Positive Non-small Cell Lung Cancer Research Articles

IC Regimen: Delaying Resistance to Lorlatinib in ALK Driven Cancers by Adding Repurposed Itraconazole and Cilostazol.

Lorlatinib is a pharmaceutical ALK kinase inhibitor used to treat ALK driven non-small cell lung cancers. This paper analyses the intersection of past published data on the physiological consequences of two unrelated drugs from general medical practice-itraconazole and cilostazol-with the pathophysiology of ALK positive non-small cell lung cancer. A conclusion from that data analysis is that adding itraconazole and cilostazol may make lorlatinib more effective. Itraconazole, although marketed worldwide as a generic antifungal drug, also inhibits Hedgehog signaling, Wnt signaling, hepatic CYP3A4, and the p-gp efflux pump. Cilostazol, marketed worldwide as a generic thrombosis preventative drug, acts by inhibiting phosphodiesterase 3, and, by so doing, lowers platelets' adhesion, thereby partially depriving malignant cells of the many tumor trophic growth factors supplied by platelets. Itraconazole may enhance lorlatinib effectiveness by (i) reducing or stopping a Hedgehog-ALK amplifying feedback loop, by (ii) increasing lorlatinib's brain levels by p-gp inhibition, and by (iii) inhibiting growth drive from Wnt signaling. Cilostazol, surprisingly, carries minimal bleeding risk, lower than that of aspirin. Risk/benefit assessment of the combination of metastatic ALK positive lung cancer being a low-survival disease with the predicted safety of itraconazole-cilostazol augmentation of lorlatinib favors a trial of this drug trio in ALK positive lung cancer.

Clinical and molecular significance of homologous recombination deficiency positive non-small cell lung cancer in Chinese population: An integrated genomic and transcriptional analysis.

The clinical significance of homologous recombination deficiency (HRD) in breast cancer, ovarian cancer, and prostate cancer has been established, but the value of HRD in non-small cell lung cancer (NSCLC) has not been fully investigated. This study aimed to systematically analyze the HRD status of untreated NSCLC and its relationship with patient prognosis to further guide clinical care. A total of 355 treatment-naïve NSCLC patients were retrospectively enrolled. HRD status was assessed using the AmoyDx Genomic Scar Score (GSS), with a score of ≥50 considered HRD-positive. Genomic, transcriptomic, tumor microenvironmental characteristics and prognosis between HRD-positive and HRD-negative patients were analyzed. Of the patients, 25.1% (89/355) were HRD-positive. Compared to HRD-negative patients, HRD-positive patients had more somatic pathogenic homologous recombination repair (HRR) mutations, higher tumor mutation burden (TMB) (P<0.001), and fewer driver gene mutations (P<0.001). Furthermore, HRD-positive NSCLC had more amplifications in PI3K pathway and cell cycle genes, MET and MYC in epidermal growth factor receptor (EGFR)/anaplastic lymphoma kinase (ALK) mutant NSCLC, and more PIK3CA and AURKA in EGFR/ALK wild-type NSCLC. HRD-positive NSCLC displayed higher tumor proliferation and immunosuppression activity. HRD-negative NSCLC showed activated signatures of major histocompatibility complex (MHC)-II, interferon (IFN)-γ and effector memory CD8+ T cells. HRD-positive patients had a worse prognosis and shorter progression-free survival (PFS) to targeted therapy (first- and third-generation EGFR-TKIs) (P=0.042). Additionally, HRD-positive, EGFR/ALK wild-type patients showed a numerically lower response to platinum-free immunotherapy regimens. Unique genomic and transcriptional characteristics were found in HRD-positive NSCLC. Poor prognosis and poor response to EGFR-TKIs and immunotherapy were observed in HRD-positive NSCLC. This study highlights potential actionable alterations in HRD-positive NSCLC, suggesting possible combinational therapeutic strategies for these patients.

Post-marketing safety of lorlatinib: a real-world study based on the FDA adverse event reporting system.

Lorlatinib displays marked systemic and intracranial efficacy against anaplastic lymphoma kinase (ALK) positive non-small cell lung cancer (NSCLC). We aimed to establish the safety profile of lorlatinib based on the Food and Drug Administration Adverse Event Reporting System (FAERS). Reports from the FAERS between 2019 and 2023 were collected to conduct the disproportionality analysis. Reporting odds ratio (ROR) was employed to detect the potential adverse events (AEs) related to lorlatinib. The clinical characteristics, age and gender differences, time to onset of AEs were also investigated. A total of 2,941 AE reports were found to be associated with lorlatinib among the 8,818,870 AE reports obtained from the FAERS database. 167 lorlatinib-related AE signals were identified. The frequently reported AEs including hypercholesterolemia, oedema, and cognitive disorder were in line with those observed in clinical trials and drug instruction. However, AEs such as interstitial lung disease and AV block indicated in the drug label require further evaluation. More attention should be paid to the new potential unexpected AEs including pulmonary arterial hypertension and radiation necrosis. Furthermore, we examined the specific high-risk AEs of different ages and genders. In addition, majority of AEs occurred within the first 2months after lorlatinib initiation with a median onset time of 51days. Our study provides valuable insight into the post-marketing safety profile of lorlatinib, which can potentially benefit the rational and safe administration of lorlatinib in the clinic. Further prospective studies are needed to validate the associations between lorlatinib and the identified AEs.

