Non-small Cell Lung Carcinoma Research Articles

LC-ESI QToF MS Non-Targeted Screening of Latex Extracts of Euphorbia seguieriana ssp. seguieriana Necker and Euphorbia cyparissias and Determination of Their Potential Anticancer Activity.

Euphorbia seguieriana ssp. seguieriana Necker (ES) and Euphorbia cyparissias (EC) with a habitat in the Deliblato Sands were the subject of this examination. The latexes of these so far insufficiently investigated species of the Euphorbia genus are used in traditional medicine for the treatment of wounds and warts on the skin. To determine their chemical composition, non-targeted screening of the latexes' chloroform extracts was performed using liquid chromatography coupled with quadrupole time-of-flight mass spectrometry employing an electrospray ionization source (LC-ESI QTOF MS). The analysis of the obtained results showed that the latexes of ES and EC represent rich sources of diterpenes, tentatively identified as jatrophanes, ingenanes, tiglianes, myrsinanes, premyrsinanes, and others. Examination of the anticancer activity of the ES and EC latex extracts showed that both extracts significantly inhibited the growth of the non-small cell lung carcinoma NCI-H460 and glioblastoma U87 cell lines as well as of their corresponding multi-drug resistant (MDR) cell lines, NCI-H460/R and U87-TxR. The obtained results also revealed that the ES and EC extracts inhibited the function of P-glycoprotein (P-gp) in MDR cancer cells, whose overexpression is one of the main mechanisms underlying MDR.

Radiotranscriptomics of non-small cell lung carcinoma for assessing high-level clinical outcomes using a machine learning-derived multi-modal signature

BackgroundMulti-omics research has the potential to holistically capture intra-tumor variability, thereby improving therapeutic decisions by incorporating the key principles of precision medicine. The purpose of this study is to identify a robust method of integrating features from different sources, such as imaging, transcriptomics, and clinical data, to predict the survival and therapy response of non-small cell lung cancer patients.Methods2996 radiomics, 5268 transcriptomics, and 8 clinical features were extracted from the NSCLC Radiogenomics dataset. Radiomics and deep features were calculated based on the volume of interest in pre-treatment, routine CT examinations, and then combined with RNA-seq and clinical data. Several machine learning classifiers were used to perform survival analysis and assess the patient’s response to adjuvant chemotherapy. The proposed analysis was evaluated on an unseen testing set in a k-fold cross-validation scheme. Score- and concatenation-based multi-omics were used as feature integration techniques.ResultsSix radiomics (elongation, cluster shade, entropy, variance, gray-level non-uniformity, and maximal correlation coefficient), six deep features (NasNet-based activations), and three transcriptomics (OTUD3, SUCGL2, and RQCD1) were found to be significant for therapy response. The examined score-based multi-omic improved the AUC up to 0.10 on the unseen testing set (0.74 ± 0.06) and the balance between sensitivity and specificity for predicting therapy response for 106 patients, resulting in less biased models and improving upon the either highly sensitive or highly specific single-source models. Six radiomics (kurtosis, GLRLM- and GLSZM-based non-uniformity from images with no filtering, biorthogonal, and daubechies wavelets), seven deep features (ResNet-based activations), and seven transcriptomics (ELP3, ZZZ3, PGRMC2, TRAK1, ATIC, USP7, and PNPLA2) were found to be significant for the survival analysis. Accordingly, the survival analysis for 115 patients was also enhanced up to 0.20 by the proposed score-based multi-omics in terms of the C-index (0.79 ± 0.03).ConclusionsCompared to single-source models, multi-omics integration has the potential to improve prediction performance, increase model stability, and reduce bias for both treatment response and survival analysis.

SAMPLER: unsupervised representations for rapid analysis of whole slide tissue images

