Lung Cancer Biopsies Research Articles

Unveiling the influence of tumor and immune signatures on immune checkpoint therapy in advanced lung cancer.

This study investigates the variability among patients with non-small cell lung cancer (NSCLC) in their responses to immune checkpoint inhibitors (ICIs). Recognizing that patients with advanced-stage NSCLC rarely qualify for surgical interventions, it becomes crucial to identify biomarkers that influence responses to ICI therapy. We conducted an analysis of single-cell transcriptomes from 33 lung cancer biopsy samples, with a particular focus on 14 core samples taken before the initiation of palliative ICI treatment. Our objective was to link tumor and immune cell profiles with patient responses to ICI. We discovered that ICI non-responders exhibited a higher presence of CD4+ regulatory T cells, resident memory T cells, and TH17 cells. This contrasts with the diverse activated CD8+ T cells found in responders. Furthermore, tumor cells in non-responders frequently showed heightened transcriptional activity in the NF-kB and STAT3 pathways, suggesting a potential inherent resistance to ICI therapy. Through the integration of immune cell profiles and tumor molecular signatures, we achieved an discriminative power (area under the curve [AUC]) exceeding 95% in identifying patient responses to ICI treatment. These results underscore the crucial importance of the interplay between tumor and immune microenvironment, including within metastatic sites, in affecting the effectiveness of ICIs in NSCLC.

Unveiling the influence of tumor and immune signatures on immune checkpoint therapy in advanced lung cancer

This study investigates the variability among patients with non-small cell lung cancer (NSCLC) in their responses to immune checkpoint inhibitors (ICIs). Recognizing that patients with advanced-stage NSCLC rarely qualify for surgical interventions, it becomes crucial to identify biomarkers that influence responses to ICI therapy. We conducted an analysis of single-cell transcriptomes from 33 lung cancer biopsy samples, with a particular focus on 14 core samples taken before the initiation of palliative ICI treatment. Our objective was to link tumor and immune cell profiles with patient responses to ICI. We discovered that ICI non-responders exhibited a higher presence of CD4+ regulatory T cells, resident memory T cells, and TH17 cells. This contrasts with the diverse activated CD8+ T cells found in responders. Furthermore, tumor cells in non-responders frequently showed heightened transcriptional activity in the NF-kB and STAT3 pathways, suggesting a potential inherent resistance to ICI therapy. Through the integration of immune cell profiles and tumor molecular signatures, we achieved an discriminative power (area under the curve [AUC]) exceeding 95% in identifying patient responses to ICI treatment. These results underscore the crucial importance of the interplay between tumor and immune microenvironment, including within metastatic sites, in affecting the effectiveness of ICIs in NSCLC.

Characterizing dynamic tumor-immune interactions in lung adenocarcinoma through orthotopic allograft modeling.

The major clinical challenge in lung cancer immunotherapy is drug resistance. Therefore, establishing efficient orthotopic lung cancer mouse models to explore the mechanisms of drug immunotherapy resistance is highly important. In this study, we generated multiple fluorescently labeled lung adenocarcinoma cell lines from agenetically engineered KPZ mice model. Orthotopic transplantation of the primary 1F3 cell line induced a strong immune response, causing many small tumors to disappear, but some tumors evaded the immune attack and eventually formed large tumors. Tumor microenvironment analysis demonstrated that M2 macrophages play key roles in the immune response. Further mechanistic studies revealed that the chemokine CCL7 promoted the infiltration of M2 macrophages to facilitate immune escape, thereby promoting tumor growth in the orthotopic mouse model. Moreover, CCL7 levels were elevated in human lung cancer biopsies and positively correlated with M2 macrophage infiltration, and high CCL7 levels predicted advanced pathological stage and poor survival in lung cancer patients. Overall, we established a visualized and orthotopic mouse model with fluorescently labeled cells to better dissect the tumor microenvironment of lung cancer and define the critical role of CCL7 in promoting M2 macrophage polarization and tumorigenesis, providing new preclinical tools and potential targets for lung cancer immunotherapy.