Real-world osimertinib pretreatment experience in patients with epidermal growth factor receptor T790M mutation-positive locally advanced or metastatic non-small cell lung cancer.

Osimertinib has demonstrated efficacy in patients with epidermal growth factor receptor (EGFR) T790M-positive non-small cell lung cancer (NSCLC) in clinical trials. However, real-world data on its effectiveness remain scarce. Taiwanese patients with T790M-positive locally advanced or metastatic NSCLC and progressive disease following treatment with at least one EGFR tyrosine kinase inhibitor (TKI) were enrolled from the osimertinib early access program. Of the 419 patients (mean age, 63 years; female, 67%), 53% were heavily pretreated (≥ third-line [3L]), making osimertinib a fourth-line (4L) intervention. The median progression-free survival (PFS) was 10.5 months (95% confidence interval [CI]: 8.95-11.41); the 18-month PFS rate was 26.5%. The median overall survival (OS) was 19.0 months (95% CI: 16.30-20.95); the 24-month OS rate was 40.9%. The objective response rate was 32.46%, and the disease control rate was 86.38%. The median time to treatment discontinuation of osimertinib monotherapy was 11.9 months (95% CI: 10.49-13.11). Subgroup analyses of median PFS and OS in the chemotherapy combination group vs. the osimertinib monotherapy group yielded no difference. Central nervous system (CNS) metastasis, number of prior lines of therapy, and types of initial EGFR-TKIs did not significantly impact outcomes. The median PFS values were 9.0 (95% CI: 5.18-11.34) and 10.9 (95% CI: 9.18-11.90) months with and without CNS metastasis, respectively, and 10.8 (95% CI: 8.59-12.69), 13.6 (95% CI: 10.89-16.3), and 9.2 (95% CI: 7.8-10.62) months for second-line (2L), 3L, and ≥4L therapy, respectively. In patients who received osimertinib as 2L therapy, the median PFS values in response to prior afatinib, erlotinib and gefitinib treatment were 11.2 (95% CI: 4.85-4.79), 10.5 (95% CI: 8.59-20.26) and 8.7 (95% CI: 7.21-16.79) months, respectively. Overall, real-world data from Taiwan support the clinical benefits of osimertinib in EGFR T790M -positive NSCLC.

Prognostic value of skeletal muscle measured by CT at the T4 level in advanced EGFR-positive non-small cell lung cancer patients treated with ecotinib

Objective: To investigate the prognostic value of skeletal muscle measured by CT at the level of the fourth thoracic vertebra (T4) in advanced epidermal growth factor receptor (EGFR)-positive non-small cell lung cancer (NSCLC) patients treated with ecotinib. Methods: The study retrospectively reviewed clinical and pathological characteristics of 176 patients with advanced EGFR-positive NSCLC who received ecotinib and underwent chest CT scans at Wuhan Union Hospital between January 2017 and October 2020. Among them, 70 were male and 106 were female, with ages ranging from 27 to 80 (58.6±10.6) years. As of August 21, 2021, the median follow-up duration was 19.2 months (95%CI: 15.3 to 23.7 months). The optimal cut-off value of skeletal muscle density (T4-SMD) on CT images at the T4 level were determined using X-tile software. Kaplan-Meier analysis and log-rank test were used to plot progression-free survival curves. Cox proportional hazards regression models were employed to analyze factors influencing 1-year progression-free survival (PFS), and a nomogram prognostic model was constructed accordingly. Receiver operating characteristic (ROC) curves, calibration curves, and decision curve analysis (DCA) were utilized to evaluate the predictive value of the nomogram. Results: The T4-SMD [M (Q1,Q3)] of 176 patients was 42.56 (37.05, 45.93) HU. Patients were divided into low T4-SMD group (n=122) and high T4-SMD group (n=54) based on the cut-off value (The values for males and females were 49.44 and 41.41 HU, respectively) of T4-SMD. The median PFS time and 1-year PFS rate in the low T4-SMD group were significantly lower than those in the high T4-SMD group [10.4 (95%CI: 9.3-11.8) vs 13.7 (95%CI: 11.1-18.5) months, 36.1% vs 59.3%, respectively, P=0.034]. Eastern Cooperative Oncology Group performance status (HR=3.308, 95%CI: 1.183-9.247, P=0.023), lactate dehydrogenase level (HR=1.852, 95%CI: 1.037-3.307, P=0.037), systemic immune-inflammation index (HR=1.772, 95%CI: 1.019-3.080, P=0.043), and T4-SMD (HR=0.563, 95%CI: 0.325-0.974, P=0.040) were prognostic factors for 1-year PFS in advanced EGFR-positive NSCLC patients treated with ecotinib. A nomogram for predicting 1-year PFS of advanced EGFR-positive NSCLC patients treated with ecotinib was constructed based on the four indicators selected by multivariate Cox regression analysis. The area under the ROC curve of the nomogram was 0.775 (95%CI: 0.676-0.874). The calibration curve showed good consistency between the predicted and actual 1-year PFS. DCA demonstrated good clinical prediction effectiveness of the nomogram. Conclusion: Low T4-SMD is a prognostic risk factor for patients with advanced EGFR-positive NSCLC receiving icotinib therapy.

