Non-small Cell Lung Cancer Cancers Research Articles

Serum Splicing Factor Proline- and Glutamine-Rich Is a Diagnostic Marker for Non-Small-Cell Lung Cancer and Other Solid Cancers.

Cancer markers are measurable molecules in blood or tissues that are produced by tumor cells or immune cells in response to cancer progression. They play an important role in clinical diagnosis, prognosis, and therapy monitoring. Splicing factor proline- and glutamine-rich (SFPQ) plays an important role in cancer growth and metastasis. SFPQ is not only more highly expressed in non-small-cell lung cancer (NSCLC) cells than it is in controls, but also highly expressed in cancer cells in patients with other solid cancers. Thus, a new enzyme-linked immunosorbent assay (ELISA) for detecting SFPQ was developed, in which the SFPQ protein is trapped by the first specific mAb coated on a microplate, and then recognized by a second specific mAb. This assay allows for the specific detection of SFPQ in the serum of patients with solid cancer. Regarding NSCLC, the serum SFPQ levels distinguished the non-cancer controls from the patients with NSCLC, with an area under the curve of 0.876, a sensitivity of 87%, and a specificity of 94%. The serum SFPQ levels were significantly elevated in the patients with NSCLC or other solid cancers. In conclusion, serum SFPQ could be a promising novel diagnostic biomarker for NSCLC and other malignancies.

Population pharmacokinetics of trastuzumab deruxtecan (T-DXd) in subjects with HER2-mutant and HER2-overexpressing non-small cell lung cancer (NSCLC).

e20537 Background: T-DXd is a novel human epidermal growth factor receptor 2 (HER2) targeting antibody drug conjugate, approved globally at the dose of 5.4 mg/kg in HER2-positive or HER2-low breast cancer (BC) and HER2-mutant (HER2m) NSCLC1,2. The aim of the analysis was to characterize the populaton pharmacokinetics (PopPK) of T-DXd and its released payload, deruxtecan (DXd), in HER2m or HER2-overexpressing (HER2-OE) NSCLC subjects using data across tumor types (BC, NSCLC and others) from 12 Phase 1 to 3 studies with T-DXd doses ranging from 0.8-8.0 mg/kg. Methods: Analysis wasperformed using nonlinear mixed-effects modeling methods in NONMEM (version 7.4.3), similar to the previous analysis in HER2-positive BC1. Covariate effects were explored on the PK parameters of T-DXd and DXd. Results: Analysis included data across 1822 subjects, including 346, 1128, and 348 subjects with NSCLC, BC, and other cancers, respectively. The PK of T-DXd and DXd were adequately described by a PopPK model, judging by standard model diagnostics3. Covariate effects on T-DXd and DXd steady-state area under the concentration-time curve (AUC) were not clinically meaningful (within 0.8 to 1.25 AUC ratio interval for tested covariate level versus reference), except for subjects with high body weight (e.g., 90 kg; 95th percentile) showing a 31% and 37% higher T-DXd and DXd AUC, respectively, relative to typical subject with 58 kg body weight (median).The T-DXd and DXd AUC were also similar across subjects with BC, HER2m or HER2-OE NSCLC, with the same T-DXd dose. T-DXd and DXd AUC in NSCLC subjects were similar across subjects with mild hepatic or mild or moderate renal impairment (vs. normal counterparts), region (Asia, North America, Europe, and rest of the world), and race-country (Asian-Japan, Asian-Non-Japan, and Non-Asian) groups. Conclusions: T-DXd and DXd exposures were similar across subjects with BC and HER2m or HER2-OE NSCLC, as well as across hepatic function, renal function, region, and race-country groups within NSCLC supporting the use of 5.4 mg/kg dose (approved dose in BC) in HER2m or HER2-OE NSCLC from PK perspective. Reference: Enhertu [package insert]. Basking Ridge, NJ: Daiichi Sankyo, Inc.; 2022. Enhertu [summary of product characteristics]. Daiichi Sankyo Europe GmbH, Munich; 2022 Yin O et al. Clin Pharmacol Ther. 2021;109(5):1314-1325. Clinical trial information: NCT04644237 , NCT03505710 .

Assessing secular trends in lung cancer stage in the United States community oncology setting from 2013 to 2023.

