Non-small Cell Lung Cancer Biopsies Research Articles

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.

Liquid biopsy for breast cancer and other solid tumors: a review of recent advances.

Liquid biopsy using circulating tumor DNA (ctDNA) has been reported to be less invasive and effective for comprehensive genetic analysis of heterogeneous solid tumors, including decision-making for therapeutic strategies, predicting recurrence, and detecting genetic factors related to treatment resistance in various types of cancers. Breast cancer, colorectal cancer, and lung cancer are among the most prevalent malignancies worldwide, and clinical studies of liquid biopsy for these cancers are ongoing. Liquid biopsy has been used as a companion diagnostic tool in clinical settings, and research findings have accumulated, especially in cases of colorectal cancer after curative resection and non-small cell lung cancer (NSCLC) after curative chemoradiotherapy, in which ctDNA detection helps predict eligibility for adjuvant chemotherapy. Liquid biopsy using ctDNA shows promise across a wide range of cancer types, including breast cancer, and its clinical applications are expected to expand further through ongoing research. In this article, studies on liquid biopsy in breast cancer, colorectal cancer, and NSCLC are compared focusing on ctDNA.

Benefits of Implementing Reflex Genomic Analysis for Nonsmall Cell Lung Cancer.

Molecular biomarker analysis is standard of care in advanced nonsmall cell lung cancer (NSCLC). Pathologist-driven reflex testing protocols are one approach to initiating this analysis. Two years after insourcing genomic analysis at our institution, a reflex testing protocol for advanced NSCLC was initiated. A retrospective review of the records of 578 NSCLC biopsies was performed to assess the impact of 3 genomic testing workflows (send-out, in-house clinician-ordered, and in-house reflex) on time to initiation of molecular testing [initiation time (IT)], reporting time (RT), proportion of test failures, and test ordering practices. The proportion of test failures by test methodology was also assessed. IT was lowest for reflex protocol orders (mean weekdays: 30.0 send-out, 27.4 in-house clinician-ordered, 0.95 reflex). Test failure was highest for send-out testing (31.7% vs. 10% each for in-house clinician-ordered and reflex). RT remained consistent across the 3 workflows (mean weekdays: 11.1 send-out, 11.9 in-house clinician-ordered, and 11.4 reflex). Guideline-congruent molecular testing increased upon insourcing genomic analysis and again upon implementing reflex testing with a reduction in nonbiomarker informed care (58.8% send-out, 19.5% in-house clinician-ordered, 11.5% reflex). Implementation of reflex in-house genomic analysis for advanced NSCLC ensured consistency in RT and significantly decreased IT and proportion of test failures. Insourcing genomic analysis and thoughtful care pathway design improve equitable access to molecular biomarker analysis and mitigate nonbiomarker informed cancer care in NSCLC.

Abstract 6768: The potential predictive role of spatial phenotyping in non-small cell lung cancer

Abstract Background Lung cancers are the leading cause of cancer-related deaths with a 5-year-survival of only ~20%. Whilst immunotherapies have led to durable and prolonged survival, only a subset of patients remain responsive. Additional biomarkers are thus needed to better predict if patients will respond or develop resistance against immune checkpoint inhibitor (ICI) therapies. Spatial phenotyping of the tumor microenvironment (TME) is now recognized as a proxy for ICI therapy outcomes. Our study employs an end-to-end spatial biology strategy to survey non-small-cell lung cancer (NSCLC) tissues for new biomarkers that could guide immunotherapy treatments. Methods We phenotyped pre-treatment biopsies from non-small-cell lung cancer (NSCLC) patients treated with single-agent Nivolumab. We first performed 57-plex whole-slide Single Cell Spatial Phenotyping on the PhenoCycler®-Fusion platform. Next, we developed the spatial analysis of a larger NSCLC cohort using customizable PhenoCode Signature Panels (PSP) for high-throughput immune profile (CD3/CD8/CD20/CD68/PanCK + CD4 add-in) and immuno-contexture (CD8/CD68/PD-L1/FoxP3/PanCK + PD-1 add-in) imaging. The PSP panel content combines the barcode-based antibody chemistry from the PhenoCycler platform with the signal amplification of Opal chemistry from the PhenoImager platform. PSP panels were profiled across n=27 NSCLC biopsies and provided a wider breadth of tissue profiling. Results Our whole-slide single-cell spatial phenotyping analyses revealed high phenotypic diversity in the TME of patients responsive and resistant to ICI therapy. However, higher-throughput analyses of the same tissues using targeted PSP panels revealed no significant differences in quantities of immune cell lineages, including T-cells and macrophages. On the contrary, we discovered multiple quantifiable and statistically significant spatial signatures that appear to be predictive of treatment benefit, which confirms the potential biomarker value of spatial associations in patient tissues. Conclusions This study amounts to a uniquely comprehensive Single Cell Spatial Phenotyping analysis of pre-treatment NSCLC biopsies from a single-agent Nivolumab study. Our data catalogue the diversity in the immune microenvironment of NSCLC but highlighted that immune cell quantification is insufficient to stratify patient cohorts. Single-cell spatial phenotyping, on the other hand, promises to reveal new biomarkers that may aid in better stratification of patients in pre-treatment evaluations. Citation Format: Ning Ma, Aditya Pratapa, James Monkman, Ken O’Byrne, Oliver Braubach, Arutha Kulasinghe. The potential predictive role of spatial phenotyping in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6768.

