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Immunotherapy has shown limited efficacy in patients with EGFR-mutated lung cancer. Efforts to enhance the immunogenicity of EGFR-mutated lung cancer have been unsuccessful to date. Here, we discover that MET amplification, the most common mechanism of resistance to third-generation EGFR tyrosine kinase inhibitors (TKI), activates tumor cell STING, an emerging determinant of cancer immunogenicity (1). However, STING activation was restrained by ectonucleosidase CD73, which is induced in MET-amplified, EGFR-TKI–resistant cells. Systematic genomic analyses and cell line studies confirmed upregulation of CD73 in MET-amplified and MET-activated lung cancer contexts, which depends on coinduction of FOSL1. Pemetrexed (PEM), which is commonly used following EGFR-TKI treatment failure, was identified as an effective potentiator of STING-dependent TBK1-IRF3-STAT1 signaling in MET-amplified, EGFR-TKI–resistant cells. However, PEM treatment also induced adenosine production, which inhibited T-cell responsiveness. In an allogenic humanized mouse model, CD73 deletion enhanced immunogenicity of MET-amplified, EGFR-TKI–resistant cells, and PEM treatment promoted robust responses regardless of CD73 status. Using a physiologic antigen recognition model, inactivation of CD73 significantly increased antigen-specific CD8+ T-cell immunogenicity following PEM treatment. These data reveal that combined PEM and CD73 inhibition can co-opt tumor cell STING induction in TKI-resistant EGFR-mutated lung cancers and promote immunogenicity. Significance: MET amplification upregulates CD73 to suppress tumor cell STING induction and T-cell responsiveness in TKI-resistant, EGFR-mutated lung cancer, identifying a strategy to enhance immunogenicity and improve treatment.

Although BCL2 mutations are reported as later occurring events leading to venetoclax resistance, many other mechanisms of progression have been reported but remain poorly understood. Here we analyze longitudinal tumor samples from eleven patients with disease progression on venetoclax to characterize the clonal evolution of resistance. All patients tested showed increased in vitro resistance to venetoclax at their post-treatment timepoint. We found the previously described acquired BCL2-G101V mutation in only 4/11 patients with 2 patients showing very low variant allele fraction (VAF; 0.03-4.68%). Whole exome sequencing (WES) revealed acquired loss(8p) in 4/11 patients of which 2 patients also have gain (1q21.2-21.3) in the same cells, affecting the MCL-1 gene. In vitro experiments showed that CLL cells from the four patients with loss(8p) were more resistant to venetoclax than those without it, while the cells from two patients also carrying gain (1q21.2-21.3) showed increased sensitivity to MCL-1 inhibition. Progression samples with gain (1q21.2-21.3) were more susceptible to combination MCL-1 inhibitor with venetoclax. Differential gene expression analysis comparing bulk RNAseq data from pre-treatment and progression time points of all patients showed upregulation of proliferation, BCR and NFKB gene sets including MAPK genes. Cells from progression timepoints demonstrated upregulation of surface immunoglobulin M (sIgM) and higher pERK levels compared to the pre-timepoint, 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 venetoclax resistant CLL patients.

Activation of the stimulator of interferon genes (STING) pathway promotes antitumor immunity but STING agonists have yet to achieve clinical success. Increased understanding of the mechanism of action of STING agonists in human tumors is key to developing therapeutic combinations that activate effective innate antitumor immunity. Here, we report that malignant pleural mesothelioma cells robustly express STING and are responsive to STING agonist treatment ex vivo. Using dynamic single-cell RNA sequencing of explants treated with a STING agonist, we observed CXCR3 chemokine activation primarily in tumor cells and cancer-associated fibroblasts, as well as T-cell cytotoxicity. In contrast, primary natural killer (NK) cells resisted STING agonist–induced cytotoxicity. STING agonists enhanced migration and killing of NK cells and mesothelin-targeted chimeric antigen receptor (CAR)-NK cells, improving therapeutic activity in patient-derived organotypic tumor spheroids. These studies reveal the fundamental importance of using human tumor samples to assess innate and cellular immune therapies. By functionally profiling mesothelioma tumor explants with elevated STING expression in tumor cells, we uncovered distinct consequences of STING agonist treatment in humans that support testing combining STING agonists with NK and CAR-NK cell therapies.

