Elaina Mann

Abstract B136: Plasma EGFR mutation detection using a combined exosomal RNA and circulating tumor DNA approach in patients with acquired resistance to first-generation EGFR-TKIs

Background: After initial responses to tyrosine kinase inhibitors (TKIs), NSCLC patients (pts) harboring EGFR activating mutations inevitably progress, with the “gatekeeper” EGFR T790M resistance mutation accounting for approximately 60% of cases of acquired resistance (AR) to TKIs. EGFR activating and T790M resistance mutations can be found in plasma on both circulating free tumor DNA (ctDNA) and on RNA contained within exosomes. While ctDNA is thought to be primarily released by dying cells, exosome RNA is actively released by many living cells (Jahr et al. Cancer Res 2001; Thery et al. Nat Rev Immunol 2009). Some pts, particularly those with either early stage or intra-thoracic disease, do not seem to release mutations on ctDNA into circulation that is detectable by any current method. Here we present data demonstrating the detection of activating and AR EGFR mutations using a combined single-step exosomal RNA (exoRNA) and ctDNA approach to maximize sensitivity and demonstrate the ability to detect mutations using exosomal RNA on pts previously described as negative by ctDNA methods alone. Methods: Matched pretreatment tumor tissue and plasma were collected from 81 NSCLC pts enrolled in TIGER-X, a Ph1/2 study of rociletinib in previously treated mutant EGFR pts with advanced NSCLC. Among the 81 pts (all enrolled before Dec 2014), 56 cases were randomly chosen from the clinical patient population (including 35 cases ≥10 mutant copies/mL and 21 cases Results: For the 56 cases randomly chosen from the clinical patient population, 54 had valid tumor tissue results. The positive percent agreement (PPA) between plasma and tumor was 96% (52/54) for activating mutations and 86% (42/49) for T790M with tumor as the reference method. For most cases analyzed, the combined mutation signal from exoRNA and ctDNA was greater than the signal from ctDNA alone. Furthermore, we detect mutations in the circulation of some pts who were previously called negative by analysis of ctDNA alone, suggesting improved sensitivity from addition of exoRNA to the analysis. In the subset of pts with low or undetectable levels of ctDNA and valid tumor results (N = 45), we detect activating mutations in 38 of 45 cases (PPA 84%) compared to 27 of 45 (PPA 60%) by ctDNA alone, as well as T790M in 22 of 35 evaluable cases (PPA 63%) compared to 19 of 35 in ctDNA alone (PPA 54%). Conclusions: Our data demonstrate the ability to detect low copy numbers of activating and AR mutations in plasma of lung cancer pts by combining the mutation signal from exoRNA and ctDNA isolated by EXO52 and using the EXO1000 targeted NGS gene panel. By combining these two analytes, a higher sensitivity of mutation detection may be possible compared to analysis by ctDNA alone. Citation Format: Anne K. Krug, Chris Karlovich, Tina Koestler, Kay Brinkmann, Alexandra Spiel, Jennifer Emenegger, Mikkel Noerholm, Vince O9Neill, Lecia V. Sequist, Jean-Charles Soria, Jonathan W. Goldman, D. Ross Camidge, Heather A. Wakelee, Shirish M. Gadgeel, Elaina Mann, Shannon Matheny, Lindsey Rolfe, Mitch Raponi, Daniel Enderle, Johan Skog. Plasma EGFR mutation detection using a combined exosomal RNA and circulating tumor DNA approach in patients with acquired resistance to first-generation EGFR-TKIs. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B136.

Abstract 927: Pretreatment and serial plasma assessments of EGFR mutations in NSCLC patients treated with rociletinib (CO-1686)

