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R.A. Tsai
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MINI 14 - Pre-Clinical Therapy (ID 119)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:L. Fernandez-Cuesta, A.F. Gazdar
- Coordinates: 9/08/2015, 10:45 - 12:15, Mile High Ballroom 2c-3c
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MINI14.01 - EGFR-Mutated PDX in NSCLC: Molecular Fidelity and Correlation of PDX and Patient Response to EGFR Inhibition (ID 2191)
10:45 - 10:50 | Author(s): R.A. Tsai
- Abstract
- Presentation
Background:
Inevitable emergence of resistance to tyrosine kinase inhibitor (TKI) therapy in EGFR-mutated NSCLC warrants development of pro-active therapeutic strategies to delay or circumvent this evolution. To model such approaches, we are employing a clinically and genomically annotated patient derived xenotransplant (PDX) resource designed to duplicate relevant known mechanisms of resistance to TKI therapy. This analysis examines molecular fidelity and correlates response between patient and PDX in EGFR-mutant NSCLC.
Methods:
Six EGFR-mutated NSCLC, 1 EGFR-TKI naïve and 5 after progressive disease on erlotinib, were implanted subcutaneously into the flank of NOD.Cg-Prkdc[scid] Il2rg[tm1Wjl]/SzJ (NSG) mice as previously described (DR Gandara, Clin Lung Cancer 2015). Models were considered established when PDX growth was confirmed in passage 1 (P1); tumor growth studies were conducted in P3-P5. The donor patient tumor (PT) and the resultant PDX were analyzed for driver mutations (Response Genetics Inc., and Illumina TSCAP), copy number variants (CNV) and global RNA expression (Affymetrix arrays). Informed consent was obtained from all patients. EGFR-mutant PDX treatments included: erlotinib, afatinib, cetuximab, and afatinib+cetuximab. Patient response was graded by RECIST 1.1 and measured in PDX by tumor shrinkage from pre-treatment baseline. In select models, pharmacodynamic studies (kinase arrays; immunoblotting) were also performed.
Results:
The EGFR mutation subtypes identified in the donor PT were preserved in all PDX models (4 EGFR E19del and 2 EGFR L858R). Corresponding putative mechanisms of resistance were identical in both PT and PDX in 3 cases: EGFR T790M (2 of 5) and MET amplification (1 of 5). Of 5 post-erlotinib progression PDX models, 3 had progressive disease (PD) and 2 had transient tumor shrinkage to erlotinib. The PDX derived from an erlotinib-naïve patient (EGFR E19del) demonstrated sustained tumor shrinkage to erlotinib. Patient-PDX treatment correlations were possible in 3 post erlotinib-progression models. Two of these patients received afatinib-cetuximab: 1 with partial response (PR) and 1 with PD. The two models corresponding to these patients, when treated with afatinib-cetuximab, underwent complete regression of tumor (CR) and PD, respectively. Pharmacodynamic assessment of the responding model at 24h showed near complete diminishment of pEGFR following afatinib-cetuximab, concomitant with decreased pHer2, pERK, pAKT and p38. Erlotinib showed transient inhibition on signaling in this model at 6h, returning to baseline by 24h. In contrast, the non-responding model showed minimal effects on target inhibition and signal transduction following treatment with any EGFR inhibitor.
Conclusion:
Genomic fidelity was preserved in EGFR-mutant PDX, including putative mechanisms of resistance in the post-erlotinib progression models. The majority (3/5) of the EGFR-mutant PDXs created after erlotinib resistance demonstrated PD. In the other post-erlotinib progression models transient tumor shrinkage was noted, which may reflect PDX passaging in the absence of selective pressure of EGFR-inhibition or pharmacokinetic considerations. Overall, the PDX response to treatment reflected the corresponding patient’s clinical course. Pharmacodynamic studies of select models informed PDX response to treatment.
