RAS/RAF Comutation and ERBB2 Copy Number Modulates HER2 Heterogeneity and Responsiveness to HER2-directed Therapy in Colorectal Cancer.

PURPOSE: ERBB2-amplified colorectal cancer is a distinct molecular subtype with expanding treatments. Implications of concurrent oncogenic RAS/RAF alterations are not known. EXPERIMENTAL DESIGN: Dana-Farber and Foundation Medicine Inc. Colorectal cancer cohorts with genomic profiling were used to identify ERBB2-amplified cases [Dana-Farber, n = 47/2,729 (1.7%); FMI, n = 1857/49,839 (3.7%)]. Outcomes of patients receiving HER2-directed therapies are reported (Dana-Farber, n = 9; Flatiron Health-Foundation Medicine clinicogenomic database, FH-FMI CGDB, n = 38). Multisite HER2 IHC and genomic profiling were performed to understand HER2 intratumoral and interlesional heterogeneity. The impact of concurrent RAS comutations on the effectiveness of HER2-directed therapies were studied in isogenic colorectal cancer cell lines and xenografts. RESULTS: ERBB2 amplifications are enriched in left-sided colorectal cancer. Twenty percent of ERBB2-amplified colorectal cancers have co-occurring oncogenic RAS/RAF alterations. While RAS/RAF WT colorectal cancers typically have clonal ERBB2 amplification, colorectal cancers with co-occurring RAS/RAF alterations have lower level ERRB2 amplification, higher intratumoral heterogeneity, and interlesional ERBB2 discordance. These distinct genomic patterns lead to differential responsiveness and patterns of resistance to HER2-directed therapy. ERBB2-amplified colorectal cancer with RAS/RAF alterations are resistant to trastuzumab-based combinations, such as trastuzumab/tucatinib, but retain sensitivity to trastuzumab deruxtecan in in vitro and murine models. Trastuzumab deruxtecan shows clinical efficacy in cases with high-level ERBB2-amplified RAS/RAF coaltered colorectal cancer. CONCLUSIONS: Co-occurring RAS/RAF alterations define a unique subtype of ERBB2-amplified colorectal cancer that has increased intratumoral heterogeneity, interlesional discordance, and resistance to trastuzumab-based combinations. Further examination of trastuzumab deruxtecan in this previously understudied cohort of ERBB2-amplified colorectal cancer is warranted.

Atypical Droplet Digital Polymerase Chain Reaction Patterns That Indicate Uncommon but Clinically Actionable EGFR Mutations in Lung Cancer.

CONTEXT.­: Droplet digital polymerase chain reaction (ddPCR) is a sensitive method to detect common pathogenic EGFR mutations in non-small cell lung cancer. Although targeted assays have not been specifically designed to detect them, uncommon EGFR mutations have been linked to response to targeted therapy. OBJECTIVE.­: To describe atypical ddPCR patterns that correspond to uncommon but clinically actionable EGFR mutations. DESIGN.­: A cohort of 1134 consecutive non-small cell lung cancers that underwent targeted next-generation sequencing was reviewed. Uncommon EGFR mutations involving probe binding sites were evaluated by ddPCR. RESULTS.­: Two hundred fifty-five of 1134 cancers (22.5%) harbored pathogenic EGFR mutations. One hundred eighty-six of 255 (72.9%) had canonical EGFR exon 19 deletion or exon 21 p.L858R variants designed for detection by ddPCR. An additional 25 of 255 cases (9.8%) had uncommon EGFR mutations within the probe-binding site, including one case with concurrent uncommon mutations in both exon 19 and exon 21. These mutations included uncommon EGFR exon 19 deletions (n = 6), EGFR exon 19 substitutions p.L747P (n = 3) and p.L747A (n = 1), dinucleotide substitutions leading to EGFR p.L858R (n = 5), EGFR exon 21 substitutions p.K860I (n = 1) and p.L861Q (n = 9), and EGFR p.[L858R;K860I] (n = 1). Droplet digital polymerase chain reaction generated atypical but reproducible signal for each of these uncommon variants. CONCLUSIONS.­: Droplet digital polymerase chain reaction analysis of uncommon pathogenic EGFR variants can yield unique and reproducible results. Recognition of atypical patterns in EGFR ddPCR testing can prompt confirmatory molecular testing and aid appropriate targeted therapy selection for patients with non-small cell lung cancer.

Early Changes in Circulating Cell-Free KRAS G12C Predict Response to Adagrasib in KRAS Mutant Non-Small Cell Lung Cancer Patients.