Liquid Biopsy in Lung Cancer: Nano–Flow Cytometry Detection of Non–Small Cell Lung Cancer in Blood

Non–small cell lung cancer (NSCLC) remains a leading cause of global mortality, with current screening and diagnostic methods often lacking in sensitivity and specificity. In our endeavor to develop precise, objective, and easily accessible diagnostic biomarkers for NSCLC, this study aimed to leverage rapidly evolving liquid biopsy techniques in the field of pathology to differentiate NSCLC patients from healthy controls by isolating peripheral blood samples and enriching extracellular vesicles (EVs) containing lung-derived proteins (thyroid transcription factor-1 [TTF-1] and surfactant protein B [SFTPB]), along with the cancer-associated protein CD151+ EVs. Additionally, for practical applications, we established a nano–flow cytometry assay to detect plasma EVs readily. NSCLC patients demonstrated significantly reduced counts of TTF-1+ EVs and CD151+ EVs in plasma compared with healthy controls (P < .0001), whereas SFTPB+ EVs showed no significant difference (P > .05). Integrated analysis of TTF-1+, CD151+, and SFTPB+ EVs yielded an area under the curve values of 0.913 and 0.854 in the discovery and validation cohorts, respectively. Thus, although further validation is essential, the newly developed technologies are of great significance for the robust detection of NSCLC biomarkers.

P3.06D.08 ctDNA-NGS Assay (Liquid Biopsy) for Non-Small Cell Lung Cancer: Utility of Plasma Cell-Free DNA and Tissue NGS in Detecting Genetic Mutations

P3.06D.08 ctDNA-NGS Assay (Liquid Biopsy) for Non-Small Cell Lung Cancer: Utility of Plasma Cell-Free DNA and Tissue NGS in Detecting Genetic Mutations

Clinical utility of upfront liquid biopsy in non-small cell lung cancer in the community setting.

316 Background: Molecular diagnosis of advanced non-small cell lung cancer (NSCLC) is critical to guide therapeutic decisions. Blood next generation sequencing (NGS) allows for non-invasive screening for targetable genetic abnormalities. Many studies have validated using the liquid biopsies to screen for resistant mutations at the emergence of treatment failure. However, the clinical utility of liquid biopsy for newly diagnosed NSCLC, as part of a comprehensive molecular profiling, is an area of active research. Methods: Retrospective analysis of the molecular landscape and clinical outcomes in NSCLC patients who had liquid biopsy done between December of 2017 to February of 2022. Patients had EGFR, ALK, ROS-1, and BRAF tested in the tissue, and underwent liquid biopsy for broad molecular profiling. The plasma genotyping was conducted using a 73-gene panel by Guardant 360 test. Actionable mutations were stratified accordingly to the OncoKB database based on the level of preclinical and clinical evidence of drug targetability. Survival analysis was conducted by August of 2023. Results: We enrolled 122 NSCLC patients who 84% were treatment-naïve and 78% had the liquid biopsy performed at the initial diagnosis. We found that 86 patients had oncogenic mutations and 43 (35%) had actionable mutations, with 37 (29%) falling under level 1 of evidence mutations. EGFR and KRAS G12C mutations were the two most common level 1 genetic abnormalities (23% and 11% of all actionable mutations, respectively). We identified two germline BRCA carriers who were referred for genetic counselling. 25 patients (20%) received treatment based on the liquid biopsy results, with 15 (12%) achieving partial response. Six patients had liquid biopsy done at progression of EGFR or MET therapies, which identified a resistance mechanism in three of them. Conclusions: Upfront liquid biopsy identified a significant proportion of patients with targetable mutations. Here, we reported actionable mutations in 35% of our patients who 84% had no prior systemic treatment. Our results complement prior literature and shows that blood next generation sequencing can be a useful tool to guide upfront therapeutical options at the community healthcare setting.

