Mechanisms Of Acquired Resistance Research Articles

Multifunctional Nanoassemblies for Cytotoxic Drug and Therapeutic Enzymes Delivery in Neuroblastoma Therapy

AbstractNeuroblastoma cells can acquire resistance mechanisms that make them invulnerable to chemotherapeutic agents. The use of nanoparticles as drug carriers provides the possibility to deliver several drugs simultaneously to specific tumoral cell populations, improving their therapeutic outcome. Herein, the development of a multifunctional nanoplatform based on the assembly of protocells (PC) and polymeric nanocapsules (PNC) is reported. PC provides the ability to transport and release cytotoxic drugs while PNC offers the capacity to transport enzymes to the tumoral tissues preserving their catalytic activity. Doxorubicin (Dox) and Glucose Oxidase (Gox) are housed within PC and PNC, respectively. The external surface of these nanoassemblies is decorated with synthetic targeting moieties, providing selectivity to neuroblastoma cells. Thus, the nanoplatform is endowed with the ability to generate multiple insults within neuroblastoma cells as cytotoxic drug release, glucose starvation, and oxidative damage. This nanoplatform exhibits significantly higher cytotoxic activity in comparison with only drug‐loaded protocells or empty protocells decorated with glucose oxidase nanocapsules, which points out the existence of a potent synergic effect between the action of both therapeutic agents: Dox and Gox. This strategy can be adapted to the production of multifunctional nanoassemblies, improving the arsenal against different types of tumors.

Abstract A04: Targeted protein degradation as a strategy to overcome non-covalent BTK inhibitor resistance in lymphoma

Abstract Bruton’s tyrosine kinase (BTK) is a target for multiple generations of covalent (irreversible) and non-covalent (reversible) inhibitors due to its critical role in the proliferation and survival of B-cell malignancies. Similar to drug resistance mechanisms in other cancers, resistance to covalent BTK inhibitors (BTKi) in chronic lymphocytic leukemia (CLL), such as ibrutinib, arise through on-target BTK mutations at the BTK C481 residue (the binding site of ibrutinib), which allow escape from BTK inhibition. Non-covalent BTKi represent a new avenue to overcome resistance to the clinically approved covalent BTKi; however, we have recently discovered mechanisms of resistance to non-covalent BTK inhibition in patients with CLL (Wang, Xi, Thompson, Montoya et al NEJM 2022). We used bulk and single cell genomic analyses that identified a series of acquired BTK mutations in a cohort of CLL patients that relapsed on the phase I/II clinical trial of pirtobrutinib. We discovered mutations (BTK V416L, A428D, M437R, T474I, L528W) that occur at critical residues within the catalytic kinase domain of BTK and conferred resistance to both non-covalent and covalent BTKis. Based on structural models of BTK, we observed that these mutations physically impede drug binding, and disrupt the normal kinase activity of BTK but can, upon B-cell receptor stimulation, sustain AKT, ERK, and NF𝜅B signaling as well intracellular Ca2+ release in the presence of pirtobrutinib. In addition, CITE-seq analyses of 53,722 cells from this cohort identified pre-existing leukemic and immune cell states associated with development of resistance in patients. These findings identify novel mechanisms to engender clinical resistance to non-covalent BTKis, including contribution of the immune microenvironment to response to BTK inhibition. Together our data suggest that mutations at the BTK kinase domain may alter conformation of BTK’s non-kinase protein interaction domains thus allowing BTK to be used as a scaffold for other signaling molecules to phosphorylate phospholipase C gamma 2 (PLC𝛾2), the direct downstream target of BTK. We have performed mass spectrometry phosphoproteomics to evaluate potential signaling molecules that are active and could bypass BTK in catalytically inactive BTK mutant cells. Given the above, we decided to target BTK’s non-kinase function by investigating targeted protein degraders of BTK using BTK-degrading protein targeting chimeras (PROTACS). The immediate advantages of using protein degraders are (1) Binding may occur at any site of the target protein, and (2) PROTACS can act catalytically to bind to and degrade multiple target proteins. Our studies have shown that several of the BTK mutants that engendered clinical resistance to covalent and non-covalent BTKi are sensitive to these degraders, elucidating an exciting alternative therapeutic approach for patients who become resistant to BTKi therapies. We are continuing to test the efficacy of BTK PROTAC degraders in overcoming acquired resistance mechanisms in CLL as well as other B-cell lymphomas. Citation Format: Skye Montoya, Eric Wang, Jessie Bourcier, Sana Chaudhry, Tulasigeri Totiger, Alejandro Pardo, Gabriel Pardo, Maurizio Affer, Jacob Jahn, Anthony Mato, Omar Abdel-Wahab, Justin Taylor. Targeted protein degradation as a strategy to overcome non-covalent BTK inhibitor resistance in lymphoma [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A04.

