EGFR T790M Research Articles

Variations in Genomic Testing in Non-small Cell Lung Carcinoma: A Healthcare Professional Survey of Current Practices in the UK.

This survey aims to understand the current UK practice for non-small cell lung carcinoma (NSCLC) and identify barriers that may impact patient treatment and outcomes. In March-June 2021, 57 interviews were conducted with healthcare professionals involved in the secondary care management of patients with NSCLC. Most respondents performed genetic testing at onsite and non-genomic laboratory hub (GLH) offsite locations. The most common genetic tests were EGFR T790M variant (100%), EGFR exon 18-21 covered (95%) and BRAF (93%). No targeted therapy (TT) available (69%), lack of access to a TT (54%) or excessive molecular testing turnaround times (39%) were the most common reasons for using an immuno-oncology therapy over a TT in the first-line setting. The survey highlights variation in mutation testing practices across the UK, which may impact treatment decisions and contribute to health outcome inequality.

Overall survival analysis from the ADAURA trial of adjuvant osimertinib in patients with resected EGFR-mutated (EGFRm) stage IB–IIIA non-small cell lung cancer (NSCLC).

LBA3 Background: Osimertinib is a third-generation, central nervous system (CNS) active EGFR-TKI, that potently and selectively inhibits EGFR-TKI sensitizing and EGFR T790M resistance mutations. In the primary analysis from the Phase III ADAURA study (NCT02511106), adjuvant osimertinib demonstrated a clinically meaningful, statistically significant, and practice-changing disease-free survival (DFS) benefit vs placebo in patients with completely resected EGFRm (ex19del/L858R) NSCLC ± adjuvant chemotherapy. In an updated DFS analysis, with 2 years additional follow-up, DFS and CNS DFS benefit with adjuvant osimertinib were sustained (stage II–IIIA DFS hazard ratio [HR] 0.23; 95% confidence interval [CI] 0.18, 0.30; stage IB–IIIA DFS HR 0.27; 95% CI 0.21, 0.34; stage II–IIIA CNS DFS HR 0.24; 95% CI 0.14, 0.42), with a tolerable safety profile observed over the extended treatment duration. Here, we report the planned final overall survival (OS) analysis from ADAURA. Methods: Eligible patients (aged ≥18 years [≥20 in Japan and Taiwan], WHO PS 0/1 with completely resected EGFRm (ex19del/L858R) stage IB, II or IIIA [AJCC/UICC 7th edition] NSCLC; adjuvant chemotherapy allowed) were randomized 1:1 to osimertinib 80 mg once daily or placebo until disease recurrence, treatment completion (3 years), or a discontinuation criterion was met. The primary endpoint was investigator-assessed DFS in stage II–IIIA. Key secondary endpoints: DFS in stage IB–IIIA, OS and safety. Data cut-off: January 27, 2023. Results: Globally, 682 patients were randomized; osimertinib n=339, placebo n=343. Adjuvant osimertinib significantly improved OS vs placebo. In patients with stage II–IIIA disease, OS HR was 0.49 (95% CI 0.33, 0.73; p=0.0004; 100/470 events, 21% maturity); 5-year OS rate was 85% with osimertinib vs 73% with placebo. Median follow-up for OS in stage II–IIIA was 59.9 months (osimertinib) and 56.2 months (placebo). In the overall population (stage IB–IIIA), OS HR was 0.49 (95% CI 0.34, 0.70; p<0.0001; 124/682 events, 18% maturity); 5-year OS rate was 88% with osimertinib vs 78% with placebo, with a median follow-up for OS of 60.4 months (osimertinib) and 59.4 months (placebo). Median OS was not reached in either population or treatment group. Conclusions: Adjuvant osimertinib demonstrated an unprecedented, highly statistically significant and clinically meaningful OS benefit in patients with EGFRm stage IB–IIIA NSCLC after complete tumor resection, with or without adjuvant chemotherapy. ADAURA is the first global Phase III study to demonstrate a statistically significant DFS and OS benefit with targeted treatment for patients with EGFRm stage IB–IIIA NSCLC. Clinical trial information: NCT02511106 .

Deciphering the molecular mechanism of enhanced tumor activity of the EGFR variant T790M/L858R using melanoma cell lines.

The abnormal expression and mutagenesis of EGFR drives both the development and progression of a multitude of human cancers. Further mutations within the tyrosine kinase region of the EGFR subsequently contribute to resistance to targeted drugs. What is not known is how these mutations affect progression-related behaviors of cancer cells. The mutagenesis of EGFR T790M, L858R, and T790M/L858R was performed via oligo primer-guided polymerase chain reaction (PCR). GFP-tagged mammalian expression vectors were constructed and confirmed. Stable melanoma cell lines WM983A and WM983B expressing WT or mutant EGFRs were generated for determining the functions of WT and mutant EGFRs in migration, invasion, and resistance to doxorubicin. Immunoblotting and immunofluorescence were performed to detect the transphosphorylation and autophosphorylation of WT and mutant EGFRs and other molecules. The EGFR mutant T790M/L858R showed significantly higher basal autophosphorylation in melanoma cell lines WM983A and WM983B. Overexpression of WT EGFR significantly enhanced the protein level of E-cadherin (E-cad) via upregulating its mRNA. In contrast, L858R significantly downregulated E-cad. Biological activity assays show that T790M/L858R presented significant enhancement in vitro in invasion and migration, while WT and T790M moderately inhibited invasion and migration. In WM983A cells, enhanced invasion and migration by T790M/L858R required the downstream signaling pathways through Akt and p38. T790M/L858R dramatically triggers phosphorylation of actin cross-linking protein alpha-actinin-4 in the absence of EGF. This double mutant also conferred resistance to a general chemotherapy doxorubicin through Akt but not the p38 signaling pathway. These findings suggest that T790M/L858R not only confers enhanced therapeutic resistance in cancer cell lines but also may promote tumor metastasis via its boosted downstream signaling pathways and/or direct phosphorylation of other key proteins.