11178 Background: Detecting disease at an early stage is critical to improving lung cancer (LC) survival, as reflected in guidelines recommending screening with low dose computed tomography for individuals at high risk by clinical societies including the NCCN (2011) and US Preventive Services Task Force (2013, updated 2021). Presenting stage distribution may also be impacted by healthcare utilization changes following the COVID-19 pandemic. Real-world data (RWD)-based investigations are important complements to national cancer registries to provide insight into this potentially changing diagnostic landscape, especially given utilization, practice and referral patterns unique to community settings. We thus assessed LC stage from 2013-23 in a large, nationally representative sample of community oncology practices leveraging RWD. Methods: This is a retrospective observational cohort study of patients within The US Oncology Network and non-Network practices, which include a nationally representative network of over 3,700 providers and more than 1 million patients seen annually in community-based oncology practices. All adult (≥18 years) patients diagnosed with non-small cell LC (NSCLC) or small-cell LC (SCLC) who had a first observed stage available in the community oncology setting within 2013 to 2023 were included. Demographics and medical history data were sourced from structured data fields in iKnowMed, an oncology-specific electronic health record system. Time trends of patient diagnosis and characteristics were descriptively evaluated. Results: The analysis included 98,806 patients with LC (84,023 [85.0%] NSCLC and 14,783 [15.0%] SCLC). Approximately half were female (49.7% and 50.3% in each disease), three-fourths were White (72.7% and 77.0%, respectively), and mean ages at diagnosis were 69.9 and 68.1 years. From 2013 to 2023, there was an increase in the total number of patients observed in the database with a documented stage, from 7,159 to 9,115 (27.3% increase) for NSCLC and from 1,162 to 1,595 (37.3% increase) for SCLC. This was largely driven by an absolute increase in advanced stage diagnoses. The proportion of patients diagnosed with Stage IV NSCLC increased from 43.3% in 2013 to 49.3% in 2023, while the proportions of Stage 0/I and II diagnoses decreased. A similar trend was observed for SCLC, with 60.3% of patients diagnosed with Stage IV in 2013 compared to 68.2% in 2023. The proportion of Stage IV NSCLC cancers increased in the pandemic period, from 48.0% in 2019 to 51.6% in 2020 and 50.6% in 2021. Conclusions: Over the last decade, the proportion of advanced stage LC cases has increased, possibly reflecting changes in referral patterns and utilization specific to the community setting, including those related to the COVID-19 pandemic. The burden of advanced disease highlights the need for continuous investment in advanced-stage treatments as well as early detection efforts.

Abstract 4007: AIC100 CAR-T cells targeting ICAM-1 are efficacious against solid tumors in xenograft mouse models of non-small cell lung cancer (NSCLC) and cervical cancer (CC)

Abstract Purpose: Establish preclinical proof of concept efficacy with CAR T cells (AIC100) in xenograft models of solid tumors (NSCLC and CC) expressing high levels of surface ICAM-1. Experimental: AIC100 is a third generation CAR T cell with micromolar affinity to ICAM-1, tuned lower than most CARs used to date in pre-/clinical studies. Affinity tuned AIC100 drug product is expected to selectively bind and kill tumor cells while safely sparing healthy cells. AIC100 also co-expresses somatostatin receptor 2 (SSTR2), which enables in vivo monitoring of AIC100 distribution and expansion by DOTATATE PET/CT imaging. To test preclinical efficacy of AIC100 in NSCLC and CC, four xenograft mouse models were generated using tumor cell lines known to have ICAM-1 expression (verified by flow cytometry) to test efficacy of AIC100. Reporter cell lines transfected with Luciferase were H226 NSCLC adenocarcinoma, H441 NSCLC squamous cell carcinoma, HeLa CC adenocarcinoma and SW756 CC squamous cell carcinoma. AIC100 efficacy was established by in vitro cytotoxicity prior to in vivo studies. NSG mice (n=10 per cell line) were inoculated via tail vein injection (1.0x106 cells) in cell growth media. Mice were imaged to ensure solid tumor formation which occurred in all mice within 3-5 days in the lung. At D0, mice were randomized into 2 groups (n=5 per group) of vehicle (PBS) and AIC100. At D1, mice were injected with AIC100 (1.0x107 live T cells) or vehicle. Mice were followed by weight and clinical observations 2x weekly and imaging by IVIS weekly. The study continued until vehicle group was euthanized due to tumor related co-morbidities at which time the study concluded. Results: AIC100 showed clear treatment benefit in both models of NSCLC and CC. In CC HeLa model, in mice treated with AIC100, primary tumors were significantly reduced by D8 and eliminated in 5/5 mice by D22. In CC SW756 model, in mice treated with AIC100, tumor growth was reduced in size by D22. In NSCLC H441 model, in mice treated with AIC100, primary tumors were largely eliminated in 5/5 mice by D15. In NSCLC H226 model, in mice treated with AIC100, tumors were significantly reduced by D8 and largely eliminated by D57 (4/5 mice). A clear survival benefit was observed in AIC100 treated mice in NSCLC H226 xenograft model. These results show treatment benefit by AIC100 in 2 tumor models per indication and establish proof of concept for the potential development of AIC100 as a treatment for ICAM-1 expressing NSCLC and CC cancers. Conclusions: AIC100 has significant treatment benefit in NSCLC and CC tumors with ICAM-1 expression in in vivo preclinical studies. Citation Format: Alyssa Birt, Serene Josiah, Kelsey L. Joyce, Srinija Alla, Michael P. Gallagher, Janusz Puc, Hanh Nguyen, Emily Walsh Martin, Sonal Gupta. AIC100 CAR-T cells targeting ICAM-1 are efficacious against solid tumors in xenograft mouse models of non-small cell lung cancer (NSCLC) and cervical cancer (CC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4007.