1078P Correlation between T cell immunity and EGFR-TKI treatment in patients harboring EGFR driver mutation

Although osimertinib provides a longer survival time than first/second-generation EGFR tyrosine kinase inhibitor (TKI) for non-small cell lung cancer (NSCLC) harboring EGFR mutations, acquired resistance is inevitable. Since the time to acquire resistance to TKI is shorter for cancers with a higher mutational burden and mutations increase after TKI resistance is acquired, the acquired resistance mechanism depends primarily on acquired mutations. Although the cancer immunoediting theory indicates that the process of genetic mutation accumulation by cancer cells involves a conflict with T-cell immunity, which recognizes gene mutation products as neoantigens and destroys them, the relationship between TKI treatment effect and immunological response in patients with EGFR-bearing NSCLC is unknown.To identify the correlation between T-cell immunity and the duration of TKI response, we examined the peripheral blood mononuclear cells (PBMCs) of patients with lung cancer prior to osimertinib therapy.

EP08.02-138 SAFFRON: Ph3 Savolitinib + Osimertinib vs Chemotherapy in EGFRm NSCLC with MET Overexpression/Amplification Post-Osimertinib

Osimertinib is a potent, third-generation, irreversible, oral EGFR-TKI which has demonstrated efficacy in NSCLC and related central nervous system metastases. Osimertinib is the preferred first-line treatment in advanced EGFRm NSCLC; however, patients may develop resistance to osimertinib. MET overexpression and/or amplification is the most common acquired resistance mechanism to osimertinib. However, there is no approved targeted therapy for these patients; platinum-based chemotherapy remains the standard of care but has limited efficacy.

The drug efflux pump MDR1 promotes intrinsic and acquired resistance to PROTACs in cancer cells.

Proteolysis-targeting chimeras (PROTACs) are a promising new class of drugs that selectively degrade cellular proteins of interest. PROTACs that target oncogene products are avidly being explored for cancer therapies, and several are currently in clinical trials. Drug resistance is a substantial challenge in clinical oncology, and resistance to PROTACs has been reported in several cancer cell models. Here, using proteomic analysis, we found intrinsic and acquired resistance mechanisms to PROTACs in cancer cell lines mediated by greater abundance or production of the drug efflux pump MDR1. PROTAC-resistant cells were resensitized to PROTACs by genetic ablation of ABCB1 (which encodes MDR1) or by coadministration of MDR1 inhibitors. In MDR1-overexpressing colorectal cancer cells, degraders targeting either the kinases MEK1/2 or the oncogenic mutant GTPase KRASG12C synergized with the dual epidermal growth factor receptor (EGFR/ErbB)/MDR1 inhibitor lapatinib. Moreover, compared with single-agent therapies, combining MEK1/2 degraders with lapatinib improved growth inhibition of MDR1-overexpressing KRAS-mutant colorectal cancer xenografts in mice. Together, our findings suggest that concurrent blockade of MDR1 will likely be required with PROTACs to achieve durable protein degradation and therapeutic response in cancer.

Circulating Tumor DNA Identifies Diverse Landscape of Acquired Resistance to Anti-Epidermal Growth Factor Receptor Therapy in Metastatic Colorectal Cancer.

Anti-epidermal growth factor receptor (EGFR) antibodies are effective treatments for metastatic colorectal cancer. Improved understanding of acquired resistance mechanisms may facilitate circulating tumor DNA (ctDNA) monitoring, anti-EGFR rechallenge, and combinatorial strategies to delay resistance. Patients with treatment-refractory metastatic colorectal cancer (n = 169) enrolled on the CO.26 trial had pre-anti-EGFR tissue whole-exome sequencing (WES) compared with baseline and week 8 ctDNA assessments with the GuardantOMNI assay. Acquired alterations were compared between patients with prior anti-EGFR therapy (n = 66) and those without. Anti-EGFR therapy occurred a median of 111 days before ctDNA assessment. ctDNA identified 12 genes with increased mutation frequency after anti-EGFR therapy, including EGFR (P = .0007), KRAS (P = .0017), LRP1B (P = .0046), ZNF217 (P = .0086), MAP2K1 (P = .018), PIK3CG (P = .018), BRAF (P = .048), and NRAS (P = .048). Acquired mutations appeared as multiple concurrent subclonal alterations, with most showing decay over time. Significant increases in copy-gain frequency were noted in 29 genes after anti-EGFR exposure, with notable alterations including EGFR (P < .0001), SMO (P < .0001), BRAF (P < .0001), MET (P = .0002), FLT3 (P = .0002), NOTCH4 (P = .0006), ERBB2 (P = .004), and FGFR1 (P = .006). Copy gains appeared stable without decay 8 weeks later. There were 13 gene fusions noted among 11 patients, all but one of which was associated with prior anti-EGFR therapy. Polyclonal resistance was common with acquisition of ≥ 10 resistance related alterations noted in 21% of patients with previous anti-EGFR therapy compared with 5% in those without (P = .010). Although tumor mutation burden (TMB) did not differ pretreatment (P = .63), anti-EGFR exposure increased TMB (P = .028), whereas lack of anti-EGFR exposure resulted in declining TMB (P = .014). Paired tissue and ctDNA sequencing identified multiple novel mutations, copy gains, and fusions associated with anti-EGFR therapy that frequently co-occur as subclonal alterations in the same patient.