Length-scale study in deep learning prediction for non-small cell lung cancer brain metastasis

Deep learning-assisted digital pathology has demonstrated the potential to profoundly impact clinical practice, even surpassing human pathologists in performance. However, as deep neural network (DNN) architectures grow in size and complexity, their explainability decreases, posing challenges in interpreting pathology features for broader clinical insights into physiological diseases. To better assess the interpretability of digital microscopic images and guide future microscopic system design, we developed a novel method to study the predictive feature length-scale that underpins a DNN’s predictive power. We applied this method to analyze a DNN’s capability in predicting brain metastasis from early-stage non-small-cell lung cancer biopsy slides. This study quantifies DNN’s attention for brain metastasis prediction, targeting features at both the cellular scale and tissue scale in H&E-stained histological whole slide images. At the cellular scale, the predictive power of DNNs progressively increases with higher resolution and significantly decreases when the resolvable feature length exceeds 5 microns. Additionally, DNN uses more macro-scale features associated with tissue architecture and is optimized when assessing visual fields greater than 41 microns. Our study computes the length-scale requirements for optimal DNN learning on digital whole-slide microscopic images, holding the promise to guide future optical microscope designs in pathology applications and facilitating downstream deep learning analysis.

Nanoscale Extracellular Vesicle-Enabled Liquid Biopsy: Advances and Challenges for Lung Cancer Detection.

Lung cancer is responsible for the death of over a million people worldwide every year. With its high mortality rate and exponentially growing number of new cases, lung cancer is a major threat to public health. The high mortality and poor survival rates of lung cancer patients can be attributed to its stealth progression and late diagnosis. For a long time, intrusive tissue biopsy has been considered the gold standard for lung cancer diagnosis and subtyping; however, the intrinsic limitations of tissue biopsy cannot be overlooked. In addition to being invasive and costly, it also suffers from limitations in sensitivity and specificity, is not suitable for repeated sampling, provides restricted information about the tumor and its molecular landscape, and is inaccessible in several cases. To cope with this, advancements in diagnostic technologies, such as liquid biopsy, have shown great prospects. Liquid biopsy is an innovative non-invasive approach in which cancer-related components called biomarkers are detected in body fluids, such as blood, urine, saliva and others. It offers a less invasive alternative with the potential for applications such as routine screening, predicting treatment outcomes, evaluating treatment effectiveness, detecting residual disease, or disease recurrence. A large number of research articles have indicated extracellular vesicles (EVs) as ideal biomarkers for liquid biopsy. EVs are a heterogeneous collection of membranous nanoparticles with diverse sizes, contents, and surface markers. EVs play a critical role in pathophysiological states and have gained prominence as diagnostic and prognostic biomarkers for multiple diseases, including lung cancer. In this review, we provide a detailed overview of the potential of EV-based liquid biopsy for lung cancer. Moreover, it highlights the strengths and weaknesses of various contemporary techniques for EV isolation and analysis in addition to the challenges that need to be addressed to ensure the widespread clinical application of EV-based liquid biopsies for lung cancer. In summary, EV-based liquid biopsies present interesting opportunities for the development of novel diagnostic and prognostic platforms for lung cancer, one of the most abundant cancers responsible for millions of cancer-related deaths worldwide.

The probability of implantation metastasis after peripheral lung cancer biopsy.

At present, it is known that there is a possibility of pleural cavity and needle tract implantation metastasis after lung cancer puncture biopsy, but clinicians have not paid attention to this phenomenon, and the probability of occurrence is unknown. In this study, we aimed to study the probability of implantation metastasis after peripheral lung cancer biopsy. The intraoperative isolated completely collapsed fresh intact human lung lobes of 30 patients with peripheral lung cancer were taken, and the tumor body was punctured. The pleural pinholes and puncture needle tips were repeatedly rinsed with normal saline respectively. The flushing solution was prepared as inoculum, and then the inoculum was smeared for microscopic examination to find cancer cells. The inoculum was inoculated subcutaneously into nude mice, and then the probability of pleural cavity and needle tract implantation metastasis after lung biopsy was indirectly obtained by obtaining the nude mice tumorigenesis rate. The tumorigenesis rate of nude mice in the pleural pinholes group was 3.3%, and the tumorigenesis rate of nude mice in the puncture needle tip group was 3.3%. Patients with lung nodules suspected of being early stage cancer and who are fit for surgical resection may benefit from forgoing lung biopsies to avoid pleural cavity or needle tract seeding with tumor cells.

Recent progress of exosomal lncRNA/circRNA-miRNA-mRNA axis in lung cancer: implication for clinical application.