Osimertinib in combination with anti-angiogenesis therapy presents a promising option for osimertinib-resistant non-small cell lung cancer

BackgroundOsimertinib has become standard care for epidermal growth factor receptor (EGFR)-positive non-small cell lung cancer (NSCLC) patients whereas drug resistance remains inevitable. Now we recognize that the interactions between the tumor and the tumor microenvironment (TME) also account for drug resistance. Therefore, we provide a new sight into post-osimertinib management, focusing on the alteration of TME.MethodsWe conducted a retrospective study on the prognosis of different treatments after osimertinib resistance. Next, we carried out in vivo experiment to validate our findings using a humanized mouse model. Furthermore, we performed single-cell transcriptome sequencing (scRNA-seq) of tumor tissue from the above treatment groups to explore the mechanisms of TME changes.ResultsTotally 111 advanced NSCLC patients have been enrolled in the retrospective study. The median PFS was 9.84 months (95% CI 7.0–12.6 months) in the osimertinib plus anti-angiogenesis group, significantly longer than chemotherapy (P = 0.012) and osimertinib (P = 0.003). The median OS was 16.79 months (95% CI 14.97–18.61 months) in the osimertinib plus anti-angiogenesis group, significantly better than chemotherapy (P = 0.026), the chemotherapy plus osimertinib (P = 0.021), and the chemotherapy plus immunotherapy (P = 0.006).The efficacy of osimertinib plus anlotinib in the osimertinib-resistant engraft tumors (R-O+A) group was significantly more potent than the osimertinib (R-O) group (P<0.05) in vitro. The combinational therapy could significantly increase the infiltration of CD4+ T cells (P<0.05), CD25+CD4+ T cells (P<0.001), and PD-1+CD8+ T cells (P<0.05) compared to osimertinib.ScRNA-seq demonstrated that the number of CD8+ T and proliferation T cells increased, and TAM.mo was downregulated in the R-O+A group compared to the R-O group. Subtype study of T cells explained that the changes caused by combination treatment were mainly related to cytotoxic T cells. Subtype study of macrophages showed that proportion and functional changes in IL-1β.mo and CCL18.mo might be responsible for rescue osimertinib resistance by combination therapy.ConclusionsIn conclusion, osimertinib plus anlotinib could improve the prognosis of patients with a progressed disease on second-line osimertinib treatment, which may ascribe to increased T cell infiltration and TAM remodeling via VEGF-VEGFR blockage.

Osimertinib plus local treatment for brain metastases versus osimertinib alone in patients with EGFR-Mutant Non-Small Cell Lung Cancer

ObjectivesOsimertinib is a standard treatment for patients with EGFR-mutant non-small cell lung cancer (NSCLC) and is highly effective for brain metastases (BMs). However, it is unclear whether local treatment (LT) for BMs prior to osimertinib administration improves survival in EGFR-mutant NSCLC. We aimed to reveal the survival benefit of upfront local treatment (LT) for BMs in patients treated with osimertinib. Materials and MethodsThis multicenter retrospective study included consecutive patients with EGFR mutation (19del or L858R)-positive NSCLC who had BMs before osimertinib initiation between August 2018 and October 2021. We compared overall survival (OS) and central nervous system progression-free survival (CNS-PFS) between patients who received upfront LT for BMs (the upfront LT group), and patients who received osimertinib only (the osimertinib-alone group). Inverse-probability treatment weighting (IPTW) analysis was performed to adjust for potential confounding factors. ResultsOf the 121 patients analyzed, 57 and 64 patients had 19del and L858R, respectively. Forty-five and 76 patients were included in the upfront LT group and the osimertinib-alone groups, respectively. IPTW-adjusted Kaplan–Meier curves showed that the OS of the upfront LT group was significantly longer than that of the osimertinib-alone group (median, 95 % confidence intervals [95 %CI]: Not reached [NR], NR–NR vs. 31.2, 21.7–33.2; p = 0.021). The hazard ratio (HR) for OS and CNS-PFS was 0.37 (95 %CI, 0.16–0.87) and 0.36 (95 %CI, 0.15–0.87), respectively. ConclusionsThe OS and CNS-PFS of patients who received upfront LT for BMs followed by osimertinib were significantly longer than those of patients who received osimertinib alone. Upfront LT for BMs may be beneficial in patients with EGFR-mutant NSCLC treated with osimertinib.