Successful Retreatment With Venetoclax in a Patient With Chronic Lymphocytic Leukemia.

Successful Retreatment With Venetoclax in a Patient With Chronic Lymphocytic Leukemia.

Results of monitoring studies of caecal samples with animal contents for antimicrobial resistance in 2021

The article presents the results of studies of caecum samples (cecal appendages) with contents from cattle, pigs, and poultry following the State Monitoring Plan for Antimicrobial Resistance in Veterinary Medicine for 2021, isolated and identified isolates of zoonoses and commensal microorganisms, namely: Escherichia coli, Salmonella spp., Enterococcus faecium, Enterococcus faecalis, Campylobacter spp. The disk diffusion method conducted a study on determining antimicrobial sensitivity to antibacterial drugs. The results of studies on identifying acquired resistance mechanisms to antibacterial drugs are also presented. Interpretation of growth retardation zones was carried out following EUCAST requirements. As a result of the obtained data, 448 isolates were isolated and identified among 2120 samples submitted for research: E coli accounted for 37.7 %, Salmonella spp. – 4.24 %, Enterococcus faecium – 12.7 %, Enterococcus faecalis – 37.9 %, Campylobacter spp. – 7.4 % of all isolated isolates. When determining the sensitivity to antibacterial drugs, eight sensitive isolates were found. 237 isolates were monoresistant (sensitive to 1–2 ABP), and 203 were polyresistant (sensitive to 3 or more ABP). As a result of the research, production (ESBL) was detected and confirmed in ten Escherichia coli strains. Three vancomycin-resistant strains of Enterococcus faecium, and Enterococcus faecalis were identified. Study the prevalence of Escherichia coli, Salmonella spp., Enterococcus faecium, Enterococcus faecalis, Campylobacter spp. circulating in Ukraine. will promote a standardized approach to data collection, analysis, and sharing on a global scale and will ensure the fulfillment of the National Action Plan for Combating Antibiotic Resistance to Antimicrobial Drugs tasks. Ukraine has developed a National Action Plan to combat antimicrobial resistance. In the countries of the European Union, there is constant monitoring for Escherichia coli, Salmonella spp., Enterococcus faecium, Enterococcus faecalis, Campylobacter spp. The prospect of further research is to continue monitoring antimicrobial resistance against zoonoses and commensal bacteria, namely: isolates of Escherichia coli, Salmonella spp., Enterococcus faecium, Enterococcus faecalis, Campylobacter spp., as they play an important epidemiological role among infectious diseases common to humans and animals, determining their sensitivity and identifying acquired mechanisms of resistance to antibacterial drugs.

Using Circulating Tumor DNA in Colorectal Cancer: Current and Evolving Practices.

There exists a tremendous opportunity in identifying and determining the appropriate predictive and prognostic biomarker(s) for risk stratification of patients with colorectal cancers (CRCs). Circulating tumor DNA (ctDNA) has emerged as a promising prognostic and possibly predictive biomarker in the personalized management of patients with CRCs. The disease is particularly suited to a liquid biopsy–based approach since there is a great deal of shedding of circulating tumor fragments (cells, DNA, methylation markers, etc). ctDNA has been shown to have several potential applications, including detecting minimal residual disease (MRD), monitoring for early recurrence, molecular profiling, and therapeutic response prediction. The utility of ctDNA has broadened from its initial use in the advanced/metastatic setting for molecular profiling and detection of acquired resistance mechanisms, toward identifying MRD, as well as early detection. Prospective studies such as CIRCULATE, COBRA, Dynamic II/III, and ACT3 are underway in the MRD setting to further understand how ctDNA may be used to inform clinical decision making using both tumor-informed and tumor-agnostic platforms. These prospective studies use ctDNA to guide management of patients with CRC and will be critical to help guide how and where ctDNA should or should not be used in clinical decision making. It is also important to understand that there are different types of ctDNA liquid biopsy platforms, each with advantages and disadvantages in different clinical indications. This review provides an overview of the current and evolving use of ctDNA in CRC.