Deep learning has revolutionized digital pathology, allowing automatic analysis of hematoxylin and eosin (H&E) stained whole slide images (WSIs) for diverse tasks. WSIs are broken into smaller images called tiles, and a neural network encodes each tile. Many recent works use supervised attention-based models to aggregate tile-level features into a slide-level representation, which is then used for downstream analysis. Training supervised attention-based models is computationally intensive, architecture optimization of the attention module is non-trivial, and labeled data are not always available. Therefore, we developed an unsupervised and fast approach called SAMPLER to generate slide-level representations. Slide-level representations of SAMPLER are generated by encoding the cumulative distribution functions of multiscale tile-level features. To assess effectiveness of SAMPLER, slide-level representations of breast carcinoma (BRCA), non-small cell lung carcinoma (NSCLC), and renal cell carcinoma (RCC) WSIs of The Cancer Genome Atlas (TCGA) were used to train separate classifiers distinguishing tumor subtypes in FFPE and frozen WSIs. In addition, BRCA and NSCLC classifiers were externally validated on frozen WSIs. Moreover, SAMPLER's attention maps identify regions of interest, which were evaluated by a pathologist. To determine time efficiency of SAMPLER, we compared runtime of SAMPLER with two attention-based models. SAMPLER concepts were used to improve the design of a context-aware multi-head attention model (context-MHA). SAMPLER-based classifiers were comparable to state-of-the-art attention deep learning models to distinguish subtypes of BRCA (AUC=0.911±0.029), NSCLC (AUC=0.940±0.018), and RCC (AUC=0.987±0.006) on FFPE WSIs (internal test sets). However, training SAMLER-based classifiers was >100 times faster. SAMPLER models successfully distinguished tumor subtypes on both internal and external test sets of frozen WSIs. Histopathological review confirmed that SAMPLER-identified high attention tiles contained subtype-specific morphological features. The improved context-MHA distinguished subtypes of BRCA and RCC (BRCA-AUC=0.921±0.027, RCC-AUC=0.988±0.010) with increased accuracy on internal test FFPE WSIs. Our unsupervised statistical approach is fast and effective for analyzing WSIs, with greatly improved scalability over attention-based deep learning methods. The high accuracy of SAMPLER-based classifiers and interpretable attention maps suggest that SAMPLER successfully encodes the distinct morphologies within WSIs and will be applicable to general histology image analysis problems. This study was supported by the National Cancer Institute (Grant No. R01CA230031 and P30CA034196).

USP7-mediated ERβ stabilization mitigates ROS accumulation and promotes osimertinib resistance by suppressing PRDX3 SUMOylation in non-small cell lung carcinoma

Osimertinib resistance is regarded as a major obstacle limiting survival benefits for patients undergoing treatment of epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC). However, the underlying mechanisms of acquired resistance remain unclear. In this study, we report that estrogen receptor β (ERβ) is highly expressed in osimertinib-resistant NSCLC and plays a pivotal role in promoting osimertinib resistance. We further identified ubiquitin-specific protease 7 (USP7) as a critical binding partner that deubiquitinates and upregulates ERβ in NSCLC. ERβ promotes osimertinib resistance by mitigating reactive oxygen species (ROS) accumulation. We found that ERβ mechanistically suppresses peroxiredoxin 3 (PRDX3) SUMOylation and thus confers osimertinib resistance onto NSCLC. Furthermore, we provide evidence showing that depletion of ERβ induces ROS accumulation and reverses osimertinib resistance in NSCLC both in vitro and in vivo. Thus, our results demonstrate that USP7-mediated ERβ stabilization suppresses PRDX3 SUMOylation to mitigate ROS accumulation and promote osimertinib resistance, suggesting that targeting ERβ may be an effective therapeutic strategy to overcome osimertinib resistance in NSCLC.

A Rare Case of Co-existing Non-small Cell Lung Carcinoma and Non-tuberculous Mycobacteria

A Rare Case of Co-existing Non-small Cell Lung Carcinoma and Non-tuberculous Mycobacteria

Non-Small Cell Lung Carcinoma With Clear Cell Features and FGFR3::TACC3 Gene Rearrangement : Clinicopathologic and Next Generation Sequencing Study of 7 Cases.

Seven cases of primary lung tumors characterized histologically by clear cell morphology and a distinctive FGFR3::TACC3 gene rearrangement are described. The tumors arose in 4 women and 3 men, aged 47 to 81 years (mean=68). They occurred in peripheral locations, predominantly subpleural, and ranged in size from 1.4 to 6.5cm (mean=4.1cm). All tumors showed a solid growth pattern with abundant central areas of necrosis and marked nuclear pleomorphism. The tumors demonstrated clear cell histology, with large cohesive tumor cells displaying atypical nuclei and abundant clear cytoplasm. Immunohistochemical stains identified a squamous phenotype in 5 cases and an adenocarcinoma phenotype in 2 cases. One case was a squamous cell carcinoma with focal glandular component, and one of the squamous cell carcinomas showed focal sarcomatoid changes. Next generation sequencing identified FGFR3::TACC3 gene rearrangements in all 7 cases. One case demonstrated a concurrent activating FGFR3 mutation and a second case demonstrated concurrent FGFR3 amplification. Two cases harbored a concurrent KRAS G12D mutation. One case harbored both KRAS and EGFR mutations, and 1 case had a concurrent TP53 mutation. Non-small cell lung carcinoma harboring FGFR3::TACC3 gene rearrangements is extremely rare, and this rearrangement may potentially be enriched in tumors that demonstrate clear cell histology. Identification of FGFR3::TACC3 in patients with lung carcinomas with clear cell features may be of importance as they could potentially be candidates for therapy with tyrosine kinase inhibitors.

Acquired Radiation Resistance Induces Thiol-dependent Cisplatin Cross-resistance.