Abstract 1875: Enhanced lung cancer treatment using AOH1996, a potent PCNA inhibitor

Abstract AOH1996 targets a cancer-associated form of PCNA (caPCNA) by altering the protein-protein interface between caPCNA and its many binding partners. AOH1996 does this by inserting into the PCNA-interacting protein-box (PIP-box) pocket that is, in part, defined by the interdomain connecting loop (IDCL), the site of interaction between PCNA and most of its many binding partners. As a result, AOH1996 inhibits PCNA functions such as DNA replication, DNA repair, and transcription-replication conflict (TRC) resolution leading to cell cycle arrest and apoptosis. These effects are cancer specific and AOH1996 has little effect on non-cancerous cells even at 6-fold the effective dose in cancer cells. AOH1996 was developed in our lab and is currently in Phase I human clinical trials. Six patients have been enrolled in the trial and have not experienced AOH1996-related toxicities as the doses have been escalated to levels found to be efficacious in preclinical animal studies. Resistance to EGFR tyrosine kinase inhibitors (TKIs) poses a significant challenge in the treatment of non-small cell lung cancer (NSCLC) patients harboring EGFR activating mutations. Initially, EGFR TKIs like gefitinib, erlotinib, afatinib and osimertinib deliver remarkable responses, inducing tumor regression and improving overall survival. However, the emergence of resistance mechanisms, such as secondary EGFR mutations and bypass signaling pathway activation, hampers the efficacy of these drugs over time. The functional relationship between PCNA and EGFR has implications for the treatment of lung cancer patients with activating EGFR mutations. Membrane-localized EGFR modulates the PCNA-centric process of DNA replication through signaling pathways such as the Ras-Raf-Mek-Erk, PI3K/AKT, and PLCγ1/PKC signaling pathways. Nucleus-localized EGFR enhances PCNA stability by phosphorylating PCNA on tyrosine 211 (Y211), which prevents its degradation. In addition, phosphorylation at Y211 by EGFR results in decreased association of PCNA with MutS, which results in reduced mismatch repair (MMR). In this study, we combine AOH1996 and osimertinib as an innovative approach to treating NSCLC cancers with activating EGFR mutations and find that the drug combination: 1.) enhances the killing of NSCLC cell lines with activating EGFR mutations and acquired resistance to TKIs; 2.) enhances the destabilization of PCNA on chromatin; and 3.) causes changes in the localization and colocalization of PCNA and EGFR. We conclude that the combination of AOH1996 and osimertinib holds much potential as an improved treatment regimen for lung cancer patients with activating EGFR mutations. Citation Format: Robert G. Lingeman, Robert J. Hickey, Linda H. Malkas. Enhanced lung cancer treatment using AOH1996, a potent PCNA inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1875.

Abstract PR011: Discovery of VVD-065, a first-in-class allosteric molecular glue of the Keap1-Cul3 E3-ligase complex for the treatment of NRF2-activated cancers

Abstract Somatic gain of function mutations in nuclear factor erythroid 2-related factor 2 (NRF2) transcription factor or loss of function mutations in Kelch-like ECH Associated Protein 1 (KEAP1) E3 ligase, which regulates NRF2 protein levels, are frequently identified in many solid cancers such as non-small cell lung cancer (NSCLC), esophageal squamous cell carcinoma (ESCC), and head and neck squamous cell carcinoma (HNSCC). These genomic alterations drive the activation of cytoprotective NRF2 transcriptional programs. In addition to mutational activation, NRF2 pathway activation has been documented with high frequency in patients without mutations in the pathway. Since tumor cells with hyperactivated NRF2 have been demonstrated to be dependent on this pathway for survival, targeting NRF2 represents an attractive therapeutic approach in tumors with aberrant NRF2 activation. Here, we report the identification of VVD-065, a first-in-class NRF2 inhibitor that acts via an unprecedented mechanism of action. Covalent modification of sensor cysteines on KEAP1 during electrophilic or oxidative stress is known to inhibit KEAP1 mediated NRF2 degradation. In complete contrast, covalent modification of a KEAP1 sensor cysteine by VVD-065 dramatically enhances NRF2 degradation. At low nM concentrations, VVD-065 covalently targets KEAP1 E3 ligase, and allosterically increases the affinity between KEAP1 and CUL3, thereby promoting the formation of active KEAP1-CUL3 E3 ligase complexes and increasing the rate of NRF2 degradation. VVD-065 profoundly reduces NRF2 protein levels in WT KEAP1/NRF2 settings, as well as various KEAP1 and NRF2 mutant settings. Consequently, VVD-065 substantially inhibits NRF2-dependent gene expression and proliferation of NRF2-dependent cell lines. In vivo, oral administration of VVD-065 results in robust degradation of NRF2 and decreased expression of NRF2 target genes. Tumor growth inhibition studies with cell-line derived, and patient derived NSCLC/ESCC xenograft (CDX/PDX) models revealed robust dose-dependent tumor growth inhibition and tumor regression in the absence of any overt toxicity. Since constitutive NRF2 activation often contributes to chemotherapeutic resistance, we also conducted TGI studies with 100+ PDX models to assess combination opportunities with chemotherapeutic agents. A marked combination response was achieved with cisplatin and nab-paclitaxel in various solid tumor types such as NSCLC, ESCC, HNSCC, and uterine cancers. Combination with chemotherapy was safe and well-tolerated in these mouse studies. In summary, we have identified VVD-065, a potent and selective KEAP1 activator that promotes the degradation of NRF2. Degradation of NRF2 led to robust monotherapy response in NRF2-activated cancers and also chemo-sensitized chemo-refractory tumors. A related molecule, acting through the same mechanism of action has now entered into Phase I clinical trials. Citation Format: Nil Roy, Tine Wyseure, I-Chung Lo, Jordon Inloes, Aaron Snead, Steffen Bernard, Justine Metzger, Jonathan Pollock, Stephanie Grabow, Christie Eissler, Melaminah Williams, Sarah Jacinto, Gabe Simon, Todd Kinsella, David Weinstein, Matt Patricelli. Discovery of VVD-065, a first-in-class allosteric molecular glue of the Keap1-Cul3 E3-ligase complex for the treatment of NRF2-activated cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr PR011.

