Ryan S. Alden

Association Between Plasma Genotyping and Outcomes of Treatment With Osimertinib (AZD9291) in Advanced Non–Small-Cell Lung Cancer

PURPOSE Third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have demonstrated potent activity against TKI resistance mediated by EGFR T790M. We studied whether noninvasive genotyping of cell-free plasma DNA (cfDNA) is a useful biomarker for prediction of outcome from a third-generation EGFR-TKI, osimertinib. METHODS Plasma was collected from all patients in the first-in-man study of osimertinib. Patients who were included had acquired EGFR-TKI resistance and evidence of a common EGFR-sensitizing mutation. Genotyping of cell-free plasma DNA was performed by using BEAMing. Plasma genotyping accuracy was assessed by using tumor genotyping from a central laboratory as reference. Objective response rate (ORR) and progression-free survival (PFS) were analyzed in all T790M-positive or T790M-negative patients. RESULTS Sensitivity of plasma genotyping for detection of T790M was 70%. Of 58 patients with T790M-negative tumors, T790M was detected in plasma of 18 (31%). ORR and median PFS were similar in patients with T790M-positive plasma (ORR, 63%; PFS, 9.7 months) or T790M-positive tumor (ORR, 62%; PFS, 9.7 months) results. Although patients with T790M-negative plasma had overall favorable outcomes (ORR, 46%; median PFS, 8.2 months), tumor genotyping distinguished a subset of patients positive for T790M who had better outcomes (ORR, 69%; PFS, 16.5 months) as well as a subset of patients negative for T790M with poor outcomes (ORR, 25%; PFS, 2.8 months). CONCLUSION In this retrospective analysis, patients positive for T790M in plasma have outcomes with osimertinib that are equivalent to patients positive by a tissue-based assay. This study suggests that, upon availability of validated plasma T790M assays, some patients could avoid a tumor biopsy for T790M genotyping. As a result of the 30% false-negative rate of plasma genotyping, those with T790M-negative plasma results still need a tumor biopsy to determine presence or absence of T790M.

Prospective Validation of Rapid Plasma Genotyping for the Detection of EGFR and KRAS Mutations in Advanced Lung Cancer

IMPORTANCE Plasma genotyping of cell-free DNA has the potential to allow for rapid noninvasive genotyping while avoiding the inherent shortcomings of tissue genotyping and repeat biopsies. OBJECTIVE To prospectively validate plasma droplet digital PCR (ddPCR) for the rapid detection of common epidermal growth factor receptor (EGFR) and KRAS mutations, as well as the EGFR T790M acquired resistance mutation. DESIGN, SETTING, AND PARTICIPANTS Patients with advanced nonsquamous non-small-cell lung cancer (NSCLC) who either (1) had a new diagnosis and were planned for initial therapy or (2) had developed acquired resistance to an EGFR kinase inhibitor and were planned for rebiopsy underwent initial blood sampling and immediate plasma ddPCR for EGFR exon 19 del, L858R, T790M, and/or KRAS G12X between July 3, 2014, and June 30, 2015, at a National Cancer Institute-designated comprehensive cancer center. All patients underwent biopsy for tissue genotyping, which was used as the reference standard for comparison; rebiopsy was required for patients with acquired resistance to EGFR kinase inhibitors. Test turnaround time (TAT) was measured in business days from blood sampling until test reporting. MAIN OUTCOMES AND MEASURES Plasma ddPCR assay sensitivity, specificity, and TAT. RESULTS Of 180 patients with advanced NSCLC (62% female; median [range] age, 62 [37-93] years), 120 cases were newly diagnosed; 60 had acquired resistance. Tumor genotype included 80 EGFR exon 19/L858R mutants, 35 EGFR T790M, and 25 KRAS G12X mutants. Median (range) TAT for plasma ddPCR was 3 (1-7) days. Tissue genotyping median (range) TAT was 12 (1-54) days for patients with newly diagnosed NSCLC and 27 (1-146) days for patients with acquired resistance. Plasma ddPCR exhibited a positive predictive value of 100% (95% CI, 91%-100%) for EGFR 19 del, 100% (95% CI, 85%-100%) for L858R, and 100% (95% CI, 79%-100%) for KRAS, but lower for T790M at 79% (95% CI, 62%-91%). The sensitivity of plasma ddPCR was 82% (95% CI, 69%-91%) for EGFR 19 del, 74% (95% CI, 55%-88%) for L858R, and 77% (95% CI, 60%-90%) for T790M, but lower for KRAS at 64% (95% CI, 43%-82%). Sensitivity for EGFR or KRAS was higher in patients with multiple metastatic sites and those with hepatic or bone metastases, specifically. CONCLUSIONS AND RELEVANCE Plasma ddPCR detected EGFR and KRAS mutations rapidly with the high specificity needed to select therapy and avoid repeat biopsies. This assay may also detect EGFR T790M missed by tissue genotyping due to tumor heterogeneity in resistant disease.

