An approach for developing a blood-based screening panel for lung cancer based on clonal hematopoietic mutations.

Micro-RNAs are approximately 21-25-nucleotide-long noncoding RNAs that regulate gene expression primarily at the post-transcriptional level in animals. Here, we report that micro-RNA-1 (miR-1), abundant in the cardiac and smooth muscles, is expressed in the lung and is down-regulated in human primary lung cancer tissues and cell lines. In situ hybridization demonstrated localization of miR-1 in bronchial epithelial cells. The tumor suppressor C/EBPalpha, frequently suppressed in lung cancer, reactivated miR-1 expression in the lung cancer cells. Repressed miR-1 was also activated in lung cancer cells upon treatment with a histone deacetylase inhibitor. These observations led us to examine the antitumorigenic potential of miR-1 in lung cancer cells. Expression of miR-1 in nonexpressing A549 and H1299 cells reversed their tumorigenic properties, such as growth, replication potential, motility/migration, clonogenic survival, and tumor formation in nude mice. Exogenous miR-1 significantly reduced expression of oncogenic targets, such as MET, a receptor tyrosine kinase, and Pim-1, a Ser/Thr kinase, frequently up-regulated in lung cancer. Similarly, the levels of two additional targets, FoxP1, a transcription factor with oncogeneic property, and HDAC4 that represses differentiation-promoting genes, were reduced in miR-1-expressing cells. Conversely, depletion of miR-1 facilitated N417 cell growth with concomitant elevation of these targets. Further, ectopic miR-1 induced apoptosis in A549 cells in response to the potent anticancer drug doxorubicin. Enhanced activation of caspases 3 and 7, cleavage of their substrate PARP-1, and depletion of anti-apoptotic Mcl-1 contributed to the sensitivity of miR-1-expressing cells to doxorubicin. Thus, miR-1 has potential therapeutic application against lung cancers.

Mass spectrometry-based analyses of the low-molecular-weight fraction of serum proteome allow identifying proteome profiles (signatures) that are potentially useful in detection and diagnostics of cancer. Here we compared serum proteome profiles of healthy donors and patients with three different types of cancer aiming to identify peptide signatures that were either common for all cancer samples or specific for cancer type. Blood samples were collected before start of the therapy from patients with head and neck squamous cell cancer, colorectal adenocarcinoma and non-small cell lung cancer, and from a corresponding group of healthy volunteers. Mass profiles of the serum proteome were recorded in the range between 2 and 13 kDa using MALDI-ToF spectrometry and 131 identified peptide ions were used for statistical analyses. Similar degrees of overall similarities were observed in all intra-group and inter-group analyses when general features of serum proteome profiles were compared between individual samples. However, classifiers built of selected spectral components allowed differentiation between healthy donors and three groups of cancer patients with 69-74% sensitivity and 82-84% specificity. There were two common peptide species (3766 and 5867 Da) with increased levels in all cancer samples. Several spectral components permitted differentiation between lung cancer samples and either head and neck cancer or colorectal cancer samples, but two latter types of samples could not be properly discriminated. Abundance of spectral components that putatively corresponded to fragments of serum amyloid A (11511 and 11667 Da) was highest in lung cancer samples, yet increased levels of these peptides appeared to generally associate with more advanced cancer cases. We concluded that certain components of serum peptide signatures are common for different cancer signatures and putatively reflect general response of organism to the disease, yet other components of such signatures are more specific and most likely correspond to clinical stage of the malignancy.