Abstract 5839: Identification of low-dose ligand dependent drug-resistance in ALK/ROS1 positive NSCLC via CRISPR library screening

Abstract Tyrosine kinase inhibitor (TKI) therapy has displayed significant effectiveness in treating patients with anaplastic lymphoma kinase (ALK) and ROS proto-oncogene 1 (ROS1)-positive non-small cell lung cancer (NSCLC). Nonetheless, the emergence of acquired resistance in most patients presents a major challenge. Drug-tolerant persister (DTP) cells have recently gained attention as potential sources of resistance, highlighting the need for a deeper understanding of drug resistance mechanisms. To address this issue, a comprehensive genome-wide CRISPR-Cas9 knockout screening was conducted using an ALK-positive NSCLC cell line derived from pleural effusion in a patient who had not been exposed to ALK-TKI treatment. Following a 9-day ALK-TKI treatment, sequencing analysis was performed to identify sgRNAs in DTP cells. Notably, this analysis detected the involvement of tumor suppressor genes, including NF2, BAX, and ERRFI1.NF2 depletion was found to induce a substantial resistance to ALK-TKIs, and a combination therapy involving mTOR inhibitors showed promise in partially overcoming this resistance. Moreover, knockout of ERRFI1 or BAX led to an increase in DTP cells, drawing particular attention to ERRFI1, also known as MIG6, which is known for negatively regulating EGFR signaling. One intriguing discovery was that the loss of MIG6 induced resistance to ALK-TKIs when exposed to a relatively low dose of EGF, comparable to concentrations in plasma of healthy adults. This resistance was attributed to the upregulation of the MAPK and PI3K/Akt/mTOR pathways. Similar to ALK-rearranged NSCLC, ROS1 fusion-positive NSCLC cell lines also showed resistance by MIG6 knockout. Interestingly, some cell lines with reduced MIG6 expression tended to develop resistance with low-dose EGF, even without prior treatment, and overexpression of MIG6 restored sensitivity to TKIs. We further explored the potential of combination therapy, combining ALK-TKIs with anti-EGFR antibodies, and this approach effectively addressed acquired resistance in both in vivo and in vitro models. Furthermore, an analysis of clinical samples revealed that individuals resistant to ALK-TKIs displayed decreased mRNA MIG6 levels in comparison to their sensitive counterparts. Investigating the mechanisms behind MIG6 expression, we confirmed that ALK-TKI therapy suppressed MIG6 expression levels, while factors such as corticosteroid usage or exposure to hypoxia induced the upregulation of MIG6, potentially influencing the formation of DTP cells. In conclusion, this research has unveiled a novel factor contributing to resistance in ALK and ROS1-TKI therapies by activating the EGFR pathway through exposure to low-dose ligands, shedding light on potential therapeutic strategies to combat acquired resistance. Citation Format: Nobuyuki Kondo, Takahiro Utsumi, Ken Uchibori, Yasunari Miyazaki, Ryohei Katayama. Identification of low-dose ligand dependent drug-resistance in ALK/ROS1 positive NSCLC via CRISPR library screening [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 5839.

Abstract 7504: Longitudinal multi-omics characterization of circulating tumor cells from ALK positive NSCLC patients