PURPOSE: Non-invasive monitoring of circulating tumor DNA (ctDNA) has the potential to be a readily available measure for early prediction of clinical response. Here, we report on early ctDNA changes of KRAS G12C in a Phase 2 trial of adagrasib in patients with advanced, KRAS G12C-mutant lung cancer. EXPERIMENTAL DESIGN: We performed serial droplet digital PCR (ddPCR) and plasma NGS on 60 KRAS G12C-mutant patients with lung cancer that participated in cohort A of the KRYSTAL-1 clinical trial. We analyzed the change in ctDNA at 2 specific intervals: Between cycles 1 and 2 and at cycle 4. Changes in ctDNA were compared with clinical and radiographic response. RESULTS: We found that, in general, a maximal response in KRAS G12C ctDNA levels could be observed during the initial approximately 3-week treatment period, well before the first scan at approximately 6 weeks. 35 patients (89.7%) exhibited a decrease in KRAS G12C cfDNA >90% and 33 patients (84.6%) achieved complete clearance by cycle 2. Patients with complete ctDNA clearance at cycle 2 showed an improved objective response rate (ORR) compared with patients with incomplete ctDNA clearance (60.6% vs. 33.3%). Furthermore, complete ctDNA clearance at cycle 4 was associated with an improved overall survival (14.7 vs. 5.4 months) and progression-free survival (HR, 0.3). CONCLUSIONS: These results support using early plasma response of KRAS G12C assessed at approximately 3 weeks to anticipate the likelihood of a favorable objective clinical response.

Genetic events associated with venetoclax resistance in CLL identified by whole-exome sequencing of patient samples.

Although BCL2 mutations are reported as later occurring events leading to venetoclax resistance, many other mechanisms of progression have been reported though remain poorly understood. Here, we analyze longitudinal tumor samples from 11 patients with disease progression while receiving venetoclax to characterize the clonal evolution of resistance. All patients tested showed increased in vitro resistance to venetoclax at the posttreatment time point. We found the previously described acquired BCL2-G101V mutation in only 4 of 11 patients, with 2 patients showing a very low variant allele fraction (0.03%-4.68%). Whole-exome sequencing revealed acquired loss(8p) in 4 of 11 patients, of which 2 patients also had gain (1q21.2-21.3) in the same cells affecting the MCL1 gene. In vitro experiments showed that CLL cells from the 4 patients with loss(8p) were more resistant to venetoclax than cells from those without it, with the cells from 2 patients also carrying gain (1q21.2-21.3) showing increased sensitivity to MCL1 inhibition. Progression samples with gain (1q21.2-21.3) were more susceptible to the combination of MCL1 inhibitor and venetoclax. Differential gene expression analysis comparing bulk RNA sequencing data from pretreatment and progression time points of all patients showed upregulation of proliferation, B-cell receptor (BCR), and NF-κB gene sets including MAPK genes. Cells from progression time points demonstrated upregulation of surface immunoglobulin M and higher pERK levels compared with those from the preprogression time point, suggesting an upregulation of BCR signaling that activates the MAPK pathway. Overall, our data suggest several mechanisms of acquired resistance to venetoclax in CLL that could pave the way for rationally designed combination treatments for patients with venetoclax-resistant CLL.

Combination of Type I and Type II MET Tyrosine Kinase Inhibitors as Therapeutic Approach to Prevent Resistance.

MET-targeted therapies are clinically effective in MET-amplified and MET exon 14 deletion mutant (METex14) non-small cell lung cancers (NSCLCs), but their efficacy is limited by the development of drug resistance. Structurally distinct MET tyrosine kinase inhibitors (TKIs) (type I/II) have been developed or are under clinical evaluation, which may overcome MET-mediated drug resistance mechanisms. In this study, we assess secondary MET mutations likely to emerge in response to treatment with single-agent or combinations of type I/type II MET TKIs using TPR-MET transformed Ba/F3 cell mutagenesis assays. We found that these inhibitors gave rise to distinct secondary MET mutant profiles. However, a combination of type I/II TKI inhibitors (capmatinib and merestinib) yielded no resistant clones in vitro The combination of capmatinib/merestinib was evaluated in vivo and led to a significant reduction in tumor outgrowth compared with either MET inhibitor alone. Our findings demonstrate in vitro and in vivo that a simultaneous treatment with a type I and type II MET TKI may be a clinically viable approach to delay and/or diminish the emergence of on target MET-mediated drug-resistance mutations.

Oncogenic switch and single-agent MET inhibitor sensitivity in a subset of EGFR-mutant lung cancer.