Supplementary Data from Activation of Tumor-Cell STING Primes NK-Cell Therapy

Application of Bliss independence to test for synergy effect of TKI combination treatment in vivo

Supplementary Table S2 shows type I and II MET kinase inhibitors and key trials studying their activity lung cancer populations enriched for MET-dependence.

Small cell lung carcinoma (SCLC) is highly mutated, yet durable response to immune checkpoint blockade (ICB) is rare. SCLC also exhibits cellular plasticity, which could influence its immunobiology. Here we discover that a distinct subset of SCLC uniquely upregulates MHC I, enriching for durable ICB benefit. In vitro modeling confirms epigenetic recovery of MHC I in SCLC following loss of neuroendocrine differentiation, which tracks with derepression of STING. Transient EZH2 inhibition expands these nonneuroendocrine cells, which display intrinsic innate immune signaling and basally restored antigen presentation. Consistent with these findings, murine nonneuroendocrine SCLC tumors are rejected in a syngeneic model, with clonal expansion of immunodominant effector CD8 T cells. Therapeutically, EZH2 inhibition followed by STING agonism enhances T-cell recognition and rejection of SCLC in mice. Together, these data identify MHC I as a novel biomarker of SCLC immune responsiveness and suggest novel immunotherapeutic approaches to co-opt SCLC's intrinsic immunogenicity.Significance:SCLC is poorly immunogenic, displaying modest ICB responsiveness with rare durable activity. In profiling its plasticity, we uncover intrinsically immunogenic MHC Ihi subpopulations of nonneuroendocrine SCLC associated with durable ICB benefit. We also find that combined EZH2 inhibition and STING agonism uncovers this cell state, priming cells for immune rejection.This article is highlighted in the In This Issue feature, p. 1861

Supplementary Figure S2 shows expression of D1228V mutation in PC9 cells as confirmed by RT-PCR and cDNA sequencing.

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.

Supplementary Table S1

Table S3 shows list of KRAS WT; LKB1 WT and KRAS WT; LKB1 Mut lung adenocarcinoma cell lines related with supplementary figure S1.

Figure S1. Characterization of cells and CDK4/6 inhibitors; Figure S2. CDK6 phosphorylates serine residues of the regulatory domain of NFAT4 (NFATc3); Figure S3. Analysis of lung tumor immune infiltrates after CDK4/6 inhibition from KrasG12D (Kras), KrasG12DLkb1 (KL) or KrasG12DTrp53fl/fl (KP) mice; Figure S4. T cell proliferation and cytokine/chemokine profiling of KrasG12DTrp53fl/fl GEMM mice; Figure S5. Tumor antigen experienced T cells are more sensitive to CDK4/6 inhibition; Figure S6. Short-term CDK4/6 inhibition alters the cell cycle status of tumor infiltrating T cells; Figure S7. CDK4/6 inhibition induces changes in the expression of activation and suppression marker genes in tumor-infiltrating T cells; Figure S8. Combination treatment of CDK4/6 inhibitor and anti-PD-1 antibody elicits anti-tumor immunity; Figure S9. Combination treatment of CDK4/6 inhibitor and anti-PD-1 antibody on established tumor; Figure S10. Effect of TCR stimulation and CDK4/6 inhibition on phosphorylation of NFkB; Supplementary Table S1; Supplmentary Table S2; Supplementary Table S3: Selected genes reported to be regulated by NFAT

Supplementary Figures S1-3; Supplementary Tables S1-3

Supplementary methods, including croplet digital PCR for MET D1228V; molecular docking study; Molecular Dynamics Simulations.