Abstract Introduction: Anaplastic lymphoma kinase positive non-small cell lung cancer (ALK+NSCLC) represents a distinct subtype of NSCLC characterized by oncogenic ALK gene mutations leading to ALK overexpression and activation in tumor cells. ALK+ NSCLC will frequently develop mutations during treatment, leading to nonresponse and eventually cancer relapse. Therefore, there is a critical need for sensitive technologies to monitor ALK+ NSCLC disease status in patients over time. To facilitate longitudinal monitoring, there has been a rapid evolution in diagnostic technologies leveraging circulating tumor cells (CTCs) as a liquid biopsy tool. CTCs can be collected non-invasively at various intervals throughout patient treatment. Moreover, they shed from solid tumors and disseminate into the bloodstream, which makes isolating CTCs a valuable source of genomic, transcriptomic, and proteomic insights pertaining to the primary tumor. In this study, we present a comprehensive workflow for conducting multi-omic analysis on CTCs derived from ALK+ NSCLC patients. Methods: Blood samples were collected from a cohort of ALK+ NSCLC patients, and CTCs were isolated using a high throughput, label free microfluidic device, Labyrinth. Enriched CTCs were immunofluorescently stained for cytokeratin, CD45, DAPI, epithelial cell adhesion molecule (EpCAM), and vimentin markers. Heterogeneous CTC populations with hybrid epithelial/mesenchymal (EM) phenotypes are reported. Fluorescence in situ hybridization (FISH) was performed on enriched CTC samples. DNA was extracted and sequenced using a 523 gene targeted sequencing panel. Single cell RNA-seq technology was performed in parallel on CTC samples to investigate the gene expression throughout treatment. Results: The median concentration of CTCs in the ALK+ NSCLC cohort (n=33) is 137 CTCs per ml of blood, and 64% of CTCs are in an EM hybrid state. In ALK+ NSCLC patients (n=3) with an average of 4.6 visits per sample, we observed a trend of decreasing CTC concentration for patients with partial response to the treatment. The presence of ALK fusion in ALK+ NSCLC patient’s CTC is confirmed by FISH. Oncogenic mutations (JAK1, SPEN, ERBB4, NTRK3) are observed in bulk CTC samples from two NSCLC patients. Single cell RNA sequencing was performed on three ALK+ NSCLC patients; 58, 199, and 700 potential CTCs were detected with positive cytokeratin expression and no white blood cell marker expression. Conclusions: This study examined the feasibility of using CTCs for studying and monitoring the clinical status of the ALK+ NSCLC patients as well as the genomic and transcriptomic signatures within their tumors. More patients will need to be processed in order to validate our workflow and reinforce our conclusions. Citation Format: Yuru Chen, Shamileh Fouladdel, Harrison Ball, Zhaoping Qin, Albert Liu, Xu Cheng, Liwei Bao, Habib Serhan, Bryce Vandenburg, Varun Kathawate, Larua Goo, Peter Ulintz, Nathan Merrill, Aaron Udager, Angel Qin, Sofia D. Merajver, Sunitha Nagrath. Longitudinal multi-omics characterization of circulating tumor cells from ALK positive NSCLC patients [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 7504.

Abstract 1958: EGFR and mTOR signaling facilitate RET-independent resistance to selective RET-TKIs

Abstract RET fusions are an oncogenic driver mutation occurring in 1-2% non-small cell lung cancer (NSCLC). Currently, RET-selective tyrosine kinase inhibitors (TKIs) selpercatinib and pralsetinib are approved for this patient population, demonstrating favorable antitumor activity and safety profiles in advanced RET fusion-positive NSCLC. However, tumors eventually become refractory to selective RET inhibitor monotherapy. We sought to identify signaling pathways which mediate RET-independent acquired resistance to RET inhibitors and effective therapeutic combinations to overcome resistance. Using LC-2/ad (CCDC6-RET) NSCLC cells, we assessed the expression and activation of critical signaling pathways at a proteomic level following RET knockdown using reverse-phase protein array. RET knockdown resulted in rapidly increased adaptive upregulation of EGFR, HER2, MET, mTOR and MEK pathway signaling; observations were confirmed by Western Blotting. To determine whether EGFR or MET signaling facilitate RET inhibitor resistance, LC-2/ad and NCCE-TH1101 (KIF5B-RET) NSCLC cells were treated with increasing concentrations of selpercatinib or pralsetinib with or without ligands for these receptors, EGF or HGF respectively, and after five days cell viability was measured by CellTiter Glo. EGFR and MET signaling promoted RET TKI resistance in both cell lines. Next, to evaluate whether blockade of EGFR or MET signaling could enhance the activity of RET inhibitors, we treated LC-2/ad and NCCE-TH1101 cells with RET TKIs alone or in combination with the EGFR TKI erlotinib or poziotinib, or the MET inhibitor SU11274. We observed an additive effect with EGFR but not MET inhibitors when combined with RET TKIs. Western blot analysis revealed that EGFR signaling could reactivate mTOR and MAPK signaling after RET TKI treatment. Therefore, we next tested whether inhibition of these downstream pathways might enhance RET TKI sensitivity. We observed that treatment of LC-2/ad and NCCE-TH1101 cells with an mTOR inhibitor (everolimus) or a MEK inhibitor (trametinib) in combination with RET TKIs increased the anti-tumor activity or RET inhibitors. Finally, we established cells with acquired resistance to selpercatinib and pralsetinib. RET TKI resistant cells did not harbor secondary RET fusion mutations but underwent epithelial to mesenchymal transition and exhibited pan-RET inhibitor resistance. Moreover, RET TKI resistant cells showed increased cell surface expression of EGFR and MET by flow cytometry. RET TKI resistant cells were sensitive to mTOR or MEK inhibitors in combination with RET TKIs. Collectively, our findings indicate that in RET-fusion positive NSCLC tumor cells, activation of bypass pathways including the EGFR, MAPK, mTOR pathways may facilitate RET inhibitor resistance and that blockade of bypass pathways may enhance the efficacy of selective RET inhibitors and overcome acquired RET TKI resistance. Citation Format: Tomohiro Takehara, Monique B. Nilsson, Ximeng Liu, Hibiki Udagawa, Naoto Morikawa, Alissa Poteete, Junqin He, Xiaoxing Yu, Thiru Arumugam, Koichi Goto, John V. Heymach. EGFR and mTOR signaling facilitate RET-independent resistance to selective RET-TKIs [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 1958.