Liquid and tissue profiling of somatic mutational landscape of non-small cell lung cancer from an Indian cohort.

e21202 Background: Circulating tumor DNA (ctDNA) encompasses spatial and temporal heterogeneity, tumor evolution, and is ideal for repeat sampling and predictive therapeutic course corrections whereas tissue is the first choice for molecular profiling. We report the mutational landscape of an Indian NSCLC cohort, with focus on liquid biopsy and value addition offered by paired tissue and liquid profiling. Methods: The study comprised 507 tumor tissue and 1271 liquid biopsies from NSCLC patients. Evaluation of single nucleotide alterations, copy number alterations and fusions was conducted using targeted next generation sequencing with paired tissue and liquid profiling in a sub cohort (n = 45). Results: In tissue based analysis (n = 507), EGFR-sensitizing mutations were the most common alterations, detected in 41.4% of samples. Among these, Exon 19 deletion was seen in 46.3%, Exon 21 L858R in 23.9%, Exon 20 insertion in 4.6%, Exon 18 G719X in 3.7% and compound mutations in 5%. TP53, KRAS and PIK3CA mutations were detected in 34.5%,13.4% and 6.3% respectively. MYC was commonly amplified (15.4%) gene followed by PIK3CA (4.1%), CCNE1 (3.3%), EGFR (2.9%), and MET (2.9%) amplifications. Tissue fusion analysis (n = 398) detected ALK in 6.3%, ROS1 in 3.3%, and RET in 1.8%. METEx14 was detected in 4.5%. Liquid biopsy (n = 1271) showed comparable mutation spectrum as tissue, with the EGFR gene being the most altered (32.9%). Mutations in TP53 were observed in 26.4%, KRAS in 7.4%, PIK3CA in 4.4% and BRAF in 3%. In EGFR positive individuals, EGFR T790M was detected at higher frequency in liquid (16.7%) than tissue (8.6%), indicating this acquired resistance alteration being better captured in liquid biopsy than tissue due to tumor heterogeneity and tumor evolution. 2 liquid biopsy samples identified additional KRAS driver mutations not seen in the paired tissue analysis, highlighting the utility of simultaneous tissue and liquid analysis. Concurrent actionable alterations were detected in 11% of tissue and 5% of liquid samples. Conclusions: We report the largest liquid biopsy study reporting the genomic landscape from NSCLC cases of Indian origin along with tissue based molecular landscape. Presence of alterations like KRAS and EGFR T790M in liquid but absence in concurrent tissue biopsy shows utility of paired tissue and liquid biopsy profiling. Higher incidence of targetable and co-existing alterations in the Indian population highlight the utility of broad molecular profiling for successful utilization of targeted therapies. [Table: see text]

Oncomine Dx Express CE-IVD liquid biopsy assay for non-small cell lung cancer: Performance review and analytical validation.

e21218 Background: Clinical diagnostics assays for oncology are becoming more readily available due to the advancement and democratization of Next-Generation Sequencing (NGS). Additionally, liquid biopsy can be used in NGS to detect genetic variants in circulating tumor DNA and RNA. Liquid biopsy is a less invasive option compared to traditional biopsy methods for early detection and continuous monitoring of cancer treatment outcomes. Here, we discuss screening of over 3,500 non-small cell lung cancer (NSCLC) samples, and the analytical validation of the Oncomine Dx Express Test (ODxET) and Genexus Dx Integrated Sequencer for detection of clinically significant variants in liquid biopsy samples. Methods: Our team screened over 3,500 NSCLC liquid biopsy samples in search of high priority clinical variants. Variants selected for analytical validation studies included ERBB2 exon 20 insertion, EGFR exon 20 insertion, EGFR exon 19 deletion, EGFR T790M, KRAS G12C, BRAF V600E, and RNA fusion isoforms including ALK, NTRK1/2/3, RET, and ROS1 oncogenic drivers. Over 550 screened samples were found to be positive for these variants of interest. This screening was performed on the Genexus Dx Integrated Sequencer according to the user guide. Results: The Genexus Dx Integrated Sequencer automates library preparation, sequencing, analysis, and reporting QC metrics and variant calls. Sequencing run setup is quick and straightforward, taking less than 15 minutes to start a run and just over 24 hours to go from nucleic acid to report. Limit of detection (LoD) for DNA SNVs, insertions, and deletions at 5 ng DNA input level ranged from 0.65% to 1.82% allelic frequency (AF), depending on the variant. The higher DNA input of 30 ng resulted in a lower LoD range, from 0.31% to 0.42% AF. RNA fusion and splice variant LoD at 5 ng sample input ranged from 9.9 to 19.6 molecular counts. The higher 30 ng input resulted in a lower LoD range for RNA variants as well, ranging from 6.4 to 8.0 molecular counts. In the analytical accuracy study, the false positive rate was found to be 0.2% for SNVs, 0% for insertions/deletions, and 0% for fusion targets. In the analytical reproducibility study, the average with-in run repeatability call rate (No Calls excluded) was 99.64% for DNA variants and 98.75% for RNA variants. Conclusions: Here, we demonstrated that the ODxET workflow on Genexus Dx Integrated Sequencer is a fast and efficient tool for testing clinical NSCLC liquid biopsy samples with high sensitivity and specificity.

