Plasma Droplet Digital PCR for Detection of EGFR and KRAS Mutations in Patients With Advanced Nonsquamous NSCLC Slideset on: Sacher AG, Paweletz C, Dahlberg SE, et al. Prospective validation of rapid plasma genotyping for the detection of EGFR and KRAS mutations in advanced lung cancer. JAMA Oncol. 2016;[Epub ahead of print]. NSCLC, non-small-cell lung cancer. This activity is supported by educational grants from Genentech, Lilly, and Novartis Pharmaceuticals Corporation.

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Background: Genotyping of Targetable EGFR Mutations in Advanced NSCLC Choice of optimal therapy for advanced NSCLC pts depends on rapid, accurate detection of targetable mutations[1] Standard tissue genotyping can have slow turnaround time, is invasive, and is limited by tissue availability, heterogeneity, and potential for failed biopsies[2,3] Plasma genotyping is rapid, noninvasive alternative to tissue biopsy; however, careful validation imperative for clinical application[2,3] ddPCR-based assay effective for detection of EGFR and KRAS mutations in cell-free plasma from pts with advanced lung cancer[4] Current study prospectively evaluated feasibility, accuracy of ddPCR- based plasma genotyping for clinical detection of EGFR and KRAS mutations in pts with advanced NSCLC[5] ddPCR, digital droplet polymerase chain reaction; NSCLC, non-small-cell lung cancer. 1. Li T, et al. J Clin Oncol. 2013;31:1039-1049. 2. Diaz LA Jr, et al. J Clin Oncol. 2014;32:579-586. 3. Haber DA, et al. Cancer Discov. 2014;4:650-661. 4. Oxnard GR, et al. Clin Cancer Res. 2014;15:1698-1705. 5. Sacher AG, et al. JAMA Oncol. 2016;[Epub ahead of print]. Slide credit: clinicaloptions.com

Study Design: Prospective Validation of Plasma ddPCR Genotyping for NSCLC Prospective plasma genotyping validation study of 2 cohorts from a NCI-designated comprehensive cancer center (N = 180) Eligible patients (n = 180) Excluded missed blood draw (n = 3) tissue genotyping failed (n = 3) TAT analysis (n = 174) Newly Diagnosed prior to systemic therapy (n = 115) Acquired EGFR Resistance scheduled for rebiopsy/retreatment (n = 59) Excluded tissue KRAS failed (n = 28) Excluded tissue T790M biopsy failed ddPCR, digital droplet polymerase chain reaction; NCI, National Cancer Institute; NSCLC, non-small-cell lung cancer. KRAS G12X analysis (n = 87) EGFR exon 19/L858R analysis (n = 174) EGFR T790M analysis (n = 54) *Assay turnaround time (TAT): from blood draw/test order to time of reporting. Slide credit: clinicaloptions.com Sacher AG, et al. JAMA Oncol. 2016;[Epub ahead of print].

Tissue Genotyping as Reference Standard for Validation of Plasma ddPCR Assay Tissue Genotype, % Cohort 1, Newly Diagnosed (n = 120) Cohort 2, Acquired Resistance* (n = 60) EGFR exon 19 del 12 62 EGFR L858R 11 30 Rare EGFR mutations 8 EGFR T790M 58 KRAS G12X 22 EGFR/KRAS wild type 53 Failed genotyping 3 ddPCR, digital droplet polymerase chain reaction. *May not total to 100% as T790M may occur with other EGFR mutations. True positive/negative: positive/negative in both tissue, plasma False positive: positive in plasma, negative in tissue False negative: negative in plasma, positive in tissue Slide credit: clinicaloptions.com Sacher AG, et al. JAMA Oncol. 2016;[Epub ahead of print].

Positive Predictive Value, Plasma ddPCR Assay Sensitivity, Specificity, and Positive Predictive Value Assay Sensitivity, % (95% CI) True Positive, n False Negative, Specificity, Positive Predictive Value, EGFR exon 19 del Newly diagnosed 86 (57-98) 12 2 100 (96-100) 101 100 (74-100) Acquired resistance 81 (64-92) 29 7 100 (85-100) 23 100 (88-100) Overall 82 (69-91) 41 9 100 (97-100) 124 100 (91-100) EGFR L858R 69 (39-91) 4 102 100 (66-100) 78 (52-94) 14 100 (77-100) 74 (55-88) 8 143 EGFR T790M 77 (60-90) 27 63 (38-84) 79 (62-91) KRAS G12X 64 (43-82) 16 100 (94-100) 62 100 (79-100) ddPCR, digital droplet polymerase chain reaction. Slide credit: clinicaloptions.com Sacher AG, et al. JAMA Oncol. 2016;[Epub ahead of print].

Sensitivity of Plasma ddPCR Higher in Pts With Metastases Increasing number of metastatic sites (P = .001) and presence of bone (P = .007), hepatic (P = .001) metastases significantly associated with assay sensitivity 100 80 60 Assay Sensitivity (%) 40 ddPCR, digital droplet polymerase chain reaction. 20 1 2 3 ≥ 4 Number of Metastatic Sites Slide credit: clinicaloptions.com Sacher AG, et al. JAMA Oncol. 2016;[Epub ahead of print].