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ORAL 38 - Liquid Biopsies (ID 147)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:M.S. Tsao, J. Wang
- Coordinates: 9/09/2015, 16:45 - 18:15, Mile High Ballroom 2a-3b
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ORAL38.06 - Identification of Actionable Tumor Alterations in Circulating Cell-Free Tumor DNA (cf DNA) Using Digital Sequencing from NSCLC Patients (ID 1706)
17:39 - 17:50 | Author(s): R.A. Tsai
- Abstract
- Presentation
Background:
To fully implement precision therapy in lung cancer, transition to a re-biopsy policy will be required at baseline and at progression after each line of therapy. The molecular testing paradigm is shifting toward next generation sequencing (NGS). As tissues are limited and repeat invasive biopsy introduces cost and risk, novel technologies sensitive and specific enough for multiplexed assessment in cell-free DNA (cfDNA) isolated from patient blood would represent a significant advance. Preliminary experience from investigators suggest a high degree of correlation between repeat tumor biopsy and plasma NGS. Here, we present the Guardant Health (GH) digital sequencing approach in a consecutive series of NSCLC cases.
Methods:
225 consecutive blood specimens from NSCLC patients, collected February–March 2015, were evaluated for cfDNA tumor alterations by digital sequencing using the GH panel of 68 genes. The test includes all reported fusion partners for ALK, RET, ROS1, and NTRK1 and cfDNA amplification for 16 genes. The mutant allele fraction (MAF) was calculated relative to WT in cfDNA. The test is sensitive to a single fragment of mutated cfDNA in a 10 ml blood sample and analytic specificity is >99.9999%.
Results:
Canonical EGFR activating mutations were detected in 20 cases (14 E19del, 3 L858R, 2 E20ins, 1 G719A). EGFR T790M co-occurred in 7 cases (6 E19del, 1 L858R), with EGFR amplification observed in 6 of the 20. Median age for patients with EGFR mut+ was 62.5; 18 female(90%), compared to nonEGFR-mutant cases. Four cases had driver fusions (2EML4-ALK, 2 KIF5B-RET) and five cases harbored an ERBB2 E20ins. KRAScodon 12/13 mutations were detected in 23 patients, while 3 harbored mutations in HRAS(Q61L) and NRAS(Q61L, G13R), and 6 had BRAF mutations (4 V600E, 2 G466X). All putative drivers were mutually exclusive. Mutations in signal transduction factors with confirmed gain-of-function activity included AKT1(E17K), MEK1(K57N, C121S), PIK3CA(E542K, E545K x2, H1047L, M1043V, R93W) and JAK2(V617F x2); truncating or missense mutations (>3% MAF) were observed in NF1 (6 cases), PTEN(1 case), SMAD4(4 cases) and STK11(4 cases). TP53 mutations were detected in 116/225 (51%). Evidence of gene amplification was seen in 32 cases, with 11 harboring multiple events. By function, amp events were observed for G1 cell cycle factors:11, RTKs: 17, MYC: 2; and signal transduction: 21. MAF ranged from 0.06% to 83.4% (av 5.1%; median: 9.8%), reflecting clinical and biologic diversity of patients. In a clinical subset at UC Davis, 27 patients were evaluated and alterations were detected in 18 (66.7%). Actionable findings were identified in 14 (77.8%) including 2 with EGFRL858R, 1 with EGFR E19del, and 1 interesting case with EGFR E19del at 45% MAF, EGFR amplification, and an emerging EGFR T790M clone at 0.54% MAF.
Conclusion:
In a series of NSCLC cases, high-sensitivity, high-specificity cfDNA analysis demonstrated the ability to identify somatic tumor alterations, including clinically actionable predictors, in a majority of patients via a simple blood draw, suggesting that this approach can be used for guiding therapeutic decision-making when repeat biopsy is high risk or not possible. Assuming validation, plasma cfDNA analysis may supplant invasive tumor biopsy in the near future.
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