Abstract 5833: Genomic mechanisms of RET inhibitor resistance in RET-fusion positive NSCLC

Abstract RET fusions are a driver oncogene detected in 1-2% of non-small cell lung cancer (NSCLC) cases. Currently, two selective RET inhibitors (RETi), selpercatinib and pralsetinib have received FDA approval for RET fusion-positive NSCLC. However, RET fusions are highly heterogeneous and the impact of specific fusion partners and breakpoints on RETi sensitivity is not well understood. Moreover, most patients eventually develop resistance to RETi, and secondary RET mutations are thought to be a key mechanism of acquired resistance. Thus, we aimed to identify RET-dependent mechanism of RETi resistance in effort to improve treatment options and clinical outcomes. We engineered a panel of Ba/F3 cells expressing various RET fusion partners and breakpoints observed in clinical cases of NSCLC and evaluated their drug sensitivity profile against a panel of RETi including selpercatinib, pralsetinib, TPX-0046, lovatinib, zeteletinib and ponatinib. We determined that individual RET fusions impart distinct sensitivities to RETi. For example, cells expressing KIF-RET (K22, R12) were sensitive to selpercatinib and pralsetinib but less sensitive to ponatinib, whereas cells expressing KIF-RET (K15, R12) were highly sensitive to selpercatinib, pralsetinib, and ponatinib. Moreover, using RET-fusion positive NSCLCs, we observed that the RETi sensitivity profile of human cell lines was similar to that observed using Ba/F3 models. To identify potential resistance mutations, we utilized the LentiMutate approach. In KIF5B-RET (K15, R12) expressing cells, we identified G810S as being associated with resistance to both selpercatinib and pralsetinib, a finding consistent with previous reports and clinical observations. In addition, we identified Y806H/N, V804M/E, and M918T as being associtated with selpercatinib-resistance and L730I/V, E732K, and Y806H/N as being associated with pralsetinib-resistance. We next constructed Ba/F3 cells expressing KIF5B-RET in combination with each secondary mutation and evaluated RETi sensitivity. G810S and Y806N conferred resistance to selpercatinib and pralsetinib, as well as to RET-targeting multi-kinase inhibitors. V804M/E mutations conferred resistance to selpercatinib but not pralsetinib, whereas L730I/V and E732K mutations conferred resistance to pralsetinib but not selpercatinib, demonstrating RETi-specific resistance mutations. Cells expressing G810S secondary mutations were resistant to selpercatinib and pralsetinib, but sensitive to zeteletinib. Collectively, we show that individual RET fusion variants have distinct drug sensitivity profiles and that secondary resistance mutations are non-overlapping between RETi. The comprehensive characterization of RET-dependent mechanisms of resistance to RETi may provide therapeutic guidance for treating RET-fusion driven NSCLC and provide structural insights that can guide the development of new therapeutic regimens. Citation Format: Ximeng Liu, Monique Nilsson, Tomohiro Takehara, Junqin He, Xiaofang Huo, Ashwani Kumar, John Heymach, Ralf Kittler. Genomic mechanisms of RET inhibitor resistance in RET-fusion positive NSCLC [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 5833.

Targeting RET alterations in non-small cell lung cancer

Rearranged during transfection (RET) alterations, which lead to aberrant activation of the RET proto-oncogene, have been identified in various cancers. In non-small cell lung cancer (NSCLC), RET mutations often manifest as RET fusion genes and are observed in 1–2 % of patients with NSCLC. In recent years, selective RET inhibitors such as selpercatinib and pralsetinib, approved by the Food and Drug Administration (FDA) in 2020, have been part of the revolutionary changes in the treatment landscape for non-small cell lung cancer. While first-generation RET inhibitors have become part of the standard of care for RET-fusion positive NSCLC, a new challenge has emerged: acquired resistance to RET inhibitors. RET resistance is a complex phenomenon that can manifest as either on-target or off-target resistance. Numerous studies have been conducted to identify the mechanisms behind this resistance. This review provides an overview of the biology of RET in NSCLC, methods of RET testing, and a comprehensive analysis of the clinical outcomes associated with multikinase and selective RET inhibitors for NSCLC. Additionally, we will explore future perspectives for RET fusion-positive NSCLC, including ongoing trials and the challenges involved in overcoming resistance to RET inhibitors.

