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Purpose: Tumor genotyping using cell-free plasma DNA (cfDNA) has the potential to allow noninvasive assessment of tumor biology, yet many existing assays are cumbersome and vulnerable to false-positive results. We sought to determine whether droplet digital PCR (ddPCR) of cfDNA would allow highly specific and quantitative assessment of tumor genotype.Experimental Design: ddPCR assays for EGFR, KRAS, and BRAF mutations were developed using plasma collected from patients with advanced lung cancer or melanoma of a known tumor genotype. Sensitivity and specificity were determined using cancers with nonoverlapping genotypes as positive and negative controls. Serial assessment of response and resistance was studied in patients with EGFR-mutant lung cancer on a prospective trial of erlotinib.Results: We identified a reference range for EGFR L858R and exon 19 deletions in specimens from KRAS-mutant lung cancer, allowing identification of candidate thresholds with high sensitivity and 100% specificity. Received operative characteristic curve analysis of four assays demonstrated an area under the curve in the range of 0.80 to 0.94. Sensitivity improved in specimens with optimal cfDNA concentrations. Serial plasma genotyping of EGFR-mutant lung cancer on erlotinib demonstrated pretreatment detection of EGFR mutations, complete plasma response in most cases, and increasing levels of EGFR T790M emerging before objective progression.Conclusions: Noninvasive genotyping of cfDNA using ddPCR demonstrates assay qualities that could allow effective translation into a clinical diagnostic. Serial quantification of plasma genotype allows noninvasive assessment of response and resistance, including detection of resistance mutations up to 16 weeks before radiographic progression. Clin Cancer Res; 20(6); 1698–705. ©2014 AACR.

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XLSX file - 25K, cfDNA results for figures 2, 4, 5, calculated both as concentration per mL plasma and as percent mutant.

PDF file - 365K, Supplemental Figure 1: Assay optimization. For each TaqMan probe, the optimal annealing temperature was determined by testing each assay across a temperature gradient of 55.0 - 65{degree sign}C. Typical FAM plots for EGFR L858R (A) and EGFR exon 19 deletion (B) are shown. Supplemental Figure 2: Assay characteristics. As the sample input increases, the copies/μL output increases in a linear fashion across a wide dynamic range for both the L858R assay (A) and the exon 19 deletion assay (B). Testing for 10 and 50 copies of mutant EGFR in a background of 1000 and 50,000 genome equivalents (GE), the L858R assay demonstrates more consistent sensitivity (C) than the exon 19 deletion assay (D). Supplemental Figure 3: Detection of BRAF V600E in cfDNA from patients with advanced melanoma. Supplemental Figure 4: Correlation of LINE-1 quantitative PCR (QPCR) levels with DNA concentration as measured with PicoGreen across 69 plasma specimens (R2 = 0.94, p

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Purpose: Tumor genotyping using cell-free plasma DNA (cfDNA) has the potential to allow noninvasive assessment of tumor biology, yet many existing assays are cumbersome and vulnerable to false-positive results. We sought to determine whether droplet digital PCR (ddPCR) of cfDNA would allow highly specific and quantitative assessment of tumor genotype. Experimental Design: ddPCR assays for EGFR, KRAS, and BRAF mutations were developed using plasma collected from patients with advanced lung cancer or melanoma of a known tumor genotype. Sensitivity and specificity were determined using cancers with nonoverlapping genotypes as positive and negative controls. Serial assessment of response and resistance was studied in patients with EGFR-mutant lung cancer on a prospective trial of erlotinib. Results: We identified a reference range for EGFR L858R and exon 19 deletions in specimens from KRAS-mutant lung cancer, allowing identification of candidate thresholds with high sensitivity and 100% specificity. Received operative characteristic curve analysis of four assays demonstrated an area under the curve in the range of 0.80 to 0.94. Sensitivity improved in specimens with optimal cfDNA concentrations. Serial plasma genotyping of EGFR-mutant lung cancer on erlotinib demonstrated pretreatment detection of EGFR mutations, complete plasma response in most cases, and increasing levels of EGFR T790M emerging before objective progression. Conclusions: Noninvasive genotyping of cfDNA using ddPCR demonstrates assay qualities that could allow effective translation into a clinical diagnostic. Serial quantification of plasma genotype allows noninvasive assessment of response and resistance, including detection of resistance mutations up to 16 weeks before radiographic progression. Clin Cancer Res; 20(6); 1698–705. ©2014 AACR.