Resistance to radiation remains a significant clinical challenge in non-small cell lung carcinoma (NSCLC). It is therefore important to identify the underlying molecular and cellular features that drive acquired resistance. We generated genetically matched NSCLC cell lines to investigate characteristics of acquired resistance. Murine Lewis lung carcinoma (LLC) and human A549 cells acquired an approximate 1.5-2.5-fold increase in radiation resistance as compared to their parental match, which each had unique intrinsic radio-sensitivities. The radiation resistance (RR) was reflected in higher levels of DNA damage and repair marker γH2AX and reduced apoptosis induction after radiation. Morphologically, we found that radiation resistance A549 (A549-RR) cells exhibited a greater nucleus-to-cytosol (N/C) ratio as compared to its parental counterpart. Since the N/C ratio is linked to the differentiation state, we next investigated the epithelial-to-mesenchymal transition (EMT) phenotype and cellular plasticity. We found that A549 cells had a greater radiation-induced plasticity, as measured by E-cadherin, vimentin and double-positive (DP) modulation, as compared to LLC. Additionally, migration was suppressed in A549-RR cells, as compared to A549 cells. Subsequently, we confirmed invivo that the LLC-RR and A549-RR cells are also more resistance to radiation than their isogenic-matched counterpart. Moreover, we found that the acquired radiation resistance also induced resistance to cisplatin, but not carboplatin or oxaliplatin. This cross-resistance was attributed to induced elevation of thiol levels. Gamma-glutamylcysteine synthetase inhibitor buthionine sulfoximine (BSO) sensitized the resistant cells to cisplatin by decreasing the amount of thiols to levels prior to obtaining acquired radiation resistance. By generating radiation-resistance genetically matched NSCLC we were able to identify and overcome cisplatin cross-resistance. This is an important finding arguing for combinatorial treatment regimens including glutathione pathway disruptors in patients with the potential of improving clinical outcomes in the future.

Clinicopathological Characteristics and Real-life Data of Patients Receiving Tyrosine Kinase Inhibitor in Metastatic EGFR Mutant Non-small Cell Lung Carcinoma

Clinicopathological Characteristics and Real-life Data of Patients Receiving Tyrosine Kinase Inhibitor in Metastatic EGFR Mutant Non-small Cell Lung Carcinoma

Immunofluorescence-Based Assay for High-Throughput Analysis of Multidrug Resistance Markers in Non-Small Cell Lung Carcinoma Patient-Derived Cells.

Lung cancer remains the leading cause of cancer death globally, with non-small cell lung cancer (NSCLC) accounting for the majority of cases. Multidrug resistance (MDR), often caused by ATP-binding cassette (ABC) transporters, represents a significant obstacle in the treatment of NSCLC. While genetic profiling has an important role in personalized therapy, functional assays that measure cellular responses to drugs are gaining in importance. We developed an automated microplate-based immunofluorescence assay for the evaluation of MDR markers ABCB1, ABCC1, and ABCG2 in cells obtained from NSCLC patients through high-content imaging and image analysis, as part of a functional diagnostic approach. This assay effectively discriminated cancer from non-cancer cells within mixed cultures, which is vital for accurate assessment of changes in MDR marker expression in different cell populations in response to anticancer drugs. Validation was performed using established drug-sensitive (NCI-H460) and drug-resistant (NCI-H460/R) NSCLC cell lines, demonstrating the assay's capacity to distinguish and evaluate different MDR profiles. The obtained results revealed wide-ranging effects of various chemotherapeutic agents on MDR marker expression in different patient-derived NSCLC cultures, emphasizing the need for MDR diagnostics in NSCLC. In addition to being a valuable tool for assessing drug effects on MDR markers in different cell populations, the assay can complement genetic profiling to optimize treatment. Further assay adaptations may extend its application to other cancer types, improving treatment efficacy while minimizing the development of resistance.

Comprehensive molecular analysis of driver mutations in non-small cell lung carcinomas and its correlation with PD-L1 expression, An Indian perspective