Supplementary Figures 1-12

The supplementary file shows Supplementary figure S1-S4 and their legends, and the legends for Supplementary table S1-S4.

Supplementary Figure Legends.

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. 1306.This article is highlighted in the In This Issue feature, p. 1293

Tamoxifen is widely used in the adjuvant treatment of estrogen receptor–positive (ER+) breast cancer and is an important drug for pre-menopausal women and post-menopausal patients who cannot tolerate aromatase inhibitors. Despite the clear clinical benefit in improving relapse-free and overall survival in these patients, an adverse effect of tamoxifen is a 2- to 7-fold increased risk of uterine cancer (UC) after 2-5 years of treatment. To date, the mechanism of tamoxifen-driven tumorigenesis is not well understood, and preventive approaches are lacking. Here, to molecularly characterize tamoxifen-associated uterine cancers (TA-UCs) and gain insights into their unique evolution, we performed whole-exome sequencing of 21 TA-UCs (discovery cohort) and droplet digital PCR (ddPCR) of an additional 40 TA-UCs (validation cohort) obtained from the ‘Tamoxifen Associated Malignancies: Aspects of Risk’ (TAMARISK) study. In addition, we used in vivo mouse models to: (i) further investigate tamoxifen-activated molecular pathways that may be involved in TA-UC tumorigenesis; and (ii) offer mechanistic insights. Overall, we discovered that TA-UCs were genomically similar to non–TA-UCs from The Cancer Genome Atlas (TCGA) project, with one profound exception: TA-UCs are characterized by a lower-than-expected frequency of mutations in two highly prevalent UC driver genes in the PI3K pathway: PIK3CA (14% [3/21] vs 48% [265/554] in non–TA-UC; P=0.003, Fisher’s exact test; Q=0.02, Benjamini-Hochberg FDR) and PIK3R1 (0%, [0/21] vs 31% [174/554]; P=0.001; Q=0.01). We used ddPCR in the independent TA-UC validation cohort and confirmed the low frequency of mutations in PIK3CA (7.5% [3/40] vs 21% [144/685] in control UCs from the Dana-Farber contribution to the AACR GENIE project; P=0.04). We next performed mouse in vivo studies and demonstrated that tamoxifen activated the PI3K pathway and increased cell proliferation in normal mouse uterine tissue through paracrine and autocrine effects, both of which were abrogated by the PI3K inhibitor alpelisib. Taken together, we describe a distinct and novel pathway of carcinogenesis in which tamoxifen acts as a driver event in the uterus and promotes tumor development in a mutation-independent manner. Indeed, tamoxifen may increase the risk of UC by activating the PI3K pathway, which can substitute for the early acquisition of oncogenic PIK3CA or PIK3R1 mutations observed in non–TA-UC tumors. Furthermore, the ability of a PI3K inhibitor to reduce cell proliferation in our mouse model raises the possibility that downregulating the PI3K pathway may prevent or significantly reduce TA-UC development, offering a potential future therapeutic and prevention strategy for specific high-risk patients undergoing tamoxifen therapy. Citation Format: Kirsten Kubler, Agostina Nardone, Shankara Anand, Daniel Gorvich, Marjolein Droog, Francisco Hermida-Prado, Tara Akshi, Avery S Feit, Gabriella Cohen, Gwen Dackus, Matthew Pun, Yanan Kuang, Justin Cha, Mendy Miller, William J Gibson, Cloud P Paweletz, Eliezer M Van Allen, Flora E van Leeuwen, Petra Nederlof, Harry Hollema, Quang-Dé Nguyen, Marian JE Mourits, Ignaty Leshchiner, Chip Stewart, Ursula A Matulonis, Wilbert Zwart, Yosef E Maruvka, Gad Getz, Rinath Jeselsohn. Tamoxifen instigates uterine cancer development by activating PI3K signaling and supersedes PIK3CA driver mutations [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS2-09.

