EGFR TKI Research Articles

Development and validation of a UPLC–MS/MS method for quantification of C-005, a novel third-generation EGFR TKI, and its major metabolite in plasma: Application to its first-in-patient study

C-005 is a novel third-generation EGFR tyrosine kinase inhibitor for the treatment of non-small cell lung cancer (NSCLC). To support its clinical trial, we developed a rapid and sensitive bioanalytical method based on ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) technique for the quantification of C-005 and its major metabolite in NSCLC patients following international bioanalytical guidelines. After a simple and quick protein precipitation step, the supernatant was injected to a Waters Acquity BEH C18 column (2.1 × 50 mm i.d., 1.7 mm), and the column was eluted with a gradient of buffer A (5 mM ammonium acetate and 0.1% formic acid in water) and buffer B (formic acid-acetonitrile (1:1000, v/v)). The eluates were subsequently detected by an AB QTRAP 5500 mass spectrometer with electrospray ionization using multiple-reaction monitoring mode. The method showed good linearity from 2.00 to 1000 ng/mL for C-005 and 1.00 to 500 ng/mL for M1. In conclusion, the validation results demonstrated the robustness of the method and its well-poised to support the first-in-patient study of C-005 in NSCLC patients.

PCN102 Healthcare Resource Utilization (HCRU) and Costs with Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors (EGFR TKIS) and Subsequent Chemotherapy Among Patients with LUNG Cancer

The study objective was to describe the HCRU and costs among patients with lung cancer during treatment with EGFR TKIs and during subsequent chemotherapy. This retrospective study used medical and pharmacy claims from the Optum® Clinformatics® Data Mart for adults with US commercial or Medicare Advantage insurance with lung cancer initiating first-line therapy with EGFR TKIs (gefitinib, erlotinib, afatinib, or osimertinib) from June 2013 - June 2019 who subsequently initiated chemotherapy. Continuous enrollment from 6 months prior to EGFR TKI initiation (baseline period) through ≥1 month after chemotherapy initiation was required. HCRU and costs per patient month were compared while on EGFR TKI therapy and while on subsequent chemotherapy using descriptive statistics. In the study sample of 192 patients, mean±SD age was 71 ±9 years, and 63% were female. Patients had a mean [median] of 250 [172] days on EGFR TKI therapy and 178 [124] days after chemotherapy start until chemotherapy cessation or end of data. During the EGFR TKI period, lung cancer patients had a lower prevalence of all-cause inpatient hospitalization (24% vs. 41% p=0.0005) compared to the chemotherapy period. During the EGFR TKI period there were lower mean monthly counts of all-cause inpatient hospitalizations (0.07 vs. 0.21 p<0.0001), outpatient ER visits (0.07 vs. 0.12 p=0.0134), outpatient office visits (1.77 vs. 2.45 p<0.0001), and other outpatient visits (4.61 vs. 8.62 p<0.0001) compared with the chemotherapy period. Total all-cause costs per month were lower during the EGFR TKI period ($15,811 vs. $40,730 p<0.0001) compared with the chemotherapy period. Healthcare utilization and costs per month were significantly lower for lung cancer patients while receiving EGFR TKIs than with subsequent chemotherapy. Prolonging time on EGFR TKI treatment before chemotherapy could reduce healthcare utilization and costs in EGFR mutation positive non-small cell lung cancer.

PTEN, ATM, IDH1 mutations and MAPK pathway activation as modulators of PFS and OS in patients treated by first line EGFR TKI, an ancillary study of the French Cooperative Thoracic Intergroup (IFCT) Biomarkers France project

ObjectivesTumor mutation screening is standard of care for patients with stage IV NSCLC. Since a couple of years, widespread NGS approaches used in routine diagnostics to detect driver mutations such as EGFR, KRAS, BRAF or MET allows the identification of other alterations that could modulated the intensity or duration of response to targeted therapies. The prevalence of co-occurring alterations that could affect response or prognosis as not been largely analyzed in clinical settings and large cohorts of patients. Thanks to the IFCT program “Biomarkers France“, a collection of samples and data at a nation-wide level was available to test the impact of co-mutations on first line EGFR TKI in patients with EGFR mutated cancers. Materials and methodsTargeted NGS was assessed on available (n = 208) samples using the Ion AmpliSeq™ Cancer Hotspot Panel v2 to screen for mutations in 50 different cancer genes. ResultsThis study showed that PTEN inactivating mutations, ATM alterations, IDH1 mutations and complex EGFR mutations were predictors of short PFS in patients with a stage 4 lung adenocarcinoma receiving first line EGFR TKI and that PTEN, ATM, IDH1 and KRAS mutations as well as alterations in the MAPK pathway were related to shorter OS. ConclusionThese findings may lead to new treatment options in patients with unfavorable genotypes to optimize first line responses.