Examination of the Functional Relationship between PD-L1 DNA Methylation and mRNA Expression in Non-Small-Cell Lung Cancer

Immunotherapy targeting the interaction between programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) is a treatment option for patients with non-small-cell lung cancer (NSCLC). The expression of PD-L1 by the NSCLC cells determines treatment effectiveness, but the relationship between PD-L1 DNA methylation and expression has not been clearly described. We investigated PD-L1 DNA methylation, mRNA expression, and protein expression in NSCLC cell lines and tumor biopsies. We used clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) to modify PD-L1 genetic contexts and endonuclease deficient Cas9 (dCas9) fusions with ten-eleven translocation methylcytosine dioxygenase 1 (TET1) and DNA (cytosine-5)-methyltransferase 3A (DNMT3A) to manipulate PD-L1 DNA methylation. In NSCLC cell lines, we identified specific PD-L1 CpG sites with methylation levels inversely correlated with PD-L1 mRNA expression. However, inducing PD-L1 mRNA expression with interferon-γ did not decrease the methylation level for these CpG sites, and using CRISPR-Cas9, we found that the CpG sites did not directly confer a negative regulation. dCas9-TET1 and dCas9-DNMT3A could induce PD-L1 hypo- and hyper-methylation, respectively, with the latter conferring a decrease in expression showing the functional impact of methylation. In NSCLC biopsies, the inverse correlation between the methylation and expression of PD-L1 was weak. We conclude that there is a regulatory link between PD-L1 DNA methylation and expression. However, since these measures are weakly associated, this study highlights the need for further research before PD-L1 DNA methylation can be implemented as a biomarker and drug target for measures to improve the effectiveness of PD-1/PD-L1 immunotherapy in NSCLC.

Matched Analyses of Brain Metastases versus Primary Non-Small Cell Lung Cancer Reveal a Unique microRNA Signature.

Distant spreading of tumor cells to the central nervous system in non-small cell lung cancer (NSCLC) occurs frequently and poses major clinical issues due to limited treatment options. RNAs displaying differential expression in brain metastasis versus primary NSCLC may explain distant tumor growth and may potentially be used as therapeutic targets. In this study, we conducted systematic microRNA expression profiling from tissue biopsies of primary NSCLC and brain metastases from 25 patients. RNA analysis was performed using the nCounter Human v3 miRNA Expression Assay, NanoString technologies, followed by differential expression analysis and in silico target gene pathway analysis. We uncovered a panel of 11 microRNAs with differential expression and excellent diagnostic performance in brain metastasis versus primary NSCLC. Five microRNAs were upregulated in brain metastasis (miR-129-2-3p, miR-124-3p, miR-219a-2-3p, miR-219a-5p, and miR-9-5p) and six microRNAs were downregulated in brain metastasis (miR-142-3p, miR-150-5p, miR-199b-5p, miR-199a-3p, miR-199b-5p, and miR-199a-5p). The differentially expressed microRNAs were predicted to converge on distinct target gene networks originating from five to twelve core target genes. In conclusion, we uncovered a unique microRNA profile linked to two target gene networks. Our results highlight the potential of specific microRNAs as biomarkers for brain metastasis in NSCLC and indicate plausible mechanistic connections.