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.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) are the standard-of-care treatment for EGFR-mutant non–small cell lung cancers (NSCLC). However, most patients develop acquired drug resistance to EGFR TKIs. HER3 is a unique pseudokinase member of the ERBB family that functions by dimerizing with other ERBB family members (EGFR and HER2) and is frequently overexpressed in EGFR-mutant NSCLC. Although EGFR TKI resistance mechanisms do not lead to alterations in HER3, we hypothesized that targeting HER3 might improve efficacy of EGFR TKI. HER3–DXd is an antibody–drug conjugate (ADC) comprised of HER3-targeting antibody linked to a topoisomerase I inhibitor currently in clinical development. In this study, we evaluated the efficacy of HER3–DXd across a series of EGFR inhibitor–resistant, patient-derived xenografts and observed it to be broadly effective in HER3-expressing cancers. We further developed a preclinical strategy to enhance the efficacy of HER3–DXd through osimertinib pretreatment, which increased membrane expression of HER3 and led to enhanced internalization and efficacy of HER3–DXd. The combination of osimertinib and HER3–DXd may be an effective treatment approach and should be evaluated in future clinical trials in EGFR-mutant NSCLC patients. Significance: EGFR inhibition leads to increased HER3 membrane expression and promotes HER3–DXd ADC internalization and efficacy, supporting the clinical development of the EGFR inhibitor/HER3–DXd combination in EGFR-mutant lung cancer. See related commentary by Lim et al., p. 18

Immunotherapy has shown limited efficacy in patients with EGFR-mutated lung cancer. Efforts to enhance the immunogenicity of EGFR-mutated lung cancer have been unsuccessful to date. Here, we discover that MET amplification, the most common mechanism of resistance to third-generation EGFR tyrosine kinase inhibitors (TKI), activates tumor cell STING, an emerging determinant of cancer immunogenicity (1). However, STING activation was restrained by ectonucleosidase CD73, which is induced in MET-amplified, EGFR-TKI–resistant cells. Systematic genomic analyses and cell line studies confirmed upregulation of CD73 in MET-amplified and MET-activated lung cancer contexts, which depends on coinduction of FOSL1. Pemetrexed (PEM), which is commonly used following EGFR-TKI treatment failure, was identified as an effective potentiator of STING-dependent TBK1-IRF3-STAT1 signaling in MET-amplified, EGFR-TKI–resistant cells. However, PEM treatment also induced adenosine production, which inhibited T-cell responsiveness. In an allogenic humanized mouse model, CD73 deletion enhanced immunogenicity of MET-amplified, EGFR-TKI–resistant cells, and PEM treatment promoted robust responses regardless of CD73 status. Using a physiologic antigen recognition model, inactivation of CD73 significantly increased antigen-specific CD8+ T-cell immunogenicity following PEM treatment. These data reveal that combined PEM and CD73 inhibition can co-opt tumor cell STING induction in TKI-resistant EGFR-mutated lung cancers and promote immunogenicity.Significance:MET amplification upregulates CD73 to suppress tumor cell STING induction and T-cell responsiveness in TKI-resistant, EGFR-mutated lung cancer, identifying a strategy to enhance immunogenicity and improve treatment.

Quantitative precision of Digital Sequencing results. (a) SNV VAFs in 375 consecutive SNV control runs. (b) Indel and fusion VAFs (left y-axis) and CNA copy number (right y-axis) in consecutive CFI control runs. (c) Variation distribution of DS results in 375 consecutive analyses of a single SNV control lot (blue) and multiple consecutive lots of indel (red), fusion (grey), and CNA (orange) controls.

Detailed results of Digital Sequencing and clinical ddPCR comparison. Results shown in individual 2x2 tables for each biomarker tested. *Note, all discordances were below DFCI ddPCR's reportable range. Detailed clinical information for each is presented in Supplemental Table 3.

Supplementary Data from MET-Induced CD73 Restrains STING-Mediated Immunogenicity of EGFR-Mutant Lung Cancer

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.