The clinical efficacy of epidermal growth factor receptor (EGFR)­targeted therapy in EGFR-mutant non­small cell lung cancer is limited by the development of drug resistance. One mechanism of EGFR inhibitor resistance occurs through amplification of the human growth factor receptor (MET) proto-oncogene, which bypasses EGFR to reactivate downstream signaling. Tumors exhibiting concurrent EGFR mutation and MET amplification are historically thought to be codependent on the activation of both oncogenes. Hence, patients whose tumors harbor both alterations are commonly treated with a combination of EGFR and MET tyrosine kinase inhibitors (TKIs). Here, we identify and characterize six patient-derived models of EGFR-mutant, MET-amplified lung cancer that have switched oncogene dependence to rely exclusively on MET activation for survival. We demonstrate in this MET-driven subset of EGFR TKI-refractory cancers that canonical EGFR downstream signaling was governed by MET, even in the presence of sustained mutant EGFR expression and activation. In these models, combined EGFR and MET inhibition did not result in greater efficacy in vitro or in vivo compared to single-agent MET inhibition. We further identified a reduced EGFR:MET mRNA expression stoichiometry as associated with MET oncogene dependence and single-agent MET TKI sensitivity. Tumors from 10 of 11 EGFR inhibitor­resistant EGFR-mutant, MET-amplified patients also exhibited a reduced EGFR:MET mRNA ratio. Our findings reveal that a subset of EGFR-mutant, MET-amplified lung cancers develop dependence on MET activation alone, suggesting that such patients could be treated with a single-agent MET TKI rather than the current standard-of-care EGFR and MET inhibitor combination regimens.

Reduced Mitochondrial Apoptotic Priming Drives Resistance to BH3 Mimetics in Acute Myeloid Leukemia.

Acquired resistance to BH3 mimetic antagonists of BCL-2 and MCL-1 is an important clinical problem. Using acute myelogenous leukemia (AML) patient-derived xenograft (PDX) models of acquired resistance to BCL-2 (venetoclax) and MCL-1 (S63845) antagonists, we identify common principles of resistance and persistent vulnerabilities to overcome resistance. BH3 mimetic resistance is characterized by decreased mitochondrial apoptotic priming as measured by BH3 profiling, both in PDX models and human clinical samples, due to alterations in BCL-2 family proteins that vary among cases, but not to acquired mutations in leukemia genes. BCL-2 inhibition drives sequestered pro-apoptotic proteins to MCL-1 and vice versa, explaining why in vivo combinations of BCL-2 and MCL-1 antagonists are more effective when concurrent rather than sequential. Finally, drug-induced mitochondrial priming measured by dynamic BH3 profiling (DBP) identifies drugs that are persistently active in BH3 mimetic-resistant myeloblasts, including FLT-3 inhibitors and SMAC mimetics.

Acquired resistance to combined BET and CDK4/6 inhibition in triple-negative breast cancer.

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.

Phase I Study of Rapid Alternation of Sunitinib and Regorafenib for the Treatment of Tyrosine Kinase Inhibitor Refractory Gastrointestinal Stromal Tumors.

PURPOSE: Polyclonal emergence of KIT secondary mutations is a main mechanism of imatinib progression in gastrointestinal stromal tumor (GIST). Approved KIT inhibitors sunitinib and regorafenib have complementary activity against KIT resistance mutations. Preclinical evidence suggests that rapid alternation of sunitinib and regorafenib broadens the spectrum of imatinib-resistant subclones targeted. PATIENTS AND METHODS: Phase Ib study investigating continuous treatment with cycles of sunitinib (3 days) followed by regorafenib (4 days) in patients with tyrosine kinase inhibitor (TKI)-refractory GIST. A 3+3 dosing schema was utilized to determine the recommended phase II dose (RP2D). Plasma samples were analyzed for pharmacokinetics and circulating tumor DNA (ctDNA) studies using targeted error correction sequencing (TEC-seq) and droplet digital PCR (ddPCR). RESULTS: Of the 14 patients enrolled, 2 experienced dose-limiting toxicities at dose level 2 (asymptomatic grade 3 hypophosphatemia). Sunitinib 37.5 mg/day and regorafenib 120 mg/day was the RP2D. Treatment was well-tolerated and no unexpected toxicities resulted from the combination. Stable disease was the best response in 4 patients, and median progression-free survival was 1.9 months. Combined assessment of ctDNA with TEC-seq and ddPCR detected plasma mutations in 11 of 12 patients (92%). ctDNA studies showed that KIT secondary mutations remain the main mechanism of resistance in TKI-refractory GIST, revealing effective suppression of KIT-mutant subpopulations in patients benefiting from the combination. CONCLUSIONS: Sunitinib and regorafenib combination is feasible and tolerable. Rapid alternation of TKIs with complementary activity might be effective when combining drugs with favorable pharmacokinetics, potentially allowing active doses while minimizing adverse events. Serial monitoring with ctDNA may guide treatment in patients with GIST.