Distribution of Leukocidins, Exfoliative Toxins, and Selected Resistance Genes Among Methicillin-resistant and Methicillin-sensitive Staphylococcus aureus Clinical Strains in Egypt

Background: Infection with Staphylococcus aureus (S.aureus) is an increasing health problem worldwide. This pathogen has multiple virulence factors that contribute to its pathogenesis in a wide range of diseases. The present study aimed to investigate the prevalence of leukocidins, exfoliative toxins, and common antimicrobial resistance genes among Methicillin-Resistant Staphylococcus aureus (MRSA) and Methicillin-Sensitive Staphylococcus aureus (MSSA) strains collected from various clinical sources in Egypt. Methods: Isolates were identified as S.aureus by the standard microbiological methods. Methicillin resistance was detected phenotypically by cefoxitin disc diffusion method and genotypically by PCR for detection of mecA gene. PCR was also used to detect the presence of leukocidin genes (LukD, LukE, LukF-PV, and LukS-PV), exfoliative toxin genes (eta and etb), and antibiotic resistance genes (tetK, tetM, ermA, ermC, msrA, and aacA-aphD). Results: About 50.5% of tested isolates were methicillin resistant by cefoxitin disc assay, while mecA gene was amplified in 64.6% of isolates. The highest prevalent toxin gene was lukE (93%) and the least prevalent one was eta (1%). The resistance genes tetK and tetM were detected in nearly 50% of the tested strains but lower prevalence rates were recorded for aacA-aphD, msrA, ermA, and ermC genes. Conclusion: Methicillin resistance was highly prevalent among tested S.aureus strains. Regarding the studied virulence and resistance genes, no significant difference was detected between MRSA and MSSA strains, except for ermA gene p&lt;0.05 which was highly prevalent in MRSA strains. So, the variation between MRSA and MSSA strains in the response to treatment may be attributed to the resistance of MRSA strains to all β-lactams in addition to other possible acquired resistance mechanisms. Accordingly, fewer options of antimicrobial medications are available to treat MRSA infections.

Abstract 5335: Elucidation of initial resistance mechanisms in EGFR mutation-positive lung cancer focusing on YAP1 and cancer stem cells

Abstract Background: EGFR mutation-positive lung cancer has high response rates to EGFR-tyrosine kinase inhibitors (TKIs), but eventually acquires resistance to EGFR-TKIs. Acquired resistance mechanisms are diverse because of tumor heterogeneity, which makes it difficult to overcome acquired resistance. Initial resistance is the resistance mechanism of tumor cells that survive the initial treatment. If we could overcome the initial resistance, we could prevent cancer cells from developing various types of acquired resistance. We are now focusing on YAP1 and cancer stem cells, which have been also reported as acquired resistance mechanisms in various types of cancers. Methods: PC-9 cells and HCC827 cells which were EGFR mutation-positive lung cancer cell lines were mainly utilized in our experiments; they were purchased from ECACC and ATCC, respectively. We mainly utilized osimertinib as an EGFR-TKI and verteporfin as a YAP1 inhibitor. The nuclear translocation of YAP1 was confirmed by fluorescence immunostaining. siRNA was utilized to knock down YAP1. Cell viability assays were performed using CellTiter-Glo reagent. qRT-PCR was performed to confirm the gene expression of cancer stem cells. Results: YAP1 is activated by nuclear translocation. In PC-9 cells, YAP1 was localized in the nucleus after osimertinib exposure. In contrast, YAP1 was localized in the nucleus of HCC827 cells before osimertinib exposure and remained in the nucleus even after osimertinib exposure. These data suggest that YAP1 activation is correlated with initial resistance. Cell viability assay showed that both PC-9 cells and HCC827 cells became more sensitive to osimertinib with the knocking down of YAP1. In addition, cell viability assays using PC-9 cells with verteporfin and osimertinib showed increased sensitivity to osimertinib. The gene expression of ALDH1A1 and SOX2 which are involved in cancer stem cells were increased in PC-9 cells after osimertinib exposure. On the other hand, the gene expression of ALDH1A1 was up-regulated but SOX2 was down-regulated in HCC827 cells after osimertinib exposure. We hypothesized that increased gene expression of cancer stem cells occurred downstream of YAP1 followed by initial resistance, thus we are currently confirming the gene alteration of cancer stem cells associated with osimertinib exposure with the knocking down YAP1. We are also considering the investigation of combination therapy of EGFR-TKIs and YAP1 inhibitors in vivo studies. Conclusions: YAP1 and cancer stem cells may be involved in initial resistance mechanisms to EGFR-TKIs in EGFR mutation-positive lung cancer. Our results suggest that especially YAP1 can be a potential therapeutic target, thus we will continue additional studies of combination therapy of EGFR-TKIs and YAP1 inhibitors. In addition, we will continue to investigate whether the gene for cancer stem cells acts downstream of YAP1. Citation Format: Tatsuya Ogimoto, Hiroaki Ozasa, Kentaro Hashimoto, Hiroshi Yoshida, Kazutaka Hosoya, Masatoshi Yamazoe, Hitomi Ajimizu, Tomoko Funazo, Takahiro Tsuji, Hironori Yoshida, Ryo Itotani, Yuichi Sakamori, Toyohiro Hirai. Elucidation of initial resistance mechanisms in EGFR mutation-positive lung cancer focusing on YAP1 and cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5335.