Lung cancer is the leading cause of death among malignant tumors in the world. High lung cancer mortality rate is due to most of patients diagnosed at advanced stage. The Liquid biopsy of lung cancer have received recent interest for early diagnosis. One of the components of liquid biopsy is the exosome. The exosome cargos non-coding-RNAs, especially long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), and microRNAs (miRNAs). The lung cancer derived exosomal non-coding RNAs play the pivotal roles of lung cancer in carcinogenesis, diagnosis, therapy, drug resistance and prognosis of lung cancer. Given ceRNA (competitive endogenous RNA) mechanism, lncRNA or circRNA can act as ceRNA to compete to bind miRNAs and alter the expression of the targeted mRNA, contributing to the development and progression of lung cancer. The current research progress of the roles of the exosomal non-coding-RNAs and the interplay of ceRNAs and miRNAs in mediated lung cancer is illustrated in this article. Hence, we presented an experimentally validated lung cancer derived exosomal non-coding RNAs-regulated target gene axis from already existed evidence in lung cancer. Then LncRNA/circRNA-miRNA-mRNA axis may be a potential target for lung cancer treatment and has great potential in the diagnosis and prognosis of lung cancer.

Evaluating predictors of kinase activity of STK11 variants identified in primary human non-small cell lung cancers.

Critical evaluation of computational tools for predicting variant effects is important considering their increased use in disease diagnosis and driving molecular discoveries. In the sixth edition of the Critical Assessment of Genome Interpretation (CAGI) challenge, a dataset of 28 STK11 rare variants (27 missense, 1 single amino acid deletion), identified in primary non-small cell lung cancer biopsies, was experimentally assayed to characterize computational methods from four participating teams and five publicly available tools. Predictors demonstrated a high level of performance on key evaluation metrics, measuring correlation with the assay outputs and separating loss-of-function (LoF) variants from wildtype-like (WT-like) variants. The best participant model, 3Cnet, performed competitively with well-known tools. Unique to this challenge was that the functional data was generated with both biological and technical replicates, thus allowing the assessors to realistically establish maximum predictive performance based on experimental variability. Three out of the five publicly available tools and 3Cnet approached the performance of the assay replicates in separating LoF variants from WT-like variants. Surprisingly, REVEL, an often-used model, achieved a comparable correlation with the real-valued assay output as that seen for the experimental replicates. Performing variant interpretation by combining the new functional evidence with computational and population data evidence led to 16 new variants receiving a clinically actionable classification of likely pathogenic (LP) or likely benign (LB). Overall, the STK11 challenge highlights the utility of variant effect predictors in biomedical sciences and provides encouraging results for driving research in the field of computational genome interpretation.

Overview of the Role of Liquid Biopsy in Non-small Cell Lung Cancer (NSCLC)

Solid tumour tissue has traditionally been used for cancer molecular diagnostics. Recently, biomarker assessment in blood or liquid biopsies has become relevant because it allows genotyping in a less invasive and costly manner. In addition, it is a very useful technique in cases with insufficient tumour samples.Recent data have shown that this method can provide the baseline molecular characteristics of the tumour and resistance changes that emerge during cancer treatment. In terms of diagnostic application, the platforms available for clinical use in lung cancer focus on the isolation and detection of circulating DNA (ctDNA) and generally cover a limited number of mutations in genes such as epidermal growth factor receptor (EGFR), Kirsten rat sarcoma viral oncogene homolog (KRAS) and BRAF, as well as anaplastic lymphoma kinase (ALK) rearrangements. In parallel, there are plasma genotyping platforms based on next-generation sequencing (NGS) techniques, which are much broader in scope, allowing multiple genes to be studied simultaneously in a more efficient manner.More recently, promising research scenarios for liquid biopsy have emerged, such as its utility for early diagnosis and evaluation of minimal residual disease after oncological treatment.In light of these advances, knowledge of the benefits and limitations of liquid biopsy, as well as awareness of emerging information on new indications for this technique in non-small cell lung cancer (NSCLC), are of paramount importance in developing more effective management strategies for patients with this neoplasm.

