1. 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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3. 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:
2. Diaz LA Jr, et al. J Clin Oncol. 2014;32:
3. Haber DA, et al. Cancer Discov. 2014;4:
4. Oxnard GR, et al. Clin Cancer Res. 2014;15:
5. Sacher AG, et al. JAMA Oncol. 2016;[Epub ahead of print].
Slide credit: clinicaloptions.com

4. 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].

5. 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].

6. 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].

7. 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].

8. 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].

9. 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].

10. 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].

11. 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].

12. 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 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].

13. 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].

14. 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