BackgroundThe understanding of molecular mechanisms involved in non-small cell lung carcinoma (NSCLC) has revolutionized significantly in the recent years. These have helped to develop personalized management strategies by identifying specific molecular alterations such as mutations in EGFR, ROS1, BRAF, ERBB2, MET, ALK, and KRAS genes. These mutations are targetable ensuring a better clinical outcome. Next-generation sequencing (NGS) methodology is the recommended technique for the identification of driver mutations in the five hot-spot genes (EGFR, ALK, ROS1, MET, and BRAF) involved in the NSCLC. NGS has numerous advantages including multiplexing, tissue conservation, identification of rare and novel variants, and reduced cost over the sequential single gene testing. Herein, we sought to demonstrate the mutational profile in NSCLC and their clinicopathologic correlation in a contemporary cohort of Indian NSCLC patients. Additionally, we studied the correlation of oncogenic driver mutations with PD-L1 status in these patients. Materials and MethodsFive fifty-two stage IV NSCLC patients (adenocarcinoma=490; squamous cell carcinoma=51; adenosquamous carcinoma=5; large cell carcinoma=2; sarcomatoid carcinoma=3; spindle cell carcinoma=1) underwent broad molecular profiling by a custom-made, targeted DNA- and RNA-based five hot-spot genes lung cancer panel (EGFR, ALK, ROS1, BRAF, and MET), compatible with the NGS Ion S5 system. The mutations were correlated with the clinicopathologic characteristics. Additionally, PD-L1 expression status, available on 252 tumors, was correlated with the oncogenic drivers. ResultsValidation of the 5 gene panel yielded the following results: a) specificity of 99.74%; b) sensitivity of 100% for single nucleotide variants (SNVs) (>5% variant allele frequency, VAF), indels (>10% VAF) and fusions; c) 100% intra- and inter-run reproducibility; d) 88% inter-laboratory agreement. Validated panel was then used to analyze clinical samples. Sixty percentage tumors harbored either one (54.71%) or multiple (3.26%) mutations. EGFR and BRAF V600E mutations, ALK and ROS1 rearrangements, and MET exon 14 skipping mutation were observed in 38.41% (n = 212) and 2.72% (n = 15) patients, 12.14% (n = 67) and 3.62% (n = 20) patients, and 1.09% (n = 6) patients, respectively. EGFR exon 19 deletion accounted for 52.83% of all mutations, followed by L858R (35.85%), T790M (5.19%), exon 20 insertions (6.6%), and other rare mutations (G719X, L861Q, S768I) (9.91%). Concurrent EGFR with ALK, EGFR with ROS1, EGFR with MET, and EGFR with BRAF were observed in 10, 4, 1, and 3 patients, respectively. PD-L1 was expressed in 134 patients (53.2%). Exon 19 deletion was more prevalent in PD-L1 negative tumors whereas exon 21 substitution (L858R) was seen more in PD-L1 positive tumors. ConclusionsThis is one of the largest cohorts of NSCLC for comprehensive targeted mutational profiling and correlation with the PD-L1 expression. The mutations are more prevalent in non-smoker females for all genes, except ALK (non-smoker males). MET and BRAF mutations are more common in elderly population whereas EGFR mutations, and ALK and ROS1 genes rearrangements are more prevalent in younger population. The most common histopathologic subtype/feature associated with various mutations was as follows: acinar with EGFR, solid with ALK, macronucleoli with ROS1, signet ring with MET, and micropapillary with BRAF.

Concordance of Immunohistochemistry and Fluorescence In Situ Hybridization in the Detection of Anaplastic Lymphoma Kinase (ALK) and Ros Proto-oncogene 1 (ROS1) Gene Rearrangements in Non-Small Cell Lung Carcinoma: A 4.5-Year Experience Highlighting Challenges and Pitfalls.

ALK and ROS1 rearrangements are essential biomarkers to be tested in advanced lung adenocarcinomas. While D5F3 Ventana assay is a companion diagnostic for anaplastic lymphoma kinase-targeted therapy, immunohistochemistry is only a screening tool for detecting ROS1 rearrangement. Confirmation by cytogenetic or molecular techniques is necessary. To evaluate the utility of ALK and ROS1 fluorescence in situ hybridization as a complement to immunohistochemistry in routine predictive biomarker testing algorithms. The study was ambispective, spanning 4.5 years during which lung adenocarcinoma samples were subjected to EGFR mutation testing by real-time polymerase chain reaction and ALK/ROS1 rearrangement testing by immunohistochemistry (Ventana D5F3 assay for anaplastic lymphoma kinase protein; manual assay with D4D6 clone for Ros proto-oncogene 1 protein). Fluorescence in situ hybridization was performed in all anaplastic lymphoma kinase equivocal and Ros proto-oncogene 1 immunopositive cases. Of 1874 samples included, EGFR mutations were detected in 27% (481 of 1796). Anaplastic lymphoma kinase immunohistochemistry was positive in 10% (174 of 1719) and equivocal in 3% (58 of 1719) of samples tested. ALK fluorescence in situ hybridization showed 81% (77 of 95) concordance with immunohistochemistry. Ros proto-oncogene 1 immunopositivity was noted in 13% (190 of 1425) of cases, with hybridization-confirmed rearrangements in 19.3% (26 of 135) of samples, all of which showed diffuse, strong- to moderate-intensity, cytoplasmic staining in tumor cells. Ros proto-oncogene 1 protein overexpression without rearrangement was significantly common in EGFR-mutant and ALK-rearranged adenocarcinomas. Immunostaining is a robust method for ALK-rearrangement testing, with fluorescence in situ hybridization adding value in the rare equivocal stained case. ROS1-rearrangement testing is more cost-effective if immunohistochemistry is followed by fluorescence in situ hybridization after excluding EGFR-mutant and ALK-rearranged adenocarcinomas.