Potential clinical utility of liquid biopsy in early-stage non-small cell lung cancer

BackgroundLiquid biopsy has been widely researched for early diagnosis, prognostication and disease monitoring in lung cancer, but there is a need to investigate its clinical utility for early-stage non-small cell lung cancer (NSCLC).MethodsWe performed a meta-analysis and systematic review to evaluate diagnostic and prognostic values of liquid biopsy for early-stage NSCLC, regarding the common biomarkers, circulating tumor cells, circulating tumor DNA (ctDNA), methylation signatures, and microRNAs. Cochrane Library, PubMed, EMBASE databases, ClinicalTrials.gov, and reference lists were searched for eligible studies since inception to 17 May 2022. Sensitivity, specificity and area under the curve (AUC) were assessed for diagnostic values. Hazard ratio (HR) with a 95% confidence interval (CI) was extracted from the recurrence-free survival (RFS) and overall survival (OS) plots for prognostic analysis. Also, potential predictive values and treatment response evaluation were further investigated.ResultsIn this meta-analysis, there were 34 studies eligible for diagnostic assessment and 21 for prognostic analysis. The estimated diagnostic values of biomarkers for early-stage NSCLC with AUCs ranged from 0.84 to 0.87. The factors TNM stage I, T1 stage, N0 stage, adenocarcinoma, young age, and nonsmoking contributed to a lower tumor burden, with a median cell-free DNA concentration of 8.64 ng/ml. For prognostic analysis, the presence of molecular residual disease (MRD) detection was a strong predictor of disease relapse (RFS, HR, 4.95; 95% CI, 3.06–8.02; p < 0.001) and inferior OS (HR, 3.93; 95% CI, 1.97–7.83; p < 0.001), with average lead time of 179 ± 74 days between molecular recurrence and radiographic progression. Predictive values analysis showed adjuvant therapy significantly benefited the RFS of MRD + patients (HR, 0.27; p < 0.001), while an opposite tendency was detected for MRD − patients (HR, 1.51; p = 0.19). For treatment response evaluation, a strong correlation between pathological response and ctDNA clearance was detected, and both were associated with longer survival after neoadjuvant therapy.ConclusionsIn conclusion, our study indicated liquid biopsy could reliably facilitate more precision and effective management of early-stage NSCLC. Improvement of liquid biopsy techniques and detection approaches and platforms is still needed, and higher-quality trials are required to provide more rigorous evidence prior to their routine clinical application.

New methodology of TMB assessment from tissue and liquid biopsy in NSCLC.

Immunotherapy has dramatically influenced and changed therapeutical approach in non-small cell lung cancer (NSCLC) in recent five years. Even though we can reach long-term response to this treatment in approximately 20% of patients with NSCLC, we are still not able to identify this cohort of patients based on predictive biomarkers. In our study we have focused on tumor mutation burden (TMB), one of the potential biomarkers which could predict effectiveness of check-point inhibitors, but has several limitations, especially in multiple approaches to TMB quantification and ununiform threshold. We determined the value of TMB in tumor tissue (tTMB) and blood (bTMB) in 20 patients with early stage NSCLC using original custom gene panel LMB_TMB1. We evaluated various possibilities of TMB calculation and concluded that TMB should be counted from both somatic non-synonymous and synonymous mutations. Considering various factors, we established cut-offs of tTMB in/excluding HLA genes as ≥22 mut/Mb and 12 mut/Mb respectively, and cut-offs of bTMB were defined as ≥21 mut/Mb and ≥5 mut/Mb, respectively. We also observed trend in correlation of somatic mutations in HLA genes with overall survival of patients.

Prevalence and breakdown of non-small cell lung cancer BRAF driver mutations in a large UK cohort

BRAF inhibitors have been shown in clinical trials to improve patient outcomes in non-small cell lung cancer (NSCLC) patients harbouring selected BRAF driver mutations with a limited side effect profile, and therefore show potential as therapeutics in clinical practice. To utilise BRAF inhibitors effectively, understanding the prevalence of BRAF mutations within the local patient population is crucial, especially since NSCLC driver mutation rates have been observed to vary in different populations around the world. We interrogated a clinical archive of next generation sequencing (NGS) data representative of 7 years of routine UK practice in the National Health Service (NHS) to investigate the frequency of BRAF mutations, the breakdown of mutation classes and co-occurrence of other oncogenic driver mutations.Tissue biopsies from NSCLC cases referred to the Sarah Cannon Molecular Diagnostics Laboratory between January 2015 and February 2022 from multiple centres across UK were included in this study. Somatic mutation hotspots in relevant cancer-associated genes were analysed using amplicon/ion-torrent based NGS assays, and all NSCLC samples which harboured recognised BRAF driver mutations were identified through a combination of automated and manual data retrieval. Data regarding any other detected mutations and basic demographic information were also collected.Over the 7-year period, 5384 NSCLC samples were sequenced, with BRAF mutation identified in 185 (3.44%) of cases. These 185 cases represented a total of 73 Class I BRAF mutations (39.5%), 61 Class II mutations (33.0%) and 51 Class III mutations (27.6%). Of the 73 identified Class I mutations, 69 (69/185, 37.3%) were V600E and four (4/185, 2.16%) were non-V600E mutations. Five V600E cases had co-mutations (5/185, 2.7%). Various other known driver mutations were also identified in these 185 tumour samples, with KRAS (18/185, 9.73%) and PIK3CA (7/185, 3.78%) occurring at the highest frequency.This is the first large cohort-level study in the UK to profile the breakdown of BRAF-positive NSCLC biopsy samples using NGS in routine clinical practice. This study defines the proportion of NSCLC patients that may be expected to benefit from BRAF inhibitors and highlights the utility of using NGS as a diagnostic tool to improve targeted therapy stratification for NSCLC patients.