Background: Genotype directed treatment is the standard of care for patients with advanced non-small cell lung cancer (NSCLC). However, acquired drug resistance invariably develops. There is a need to develop clinically relevant animal models from patients who have developed acquired resistance in order to study mechanisms of resistance and validate new therapeutic strategies. We have generated a portfolio of clinically relevant, early passage, patient-derived xenograft (PDX) models of lung cancer. Methods: Tumor biopsies (core biopsies (n = 8), pleural effusions (n = 28) and surgical samples (n = 11)) were implanted subcutaneously (n = 39) or into the sub-renal capsule (n = 8) of NSG mice under an IRB approved protocol. After implanting 35 specimens, mostly from pleural effusions, several mice exhibited weight and fur loss. Histopathology revealed infiltration of the liver, skin and lungs by human derived T-cells consistent with graft-versus-host disease (GVHD). Subsequent pleural effusions were then subjected to CD45 depletion using a human CD45 microbead separation kit which eliminated the incidence of GVHD. Tumors from all three sources were allowed to grow and subsequently passaged only as subcutaneous implants in NSG mice. Molecular characterization of PDX models was performed using targeted next generation sequencing and tumor histology confirmed by H&E staining. Some established tumor models have also been propagated as cell lines in vitro. Results: 47 specimens were implanted with an overall take rate of ∼30%. The initial take rate was 22% but increased to 66% following CD45 depletion. We have established 11 NSCLC adenocarcinoma and 3 small-cell lung cancer (SCLC) PDX models from patient samples. The models established thus far range from erlotinib resistant EGFR mutant models with T790M mutation (n = 3), MET amplification (n = 1), SCLC transformation (n = 2) or an unknown mechanism (n = 1); to models including SCLC with an NRAS Q61K mutation (n = 1); EML4-ALK resistant to crizotinib and ceritinib (n = 1); KRAS G12C mutation (n = 1); NUT-mid line carcinoma with BRD4 rearrangement (n = 1); pan wild-type (n = 2) and a PIK3CA mutation (n = 1). We have used these models to study therapeutic interventions. Treatment of the NRAS Q61K mutant SCLC with trametinib resulted in complete tumor regression. Pharmacodynamic analyses revealed inhibition of pERK 1/2 and induction of apoptosis. Data will be presented with further characterization and platform development of the lung PDX models. Notably, >50% of the models do show pulmonary metastases from the subcutaneous implant site. Conclusions: PDXs can be established from lung cancer patients undergoing routine clinical biopsies. Establishment of models from pleural effusions is also feasible following CD45 depletion to prevent GVHD. These models will provide unique information on mechanisms of drug resistance and can be used to evaluate pre-clinical therapeutic strategies. Citation Format: Prafulla C. Gokhale, Sangeetha Palakurthi, Man Xu, Antonio Calles, Shruti D. Shah, Allison O'Connell, Melissa Messineo, Yanan Kuang, Masahiko Yanagita, Mosab Ali, Cam Anh Tran, Jihyun Choi, Atsuko Ogino, Mohit Butaney, Amanda J. Redig, Cloud Paweletz, Paul T. Kirschmeier, Pasi A. Jänne. Modeling patient-derived lung cancer in mice: Preclinical tool for drug development. [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 1451. doi:10.1158/1538-7445.AM2015-1451

Tumor genotyping has become standard in the care of non-small cell lung cancer (NSCLC), colorectal cancer, and melanoma because of its power in guiding the personalization of treatment with molecularly targeted therapies. A major limitation of cancer genotyping is the availability of pre- and post-treatment tumor biopsy specimens for accurate assessment of cancer biology. In addition, serial tumor biopsies from NSCLC patients at time of disease response or progression are not always desirable from the patient perspective and feasibility can be limited due to accessible tumor tissue and cost. The analysis of circulating cell-free DNA (cfDNA) has demonstrated potential to be a non-invasive means of interrogating the biology underlying response and resistance. Here we report the detection, quantification and monitoring of EGFR mutations by droplet digital PCR in cfDNA on a prospective clinical trial of EGFR-mutant NSCLC patients on erlotinib. Droplet digital PCR, a novel technology that emulsifies DNA input into ∼20,000 consistently sized droplets, each individually genotyped, represents a promising approach for non-invasive quantification of specific tumor mutations. EGFR L858R, Del(19) and T790M were serially measured in over 300 samples from 40 lung cancer patients treated on erlotinib. Complete plasma response was seen in 8 cases; in 6, rising levels of inhibitor sensitizing EGFR Del(19) and L858R and acquired EGFR T790M mutations were detected up to 4 months prior to objective radiographic progression. An analysis of patients that progressed showed that 53% had tumor-genotype-confirmed EGFR sensitizing mutations in their plasma at diagnosis. Four of these patients had a tumor rebiopsy adequate for EGFR genotyping at progression, and EGFR T790M was confirmed in each. This contrasts to the same analysis on circulating tumor cells (CTC), collected simultaneously: No CTCs were identified at diagnosis and only 22% of patients had any detectable CTCs at progression. Our results strongly suggest that cfDNA genotyping has clinical utility in the molecular assessment of patients at diagnosis, and providing molecular understanding of patient's tumor evolution, in real time, by serial monitoring for resistance mutations. Citation Format: Cloud P. Paweletz, Geoffrey R. Oxnard, Yanan Kuang, Allison O'Connell, Masahiko Yanagita, Melissa M. Messineo, Paul Kirschmeier, Jessie M. English, David M. Jackman, Pasi A. Jänne. Serial monitoring of EGFR mutations in plasma and matched tissue from EGFR mutant non-small cell lung cancer patients on erlotinib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 939. doi:10.1158/1538-7445.AM2014-939