Abstract B047: Tertiary lymphoid structures and B cells signatures determine clinically relevant T cell phenotype in ovarian cancer

Abstract Tertiary lymphoid structures (TLSs) are ectopic lymphoid formations that arise at sites with persistent inflammatory conditions, including tumors. TLSs are composed predominantly of B cells, T cells, and CD21+ CD23+ follicular dendritic cells and exhibit different levels of the organization, ranging from locally concentrated aggregates of immune cells to well-defined B cell follicles to mature follicles with germinal center formation. Supporting key relevance for TLSs in natural and immunotherapy-driven immunosurveillance, intratumoral TLS density has been associated with improved disease outcome and response to cancer immunotherapy, mainly check-point inhibitors (ICIs) in solid carcinomas. Here, we demonstrate that compared to the immunologically hot tumors like non-small cell lung carcinoma (NSCLC), human high-grade serous ovarian carcinoma (HGSOC) contains only a limited number of TLS with germinal center formation. In parallel, TLS frequency and maturation positively correlate with progenitor stem-like CD8+ T cell phenotype. In line with this notion, B cell depletion in HGSOC biopsies promoted terminally exhausted, poorly ICI-sensitive CD8+ T cells, suggesting a critical role of intratumoral B cells in maintaining T cell effector functions. These findings point to key numerical and functional differences between mature TLSs in ICI-responsive vs ICI-irresponsive neoplasms that may guide the development of alternative ICI-based immunotherapies for patients with HGSOC. Citation Format: Irena Kusova Moserova, Lenka Kasikova, Jana Rakova, Jana Tomankova, Michal Henser, Tereza Lanickova, Katerina Mojzisova, Jana Drozenova, Jan Laco, Lukas Rob, Michael Halaska, Ales Ryska, Robert Lischke, Jiri Vachtenheim, David Cibula, Lorenzo Galluzzi, Radek Spisek, Jitka Fucikova. Tertiary lymphoid structures and B cells signatures determine clinically relevant T cell phenotype in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B047.

Abstract B074: Discovery of MK-1084, a low dose selective clinical stage KRAS G12C inhibitor

Abstract KRAS mutations are the predominant oncogenic drivers in multiple indications including non-small cell lung carcinoma (NSCLC), colorectal carcinoma (CRC) and pancreatic ductal adenocarcinoma (PDAC). KRAS has long been considered a challenging target due to the perceived lack of druggable binding pockets and high flexibility of the protein. However, recent progress in discovering and developing covalent inhibitors against the GDP-loaded form of G12C mutant KRAS has provided a strategy for targeting mutant-specific KRAS as the cysteine residue in the switch II pocket offers a handle for covalent modification. With encouraging efficacy data observed with KRAS G12C-GDP covalent inhibitors in the clinic, attention has shifted to increasing response rate and durability of response. Achieving these goals will likely require combination therapies with different mechanisms of action such as PD-1 or EGFR inhibitors, or other inhibitors within the MAPK pathway (e.g. MEKi, SHP2i). Excellent tolerability of a KRAS G12C inhibitor in such combination settings will be a pre-requisite to success of this strategy. In 2019, Merck & Co., Inc., entered into a research collaboration and license agreement with Taiho Pharmaceutical Co, Ltd. and Astex Pharmaceuticals (UK), to research, develop and commercialize small molecule modulators of mutant KRAS oncogenes, including MK-1084, an inhibitor of G12C-GDP which was advanced under the collaboration. MK-1084 is a highly potent and specific KRAS G12C-GDP macrocyclic inhibitor that was discovered from a structure-guided lead optimization effort. It exhibits a favorable preclinical potency, PK, PK/PD, efficacy, off-target and tolerability profile and has QD oral dosing potential with a projected clinical dose that compares favorably to approved competitors. The significantly lower dose projection along with superior off-target profile puts MK-1084 in a position to differentiate in the clinic regarding dose/pill burden, potential for drug-drug interactions (DDIs) and/or risks associated with drug induced liver injury (DILI) in a combination setting. MK-1084 is currently being evaluated in the clinic for solid tumors as a monotherapy and in combination with pembrolizumab. We will describe the research efforts that led to the discovery of MK-1084, its pre-clinical profile, and disclose its structure. Citation Format: Matthew L Maddess, David L Sloman, Xiaoshen Ma, Anandan Palani, Alexander Stoeck, Aaron C Sather, Nicolas Solban, Ruchia Duggal, Jeanine Ballard, Erik V Munsell, Lin-Chi Chen, Yongxin Han, Richard Miller, Symon Gathiaka, Christopher Brynczka, Hongming Li, Ruojing Yang, Christopher Brynczka, Phil Day. Discovery of MK-1084, a low dose selective clinical stage KRAS G12C inhibitor [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 B074.