Supplemental Figure 1. Characterization of DFCI 359 and DFCI 315.

DFCI358 signaling with respect to mTOR; crizotinib+trametinib combination;RTK activation

Detailed description of methodology and reagents

Sunitinib (A) and Regorafenib (B) plasma concentration (ng/mL) across several timepoints during cycle 1.

PDF file - 71K

Supplementary Figure Legend

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 AACR.See related commentary by Bauml and Levy, p. 4352

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.

Alterations detectable by Guardant360 gene panel

Summary of EGFR inhibitor resistant patient-derived xenografts.

Purpose:Surgery often represents the best chance for disease control in locoregionally recurrent squamous cell carcinoma of the head and neck (SCCHN). We investigated dual immune-checkpoint inhibition [anti–PD-1, nivolumab (N), and anti-KIR, lirilumab (L)] before and after salvage surgery to improve disease-free survival (DFS).Patients and Methods:In this phase II study, patients received N (240 mg) + L (240 mg) 7 to 21 days before surgery, followed by six cycles of adjuvant N + L. Primary endpoint was 1-year DFS; secondary endpoints were safety, pre-op radiologic response, and overall survival (OS). Correlatives included tumor sequencing, PD-L1 scoring, and immunoprofiling.Results:Among 28 patients, the median age was 66, 86% were smokers; primary site: 9 oral cavity, 9 oropharynx, and 10 larynx/hypopharynx; 96% had prior radiation. There were no delays to surgery. Grade 3+ adverse events: 11%. At the time of surgery, 96% had stable disease radiologically, one had progression. Pathologic response to N + L was observed in 43% (12/28): 4/28 (14%) major (tumor viability, TV ≤ 10%) and 8/28 (29%) partial (TV ≤ 50%). PD-L1 combined positive score (CPS) at surgery was similar regardless of pathologic response (P = 0.71). Thirteen (46%) recurred (loco-regional = 10, distant = 3). Five of 28 (18%) had positive margins, 4 later recurred. At median follow-up of 22.8 months, 1-year DFS was 55.2% (95% CI, 34.8–71.7) and 1-year OS was 85.7% (95% CI, 66.3–94.4). Two-year DFS and OS were 64% and 80% among pathologic responders.Conclusions:(Neo)adjuvant N + L was well tolerated, with a 43% pathologic response rate. We observed favorable DFS and excellent 2-year OS among high-risk, previously treated patients exhibiting a pathologic response. Further evaluation of this strategy is warranted.

Antibodies used in the study

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) are the standard-of-care treatment for EGFR-mutant non–small cell lung cancers (NSCLC). However, most patients develop acquired drug resistance to EGFR TKIs. HER3 is a unique pseudokinase member of the ERBB family that functions by dimerizing with other ERBB family members (EGFR and HER2) and is frequently overexpressed in EGFR-mutant NSCLC. Although EGFR TKI resistance mechanisms do not lead to alterations in HER3, we hypothesized that targeting HER3 might improve efficacy of EGFR TKI. HER3–DXd is an antibody–drug conjugate (ADC) comprised of HER3-targeting antibody linked to a topoisomerase I inhibitor currently in clinical development. In this study, we evaluated the efficacy of HER3–DXd across a series of EGFR inhibitor–resistant, patient-derived xenografts and observed it to be broadly effective in HER3-expressing cancers. We further developed a preclinical strategy to enhance the efficacy of HER3–DXd through osimertinib pretreatment, which increased membrane expression of HER3 and led to enhanced internalization and efficacy of HER3–DXd. The combination of osimertinib and HER3–DXd may be an effective treatment approach and should be evaluated in future clinical trials in EGFR-mutant NSCLC patients.Significance:EGFR inhibition leads to increased HER3 membrane expression and promotes HER3–DXd ADC internalization and efficacy, supporting the clinical development of the EGFR inhibitor/HER3–DXd combination in EGFR-mutant lung cancer.See related commentary by Lim et al., p. 18

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.

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.