Early detection provides the best opportunity for lung cancer survival; however, lung cancer is difficult to detect early because symptoms do not often appear until later stages. Current screening methods such as x-ray and computed tomographic imaging lack the sensitivity and specificity needed for effective early diagnosis. Dogs have highly developed olfactory systems and may be able to detect cancer in its primary stages. Their scent detection could be used to identify biomarkers associated with various types of lung cancer. To determine the accuracy of trained beagles' ability to use their olfactory system to differentiate the odor of the blood serum of patients with lung cancer from the blood serum of healthy controls. Over the course of 8 weeks, operant conditioning via clicker training was used to train dogs to use their olfactory system to distinguish blood serum from patients with malignant lung cancer from blood serum from healthy controls in a double-blind study. After training, non-small cell lung cancer and healthy control blood serum samples were presented to the dogs, and the sensitivity and specificity of each dog were analyzed. Four dogs were trained for the study, but 1 was unmotivated by training and removed from the study. Three dogs were able to correctly identify the cancer samples with a sensitivity of 96.7%, specificity of 97.5%, positive predictive value of 90.6%, and negative predictive value of 99.2%. Trained dogs were able to identify non-small cell lung cancer samples from healthy controls. The findings of this study provide a starting point for a larger-scale research project designed to explore the use of canine scent detection as a tool for cancer biomarkers.

Lung cancer is one of the most common cancers in the world. It is a leading cause of cancer death in men and women in the United States, cigarette smoking being leading cause of lung cancer. There are two main types of lung cancer small cell and non-small cell lung cancer (NSCLC). The most common type being NSCLC which accounts for 80-85% of all reported cases. The ability to rapidly detect / diagnose lung cancer at an early stage is critical to successful treatment selection and patient survival. The use of metabolic profiling in biomedical applications metabolic profiling is being deployed as a method for finding novel, mechanistic, biomarkers of disease with obvious potential for improving diagnosis, and patient stratification. Here we present a rapid, simple and reliable high throughput targeted LC/MS single platform, for the quantification/monitoring of small molecule metabolites, lipids and peptides. The methodology employs a single set of LC/MS conditions which facilitate TCA cycle, bile acids, biogenic amine, free fatty acids, acyl carnitines, lipids and 100 protein panel without need for user intervention. Plasma from pilot cohort of lung cancer a health control samples were evaluated using this new methodology. The methodology showed excellent reproducibility and accuracy. The peptide analysis showed that 10 peptides were shown to be markers of lung cancer, these are listed in Table 1. Acylcarnitines were quantified over a range of 5 - 625ng/mL data showed that the C14:2 tetradecadienoyl carnitine and C16:1 palmitoleoyl carnitine were elevated in lung cancer samples whereas the C8:1 octenoyl carnitine level was reduced in the lung cancer samples . Although deoxycholic acid and chendeoxycholic acid appeared to be reduced in the lung cancer samples this was determined to be not statistically relevant. A total of 29 amino acids were measured during the analysis. Using t-test with p-value FDR cutoff adjusted to 0.01 the data clearly showed that sarcosine was highly over-expressed in lung cancer samples. Table 1P02749Apolipoprotein HP19652Alpha-1-acid glycoprotein 2P68871Hemoglobin subunit betaP02671Fibrogen alpha chainP02656Apolipoprotein C-IIIP02750Leucine-rich alpha 2-glycoprotein (LRG)P00738HaptoglobinP02748Complement component C9P02763Alpha-1-acid glycoprotein 1P01011Alpha-1-antichymotrypsin Note: This abstract was not presented at the meeting. Citation Format: Robert S. Plumb, Lee Gethings, Andrew Peck. Diagnostic screen for lung cancer using high throughput metabolomics screen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 663.