KRASG12C inhibition using MRTX1257: a novel radio-sensitizing partner

BackgroundKRAS activating mutations are considered the most frequent oncogenic drivers and are correlated with radio-resistance in multiple cancers including non-small cell lung cancer (NSCLC) and colorectal cancer. Although KRAS was considered undruggable until recently, several KRAS inhibitors have recently reached clinical development. Among them, MRTX849 (Mirati Therapeutics) showed encouraging clinical outcomes for the treatment of selected patients with KRASG12C mutated NSCLC and colorectal cancers. In this work, we explore the ability of MRTX1257, a KRASG12C inhibitor analogous to MRTX849, to radio-sensitize KRASG12C+/+ mutated cell lines and tumors.MethodsBoth in vitro and in vivo models of radiotherapy (RT) in association with MRTX1257 were used, with different RAS mutational profiles. We assessed in vitro the radio-sensitizing effect of MRTX1257 in CT26 KRASG12C+/+, CT26 WT, LL2 WT and LL2 NRAS KO (LL2 NRAS−/−) cell lines. In vivo, we used syngeneic models of subcutaneous CT26 KRASG12C+/+ tumors in BALB/c mice and T cell deficient athymic nu/nu mice to assess both the radio-sensitizing effect of MRTX1257 and its immunological features.ResultsMRTX1257 was able to radio-sensitize CT26 KRASG12C+/+ cells in vitro in a time and dose dependent manner. Moreover, RT in association with MRTX1257 in BALB/c mice bearing CT26 KRASG12C+/+ subcutaneous tumors resulted in an observable cure rate of 20%. However, no durable response was observed with similar treatment in athymic nude mice. The analysis of the immune microenvironment of CT26 KRASG12C+/+ tumors following RT and MRTX1257 showed an increase in the proportion of various cell subtypes including conventional CD4 + T cells, dendritic cells type 2 (cDC2) and inflammatory monocytes. Furthermore, the expression of PD-L1 was dramatically down-regulated within both tumor and myeloid cells, thus illustrating the polarization of the tumor microenvironment towards a pro-inflammatory and anti-tumor phenotype following the combined treatment.ConclusionThis work is the first to demonstrate in vitro as in vivo the radio-sensitizing effect of MRTX1257, a potent KRASG12C inhibitor compatible with oral administration, in CT26 KRASG12C mutated cell lines and tumors. This is a first step towards the use of new combinatorial strategies using KRAS inhibitors and RT in KRASG12C mutated tumors, which are the most represented in NSCLC with 14% of patients harboring this mutational profile.

ALKing the flames of lung cancer immunosensitivity.

Immune checkpoint inhibitors (ICIs) are utilised in treating non-small cell lung cancer (NSCLC) by enhancing the immune response against cancer cells. However, they are not effective against cancers with certain genetic alterations. A recent study by Mota etal. focussed on understanding why ALK+ NSCLC cancers are immune cold and making them more receptive to ICIs using a vaccine-based approach. The study highlighted cell-specific differences in the presentation of immunogenic peptides and the location of tumours as factors in the poor immune response. Vaccines based on ALK peptides improved immune response, and when combined with ICIs, this led to a striking improvement in survival in a mouse model of ALK+ NSCLC.

TRIM11 promotes cell proliferation of non-small cell lung cancer through the inhibition of ferroptosis by AMPK.

Lung cancer is one of the leading causes of cancer-related deaths worldwide, with non-small cell lung cancer (NSCLC) being the most prevalent type. This study investigates the role of TRIM11 gene in NSCLC and its underlying mechanism. NSCLC patients were recruited from our hospital and showed upregulated TRIM11 mRNA and protein expressions. Patients with high TRIM11 expression had lower survival rates. TRIM11 gene was found to promote cell proliferation and reduce ROS-induced ferroptosis in NSCLC. Additionally, TRIM11 gene induced AMPK expression and its regulation affected TRIM11's effects on cell proliferation and ferroptosis in NSCLC. IP analysis revealed that TRIM11 protein interacted with AMPK protein in NSCLC. These data confirmed that TRIM11 promotes cell proliferation and reduces ROS-induced ferroptosis in NSCLC through AMPK. Hence, TRIM11 is a potential target for the treatment of NSCLC and other cancers.

Friendly-user score assessing gut dysbiosis and resistance to immune checkpoint inhibitors (ICI).