Systematic study of tissue factor expression in solid tumors.

Elevated tissue factor (TF) expression, although restricted in normal tissue, has been reported in multiple solid cancers, and expression has been associated with poor prognosis. This manuscript compares TF expression across various solid tumor types via immunohistochemistry in a single study, which has not been performed previously. To increase insight in the prevalence and cellular localization of TF expression across solid cancer types, we performed a detailed and systematic analysis of TF expression in tumor tissue obtained from patients with ovarian, esophageal, bladder, cervical, endometrial, pancreatic, prostate, colon, breast, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), and glioblastoma. The spatial and temporal variation of TF expression was analyzed over time and upon disease progression in patient-matched biopsies taken at different timepoints. In addition, TF expression in patient-matched primary tumor and metastatic lesions was also analyzed. TF expression was detected via immunohistochemistry (IHC) using a validated TF-specific antibody. TF was expressed in all cancer types tested, with highest prevalence in pancreatic cancer, cervical cancer, colon cancer, glioblastoma, HNSCC, and NSCLC, and lowest in breast cancer. Staining was predominantly membranous in pancreatic, cervical, and HNSCC, and cytoplasmic in glioblastoma and bladder cancer. In general, expression was consistent between biopsies obtained from the same patient over time, although variability was observed for individual patients. NSCLC biopsies of primary tumor and matched lymph node metastases showed no clear difference in TF expression overall, although individual patient changes were observed. This study shows that TF is expressed across a broad range of solid cancer types, and expression is present upon tumor dissemination and over the course of treatment.

Abstract 1102: Fyn and Src cooperate with ERBB family to drive resistance to crizotinib in ROS1+ and ALK+ NSCLC

Abstract ALK/ROS1 TKIs have significantly improved outcomes for patients with ALK/ROS1+ lung adenocarcinoma. However, drug resistance inevitably develops, leading to disease progression. Approximately 1/3 of patients resistant to ROS1 TKIs demonstrate on-target ROS1 kinase domain (KD) mutations (Dziadziuszko et al. ESMO 2019), but the mechanism of resistance in most patients remains unknown/poorly characterized. To model and characterize acquired resistance to crizotinib, we used ROS1+ and ALK+ primary, patient tumor-derived non-small cell lung cancer (NSCLC) cell lines to generate acquired resistance models to the ALK/ROS1 inhibitor crizotinib. We used several techniques to probe the mechanisms driving resistance: DNA and RNA sequencing (seq), fluorescence in-situ hybridization (FISH), cell proliferation assays, and western blotting. The crizotinib-resistant line initially driven by a TPM3-ROS1 fusion (CUTO28-CR) displayed exquisite sensitivity to Src family kinase (SFK) TKIs in proliferation assays, which was modestly improved by addition of ERBB TKI afatinib. Bulk RNA seq revealed upregulation of SFKs Fyn and Src transcripts (141- and 2.31-fold, respectively). Interphase FISH revealed modest EGFR gene amplification in CUTO28-CR cells vs parental (EGFR:CEP7 ratio 2.1 vs 1.0), and RNA seq revealed upregulation of EGFR and HER2 transcripts (1.79- and 2.45-fold, respectively). Western blot analysis confirmed upregulation of Fyn at the protein level. CUTO28-CR cells treated with a SFK TKI revealed dose-dependent deactivation of Akt signaling, and addition of afatinib caused inhibition of MAPK signaling. Stimulation with epidermal growth factor (EGF) enhanced MAPK signaling in CUTO28-CR cells more than in parental cells, consistent with EGFR/HER2 dependence. Because of a noted propensity for the EML4-ALK line (H3122) to upregulate ERBB kinase activity under sustained ALK TKI treatment, we maintained these cells in low dose (25nM) afatinib while driving crizotinib resistance to identify additional bypass signaling pathways. These H3122-CAR cells were partially resensitized to crizotinib with the addition of SFK TKIs, and the addition of afatinib with SFK TKIs further resensitized these resistant cells to crizotinib. Bulk RNA seq of H3122-CAR cells revealed upregulation of Fyn and Src (2.64- and 3.63-fold, respectively), and western blot analysis confirmed upregulation at the protein level. In conclusion, we demonstrated increased SFK (particularly Fyn and/or Src) expression and/or activation is a mechanism utilized to drive bypass signaling, in cooperation with ERBB family kinases, to circumvent treatment with ALK/ROS1 TKI crizotinib. To our knowledge, this is the first report of SFK-driven bypass signaling as a mechanism of acquired resistance to an ALK/ROS1 TKI in NSCLC. The upregulation of Src and/or Fyn in NSCLC biopsies may guide the exploration of combination SFK TKIs with ALK/ROS1 TKIs. Citation Format: Logan C. Tyler, Nan Chen, Anh T. Le, Hala Nijmeh, Liming Bao, Robert C. Doebele. Fyn and Src cooperate with ERBB family to drive resistance to crizotinib in ROS1+ and ALK+ NSCLC [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 1102.