Abstract C128: PT-112, a novel immunogenic cell death inducer, causes ribosomal biogenesis inhibition and organelle stress in cancer cells

Abstract PT-112 is a novel immunogenic small molecule under Phase 2 development in metastatic castration-resistant prostate cancer and thoracic malignancies. PT-112 has been shown to release damage-associated molecular patterns, essential immune signaling components of immunogenic cell death (ICD), and to elicit anticancer immune responses. In addition, PT-112 causes mitochondrial and endoplasmic reticulum (ER) stress in vitro. Here, we sought to elucidate early molecular effects of PT-112 that culminate in cancer cell death. We assessed nucleolar protein phenotypes in human prostate cancer cells treated with PT-112 using antibody labeling, followed by confocal microscopy. We also conducted nascent RNA sequencing in human cancer cells (non-small cell lung, prostate, and renal cell carcinoma) following 1- and 6-hour PT-112 treatment. We then performed an analysis using Enrichr across multiple databases to identify biological pathways linked to clusters of differentially expressed genes (p ≤ 0.001) in PT-112-treated cells. Pathways affected by PT-112 (p ≤ 0.05) were sorted by statistical significance. In prostate cancer cells, PT-112 caused changes in nucleolar protein labeling. The most pronounced effect, observed after 8 or more hours of treatment, was rapid NPM1 relocation from nucleoli to nucleoplasm, a hallmark of nucleolar stress and disruption of ribosomal biogenesis (RiBi). PT-112 also induced gene expression changes in the treated cancer cell lines, resulting in statistically significant repression of pathways related to RiBi, ribosomal RNA (rRNA) processing and translation. Notably, a pattern across these pathways was the reduced expression of many ribosomal genes, consistent with RiBi inhibition. Additional analysis identified oncogenic pathways such as Myc and NF-κB significantly inhibited by PT-112. The degree of pathway suppression increased with treatment time. These results demonstrate that PT-112 rapidly causes nucleolar stress and RiBi inhibition, suggesting these are central to PT-112-induced cancer cell death. This mechanism could also explain the selective killing of cancer cells by PT-112, given that aberrant nucleolar size and number as well as ribosomal activity in cancer cells make them more sensitive to these drug effects. The broad consequences of impaired ribosome production and downstream protein translation may explain PT-112’s anticancer and immunogenic effects. Additionally, these disruptions have been reported to cause an increase in free ribosomal proteins and changes in nucleolar protein localization, which have been shown to induce ER stress and shuttle pro-apoptotic proteins to the mitochondria. These additional consequences of RiBi inhibition and nucleolar stress may contribute to or drive previously observed PT-112-induced cancer organelle stress and ICD. While further validation of these findings is needed, this work deepens our understanding of PT-112’s mechanism of action. Present findings, together with demonstrated clinical activity of PT-112, position it as a promising small molecule cancer immunotherapy. Citation Format: Christina Y. Yim, Maria T. Congenie, Hannah L. Johnson, Maureen G. Mancini, Fabio Stossi, Michael A. Mancini, Joey Azofeifa, Matthew R. Price, Johan Baeck, Tyler D. Ames. PT-112, a novel immunogenic cell death inducer, causes ribosomal biogenesis inhibition and organelle stress in cancer cells [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 C128.

Abstract B025: The next generation farnesyltransferase inhibitor, KO-2806, blocks oncogenic signaling at multiple nodes to enhance the antitumor efficacy of KRASG12C inhibitor adagrasib in KRASG12C non-small cell lung carcinoma

Abstract Emerging clinical and preclinical data have shown that responses to KRASG12C inhibition are limited by the activation of compensatory signaling proteins, including receptor tyrosine kinases (RTKs) and mTOR. Combination therapeutic strategies co-targeting these nodes are needed to fully realize the potential of breakthrough KRASG12C-selective inhibitors. We propose that, given its multipronged mechanism of action, KO-2806, a next-generation farnesyltransferase inhibitor (FTI) with increased potency and improved pharmacokinetic properties relative to earlier FTI candidates that is poised to enter a first-in-human clinical trial, is a uniquely suited combination partner for KRASG12C inhibitors. Here, we evaluate the therapeutic potential of combined KO-2806 and adagrasib in preclinical models of KRASG12C non-small cell lung cancer (NSCLC). Our previous work indicated that FTIs amplify the antitumor effects of PI3Kα inhibition by blocking compensatory mTOR signaling. As activation of compensatory signaling is a well-described bypass mechanism to escape KRAS inhibition, we asked whether KO-2806 could improve the in vivo efficacy of adagrasib. Combination of KO-2806 with adagrasib led to markedly enhanced antitumor effects in KRASG12C-mutant NSCLC cell line- and patient-derived xenograft models compared to adagrasib alone. Tumor regression in the combination group corresponded to increased apoptosis, as evidenced by higher cleaved caspase 3 staining in combination-treated tumors compared to single agent groups. Intriguingly, adagrasib induced expression of the RTK HER3, a potential driver of bypass signaling, while HER3 expression in combination-treated tumors was lower than in vehicle-treated tumors. To further interrogate the mechanistic basis of the observed in vivo synergy, we cultured KRASG12C-mutant cell lines as 3D tumor spheroids and exposed them to KO-2806 and/or adagrasib. Consistent with the xenograft tumor data, spheroids exposed to the combination expressed lower levels of the upstream receptor HER3 (total and phosphorylated forms) and higher levels of apoptotic markers cleaved PARP and cleaved caspase 3. Concurrently, activity of the downstream effectors ERK and mTOR was reduced. Co-exposure to KO-2806 and adagrasib potently inhibited phosphorylation of ERK and its substrate p90 RSK as well as phosphorylation of mTOR substrates p70 S6K, 4EBP1, and S6. The mTOR signaling reduction paralleled defarnesylation of the farnesylation-dependent mTOR activator RHEB, so we depleted RHEB expression in spheroids and assayed their growth. Genetic depletion of RHEB phenocopied the antiproliferative effects of KO-2806 in combination with adagrasib, implicating RHEB as an important farnesylated target in KRASG12C NSCLC. These preclinical results suggest that KO-2806 is an effective combination partner for KRASG12C inhibitors by simultaneously targeting an upstream activator (HER3) and a downstream effector (RHEB/mTOR) of KRAS, leading to improved antitumor activity in KRASG12C-mutant NSCLC models. Citation Format: Hetika V Patel, Alison Smith, Stacia Chan, Linda Kessler, Francis Burrows, Shivani Malik. The next generation farnesyltransferase inhibitor, KO-2806, blocks oncogenic signaling at multiple nodes to enhance the antitumor efficacy of KRASG12C inhibitor adagrasib in KRASG12C non-small cell lung carcinoma [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 B025.