BackgroundEvidence from observational studies suggests an association between chronic obstructive pulmonary disease (COPD) and lung cancer. The potential interactions between the immune system and the lungs may play a causative role in COPD and lung cancer and offer therapeutic prospects. However, the causal association and the immune-mediated mechanisms between COPD and lung cancer remain to be determined.MethodsWe employed a two-sample Mendelian randomization (MR) approach to investigate the causal association between COPD and lung cancer. Additionally, we examined whether immune cell signals were causally related to lung cancer, as well as whether COPD was causally associated with immune cell signals. Furthermore, through two-step Mendelian randomization, we investigated the mediating effects of immune cell signals in the causal association between COPD and lung cancer. Leveraging publicly available genetic data, our analysis included 468,475 individuals of European ancestry with COPD, 492,803 individuals of European ancestry with lung cancer, and 731 immune cell signatures of European ancestry. Additionally, we conducted single-cell transcriptome sequencing analysis on COPD, lung cancer, and control samples to validate our findings.FindingsWe found a causal association between COPD and lung cancer (odds ratio [OR] = 1.63, 95% confidence interval [CI] = 1.31–2.02, P-value < 0.001). We also observed a causal association between COPD and regulatory T cells (odds ratio [OR] = 1.19, 95% confidence interval [CI] = 1.01–1.40, P-value < 0.05), as well as a causal association between regulatory T cells and lung cancer (odds ratio [OR] = 1.02, 95% confidence interval [CI] = 1.002–1.045, P-value < 0.05). Furthermore, our two-step Mendelian randomization analysis demonstrated that COPD is associated with lung cancer through the mediation of regulatory T cells. These findings were further validated through single-cell sequencing analysis, confirming the mediating role of regulatory T cells in the association between COPD and lung cancer.InterpretationAs far as we are aware, we are the first to combine single-celled immune cell data with two-sample Mendelian randomization. Our analysis indicates a causal association between COPD and lung cancer, with regulatory T cells playing an intermediary role.

Simple SummaryExosomes (or extracellular vesicles) are known to mediate intercellular communication and to transmit molecular signals between cells. Molecules carried by exosomes have their own molecular roles in affecting surrounding and distant environment, as well as recipient cells. Molecular components of exosomes can be used as cancer biomarkers for diagnosis and prognosis, being promising therapeutic targets for the interruption of cellular signals. Therefore, the understanding of the molecular compositions and their functional indications of exosomes has the potential to help doctors to diagnose and monitor diseases and to allow researchers to design and develop potential targeted therapies. This review aims to provide a comprehensive protein and lipid characterization of lung cancer exosomes and to explore their molecular functions and mechanisms regulating physiological and pathological processes. This organization offers informative insight for lung cancer diagnosis and treatment.Exosomes participate in cell–cell communication by transferring molecular components between cells. Previous studies have shown that exosomal molecules derived from cancer cells and liquid biopsies can serve as biomarkers for cancer diagnosis and prognosis. The exploration of the molecules transferred by lung cancer-derived exosomes can advance the understanding of exosome-mediated signaling pathways and mechanisms. However, the molecular characterization and functional indications of exosomal proteins and lipids have not been comprehensively organized. This review thoroughly collected data concerning exosomal proteins and lipids from various lung cancer samples, including cancer cell lines and cancer patients. As potential diagnostic and prognostic biomarkers, exosomal proteins and lipids are available for clinical use in lung cancer. Potential therapeutic targets are mentioned for the future development of lung cancer therapy. Molecular functions implying their possible roles in exosome-mediated signaling are also discussed. Finally, we emphasized the importance and value of lung cancer stem cell-derived exosomes in lung cancer therapy. In summary, this review presents a comprehensive description of the protein and lipid composition and function of lung cancer-derived exosomes for lung cancer diagnosis, prognosis, and treatment.