The utilization of artificial intelligence to produce clinically relevant scores on PD-L1 immunostained non-small cell lung cancer biopsies.

e14576 Background: Immunotherapies targeting complex programmed death-ligand 1 (PD-L1) interactions have been groundbreaking in the treatment of non-small cell lung cancer (NSCLC), offering new strategies for patients who are likely to respond. PD-L1 binds to the checkpoint PD-1 to regulate T cells, which has an important role in boosting the immune response. Identifying patients who may benefit from PD-L1 focused care is dependent on the interpretation of the drug’s associated companion diagnostic (CDx) immunohistochemistry (IHC) assay. PD-L1 IHC assays stratify NSCLC patients using a Tumor Proportion Score (TPS), a manual scoring paradigm which must be applied by a clinical pathologist. Application of the score is complex as it requires the reader to visually exclude stromal and tumor-infiltrating immune cells across a full sample, retaining PD-L1 expression information for true tumor cells only. Methods: In this study, we utilized the PD-L1 [28-8] immunohistochemistry (IHC) assay to stain 10 NCSLC specimens. Stained slides were then imaged using the AT2 scanner (Leica Biosystems, Buffalo Grove, IL) scanner and analyzed using the Visiopharm Image Analysis platform. Results: The intricacy of TPS application is time consuming for the pathologist and introduces opportunity for inter- and intra-reader variability. Our novel image analysis approach utilizes artificial intelligence (AI) to automatically denote tumor nests and exclude tumor-infiltrating immune cells. The remaining true tumor cells may then be quantified for PD-L1 expression across the full biopsy, producing a TPS for each sample. Conclusions: This method allows for great accuracy and time-efficiency in providing a TPS than traditional methods, which may result in improved patient care.

Functional assessment of somatic STK11 variants identified in primary human non-small cell lung cancers.

Serine/Threonine Kinase 11 (STK11) encodes an important tumor suppressor that is frequently mutated in lung adenocarcinoma. Clinical studies have shown that mutations in STK11 resulting in loss of function correlate with resistance to anti-PD-1 monoclonal antibody therapy in KRAS-driven non-small cell lung cancer (NSCLC), but the molecular mechanisms responsible remain unclear. Despite this uncertainty, STK11 functional status is emerging as a reliable biomarker for predicting non-response to anti-PD-1 therapy in NSCLC patients. The clinical utility of this biomarker ultimately depends upon accurate classification of STK11 variants. For nonsense variants occurring early in the STK11 coding region, this assessment is straightforward. However, rigorously demonstrating the functional impact of missense variants remains an unmet challenge. Here we present data characterizing four STK11 splice-site variants by analyzing tumor mRNA, and 28 STK11 missense variants using an in vitro kinase assay combined with a cell-based p53-dependent luciferase reporter assay. The variants we report were identified in primary human NSCLC biopsies in collaboration with the University of Vermont Genomic Medicine group. Additionally, we compare our experimental results with data from 22 in silico predictive algorithms. Our work highlights the power, utility and necessity of functional variant assessment and will aid STK11 variant curation, provide a platform to assess novel STK11 variants and help guide anti-PD-1 therapy utilization in KRAS-driven NSCLCs.

Proteogenomics of non-small cell lung cancer reveals molecular subtypes associated with specific therapeutic targets and immune evasion mechanisms.