Tyrosine kinase inhibitors as induction therapy in nonsmall-cell lung cancer.

Purpose of reviewTKI therapy has shown excellent efficacy and favorable tolerability in patients with mutation-positive nonsmall cell lung cancer. However, there is no clear consensus on the role of TKI as induction therapy. In this article, we reviewed recently published studies to analyze the benefits of tyrosine kinase inhibitors, in particular, EGFR TKIs and ALK TKIs, as inducible treatments for NSCLC.Recent findingsSeveral clinical trials have recently presented their latest data, giving analysis of patient's survival benefits and adverse events. Initial results have demonstrated promising efficacy and safety data. Some clinical case reports and retrospective analysis demonstrated that EGFR/ALK TKIs can significantly improve PFS and the rate of radical surgery. However, there was no statistically significant difference in overall survival time of almost all clinical trials.SummaryTKIs are increasingly accepted by clinicians as induction therapy in NSCLC. Many studies have demonstrated that neoadjuvant therapy increases the likelihood of surgery and is associated with good resection rates, as evidenced by high prospective downstaging rates in patients with locally advanced NSCLC. However, the risk of recurrence remains high with no evidence of overall survival benefits being reported. Now that more clinical trials are being conducted and more data will be available for analysis, a clearer and more comprehensive view of what role TKIs play in induction therapy will emerge.

Emergence of NOTCH2-NTRK1 After Osimertinib in a Patient With Lung Adenocarcinoma With Neuroendocrine Differentiation

Clinical Practice Points • Osimertinib is widely used in patients with lung cancer with EGFR T790M after EGFR TKI treatment with high efficacy and approved in first-line treatment for all EGFR positive patients. However, the resistance profile to osimertinib is not well-elucidated. NTRK fusions are rare oncogenic drivers in lung cancer. • Here, we report a NOTCH2–NTRK1 rearranged fusion in lung adenocarcinoma with neuroendocrine differentiation after osimertinib treatment. The patient had significantly improved clinical symptoms and had stable disease for 2 months after larotrectinib and osimertinib treatment, with significant decrease in NOTCH2-NTRK1 fusion (AF from 24.85% to 0.63%). • The emergence of NTRK fusions after osimertinib treatment may suggest a therapeutic target. NTRK fusions screening is important after osimertinib resistance.

Preclinical Characterization of Mobocertinib Highlights the Putative Therapeutic Window of This Novel EGFR Inhibitor to EGFR Exon 20 Insertion Mutations

IntroductionEGFR exon 20 insertion mutations account for 10% of all EGFR mutations and are mostly insensitive to approved EGFR tyrosine kinase inhibitors (EGFR TKIs). Novel EGFR TKIs have been developed or repurposed for these mutants. A limited number of preclinical studies have detailed these EGFR TKIs. We sought to use commercially available mobocertinib (TAK-788) to characterize the preclinical therapeutic window of this EGFR TKI against EGFR mutations and to probe possible on-target mechanisms of resistance (EGFR-C797S). MethodsWe used models of EGFR mutations to probe representative first, second, third generation, and in-development EGFR exon 20-active (poziotinib, mobocertinib) TKIs. We also introduced EGFR-C797S to these models to identify mechanisms of resistance. ResultsCells driven by the most common EGFR exon 20 insertion mutations (A767_V769dupASV, D770_N771insSVD, H773_V774insH, and others) were inhibited by in-development EGFR TKIs at doses below those affecting EGFR-wildtype; albeit more common EGFR mutations (exon 19 deletions and L858R) were inhibited more readily by mobocertinib and poziotinib. Mobocertinib was able to inhibit the phosphorylation of EGFR in multiple preclinical models. The presence of EGFR-C797S led to greater than 200-fold resistance in proliferation assays probing mobocertinib and osimertinib. A review of clinical studies of mobocertinib disclosed responses that could be lasting. ConclusionsThis is one of the initial reports to characterize the novel EGFR TKI mobocertinib and highlights its broad activity against EGFR mutants plus the therapeutic window to EGFR exon 20 insertion mutations; and EGFR-C797S as a possible mechanism of resistance. Further clinical development of mobocertinib merits continuation.

Beyond EGFR, ALK and ROS1: Current evidence and future perspectives on newly targetable oncogenic drivers in lung adenocarcinoma.