BackgroundS‐phase kinase‐associated protein 2 (Skp2) takes great part in the development of multiple tumors. However, the post‐transcriptional modulation mechanism of Skp2 remains unclear. Here, we present a new regulatory microRNA of Skp2, miR‐339, which directly targets Skp2 to inhibit cell proliferation in lung cancer.MethodsThe expression of miR‐339 or Skp2 in lung cancer samples was tested by real time‐PCR. The correlation between miR‐339 and Skp2 in lung cancer samples was analyzed by Pearson's correlation coefficient. The effect of miR‐339 or anti‐miR‐339 on Skp2 was evaluated by immunoblotting. The luciferase reporter gene assay was used to test the targeting of miR‐339 on Skp2. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5 diphenyltetrazolium bromide and colony formation analysis were applied to examine the function of miR‐339 targeting Skp2 in lung cancer cells.ResultsThe negative correlation of miR‐339 with Skp2 was found in clinical human lung cancer tissues. Furthermore, Skp2 expression was obviously abated by miR‐339 in lung cancer A549 cells. Mechanistically, we used bioinformatics to predict that miR‐339 could target the 3′‐untranslated region of Skp2 mRNA. Luciferase reporter gene assay demonstrated that miR‐339 could decrease the luciferase activities of the 3′‐untranslated region vector of Skp2. In terms of function, ectopic miR‐339 expression significantly suppressed cell proliferation in lung cancer. Overexpressed Skp2 accelerated miR‐339‐bated proliferation of lung cancer cells. MiR‐339 inhibitor promoted cell proliferation in lung cancer, but Skp2 RNA interference reversed miR‐339 inhibitor‐driven cell proliferation.ConclusionMiR‐339 targets the 3′‐untranslated region of Skp2 mRNA to depress the proliferation of lung cancer cells.

Detection of Mutant BRAF Alleles in the Plasma of Patients with Metastatic Melanoma

BackgroundThe tumor microenvironment (TME) is composed of highly heterogeneous cell types that dynamically interact with each other. To more precisely distinguish and characterize cellular diversity and disease states within tissues,...

Objective: PD-L1 expression and Tumor Mutation Burden (TMB) have independently emerged as prospective biomarkers of response to anti PD1-/PDL1 checkpoint inhibitors. However, TMB has not fully proven its value as a biomarker of Immunotherapy response in lung cancer. Moreover, FDA-approved CDx PD-L1 expression alone is not an optimal biomarker for checkpoint inhibitors and combined use of MSI, TMB and PD-L1 protein levels has been proposed. Here we present the correlation between genomic landscape, including TMB and MSI with PDL1 IHC in lung cancer specimens to help identify immunogenomic profiles for stratification of patients for check-point inhibitors therapies evaluation. Methods: 874 FFPE clinical samples across cancer types were characterized in our CLIA/CAP accredited clinical laboratory using a CLIA-validated NGS-based assay that interrogates SNVs, indels, TMB and Microsatellite Status (27 MS markers) using a 323 gene panel. TMB (mutations/Mb) is categorized as low (≤7), intermediate (7&amp;lt;TMB≤15) and high: (greater than 15). The de-identified aggregated results paired with PD-L1 IHC data from 424 lung cancer samples were analyzed and correlations between PD-L1 tumor proportion scores (TPS) and TMB results were made. In silico analyses were also performed on 5939 lung cancer samples from public databases. Results: The detected TMB median in lung cancer was 8.6 (range 0-126.4). Median PD-L1 TPS was 7%, with 26% of samples being PD-L1 negative (TPS&amp;lt;1%), 44% Low expressing (≥1-49%) and 31% High (≥50%). All samples were MSS. We found poor correlation between PD-L1 expression and TMB in NSCLC (r2=0.266). We classified samples based on TMB and PD-L1 TPS and found mutational correlations specific to in each of the following groups: PD-L1 High/TMB Low, PD-L1 High/TMB High, PD-L1 Low/TMB High, PD-L1 High/TMB High, and PD-L1 Neg/TMB High. Of note 67% (34/51) of PD-L1 High/TMB Low samples presented mutations either on EGFR (12%), KRAS (23.5%) or in genes from known driver TRK/MAPK pathways, whereas only KRAS was part of the frequently mutated gene signature with 36.5% (13/36) samples mutated on PD-L1 High/TMB High samples. Neither EGFR nor KRAS were found frequently mutated on PD-L1 Low/TMB High group (n=46). Strikingly the top 12 most frequently mutated genes on PD-L1 Neg/TMB High tumors were TP53, LRP1B, SPTA1, SMARCA4, GNAS, ALK, FGFR2, SLIT2, ROS1, AMER1, FAT1, and MED12. We found statistically significant co-occurrences between LRP1B and 7 other genes from this signature in additional 5939 lung cancer patients assessed. Conclusions: TMB and PD-L1 results do not correlate in lung cancer specimens. Common genomic alteration signatures might define subsets of lung cancer tumors with no PD-L1 expression to complement TMB and PD-L1 on the selection criteria for patients whom may benefit from checkpoint inhibitors. Citation Format: Fernando J. Lopez-Diaz, Lauryn Keeler, Forrest Blocker, Shiloh Sprankles, Ryan Bender, Sally Agersborg, Lawrence Weiss, Vincent Funari. Different genomic mutations signatures are associated to specific PD-L1/TMB states on lung cancer with potential value for patients screening for immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3170.