Bias-Corrected Targeted Next-Generation Sequencing for Rapid, Multiplexed Detection of Actionable Alterations in Cell-Free DNA from Advanced Lung Cancer Patients

Purpose: Tumor genotyping is a powerful tool for guiding non–small cell lung cancer (NSCLC) care; however, comprehensive tumor genotyping can be logistically cumbersome. To facilitate genotyping, we developed a next-generation sequencing (NGS) assay using a desktop sequencer to detect actionable mutations and rearrangements in cell-free plasma DNA (cfDNA). Experimental Design: An NGS panel was developed targeting 11 driver oncogenes found in NSCLC. Targeted NGS was performed using a novel methodology that maximizes on-target reads, and minimizes artifact, and was validated on DNA dilutions derived from cell lines. Plasma NGS was then blindly performed on 48 patients with advanced, progressive NSCLC and a known tumor genotype, and explored in two patients with incomplete tumor genotyping. Results: NGS could identify mutations present in DNA dilutions at ≥0.4% allelic frequency with 100% sensitivity/specificity. Plasma NGS detected a broad range of driver and resistance mutations, including ALK, ROS1, and RET rearrangements, HER2 insertions, and MET amplification, with 100% specificity. Sensitivity was 77% across 62 known driver and resistance mutations from the 48 cases; in 29 cases with common EGFR and KRAS mutations, sensitivity was similar to droplet digital PCR. In two cases with incomplete tumor genotyping, plasma NGS rapidly identified a novel EGFR exon 19 deletion and a missed case of MET amplification. Conclusions: Blinded to tumor genotype, this plasma NGS approach detected a broad range of targetable genomic alterations in NSCLC with no false positives including complex mutations like rearrangements and unexpected resistance mutations such as EGFR C797S. Through use of widely available vacutainers and a desktop sequencing platform, this assay has the potential to be implemented broadly for patient care and translational research. Clin Cancer Res; 22(4); 915–22. ©2015 AACR. See related commentary by Tsui and Berger, p. 790

Acquired METD1228V Mutation and Resistance to MET Inhibition in Lung Cancer

Amplified and/or mutated MET can act as both a primary oncogenic driver and as a promoter of tyrosine kinase inhibitor (TKI) resistance in non-small cell lung cancer (NSCLC). However, the landscape of MET-specific targeting agents remains underdeveloped, and understanding of mechanisms of resistance to MET TKIs is limited. Here, we present a case of a patient with lung adenocarcinoma harboring both a mutation in EGFR and an amplification of MET, who after progression on erlotinib responded dramatically to combined MET and EGFR inhibition with savolitinib and osimertinib. When resistance developed to this combination, a new MET kinase domain mutation, D1228V, was detected. Our in vitro findings demonstrate that METD1228V induces resistance to type I MET TKIs through impaired drug binding, while sensitivity to type II MET TKIs is maintained. Based on these findings, the patient was treated with erlotinib combined with cabozantinib, a type II MET inhibitor, and exhibited a response. SIGNIFICANCE With several structurally distinct MET inhibitors undergoing development for treatment of NSCLC, it is critical to identify mechanism-based therapies for drug resistance. We demonstrate that an acquired METD1228V mutation mediates resistance to type I, but not type II, MET inhibitors, having therapeutic implications for the clinical use of sequential MET inhibitors. Cancer Discov; 6(12); 1334-41. ©2016 AACR.See related commentary by Trusolino, p. 1306This article is highlighted in the In This Issue feature, p. 1293.

