Parul Patel

Prevalence of ESR1 Mutations in Cell-Free DNA and Outcomes in Metastatic Breast Cancer: A Secondary Analysis of the BOLERO-2 Clinical Trial

Importance Estrogen receptor α (ESR1) mutations found in metastatic breast cancer (MBC) promote ligand-independent receptor activation and resistance to estrogen-deprivation therapy in laboratory models. The prevalence of these mutations and their potential impact on clinical outcomes has not been established. Objective To determine the prevalence of ESR1 mutations (Y537S and D538G) in estrogen receptor (ER)-positive MBC and determine whether mutation is associated with inferior outcomes. Design, Setting, and Participants From December 16, 2014, to August 26, 2015, we analyzed cell-free DNA (cfDNA) from baseline plasma samples from participants in the BOLERO-2 double-blind phase 3 study that randomized patients from 189 centers in 24 countries with MBC to exemestane plus placebo or exemestane plus everolimus. The study enrolled postmenopausal women with a diagnosis of MBC and prior exposure to an aromatase inhibitor. Baseline plasma samples were available from 541 of 724 patients (74.7%). We assessed the effect of mutation on overall survival of the population and the effect of mutation on progression-free survival (PFS) by treatment arm. Interventions Patients were randomized to treatment with exemestane (25 mg oral daily) together with everolimus (10 mg oral daily) or with placebo. Main Outcomes and Measures The 2 most frequent mutations in ESR1 (Y537S and D538G) were analyzed from cfDNA using droplet digital polymerase chain reaction and samples scored as wild-type, D538G, Y537S, or double mutant. Cox-proportional hazards model was used to assess PFS in patient subgroups defined by mutations, and the effect of each mutation on overall survival. Results Of 541 evaluable patients, 156 (28.8%) had ESR1 mutation D538G (21.1%) and/or Y537S (13.3%), and 30 had both. These mutations were associated with shorter overall survival (wild-type, 32.1 months [95% CI, 28.09-36.40 months]; D538G, 25.99 months [95% CI, 19.19-32.36 months]; Y537S, 19.98 months [13.01-29.31 months]; both mutations, 15.15 months [95% CI, 10.87-27.43 months]). The D538G group (hazard ratio, 0.34 [95% CI, 0.02-0.57]) derived a similar PFS benefit as wild type from addition of everolimus to exemestane. Conclusions and Relevance ESR1 mutations are prevalent in ER-positive aromatase inhibitor-treated MBC. Both Y537S and D538G mutations are associated with more aggressive disease biology. Trial Registration clinicaltrials.gov Identifier: NCT00863655.

Genomic Biomarkers of a Randomized Trial Comparing First-line Everolimus and Sunitinib in Patients with Metastatic Renal Cell Carcinoma

BACKGROUND Metastatic renal cell carcinoma (RCC) patients are commonly treated with vascular endothelial growth factor (VEGF) inhibitors or mammalian target of rapamycin inhibitors. Correlations between somatic mutations and first-line targeted therapy outcomes have not been reported on a randomized trial. OBJECTIVE To evaluate the relationship between tumor mutations and treatment outcomes in RECORD-3, a randomized trial comparing first-line everolimus (mTOR inhibitor) followed by sunitinib (VEGF inhibitor) at progression with the opposite sequence in 471 metastatic RCC patients. DESIGN, SETTING, AND PARTICIPANTS Targeted sequencing of 341 cancer genes at ∼540× coverage was performed on available tumor samples from 258 patients; 220 with clear cell histology (ccRCC). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Associations between somatic mutations and median first-line progression free survival (PFS1L) and overall survival were determined in metastatic ccRCC using Cox proportional hazards models and log-rank tests. RESULTS AND LIMITATIONS Prevalent mutations (≥ 10%) were VHL (75%), PBRM1 (46%), SETD2 (30%), BAP1 (19%), KDM5C (15%), and PTEN (12%). With first-line everolimus, PBRM1 and BAP1 mutations were associated with longer (median [95% confidence interval {CI}] 12.8 [8.1, 18.4] vs 5.5 [3.1, 8.4] mo) and shorter (median [95% CI] 4.9 [2.9, 8.1] vs 10.5 [7.3, 12.9] mo) PFS1L, respectively. With first-line sunitinib, KDM5C mutations were associated with longer PFS1L (median [95% CI] of 20.6 [12.4, 27.3] vs 8.3 [7.8, 11.0] mo). Molecular subgroups of metastatic ccRCC based on PBRM1, BAP1, and KDM5C mutations could have predictive values for patients treated with VEGF or mTOR inhibitors. Most tumor DNA was obtained from primary nephrectomy samples (94%), which could impact correlation statistics. CONCLUSIONS PBRM1, BAP1, and KDM5C mutations impact outcomes of targeted therapies in metastatic ccRCC patients. PATIENT SUMMARY Large-scale genomic kidney cancer studies reported novel mutations and heterogeneous features among individual tumors, which could contribute to varied clinical outcomes. We demonstrated correlations between somatic mutations and treatment outcomes in clear cell renal cell carcinoma, supporting the value of genomic classification in prospective studies.