Multidrug resistance (MDR) due to enhanced drug efflux activity of tumor cells can severely impact the efficacy of antitumor therapies. We recently showed that increased activity of the efflux transporter P-glycoprotein (P-gp) associated with activation of Snail transcriptional regulators may be mediated mainly by moesin in lung cancer cells. Here, we aimed to systematically evaluate the relationships among mRNA expression levels of efflux transporters (P-gp, breast cancer resistance protein (BCRP), and multidrug resistance-associated protein 2 (MRP2)), scaffold proteins (ezrin (Ezr), radixin (Rdx), and moesin (Msn); ERM proteins), and SNAI family members (Snail, Slug, and Smac) in clinical lung cancer and noncancer samples. We found high correlations between relative (cancer/noncancer) mRNA expression levels of Snail and Msn, Msn and P-gp, Slug and MRP2, and Smuc and BCRP. These findings support our previous conclusion that Snail regulates P-gp activity via Msn and further suggest that Slug and Smuc may contribute to the functional regulation of MRP2 and BCRP, respectively, in lung cancer cells. This trial is registered with UMIN000023923.

Lung cancer (LC) is a highly lethal cancer worldwide. Research on the distribution and nature of extrachromosomal DNA molecules (EcDNAm) in early LC is scarce. In this study, after removing linear DNA and mitochondrial circular DNA, EcDNAm were extracted from two paired LC tissue samples and amplified using rolling circle amplification. High throughput extrachromosomal DNA (EcDNA) or RNA sequencing and bioinformatics analysis were subsequently utilized to explore the distribution and nature of the EcDNAm. Additionally, to elucidate the role of oncogenes with large EcDNAm sizes, gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed. The RNA sequencing results revealed significant differences in certain genes between tumors and corresponding normal samples. At the same time, slight distinctions were observed between relapsed and non-relapsed tumor samples. The nature of the EcDNAm was compared between LC samples and matched normal samples. There was a tendency for the number of EcDNAm with longer size (EcDNA) and its containing driver oncogenes to be higher in cancer samples. Enrichment analysis of the cancer samples revealed enrichment in biological processes, such as positive regulation of protein localization, axon development, and in-utero embryonic development. This study highlights the universal distribution and characteristics of EcDNAm in early LC. Moreover, our work fills the investigation of the EcDNAm gap and future studies should focus on the application of EcDNA as a potential biomarker in patients with early LC.

TP53 mutations in Pap test DNA are a potential biomarker of ovarian cancer risk in high-risk women (226)