Unraveling the clinicopathological features driving the emergence of ESR1 mutations in metastatic breast cancer.

ESR1 mutations were recently found to be an important mechanism of endocrine resistance in ER-positive (ER + ) metastatic breast cancer. To determine the clinicopathological features driving the emergence of the ESR1 mutations we studied plasma cfDNA and detailed clinical data collected from patients with metastatic breast cancer. Droplet Digital PCR was performed for the detection of the most common ESR1 mutations and PIK3CA mutations. Among the patients with ER + /HER2- disease, ESR1 mutations were detected in 30% of the patients. There were no associations between the pathological features of the primary disease or time to distant recurrence and the emergence of ESR1 mutations in metastatic disease. The prevalence of the ESR1 mutations was significantly associated with prior treatment with an aromatase inhibitor in the adjuvant or metastatic setting. The prevalence of the ESR1 mutations was also positively associated with prior fulvestrant treatment. Conversely, the prevalence of ESR1 mutations was lower after treatment with a CDK4/6 inhibitor. There were no significant associations between specific systemic treatments and the prevalence of PIK3CA mutations. These results support the evolution of the ESR1 mutations under the selective pressure of treatment with aromatase inhibitors in the adjuvant and metastatic settings and have important implications in the optimization of adjuvant and metastatic treatment in ER + breast cancer.

Tumor innate immunity primed by specific interferon-stimulated endogenous retroviruses.

Mesenchymal tumor subpopulations secrete pro-tumorigenic cytokines and promote treatment resistance1-4. This phenomenon has been implicated in chemorefractory small cell lung cancer and resistance to targeted therapies5-8, but remains incompletely defined. Here, we identify a subclass of endogenous retroviruses (ERVs) that engages innate immune signaling in these cells. Stimulated 3 prime antisense retroviral coding sequences (SPARCS) are oriented inversely in 3' untranslated regions of specific genes enriched for regulation by STAT1 and EZH2. Derepression of these loci results in double-stranded RNA generation following IFN-γ exposure due to bi-directional transcription from the STAT1-activated gene promoter and the 5' long terminal repeat of the antisense ERV. Engagement of MAVS and STING activates downstream TBK1, IRF3, and STAT1 signaling, sustaining a positive feedback loop. SPARCS induction in human tumors is tightly associated with major histocompatibility complex class 1 expression, mesenchymal markers, and downregulation of chromatin modifying enzymes, including EZH2. Analysis of cell lines with high inducible SPARCS expression reveals strong association with an AXL/MET-positive mesenchymal cell state. While SPARCS-high tumors are immune infiltrated, they also exhibit multiple features of an immune-suppressed microenviroment. Together, these data unveil a subclass of ERVs whose derepression triggers pathologic innate immune signaling in cancer, with important implications for cancer immunotherapy.

Monitoring of Response and Resistance in Plasma of EGFR-Mutant Lung Cancer Using Droplet Digital PCR.

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.

False-Positive Plasma Genotyping Due to Clonal Hematopoiesis.

Purpose: Plasma cell-free DNA (cfDNA) genotyping is increasingly used in cancer care, but assay accuracy has been debated. Because most cfDNA is derived from peripheral blood cells (PBC), we hypothesized that nonmalignant mutations harbored by hematopoietic cells (clonal hematopoiesis, CH) could be a cause of false-positive plasma genotyping.Experimental Design: We identified patients with advanced non-small cell lung cancer (NSCLC) with KRAS, JAK2, or TP53 mutations identified in cfDNA. With consent, PBC DNA was tested using droplet digital PCR (ddPCR) or next-generation sequencing (NGS) to test for CH-derived mutations.Results: We first studied plasma ddPCR results from 58 patients with EGFR-mutant NSCLC. Two had KRAS G12X detected in cfDNA, and both were present in PBC, including one where the KRAS mutation was detected serially for 20 months. We then studied 143 plasma NGS results from 122 patients with NSCLC and identified 5 JAK2 V617F mutations derived from PBC. In addition, 108 TP53 mutations were detected in cfDNA; for 33 of the TP53 mutations, PBC and tumor NGS were available for comparison, and 5 were present in PBC but absent in tumor, consistent with CH.Conclusions: We find that most JAK2 mutations, some TP53 mutations, and rare KRAS mutations detected in cfDNA are derived from CH not tumor. Clinicians ordering plasma genotyping must be prepared for the possibility that mutations detected in plasma, particularly in genes mutated in CH, may not represent true tumor genotype. Efforts to use plasma genotyping for cancer detection may need paired PBC genotyping so that CH-derived mutations are not misdiagnosed as occult malignancy. Clin Cancer Res; 24(18); 4437-43. ©2018 AACRSee related commentary by Bauml and Levy, p. 4352.