Patients harboring uncommon EGFR exon 19delins variants of advanced non-small-cell lung cancer and response to osimertinib: A multi-center retrospective study.

e21184 Background: This study aims to investigate the efficacy of different EGFR inhibitors therapy in patients with advanced non-small-cell lung cancer (NSCLC) harboring atypical EGFR exon 19delins variants. Methods: Targeted sequencing database of 1426 treatment-naive patients with advanced NSCLC were retrospectively screened for EGFR exon 19 deletion (19del) variants in a multi-center study from January 2017 to January 2022, and 34 patients with uncommon EGFR exon 19 deletion-insertions (19delins) were included. The response rate, and progression-free survival (PFS) were analyzed according to the radiographic images. Results: We identified 3 previously unreported uncommon EGFR exon 19delins variants. Among the 34 patients who had uncommon EGFR19delins, 24 patients received first-generation, and 10 patients received third-generation as front-line EGFR TKI therapy, the median progression-free survival (mPFS) was significantly longer for patients who received first-generation than those with third-generation (19.1 months vs 6.9 months; HR: 0.18, 95% CI: 0.062-0.507; p < 0.001). At progression, EGFR T790M was the main acquired mechanism of resistance (8/14; 57.2%). There were 22 patients who received osimertinib therapy, the overall ORR and DCR were 54.5% and 81.8%, respectively, and the overall mPFS was 6.9 months. But no significant difference was observed in mPFS between first-line osimertinib and second-line osimertinib therapy (6.9 months vs. 3.8 months; p = 0.206). Conclusions: The uncommon EGFR exon 19delins variants of advanced NSCLC have longer mPFS for the first-generation compared with the third-generation as front-line EGFR TKI therapy, and respond poor to third-generation EGFR inhibitors, regardless of whether osimertinib was used as first-line or second-line therapy. Patients with uncommon EGFR exon 19delins may be a biomarker of poor efficacy with the third-generation EGFR TKI therapy.

A nation-wide genomic screening project for further development of targeted therapies in treatment-refractory non–small-cell lung cancer (LC-SCRUM-TRY).

9097 Background: Targeted therapies based on oncogenic drivers demonstrate dramatic and durable response in patients with non-small cell lung cancer (NSCLC). However, acquired resistance inevitably develop by diverse genomic resistance mechanisms. Genotype-matched targeted therapies to overcome treatment-resistance have not been established, except osimertinib against EGFR T790M mutation. Methods: We established a nation-wide genomic screening project in treatment-resistant NSCLC (LC-SCRUM-TRY; UMIN000041957) in September 2020 to identify the genetic resistant alterations, and to promote clinical therapeutic development. Enrolled patients have been screened for genetic alterations using a rapid next-generation sequencing (NGS) system, Oncomine Precision Assay (OPA) for tissue samples, and Guardant 360 (G360) or OPA for plasma samples. MET amplification was also evaluated by FISH for NSCLC post EGFR-TKI treatment. Results: As of January 2023, 129 institutions in Japan were participating, and 1252 patients had been enrolled. Sample types were tissue (84%), and plasma (16%), respectively. Turn-around-time (median [interquartile range]) of OPA and G360 was 5 (4-6), and 9 (8-10.5) days, respectively. Of these, a total of 711 (57%) were already identified to have oncogenic driver at enrollment ( EGFR, 556 [43%]; ALK, 70 [6%]; RET, 22 [2%]; ROS1, 21 [2%]; KRAS, 15 [1%]; ERBB2 exon 20 insertion [ex20ins], 8 [0.6%]; MET exon 14 skipping [ex14skip], 8 [0.6%]; BRAF V600E, 7 [0.6%]; NTRK3, 2 [0.2%]; others, 2 [0.2%]). Of 556 EGFR-mutated NSCLC, 537 (97%) were enrolled post EGFR-TKIs. EGFR mutation subtypes were exon 19 deletion (52%), L858R (41%), ex20ins (1%), others (6%), respectively. Of 537 EGFR-TKI resistant tumors, 326 (40%) had at least one genetic alteration related with drug resistance, including EGFR alterations (amplification [13%], C797S [4%], A750P [3%], E709X [2%], L792X [1%], L718Q [1%]), MET amp (16%), and other driver mutation/rearrangement (8%). Through this screening, 37 (7%) of patients resistant to EGFR-TKI were enrolled into clinical trials, with 16/102 (16%) patients with targetable alterations ( MET amp or EGFR C797S). In addition, of 541 NSCLC without oncogenic drivers at enrollment, 116 (21%) were identified to have actionable oncogenic drivers with FDA-approved drug (KRAS G12C, 23 [4%]; ERBB2 ex20ins, 23 [4%]; RET, 13 [2%]; MET ex14skip, 13 [2%]; EGFR except ex20ins, [4%]; EGFR ex20ins, 10[2%]; ALK, 6 [1%]; ROS1, 5 [1%]). Conclusions: LC-SCRUM-TRY contributes to the clinical development of precision medicine to overcome drug resistance, especially for EGFR-mutated NSCLC resistant to EGFR-TKI. This screening platform also help to practice precision medicine for patients initially diagnosed as driver-negative. Clinical trial information: UMIN000041957 .

Clinical validity of oncogenic driver genes detected from circulating tumor DNA in the blood of lung cancer patients.

This study aimed to evaluate the concordance of oncogenic driver mutations between tumor tissues and circulating tumor DNA (ctDNA) in patients with lung cancer. In addition, this study attempted to reveal the clinical utility of ctDNA in lung cancer treatment. Recurrent or metastatic non-small cell lung cancer (NSCLC) patients were prospectively enrolled in this study. Tumor tissue and serial blood samples were obtained from newly diagnosed patients (Cohort A) or patients treated with targeted therapy (Cohort B) and targeted gene panel sequencing was conducted to identify tumor mutational profiles. At the time of diagnosis, patients in Cohort A with a high cell-free DNA (cfDNA) concentration had poorer overall survival than those with a low cfDNA concentration. The sensitivity and precision of ctDNA analysis in pre-treatment patients compared with those of tissue sequencing were 58.4% and 61.5%, respectively. Known lung cancer-associated variants of oncogenic driver genes, including EGFR and KRAS, and tumor suppressor genes, including TP53 and APC, were frequently detected in the ctDNA of the patients (76.9%). An association between smoking and TP53 mutation status was observed in both tissues and ctDNA (P=0.005 and 0.037, respectively). In addition, the EGFR T790M resistance mutation was detected solely from the ctDNA of two patients after treatment with an EGFR tyrosine kinase inhibitor. ctDNA may be a reliable prognostic biomarker with an additional role in treating patients with lung cancer. Further analyses are necessary to understand the properties of ctDNA and widen its clinical use.