Abstract 657: Impact of therapy induced APOBEC3A mutagenesis on tumor evolution in non small cell lung cancer

Abstract Acquired drug resistance to even the most effective anti cancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified, the underlying molecular mechanisms shaping tumor evolution during treatment are incompletely understood. We have seen that lung cancer targeted therapies commonly used in the clinic induce the expression of cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug tolerant cancer cells persisting during therapy. Preventing therapy induced A3A mutagenesis by gene deletion delayed the emergence of drug resistance. Here, we show that therapy induced A3A mutagenesis contributes to tumor evolution in NSCLC. Whole genome sequencing revealed that resistant clones that evolved from persistent drug tolerant cells (late clones) harbored more A3A mutations compared to the parental cell population than pre existing resistant clones (early clones). In a subset of NSCLC patients who received targeted therapies, we observed A3A mutations accompanied clonal evolution during treatment. Comparison of APOBEC mutation fractions in short vs long term responders suggests that short responders with acquired resistance mechanisms that evolved from pre existing resistant clones have less accumulation of APOBEC mutations. Collectively, these findings insist that an increase in mutagenic processes drives tumor evolution during targeted therapy treatment and leads to acquired resistance. Thus, suppressing expression or enzymatic activity of A3A may represent a potential therapeutic strategy to halt the evolution of resistant clones and prevent acquired resistance to lung cancer targeted therapy. Citation Format: Hideko Isozaki, Ammal Abbasi, Naveed Nikpour, Marcello Stanzione, Ramin Sakhtemani, Susanna L. Monroe, Alice T. Shaw, Jessica J. Lin, Lecia V. Sequist, Zofia Piotrowska, Rémi Buisson, Michael S. Lawrence, Aaron N. Hata. Impact of therapy induced APOBEC3A mutagenesis on tumor evolution in non small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 657.

Abstract 5336: Dissecting the molecular mechanism of trastuzumab resistance in stage IV HER2-positive gastric cancer patients

Abstract Introduction: The effect of trastuzumab in targeted therapy for HER2-positive gastric cancer (GC) is limited by the eventual development of resistance within patients. Although numerous studies have been published on this topic, both the mechanism of innate and acquired trastuzumab resistance are still not fully understood. In this study, we compare the mutational landscape of paired solid tumor samples before and after treatment in an effort to identify acquired events associated with resistance. Methods: We performed whole-exome sequencing on 46 tumor and 23 whole blood samples from patients with stage IV gastric cancer were obtained from before treatment (n = 23) and at disease progression (PD) (n = 23). HER2 status was checked using immunohistochemistry and validated with florescence in situ hybridization (FISH). Correlation analysis was then conducted between the mutational profile and clinical data of patients. Results: Comparisons between innate resistance samples and acquired resistance samples at baseline revealed three copy number variation events (CDK12 amplification, ERBB2 amplification, MECOM amplification) that occurs in significantly different proportions (p &amp;lt; 0.05). In addition, for baseline samples, MECOM was shown to be associated with poor progression-free survival (PFS) (Hazard Ratio (HR), 12; 95% CI, 1.6 - 85; p = 0.027). Commonly acquired events (i.e., lacking in before treatment samples and present at PD) within patients include TP53 mutation, MYC amplification, MCL1 amplification, CDKN2A/2B deletion, and CCNE1 amplification. Of these, patients with MCL1 gene amplification had worse survival (HR, 4.8; 95% CI, 1.2 - 20; p = 0.050) than patients with wild-type MCL1. In addition, the NOTCH pathway acquired mutations in 4 of 23 patients and was found to be correlated with a worse PFS (HR, 5.1; 95% CI, 1.4 - 19; p = 0.023), indicating that this pathway is possibly involved with trastuzumab resistance to some degree. Conclusion: Overall, our results show that comparisons of samples obtained before treatment and at PD can give insight about innate and acquired resistance mechanisms to trastuzumab in stage IV, HER2-positive GC patients. Citation Format: Qi Xu, Jingjing Li, Haimeng Tang, Hua Bao, Junrong Yan, Xue Wu, Yang Shao, Jianying Jin, Cong Luo, Haimin Weng, Jieer Ying. Dissecting the molecular mechanism of trastuzumab resistance in stage IV HER2-positive gastric cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5336.