ROS1 Positive Non-Small Cell Lung Cancer Patient with Brain Metastases Who Overcame Crizotinib Resistance with Lorlatinib: Case Report

Background: ROS1-positive patients account for 1-2% of non small cell lung cancer (NSCLC) cases. Crizotinib is the first tyrosine kinase inhibitor (TKI) indicated in first line treatment of ROS1-rearranged NSCLC. However, crizotinib resistance is frequent within the first 12 months of treatment. Lorlatinib is a novel tyrosine kinase inhibitor of ROS-1 recently indicated for metastatic or locally advanced crizotinib-resistant NSCLC. Case report: We report a case of a 43-year-old female patient with no medical history, diagnosted with stage IVB NSCLC including asymptomatic brain metastasis, and harboring ROS1 rearrangement. Within the first three months of starting crizotinib, the patient showed on CT scan evaluation new bone and brain metastases. Lorlatinib in the second line revealed a noteworthy improvement in metastasis and primary lesion. Conclusion: Brain metastases frequently occur in ROS1-rearranged NSCLC at the time of diagnosis and treatment course. However the efficacy of crizotinib might not translate to intracranial control of the disease, one of the cause of resistance to this drug.

152P Treatment (tx) patterns and clinical outcomes in unresectable stage III epidermal growth factor receptor-mutation positive (EGFRm) non-small cell lung cancer (NSCLC) treated with chemoradiotherapy (CRT): Interim analysis of a global real-world (RW) study

152P Treatment (tx) patterns and clinical outcomes in unresectable stage III epidermal growth factor receptor-mutation positive (EGFRm) non-small cell lung cancer (NSCLC) treated with chemoradiotherapy (CRT): Interim analysis of a global real-world (RW) study

38P Real-world treatment patterns and effectiveness of subsequent treatments following first-line (1L) brigatinib for patients with anaplastic lymphoma kinase positive (ALK+) non-small cell lung cancer (NSCLC) from ALTA-1L

38P Real-world treatment patterns and effectiveness of subsequent treatments following first-line (1L) brigatinib for patients with anaplastic lymphoma kinase positive (ALK+) non-small cell lung cancer (NSCLC) from ALTA-1L

A Phase II trial of alternating osimertinib and gefitinib therapy in advanced EGFR-T790M positive non-small cell lung cancer: OSCILLATE

In this phase II, single arm trial (ACTRN12617000720314), we investigate if alternating osimertinib and gefitinib would delay the development of resistance to osimertinib in advanced, non-small cell lung cancer (NSCLC) with the epidermal growth factor receptor (EGFR) T790M mutation (n = 47) by modulating selective pressure on resistant clones. The primary endpoint is progression free-survival (PFS) rate at 12 months, and secondary endpoints include: feasibility of alternating therapy, overall response rate (ORR), overall survival (OS), and safety. The 12-month PFS rate is 38% (95% CI 27.5–55), not meeting the pre-specified primary endpoint. Serial circulating tumor DNA (ctDNA) analysis reveals decrease and clearance of the original activating EGFR and EGFR-T790M mutations which are prognostic of clinical outcomes. In 73% of participants, loss of T790M ctDNA is observed at progression and no participants have evidence of the EGFR C797S resistance mutation following the alternating regimen. These findings highlight the challenges of treatment strategies designed to modulate clonal evolution and the clinical importance of resistance mechanisms beyond suppression of selected genetic mutations in driving therapeutic escape to highly potent targeted therapies.

Sialyltransferase ST3GAL4 confers osimertinib resistance and offers strategies to overcome resistance in non-small cell lung cancer

The third-generation EGFR-TKI osimertinib is widely used in EGFR-mutated positive non-small cell lung cancer (NSCLC) patients, but drug resistance is inevitable. The currently known mechanisms only explain resistance in a small proportion of patients. For most patients, the mechanism of osimertinib resistance is still unclear, especially for EGFR-independent resistance. Herein, we thoroughly investigated the novel mechanism of osimertinib resistance and treatment strategies. We identified that ST3GAL4, a sialyltransferase, catalyzes terminal glycan sialylation of receptor protein tyrosine kinases, which induces acquired resistance to osimertinib in vitro and in vivo. In addition, ST3GAL4 is generally overexpressed in osimertinib-resistant patients with unknown resistance mechanisms. ST3GAL4 modifies MET glycosylation on N785 with sialylation, which antagonizes K48-related ubiquitin-dependent MET degradation and subsequently activates MET and its downstream proliferation signaling pathways. Meanwhile, ST3GAL4 knockdown or inhibition by brigatinib resensitizes resistant non-small cell lung cancer cells to osimertinib in vitro and in vivo This study suggests that ST3GAL4 can induce acquired resistance to osimertinib, which may be an important EGFR-independent resistance mechanism Furthermore, targeting ST3GAL4 with brigatinib provides new strategies to overcome osimertinib resistance.

