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S1,S2,S3,S4,S5,S6,S7, Supplemental table 1,supplemental table 2

Supplementary Table 1

Targeted monoclonal antibody therapy is a promising therapeutic strategy for cancer, and antibody-dependent cell-mediated cytotoxicity (ADCC) represents a crucial mechanism underlying these approaches. The majority of patients have limited responses to monoclonal antibody therapy due to the development of resistance. Models of ADCC provide a system for uncovering immune-resistance mechanisms. We continuously exposed epidermal growth factor receptor (EGFR+) A431 cells to KIR-deficient NK92-CD16V effector cells and the anti-EGFR cetuximab. Persistent ADCC exposure yielded ADCC-resistant cells (ADCCR1) that, compared with control ADCC-sensitive cells (ADCCS1), exhibited reduced EGFR expression, overexpression of histone- and interferon-related genes, and a failure to activate NK cells, without evidence of epithelial-to-mesenchymal transition. These properties gradually reversed following withdrawal of ADCC selection pressure. The development of resistance was associated with lower expression of multiple cell-surface molecules that contribute to cell–cell interactions and immune synapse formation. Classic immune checkpoints did not modulate ADCC in this unique model system of immune resistance. We showed that the induction of ADCC resistance involves genetic and epigenetic changes that lead to a general loss of target cell adhesion properties that are required for the establishment of an immune synapse, killer cell activation, and target cell cytotoxicity.

Legends for Supplemental Figures 1-7 in Supplementary Data file

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.

Supplemental Figure 1: Comparison of targeted sequencing of cfDNA using standard hybridization selection versus Resolution methods. (A) UCSC genome browser depiction of read coverage in the TP53 vicinity. The coverage track representing hybridization selection of cfDNA was created by mapping the reads from sample SRR1197557 of Newman et al 2014. The other coverage track represents unique reads from Sample 511 in this paper. The off-target noise outside of the TP53 gene is higher in SRR1197557 compared with Sample 511. (B) Genome-wide calculation of off-target versus on-target coverage for the two samples. The percentage of read coverage overlapping the declared target regions (widened by an extra 100 nucleotides on each side) was calculated using bedtools. Supplemental Figure 2: Sensitivity of mutant allele detection as a function of dilution. A total of four gene fusions (orange open squares), thirteen point mutations (black circles) and one indels (orange open squares), were assayed at varying mutant allele concentrations (Supplemental Table 1). All 18 lesions were detected in the 2.5%, 1.0%, and 0.4% pools, while fourteen out of the 18 were detected at 0.1%. We expect gene fusions to be more difficult to detect because adequate sequence coverage demands a larger assay footprint than point mutations and probe orientation is unidirectional rather than bidirectional. Consistent with this expectation, the ROS1 fusion in cell line HCC78 and the ALK fusion in cell line H3122 were underrepresented. Supplemental Figure 3: Schematic of predicted genomic junctions for 5 translocations identified in plasma specimens.

PDF file - 71K

Supplementary Figure Legend

Supplemental Table 3: Summary of ddPCR and NGS findings. The table shows the tissue genotype, ddPCR results, ddPCR allele frequency (mut / mut +wt allele frequency), NGS mutation calls and allele frequencies, and sequencing reads.

Supplemental Table 1: Summary of probe sequences

Shown is the schema of study design and enrollment.(30) As noted, the cut-off for data analysis was May 15, 2015.

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

Supplemental Table 2: Compilation of cell line admixture experimental data. The table shows the number of detected mutant reads, the total number of wild-type reads that correspond to the genomic coordinates of the mutant allele, and the frequency of mutant allele detection.

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Table S1: Inclusion and exclusion criteria for study eligibility; Table S2: PCR primer and probe sequences; Table S3: Patient and tumor characteristics.

Purpose: Genotype-directed therapy is the standard of care for advanced non–small cell lung cancer (NSCLC), but obtaining tumor tissue for genotyping remains a challenge. Circulating tumor cell (CTC) or cell-free DNA (cfDNA) analysis may allow for noninvasive evaluation. This prospective trial evaluated CTCs and cfDNA in EGFR-mutant NSCLC patients treated with erlotinib until progression.Experimental Design: EGFR-mutant NSCLC patients were enrolled in a phase II trial of erlotinib. Blood was collected at baseline, every 2 months on study, and at disease progression. Plasma genotyping was performed by droplet digital PCR for EGFR19del, L858R, and T790M. CTCs were isolated by CellSave, enumerated, and analyzed by immunofluorescence for CD45 and pan-cytokeratin and EGFR and MET FISH were also performed. Rebiopsy was performed at disease progression.Results: Sixty patients were enrolled; 44 patients discontinued therapy for disease progression. Rebiopsy occurred in 35 of 44 patients (80%), with paired CTC/cfDNA analysis in 41 of 44 samples at baseline and 36 of 44 samples at progression. T790M was identified in 23 of 35 (66%) tissue biopsies and 9 of 39 (23%) cfDNA samples. CTC analysis at progression identified MET amplification in 3 samples in which tissue analysis could not be performed. cfDNA analysis identified T790M in 2 samples in which rebiopsy was not possible. At diagnosis, high levels of cfDNA but not high levels of CTCs correlated with progression-free survival.Conclusions: cfDNA and CTCs are complementary, noninvasive assays for evaluation of acquired resistance to first-line EGFR TKIs and may expand the number of patients in whom actionable genetic information can be obtained at acquired resistance. Serial cfDNA monitoring may offer greater clinical utility than serial monitoring of CTCs. Clin Cancer Res; 22(24); 6010–20. ©2016 AACR.