Abstract 1106: Detection of MET alterations at the DNA, RNA and protein levels in NSCLC patients progressing on ALK and ROS1 targeted therapies

Abstract Background: ALK and ROS1 fusion-positive NSCLC patients derive clinical benefit from tyrosine kinase inhibitors (TKIs) but ultimately relapse. Acquired resistance mechanisms include on-target secondary mutations or copy number gains and activation of bypass signaling. Although MET amplification has been described as a bypass resistance mechanism to ALK and ROS1 inhibitors, there are limited data on MET gene and protein overexpression. Aims: (i) detection of MET alterations at the DNA, mRNA and protein levels in ALK and ROS1 fusion-positive NSCLC patients progressing on TKIs, and (ii) establishment of primary cultures using samples from those patients. Methods: MET alterations were studied in 12 patients after progression on TKIs, 10 ALK and 2 ROS1 fusion-positive. Informed consent was obtained from all patients. NGS and FISH were used to detect resistance mutations and amplifications. MET mRNA expression levels were determined by nCounter. Total and phospho-MET levels were assessed by IHC and Western blotting. Results: A total of 21 samples were available from the 12 patients, including tumor biopsies (n=5), plasma (n=10), pleural effusions (n=3) and cerebrospinal fluids (n=3). MET alterations were detected in 4 patients, 3 ALK fusion-positive progressing on lorlatinib and one ROS1 fusion-positive patient progressing on crizotinib. The 3 ALK fusion-positive patients had MET amplification detected in liquid biopsy (n=2) or tumor tissue (n=1), collected after progression on lorlatinib. In the patient with tissue biopsy available, the increased MET copy number was in line with the presence of MET protein and RNA overexpression. MET amplification was not present on the pre-treatment biopsy available for one of the 3 patients. In primary cultures established from pleural effusion samples of 2 ALK fusion-positive patients, MET amplification was maintained, particularly if the cells were cultured in presence of an ALK TKI. Although the ROS1 fusion-positive patient had no MET amplification after progression on crizotinib, MET and phospho-MET upregulation were detected by IHC. These alterations were also present in primary culture that could be established from a pleural effusion sample of the patient. Conclusions: We found MET alterations in 4 out of 12 (33%) fusion-positive patients after progression on TKIs. Three of them had MET amplification and one MET protein overexpression in the absence of MET copy number gain. Despite the small size of the cohort, our results suggest that testing not only at the DNA but also at the RNA and protein levels, discovers amplification-negative patients with MET alterations who may derive benefit from a MET targeted therapy. Finally, our findings highlight the potential of pleural effusion as a source of material for the establishment of primary cultures. Citation Format: Núria Jordana-Ariza, Nadine Reischmann, Carlos Esparré, Ruth Román, Cristina Aguado-Esteban, Silvia García-Román, Christopher Stroh, Andrés Aguilar Aguilar, Rafael Rosell, Niki Karachaliou, Miguel A. Molina-Vila. Detection of MET alterations at the DNA, RNA and protein levels in NSCLC patients progressing on ALK and ROS1 targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1106.

Foretinib can overcome common on-target resistance mutations after capmatinib/tepotinib treatment in NSCLCs with MET exon 14 skipping mutation

BackgroundCapmatinib and tepotinib are guideline-recommended front-line treatments for non-small-cell lung cancer (NSCLC) patients with MET exon 14 skipping mutations (METex14). However, the emergence of acquired resistance to capmatinib/tepotinib is almost inevitable partially due to D1228X or Y1230X secondary mutations of the MET. In this study, we explored agents that are active against both D1228X and Y1230X MET to propose an ideal sequential treatment after capmatinib/tepotinib treatment failure in NSCLC patients with METex14.MethodsThe inhibitory effects of 300 drugs, including 33 MET-TKIs, were screened in Ba/F3 cells carrying METex14 plus MET D1228A/Y secondary mutations. The screen revealed four-candidate type II MET-TKIs (altiratinib, CEP-40783, foretinib and sitravatinib). Therefore, we performed further growth inhibitory assays using these four MET-TKIs plus cabozantinib and merestinib in Ba/F3 cells carrying MET D1228A/E/G/H/N/V/Y or Y1230C/D/H/N/S secondary mutations. We also performed analyses using Hs746t cell models carrying METex14 (with mutant allele amplification) with/without D1228X or Y1230X in vitro and in vivo to confirm the findings. Furthermore, molecular dynamics (MD) simulations were carried out to examine differences in binding between type II MET-TKIs.ResultsAll 6 type II MET-TKIs were active against Y1230X secondary mutations. However, among these 6 agents, only foretinib showed potent activity against D1228X secondary mutations of the MET in the Ba/F3 cell and Hs746t in vitro model and Hs746t in vivo model, and CEP-40783 and altiratinib demonstrated some activity. MD analysis suggested that the long tail of foretinib plays an important role in binding D1228X MET through interaction with a residue at the solvent front (G1163). Tertiary G1163X mutations, together with L1195F/I and F1200I/L, occurred as acquired resistance mechanisms to the second-line treatment foretinib in Ba/F3 cell models.ConclusionsThe type II MET-TKI foretinib may be an appropriate second-line treatment for NSCLCs carrying METex14 after campatinib/tepotinib treatment failure by secondary mutations at residue D1228 or Y1230.