Liquid and tissue profiling of targetable co-mutations with KRAS in non-small cell lung cancer.

e21201 Background: The primary objective of this study was to understand the co-occurrence of KRAS with other actionable drivers in Indian lung cancers patients, and to assess the utility of liquid biopsy in this cohort. Methods: We evaluated single nucleotide alterations, copy number alterations, and fusions in 507 tumor tissue and 1271 blood specimens with targeted NGS panels in NSCLC patients. Results: In tissue-based analysis (n = 507), KRAS mutations were detected in 68 (13.4%) samples. Of these, 63 (92.6%) samples did not show the presence of any targetable co-mutations. Targetable co-mutations were detected in 5 (7.4%), with concomitant EGFR mutations in all 5 samples(100%) and BRAF mutations in 2 samples (33.3%) In the liquid biopsy (n = 1271) analysis, KRAS mutations were observed in 94 (7.4%) samples. The liquid biopsy analysis showed higher presence of targetable co-mutations in 11.7% (n = 11). EGFR was the most common co-occurring targetable gene observed in 9 samples (81.8%). Mutations in BRAF were detected in 3 (27.3%) samples. ERBB2 Exon 20 insertion was detected in 1 sample (9.1%). No ALK, RET or ROS1 fusions were seen in our KRAS positive cohort. In the tissue cohort, one newly diagnosed adenocarcinoma case showed primary co-mutations in EGFR (Exon 19del VAF 76.8%) and KRAS (G13D VAF 10.5%), impacting utility of EGFR TKI as the first line therapy. Another EGFR Exon 19 del patient developed KRAS G12D mutation as an acquired resistance mutation post Osimertinib therapy. In the liquid biopsy cohort (n = 11), the KRAS positive cohort had four newly diagnosed patients with 3 having EGFR and 1 with BRAF co-mutation. Among the patients with prior therapy, one patient at presentations had EGFR Exon 19 deletion and developed EGFR T790M at progression on Afatinib. The baseline tissue did not have coexisting KRAS mutation. Fresh tissue biopsy was performed at the progression on Osimertinib. The noteworthy finding was the KRAS G12C (VAF 0.36%) presence in liquid biopsy but absences in the simultaneous fresh tissue sample. The EGFR Exon 19 was present at lesser frequency than KRAS in the liquid (VAF 0.23%). In the patients from liquid biopsy cohort with Osimertinib therapy (n = 4), all presented with KRAS as acquired resistance mutation. One among these also showed concomitant EGFR C797S mutation. Conclusions: KRAS mutation virtually makes it unlikely for other targetable mutations to coexist. It makes it thus compelling to further study the role of liquid biopsy with KRAS first and decide if chemotherapy with IO can be started considering the standard of care is to wait for the entire targeted panel before initiating IO. Equally its important to include KRAS in the lung panel and although small the concomitant KRAS may compromise the efficacy to TKI. Liquid biopsy is a promising alternative for detection of actionable alterations, including KRAS and can be harnessed for longitudinal profiling, monitoring tumor evolution and treatment outcome.

BLU-945 monotherapy and in combination with osimertinib (OSI) in previously treated patients with advanced EGFR-mutant (EGFRm) NSCLC in the phase 1/2 SYMPHONY study.

9011 Background: Despite improved frontline OSI treatment outcomes, patients (pts) with advanced EGFRm NSCLC inevitably progress, with worse outcomes in the EGFR L858R subgroup (median PFS of 14.4 vs 21.4 mo in pts with exon 19 deletions). BLU-945 is an investigational, next-generation, oral tyrosine kinase inhibitor (TKI) that targets common EGFR-activating and T790M and C797X resistance mutations, while being selective against EGFR wild type (WT). Preclinically, BLU-945 in combination (combo) with OSI had enhanced potency on L858R and extended antitumor response durability versus OSI alone. SYMPHONY (NCT04862780) is an ongoing first-in-human phase 1/2 study of BLU-945 monotherapy (mono) or combo with OSI. Methods: In the phase 1 dose escalation, pts (aged ≥18 y; metastatic EGFRm NSCLC; ECOG PS 0–1; treated with ≥1 EGFR TKI) received BLU-945 mono starting at 25 mg QD. Pts progressing on OSI could receive BLU-945 with 80 mg OSI starting at 50% of the highest safe BLU-945 mono dose. Each dose escalation followed a Bayesian Optimal Interval design. Safety, including dose-limiting toxicities (DLT), PK, and circulating tumor DNA (ctDNA) mutational status were assessed. Results: As of Jan 6, 2023, 108 pts received BLU-945 mono (25–600 mg QD; 100–300 mg BID). Pts had a median of 3 lines of prior therapy and were genomically complex with ctDNA detectable EGFR on-target and/or off-target resistance alterations (46%) at baseline. DLTs included alanine transaminase (ALT) and aspartate transaminase (AST) elevation, hepatic cytolysis, fatigue, nausea, vomiting, and hyponatremia, occurring at doses of ≥400 mg/d. The maximum tolerated dose was 500 mg/d. The most common AEs were nausea (42%), headache (40%), increased ALT (38%), increased AST (37%), and vomiting (32%). Robust on-target EGFR activity was observed with ctDNA reduction on day 15 in 90%, 85% and 70% of EGFR T790M, C797S, and L858R alleles at ≥400 mg/d doses, respectively. At ≥400 mg/d, 48% had tumor shrinkage, including partial responses (PRs). Twenty-five pts received BLU-945 (200–400 mg QD; 100–200 mg BID) combo with OSI. Pts had a median of 2 lines of prior therapy. The most common combo AEs were fatigue (36%), diarrhea (32%), headache (32%), and nausea (28%). Other EGFR WT AEs were dry skin (20%) and rash (8%). One DLT (Grade 4 pneumonitis) was reported in the BLU-945 300 mg QD with OSI group. Tumor reductions, including PRs in 2 pts progressing on OSI, were observed at ≥300 mg/d. Dose escalation is ongoing. Conclusions: BLU-945 mono and combo with OSI were generally well tolerated and showed robust on-target EGFR ctDNA reduction, with tumor shrinkage in genomically heterogeneous, heavily pretreated pts. Combo showed responses at BLU-945 doses lower than in mono, consistent with additive benefit. The combo safety profile and on-target activity provide rationale for further development in front line. Clinical trial information: NCT04862780 .

