Recently, somatic cancer-associated mutations have been identified in circulating cell free plasma tumor DNA (ptDNA). Less is known about the mutation profile of DNA extracted from CTC (CTC-DNA). Since CTC-DNA provides mutational information of single cells, we hypothesize CTC-DNA will complement ptDNA to give greater insight into tumor heterogeneity. Methods: Patients with ER positive MBC who were enrolled in the Mi CTC-ONCOSEQ, a companion trial to Mi-ONCOSEQ (the Michigan Oncology Sequencing Program), and who had ≥5CTC/7.5 ml whole blood were included. CTC were enriched from white blood cells (WBC) with CellSearch

Abstract 3151: Genetic profiling of circulating tumor cells (CTC) in metastatic breast cancer (MBC) patients

Introduction: Cancer-associated mutations are present in circulating cell free plasma tumor DNA (ptDNA). We have previously reported mutation profiles of DNA extracted from CTC (CTC-DNA) from two patients with MBC (#2 and 24 in table). Here, we report an expanded cohort with an updated gene panel. Methods: We studied seven patients (two previously reported, along with five additional patients) with MBC who were enrolled in Mi-CTC-ONCOSEQ, had ≥5 CTC/7.5 ml whole blood (WB), and had at least one CTC with high quality DNA determined by the Ampli1™ quality control kit. CTC were enriched from WB with CellSearch© and purified from white blood cells (WBC) (DEPArray™). DNA from individual CTC and WBC was isolated and subjected to whole genomic amplification (Ampli 1™ WGA) and genotyped by multiplexed PCR-based next generation sequencing with the Oncomine Comprehensive Panel (OCP) on the Ion Torrent Proton. Exome sequencing of research biopsies of metastatic tissue was performed using an Illumina HiSeq 2500 platform. Previously reported patients (#2 and 24) sequenced with a beta version of the OCP were re-run, and updated results are provided. Results: Six of seven patients were ER positive. Patients #2, 12, and 24 had CTC with mutations also found in the research biopsy (table). Novel alterations were found in comparison to research biopsy in five of the seven patients (table). In two patients (#19, 24), two potential actionable mutations (PTCH1 and NOTCH1) were found in CTC-DNA but not in tissue-DNA. No mutations were detected in any WBC. Conclusions: We demonstrate the ability to purify CTC, and to isolate and amplify DNA of suitable quality for genetic analysis using a comprehensive targeted sequencing panel. Mutations found in tissue as well as novel mutations were found in CTC-DNA. Two potential actionable mutations were identified in CTC, but not in tissue, opening potentially new therapeutic opportunities. We conclude that mutational analysis of CTC-DNA and of tissue may be complementary. Citation Format: Costanza Paoletti, Andi K. Cani, Kimberly Aung, Elizabeth P. Darga, Emily M. Cannell, Daniel H. Hovelson, Maryam Yazdani, Allen R. Blevins, Nahomi Tokudome, Paul J. Baratta, Jose’ M. Larios, Dafydd G. Thomas, Martha E. Brown, Christina Gersch, Anne F. Schott, Daniel Robinson, Arul M. Chinnaiyan, Farideh Bischoff, Daniel F. Hayes, James M. Rae, Scott A. Tomlins. Genetic profiling of circulating tumor cells (CTC) in metastatic breast cancer (MBC) patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3151.

Abstract P5-12-05: CYP3A4*22 polymorphism is associated with increased exemestane concentrations in postmenopausal breast cancer patients

Background: Exemestane is a second generation steroidal aromatase inhibitor (AI) used for the treatment of estrogen receptor (ER) positive breast cancer in postmenopausal women. Variability in AI treatment efficacy and side effects seen across patients may be due, in part, to inter-patient differences in drug exposure. This exposure variability is likely caused by patient genetics factors, such as single nucleotide polymorphisms (SNPs) in drug metabolizing enzymes, or clinical factors such as patient body size, organ function, and comorbidities. The objective of this secondary correlative analysis was to identify genetic and clinical characteristics that affect steady state exemestane concentration, with a specific focus on the influence of inherited genetic variants and baseline hepatic function. Methods: 500 patients were enrolled on the Exemestane and Letrozole Pharmacogenetics (ELPh) Study and randomized to either drug. Clinical data and DNA were collected at baseline and blood samples were collected after 1 or 3 months of treatment to measure steady-state exemestane concentration via HPLC/MS. Genotyping was performed on a custom Sequenom MassARRAY iPLEX that included the recently discovered low activity CYP3A4*22 (rs35599367) SNP and several other SNPs with putative functional consequence in enzymes thought to be involved in exemestane metabolism (CYP1A1/2, CYP1B1, CYP3A4, CYP4A11, AKR1C3/4, AKR7A2). Our primary hypothesis was that patients carrying CYP3A4*22 variants would have higher serum exemestane concentrations. Other SNPs and clinical characteristics (hepatic and renal function, age, body mass index (BMI), time of sample collection, prior chemotherapy) were assessed for independent association, and then adjusted for in a multivariable tobit regression model for CYP3A4*22 on log-transformed censored exemestane concentration. Results: 246 (225 randomized to exemestane arm, 21 crossed-over from letrozole arm) patients had exemestane steady state levels and were evaluable in this analysis. As hypothesized, the CYP3A4*22 polymorphism (minor allele frequency=0.06) was associated with a 54% increase in exemestane concentration (95% CI: 14% - 109%, p 40) at baseline (95% CI: 2% - 104%, p=0.02), 1% lower per unit increase in BMI (95% CI: 0% - 3%, p=0.05), and 20% lower in patients who received prior chemotherapy (95% CI: 4% - 34%, p=0.03). Age, renal impairment, and other SNPs were not associated with exemestane concentration. After adjustment for significant clinical covariates the CYP3A4*22 SNP remained significant (p Conclusions: Genetic and clinical predictors of exemestane concentration were discovered in a large cohort of prospectively enrolled estrogen responsive breast cancer patients. Ongoing analyses will determine whether the variability in exemestane concentration was associated with downstream effects on estrogen depletion or treatment-related toxicity. If so, these genetic and clinical characteristics could be useful for individualizing dosing of exemestane to ensure that all patients are receiving maximal benefit with minimal toxicity. Citation Format: Hertz DL, Kidwell KM, Seewald NJ, Gersch CL, Desta Z, Flockhart DA, Storniolo AM, Stearns V, Skaar TC, Hayes DF, Henry NL, Rae JM. CYP3A4*22 polymorphism is associated with increased exemestane concentrations in postmenopausal breast cancer patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-12-05.