KRAS G12V mutation as an acquired resistance mechanism after treatment with dabrafenib and trametinib in non-small cell lung cancer harbouring the BRAF V600E mutation: a case report

KRAS G12V mutation as an acquired resistance mechanism after treatment with dabrafenib and trametinib in non-small cell lung cancer harbouring the BRAF V600E mutation: a case report

Resistance mechanisms to anti-EGFR therapy in RAS/RAF wildtype colorectal cancer varies by regimen and line of therapy.

3554 Background: The conventional theory for the development of treatment resistance to anti-EGFR for metastatic colorectal cancer (mCRC) is the selective growth advantage of pre-existing therapy-resistant subclones with genomic mechanisms such as RAS mutations, leading to treatment resistance and disease progression. However, the impact of cytotoxic chemotherapy in combination with anti-EGFR on the mechanisms of resistance has not been assessed. Methods: We analyzed paired plasma samples from RAS/BRAF/EGFR wild-type mCRC patients enrolled in three large randomized phase 3 trials of anti-EGFR rechallenge in whom paired baseline and time of progression plasma samples had been collected for sequencing of ctDNA on a platform optimized for very low allele frequencies. 569 patients had paired baseline and progression ctDNA samples analyzed, including 147 in the first line study of FOLFOX +/- panitumumab, 91 patients in third line with panitumumab vs best supportive care, and 331 patients in the third line study of cetuximab vs. panitumumab. The mutational signature of the alterations acquired with therapy was evaluated. We also established colon cancer cell lines with resistance to cetuximab, FOLFOX, and SN38, and profiled transcriptional changes. Results: Using serial plasma samples, we demonstrate that patients whose tumors were treated with and responded to anti-EGFR alone were approximately 5-times more likely to develop acquired mutations at progression compared to those treated with an EGFR inhibitor in combination with cytotoxic chemotherapy (46% vs. 9%, respectively; p &lt; 0.001). Consistent with this clinical finding, cell lines with non-genomic acquired resistance to cetuximab were cross-resistant to cytotoxic chemotherapy and vice-versa, with transcriptomic profiles consistent with epithelial to mesenchymal transition. In contrast, common acquired genomic alterations in the MAPK pathway that drive resistance to EGFR monoclonal antibodies do not impact sensitivity to cytotoxic chemotherapy. Further, contrary to the generally accepted hypothesis of clonal expansion of acquired resistance, in our work we demonstrate that baseline resistant subclonal mutations rarely expanded to become clonal at the time of progression (8%), and most remained subclonal (44%) or disappeared (49%). Conclusions: Collectively, this work outlines a model of resistance where non-genomic mechanisms of resistance common to both EGFR inhibitors and cytotoxic chemotherapy predominate in patients treated with EGFR and chemotherapy combinations. With EGFR inhibitor monotherapy, genomic acquired resistance mechanisms predominate, although only rarely through expansion of pre-existing subclones. These findings have important implications for strategies of EGFR-inhibitor rechallenge studies.

Use of real-world data (RWD) to assess the utility of cell-free circulating tumor DNA (cfDNA) in identifying resistance to early treatment in advanced breast cancer (aBC).

1011 Background: The approvals of CDK4/6 inhibitors (CDK4/6i) and alpelisib (a PI3Ka inhibitor, PI3Ki) have overhauled early treatment of hormone positive aBC. While some clinical trials have investigated mechanisms of resistance to these drugs, their impact on tumor evolution requires further exploration. Here we use cfDNA to examine molecular changes pre- and post-CDK4/6i or PI3Ki treatment and use RWD to assess the impact of putative resistance alterations on response to treatment. Methods: Patients (pts) with aBC were identified via the Guardant INFORM database and included if they had a cfDNA test within 90 days prior to therapy initiation and/or 90 days after therapy discontinuation with CDK4/6i or PI3Ki. Pts with RB1 loss of function (LOF) alterations (alts) who received CDK4/6i and pts with PTEN LOF alts who received PI3Ki were separately identified, and these cohorts were matched 1:3 with a RB1/PTEN negative population respectively, by age (+/- 5 years), sex, year of cfDNA test, and line of therapy. Log-rank tests were used to assess differences in time to discontinuation (TTD) and time to next treatment (TTNT). Results: Differences in the frequencies of certain alts detected in pts pre- and post-CDK4/6i or PI3Ki treatment are shown (Table). Pts with RB1 LOF alts prior to the start of CDK4/6i had significantly worse TTD and numerically worse TTNT versus controls (TTD = 3 mos vs 4.7 mos, p=0.018; TTNT = 7.3 mos vs 8 mos, p=0.082). Pts with PTEN LOF alts prior to start of PI3Ki had no significant difference in TTD or TTNT versus controls (TTD = 4.1 mos vs 4.1 mos, p=0.92; TTNT = 7.4 mos vs 7 mos, p=0.32). Notably, 54% of pts receiving CDK4/6i and 84% of pts receiving PI3Ki were on their third or later line of therapy. Conclusions: Using cfDNA, we were able to further characterize the resistance landscape of both CDK4/6i and PI3Ki, and identified specific ESR1, RB1 and PTEN alterations that appear likely to occur under the pressure of therapy. Our real-world analysis examining RB1 LOF alts added further evidence to suggest it may be both a primary and acquired resistance mechanism to CDK4/6i. As a non-invasive alternative to tissue biopsies, this data further illustrates that cfDNA can provide unique insight into tumor evolution and disease progression in the aBC setting. [Table: see text]

