Here we studied cell-free plasma DNA (cfDNA) collected from subjects with advanced lung cancer whose tumors had developed resistance to the epidermal growth factor receptor (EGFR) tyrosine kinase ...inhibitor (TKI) AZD9291. We first performed next-generation sequencing of cfDNA from seven subjects and detected an acquired EGFR C797S mutation in one; expression of this mutant EGFR construct in a cell line rendered it resistant to AZD9291. We then performed droplet digital PCR on serial cfDNA specimens collected from 15 AZD9291-treated subjects. All were positive for the T790M mutation before treatment, but upon developing AZD9291 resistance three molecular subtypes emerged: six cases acquired the C797S mutation, five cases maintained the T790M mutation but did not acquire the C797S mutation and four cases lost the T790M mutation despite the presence of the underlying EGFR activating mutation. Our findings provide insight into the diversity of mechanisms through which tumors acquire resistance to AZD9291 and highlight the need for therapies that are able to overcome resistance mediated by the EGFR C797S mutation.
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.
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.
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.
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.
BET inhibitors are promising therapeutic agents for the treatment of triple-negative breast cancer (TNBC), but the rapid emergence of resistance necessitates investigation of combination therapies ...and their effects on tumor evolution. Here, we show that palbociclib, a CDK4/6 inhibitor, and paclitaxel, a microtubule inhibitor, synergize with the BET inhibitor JQ1 in TNBC lines. High-complexity DNA barcoding and mathematical modeling indicate a high rate of de novo acquired resistance to these drugs relative to pre-existing resistance. We demonstrate that the combination of JQ1 and palbociclib induces cell division errors, which can increase the chance of developing aneuploidy. Characterizing acquired resistance to combination treatment at a single cell level shows heterogeneous mechanisms including activation of G1-S and senescence pathways. Our results establish a rationale for further investigation of combined BET and CDK4/6 inhibition in TNBC and suggest novel mechanisms of action for these drugs and new vulnerabilities in cells after emergence of resistance.
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).
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.
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.
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.
Circulating tumor cells (CTCs) likely derive from clones in the primary tumor, suggesting that they can be used for all biological tests applying to the primary cells.
The ScreenCell® devices are ...single-use and low-cost innovative devices that use a filter to isolate and sort tumor cells by size.
The ScreenCell® Cyto device is able to isolate rare, fixed, tumor cells, with a high recovery rate. Cells are well preserved morphologically. Immunocytochemistry and FISH assays can be performed directly on the filter. The ScreenCell® CC device allows isolation of live cells able to grow in culture. High quality genetic materials can be obtained directly from tumor cells isolated on the ScreenCell® MB device filter.
Due to their reduced size, versatility, and capacity to isolate CTCs within minutes, the ScreenCell® devices may be able to simplify and improve non-invasive access to tumor cells.
Non-small cell lung cancers (NSCLC) that have developed resistance to EGF receptor (EGFR) tyrosine kinase inhibitor (TKI), including gefitinib and erlotinib, are clinically linked to an ...epithelial-to-mesenchymal transition (EMT) phenotype. Here, we examined whether modulating EMT maintains the responsiveness of EGFR-mutated NSCLCs to EGFR TKI therapy. Using human NSCLC cell lines harboring mutated EGFR and a transgenic mouse model of lung cancer driven by mutant EGFR (EGFR-Del19-T790M), we demonstrate that EGFR inhibition induces TGFβ secretion followed by SMAD pathway activation, an event that promotes EMT. Chronic exposure of EGFR-mutated NSCLC cells to TGFβ was sufficient to induce EMT and resistance to EGFR TKI treatment. Furthermore, NSCLC HCC4006 cells with acquired resistance to gefitinib were characterized by a mesenchymal phenotype and displayed a higher prevalence of the EGFR T790M mutated allele. Notably, combined inhibition of EGFR and the TGFβ receptor in HCC4006 cells prevented EMT but was not sufficient to prevent acquired gefitinib resistance because of an increased emergence of the EGFR T790M allele compared with cells treated with gefitinib alone. Conversely, another independent NSCLC cell line, PC9, reproducibly developed EGFR T790M mutations as the primary mechanism underlying EGFR TKI resistance, even though the prevalence of the mutant allele was lower than that in HCC4006 cells. Thus, our findings underscore heterogeneity within NSCLC cells lines harboring EGFR kinase domain mutations that give rise to divergent resistance mechanisms in response to treatment and anticipate the complexity of EMT suppression as a therapeutic strategy.
Tumors from 50% of epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer patients that develop resistance to gefitinib or erlotinib will contain a secondary EGFR T790M mutation. ...As most patients do not undergo repeated tumor biopsies we evaluated whether EGFR T790M could be detected using plasma DNA.
DNA from plasma of 54 patients with known clinical response to gefitinib or erlotinib was extracted and used to detect both EGFR-activating and EGFR T790M mutations. Forty-three (80%) of patients had tumor EGFR sequencing (EGFR mutant/wild type: 30/13) and seven patients also had EGFR T790M gefitinib/erlotinib-resistant tumors. EGFR mutations were detected using two methods, the Scorpion Amplification Refractory Mutation System and the WAVE/Surveyor, combined with whole genome amplification.
Both EGFR-activating and EGFR T790M were identified in 70% of patients with known tumor EGFR-activating (21 of 30) or T790M (5 of 7) mutations. EGFR T790M was identified from plasma DNA in 54% (15 of 28) of patients with prior clinical response to gefitinib/erlotinib, 29% (4 of 14) with prior stable disease, and in 0% (0 of 12) that had primary progressive disease or were untreated with gefitinib/erlotinib.
EGFR T790M can be detected using plasma DNA from gefitinib- or erlotinib-resistant patients. This noninvasive method may aid in monitoring drug resistance and in directing the course of subsequent therapy.
Mutations in the epidermal growth factor receptor (EGFR) are associated with clinical and radiographic responses to EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Currently available ...methods of EGFR mutation detection rely on direct DNA sequencing, which requires isolation of DNA from a relatively pure population of tumor cells, cannot be done on small diagnostic specimens, and lack sensitivity. Here we describe the use of a sensitive screening method that overcomes many of these limitations.
We screened 178 non-small cell lung cancer specimens for mutations in exons 18 to 21 of EGFR using a DNA endonuclease, SURVEYOR, which cleaves mismatched heteroduplexed DNA. Samples were analyzed by high-performance liquid chromatography on the Transgenomic WAVE HS system. Selected specimens that produced digestion products using SURVEYOR were subsequently reanalyzed by size separation or under partially denaturing conditions, followed by fractionation and sequencing. The specimens included DNA isolated from frozen tumor specimens, dissected formalin-fixed, paraffin-embedded tumor specimens undergoing clinical sequencing, and undissected formalin-fixed, paraffin-embedded specimens. One hundred sixty specimens were independently analyzed using direct DNA sequencing in a blinded fashion.
EGFR mutations were detected in 16 of 61 fresh frozen tumor specimens, 24 of 91 dissected formalin-fixed, paraffin-embedded tumor specimens, and 11 of 26 undissected formalin-fixed, paraffin-embedded tumor specimens. Compared with sequencing, the sensitivity and specificity of the present method were 100% and 87%. The positive and negative predictive values were 74% and 100%, respectively. SURVEYOR analysis detected 7 (4%) mutations that were not previously detected by direct sequencing.
SURVEYOR analysis provides a rapid method for EGFR mutation screening with 100% sensitivity and negative predictive value. This unbiased scanning technique is superior to direct sequencing when used with undissected formalin-fixed, paraffin-embedded specimens.
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