Differences in microenvironment of lung cancer and pleural effusions by single-cell RNA sequencing

BackgroundDirect comparison of tumor microenvironment of matched lung cancer biopsies and pleural effusions (PE) from the same patients is critical in understanding tumor biology but has not been performed. This is the first study to compare the lung cancer and PE microenvironment by single-cell RNA sequencing (scRNA-seq). MethodsMatched lung cancer biopsies and PE were obtained prospectively from ten patients. We isolated CD45+ cells and performed scRNA-seq to compare the biopsies and PE. ResultsPE had a higher proportion of CD4+ T cells but lower proportion of CD8+ T cells (False detection rate, FDR = 0.0003) compared to biopsies. There was a higher proportion of naïve CD4+ T cells (FDR = 0.04) and naïve CD8+ T cells (FDR = 0.0008) in PE vs. biopsies. On the other hand, there was a higher proportion of Tregs (FDR = 0.04), effector CD8+ (FDR = 0.006), and exhausted CD8+ T cells (FDR = 0.01) in biopsies. The expression of inflammatory genes in T cells was increased in biopsies vs. PE, including TNF, IFN-ɣ, IL-1R1, IL-1R2, IL-2, IL-12RB2, IL-18R1, and IL-18RAP (FDR = 0.009, 0.013, 0.029, 0.043, 0.009, 0.013, 0.004, and 0.003, respectively). The gene expression of exhaustion markers in T cells was also increased in tumor biopsies including PDCD1, CTLA4, LAG 3, HAVCR2, TIGIT, and CD160 (FDR = 0.008, 0.003, 0.002, 0.011, 0.006, and 0.049, respectively). ConclusionsThere is a higher proportion of naïve T cells and lower proportion of exhausted T cells and Tregs in PE compared to lung cancer biopsies, which can be leveraged for prognostic and therapeutic applications.

Exploring the potential of multiomics liquid biopsy testing in the clinical setting of lung cancer.

The transformative role of artificial intelligence (AI) and multiomics could enhance the diagnostic and prognostic capabilities of liquid biopsy (LB) for lung cancer (LC). Despite advances, the transition from tissue biopsies to more sophisticated, non-invasive methods like LB has been impeded by challenges such as the heterogeneity of biomarkers and the low concentration of tumour-related analytes. The advent of multiomics - enabled by deep learning algorithms - offers a solution by allowing the simultaneous analysis of various analytes across multiple biological fluids, presenting a paradigm shift in cancer diagnostics. Through multi-marker, multi-analyte and multi-source approaches, this review showcases how AI and multiomics are identifying clinically valuable biomarker combinations that correlate with patients' health statuses. However, the path towards clinical implementation is fraught with challenges, including study reproducibility and lack of methodological standardization, thus necessitating urgent solutions to solve these common issues.

Impact and Durability of the Affordable Care Act Medicaid Expansion on Lung Cancer Treatment

BackgroundMedicaid expansion began in 2014 after passage of the Affordable Care Act; however, the impact and durability of the effects on lung cancer treatment utilization are poorly defined. We aimed to determine whether there is a persistent difference in utilization of lung resection, lung biopsy, and nonoperative treatment of lung cancer in states participating in Medicaid expansion compared with states that are not. MethodsA retrospective cohort study was completed analyzing the difference in utilization between Medicaid expansion states and non-expansion states in 2012-2013, 2016-2017, and 2019. Patients diagnosed with and treated for lung cancer in the states of North Carolina and Florida (non-expansion states) and Maryland and New Jersey (expansion states) were included. A difference-in-difference (DID) analysis was used. ResultsIn the immediate postexpansion period (2016-2017), DID analysis revealed increased utilization in expansion states with an adjusted DID of 0.50 lung resections/100,000 persons (P = .002) and an adjusted DID of 0.76 lung biopsies/100,000 persons (P = .001). A persistent increase in utilization was found in the delayed postexpansion period (2019), with an adjusted DID of 0.51 lung resections/100,000 persons (P = .008) and an adjusted DID of 0.84 lung biopsies/100,000 persons (P = .021). No significant difference between groups was observed in the utilization of stereotactic body radiation therapy or chemotherapy. ConclusionsIn our cohort, Medicaid expansion was associated with increased utilization of procedural care for the management of lung cancer, including percutaneous biopsies and surgical resection.