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

Targeted monoclonal antibody therapy is a promising therapeutic strategy for cancer, and antibody-dependent cell-mediated cytotoxicity (ADCC) represents a crucial mechanism underlying these approaches. The majority of patients have limited responses to monoclonal antibody therapy due to the development of resistance. Models of ADCC provide a system for uncovering immune-resistance mechanisms. We continuously exposed epidermal growth factor receptor (EGFR+) A431 cells to KIR-deficient NK92-CD16V effector cells and the anti-EGFR cetuximab. Persistent ADCC exposure yielded ADCC-resistant cells (ADCCR1) that, compared with control ADCC-sensitive cells (ADCCS1), exhibited reduced EGFR expression, overexpression of histone- and interferon-related genes, and a failure to activate NK cells, without evidence of epithelial-to-mesenchymal transition. These properties gradually reversed following withdrawal of ADCC selection pressure. The development of resistance was associated with lower expression of multiple cell-surface molecules that contribute to cell–cell interactions and immune synapse formation. Classic immune checkpoints did not modulate ADCC in this unique model system of immune resistance. We showed that the induction of ADCC resistance involves genetic and epigenetic changes that lead to a general loss of target cell adhesion properties that are required for the establishment of an immune synapse, killer cell activation, and target cell cytotoxicity.

The targeting of surface antigens expressed on tumor cells by monoclonal antibodies (mAbs) has revolutionized cancer therapeutics. One mechanism of action of antibody-based immunotherapy is the activation of immune effector cells to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). This review will summarize the process of ADCC, its important role in the efficacy of mAb therapy, how to measure it, and finally future strategies for antibody design that can take advantage of it to improve clinical performance.

Navigation underwater traditionally is done by keeping a safe distance from obstacles, resulting in "fly-overs" of the area of interest. Movement of an autonomous underwater vehicle (AUV) through a cluttered space, such as a shipwreck or a decorated cave, is an extremely challenging problem that has not been addressed in the past. This paper proposes a novel navigation framework utilizing an enhanced version of Trajopt for fast 3D path-optimization planning for AUVs. A sampling-based correction procedure ensures that the planning is not constrained by local minima, enabling navigation through narrow spaces. Two different modalities are proposed: planning with a known map results in efficient trajectories through cluttered spaces; operating in an unknown environment utilizes the point cloud from the visual features detected to navigate efficiently while avoiding the detected obstacles. The proposed approach is rigorously tested, both on simulation and in-pool experiments, proven to be fast enough to enable safe real-time 3D autonomous navigation for an AUV., Comment: ICRA 2020

Purpose: Genotype-directed therapy is the standard of care for advanced non–small cell lung cancer (NSCLC), but obtaining tumor tissue for genotyping remains a challenge. Circulating tumor cell (CTC) or cell-free DNA (cfDNA) analysis may allow for noninvasive evaluation. This prospective trial evaluated CTCs and cfDNA in EGFR-mutant NSCLC patients treated with erlotinib until progression. Experimental Design: EGFR-mutant NSCLC patients were enrolled in a phase II trial of erlotinib. Blood was collected at baseline, every 2 months on study, and at disease progression. Plasma genotyping was performed by droplet digital PCR for EGFR19del, L858R, and T790M. CTCs were isolated by CellSave, enumerated, and analyzed by immunofluorescence for CD45 and pan-cytokeratin and EGFR and MET FISH were also performed. Rebiopsy was performed at disease progression. Results: Sixty patients were enrolled; 44 patients discontinued therapy for disease progression. Rebiopsy occurred in 35 of 44 patients (80%), with paired CTC/cfDNA analysis in 41 of 44 samples at baseline and 36 of 44 samples at progression. T790M was identified in 23 of 35 (66%) tissue biopsies and 9 of 39 (23%) cfDNA samples. CTC analysis at progression identified MET amplification in 3 samples in which tissue analysis could not be performed. cfDNA analysis identified T790M in 2 samples in which rebiopsy was not possible. At diagnosis, high levels of cfDNA but not high levels of CTCs correlated with progression-free survival. Conclusions: cfDNA and CTCs are complementary, noninvasive assays for evaluation of acquired resistance to first-line EGFR TKIs and may expand the number of patients in whom actionable genetic information can be obtained at acquired resistance. Serial cfDNA monitoring may offer greater clinical utility than serial monitoring of CTCs. Clin Cancer Res; 22(24); 6010–20. ©2016 AACR.