Plasma ddPCR Assay Exhibits Wide Dynamic Range EGFR exon 19 del assay EGFR L858R assay EGFR T790M assay KRAS G12X assay 100,000 10,000 1,000 Mutation Conventration/mL of Plasma 100 10 ddPCR, digital droplet polymerase chain reaction. ND del19m Positive del19m Negative L858R Positive L858R Negative T790M Positive T790M Negative Kras Positive Kras Negative Tissue Genotype Each symbol represents 1 pt. False positives: none for EGFR exon 19 del/L858R and KRAS G12X; small number for EGFR T790M assay Slide credit: clinicaloptions.com Sacher AG, et al. JAMA Oncol. 2016;[Epub ahead of print].

Turnaround Time Shorter for Plasma ddPCR vs Tissue Genotyping Turnaround time shorter for plasma genotyping vs tissue genotyping (P < .001 for cohort 1) Plasma genotyping completed for all pts Repeat biopsies required for 19% of newly diagnosed pts and 21% of pts with acquired resistance Turnaround Time, Median Days (Range) Cohort 1, Newly Diagnosed (n = 115) Cohort 2, Acquired Resistance (n = 59) Plasma genotyping* 3 (1-7) 2 (1-4) Tissue genotyping† 12 (1-54) 27 (1-146) *Plasma turnaround time: business days from blood sampling to reporting. †Tissue turnaround time: date of initial order to date of first report; includes time for repeat biopsies. ddPCR, digital droplet polymerase chain reaction. Slide credit: clinicaloptions.com Sacher AG, et al. JAMA Oncol. 2016;[Epub ahead of print].

Changes in Mutant DNA in Response to Treatment Detectable by Plasma ddPCR EGFR sensitizing mutation EGFR sensitizing with T790M KRAS codon 12 mutation Undetectable at 2 Wks Decrease to Undetectable at 6 Wks Decrease but Still Detectable at 6 Wks 100,000 100,000 100,000 10,000 10,000 10,000 1,000 1,000 1,000 Mutation Concentration, copies/mL 100 100 100 10 10 10 ND ND ND Baseline 2 Wks 6 Wks Baseline 2 Wks 6 Wks Baseline 2 Wks 6 Wks Decrease With Rebound Initial Increase Then Decrease Progressive Increase 100,000 100,000 100,000 10,000 10,000 10,000 1,000 1,000 1,000 Mutation Concentration, copies/mL ddPCR, digital droplet polymerase chain reaction. 100 100 100 10 10 10 ND ND ND Baseline 2 Wks 6 Wks Baseline 2 Wks 6 Wks Baseline 2 Wks 6 Wks Discontinuation at initial and second restaging CT scans: 0 of 23 (0%) and 1 of 23 (4%) pts with complete resolution of mutant DNA; 9 of 27 (33% ) and 15 of 27 (56%) pts without complete resolution Slide credit: clinicaloptions.com Sacher AG, et al. JAMA Oncol. 2016;[Epub ahead of print].

Clinical Utility of Plasma ddPCR Genotyping: A Case Study 24-day delay in therapy initiation for an elderly woman with erlotinib- resistant metastatic NSCLC while awaiting results of tissue genotyping Day 0: plasma ddPCR, repeat biopsy ordered Day 1: positive plasma EGFR T790M genotyping result obtained Day 25: positive tissue EGFR T790M genotyping result obtained Day 31: osimertinib therapy initiated ddPCR, digital droplet polymerase chain reaction; NSCLC, non-small-cell lung cancer. Slide credit: clinicaloptions.com Sacher AG, et al. JAMA Oncol. 2016;[Epub ahead of print].

Conclusions and Faculty Assessment ddPCR-based plasma genotyping highly specific, rapid for detection of EGFR or KRAS mutations in pts with advanced nonsquamous NSCLC EGFR exon 19 del, EGFR L858R, and KRAS G12X: 100% specificity, 100% positive predictive value EGFR T790M: lower specificity (63%), positive predictive value (79%); likely due to tumor heterogeneity in setting of acquired resistance Acceptable sensitivity for all 4 ddPCR assays (64% to 86%) Sensitivity higher in pts with metastases, likely related to increased shedding of tumor-derived, cell-free DNA Turnaround time 2-3 days for plasma ddPCR genotyping vs 12-27 days for tissue genotyping Investigators concluded that plasma ddPCR genotyping is a robust, noninvasive technology ready for use in clinical decision making for pts with advanced NSCLC ddPCR, digital droplet polymerase chain reaction; NSCLC, non-small-cell lung cancer. Slide credit: clinicaloptions.com Sacher AG, et al. JAMA Oncol. 2016;[Epub ahead of print].

Conclusions and Faculty Assessment Strengths of plasma ddPCR genotyping: Rapid detection facilitates timely initiation of optimal therapy May obviate need for repeat biopsies; particularly advantageous for frail pts Potentially more reliable for the detection of EGFR T790M mutations Weaknesses of plasma ddPCR genotyping: Unable to detect ALK or ROS rearrangements, copy number alterations Future directions: Potential for monitoring disease in real time, including early prediction of treatment response or resistance emergence Multiplex detection of complex genomic alterations in combination with next-generation sequencing ddPCR, digital droplet polymerase chain reaction. Slide credit: clinicaloptions.com Sacher AG, et al. JAMA Oncol. 2016;[Epub ahead of print].

Go Online for More CCO Coverage of NSCLC! Downloadable resource summarizing current treatment of NSCLC Downloadable slidesets of key data A CME-certified text module on NSCLC with expert faculty commentary on key studies NSCLC, non-small-cell lung cancer. clinicaloptions.com/oncology