Endobronchial Ultrasound Guided Transbronchial Needle Aspiration and PD-L1 Yields

PurposeImmunotherapy is a leading approach for treating advanced non-small cell lung cancer (NSCLC) by targeting the PD-1/PD-L1 checkpoint signaling pathway, particularly in tumors expressing high levels of PD-L1 (Jug et al. in J Am Soc Cytopathol 9:485–493, 2020; Perrotta et al. in Chest 158: 1230–1239, 2020). Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a minimally invasive method to obtain tissue for molecular studies, including PD-L1 analysis, in unresectable tumors (Genova et al. in Front Immunol 12: 799455, 2021; Wang et al. in Ann Oncol 29: 1417–1422, 2018). This study aimed to assess the adequacy of PD-L1 assessment in EBUS-TBNA cytology specimens.MethodsData was collected retrospectively from patients who underwent EBUS-TBNA between 2017 and 2021 for suspected lung cancer biopsy. Samples positive for NSCLC were examined for PD-L1 expression. EBUS was performed by experienced practitioners, following institutional guidelines of a minimum of five aspirations from positively identified lesions. Sample adequacy for molecular testing was determined by the pathology department.ResultsThe analysis involved 387 NSCLC cases (149 squamous cell, 191 adenocarcinoma, 47 unspecified). Of the 263 EBUS-TBNA specimens tested for PD-L1, 237 (90.1%) were deemed adequate. While 84% adhered to the protocol, adherence did not yield better results. Significantly higher PD-L1 adequacy was observed in squamous cell carcinomas (93.2%) compared to adenocarcinoma (87.6%). The number of aspirations and sedation type did not correlate with PD-L1 adequacy in either cancer type, but lesion size and location had a significant impact in adenocarcinomas. Adenocarcinoma exhibited higher PD-L1 expression (68%) compared to squamous cell carcinoma (48%).ConclusionEBUS-TBNA offers high yields for assessing immunotherapy markers like PD-L1, with satisfactory adequacy regardless of NSCLC subtype, lesion size, or location.

The Impact of Rapid On-site Evaluation on Diagnostic Performance of Computed Tomography-Guided Core Needle Biopsy in Lung Cancer.

Rapid on-site-evaluation (ROSE) is a technique aimed at improving the diagnostic performance of computed tomography (CT)-guided core needle biopsy (CNB) in lung cancer. The aim of this retrospective study was to investigate the impact of ROSE on the rate of nondiagnostic specimens and on accuracy computed on diagnostic specimens. During a 3-year period, 417 CT-guided CNBs were performed at our center. The biopsies were retrospectively classified into 2 groups: 141 procedures were assisted by ROSE and 276 were not. All of them were reviewed for clinical, procedural, and pathological data. Pathology results were classified as diagnostic (positive or negative for malignancy) or nondiagnostic. The results were compared with the final diagnosis after surgery or clinical follow-up. Nondiagnostic rate, sensitivity/specificity/negative predictive value/positive predictive value for the ROSE and non-ROSE groups were calculated. Finally, procedural complications and the adequacy of the specimens for the molecular analysis were recorded. The study evaluated 417 CNBs (mean patients' age 71 years, 278 men). Nondiagnostic rates with and without ROSE were 4% (6/142) and 11% (29/276), respectively ( P = 0.028). Sensitivity/specificity/negative predictive value/positive predictive value with and without ROSE did not show statistically significant differences, and no difference in major/minor complication rates was observed between the 2 groups. The adequacy of specimen for subsequent molecular analysis was 100% with (42/42) and 82% without ROSE (51/62). Rapid on-site-evaluation reduced the rate of nondiagnostic specimens by 50% with no change in complication rates or accuracy and increased by 20% the chances of a successful subsequent molecular analysis.