Objective improvement in dexterity for trainees undergoing a video-assisted thoracoscopic surgery simulation program, a prospective single center study.

Video-assisted thoracoscopic surgery (VATS) is the recommended approach for the management of early-stage operable non-small cell lung carcinoma as well as for other pathologies of the thoracic cavity. Although VATS approaches have been largely adopted in Europe and North America, teaching the technique to novice thoracic surgery trainees remains challenging and non-standardized. Our objective was to assess the impact of a VATS simulation training program on the dexterity of thoracic surgery residents in a prospective single institution study. We developed a 6-month VATS simulation training program on two different dry-lab simulators (Johnson & Johnson Ethicon Stupnik® lobectomy model; CK Surgical Simulation® Crabtree perfused lobectomy model) and assessed the skills of first year thoracic surgery residents (study group, n=7) before and after this program using three standardized exercises on the Surgical Science Simball® Box (peg placement on a board, rope insertion in loops, precision circle cutting). The results were compared to those of last-year medical students who performed the same Simball® Box exercises at a 6-month interval without undergoing a training program (control group, n=5). For each participant, the travel distances of instruments, operation time and absences of periods of extreme motion were assessed for each exercise by the use of the computer-based evaluation of the Simball® Box. After the 6-month VATS training program, all residents revealed a significant increase of their performance status with respect to instrument travel distances operation times and absence of periods of extreme motion in all three exercises performed. The performance of the control group was not different from the study group prior to the training program and remained unchanged 6 months later, for all exercises and parameters assessed. Our results suggest that the implementation of a VATS simulation training program objectively increases the dexterity of thoracic surgery residents and could be an interesting training tool for their surgical education.

Circular RNA in Non-small Cell Lung Carcinoma: Identification of Targets and New Treatment Modalities.

Despite availability of several treatment options for non-small cell lung cancer (NSCLC), such as surgery, chemotherapy, radiation, targeted therapy and immunotherapy, the survival rate of patients for five years is in the range of 22%. Therefore, identification of new targets and treatment modalities for this disease is an important issue. In this context, we screened the PubMed database for up-regulated circular RNAs (circRNAs) which promote growth of NSCLC in preclinical models in vitro as well as in vivo xenograft models in immuno-compromised mice. This approach led to potential targets for further validation and inhibition with small molecules or antibody-derived entities. In case of preclinical validation, the corresponding circRNAs can be inhibited with small interfering RNAs (siRNA) or short hairpin RNAs (shRNA). The identified circRNAs act by sponging microRNAs (miRs) preventing cleavage of the mRNA of the corresponding targets. We identified nine circRNAs up-regulating transmembrane receptors, five circRNAs increasing expression of secreted proteins, nine circRNAs promoting expression of components of signaling pathways, six circRNAs involved in regulation of splicing and RNA processing, six circRNAs up-regulating actin-related and RNA processing components, seven circRNAs increasing the steady-state levels of transcription factors, two circRNAs increasing high-mobility group proteins, four circRNAs increasing components of the epigenetic modification system and three circRNAs up-regulating protein components of additional systems.

(Pro)renin Receptor Down-regulation Is Associated With a Higher Risk of Recurrence in Lung Adenocarcinoma.