Background: EGFR mutation testing is required to identify patients who may respond to TKI therapy. However, tumor biopsies from NSCLC patients can pose challenges for molecular analyses due to inadequate sample material, the inability to biopsy patients due to poor health status or inaccessible lesions, and tumor heterogeneity. We examined the detection of EGFR mutations in circulating cell-free DNA (cfDNA) from plasma and the concordance of EGFR mutation status with contemporaneously matched tumor tissue in TIGER-X, a Phase 1/2 clinical study of rociletinib (CO-1686) in previously treated advanced NSCLC patients harboring EGFR mutations in their tumors. Rociletinib is an oral, potent, small-molecule irreversible tyrosine kinase inhibitor that selectively targets mutant forms of EGFR, including T790M, L858R and Del(19), while sparing wild-type EGFR. Methods: Pretreatment matched tumor tissue and plasma from 139 Stage IIIB/IV NSCLC patients enrolled in TIGER-X were evaluated for EGFR status. Tumor tissue was processed as FFPE and tested by allele-specific PCR. Plasma was tested as a two mL aliquot using BEAMing, a quantitative and sensitive detection technology based on digital PCR. Results: Using tissue as the reference, the positive percent agreement between tumor and BEAMing plasma test results was 86% (102/119) for activating mutations and 77% (78/101) for T790M. Four plasma samples of the 23 tumor T790M+/plasma T790M- cases were also tested by three independent plasma testing platforms with claimed analytical sensitivities of Conclusions: The BEAMing plasma test identified EGFR mutations detected in tumor with high sensitivity. In addition, plasma testing identified T790M+ patients that were determined T790M- by the tumor test, which may be in part explained by tumor heterogeneity and/or inadequate biopsy. These findings demonstrate that BEAMing can be a useful tool for the non-invasive assessment of EGFR mutations in NSCLC. Citation Format: Jonathan W. Goldman, Chris Karlovich, Elaina Mann, Lindsey Rolfe, Shannon Matheny, Darrin Despain, Philipp Angenendt, Claudia Stamm, Heather A. Wakelee, Jean-Charles Soria, Benjamin Solomon, D. R. Camidge, Rafal Dziadziuszko, Leora Horn, Shirish Gadgeel, Mitch Raponi, Andrew R. Allen, Lecia V. Sequist. Pretreatment and serial plasma assessments of EGFR mutations in NSCLC patients treated with rociletinib (CO-1686). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 927. doi:10.1158/1538-7445.AM2015-927

Abstract AP27: FEASIBILITY OF MONITORING RESPONSE TO THE PARP INHIBITOR RUCAPARIB WITH TARGETED DEEP SEQUENCING OF CIRCULATING TUMOR DNA (CTDNA) IN WOMEN WITH HIGH GRADE OVARIAN CARCINOMA ON THE ARIEL2 TRIAL

BACKGROUND:TP53 mutations are present in >97% cases of high-grade serous ovarian cancer (HGSOC). Detection of TP53 mutations in ctDNA extracted from plasma has the potential to monitor disease course and treatment response. We have developed targeted amplicon deep sequencing (TADS) to detect low frequency mutations throughout the TP53 gene in ctDNA. Rucaparib is a PARP inhibitor in development for treatment of tumors with HR pathway deficiency. We used TADS to assess TP53 mutant allele fraction (MAF) in ctDNA from patients in ARIEL2, a phase 2 study of rucaparib for treatment of relapsed high-grade ovarian cancer (NCT01891344). MATERIAL AND METHODS: Plasma samples (n=65) from 18 patients were collected during screening, on day 1 of each cycle, and at the end of rucaparib treatment. DNA extracted from plasma underwent TADS of TP53 (median depth 6916×). FFPE tumor specimens were profiled using an NGS-based assay with a targeted gene panel including TP53. Investigator-assessed clinical response rates were evaluated by RECIST v1.1 and GCIG CA-125 criteria. RESULTS: Concordant TP53 mutations were detected in tumor and ctDNA from plasma for all 18 patients. Median TP53 MAF at screening and cycle 1 day 1 was 5.1% (interquartile range: 1.1–17.5, n=16) and 3.8% (IQR: 0.68–10.3, n=16), respectively. Fourteen patients were evaluable for response measured by quantification of TP53 MAF between cycle 1 and 2 (missing sample: n=2; TP53 MAF 50% reduction of TP53 MAF in ctDNA at cycle 2 achieved a RECIST confirmed PR (see Table); this included 5/6 patients with either a germline or somatic mutation in BRCA1/BRCA2. No patients with CONCLUSIONS: Noninvasive detection of TP53 mutations by TADS is feasible, using plasma samples collected from women with relapsed platinum-sensitive high-grade ovarian cancer participating in an international multicenter trial. Circulating tumor DNA is a promising biomarker for monitoring response to the PARP inhibitor rucaparib. We are now testing the pre-specified hypothesis that a >50% reduction in TP53 MAF between baseline and cycle 2 is predictive of response to rucaparib using 560 plasma samples from 139 ARIEL2 subjects. Updated results will be presented at the meeting. Citation Format: Anna Piskorz, Kevin K. Lin, James Morris, Elaina Mann, Amit Oza, Robert L. Coleman, David M. O9Malley, Michael Friedlander, Janiel M. Cragun, Ling Ma, Heidi Giordano, Nitzan Rosenfeld, Mitch Raponi, Iain A. McNeish, Elizabeth Swisher, James D. Brenton. FEASIBILITY OF MONITORING RESPONSE TO THE PARP INHIBITOR RUCAPARIB WITH TARGETED DEEP SEQUENCING OF CIRCULATING TUMOR DNA (CTDNA) IN WOMEN WITH HIGH GRADE OVARIAN CARCINOMA ON THE ARIEL2 TRIAL [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP27.