Lung cancer is the leading cause of cancer death worldwide. In the past decade EGFR, ALK and ROS1 TKIs lead to an unprecedented survival improvement of oncogene-addicted NSCLC patients, with better toxicity profile compared to chemotherapy. In recent years the implementation of high-throughput sequencing platforms led to the identification of uncommon molecular alterations in oncogenic drivers, such as BRAF, MET, RET, HER2 and NTRK. Moreover, newly developed drugs have been found to be active against hard to target drivers, such as KRAS. Specific TKIs targeting these genomic alterations are currently in clinical development and showed impressive activity and survival improvement, leading to FDA-accelerated approval for some of them. However, virtually all patients develop resistance to TKIs by on-target or off-target mechanisms. Here we review the clinicopathological features, the emerging targeted therapies and mechanisms of resistance and strategies to overcome them of KRAS, BRAF, MET, RET, HER2 and NTRK-addicted advanced NSCLCs.

Metformin Overcomes Acquired Resistance to EGFR TKIs in EGFR-Mutant Lung Cancer via AMPK/ERK/NF-κB Signaling Pathway.

Background: The major limitation of EGFR TKIs in EGFR-mutant lung cancer therapy is the development of acquired resistance. The underlying mechanisms remain unknown in about 30% of cases. NF-κB activation was encountered in the acquired resistance to EGFR TKIs. Unfortunately, none of NF-κB inhibitors has been clinically approved. The most commonly used antidiabetic drug metformin has demonstrated antitumor effects associated with NF-κB inhibition. Therefore, in this study, metformin was examined for its antitumor and antiresistance effects and underlying mechanisms.Methods: In vitro and in vivo EGFR-mutant lung cancer models with acquired resistance to EGFR TKIs were used.Results: We found that NF-κB was activated in EGFR-mutant lung cancer cells with acquired resistance to EGFR TKIs. Metformin inhibited proliferation and promoted apoptosis of lung cancer cells, especially those with acquired EGFR TKI resistance. Moreover, metformin reversed and delayed acquired resistance to EGFR TKIs as well as suppressed cancer stemness in EGFR-mutant lung cancer. Mechanistically, those effects of metformin were associated with activation of AMPK, resulting in the inhibition of downstream ERK/NF-κB signaling.Conclusions: Our data provided novel and further molecular rationale and preclinical data to support combination of metformin with EGFR TKIs to treat EGFR-mutant lung cancer patients, especially those with acquired resistance.

1369P EGFR T790M ctDNA abundance and TP53 co-mutation by next generation sequencing predicted clinical outcomes of osimertinib in non-small cell lung cancer patients from a real-world study

Osimertinib, the third-generation EGFR TKI, has been approved for non-small cell lung cancer (NSCLC) patients with EGFR T790M mutation after acquired resistance to the first-or second-generation of EGFR TKIs. However, the factors affecting the degree and duration of response to osimertinib is elusive. In this study, we aim to investigate the factors related to T790M abundance in ctDNA by next generation sequencing (NGS), and to analyze the association between the efficacy of osimertinib and T790M abundance in ctDNA. The study compromised 23 advanced EGFR T790M-mutated NSCLC patients which was detected by NGS after acquired resistance to the first-generation EGFR TKIs. The association between EGFR T790M abundance in ctDNA and clinicalpathological characteristics was performed by student t test or one-way ANOVA. Osimertinib outcomes were evaluated by osimertinib-related progression-free survival (PFS). The survival curves were calculated by the Kaplan-Meier method and analyzed by the log-rank test. Prior icotinib treatment led to the highest T790M abundance, compared with gefitinib and erlotinib. Older patients (The mPFS were 8.00±0.00 and 14.00±2.91months for patients older and younger than 68 years old, HR=0.15, 95%CI: 0.03-0.87, log-rank P=0.034), high T790M abundance (The mPFS were 9.00±1.48 and 25.00±0.00months for ctDNA T790M <4.2% and ≥4.2%, HR=0.17, 95%CI: 0.02-1.38, log-rank P=0.049) and prior icotinib (mPFS for patients who took gefinitb, erlotinib and icotinib were 14.00±7.33, 8.00±2.96 and 23.00±0.00months, HR=0.77, 95%CI: 0.43-1.39, log-rank P=0.014) were associated with improved osimertinib-related PFS. TP53 was the most common co-occurring mutations and led to shorter osimertinib-related PFS (mPFS for patients with or without TP53 co-mutation were 8.00±1.02 and 15.00±4.43months, HR=4.40, 95%CI: 0.82-23.49, log-rank P=0.042). T790M mutation abundance in ctDNA by NGS may be a novel method to predict the response and duration of osimertinib in the first-generation EGFR TKIs-resistant, T790M-mutated advanced NSCLC.