Correlation between PIK3CA mutations in cell-free DNA and everolimus efficacy in HR + , HER2 − advanced breast cancer: results from BOLERO-2

Background:The current analysis was performed to evaluate the impact of PIK3CA hotspot mutations on everolimus efficacy in BOLERO-2 participants, using cell-free DNA (cfDNA) from plasma samples collected at the time of patient randomisation.Methods:PIK3CA H1047R, E545K, and E542K mutations in plasma-derived cfDNA were analysed by droplet digital PCR (ddPCR). Median PFS was estimated for patient subgroups defined by PIK3CA mutations in each treatment arm.Results:Among 550 patients included in cfDNA analysis, median PFS in everolimus vs placebo arms was similar in patients with tumours that had wild-type or mutant PIK3CA (hazard ratio (HR), 0.43 and 0.37, respectively). Everolimus also prolonged median PFS in patients with PIK3CA H1047R (HR, 0.37) and E545K/E542K mutations (HR=0.30) with a similar magnitude.Conclusions:Mutation analysis of plasma-derived cfDNA by ddPCR suggests that PFS benefit of everolimus was maintained irrespective of PIK3CA genotypes, consistent with the previous analysis of archival tumour DNA by next-generation sequencing.

Molecular analysis of a male breast cancer patient with prolonged stable disease under mTOR/PI3K inhibitors BEZ235/everolimus

The mTORC1 inhibitor everolimus (Afinitor/RAD001) has been approved for multiple cancer indications, including ER+/HER2− metastatic breast cancer. However, the combination of everolimus with the dual PI3K/mTOR inhibitor BEZ235 was shown to be more efficacious than either everolimus or BEZ235 alone in preclinical models. Herein, we describe a male breast cancer (MBC) patient who was diagnosed with hormone receptor-positive (HR+)/HER2− stage IIIA invasive ductal carcinoma and sequentially treated with chemoradiotherapy and hormonal therapy. Upon the development of metastases, the patient began a 200 mg twice-daily BEZ235 and 2.5 mg weekly everolimus combination regimen. The patient sustained a prolonged stable disease of 18 mo while undergoing the therapy, before his tumor progressed again. Therefore, we sought to both better understand MBC and investigate the underlying molecular mechanisms of the patients sensitivity and subsequent resistance to the BEZ235/everolimus combination therapy. Genomic and immunohistochemical analyses were performed on samples collected from the initial invasive ductal carcinoma pretreatment and a metastasis postprogression on the BEZ235/everolimus combination treatment. Both tumors were relatively quiet genomically with no overlap to recurrent MBC alterations in the literature. Markers of PI3K/mTOR pathway hyperactivation were not identified in the pretreatment sample, which complements previous reports of HR+ female breast cancers being responsive to mTOR inhibition without this activation. The postprogression sample, however, demonstrated greater than fivefold increased estrogen receptor and pathogenesis-related protein expression, which could have constrained the PI3K/mTOR pathway inhibition by BEZ235/everolimus. Overall, these analyses have augmented the limited episteme on MBC genetics and treatment.

The Use of Rat Lens Explant Cultures to Study the Mechanism of Drug-induced Cataractogenesis

Lens explant cultures were used to assess the mechanism of drug-induced cataractogenic potential of NVS001, a peroxisome proliferator-activated receptor delta (PPARδ) agonist, which resulted in cataract in all treated animals during a 13-week rat study. Ciglitazone, a PPARγ agonist and cataractogenic compound, was used as a positive control to validate this model. Rat lenses were extracted and cultured in medium supplemented with antibiotics for 24-h preincubation pretreatment. Lenses showing no signs of damage at the end of the preincubation pretreatment period were randomized into five experimental groups, (1) untreated control, (2) 0.1% dimethyl sulphoxide control, (3) 10μM NVS001, (4) 10μM ciglitazone, and (5) 10μM acetaminophen (negative control). Lenses were treated every 24 h after preincubation pretreatment for up to 48 h. Samples for viability, histology, and gene expression profiling were collected at 4, 24, and 48 h. There was a time-dependent increase in opacity, which correlated to a decrease in viability measured by adenosine triphosphate levels in NVS001 and ciglitazone-treated lenses compared with controls. NVS001 and ciglitazone had comparable cataractogenic effects after 48 h with histology showing rupture of the lens capsule, lens fiber degeneration, cortical lens vacuolation, and lens epithelial degeneration. Furthermore, no changes were seen when lenses were treated with acetaminophen. Gene expression analysis supported oxidative and osmotic stress, along with decreases in membrane and epithelial cell integrity as key factors in NVS001-induced cataracts. This study suggests that in vitro lens cultures can be used to assess cataractogenic potential of PPAR agonists and to study/understand the underlying molecular mechanism of cataractogenesis in rat.