Embryonic transcription factor SOX9 drives breast cancer endocrine resistance.

The estrogen receptor (ER) drives the growth of most luminal breast cancers and is the primary target of endocrine therapy. Although ER blockade with drugs such as tamoxifen is very effective, a major clinical limitation is the development of endocrine resistance especially in the setting of metastatic disease. Preclinical and clinical observations suggest that even following the development of endocrine resistance, ER signaling continues to exert a pivotal role in tumor progression in the majority of cases. Through the analysis of the ER cistrome in tamoxifen-resistant breast cancer cells, we have uncovered a role for an RUNX2-ER complex that stimulates the transcription of a set of genes, including most notably the stem cell factor SOX9, that promote proliferation and a metastatic phenotype. We show that up-regulation of SOX9 is sufficient to cause relative endocrine resistance. The gain of SOX9 as an ER-regulated gene associated with tamoxifen resistance was validated in a unique set of clinical samples supporting the need for the development of improved ER antagonists.

Acquired METD1228V Mutation and Resistance to MET Inhibition in Lung Cancer.

Amplified and/or mutated MET can act as both a primary oncogenic driver and as a promoter of tyrosine kinase inhibitor (TKI) resistance in non-small cell lung cancer (NSCLC). However, the landscape of MET-specific targeting agents remains underdeveloped, and understanding of mechanisms of resistance to MET TKIs is limited. Here, we present a case of a patient with lung adenocarcinoma harboring both a mutation in EGFR and an amplification of MET, who after progression on erlotinib responded dramatically to combined MET and EGFR inhibition with savolitinib and osimertinib. When resistance developed to this combination, a new MET kinase domain mutation, D1228V, was detected. Our in vitro findings demonstrate that METD1228V induces resistance to type I MET TKIs through impaired drug binding, while sensitivity to type II MET TKIs is maintained. Based on these findings, the patient was treated with erlotinib combined with cabozantinib, a type II MET inhibitor, and exhibited a response. SIGNIFICANCE: With several structurally distinct MET inhibitors undergoing development for treatment of NSCLC, it is critical to identify mechanism-based therapies for drug resistance. We demonstrate that an acquired METD1228V mutation mediates resistance to type I, but not type II, MET inhibitors, having therapeutic implications for the clinical use of sequential MET inhibitors. Cancer Discov; 6(12); 1334-41. ©2016 AACR.See related commentary by Trusolino, p. 1306This article is highlighted in the In This Issue feature, p. 1293.

Bias-Corrected Targeted Next-Generation Sequencing for Rapid, Multiplexed Detection of Actionable Alterations in Cell-Free DNA from Advanced Lung Cancer Patients.

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.

In situ single-cell analysis identifies heterogeneity for PIK3CA mutation and HER2 amplification in HER2-positive breast cancer.

Detection of minor, genetically distinct subpopulations within tumors is a key challenge in cancer genomics. Here we report STAR-FISH (specific-to-allele PCR-FISH), a novel method for the combined detection of single-nucleotide and copy number alterations in single cells in intact archived tissues. Using this method, we assessed the clinical impact of changes in the frequency and topology of PIK3CA mutation and HER2 (ERBB2) amplification within HER2-positive breast cancer during neoadjuvant therapy. We found that these two genetic events are not always present in the same cells. Chemotherapy selects for PIK3CA-mutant cells, a minor subpopulation in nearly all treatment-naive samples, and modulates genetic diversity within tumors. Treatment-associated changes in the spatial distribution of cellular genetic diversity correlated with poor long-term outcome following adjuvant therapy with trastuzumab. Our findings support the use of in situ single cell-based methods in cancer genomics and imply that chemotherapy before HER2-targeted therapy may promote treatment resistance.

Intratumoral Heterogeneity in EGFR-Mutant NSCLC Results in Divergent Resistance Mechanisms in Response to EGFR Tyrosine Kinase Inhibition.

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 TGFß secretion followed by SMAD pathway activation, an event that promotes EMT. Chronic exposure of EGFR-mutated NSCLC cells to TGFß 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 TGFß 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.

Acquired EGFR C797S mutation mediates resistance to AZD9291 in non-small cell lung cancer harboring EGFR T790M.

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.