1299P Real-world treatment patterns, clinical outcomes and EGFR/T790M testing practices in patients with EGFRm advanced NSCLC and 1L EGFR TKI therapy: A retrospective multinational study (REFLECT)

1299P Real-world treatment patterns, clinical outcomes and EGFR/T790M testing practices in patients with EGFRm advanced NSCLC and 1L EGFR TKI therapy: A retrospective multinational study (REFLECT)

1296P BLU-945, a highly potent and selective 4th generation EGFR TKI for the treatment of EGFR T790M/C797S resistant NSCLC

1296P BLU-945, a highly potent and selective 4th generation EGFR TKI for the treatment of EGFR T790M/C797S resistant NSCLC

1395P Who will maintain as long-term responders more than 3 years with first- or second-generation EGFR TKI among EGFR mutant NSCLC?

1395P Who will maintain as long-term responders more than 3 years with first- or second-generation EGFR TKI among EGFR mutant NSCLC?

1195P The value of detecting resistance through liquid biopsy

Liquid biopsy has been established as an important diagnostic step in identifying EGFR T790M resistance to 1st and 2nd generation EGFR TKIs. This has led to significant interest in exploring the value of liquid biopsy in patients with other oncogene-addicted cancers and acquired resistance to targeted therapy.

Fucoxanthin extracted from Laminaria Japonica inhibits metastasis and enhances the sensitivity of lung cancer to Gefitinib

Ethnopharmacological relevanceLaminaria japonica, a brown seaweed, has been used in Traditional Chinese Medicine (TCM) to treat a variety of diseases including lung cancer. Aim of the studyTo demonstrate the effects of Fucoxanthin (FX), a major active component extracted from Laminaria japonica on metastasis and Gefitinib (Gef) sensitivity in human lung cancer cells both in vitro and in vivo. Materials and methodsInvasion and migration of lung cancer cells were detected using the wound healing assay and transwell assay. Epithelial-to-mesenchymal transition (EMT) factors and PI3K/AKT/NF-κB pathways were analyzed by western blotting. RNA interference (RNAi) technology was used to silence TIMP-2 gene expression in A549 cells. The anti-metastatic effect of FX was evaluated in vivo in an experimental lung metastatic tumor model. On the other hand, cell counting kit-8 assay was used to study the cell viability of human lung cancer PC9 cells and Gef resistant PC9 cells (PC9/G) after Gef, FX or FX combined with Gef treatment. PC9 xenograft model was established to explore the anti-tumor effect of FX or combined with Gef. Immunohistochemistry staining assay and immunofluorescence staining assay were used to reveal the effects of FX on lung cancer cell proliferation and apoptosis. ResultsFX was able to significantly inhibit lung cancer cells migration and invasion in vitro. FX suppressed the expressions of Snail, Twist, Fibronectin, N-cadherin, MMP-2, PI3K, p-AKT and NF-κB, and increased the expression of TIMP-2. Furthermore, knockdown of TIMP-2 attenuated FX-mediated invasion inhibition. Additionally, we demonstrated that FX inhibited lung cancer cells metastasis in vivo. The anti-metastatic effects of FX on lung cancer cells might be attributed to inhibition of EMT and PI3K/AKT/NF-κB pathway. We further demonstrated that the anti-tumor activity of FX was not only limited to the drug sensitive cell lines, but also prominent on lung cancer cells with Gef resistant phenotype. Furthermore, in vivo xenograft assay confirmed that FX inhibited tumor growth and enhanced the sensitivity of lung cancer cells to Gef and this effect may be due to inhibition of tumor cell proliferation and activation of apoptosis. ConclusionCollectively, our findings suggested that FX suppresses metastasis of lung cancer cells and overcomes EGFR TKIs resistance. Thus, FX is worthy of further investigation as a drug candidate for the treatment of lung cancer.

Abstract 3081: SHOC2 is a critical modulator of the sensitivity to EGFR-TKI in non-small cell lung cancer cells