New Indole Diketopiperazine Alkaloids from Soft Coral-Associated Epiphytic Fungus Aspergillus versicolor CGF 9-1-2.

Two new indole diketopiperazine alkaloids (IDAs), (+)19-epi-sclerotiamide (1) and (-)19-epi-sclerotiamide (2), along with 13 known analogs (3-15), were isolated from a soft coral-associated epiphytic fungus Aspergillus versicolor CGF 9-1-2. The structures of two new compounds were established based on the combination of HR-ESI-MS, 1D and 2D NMR spectroscopy, optical rotation measurements and quantum chemical 13 C-NMR, the absolute configurations were determined by experimental and electronic circular dichroism (ECD) calculations. The results of molecular docking showed that all the compounds had a good binding with TDP1, TDP2, TOP1, TOP2, Ache, NLRP3, EGFR, EGFR L858R, EGFR T790M and EGFR T790/L858. Biological evaluation of compounds 3, 6, 8, 11 showed that 3 exerted a strong inhibitory effect on TDP2 with a rate of 81.72 %.

3-Phosphoinositide-dependent kinase 1 drives acquired resistance to osimertinib

Osimertinib sensitive and resistant NSCLC NCI-H1975 clones are used to model osimertinib acquired resistance in humanized and non-humanized mice and delineate potential resistance mechanisms. No new EGFR mutations or loss of the EGFR T790M mutation are found in resistant clones. Resistant tumors grown under continuous osimertinib pressure both in humanized and non-humanized mice show aggressive tumor regrowth which is significantly less sensitive to osimertinib as compared with parental tumors. 3-phosphoinositide-dependent kinase 1 (PDK1) is identified as a potential driver of osimertinib acquired resistance, and its selective inhibition by BX795 and CRISPR gene knock out, sensitizes resistant clones. In-vivo inhibition of PDK1 enhances the osimertinib sensitivity against osimertinib resistant xenograft and a patient derived xenograft (PDX) tumors. PDK1 knock-out dysregulates PI3K/Akt/mTOR signaling, promotes cell cycle arrest at the G1 phase. Yes-associated protein (YAP) and active-YAP are upregulated in resistant tumors, and PDK1 knock-out inhibits nuclear translocation of YAP. Higher expression of PDK1 and an association between PDK1 and YAP are found in patients with progressive disease following osimertinib treatment. PDK1 is a central upstream regulator of two critical drug resistance pathways: PI3K/AKT/mTOR and YAP.

Synthesis and preclinical evaluation of two osimertinib isotopologues labeled with carbon-11 as PET tracers targeting the tyrosine kinase domain of the epidermal growth factor receptor

IntroductionOsimertinib is a third-generation tyrosine kinase inhibitor (TKI) that is able to inhibit the EGFR treatment resistance mutation T790M and primary EGFR mutations Del19 and L858R. The aim of the study was to evaluate the potential of carbon-11 labeled osimertinib to be used as a tracer for the PET imaging of tumors bearing the T790M mutation. MethodsOsimertinib was labeled with carbon-11 at two positions, and the effect of the labeling position on the metabolism and biodistribution was studied in female nu/nu mice. The mutation status specificity of osimertinib was confirmed in vitro in a cell growth inhibition experiment, and the tumor-targeting potential of the carbon-11 isotopologues was evaluated using female nu/nu mice xenografted with NSCLC cell lines; the wild-type EGFR expressing A549, the primary Del19 EGFR mutated HCC827 and the resistance T790M/L858R mutated H1975. One of the osimertinib tracers was selected based on the results acquired and evaluated for tracer specificity and selectivity by assessment of tumor uptake in a PET study where HCC827 tumor-bearing mice were pretreated with osimertinib or afatinib. Results[Methylindole-11C]- and [dimethylamine-11C]osimertinib were synthesized by 11C-methylation of precursors AZ5104 and AZ7550, respectively. Rapid metabolism of both analogs of [11C]osimertinib was observed. Although the tumor uptake and retention of [methylindole-11C]- and [dimethylamine-11C]osimertinib in tumors were similar, the tumor-to-muscle ratios appeared to be higher for [methylindole-11C]osimertinib. The highest uptake, tumor-to-blood, and tumor-to-muscle ratio were observed in the Del19 EGFR mutated HCC827 tumors. However, the specificity and selectivity of [methylindole-11C]osimertinib PET could not be demonstrated in HCC827 tumors. The uptake of [methylindole-11C]osimertinib was not significantly higher in T790M resistance mutated H1975 xenografts compared to the negative control cell line A549. ConclusionsOsimertinib was successfully labeled at two positions with carbon-11, yielding two EGFR PET tracers, [methylindole-11C]osimertinib and [dimethylamine-11C]osimertinib. The preclinical evaluation demonstrated uptake and retention in three NSCLC xenografts; A549, HCC827, and H1975. The highest uptake was observed in the primary Del19 EGFR mutated HCC827. The ability of [methylindole-11C]osimertinib to distinguish between the T790M resistance mutated H1975 xenografts and the wild-type EGFR expressing A549 could not be confirmed in the ex vivo study.