Despite major advancements in lung cancer treatment, long-term survival is still rare, and a deeper understanding of molecular phenotypes would allow the identification of specific cancer dependencies and immune evasion mechanisms. Here we performed in-depth mass spectrometry (MS)-based proteogenomic analysis of 141 tumors representing all major histologies of non-small cell lung cancer (NSCLC). We identified six distinct proteome subtypes with striking differences in immune cell composition and subtype-specific expression of immune checkpoints. Unexpectedly, high neoantigen burden was linked to global hypomethylation and complex neoantigens mapped to genomic regions, such as endogenous retroviral elements and introns, in immune-cold subtypes. Further, we linked immune evasion with LAG3 via STK11 mutation-dependent HNF1A activation and FGL1 expression. Finally, we develop a data-independent acquisition MS-based NSCLC subtype classification method, validate it in an independent cohort of 208 NSCLC cases and demonstrate its clinical utility by analyzing an additional cohort of 84 late-stage NSCLC biopsy samples.

Circulating circTOLLIP serves as a diagnostic biomarker for liquid biopsy in non-small cell lung cancer

BackgroundCircular RNAs (CircRNAs) have been found to possess vital functions in tumorigenesis of various cancer types, including non-small cell lung cancer (NSCLC). The aim of this study was to identify and explore the diagnostic values of the newly found Toll interacting protein (TOLLIP)-derived circRNA (circTOLLIP) for liquid biopsy in NSCLC. MethodsRNase R and actinomycin D assays were conducted to confirm the existence and stability of circTOLLIP. RT-qPCR was performed to identify the expression levels of circTOLLIP in NSCLC tumor tissues, whole blood, and cell lines. The diagnostic values were evaluated by receiver operating characteristic (ROC) curve analysis. ResultsCircTOLLIP was screened as a candidate biomarker and was found to be significantly down-regulated in both NSCLC tissues and cell lines. Interestingly, circulating circTOLLIP was also lower-expressed in the whole blood of patients with NSCLC compared to that of patients with benign lung disease and healthy controls. Importantly, the circulating circTOLLIP represented better diagnostic values in comparison to the traditional tumor markers (NSE, CYFR21-1, and CA72-4), and showed higher stability even though the whole blood was exposed to various tough conditions. ConclusionsOur findings indicate that circTOLLIP can be used as a non-invasive biomarker to distinguish early-stage NSCLC from benign lung diseases and from healthy controls, suggesting the potential application of circTOLLIP for liquid biopsy in NSCLC.

Liquid Biopsy for Biomarker Testing in Non-Small Cell Lung Cancer: A European Perspective

The development of targeted therapies has improved survival rates for patients with advanced non-small cell lung cancer (NSCLC). However, tissue biopsy is unfeasible or inadequate in many patients, limiting biomarker testing and access to targeted therapies. The increasing numbers of established and emerging biomarkers with available targeted treatments highlights the challenges associated with sequential single-gene testing and limited tissue availability. Multiplex next-generation sequencing (NGS) offers an attractive alternative and represents a logical next step, and in cases where the tumour is inaccessible, tissue biopsy yields insufficient tumour content, or when the patient’s performance status does not allow a tissue biopsy, liquid biopsy can provide valuable material for molecular diagnosis. Here, we explore the role of liquid biopsy (i.e., circulating cell-free DNA analysis) in Europe. Liquid biopsies could be used as a complementary approach to increase rates of molecular diagnosis, with the ultimate aim of improving patient access to appropriate targeted therapies. Expert opinion is also provided on potential future applications of liquid biopsy in NSCLC, including for cancer prevention, detection of early stage and minimum residual disease, monitoring of response to therapy, selection of patients for immunotherapy, and monitoring of tumour evolution to enable optimal adaptation/combination of drug therapies.

Abstract 2706: Utilizing RNAscope to assess STK11-LoF-dependent transcriptional phenotypes in human NSCLC biopsies and evaluate its potential to predict anti-PD1 therapy response