Abstract Epidermal Growth Factor Receptor tyrosine kinase inhibitors (EGFR-TKI) have become the first line treatment for patients with EGFR mutated non-small cell lung cancer (NSCLC) as a result of multiple clinical trials demonstrating superior response and less toxicity as compared to conventional cytotoxic agents. However, despite the clinical experience among nearly two decades, it is well known that EGFR-TKI monotherapy cannot cure EGFR mutation-positive lung cancer patients due to the occurrence of drug resistance. While dramatic initial tumor shrinkage is shown in most patients with EGFR-TKI treatment, therapeutic resistance is often observed in 1-2 years. This is likely caused by the outgrowth of rare pre-existing resistant clones and/or small fraction of drug tolerant persister cells (DTPs) which survive and acquire resistance to EGFR-TKIs. Several previous studies have clarified the acquired resistance mechanisms to EGFR-TKIs, but little is known how the DTPs survive during the initial phase of drug exposure and rapidly adapt to EGFR-TKIs. Utilizing the genome wide CRISPR/Cas9 screening, we have previously reported that DTPs were capable to exploit endoplasmic reticulum stress to survive EGFR-TKI exposure (Terai, et al, Cancer Research 2018). Further analysis of this data identified multiple genes which could affect the initial drug response to EGFR-TKIs, including the scaffold protein coding gene, SHOC2. In our present study we focused on this unique protein and revealed its critical function as a modulator of drug response to EGFR-TKIs in lung cancer cells. We confirmed that depletion/overexpression of SHOC2 in PC9 cells, an EGFR-dependent NSCLC cell line, renders the cells sensitive/resistant to all 1st to 3rd generation EGFR-TKIs. We have also confirmed similar findings with another EGFR dependent NSCLC cell line named H1975. Reduction of phosphorylation levels of ERK1/2 induced by EGFR-TKIs were stronger in PC9 or H1975 cells with SHOC2 knockout, indicating that SHOC2 can facilitate ERK1/2 reactivation during EGFR-TKI treatment. The above data indicate that SHOC2 is essential for DTPs and its function is correlated with MAPK-ERK pathway activity. Celastrol, a candidate SHOC2 inhibitor, inhibited the emergence of DTPs and synergized with EGFR-TKIs. Additionally, we showed that SHOC2 depletion enhanced the growth inhibitory effect of osimertinib in vivo. Lastly, we confirmed the increased expression of SHOC2 in clinical human samples which experienced EGFR-TKI failure. Our findings could offer a new treatment strategy for NSCLC patients with EGFR mutation leading to the increase of the degree and duration of EGFR TKIs response. Citation Format: Hideki Terai, Junko Hamamoto, Tadashi Manabe, Katsura Emoto, Takeshi Masuda, Satoshi Kuronuma, Keigo Kobayashi, Keita Masuzawa, Shinnosuke Ikemura, Sohei Nakayama, Ichiro Kawada, Yusuke Suzuki, Osamu Takeuchi, Yukio Suzuki, Sumio Ohtsuki, Hiroyuki Yasuda, Kenzo Soejima, Koichi Fukunaga. SHOC2 is a critical modulator of the sensitivity to EGFR-TKI in non-small cell lung cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3081.

Abstract 5198: JNJ-61186372, a novel EGFR/c-Met bispecific antibody, exhibits potent antitumor activity in broad-spectrum of acquired resistance to EGFR-TKIs

Abstract Background: 1st generation (erlotinib &amp; gefitinib), 2nd generation (afatinib), and 3rd generation (osimertinib) EGFR TKIs have marked efficacy in patient with EGFR-mutant NSCLC. Unfortunately, patients who show T790M (-) mutation after treated with 1st and 2nd generation EGFR TKI or have C797S (+) mutation or other mechanisms of acquired resistance to 3rd generation EGFR TKI have no therapeutic options for curing cancer in clinic. We introduce a potent therapeutics for overcoming acquired resistance to EGFR-TKIs in several EGFR mutant NSCLC models using JNJ-61186372 (JNJ-372), a bispecific antibody that targets the EGFR and c-Met receptors. Methods: We classified PDC and PDX models into two groups: i) 1st and 2nd generation EGFR TKI acquired resistance models with T790M (-) mutation (YU-1093, YU-1152), ii) 3rd generation EGFR TKI acquired resistance models with C797S (+) mutation (YU-1097), and C797S (-) mutation with unknown resistance mechanisms (YU-1095, YU-1089, YU-1096, YUX-1024_YH1R, YHIM-1035, YHIM-1045, and YHIM-1053). We established 7 patient-derived cells (PDC) and 3 patient-derived xenografts (PDX) model from patients with broad-spectrum acquired resistance to EGFR-TKIs. We performed cell viability test and western blot to investigate antitumor activity of JNJ-372. We evaluated then in vivo antitumor activity of JNJ-372 in PDX and PDC-xenograft models. JNJ-372 was administrated i.p. twice a week at 30 mg/kg. Results: We assessed the ability of JNJ-372 to overcome broad-spectrum of EGFR-TKI acquired resistances. JNJ-372 significantly inhibited cancer cell growth in vitro and in vivo in PDC model harboring T790M (-) mutation. JNJ-372 significantly decreased cell proliferation rate and EGFR downstream signaling in BaF3 cell harboring Del19 (L858R)/T790M/C797S mutant and PDC model harboring Del19/T790M/C797S mutant with amplified KRAS and loss of Rb. C797S (-) cases of four PDCs and three PDX models had i) loss of T790M with c-Met amplification or other acquired resistance mutation and ii) T790M (+) with amplified c-Met. In these models we identified dramatic tumor regression and reduction of EGFR and c-Met expression in vitro and in vivo PDX and PDC-xenograft models. Interestingly, we observed combination treatment of JNJ-372 and c-Met inhibitor showed synergistic immune cell mediated cellular cytotoxicity in EGFR-TKI acquired resistance models harboring c-Met amplification. JNJ-372 exhibited potent inhibition of tumor growth in preclinical models through targeting EGFR and c-Met receptor, decreasing EGFR and c-Met receptor level, inhibiting EGFR downstream signaling cascade and activating ADCC effect in vivo. Conclusion: Our results suggest that JNJ-372 will provide potential and effective therapeutic options for patients with no therapeutic option to overcome broad-spectrum of acquired resistance to EGFR-TKIs. Citation Format: SooHwan Lee, Jiyeon Yun, Seo-Yoon Jeong, Chae Won Park, Seok-Young Kim, Min Hee Hong, Hye Ryun Kim, Byoung Chul Cho. JNJ-61186372, a novel EGFR/c-Met bispecific antibody, exhibits potent antitumor activity in broad-spectrum of acquired resistance to EGFR-TKIs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5198.