Central nervous system efficacy of furmonertinib (AST2818) in patients with EGFR T790M mutated non-small cell lung cancer: a pooled analysis from two phase 2 studies

BackgroundFurmonertinib (AST2818) is a brain penetrant pan-epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) targeting both EGFR sensitizing mutations and T790M mutation. We report the pooled central nervous system (CNS) efficacy data of furmonertinib in patients with EGFR T790M mutated non-small cell lung cancer (NSCLC) from two phase 2 studies.MethodsThis was a pooled, post-hoc analysis of two phase 2 studies (NCT03127449 [phase 2a study of furmonertinib], NCT03452592 [phase 2b study of furmonertinib]). In the phase 2a study, patients received furmonertinib 40 mg, 80 mg, 160 mg, or 240 mg orally once daily. In the phase 2b study, all patients received furmonertinib 80 mg orally once daily. CNS efficacy of furmonertinib was analyzed in patients with baseline CNS lesions by an independent review center per Response Evaluation Criteria in Solid Tumors version 1.1.ResultsA total of 132 patients with baseline CNS metastases were included in this analysis. In 52 patients with measurable CNS lesions, CNS objective response rates were zero (0/1), 65% (22/34), 85% (11/13), and 25% (1/4), and CNS disease control rates were zero (0/1), 97% (33/34), 100% (13/13), and 100% (4/4) in the 40 mg, 80 mg, 160 mg, and 240 mg orally once daily group, respectively. In patients with measurable or non-measurable CNS lesions, median CNS progression-free survival was 2.8 months (95% confidence interval [CI] 1.4–8.3), 11.6 months (95% CI 8.3–13.8), 19.3 months (95% CI 5.5-not available [NA]), and not reached (95% CI 2.8 months-NA) in the 40 mg, 80 mg, 160 mg, and 240 mg orally once daily group, respectively.ConclusionsFurmonertinib showed promising CNS efficacy in doses of 80 mg orally once daily or higher in patients with EGFR T790M mutated NSCLC.Trial registrationBoth studies were registered on ClinicalTrial.gov. The phase 2a study was registered with NCT03127449 on April 25, 2017; The phase 2b study was registered with NCT03452592 on March 2, 2018.

Abstract CT102: Interim results of a first-in-human, dose escalation and expansion study of PLB-1004, an irreversible inhibitor of exon 20 insertion mutations in patients with non-small cell lung cancer

Abstract Background: PLB1004, a novel mono-anilino-pyrimidine small molecule inhibitor of EGFR, potently and irreversibly targets exon 20 insertion mutations with IC50 values ranging from 25.67-316.6 nM. The molecule also potently targets classical EGFR mutations ExDel19, L858R and T790M with a high degree of selectivity over wild-type EGFR. Methods: The study is a multi-center, open-label, dose escalation and expansion study conducted entirely in China, to assess the safety, tolerability, pharmacokinetics, and anti-tumor effect of PLB1004, administered orally once per day, in patients with advanced non-small cell lung cancer. The primary objective of the study is to assess the safety profile of PLB1004 and determine the RP2D of the molecule. Results: Dose escalation ranged from a starting dose of 10 mg QD to a top dose of 480 mg QD in 11 cohorts of patients. Dose expansion is ongoing at two dose levels, 320 mg QD and 400 mg QD. At the cutoff date for this abstract, July 31, 2022, a total of 65 patients (32 in escalation and 33 in expansion) had received treatment with PLB1004. The median age of the patients is 58 years old (range 31 to 77). Most patients are women (60%) with adenocarcinoma (95%) and good performance status (ECOG 0-1 in 90%). Prior therapy for NSCLC included platinum-based chemotherapy in 54% of patients and TKI therapy in 58%. Of note 58% of patients had intra-cranial metastases at baseline. The most frequent treatment related adverse events included diarrhea in 75% of patients (18% Grade 3), rash in 60% of patients (11% Grade 3), mouth ulceration in 43% of patients (1.5% Grade 3), elevated serum creatinine in 43% of patients (0% Grade 3) and elevated aspartate aminotransferase in 41% of patients (3% Grade 3). The criteria for DLT were not reported at any dose level and thus an MTD was not determined during cycle 1 of drug administration. Beyond Cycle 1, at the highest dose levels, frequent dose interruptions and reductions due to toxicity were observed, and further dose escalation was not attempted above 480 mg QD. A more complete summary of safety data will be presented at the meeting. Across all dose groups, a total of 38 subjects had EGFR Ex20ins mutations, including 29 at doses ≥ 160 mg QD, among whom 26 completed at least 1 tumor assessment. In these 26 patients the confirmed response rate was 57.7% (15/26) and the disease control rate (DCR) was 100% (26/26). Duration of response exceeded 6 months in 40% of responders. Conclusion: In the ongoing Phase 1 study, PLB1004 appears to be safe and well-tolerated with promising anti-tumor activity in patients with NSCLC harboring EGFR exon 20 insertion mutations. Citation Format: Jinji Yang, Yilong Wu, Meijuan Huang, Yanqiu Zhao, Jun Zhao, Jianying Zhou, Ying Mao, Huimin Wang, Yun Fan, David Chung, Kevin Schaab. Interim results of a first-in-human, dose escalation and expansion study of PLB-1004, an irreversible inhibitor of exon 20 insertion mutations in patients with non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT102.