Response to immune checkpoint inhibitor combination therapy in metastatic RET-mutated lung cancer from real-world retrospective data

BackgroundThe impact of immune checkpoint inhibitors (ICIs) based treatments on non-small cell lung cancers (NSCLCs) with RET fusions remains poorly understood.MethodsWe screened patients with RET fusions at the First Affiliated Hospital of Zhengzhou University and included those who were treated with ICIs based regimens for further analysis. We evaluated clinical indicators including objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), and overall survival (OS).ResultsA total of 232 patients with RET fusions were included in the study. Of these, 129 patients had their programmed death-ligand 1 (PDL1) expression levels tested, with 22 patients (17.8%) having a PDL1 level greater than or equal to 50%. Additionally, tumor mutational burden (TMB) status was evaluated in 35 patients, with the majority (30/35, 85.8%) having a TMB of less than 10 mutations per megabase. Out of the 38 patients treated with ICI based regimens, the median PFS was 5 months (95% confidence interval [CI]: 2.4–7.6 months) and the median OS was 19 months (95% CI: 9.7–28.3 months) at the time of data analysis. Stratification based on treatment lines did not show any significant differences in OS (18 vs. 19 months, p = 0.63) and PFS (6 vs. 5 months, p = 0.86). The ORR for patients treated with ICIs was 26.3%. Furthermore, no significant differences were found for PFS (p = 0.27) and OS (p = 0.75) between patients with positive and negative PDL1 expression. Additionally, there was no significant difference in PD-L1 levels (p = 0.10) between patients who achieved objective response and those who did not.ConclusionsPatients with RET fusion positive NSCLCs may not benefit from ICI based regimens and therefore should not be treated with ICIs in clinical practice.

Overcoming Central β-Sheet #6 (Cβ6) ALK Mutation (L1256F), TP53 Mutations and Short Forms of EML4-ALK v3/b and v5a/b Splice Variants are the Unmet Need That a Re-Imagined 5th-Generation (5G) ALK TKI Must Deliver.

Despite the development and approval of seven anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) spanning over three "generations" since the discovery of ALK fusion positive (ALK+) non-small cell lung cancer (NSCLC), there remains intrinsic and acquired resistances to these approved TKIs. Currently, a fourth-generation (4G) ALK TKI, NVL-655, is being developed to attack some of the unmet needs such as compound resistance mutations in cis. However, EML4-ALK variant 3 and TP53 mutations are intrinsic genomic alterations that negatively modulate efficacy of ALK TKIs. Potentially, in the shifting landscape where lorlatinib should be the first-line ALK TKI of choice based on the CROWN trial, the central β-sheet #6 (Cβ6) mutation ALK L1256F will be the potential acquired resistance mutation to lorlatinib which may be resistant to current ALK TKIs. Here we opine on what additional capacities a putative fifth-generation (5G) ALK TKI will need to possess if it can be achieved in one single molecule. We propose randomized trial schemas targeting some of the intrinsic resistance mechanisms that will lead to approval of a prototypic fifth-generation (5G) ALK TKI and actually be beneficial to ALK+ NSCLC patients rather than just design a positive pivotal superiority trial for the sole purpose of drug approval.

Efficacy of osimertinib in patients with EGFR-mutation positive non-small cell lung cancer with malignant pleural effusion.

As an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), osimertinib has emerged as a standard EGFR-mutation positive treatment for non-small cell lung cancer (NSCLC). However, the efficacy of osimertinib for malignant pleural effusion (MPE) remains understudied. This study aimed to evaluate the impact of osimertinib on time to treatment failure (TTF) and overall survival (OS) in patients with EGFR-mutation positive NSCLC, comparing those with and without MPE. This retrospective analysis included patients with advanced or recurrent NSCLC treated with osimertinib at our hospital between April 2016 and June 2021. TTF was defined as the duration from osimertinib initiation to discontinuation, and OS as the duration until death, irrespective of the reason. Among 229 patients receiving osimertinib, 84 had MPE before administration, 39 acquired EGFR exon20 T790M mutation following previous EGFR-TKI therapy, and 45 were EGFR-TKI-naive. Among EGFR-TKI-naive patients, median TTF was 14.8 and 19.8 months for those with and without MPE, respectively (hazard ratio [HR] 1.40; 95% confidence interval [CI]: 0.90-2.18; p = 0.12). Median OS was 32.0 and 42.0 months for patients with and without MPE, respectively (HR 1.43; 95% CI: 0.86-2.38; p = 0.16). Among patients with T790M mutation, median TTF was 12.3 and 13.1 months for patients with and without MPE, respectively (HR 1.03; 95% CI: 0.69-1.55; p = 0.88). Median OS for patients with and without MPE was 23.2 and 24.7 months, respectively (HR 1.09; 95% CI: 0.72-1.67; p = 0.68). Among patients with EGFR-mutation positive NSCLC, the evidence of MPE has little effect on survival with osimertinib.