Abstract 3046: Anti-tumor efficacy of CKD-702 in EGFR TKI-resistant patient-derived xenograft model

Abstract Background: c-MET is co-expressed with EGFR in approximately 70% of EGFR mutant-positive tumors. In addition, c-Met amplification was found in 10-20% of lung cancer patients with acquired tolerance to EGFR TKI, and c-Met overexpression was found in 14-69% of patients receiving EGFR TKI. CKD-702, a double antibody, is designed to simultaneously block both EGFR and c-MET expression. Purpose: We evaluated the anti-tumor efficacy of CKD-702 with PDX model derived from lung adenocarcinoma patients. Methods: We constructed the PDX model by transplanting surgical tissue from lung adenocarcinoma patient into NOD/SCID mice after the approval of research protocol in Seoul St. Mary's Hospital IRB. We confirmed the co-existence of activating EGFR mutation and c-MET amplification with PNAclamp and FISH methods. We also checked overexpression of EGFR and c-MET protein by immunohistochemistry. Results: The characteristics of tumor tissue transplanted for PDX model were: 1) Korean, lung cancer with EGFR TKI chemotherapy failure confirmed c-MET amplification (&amp;gt; 5 copies) and overexpression (IHC score 3+), 19del Positive EGFR mutation, 2) European lung adenocarcinoma, c-MET amplification (&amp;gt; 2.5 copy) and overexpression (IHC score 3+) were confirmed, and L858R EGFR mutation positive tumor tumor confirmed. The anti-cancer efficacy of CKD-702 was reduced by more than 80% compared to the tumor size of the control group regardless of the concentration. In the CKD-702 20mg/kg group, there was no significant difference in the change in the overall tumor size until the time of disappearing or ending during drug administration. The %TGI was 93.6 and 97.5 according to the concentration of CKD-702, and the % TGD was 118.5 and 311.8, respectively. CKD-702 has a significantly higher tumor growth inhibitory effect regardless of the concentration, but the tumor growth retardation effect is 2.6 times different depending on the concentration. % TGI of CKD-702 20mg / kg group was similar to Hu8c4 group or Hu8c4 + Vectibix group. However, % TGD was 1.4 times higher than Hu8c4 group efficacy and 3.1 times higher than Hu8c4 + Vectibix group% TGD. Even after the completion of drug administration, tumor still remained at 47 days and 67 days in the CKD-702 10 mg/kg and 20 mg/kg groups, respectively. Although c-MET protein expression decreased during the administration of CKD-702, it was confirmed that c-MET protein expression increased again in tumor tissue at the time of tumor growth. Although the tumor size was rather large (605.6 ~ 816.2mm3), CKD-702 showed twice the tumor growth inhibition effect (% TGI: 32.8 vs. 68.1) and the tumor growth delay effect was 4.4 times higher depending on the drug concentration (% TGI: 2.8 vs 12.3). Conclusion: Our data showed that CKD-702 have effective anti-tumor activity in EGFR TKI-resistant PDX model harboring both activating EGFR mutation and c-MET overexpression. Citation Format: Jung-Young Shin, Min-Young Kim, Mi-Ran Lee, Jeong-Oh Kim, Eun-Ju Jeon, Ji Hye Choi, Soon-Ki Hong, Kyu-Jin Park, Jin Hyoung Kang. Anti-tumor efficacy of CKD-702 in EGFR TKI-resistant patient-derived xenograft model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3046.