103 Background: Accumulating evidence pointed to the impact of the intestinal microbiota on ICI outcomes across various cancers. Although specific gut microbial species have been associated with beneficial responses ( i.e. Akkermansia muciniphila (Akk)), no consensus exists on a gut fingerprint predicting immunoresistance to clinical routine use. Methods: NCT04567446 provided whole genome sequencing (WGS) of longitudinal fecal samples from patients (pts) with advanced non-small cell lung cancer (NSCLC) during ICI (alone or with chemotherapy) in France and Canada. Topological Pearson networks clustered into species interacting groups (SIG) correlating with overall survival (OS; OS<12=NR; OS>12=R). Forty harmful (SIG1) and thirty-four beneficial (SIG2) WGS species were associated with NR and R to ICI. A monodimensional score (TOPOSCORE) based on SIG1/SIG2 ratio combined with Akk relative abundance was calculated and compared to machine-learning (ML) algorithms. Multivariate Cox analysis (MVA) adjusted for established risk factors (ATB, gender, age, ECOG, PD-L1, LIPI score). Intraindividual dynamics of the TOPOSCORE was evaluated in pts with at least two fecal samples. Three independent cohorts of NSCLC and genitourinary (GU) cancers pts validated the data. Results: In n=245 and n=148 NSCLC pts, we could classify pts into dysbiotic (SIG1+,33%) and eubiotic (SIG2+, 67%), using the TOPOSCORE. Pts falling within the SIG2+ exhibited a significantly prolonged OS than pts falling into SIG1+ (HR: (95% CI), 0.50 (0.36-0.71), p<0.0001). TOPOSCORE also predicted OS in 277 ICI-treated NSCLC and GU pts and compared to the state-of-the-art ML algorithms, held the highest percentage of correct predictions (63%). At MVA, TOPOSCORE was independently associated with OS (HR: 0.56 (0.39-0.81), p=0.002). Analyzing the intraindividual dynamics of the TOPOSCORE (n=67), we found that 74% of SIG2+ and 68% of SIG1+ individuals remained in their initial classification during ICI treatment. We finally scaled the calculation of the TOPOSCORE down to 24WGS (instead 75WGS) and set up a qPCR-based friendly-user test capable of accurately identifying the fecal presence of the bacteria of interest within 48 hrs. We confirmed (n=323) that OS was superior in those pts harboring a 24-bacteria-qPCR-based TOPOSCORE falling within the SIG2+ category (HR: 0.65 (0.48 to 0.87), p=0.0005). Conclusions: TOPOSCORE represents a robust biomarker predicting and following the dynamic of the immunoresistance to ICI across cancers on an individual basis. By converting the WGS TOPOSCORE to a qPCR-based test with a rapid turnaround time, it will be possible to adopt this score in routine clinical practice to improve pts stratification and ICI success rates guiding the selection of dysbiotic pts amenable to microbiota-centered interventions and eubiotic fecal microbiota transplantation donors. Clinical trial information: NCT04567446 .

Amplification of wild-type RET and clinical response to selpercatinib for non–small-cell lung cancer (NSCLC).

9123 Background: RET rearrangements and RET kinase domain mutations represent targetable genomic alterations in various cancer types. However, the frequency and characteristics of RET amplification in cancer and its potential role as a targetable oncogenic driver are not well-characterized. Methods: In a pan-cancer cohort of patients at the Dana-Farber Cancer Institute (DFCI) whose tumors underwent next-generation sequencing (NGS), we evaluated the frequency of wild-type (wt) RET amplification (defined as ≥6 copies) in the absence of RET rearrangements or activating mutations. We validated our findings using merged data from the TCGA, GENIE, and China Pan-Cancer datasets. Results: The frequency of wt RET amplification across all cancers was 0.08% (28/34,463) in the DFCI cohort and 0.16% (145/91,466) in the validation cohort. In NSCLC, the frequency of RET amplification was 0.10% (5/4773) in the DFCI cohort and 0.13% (15/11,622) in the validation cohort. Other cancer types with RET amplification included hepatobiliary cancer, prostate cancer, and breast cancer, among others. For 22 RET-amplified cases with available copy number data , the median RET gene copy number was 7 (range 6-36). Among 20 RET-amplified NSCLCs, 10 had no other known driver mutations, and 10 had concurrent driver mutations (including MET exon 14 skipping, MET amplification, KRAS G12C/S/D, EGFR L858R, EGFR exon 20 insertion, and EGFR exon 19 deletion). In cases with available smoking status, 5 out of 6 patients with RET-amplified NSCLC had a history of tobacco use. A 69-year-old man with unresectable stage III NSCLC had disease recurrence after chemoradiation and durvalumab. Both the initial and recurrent biopsy samples showed focal high-level RET amplification (22 and 28 copies, respectively); genomic and RNA sequencing showed no other driver mutations or RET rearrangements. He experienced partial response to the selective RET inhibitor selpercatinib with decrease of both intracranial and systemic tumor burden. Conclusions: Amplification of wt RET represents a novel, targetable, rare molecular subset of NSCLC and other cancers.

A first-in-human phase I, dose-escalation and dose-expansion study of SY-5007, a highly potent and selective RET inhibitor, in Chinese patients with advanced RET positive solid tumors.