Abstract 349: The role of RELTfms and FLNA in human cancer and cellular apoptosis

Abstract Receptor Expressed in Lymphoid Tissues (RELT) is a Tumor Necrosis Factor Receptor family member implicated in several cancers. RELL1 and RELL2 (RELT-Like 1 and 2) are RELT homologs that bind RELT; the three proteins are collectively referred to as RELT family members (RELTfms). We sought to evaluate the role of the OXSR1 kinase and actin-binding protein Filamin A (FLNA) in modulating RELT-induced apoptosis of cancer cells. We also assessed RELTfm expression in human breast and lung cancer biopsies and their ability to sensitize breast cancer cells to chemotherapeutic agents. Methods: Empty vector or expression plasmids for RELTfms were transiently transfected into MDA-MB-231 (231) breast cancer cells with TransfeX reagent. Cell death was assessed by morphology assays and ATP luciferase assays. Apoptosis of 231 cells was assessed by Annexin V/PI staining using Flow cytometry (FC). Immunohistochemistry (IHC) was used to assess RELT expression in human breast and lung cancer biopsies. IHC and western blotting were utilized to determine RELT cellular localization. Results: All three RELLfms were co-immunoprecipitated by a C-terminal fragment of FLNA. RELT overexpression induced death in 231 cells, and this effect was increased with co-transfection of a FLNA S215A mutant, an important phosphorylation site that protects FLNA from cleavage. 231s transfected with RELT and RELL2 demonstrated increased Annexin V/PI signal in comparison to vector control, demonstrating that these RELTfms induce death via an apoptotic pathway. Co-transfection of a mutated plasmid for the OXSR1 kinase did not abrogate RELT-induced apoptosis. A RELT construct with a mutated binding site for the OXSR1 kinase induced apoptosis to a similar extent as wild-type RELT. An increase in doxorubicin-induced apoptosis was observed when 231 cells were transfected with RELL2 versus vector control. IHC results showed increased RELT expression in malignant breast cancer biopsies compared to patient-matched benign tissue. Interestingly, RELT was localized in the nucleus of some malignant lung cancer biopsies, and western blot analysis indicates that RELT translocates to the nucleus and interacts with chromatin in 231 cells. Conclusions: Collectively, these results demonstrate that RELTfms induce apoptosis of breast cancer cells, and potentially increase the sensitivity of breast cancer cells to chemotherapeutic agents. Furthermore, this work enhances our understanding of the interaction between RELT and FLN, a protein that is implicated in many cancers, including breast cancer. Phosphorylation by OXSR1 was previously shown to be required for RELT to activate the p38 pathway, yet the OXSR1 kinase is not required for RELT-induced death. RELT expression is enhanced in breast cancer biopsies, and interestingly, RELT can localize to the nucleus in cancerous cells. Collectively, these findings significantly enhance our understanding of RELTfms and cancer. Citation Format: Emily Chou, Esther Chang, Ihab Abed, Ashley Christensen, Kailey Nguyen, Anusri Yanumula, Yennie Shyu, Alyssa Abram, Jessa Alcaide, Gianne Cusick, Maryann Batiste, Yihui Shi, Eslam Mohammed, John Cusick. The role of RELTfms and FLNA in human cancer and cellular apoptosis [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 349.

Abstract 7490: Clinical utility of liquid biopsy in early stage lung cancer: A single institution experience