Abstract 2040: Virtual patient simulation improves first-line treatment selection in advanced EGFR-mutated NSCLC

Abstract Background: Patients with advanced EGFR-mutated NSCLC benefit from treatment with EGFR TKIs. However, gaps in oncologists' performance hamper their ability to integrate individualized treatments into the care of patients with this disease. This study was conducted to determine if an online, virtual patient simulation (VPS)-based continuing medical education (CME) intervention improved performance of oncologists in using appropriate strategies to diagnose and manage patients with advanced EGFR-mutated NSCLC. Methods: The CME intervention consisted of two cases presented in a VPS platform that allowed learners to order lab tests, diagnoses and treatments in a manner matching the scope and depth of actual practice. Clinical decisions made by the learners using open field entries within an EHR interface were analyzed and, after each decision, tailored clinical guidance (CG) was provided based on current evidence and expert recommendation. Learner decisions were collected post-CG and compared with each user's baseline (pre-CG) data using a 2-tailed paired T-test to determine P values. The activity posted May 29, 2019; data were collected through October 15, 2019. Results: Significant improvements (reported as absolute) were observed after CG in clinical decisions made by oncologists: Clinical Decision% Absolute Improvement (% correct pre-CG vs % correct post-CG); P valueCase 1 (N=178) 59 YO African American female recently diagnosed with lung cancer after 6-week history of fatigue, cough, dyspnea; nonsmoker with history of hypertensionCase 2 (N=93) 51 YO male recently diagnosed with stage IV lung cancer with liver metastases after presenting with hacking cough, fatigue, and weight loss for the past 3 months; 10 pack-year history of smoking and hypertensionOrder PD-L1 expression14% (69% vs 83%); P&amp;lt;.0015% (79% vs 84%); P=.02Order brain MRI with/without contrast21% (53% vs 74%); P&amp;lt;.00111% (67% vs 78%); P=.001Order EGFR mutation test via tumor biopsy6% (84% vs 90%); P&amp;lt;.0015% (85% vs 90%); P=.02Diagnose stage IV, NSCLC, adenocarcinoma, EGFR del19 with multiple brain metastases37% (26% vs 63%); P&amp;lt;.001–Diagnose stage IVB, NSCLC, adenocarcinoma, EGFR del19–29% (41% vs 70%); P&amp;lt;.001Start first-line EGFR TKI41% (45% vs 86%); P&amp;lt;.00129% (54% vs 83%); P&amp;lt;.001Selection of osimertinib as first-line treatment26% (37% vs 63%); P&amp;lt;.00123% (49% vs 72%); P&amp;lt;.001 Conclusions: This study demonstrated that VPS that immerses and engages oncologists in an authentic and practical learning experience can significantly improve evidence-based clinical decisions related to diagnosis and management of patients with advanced EGFR-mutated NSCLC. Continued educational activities are needed to address the residual gaps in appropriate treatment and increase oncologists' confidence in this clinical setting. Citation Format: Michelle A. Worst, Rich Caracio, Donna L. Topping, Suresh S. Ramalingam. Virtual patient simulation improves first-line treatment selection in advanced EGFR-mutated NSCLC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2040.

Abstract 5199: JNJ-61186372, an EGFR-cMet bispecific antibody, in EGFR Exon 20 insertion-driven advanced non-small cell lung cancer (NSCLC)