Abstract Our work demonstrates RNAscope allows simultaneous quantitative assessment of multiple transcripts in situ using formalin fixed paraffin embedded (FFPE) human non-small cell lung cancer (NSCLC) biopsies and suggests this methodology could be used as a rapid surrogate biomarker for genotype/transcriptome profiling stemming for Serine Threonine Kinase 11 (STK11) loss of function (LoF). Recent clinical studies have correlated anti-PD1 therapy resistance in NSCLC with somatic disruption of STK11. Historically, rapid assessment of STK11 status using immunohistochemistry (IHC) has been a challenge. Our work in human NSCLC cell lines suggests that STK11 loss correlates with changes in tumor intrinsic cytokine expression. Several recent studies have suggested such changes may drive immune evasion. We therefore wondered whether cytokine expression profiles from primary human NSCLC biopsies might be used as a reliable biomarker to predict anti-PD1 therapy response. Here we present our findings using multiplexed RNAscope probes on KRAS-driven primary human NSCLC biopsies with and without STK11 loss. This initial work focuses on cytokines identified using our cell culture models as a proof of principle approach. Using CRISPR-Cas9 we engineered paired STK11 KO cell lines derived from 3 independent KRAS-driven human lung adenocarcinomas (NCI-H441, NCI-H1792, NCI-H2009). RNAseq and differential gene expression analysis revealed STK11-loss-dependent increases in IL-6, IL-8 and IL-32 in all three lines. We then created FFPE cell blocks using these cell lines and validated the RNAseq expression changes with multiplexed RNAscope probes in situ. Next, we informatically searched the molecular pathology archives at the UVM medical center and identified primary KRAS-driven human NSCLC biopsies with and without predicted STK11 loss. We then performed multiplexed RNAscope analysis on these samples to quantify IL-6, IL-8 and IL-32 gene expression as a function of tumor genotype in vivo. Our data suggest RNAscope represents a valid methodology to exploit quantitative differential gene expression as a reliable and reproducible surrogate biomarker of somatic STK11 disruption in NSCLC. Our future studies aim to establish whether genotype-dependent cytokine profiles predict anti-PD1 therapy response in KRAS-driven lung adenocarcinomas. Citation Format: Hailey M. Sarausky, Sean M. Lenahan, David Joseph Seward. Utilizing RNAscope to assess STK11-LoF-dependent transcriptional phenotypes in human NSCLC biopsies and evaluate its potential to predict anti-PD1 therapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2706.

Digital PCR detection of EGFR somatic mutations in non-small-cell lung cancer formalin fixed paraffin embedded samples

BackgroundDigital PCR (dPCR) is proposed to replace real time PCR and Sanger sequencing for detection and quantification of rare mutations, frequently unnoticed in the mass of tumoral cells. Screening of endothelial growth factor receptor (EGFR) mutations is mandatory before treatment with EGFR-targeted therapy with small-molecule tyrosine kinase inhibitors, which has been approved for the treatment of advanced non-small-cell lung cancer (NSCLC). ObjectiveIn order to establish a cost-effective method for detection of mutations, we optimized dPCR identification of EGFR mutations in exons 18–21, and determined dPCR sensitivity, limits of detection (LoD) and quantification (LoQ). MethodsFor clinical validation, we compared the performance of dPCR and castPCR in 57 NSCL formalin fixed paraffin embedded samples and 10 lung cancer-free formalin fixed paraffin embedded samples. ResultsEGFR mutations DEL19, p.L858R, p.G719X, p.L861Q and p.T790 M were detected by dPCR in 27 samples versus 11 detected by castPCR (p = 0.014). LoD was determined as 100 molecules of DNA/uL and LoQ as 1%. Most of the samples (87%) identified by competitive Allele-Specific TaqMan (castPCR) as wild-type and by dPCR as mutated, presented less than 10% mutated DNA molecules (mean 4.57%). Accuracy of dPCR was 94.44%, as measured with the assay recommended by the College of American Pathologists. ConclusionThese results indicated higher sensibility and specificity of dPCR for screening EGFR mutations in NSCLC biopsies, compared to castPCR.

Changes in PD-L1 tumor proportion score are associated with CD274 gene (encoding PD-L1) copy number variation in non-small cell lung cancer.