The (pro)renin receptor [(P)RR] plays a role not only in cardiovascular and renal diseases, but also in tumorigenesis. (P)RR contributes to the activation of the Wnt/β-catenin signaling pathway, independent of the renin-angiotensin system. Accumulating evidence has shown that (P)RR is expressed in various human cancers. However, its clinical impact in lung carcinomas remains unclear. This study aimed to clarify the associations between (P)RR expression and clinical outcomes in patients with non-small cell lung carcinoma (NSCLC). We analyzed the data of 913 patients with NSCLC who underwent resection between 1999 and 2016. Tissue microarrays were constructed and the expression of (P)RR and β-catenin was investigated using immunohistochemistry. Recurrence-free probability and overall survival were analyzed using a log-rank test and Cox proportional hazards model. In adenocarcinomas, (P)RR down-regulation correlated significantly with high-grade tumors (p=0.026) and a higher risk of recurrence in all patients (p=0.001). Among patients with (P)RR-positive tumors, the nuclear expression of β-catenin was associated with a higher risk of recurrence (p=0.001). Multivariate analysis revealed that (P)RR down-regulation was an independent predictor of disease recurrence (p=0.031). Conversely, in squamous cell carcinoma, (P)RR was not associated with patient outcomes. (P)RR down-regulation is associated with a higher risk of recurrence in lung adenocarcinomas, thereby characterizing a prognostic subset within high-grade tumors.

Selpercatinib-induced hepatotoxicity: A rare case report

Abstract Introduction/Objective Selpercatinib (Retevmo) is a highly selective rearranged during transfection (RET) kinase inhibitor indicated for treatment of solid tumors with RET gene mutation or fusion, such as medullary thyroid carcinoma or non-small cell lung carcinoma. Among selpercatinib’s side effects, severe hepatotoxicity was only seen in 3% of the patients. Here we report a case of selpercatinib-induced hepatotoxicity, which has not been well characterized before. Methods/Case Report The patient was a 56-year-old female who had medullary thyroid carcinoma with RET M918T mutation (pT3aN1b) status post total thyroidectomy. Recurrence occurred and selpercatinib treatment was initiated. Then the patient complained of mouth sores, tongue sensitivity, and lower extremity cramping. The dosage was reduced to half. The magnetic resonance imaging on day 67 of selpercatinib treatment showed heterogeneous enhancement in the right hepatic lobe, compatible with parenchymal liver disease/hepatitis, trace perihepatic ascites, and mild splenomegaly. On day 71, liver function abnormalities were detected (ALK 315 IU/L, ALT 745 IU/L, AST 899 IU/L, total bilirubin 7.7 mg/dL). Workups including autoimmune hepatitis serology, viral hepatitis, or other infections were all negative. She had no history of other drug use. On day 72, a liver biopsy was performed that demonstrated mild to moderate portal lymphocyte-predominant mixed inflammation with interface activity. The lobules showed marked inflammation with hepatocyte disorganization, acidophil bodies, hepatocyte dropout, and rare lipogranulomas. Bile duct injury and patchy canalicular cholestasis were also noted. Trichrome stain showed focal bridging fibrosis. Overall, the histologic findings were acute and chronic hepatitis pattern injury, favor selpercatinib-induced hepatotoxicity. Selpercatinib was discontinued. All liver enzymes gradually returned to normal levels on day 169. Results (if a Case Study enter NA) NA Conclusion Our study suggests that screening patients for abnormal liver function tests prior to and monitoring periodically during selpercatinib treatment is necessary. Based on the severity of hepatotoxicity, withholding, reducing the dose, or permanently discontinuing selpercatinib is recommended.

Menthol induces apoptosis and inhibits proliferation and migration of nonsmall cell lung carcinoma in vitro and in vivo through Akt pathway.

About 40% of nonsmall cell lung cancers (NSCLCs) have already progressed in an advanced stage at the time of diagnosis. Development of effective prevention and therapy approaches against NSCLC is critical for reducing mortality. As a fundamental ingredient of peppermint oil, menthol has been demonstrated to possess an antitumor activity in several types of carcinomas. However, the potential role of menthol on NSCLC has not been reported. The present study aims to investigate the effect and underlying mechanism of menthol on proliferation, apoptosis, and mobility of human lung adenocarcinoma. Cell apoptosis was examined by MTT and flow cytometry. The motility of cells was determined by Transwell assay. Western blot analysis was performed to determine expression level of proteins. In vivo model of nude mice was established for evaluating the influence of menthol on tumorigenicity of A549 cells. The expression lentiviral vector of Akt was established in NSCLC cells for further verifying the inhibiting effect of menthol on survival and mobility of NSCLC cells via Akt pathway. The results showed that menthol promoted A549 cell apoptosis, suppressed cell proliferation, and motility by altering the phosphorylated protein level of Akt. Menthol enhanced the expression level of Bax while decreasing expression of Bcl-2, Caspase-3, and MMPs proteins. In vivo experiments suggested that menthol exhibited an inhibitory effect in tumor growth on xenografts. These results were further validated in Akt over-expressed A549 and H1299 cells. Menthol could display an inhibitory effect on NSCLC cells through Akt signaling pathway, making it a potential target for NSCLC treatment.