Abstract CT232: Safety, efficacy, and tolerability of FHND9041 capsules as first-line treatment in patients with EGFR sensitive mutations or second/third-line treatment in patients with T790M+ advanced non-small cell lung cancer (NSCLC): results from a phase I/II single-arm, multi-center, open-label study

Abstract Background: FHND9041 is a third-generation EGFR inhibitor independently developed by Nanjing Chuangte Pharmaceutical Technology Co., Ltd. It irreversibly binds to EGFR protein, and demonstrates strong inhibitory effect on EGFR T790M mutations as well as EGFR sensitive mutations. This is the first single-arm, multi-center, open-label phase I/II study of FHND9041 capsule, which evaluates the safety, tolerability and preliminary efficacy of FHND9041 capsule in initial treatment of NSCLC with EGFR sensitive mutations or treatment of T790M+ advanced NSCLC who progressed after prior EGFR-TKI therapy. Methods: 124 subjects tested for EGFR mutations and eligible for evaluation were randomized to dose groups of 40 mg, 80 mg (first-line treatment or second/third-line treatment with T790M+), 120 mg and 180 mg. The primary endpoint is objective response rate (ORR), and key secondary endpoints are progression-free survival (PFS), disease control rate, duration of response, and safety. Results: The ORR of FHND9041 capsule 80 mg (recommended phase 2 dose, RP2D) is 71.43% in patients newly diagnosed of NSCLC with EGFR sensitive mutations, and 63.64% in patients with T790M+ NSCLC who had progressed after prior EGFR-TKI therapy. No DLT occurred in all dose groups, 40 mg, 80 mg and 120 mg, which demonstrated satisfactory tolerability and safety of FHND9041. Detailed efficacy data of each dose group is shown in the table below. Conclusion: FHND9041 capsule 80 mg shows significant antitumor activity with favorable efficacy and safety profile in patients for both initial treatment and treatment of T790M+ NSCLC who progressed after prior therapy. For futher research of the capsule, a multicenter, randomized, positive-controlled phase III clinical research is ongoing with recommended dose of 80 mg. Group Dosage(mg) Evaluable Subjects(n) ORR% Second/Third-line T790M+ 40mg 3 33.33 80mg 39 63.64 120mg 42 58.33 180mg 3 100.00 First-line 80mg 37 71.43 Citation Format: Yongqiang Zhu, Minghong Shang, Jia Wang, Nong Yang, Xiaorong Dong. Safety, efficacy, and tolerability of FHND9041 capsules as first-line treatment in patients with EGFR sensitive mutations or second/third-line treatment in patients with T790M+ advanced non-small cell lung cancer (NSCLC): results from a phase I/II single-arm, multi-center, open-label study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT232.

Making the Best Use of Available Weapons for the Inevitable Rivalry-Resistance to EGFR-TKIs

The emergence of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) revolutionized the treatment of advanced-stage non-small cell lung cancer (NSCLC). Detected in more than 50% of late-stage lung adenocarcinoma in Asian patients, the EGFR mutation was regarded as a golden mutation for Asians. However, resistance to TKIs seems inevitable and severely hinders patients from getting further benefits from treatment. Even though resistance caused by EGFR T790M could be effectively managed by third-generation EGFR-TKIs currently, resistance to third-generation EGFR-TKIs is still a troublesome issue faced by both clinicians and patients. Various efforts have been made to maximize the benefits of patients from EGFR-TKIs therapy. Thus, new requirements and challenges have been posed to clinicians of this era. In this review, we summarized the clinical evidence on the efficacy of third-generation EGFR-TKIs in patients with EGFR-mutated NSCLC. Then, we discussed advancements in sequential treatment aiming to delay the onset of resistance. Moreover, the resistance mechanisms and features were depicted to help us better understand our enemies. Lastly, we put forward future strategies, including recent approaches involving the utilization of antibody drug conjugates against resistance and research directions about shaping the evolution of NSCLC as a core idea in the management of NSCLC.

Abstract 379: Circumvention of gefitinib resistance by repurposing flunarizine via histone deacetylase inhibition in lung cancer

Abstract Introduction: Gefitinib is an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) indicated for first-line treatment of advanced non-small cell lung cancer (NSCLC) bearing the sensitizing EGFR mutations. However, drug resistance is severely hindering its clinical efficacy. This study investigated the circumvention of gefitinib resistance by repurposing a non-oncology drug with a putative histone deacetylase (HDAC) inhibitory effect. Materials and Methods: A few clinically-approved drugs were identified by a computational drug repurposing tool called “DRUGSURV” and evaluated for HDAC inhibition. Flunarizine, indicated initially for migraine prophylaxis and vertigo treatment, was chosen for further investigation in several NSCLC cell lines exhibiting different gefitinib resistance mechanisms (H1975 - EGFR T790M; A549 - KRAS G12S; H1650 - PTEN loss; H1993 - MET amplification). Sulforhodamine B assay evaluated cell proliferation. Western blot analysis examined histone acetylation (HAC) and activation of EGFR downstream signaling. Flow cytometric assays investigated apoptosis and cell cycle effects. Chromatin immunoprecipitation explored the interaction of transcription factors to the promoter of the pro-apoptotic gene BIM. The circumvention of gefitinib resistance by flunarizine was further verified in a patient-derived tumor xenograft (PDX) from an EGFR TKI-refractory advanced-stage NSCLC patient. Results: Flunarizine was found to increase HAC in a dose- and time-dependent manner. Among the gefitinib-resistant NSCLC cell lines tested, flunarizine was shown to potentiate the anticancer effect of gefitinib the most in H1975 cells (harboring EGFR T790M). The apoptotic protein Bim was induced but the anti-apoptotic protein Bcl-2 was decreased by the gefitinib-flunarizine combination, presumably contributing to the circumvention of gefitinib resistance. Upregulation of Bim by flunarizine was associated with increased HAC and E2F1 binding to the BIM gene promoter. HDAC inhibitors (HDACIs) are known to exhibit pleiotropic cellular effects via histone-independent mechanisms. To this end, unlike the control HDACI vorinostat, flunarizine (tested at the resistance circumventing concentrations) did not appreciably affect acetylation of representative non-histone proteins (including α-tubulin and p53) relevant to drug resistance. Besides, flunarizine was found to increase E-cadherin but reduce vimentin expression which was associated with the inhibition of cancer cell migration and invasion. Flunarizine was further shown to significantly potentiate the antitumor effect of gefitinib in EGFR TKI-refractory PDX in vivo, but without any notable animal body weight loss during the treatment course. Conclusion: The findings advocate further clinical evaluation of the repurposing use of flunarizine to overcome gefitinib resistance. Citation Format: Kenneth K. To, William C. Cho. Circumvention of gefitinib resistance by repurposing flunarizine via histone deacetylase inhibition in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 379.