Discrimination of germline EGFR T790M mutations in plasma cell-free DNA allows study of prevalence across 31,414 cancer patients

Purpose: Plasma cell-free DNA (cfDNA) analysis is increasingly used clinically for cancer genotyping, but may lead to incidental identification of germline-risk alleles. We studied EGFR T790M mutations in non–small cell lung cancer (NSCLC) toward the aim of discriminating germline and cancer-derived variants within cfDNA. Experimental Design: Patients with EGFR-mutant NSCLC, some with known germline EGFR T790M, underwent plasma genotyping. Separately, deidentified genomic data and buffy coat specimens from a clinical plasma next-generation sequencing (NGS) laboratory were reviewed and tested. Results: In patients with germline T790M mutations, the T790M allelic fraction (AF) in cfDNA approximates 50%, higher than that of EGFR driver mutations. Review of plasma NGS results reveals three groups of variants: a low-AF tumor group, a heterozygous group (∼50% AF), and a homozygous group (∼100% AF). As the EGFR driver mutation AF increases, the distribution of the heterozygous group changes, suggesting increased copy number variation from increased tumor content. Excluding cases with high copy number variation, mutations can be differentiated into somatic variants and incidentally identified germline variants. We then developed a bioinformatic algorithm to distinguish germline and somatic mutations; blinded validation in 21 cases confirmed a 100% positive predictive value for predicting germline T790M. Querying a database of 31,414 patients with plasma NGS, we identified 48 with germline T790M, 43 with nonsquamous NSCLC (P < 0.0001). Conclusions: With appropriate bioinformatics, plasma genotyping can accurately predict the presence of incidentally detected germline risk alleles. This finding in patients indicates a need for genetic counseling and confirmatory germline testing. Clin Cancer Res; 23(23); 7351–9. ©2017 AACR.

Designing a definitive trial for adjuvant targeted therapy in genotype defined lung cancer: the ALCHEMIST trials

Genotype-directed targeted therapies have revolutionized the treatment of metastatic non-small cell lung cancer (NSCLC) but they have not yet been comprehensively studied in the adjuvant setting. Previous trials of adjuvant targeted therapy in unselected early stage NSCLC patients showed no benefit versus placebo, however retrospective data suggests improved disease free survival (DFS) with epidermal growth factor receptor (EGFR) inhibitors in patients with appropriate molecular alterations. A definitive prospective, randomized, placebo-controlled trial of targeted therapies for NSCLC is needed to determine the efficacy of targeted therapy following surgical resection and standard adjuvant therapy. The principal challenges facing such a trial are (I) identification of actionable alterations in early stage patients; and (II) realization of sufficient enrollment to power definitive analyses. The ALCHEMIST trial (Adjuvant Lung Cancer Enrichment Marker Identification and Sequencing Trial) was designed to overcome these challenges. Using the national clinical trials network (NCTN) of the National Cancer Institute (NCI), several thousand patients with operable NSCLC will undergo tumor genotyping for EGFR mutations or rearrangement of anaplastic lymphoma kinase (ALK). Following resection and completion of standard adjuvant therapy, patients with EGFR-mutant NSCLC will be randomized to erlotinib versus placebo (1:1), those with ALK-rearranged NSCLC will be randomized to crizotinib versus placebo (1:1), while those not enrolled onto the adjuvant trials will continue to be followed on the screening trial. ALCHEMIST also provides for the collection of tissue at baseline and at recurrence (if available) to characterize mechanisms of recurrence and of resistance to targeted therapy. Thus, ALCHEMIST is a platform for validation of targeted therapy as part of curative care in NSCLC and creates an opportunity to advance our understanding of disease biology.

135O_PR: Plasma genotyping for predicting benefit from osimertinib in patients (pts) with advanced NSCLC