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

The identification of oncogenic driver mutations has led to the rapid rise of genotype-directed treatments. However, genetic analysis of tumors remains cumbersome and a morbid experience for patients. Noninvasive assessment of tumor genotype, so-called "liquid biopsy," such as plasma genotyping represents a potentially transformative tool. Here we describe a genotyping protocol of cell-free plasma DNA (cfDNA) using Droplet Digital™ PCR (ddPCR™). ddPCR emulsifies DNA into ~20,000 droplets in which PCR is performed to endpoint in each droplet for both mutant and wild-type DNA. Droplets are run through a modified flow cytometer where mutant and wild-type DNA emit different colored signals. The count of these signals upon Poisson distribution analysis allows sensitive quantification of allelic prevalence.

The identification of oncogenic driver mutations has led to the rapid rise of genotype-directed treatments. However, genetic analysis of tumors remains cumbersome and a morbid experience for patients. Noninvasive assessment of tumor genotype, so-called "liquid biopsy," such as plasma genotyping represents a potentially transformative tool. Here we describe a genotyping protocol of cell-free plasma DNA (cfDNA) using Droplet Digital™ PCR (ddPCR™). ddPCR emulsifies DNA into ~20,000 droplets in which PCR is performed to endpoint in each droplet for both mutant and wild-type DNA. Droplets are run through a modified flow cytometer where mutant and wild-type DNA emit different colored signals. The count of these signals upon Poisson distribution analysis allows sensitive quantification of allelic prevalence.

Fibroblast activation protein (FAP) is a type II transmembrane serine protease that functions as both a dipeptidyl peptidase and an endopeptidase. FAP is minimally expressed in normal pancreas but overexpressed in 90% of pancreatic ductal adenocarcinoma (PDAC) specimens. A meta-analysis of PDAC studies demonstrated elevated tumor FAP expression is associated with worse clinical outcomes. While immunotherapy offers remarkable results for certain cancer types, it has been largely ineffective in PDAC. This lack of efficacy may be attributed to the dense stromal fibrosis, comprised largely of pancreatic stellate cells (PSCs), that is characteristic of PDAC lesions. Here we demonstrate that human NK cell line (NK92) is activated by and kill PSCs. Upon direct contact with PSCs, NK92 cells upregulate FAP. FAP expression by NK92 cells is associated with an inactivation phenotype. Talabostat, a non-specific inhibitor of FAP, enhances NK92 killing of PSCs in vitro and enhances PDAC tumor clearance in vivo. This suggests that FAP may be a novel NK cell immune checkpoint that can be pharmacologically modulated to enhance NK cell antitumor activity. Citation Format: Allison O'Connell, Shangzi Wang, Louis M. Weiner. The potential role of fibroblast activation protein as a natural killer cell immune checkpoint [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A096.

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.

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

11068 Background: Genotype directed therapy is now standard of care for advanced NSCLC pts. However, adequate tumor tissue for comprehensive genotyping remains a challenge. Recent research suggests that CTC capture or cfDNA analysis allows for non-invasive diagnosis and monitoring of treatment. This prospective trial was designed to quantify the predictive value of CTC and cfDNA analyses of EGFR-mutant NSCLC pts treated with first-line erlotinib. Methods: TKI naive EGFR mutant NSCLC pts were enrolled in a phase II trial of erlotinib treatment. Paired blood for cfDNA and CTC analysis was collected at baseline prior to therapy and every 2 months during follow ups. Plasma genotyping was performed by ddPCR for EGFR19del, L858R, T790M while CTCs were isolated by CellSearch and analyzed by IF and MET-FISH. Repeat biopsies at progression were performed when feasible. Results: Between 2/10 and 1/15, 60 EGFRmutant pts (L858R:17, 19del:38, other:5) were enrolled. As of 1/15, 44 patients have discontinued therapy (3...

11089 Background: Plasma genotyping of cell-free DNA (cfDNA) has the potential to allow for noninvasive genotyping while avoiding the inherent shortcomings of tissue genotyping and repeat biopsies....

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