Abstract INTRODUCTION: Liquid biopsy (LB) proved its clinical utility in advanced non-small cell lung cancer, but its role in early-stage disease is less clear. Here, we investigated the clinical utility of LB for molecular diagnosis in early-stage lung cancer and its correlation with cancer relapse. METHODS: This retrospective, monocentric study collected results from LB performed in patients (pts) with an early-stage lung cancer between January 2021 and May 2023, within the prospective STING study (NCT04932525). LBs were executed at different time points (before, after curative treatment, or under treatment), by using the Foundation One Liquid Dx panel. All patients had tissue molecular testing for at least EGFR, ALK and KRAS alterations. LB was considered positive (ctDNA+) if ≥ 1cancer-related alteration was identified and negative (ctDNA-) in the absence of somatic alterations or the presence of clonal hematopoiesis (CH) only. Disease-free survival (DFS) was calculated from diagnosis to cancer relapse or last follow-up, and correlated with LB results. RESULTS: A total of 52 pts were included and 52 LBs were conducted. Pts had a median age of 63 years, 40% (25/52) were females, and 84% (43/51) had a smoking history with a median pack-years of 37.5 [4-100]. Adenocarcinoma was the most common histology (48% 25/52), followed by squamous carcinoma (23% 12/52), undifferentiated (20% 10/52) and other hystologies (9% 5/52). Patients had stage I disease in 10% (5/52), stage II in 11% (6/52) and stage III in 79% (41/52). LB was performed before curative treatment in 35/52 (67%), after curative treatment in 6/52 (11.5%) and during curative treatment in 12/52 (23%). Overall, LB was positive in 57% (30/52) of cases: 63% (26/41) in stage III and 36% (4/11) in stage I/II. Major driver mutations identified in tissue were then found in 61.5% (8/13) of LBs. The concordance between LB and tissue findings was 100% in pts with ctDNA+ (7 KRAS, 4 EGFR, 1 ALK, 1 HER2 ampl). CH was present in 66.7% (20/30) of ctDNA+ LB and 47.8% (11/23) of ctDNA- LB. Median tumor mutational burden (TMB) in ctDNA+ LB was 5.5 mut/Mb, with 4 cases of TMB &amp;gt;16. Median follow-up was 12.6 months (mo). In pts with ctDNA+ versus ctDNA- LB, mDFS was 12.9 mo (95% 6.3-18.6) versus 27.7 mo (95%CI 12.7- NR), p=0.07, in the overall population and 12.9 mo (95%CI 7.8-19.2) versus 27.2 mo (12.7-NR) respectively, p=0.19, in pts with stage III disease. CONCLUSIONS: LB has informal results for molecular diagnosis in nearly half of pts with an early-stage lung cancer. The high frequency of CH may lead to LB misinterpretation as false positive and requires caution. The potential association of positive LB with earlier relapse requires further investigation in larger cohorts with a longer follow-up. Results will be updated by the time of meeting. Citation Format: Kristi Beshiri, Arianna Marinello, Damien Vasseur, Lodovica Zullo, Maria Virginia Sanchez Becerra, Arianna Pagliaro, Massimiliano Cani, Claudia Parisi, Pernelle Lavaud, Maxime Frelaut, Pamela Abdayem, Anas Gazzah, Angela Botticella, Antonin Levy, Cecile Le Pechoux, Olaf Mercier, Santiago Ponce, Fabrice Barlesi, David Planchard, Antonio Italiano, Benjamin Besse, Jordie Remon, Mihaela Aldea. Clinical utility of liquid biopsy in early stage lung cancer: A single institution experience [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 7490.

Detection of EGFR T790M mutation using liquid biopsy for non-small cell lung cancer: Utility of droplet digital polymerase chain reaction vs. cobas real-time polymerase chain reaction

BackgroundDigital platforms for mutation detection yield higher sensitivity than non-digital platforms but lack universal positive cut-off values that correlate with the outcome of osimertinib treatment. This study determined compared droplet digital polymerase chain reaction (ddPCR) to the standard cobas assay for epithelial growth factor receptor (EGFR) T790M mutation detection in patients with non-small cell lung cancer. MethodsStudy patients had EGFR-mutant tumours with disease progression on first/second generation EGFR tyrosine kinase inhibitors, and osimertinib treatment after T790M mutation detection. T790M status was tested by cobas assay using liquid biopsy, and only by ddPCR if an EGFR mutation was identified but T790M was negative. Clinical efficacy of osimertinib was compared between patients with T790M detected by cobas vs. only by ddPCR. A positive cut-off value for ddPCR was determined by assessing efficacy with osimertinib. Results61 patients had tumors with an acquired T790M mutation, 38 detected by cobas and an additional 23 only by ddPCR. The median progression-free survival (PFS) for the cobas- and ddPCR-positive groups was 9.5 and 7.8 months, respectively (p=0.43). For ddPCR, a fractional abundance (FA) of 0.1% was used as a cut-off value. The median PFS of patients with FA ≥0.1% and <0.1% was 8.3 and 4.6 months, respectively (p=0.08). FA ≥0.1% was independently associated with a longer PFS. ConclusionUsing ddPCR to follow up the cobas assay yielded more cases (38% of total) with a T790M mutation. A cut-off value of FA ≥0.1% identified patients who responded as well to osimertinib as those identified by cobas assay. This sequential approach should detect additional patients who might benefit from osimertinib treatment.