generation sequencing (NGS) analysis was also performed on these plasma samples. Methods: Agreement (positive and negative) between the cobas tissue test and the cobas plasma test, for detection of EGFR mutations, was calculated in the pooled Phase II analysis set. Agreement between the cobas plasma test and NGS analysis of plasma was calculated using samples from AURA2 pts. Results: In the pooled analysis, the positive percentage agreement (PPA) and negative percentage agreement (NPA) between the cobas tissue test and plasma test were 61.4% and 78.6%, respectively for detection of T790M. In AURA2, the PPA and NPA between the cobas plasma test and NGS analysis of plasma were 91.5% and 91.1%, respectively. As of May 2015, comparable ORR was observed in the subset of pts with a positive T790M plasma test as for all patients selected using the cobas tissue test. Common sensitising mutations were also analysed. PPA and NPA between the cobas tissue test and plasma test were 75.6% and 98.1%, respectively, for the L858R mutation, and 85.1% and 98.0%, respectively, for exon 19 deletions. Conclusions: Data indicate that approximately 60% of pts with T790M positive NSCLC, the biomarker against which treatment with osimertinib is targeted, could have avoided an invasive biopsy by use of a plasma test. However, for EGFR-TKI-resistant pts, without detectable T790M in plasma, a tissue-based test is advised to address the potential for false negative results from the plasma test. These results indicate the utility of both plasmaand tissue-based tests in the diagnostic setting. Clinical trial identification: NCT01802632 and NCT02094261 (Release dates 25 February 2013 and 17 March 2014) Legal entity responsible for the study: AstraZeneca Funding: AstraZeneca Disclosure: S. Jenkins, S. Patel, S. Weston, R. Lawrance, M. Cantarini: Employee and shareholder: AstraZeneca. J. Yang: Advisory boards: Boehringer Ingelheim, Eli Lilly, Bayer, Roche/Genentech, AstraZeneca, Astellas, MSD, Merck Serono, Pfizer, Novartis, Clovis Oncology, Celgene. S. Ramalingam: Consultancy fees: AstraZeneca, Boehringer Ingelheim, Celgene, Genentech, Novartis, Lilly, Merck, Bristol-Myers Squibb. K. Yu: Employee: Roche Molecular Systems, Inc. P. Jänne: Consultancy fees: AstraZeneca, Pfizer, Roche Research support: AstraZeneca, Astellas Pharmaceuticals Stock ownership: Gatekeeper Pharmaceuticals Other: Post marketing royalties on DFCI owned patent on EGFR mutations licensed to Lab Corp. T. Mitsudomi: Advisory board: AstraZeneca, Boehringer-Ingelheim, Chugai, Pfizer Honoraria: AstraZeneca, Chugai, Boehringer-Ingelheim, Pfizer Research fund: BoehringerIngelheim, Chugai, Pfizer.

Abstract 4342: Ultra-deep next generation sequencing (NGS) of plasma cell-free DNA (cfDNA) from patients with advanced lung cancers: results from the Actionable Genome Consortium

Introduction: Noninvasive genotyping using plasma cfDNA from cancer patients has the potential to obviate the need for some biopsies while also characterizing disease heterogeneity. This study was undertaken to develop an ultra-deep plasma NGS panel for patients with non-small cell lung cancers (NSCLC). Methods / Results: Plasma was prospectively collected from 51 patients with advanced, progressive NSCLC and a known oncogenic driver from prior tumor genotyping. We performed ultra-deep NGS on extracted cfDNA using a customized Illumina library preparation, hybrid capture panel covering 37 lung cancer related genes (complete exons and partial introns), and ultra-deep sequencing (HiSeq4000). Mean sequencing depth was ∼50,000X (150 million, 150bp reads per sample). After specialized consensus-based error correction for low allele frequency (AF) genomic alterations, the median unique DNA molecules per position were ∼3,500. The mean sequence error rate was reduced by 20-fold to 0.002%, enabling the confident call of a driver mutation as low as 0.03%. In a subset of cases, paired plasma droplet digital PCR (ddPCR) was performed for common EGFR and KRAS mutations using a validated assay. Blinded to tumor genotype, plasma NGS detected SNVs (EGFR, KRAS, BRAF), indels (EGFR, ERBB2), and fusions (ALK, ROS1) as well as significant copy number gains (CNG) (ERBB2, MET). Sensitivity of cfDNA for the detection of known oncogenic drivers was 88% (45/51). A single false positive driver mutation was identified in a case with a known EGFR mutation in tumor; plasma NGS found both EGFR exon 19 del (0.88% AF) and KRAS G12D (2.65% AF), and plasma ddPCR confirmed the presence of both mutations (2.2% and 2.0% AF). Evaluation for an occult second primary is ongoing. In 22 EGFR, ALK, or ROS1 cases with acquired resistance to targeted therapy, plasma genotyping detected a range of potential resistance mechanisms: EGFR T790M and C797S, ALK F1174C, ERBB2 CNG, MET CNG. In 16 cases with paired resistance biopsies, concordance for EGFR T790M status was 94% (15/16). 18 cases with known EGFR or KRAS mutations underwent paired ddPCR. In 14 cases the driver mutation was detected using both assays with high concordance of the%AF (r = 0.91). The remaining 4 cases were negative with ddPCR but 3 were positive with NGS at low AF (0.04%, 0.08%, and 0.29%), and the specificity for each driver was 100%. Conclusions: Ultra-deep plasma NGS can detect a wide range of oncogenic drivers in NSCLC and may be more sensitive than established ddPCR assays. In the setting of acquired resistance to targeted therapy, plasma NGS reliably captured EGFR T790M and additional somatic alterations as potential resistance mechanisms. Citation Format: Bob T. Li, Filip Janku, Pasi A. Janne, Gordon B. Mills, Kiran Madwani, Ryan S. Alden, Cloud P. Paweletz, Marc Ladanyi, Alex Aravanis, Byoungsok Jung, Sante Gnerre, Amy J. Sehnert, David B. Solit, Gregory J. Riely, Geoffrey R. Oxnard. Ultra-deep next generation sequencing (NGS) of plasma cell-free DNA (cfDNA) from patients with advanced lung cancers: results from the Actionable Genome Consortium. [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 4342.