Abstract AP28:BRCA1andRAD51CPromoter Hypermethylation Confer Sensitivity to PARP Inhibitors in Patients with Platinum Sensitive Ovarian Carcinoma

Germline and somatic mutations in BRCA1 and BRCA2 (BRCA) confer PARP inhibitor sensitivity. Promoter hypermethylation is an alternate mechanism of gene down-regulation, and BRCA1 promoter methylation is relatively common in sporadic ovarian cancer. The clinical significance of BRCA1 methylation is less clear than for mutations, as the Cancer Genome Atlas (TCGA) and others have failed to show improved survival in ovarian carcinomas with BRCA1 methylation. No one has previously tested whether BRCA1 methylation confers in vivo sensitivity to PARP inhibitors in patients with ovarian cancer. ARIEL2 is a phase 2 study of the PARP inhibitor rucaparib in patients with recurrent platinum sensitive high-grade ovarian, peritoneal or fallopian tube carcinoma. At enrollment, ARIEL2 required pre-treatment tumor biopsies with the goal of developing tissue predictors of PARP inhibitor sensitivity other than BRCA mutations. The number of women with known germline mutations was capped at 15 patients in order to predominantly enroll BRCA wildtype cases. As presented at ASCO 2016, in cases with no BRCA mutations, a high fraction of genomic loss of heterozygosity (LOH) significantly predicted a better progression-free survival (the primary endpoint), longer duration of response, and a higher fraction of responders compared to cases with low LOH. We assessed BRCA1 and RAD51C promoter hypermethylation using methylation-sensitive polymerase chain reaction in paired archival and pre-treatment biopsies from patients on ARIEL2. Of 165 cases for which methylation analyses were completed, 21 (12.7%) were methylated at the BRCA1 promoter and four (2.4%) at the RAD51C promoter. Methylation of BRCA1 and RAD51C was mutually exclusive with mutation in BRCA or other homologous recombination genes. All four cases with RAD51C methylation and 15/19 (78.9%) with BRCA1 methylation were associated with high LOH. In 90 paired samples archival and pre-treatment tissues, RAD51C methylation was 100% concordant and BRCA1 methylation was highly concordant (p Citation Format: Elizabeth Swisher, Maria Harrell, Kevin K. Lin, Clare Scott, Sandra Goble, Amit Oza, Robert L. Coleman, Gottfried Konecny, Anna V. Tinker, David M. O9Malley, Rebecca Kristeleit, Ling Ma, James Brenton, Katherine Bell-McGuinn, Ana Oaknin, Alexandra Leary, Elaina Mann, Heidi Giordano, Mitch Rapon, Iain McNeish, Scott H. Kaufmann. BRCA1 and RAD51C Promoter Hypermethylation Confer Sensitivity to PARP Inhibitors in Patients with Platinum Sensitive Ovarian Carcinoma [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP28.

Abstract A11: NGS-based tumor genomic profiling to identify ovarian cancer patients who benefit from the PARP inhibitor rucaparib.