Abstract PURPOSE: Although EGFR exon20 insertion (ex20ins) mutations account for 4~12 % of EGFR mutant NSCLC patients, there is no effective and selectable anticancer drugs targeting ex20ins mutations so far due to various variant mutations in ex20ins mutations. Moreover, most EGFR ex20ins mutants show primary resistance to EGFR TKIs. JNJ-61186372 (JNJ-372), a bispecific antibody that targets the EGFR and cMet receptors, is currently being explored in a first-in-human study in patients with NSCLC. To better understand the mechanism of JNJ-372 activity in this patient population, we conducted preclinical studies exploring the activity of JNJ-372 in different EGFR ex20ins models. METHODS: To elucidate whether JNJ-372 has antitumor effect in EGFR ex20ins mutants via EGFR and c-MET inhibition, cell viability, western blot, cell cycle, colony formation assay, FACS analysis were performed in JNJ-372 treated BaF3 cells, PDCs, PDOs, and PDX expressing EGFR ex20ins mutation. For mouse tumor models, JNJ-372 was administered i.p. twice a week at 10 mg/kg or 30 mg/kg. Antibody dependent cellular cytotoxicity (ADCC) assay was assessed to figure out whether JNJ-372 had ADCC effects. Referenced patients with ex20ins disease were administered 1050 mg JNJ-372 i.v. weekly for the first 4-week cycle, then biweekly for each subsequent cycle RESULTS: JNJ-372 inhibited the growth of BaF3 cells, PDCs, and a PDO harboring a range of ex20ins, which were resistant to osimertinib and gefitinib. Mechanistic assays revealed the reduction of EGFR and cMet receptor levels and decreases in phospho-EGFR and c-Met, as well as inhibition of their downstream signaling pathways. Cleaved caspase-3 and BIMEL were upregulated at anti-proliferative doses, suggesting caspase-mediated cell death. JNJ-372 demonstrated corresponding antitumor activity in PDC and PDX models harboring different ex20ins; inhibition of signaling and engagement of the apoptotic pathway was confirmed in tumors of JNJ-372-treated mice. In PDCs with EGFR ex20ins mutation, we verified that JNJ-372 had a significant ADCC effect compared to EGFR antibody drug cetuximab. In the first-in-human trial, CT scans from two patients treated with JNJ-372 revealed reductions in tumor burden. A 58-year patient harboring H773delinsNPY showed -63% tumor reduction with progression-free survival of &amp;gt; 20 months and a 48-year patient harboring S768_D770dup showed -38.9% tumor reduction. CONCLUSION: JNJ-372 drives antitumor activity in preclinical models of EGFR ex20ins, which have no therapeutic options in clinic, by decreasing EGFR and cMet receptor levels, inhibiting downstream signaling cascades, activating apoptotic signaling as well as ADCC. These results provide a promising therapeutic option to patients with EGFR ex20ins mutations and an understanding of the activity of JNJ-372 being observed in the first-in-human study. Citation Format: Jiyeon Yun, Han Na Kang, Soo-Hwan Lee, Seo-Yoon Jeong, Chae Won Park, Jae-Hwan Kim, Kyoung-Ho Pyo, Ji Min Lee, Seok-Young Kim, Min Hee Hong, Hye Ryun Kim, Meena Thayu, Joshua Curtin, Roland Knoblauch, Matthew Lorenzi, Byoung Chul Cho. JNJ-61186372, an EGFR-cMet bispecific antibody, in EGFR Exon 20 insertion-driven advanced non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5199.

Abstract 1883: Erlotinib resistance via a persister bottleneck results in mixed expression of EGFR T790M and EMT-like state with high AXL expression

Abstract Background: Recent studies have suggested that EGFR mutant cells exposed to high concentrations of EGFR TKI (erlotinib) develop resistance through a persister bottleneck and reflect a spectrum of resistance mechanisms. Our goal was to exploit this approach to further understand mechanisms of EGFR TKI resistance and develop new therapeutic targets. Methods: To generate erlotinib resistant “persister” cell clones (EPs), PC9 cells were seeded at low density and exposed to 2.5 µM of erlotinib for ~6 weeks. In this time approximately 99.9% of cells died, while a small surviving fraction entered a quiescent state defined as “persisters” followed by proliferation of clones. Over 20 clonal subpopulations of resistant clones were obtained, of which four have been characterized via proliferation assays, immunoblotting, DNA sequencing and RNA-Seq analysis. Results: DNA sequencing and subsequent phenotypical analysis revealed varying EGFR T790M allele frequency and osimertinib sensitivity. Two clones were sensitive to osimertinib, with their T790M allele frequencies being 3.7% and 7.0%. The other two clones were resistant to osimertinib with allele frequencies being 0% and 8.9%. Common mechanisms of EGFR TKI resistance (MET amplification, KRAS mutations, BRAF/MEK mutations) were not observed in any of the EP clones. DNA sequencing also identified 13 unique mutations across the clones compared to the parental PC9 and none of these were known oncogenes or associated with TKI resistance. Maintenance of downstream signaling despite loss of EGFR phosphorylation in the presence of erlotinib was observed in all the clones. Analysis of the RNA-seq data revealed all four clones show an increase in vimentin, a marker of EMT, with one clone showing a 25-fold increase. Three of the four clones had greater than two-fold expression of SLUG mRNA, a known transcriptional factor of EMT resistance, while no changes in other EMT transcription factors (Snail, Twist, Zeb1) were identified. Interesting, AXL, a receptor tyrosine kinase known to be associated with EMT, was the only up-regulated gene across all four clones. Treatment with AXL TKI alone (RXDX-106 and R428) did not affect cell viability, but one EP clone demonstrated sensitivity to erlotinib when combined with R428 but not RXDX-106. Downstream signaling also shows that this combination results in strong reduction in ERK phosphorylation compared to either single agent. Conclusions: Our data suggests that EPs have developed resistance through a combination of T790M and EMT-like state, with AXL upregulation as a common theme. One clone, harboring some T790M and osimertinib sensitivity, was also sensitive to erlotinib when combined with R428. Citation Format: Sina Hosseinian, Anurima Majumder, Matthew Smith, Fumi Kinose, Eric Haura. Erlotinib resistance via a persister bottleneck results in mixed expression of EGFR T790M and EMT-like state with high AXL expression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1883.