Abstract B104: Differentiating somatic and germline variants using targeted next-generation sequencing (NGS) of cell-free plasma DNA (cfDNA)

Introduction: Genomic analysis of cfDNA is emerging as a powerful tool for noninvasive characterization of advanced cancer. Some cfDNA assays are quantitative, allowing analysis of genomic subpopulations. Here, we sought to use quantitative cfDNA analysis to distinguish somatic and germline variants, gaining insight into cancer biology as well as inherited risk without needing paired germline DNA. To do this, we studied EGFR mutations in cfDNA aiming to distinguish those with somatic T790M, generally acquired after resistance to targeted therapy, from those with germline T790M, a rare allele associated with inherited lung cancer risk. Methods & Results: We first explored an institutional cohort of lung cancer patients undergoing cfDNA genotyping using droplet digital PCR (ddPCR). We identified 64 patients positive for T790M and a concurrent EGFR driver mutation by ddPCR. For the vast majority of cases, the mutant allelic fraction (MAF) of T790M was lower than the MAF of the driver mutation, resulting in a T790M/driver ratio of 0.001-1. For two cases, T790M was the predominant mutation, with T790M/driver ratios of 4.2 and 54.6; MAF of T790M was 49% and 53% for these cases. Under an IRB-approved protocol (NCT01754025), we performed sequencing of DNA extracted from PBMCs which confirmed germline T790M in both. We then submitted cfDNA from 3 patients with lung cancer and germline T790M for blinded NGS using the 68-gene Guardant360 assay. In each, NGS identified over 90 coding and non-coding variants. T790M was identified at a high MAF (56%, 50%, 49%) in the range of other recognized SNPs, while an EGFR driver mutation (L858R or L861Q) was identified at a lower level (23%, 4%, 1%) in the range of coding TP53 mutations. Each case was treated with a third-generation EGFR kinase inhibitor targeting T790M. NGS of post-treatment cfDNA demonstrated the expected response in the EGFR driver mutation (0.2%, 0%, 0%) but minimal change in the high MAF T790M (50%, 49%, 49%), consistent with persistent shed of germline DNA on therapy. To broadly screen for germline T790M carriers, we queried a cohort of 1082 lung cancer patients who had undergone NGS of cfDNA using Guardant360. 74 were positive for EGFR T790M (median MAF 3%, range 0.2%-51%). 58 also harbored a second EGFR driver mutation (median MAF 5%), with 23 also having EGFR amplification (median MAF 16%). Four cases were identified with high MAF T790M in the expected range of SNPs (51%, 49%, 49%, 48%), but no driver EGFR mutations were identified at this high MAF. These 4 cases have been referred for germline testing on a prospective study of germline T790M (NCT01754025). Conclusions: We have identified that quantitative cfDNA analysis with NGS can identify germline mutations through differentiation of high MAF germline variants from lower MAF somatic variants. In this cohort, 4 of 74 cases (5%) positive for T790M using NGS of cfDNA were consistent with a germline mutation. This ability to differentiate germline from somatic variants differentiates NGS of cfDNA from NGS of tumor specimens, where these distinctions are challenging without paired germline DNA. Diagnostic labs performing plasma NGS will need to be vigilant to identify these potential germline alterations, and will need strategies for reporting these to providers and patients as appropriate. Citation Format: Geoffrey R. Oxnard, Adrian G. Sacher, Ryan S. Alden, Nora B. Feeney, Jennifer C. Heng, Rebecca J. Nagy, Richard B. Lanman, Cloud P. Paweletz, Pasi A. Janne. Differentiating somatic and germline variants using targeted next-generation sequencing (NGS) of cell-free plasma DNA (cfDNA). [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 B104.