Natural distribution of genomic alterations correlated with the resistance to KRASG12C inhibitor in Chinese colorectal cancer.

e15550 Background: KRAS inhibitors exhibited promising efficacy as systemic therapy in KRAS G12C-mutant colorectal cancers (CRC) lately. Recent studies have revealed a number of genomic alterations related to acquired resistance to this type of drugs. The discovered resistant gene mutations include: 1. Novel acquired secondary KRAS mutations within the adagrasib-binding pocket such as Y96C, H95D/Q/R, R68S, 2. Activating mutations in genes ( KRAS, BRAF, MAP2K1) MAPK signaling pathway, 3. Oncogenic fusions involving ALK, RET, BRAF, RAF1, and FGFR3, 4. Gene amplifications in KRAS, MET, MYC. Although found as acquired resistance mechanism to KRAS inhibitors, these genomic alterations might be primary or secondary in the setting of other type of therapies. Characterizing the natural distribution of these genomic alterations in cancer patients would contribute to clarifying the application potential of KRAS inhibitors. We herein performed a large-sample size analysis in Chinese colorectal cancer. Methods: A total of 9385 early-stage, late-stage, treated or treatment-naive CRC were included in this analysis. Calling of single nucleotide variants (SNV), copy number variants (CNV), insertion/deletions (indels), fusions and evaluation of tumor mutational burden (TMB) were performed using a wide panel next-generation sequencing (NGS) testing. Results: 43.4% (4069/9385) cases were KRAS-mutant in this cohort. KRAS G12C account for 5.8% (236/4069) in all KRAS-mutant CRC, and 15.7% (37/236) were harbored the above resistance genomic alterations. Fifteen KRAS G12C-mutant CRC were detected to have activating MAPK mutations, including G12D/V (n = 6), G13D (n = 3), Q61H (n = 6), NRAS Q61K (n = 3), MAP2K1 K57T (n = 3), and BRAF V600E (n = 4). Ten KRAS G12C-mutant CRC were detected to have oncogenic fusions, including TGFBR2- CACNA1D (n = 3), TPM4- ALK (n = 1), FGFR3- TACC3 (n = 1), ETV6- SNRPC (n = 1), KAZN- PAX7 (n = 1), NRG1- TTC12 (n = 1), TCF7L2- EWSR1 (n = 1) and ERBB2 arrangement (n = 2). It is noteworthy that a single case was with 5 gene arrangement events in 17q ( ERBB2- ZPBP2, IKZF3- ERBB2, PGAP3- ERBB2, RARA- ERBB2, RARA- GRB7). Thirteen KRAS G12C-mutant CRC were detected to have amplifications in KRAS (n = 5), MYC (n = 5) and MET (n = 4) genes. TP53 (81%), APC (65%) and PIK3CA (32%) were the utmost frequent somatic mutational genes in this subgroup of CRC. In addition, all cases were microsatellite stable (MSS). Tumors harboring fusion variants showed relatively high TMB level in this cohort. Conclusions: Our analysis results indicate about 15% KRAS G12C-mutant Chinese CRC were probable resistant to KRASG12C inhibitor. One-third of these patients had co-existed oncogenic fusions and higher TMB levels, suggesting possibilities for other therapeutic choices.

Roles of natural killer cells in immunity to cancer, and applications to immunotherapy.

Great strides have been made in recent years towards understanding the roles of natural killer (NK) cells in immunity to tumours and viruses. NK cells are cytotoxic innate lymphoid cells that produce inflammatory cytokines and chemokines. By lysing transformed or infected cells, they limit tumour growth and viral infections. Whereas T cells recognize peptides presented by MHC molecules, NK cells display receptors that recognize stress-induced autologous proteins on cancer cells. At the same time, their functional activity is inhibited by MHC molecules displayed on such cells. The enormous potential of NK cells for immunotherapy for cancer is illustrated by their broad recognition of stressed cells regardless of neoantigen presentation, and enhanced activity against tumours that have lost expression of MHC class I owing to acquired resistance mechanisms. As a result, many efforts are under way to mobilize endogenous NK cells with therapeutics, or to provide populations of ex vivo-expanded NK cells as a cellular therapy, in some cases by equipping the NK cells with chimeric antigen receptors. Here we consider the key features that underlie why NK cells are emerging as important new additions to the cancer therapeutic arsenal.