9111 Background: RET gene fusions have been identified as oncogenic drivers in 1-2% of non-small-cell lung cancer (NSCLC) and 10-20% of papillary thyroid carcinomas. Activating RET mutations occur in 50-60% of medullary thyroid cancer (MTC). Recently, selective RET TKIs, selpercatinib and pralsetinib, were approved for RET positive solid tumors by FDA. SY-5007 is a highly potent RET inhibitor that selectively targets RET fusions and mutations. This study is to investigate the safety, PK characteristics and preliminary efficacy of SY-5007 in Chinese patients with advanced RET-altered solid tumors. Methods: In this first-in-human phase I study, eligible patients previously treated and with positive RET were enrolled. In the dose escalation phase, following 3+3 design, the patients were administered orally with SY-5007 20 mg once daily, or 20, 40, 80, 120, 160, 200 mg twice daily in a 28-day cycle. The expansion doses (160 and 200 mg twice daily) were executed in a dose expansion phase to determine a recommended phase II dose (RP2D). Then three cohorts ( RET fusion-positive NSCLC or thyroid cancers and RET mutant MTC) at RP2D were expanded in eligible patients. Primary endpoints included safety, tolerability, MTD, DLTs. Secondary endpoints included PK and preliminary antitumor activity of SY-5007 per RECIST v1.1. Results: As of Feb.06, 2023, a total of 60 patients including 55 RET fusion NSCLC and 5 RET mutant solid tumors were enrolled into dose escalation cohorts (n = 17) and dose expansion cohorts (n = 43).Totally, 55 (91.7%) patients experienced treatment-related adverse events (TRAEs) including increased aspartate aminotransferase (50.0%), increased alanine aminotransferase (41.7%), diarrhea (41.7%), etc. Grade ≥3 TRAEs were observed in 22 (36.7%) patients including hypertension (15%), diarrhea (5%), increased alanine aminotransferase (3.3%), etc. PK study showed SY-5007 was absorbed rapidly, exposure of SY-5007 increased in a dose-dependent manner at doses between 20 and 160 mg twice daily, and it was saturated at 160 mg twice daily. In 50 evaluable patients, the overall ORR and DCR was 62.0% (95% CI, 47.2-75.4) and 94.0 % (95% CI, 83.5-98.8), respectively. 29 patients (24 NSCLC, 4 MTC) received SY-5007 at 160 mg twice daily, 27 of 28 (96.4%) evaluable patients showed tumor regression, ORR and DCR was 72.4% (95% CI, 52.8-87.3) and 89.7% (95% CI, 72.7-97.8), respectively. For NSCLC patients, the ORR and DCR was 75.0% (95% CI, 53.3-90.2) and 91.7% (95% CI, 73.0-99.0), respectively. Conclusions: SY-5007 was well tolerated in patients. Preliminary antitumor activity was also observed in patients with advanced RET-fusion positive NSCLS and RET mutant MTC. The pivotal Phase II clinical study of SY-5007 in NSCLC patients is ongoing. Research Sponsor: Shouyao Holdings (Beijing) Co., Ltd. Clinical trial information: NCT05278364 .

Wnt/β-Catenin Signaling and Resistance to Immune Checkpoint Inhibitors: From Non-Small-Cell Lung Cancer to Other Cancers.

Lung cancer is the leading cause of cancer-related deaths worldwide. The standard of care for advanced non-small-cell lung cancer (NSCLC) without driver-gene mutations is a combination of an anti-PD-1/PD-L1 antibody and chemotherapy, or an anti-PD-1/PD-L1 antibody and an anti-CTLA-4 antibody with or without chemotherapy. Although there were fewer cases of disease progression in the early stages of combination treatment than with anti-PD-1/PD-L1 antibodies alone, only approximately half of the patients had a long-term response. Therefore, it is necessary to elucidate the mechanisms of resistance to immune checkpoint inhibitors. Recent reports of such mechanisms include reduced cancer-cell immunogenicity, loss of major histocompatibility complex, dysfunctional tumor-intrinsic interferon-γ signaling, and oncogenic signaling leading to immunoediting. Among these, the Wnt/β-catenin pathway is a notable potential mechanism of immune escape and resistance to immune checkpoint inhibitors. In this review, we will summarize findings on these resistance mechanisms in NSCLC and other cancers, focusing on Wnt/β-catenin signaling. First, we will review the molecular biology of Wnt/β-catenin signaling, then discuss how it can induce immunoediting and resistance to immune checkpoint inhibitors. We will also describe other various mechanisms of immune-checkpoint-inhibitor resistance. Finally, we will propose therapeutic approaches to overcome these mechanisms.

Insights into pralsetinib resistance to the non-gatekeeper RET kinase G810C mutation through molecular dynamics simulations.

RET (rearranged during transfection) kinase, as a transmembrane receptor tyrosine kinase, is a therapeutic target for several human cancer such as non-small cell lung cancer (NSCLC) and thyroid cancer. Pralsetinib is a recently approved drug for the treatment of RET-driven NSCLC and thyroid cancers. A single point mutation G810C at the C-lobe of the RET kinase causes pralsetinib resistance to this non-gatekeeper variant. However, the detailed mechanism remains poorly understood. Here, multiple microsecond molecular dynamics (MD) simulations, molecular mechanics/generalized born surface area (MM/GBSA) binding free energy calculations, and community network analysis were performed to reveal the mechanism of pralsetinib resistance to the RET G810C mutant. The simulations showed that the G810C mutation had a minor effect on the overall conformational dynamics of the RET kinase domain. Energetic analysis suggested that the G810C mutation reduced the binding affinity of pralsetinib to the mutant. Per-residue energy contribution and structural analyses revealed that the hydrogen bonding interactions between pralsetinib and the hinge residues Glu805 and Ala807 were disrupted in the G810C mutant, which were responsible for the decreased binding affinity of pralsetinib to the mutant. The obtained results may provide understanding of the mechanism of pralsetinib resistance to the non-gatekeeper RET G810C mutant.