9029 Background: PD-L1 tumor proportion score (TPS) is often used to determine eligibility for first line therapy with immune checkpoint inhibitors (ICIs) in advanced non-small cell lung cancer (NSCLC). However, PD-L1 expression can vary over time and between tumor sites, potentially leading to inaccurate patient stratification. Therefore, it is critical to understand the clinicopathologic and genomic factors that are associated with PD-L1 changes in NSCLC. Methods: Clinicopathologic and genomic data were collected from patients with NSCLC and quantitative PD-L1 immunohistochemistry (IHC) on at least two different biopsies. NSCLC biopsies were categorized as PD-L1 negative, low, and high if they had a PD-L1 TPS &lt; 1%, 1-49%, and ≥50%, respectively. Intrapatient changes in PD-L1 TPS between samples (DPD-L1) were defined as follows: major decrease (decrease in PD-L1 TPS from ≥50% to &lt; 50% or from ≥1% to &lt; 1%), major increase (increase in PD-L1 TPS from &lt; 1% to ≥1% or &lt; 50% to ≥50%), and non-major change (all other cases). Next-generation sequencing (NGS) was used to evaluate copy number (CN) variations at the CD274 locus, which encodes PD-L1. Wilcoxon and Kruskal-Wallis rank sum tests were used to analyze continuous variables and Fisher’s exact test was used to analyze categorical variables. Results: Among 250 patients with NSCLC with PD-L1 IHC assays performed on at least two distinct tissue samples, PD-L1 TPS of the first biopsy was &lt; 1% in 104 (41.6%), 1-49% in 80 (32.0%), and ≥50% in 66 (26.4%) samples, for a median PD-L1 TPS of 2% (range: 0% to 100%). When intrapatient DPD-L1 was examined, there were major decreases and major increases in PD-L1 TPS in 49 (19.6%) and 65 (26.0%) cases, respectively, and non-major changes were observed in the remaining 136 samples (54.4%), with a median DPD-L1 of 0% (range: -90% to +90%). Baseline PD-L1 TPS and DPD-L1 were not significantly affected by histology, smoking status, sex, or treatment. Among 219 NSCLC samples that underwent tissue NGS and had full CN data available, the median PD-L1 TPS differed significantly based on CD274 CN: PD-L1 TPS 1% with single copy deletion vs. 5% with copy neutral vs. 42.5% with low amplification vs. 97.5% with high amplification (p &lt; 0.01). Among 56 patients with paired PD-L1 TPS and NGS on both samples, there was a significant difference in median DPD-L1 according to CD274 CN change: DPD-L1 TPS -49% with acquired CD274 CN loss vs. 0% with no major change in CD274 CN vs. +1.75% with acquired CD274 CN gain (p = 0.01). Conclusions: PD-L1 TPS varies within the same patient in almost half of NSCLC cases, with few clinicopathologic correlates of change in expression. Variation in PD-L1 TPS correlates with changes in CD274 CN across biopsies. These findings suggest a genomic correlate to predict PD-L1 TPS across samples, as well as a potential complementary method in determining in ICI initiation.

TP53-Activated lncRNA GHRLOS Regulates Cell Proliferation, Invasion, and Apoptosis of Non-Small Cell Lung Cancer by Modulating the miR-346/APC Axis.

Non-small cell lung cancer (NSCLC) is the main type of lung cancer with high mortality worldwide. To improve NSCLC therapy, the exploration of molecular mechanisms involved in NSCLC progression and identification of their potential therapy targeting is important. Long noncoding RNAs (lncRNAs) have shown important roles in regulating various tumors progression, including NSCLC. We found lncRNA GHRLOS was decreased in NSCLC cell lines and tissues which correlated with poor prognosis of NSCLC patients. However, the role and underlying mechanisms of lncRNA GHRLOS in NSCLC progression remains elusive. The expression of lncRNA GHRLOS was examined in NSCLC cell lines and biopsy specimens of patients with NSCLC by quantitative real time polymerase chain reaction (qRT-PCR). The effects of GHRLOS on proliferation, invasion and apoptosis of NSCLC cells were determined by both in vitro and in vivo experiments. The interaction between GHRLOS and TP53 was determined by dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) combined with qRT-PCR analysis. RNA immunoprecipitation (RIP) was conducted to validate the binding between GHRLOS and microRNA-346 (miR-346). Dual-luciferase reporter assays were also carried out to reveal the interaction between miR-346 and the 3’ untranslated region (3’UTR) of adenomatous polyposis coli (APC) mRNA.Our data demonstrated that overexpression of lncRNA GHRLOS suppressed cancer cell proliferation and invasion as well as promoted cell apoptosis by regulating the expression of CDK2, PCNA, E-cadherin, N-cadherin, Bax, and Bcl-2 in NSCLC cells. Moreover, lncRNA GHRLOS was upregulated by the binding of TP53 to the GHRLOS promoter. The binding target of lncRNA GHRLOS was identified to be miR-346. Impressively, overexpression of miR-346 promoted cell proliferation and invasion, as well as inhibited cell apoptosis, however, these effects can be blocked by overexpression of lncRNA GHRLOS both in vitro and in vivo. In summary, this study reveals lncRNA GHRLOS, upregulated by TP53, acts as a molecule sponge of miR-346 to cooperatively modulates expression of APC, a miR-346 target, and potentially inhibits NSCLC progression via TP53/lncRNA GHRLOS/miR-346/APC axis, which represents a novel pathway that could be useful in targeted therapy against NSCLC.