BackgroundLung cancer is one of the most common malignancies globally and a significant component of cancer‐related deaths. The lack of early diagnosis accounts for detecting approximately 75% of cancer patients at an intermediate to an advanced stage, with a low 5‐year survival rate. Therefore, a more comprehensive understanding of the molecular mechanisms of lung cancer development is necessary to find reliable and effective therapeutic and diagnostic biomarkers.Methodscirc_SAR1A, miR‐21‐5p, and TXNIP in lung cancer tissues, animal xenografts, and cell lines were validated by qRT‐PCR and western blotting analyses. RNase R digestion and nuclear/cytoplasm fractionation experiments were utilized to determine the stability and localization of circ_SAR1A in lung cancer cells. The binding between miR‐21‐5p and circ_SAR1A or TXNIP was confirmed by luciferase reporter, RNA pull‐down, Spearman's correlation, and rescue assays. CCK‐8, colony formation, flow cytometry, Transwell, and western blotting were utilized to illustrate the malignant behavior of lung cancer cells.Resultscirc_SAR1A and TXNIP were down‐regulated while miR‐21‐5p was up‐regulated in lung cancer samples and cells. circ_SAR1A was located predominantly in the cytoplasm; it inhibited lung cancer growth in vitro and in vivo by sponging to miR‐21‐5p. miR‐21‐5p silencing suppressed lung cancer malignancy by targeting TXNIP.Conclusionscirc_SAR1A is a critical negative regulator of lung carcinogenesis. circ_SAR1A/miR‐21‐5p/TXNIP attenuation inhibited lung cancer progression, presenting an ideal diagnostic and a potential therapeutic target.

Substantial population and preclinical data have indicated that the insulin growth factor (IGF) pathway plays an important role in lung cancer. Although results from early clinical trials targeting the IGF1R showed some evidence of response in non-small cell lung cancer (NSCLC), larger randomized phase III trials have not shown clear clinical benefit in combination with conventional strategies. These disappointing results have focused attention on the insulin receptor isoform A (IR-A) as one intrinsic resistance factor to anti-IGF1R therapy due to its ability to mediate growth-promoting signaling through binding to IGF-II. However, the expression status of insulin-receptor isoforms in lung cancer and its subtypes is largely unknown. In order to address this issue, the mRNA expression levels of insulin-receptor isoforms were studied in 50 normal lung samples and 183 lung cancer samples from complementary DNA (cDNA) arrays. Additionally, 31 primary small cell lung cancer (SCLC) tumor samples with paired adjacent normal tissues were also evaluated. The mRNA expression levels of insulin receptor isoform B (IR-B) were significantly lower in NSCLC than normal lung samples (n=176, p &amp;lt; 0.001), including adenocarcinoma (n=85, p &amp;lt; 0.0001), squamous cell carcinoma (n=60, p &amp;lt; 0.0001) and large cell lung cancer samples (n=16, p &amp;lt; 0.0001). The mRNA expression levels of IR-B were also significantly lower in SCLC primary tumor samples (P=0.002). The mRNA levels for IR-A were significantly higher in primary SCLC samples (p &amp;lt; 0.02) than paired adjacent normal, but no significant differences between normal lung and NSCLC or its subtypes were observed (p &amp;gt; 0.05). When comparing tumor to normal lung samples, the IR-A:IR-B mRNA expression ratios were significantly higher in primary SCLC samples (p &amp;lt;0.0001). The IR-A:IR-B mRNA expression ratios were also significantly higher in all NSCLC samples compared to normal lung, (p &amp;lt; 0.001) including adenocarcinoma (p &amp;lt; 0.0001), squamous cell carcinoma (p &amp;lt; 0.0001) and large-cell lung cancer (p &amp;lt; 0.0001). Our results indicated that the reduction of the level of IR-B expression and an increase in the IR-A:IR-B expression ratio may highlight patients with active IR-A signaling, and may be more informative than identifying those with increased IR-A expression only. Additionally, these results suggested that an increased IR-A:IR-B expression ratio might be a useful biomarker in selecting patients that may have an intrinsic resistance to anti-IGF1R therapy in lung cancer. Citation Format: Jiaqi Huang, Katie Streicher, Chris Morehouse, Kim Lehmann, Philip Brohawn, Wei Zhu, Meggan Czapiga, Theresa LaVallee, Karen Mitz, Laura Richman, Bahija Jallal, Yihong Yao. Differential expression of insulin receptor isoforms in human lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5268. doi:10.1158/1538-7445.AM2013-5268