52 (PB042) - The RAF/MEK clamp VS-6766 shows strong anti-tumor activity across multiple MAPK pathway alterations, with a preferential effect on KRAS G12V

Background: KRAS is the most frequently mutated RAS/RAF family member (mutated in 15% of cancers), followed by BRAF (5%) and NRAS (3%). Several BRAF and MEK inhibitors (MEKi) are approved for BRAF V600E cancers, and the G12C inhibitor sotorasib is approved for KRAS G12C non-small cell lung cancer (NSCLC). However, there is still a need for agents targeting other RAS/RAF mutations (mt), including KRAS G12 V which is mutated in ∼7%, ∼9% and ∼19% of NSCLC, colorectal and pancreatic cancers, respectively. VS-6766 is a unique RAF/MEK clamp that potently inhibits MEK kinase activity and induces a dominant negative RAF-MEK complex preventing phosphorylation of MEK by ARAF, BRAF and CRAF. This unique mechanism allows VS-6766 to block MEK signaling without the compensatory re-activation of MEK that appears to limit the efficacy of MEKi. Material and Methods: In vitro assays were performed to evaluate the anti-proliferative activity of VS-6766 in human tumor cell lines with MAPK pathway alterations and in RAS-less mouse embryonic fibroblasts (MEFs) stably transfected with various KRAS variants. The anti-tumor activity of VS-6766 was assessed in genetically engineered mouse models (GEMM) of KRAS G12 V/Trp53 KO and KRAS G12C/Trp53 KO NSCLC. Results: VS-6766 showed strong anti-proliferative potency across tumor cell lines carrying KRAS, BRAF, CRAF, NRAS or NF1 alterations. Among KRAS mt cell lines, the anti-proliferative potency of VS-6766 differed by KRAS variant, with greatest potency observed with G12 V > G12C > G12D, which is consistent with G12 V signaling through CRAF/MEK/ERK and G12D signaling more through PI3 K/AKT/mTOR. Similarly, in MEFs stably transfected with different KRAS variants, VS-6766 showed best potency with KRAS G12 V and G12C, and least potency with G12D and KRAS wildtype. Accordingly, in the KRAS G12 V NSCLC GEMM model, previously shown to be CRAF-dependent, VS-6766 monotherapy induced stronger tumor regression than in a corresponding G12C NSCLC GEMM model. Strikingly, the combination of VS-6766 with FAK inhibition conferred tumor regressions in 87% (27/31) and 71% (36/51) of all tumors in the KRAS G12 V and KRAS G12C NSCLC models, respectively. Clinically, objective responses to VS-6766 monotherapy occurred mainly in patients with KRAS G12 V with 4 out of the 7 responders bearing KRAS G12 V (Guo, 2020). In KRAS mt NSCLC patients treated with VS-6766 in combination with the FAK inhibitor defactinib, a G12 V preference was also observed with confirmed responses in 2/2 patients with KRAS G12 V and tumor reduction in 4/6 patients with KRAS G12C (Krebs, 2021). Conclusions: These data support the ongoing registration-directed study evaluating VS-6766 ± defactinib for patients with KRAS G12 V NSCLC (NCT04620330), as well as the clinical combinations of VS-6766 with the KRAS G12C inhibitors sotorasib (NCT05074810) or adagrasib (NCT05375994) for KRAS G12C NSCLC. No conflict of interest.

A cell-free RNA-based next-generation sequencing (NGS) assay for the detection of actionable gene fusions in patients with non–small cell lung cancer (NSCLC).

3040 Background: Gene fusions and alternate RNA splice forms represent clinically actionable driver and resistance-conferring alterations in NSCLC and other cancers. Where tissue samples are inadequate for molecular testing, liquid biopsies could address the gap in detection of clinically relevant gene fusions. Targeted cell-free (cf) DNA-based NGS methods are typically used for non-invasive blood-based testing of gene fusions, but have limited sensitivity if fusion breakpoints involve long intronic regions or repetitive sequences (e.g. NTRKs and NRG1). RNA-based approaches are not affected by introns, can identify expressed fusion genes and can discriminate splicing. We developed an NGS assay optimized for cfRNA analyte for the detection of actionable gene fusions and exon deletion/skipping events in liquid biopsies. Methods: A highly multiplexed molecular-barcoded primer panel was designed for cfRNA-based detection of actionable fusion genes in NSCLC ( ALK, BRAF, FGFR2, FGFR3, MET (including exon 14 skipping), NRG1, NTRK1/2/3, RET and ROS1) covering > 90% of reported driver and partner gene exons in COSMIC database. The panel also targets housekeeping genes as endogenous sample controls. We developed a custom bioinformatics pipeline to call fusions and exon skipping based on split and spanning reads. Results: In initial analytical validation using fragmented RNA from pre-characterized reference material (representing 32 unique fusions at known copy numbers), the assay could detect as low as 10 fusion copies with a sensitivity of 97.6% and a specificity of 100%. For clinical testing, cfRNA co-eluted with cfDNA in nucleic acid extracts from plasma was used. In plasma samples (n = 103) from advanced NSCLC, 76% (15/21) of fusions (5 ALK, 3 RET, 2 ROS1, 5 MET ex14 skipping, 1 FGFR3-TACC3) detected in a clinically validated cfDNA assay (LiquidHALLMARK) were also detected in corresponding cfRNA. In a subset of samples that were driver-negative untreated or tyrosine kinase-inhibitor treated, the cfRNA assay yielded 9 gene fusions or breakpoints (1 CD74-NRG1, 4 BRAF, 2 MET, 2 FGFR3-TACC3 fusions) that could not be detected by the cfDNA assay. This represents an increase of 8.7% (9/103) of actionable alterations (driver or resistance) identified using cfRNA. Together cfRNA and cfDNA resulted in 30 fusion events to be detected compared to 21 by the cfDNA assay alone, representing a 42.8% (9/21) increase in fusion-specific detection of the combined assay. Conclusions: This novel cfRNA assay can detect actionable gene fusions and exon skipping events in liquid biopsies with high sensitivity. Combining cfRNA with more routine cfDNA testing can increase the total actionable diagnostic information from non-invasive testing in NSCLC patients where tissue samples are lacking, especially for gene fusions not amenable to detection in cfDNA.