Background: PARP inhibitors (PARPi) are synthetically lethal to tumor cells with homologous recombination deficiency (HRD). HRD can result from deleterious BRCA1/2 mutations (BRCAmut) or other mechanisms that have not been fully elucidated. Regardless of mechanism, HRD leads to a common phenotype of genome-wide loss of heterozygosity (LOH). It has been hypothesized that this genomic phenotype can be used to identify BRCA wild-type (BRCAwt) HRD tumors likely sensitive to PARPi. Using comprehensive next generation sequencing (NGS)-based tumor genomic profiling, we developed an HRD assay for potential use as a companion diagnostic for rucaparib in high-grade ovarian cancer (HGOC) by combining tumor BRCA1/2 status and quantification of genomic LOH. Methods: In the phase 2 study ARIEL2 Part 1 (NCT01891344), pre-treatment screening biopsies and archival formalin-fixed paraffin embedded tumor specimens were profiled using Foundation Medicine9s NGS-based HRD assay, which detects all classes of genomic alterations, including base substitutions, insertions/deletions, and homozygous deletions in BRCA1/2. Genomic LOH was assessed by sequencing >3,500 evenly-distributed single nucleotide polymorphisms across the genome and quantifying the extent of genomic LOH. A pre-specified genomic LOH cutoff was determined using publicly available SNP array data of ovarian tumors to predict platinum sensitivity as a surrogate marker for PARPi sensitivity. Response was assessed by RECIST v1.1 and GCIG CA-125 response criteria. Results: As of July 1 2015, 195 archival tumor and 152 screening biopsy samples (142 matched pairs) from 206 HGOC patients enrolled (204 patients treated) in ARIEL2 Part 1 were successfully profiled using the NGS-based HRD assay. Some screening biopsies were not suitable for successful NGS-based HRD assessment primarily because of insufficient tumor nuclei or inadequate tumor volume. Most matched pairs of archival and pre-trial screening samples exhibited similar genomic LOH profiles (r=0.86); however, 14% of screening samples had higher genomic LOH compared with archival samples collected more than one year earlier. All BRCA1/2 germline and somatic mutated tumors had high genomic LOH in the screening samples. Receiver operating characteristic analysis of genomic LOH showed utility in identifying RECIST/CA-125 responders to rucaparib (AUC=0.72, p Conclusions: We developed an NGS-based HRD assay that assesses tumor BRCA1/2 and genomic LOH to prospectively identify HGOC patients who may benefit from rucaparib treatment. The optimized NGS-based HRD assay will be prospectively tested in the ongoing portion of the phase 2 study (ARIEL2 Part 2, NCT01891344) and a phase 3 maintenance study (ARIEL3, NCT01968213) that will investigate rucaparib in HGOC. Citation Format: Iain A. McNeish, Kevin K. Lin, James X. Sun, Sandra Goble, Amit Oza, Robert L. Coleman, Clare L. Scott, Gottfried Konecny, Anna V. Tinker, David M. O9Malley, Rebecca Kristeleit, Ling Ma, James D. Brenton, Katherine Bell-McGuinn, Ana Oaknin, Alexandra Leary, Elaina Mann, Heidi Giordano, Roman Yelensky, Mitch Raponi, Elizabeth Swisher. NGS-based tumor genomic profiling to identify ovarian cancer patients who benefit from the PARP inhibitor rucaparib. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A11.

Abstract A31: Assessment of EGFR mutations in matched urine, plasma and tumor tissue in NSCLC patients treated with rociletinib (CO-1686)

Background: The acquisition of suitable tumor tissue is a challenge for a significant fraction of late-stage NSCLC patients who require EGFR testing to inform choice of therapy. An alternative for these patients could be the assessment of EGFR mutations in circulating tumor DNA (ctDNA). In this study, we examined the detection of EGFR T790M mutation in ctDNA from urine, assessed urine sample requirements, and compared the results with contemporaneously matched tumor tissue and plasma in TIGER-X (NCT01526928), a Phase 1/2 clinical study of rociletinib in previously treated mutant EGFR patients with advanced NSCLC. Rociletinib is an oral, potent, small-molecule irreversible tyrosine kinase inhibitor that selectively targets mutant forms of EGFR, including T790M, L858R and Del(19), while sparing wild-type EGFR. Methods: 63 Stage IIIB/IV NSCLC patients enrolled in either Phase 1 or 2 components of TIGER-X and representing all therapeutic dose groups consented to optional urine collection. Maximum sample volumes were 100 mL for urine and 2 mL for plasma. To maximize assay sensitivity in urine, samples containing the recommended sample volume of ≥90 mL (≥ 90% of maximum in this study) were evaluated; all samples received were processed to assess this recommendation. Urinary and plasma ctDNA were tested for mutations by the same EGFR assays using a sensitive and quantitative short footprint assay method that employs a mutation enrichment step followed by next generation sequencing. Results: Urine volumes ranged from 8-100 mL with a median DNA yield of 313 ng (N = 63). The median DNA yield was 299 ng for urine specimens with volume Conclusions: The analysis of ctDNA from urine identified a similar proportion of T790M+ patients as tissue-based testing with highest PPA amongst patients with urine volumes ≥90 mL. Discordant samples between urine and tissue that were not identified by the tumor test may be explained by tumor heterogeneity and/or inadequate biopsy. EGFR mutation detection from urine increases with urine volume and DNA yields and should be considered as a viable approach, particularly when tumor tissue is not available. Lastly, monitoring urine ctDNA T790M mutations longitudinally with baseline and post-therapy sampling could be clinically useful to determine benefit from therapy. Citation Format: Shirish Gadgeel, Chris Karlovich, Vlada Melnikova, Lecia V. Sequist, D. Ross Camidge, Heather Wakelee, Maurice Perol, Geoffrey R. Oxnard, Karena Kosco, Cecile Rose T. Vibat, Elaina Mann, Shannon Matheny, Lindsey Rolfe, Mitch Raponi, Mark G. Erlander, Karen Reckamp. Assessment of EGFR mutations in matched urine, plasma and tumor tissue in NSCLC patients treated with rociletinib (CO-1686). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A31.