Abstract 5601: Oncomine™ dx express CE-IVD liquid biopsy assay for non-small cell lung cancer: Performance review and analytical validation

Abstract Introduction - Clinical diagnostics assays for oncology are becoming more readily available due to the advancement and democratization of Next-Generation Sequencing (NGS). Additionally, liquid biopsy can be used in NGS to detect genetic variants in circulating tumor DNA and RNA. Liquid biopsy is a less invasive option compared to traditional biopsy methods for early detection and continuous monitoring of cancer treatment outcomes. Here, we discuss screening of over 3,500 non-small cell lung cancer (NSCLC) samples, and the analytical validation of the Oncomine™ Dx Express Test (ODxET) and Genexus™ Dx Integrated Sequencer for detection of clinically significant variants in liquid biopsy samples. Methods - Our team screened over 3,500 NSCLC liquid biopsy samples in search of high priority clinical variants. Variants selected for analytical validation studies included ERBB2 exon 20 insertion, EGFR exon 20 insertion, EGFR exon 19 deletion, EGFR T790M, KRAS G12C, BRAF V600E, and RNA fusion isoforms including ALK, NTRK1/2/3, RET, and ROS1 oncogenic drivers. Over 550 screened samples were found to be positive for these variants of interest. This screening was performed on the Genexus™ Dx Integrated Sequencer according to the user guide. Results - The Genexus™ Dx Integrated Sequencer automates library preparation, sequencing, analysis, and reporting QC metrics and variant calls. Sequencing run setup is quick and straightforward, taking less than 15 minutes to start a run and just over 24 hours to go from nucleic acid to report. Limit of detection (LoD) for DNA SNVs, insertions, and deletions at 5 ng DNA input level ranged from 0.65% to 1.82% allelic frequency (AF), depending on the variant. The higher DNA input of 30 ng resulted in a lower LoD range, from 0.31% to 0.42% AF. RNA fusion and splice variant LoD at 5 ng sample input ranged from 9.9 to 19.6 molecular counts. The higher 30 ng input resulted in a lower LoD range for RNA variants as well, ranging from 6.4 to 8.0 molecular counts. In the analytical accuracy study, the false positive rate was found to be 0.2% for SNVs, 0% for insertions/deletions, and 0% for fusion targets. In the analytical reproducibility study, the average with-in run repeatability call rate (No Calls excluded) was 99.64% for DNA variants and 98.75% for RNA variants. Conclusion - Here, we demonstrated that the ODxET workflow on Genexus™ Dx Integrated Sequencer is a fast and efficient tool for testing clinical NSCLC liquid biopsy samples with high sensitivity and specificity. For in vitro diagnostic use. Not available in all regions including the United States. Citation Format: Nicholas Siepert, Jeoffrey Schageman, Jian Gu, Thilanka Jayaweera, Madhu Jasti, Luming He, Stephen Wunch, Emilia Ostrowska, Tasha Delacour, Diarra Hassell, Thomas Bowden, Daniela Garcia, Stephanie Tong, Swasti Raut, Nader Ezzedine, Elliott Martinez, Kelli Bramlett. Oncomine™ dx express CE-IVD liquid biopsy assay for non-small cell lung cancer: Performance review and analytical validation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5601.

Abstract 6403: A self-replicating RNA precision medicine approach to overcoming resistance to endocrine therapy in ER+BC

Abstract Drug resistance remains the major driving factor behind the clinical failure of targeted therapeutics. Current oncology precision medicine approaches rely on targeting known acquired resistance mutations, such as EGFR T790M or ALK/ROS mutations in NSCLC with 2nd and 3rd generation molecules designed to overcome or prevent resistance. These next generation targeted therapeutic approaches have increasingly long and complex drug development timelines and burdensome toxicities from off target effects (e.g. wild-type receptor targeting) or drug-drug interactions (DDI). The toxicities limit tolerability, compliance and combinability of different targeted therapeutics. RNA-based immunotherapy approaches offer an increasingly attractive alternative to next generation small molecule targeted therapeutics approaches: (1) RNA-based approaches only require a known acquired resistance sequence, (2) drug development timelines, cost and complexity can be meaningfully condensed, and (3) multiple acquired resistance mutations can be targeted with the same candidate. RBI-1000 is a candidate using a novel type of self-replicating RNA (srRNA) to generate robust immunity directed against acquired resistance mutations that develop in ER+ breast cancer (ER+ BC) in response to endocrine therapy. RBI-1000 includes on-target mutations within the estrogen receptor ligand binding domain, and bypass mutations either in the form of activating mutations in the PI3K kinase domain or amplifications of HER2/HER3. Here, we demonstrate that this srRNA encapsulated in a lipid nanoparticle primes polyfunctional CD4 and CD8 T cells leading to tumor growth inhibition and improved survival in a mouse model expressing the targeted acquired resistance mutation. Priming of T cells against acquired mutations is also confirmed in human HLA-transgenic mice. The immune cell-mediated elimination of clones expressing the acquired resistance mutations is predicted to prolong endocrine control of ER+BC, in an analogous manner to small molecule or monoclonal antibody targeted therapies, but with a more favorable dosing and adverse event profile due to precise immunologic targeting and no DDI. Citation Format: Zelanna Goldberg, Christian Maine, Gabrielle P. Dailey, Christine Domingo, Gaelle Picarda, Hunter Little, Annie Chou, Jessica Sparks, Darina Spasova, Shigeki Miyake-Stoner, Zachary C. Hartman, Christopher A. Rabiola, Erika J. Crosby, Herbert K. Lyerly, Nathaniel Wang, Parinaz Aliahmad. A self-replicating RNA precision medicine approach to overcoming resistance to endocrine therapy in ER+BC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6403.