Integrative analysis of non-small cell lung cancer patient-derived xenografts identifies distinct proteotypes associated with patient outcomes

Non-small cell lung cancer (NSCLC) is the leading cause of cancer deaths worldwide. Only a fraction of NSCLC harbor actionable driver mutations and there is an urgent need for patient-derived model systems that will enable the development of new targeted therapies. NSCLC and other cancers display profound proteome remodeling compared to normal tissue that is not predicted by DNA or RNA analyses. Here, we generate 137 NSCLC patient-derived xenografts (PDXs) that recapitulate the histology and molecular features of primary NSCLC. Proteome analysis of the PDX models reveals 3 adenocarcinoma and 2 squamous cell carcinoma proteotypes that are associated with different patient outcomes, protein-phosphotyrosine profiles, signatures of activated pathways and candidate targets, and in adenocarcinoma, stromal immune features. These findings portend proteome-based NSCLC classification and treatment and support the PDX resource as a viable model for the development of new targeted therapies.

3-D Textural Analysis of 2-[¹⁸F]FDG PET and Ki67 Expression in Nonsmall Cell Lung Cancer

The immunohistochemical determination of the Ki67 expression is regarded as a reliable method to monitor non small cell lung cancer (NSCLC) proliferation. However, cancer tissues are not uniform and the use of a small tumor sample in biopsy may not be accurate. Although positron emission tomography (PET) represents an alternative to biopsy, by providing the 3D functional and morphological distribution of cancer cells across the entire tumor volume, standardized uptake values (SUVs) exhibit poor accuracy. The purpose of this study is to identify quantitative features in 2-18FFDG PET images uniquely correlated with proliferating lung cancer cells that may perform better than SUVs in the evaluation of lung cancer proliferation. By using a xenograft NSCLC model implanted in 19 mice, we found that 89 textural parameters extracted from 3D 2-18FFDG uptake can be described by 6 independent principal components accounting for 95% of the total variance. One of these components features a significant and univocal correlation with 3D-stacked Ki67 staining immunohistochemical images (|r|>0.6;P<0.05/6, including compensation for multiple comparisons). Furthermore, the values of these components extracted from the PET images of in vivo and ex vivo explanted NSCLC cancers exhibit a strong correlation, verifying the stability of the segmentation method.

Near-Infrared MAO A Inhibitor (NMI) Outperformed FDA-Approved Chemotherapeutic Agents in Brain and Other Cancers: A Bioinformatic Analysis of NCI60 Screening Data.

Our previous work has shown that monoamine oxidase A (MAO A) is overexpressed in glioma and prostate cancer. Near-infrared dye conjugate MAO A Inhibitor (NMI) inhibited the growth of these cancers. This study investigated the effects of NMI on other cancers by NCI60 screening. Our results showed that 48 out of 59 screened cell lines from nine types of cancer had 100% growth inhibition at 10 μM NMI treatment. The in vitro efficacy of NMI determined by growth inhibition (GI50 and TGI) and lethal doses (LC50) has been further studied in various cell lines of CNS cancer, prostate cancer, and non-small cell lung cancer (NSCLC), these three cancers showed increased MAO A expression in tumors compared to normal tissues. Based on the waterfall plots and the 3D scatter plot of GI50, TGI, and LC50 data, NMI showed higher potency to several CNS cancer and NSCLC cell lines than prostate cancer cell lines. In vitro efficacy of NMI outperformed FDA-approved drugs for CNS cancer, prostate cancer, and NSCLC, respectively. The Pairwise Pearson Correlation Coefficient (PCC) showed that NMI has a unique mechanism compared to the existing anticancer drugs. This study shows that NMI is a novel theragnostic drug with high potency and unique mechanisms for brain, prostate, NSCLC, and other cancers.

Metformin Potentiates the Effects of Anlotinib in NSCLC via AMPK/mTOR and ROS-Mediated Signaling Pathways.

Anlotinib is a novel multi-targeted tyrosine kinase inhibitor with activity against soft tissue sarcoma, small cell lung cancer, and non-small cell lung cancer (NSCLC). Potentiating the anticancer effect of anlotinib in combination strategies remains a clinical challenge. Metformin is an oral agent that is used as a first-line therapy for type 2 diabetes. Interesting, metformin also exerts broad anticancer effects through the activation of AMP-activated protein kinase (AMPK) and inhibition of mammalian target of rapamycin (mTOR). Here, we evaluated the possible synergistic effect of anlotinib and metformin in NSCLC cells. The results showed that metformin enhanced the antiproliferative effect of anlotinib. Moreover, anlotinib combined with metformin induced apoptosis and oxidative stress, which was associated with the activation of AMPK and inhibition of mTOR. Reactive oxygen species (ROS)- mediated p38/JNK MAPK and ERK signaling may be involved in the anticancer effects of this combination treatment. Our results show that metformin potentiates the efficacy of anlotinib in vivo by increasing the sensitivity of NSCLC cells to the drug. These data provide a potential rationale for the combination of anlotinib and metformin for the treatment of patients with NSCLC or other cancers.