Abstract 26: Biomarker analysis of a male breast cancer patient with prolonged stable disease under mTOR/PI3K inhibitors BEZ235/RAD001

The mTORC1 inhibitor RAD001 (everolimus) has been approved for multiple cancer indications, including ER+/HER2- metastatic breast cancer. The combination of RAD001 with a dual PI3K/mTOR inhibitor BEZ235 was shown to be even more efficacious than RAD001 alone in preclinical models and was evaluated in clinical trials. We identified a male breast cancer patient who experienced a prolonged stable disease with the RAD001/BEZ235 combination as 3rd line treatment for his metastatic disease and investigated molecular mechanisms to explain the extraordinary benefit and subsequent drug resistance. The 66-year old Caucasian male had stage IIIA invasive ductal carcinoma at initial surgery. He then received two sequential adjuvant therapies: a chemo-radiotherapy and letrozole. When the patient developed multiple metastases, he was treated with chemotherapy and then fulvestrant, before received a 200 mg twice daily BEZ235 and 2.5 mg weekly RAD001 combination regimen, when a left axillary nodal metastasis was developed on fulvestrant. The patient sustained a prolonged stable disease of 18 months while under the therapy before his tumor progressed again. Tumor biopsy samples (formalin fixed, paraffin embedded) were taken from the patient at diagnosis and after progression and analyzed by immunohistochemistry (IHC) and whole exome sequencing. Blood samples were collected for germline DNA pharmacokinetic (PK) analysis. PK profile of BEZ235 was collected with Cmax, AUC, and T1/2 of 766 ng/mL, 6308 ng.h/mL, and 5.53 h, respectively. The observed BEZ235 Cmin values ranged from 75.5 to 504 ng/mL over the study. The 2.5 mg weekly RAD001 dose is substantially lower than the standard 10 mg daily dose and the Cmin could not be reliably determined. IHC assays showed low ER/PR expression (ER 30% 1+; PR 20% 1+, 10% 3+) in the diagnosis sample, but an increase of their expression (60% 1+) in the post progression sample. HER2 expression was negative in both samples. Analysis the same tumor samples using more quantitative AQUA platform demonstrated a drastic 10-fold score increase of ER from 31 to 312 and a 5-fold increase of PR from 271 to 1305. All other examined signaling pathway biomarkers (e.g. pS6, pAKT, pMAPK, pMEK and pEGFR) were expressed in both tumor samples but showed minimal expression changes between the time points. Whole exome sequencing of the diagnosis and post-progression specimens provided average coverage at 142X and 179X, respectively . No functional somatic alterations resulting in hyperactive PI3K/mTOR pathway was detected in genes such as PIK3CA, PTEN, MTOR, TSC1/2 in either tumor samples. In both specimens, a somatic deletion of 16 bp was detected in SPEN, which encodes a potential negative regulator of the estrogen signaling pathway1 . In conclusion, no PI3K/mTOR pathway hyperactivity marker was identified in the diagnostic samples. However, the increased ER/PR expression in the post-progression sample suggests that the effect of PI3K/mTOR pathway blockade by RAD001/BEZ235 might have diminished when the tumor gained further dependence on the hormonal receptor signaling. This hypothesis was supported by the observation that the patient9s tumor achieved another prolonged disease control of about 13 months by exemestane, after progression from BEZ235/RAD001 treatment. 1Legare S, et al. SPEN is a novel candidate tumor suppressor gene that regulates response to tamoxifen in estrogen receptor positive breast cancers. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013. Citation Format: A. Rose Brannron, Davide Melisi, Jennifer Hummel, Carmine Carbone, Melissa Frizziero, Wing Cheung, Parul Patel, Jorge Gallo, Giampaolo Tortora, Michael Morrissey, David Chen. Biomarker analysis of a male breast cancer patient with prolonged stable disease under mTOR/PI3K inhibitors BEZ235/RAD001. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 26.