Malignant Lung Tissues Research Articles

A clinically effective model based on cell-free DNA methylation and low-dose CT for risk stratification of pulmonary nodules

Accurate, non-invasive, and cost-effective tools are needed to assist pulmonary nodule diagnosis and management due to increasing detection by low-dose computed tomography (LDCT). We perform genome-wide methylation sequencing on malignant and non-malignant lung tissues and designed a panel of 263 differential DNA methylation regions, which is used for targeted methylation sequencing on blood cell-free DNA (cfDNA) in two prospectively collected and retrospectively analyzed multicenter cohorts. We develop and optimize an integrative model for risk stratification of pulmonary nodules based on 40 cfDNA methylation biomarkers, age, and five simple computed tomography (CT) imaging features using machine learning approaches and validate its good performance in two cohorts. Using the two-threshold strategy can effectively reduce unnecessary invasive surgeries, overtreatment costs, and injury for patients with benign nodules while advising immediate treatment for patients with lung cancer, which can potentially improve the overall diagnosis of lung cancer following LDCT/CT screening.

Microwave dielectric properties of normal, fibroelastotic, and malignant human lung tissue

Technological development of microwave treatment and detection techniques for lung cancer requires accurate and comprehensive knowledge of the microwave dielectric properties of human lung tissue. We characterize the dielectric properties of room temperature human lung tissue from 0.5 to 10 GHz for three lung tissue groups: normal, fibroelastotic, and malignant. We fit a two-pole Debye model to the measured frequency-dependent complex permittivity and calculate the median Debye parameters for the three groups. We find that malignant lung tissue is approximately 10% higher in relative permittivity and conductivity compared to normal lung tissue; this trend matches previously reported normal versus malignant data for other biological tissues. There is little contrast between benign lung tissue with fibroelastosis and malignant lung tissue. We extrapolate our data from room temperature to 37 °C using a temperature-dependence model for animal lung tissue and use the Maxwell-Garnett dielectric mixing model to predict the dielectric properties of inflation-dynamic human lung tissue; both approximations correspond with previously reported dielectric data of bovine and porcine lung tissue.

Lung and colon cancer detection from CT images using Deep Learning

Cancer is a deadly disease that has gained a reputation as a global health concern. Further, lung cancer has been widely reported as the most deadly cancer type globally, while colon cancer comes second. Meanwhile, early detection is one of the primary ways to prevent lung and colon cancer fatalities. To aid the early detection of lung and colon cancer, we propose a computer-aided diagnostic approach that employs a Deep Learning (DL) architecture to enhance the detection of these cancer types from Computed Tomography (CT) images of suspected body parts. Our experimental dataset (LC25000) contains 25000 CT images of benign and malignant lung and colon cancer tissues. We used weights from a pre-trained DL architecture for computer vision, EfficientNet, to build and train a lung and colon cancer detection model. EfficientNet is a Convolutional Neural Network architecture that scales all input dimensions such as depth, width, and resolution at the same time. Our research findings showed detection accuracies of 99.63%, 99.50%, and 99.72% for training, validation, and test sets, respectively.

Quantitative proteomics identifies tumour matrisome signatures in patients with non-small cell lung cancer.

The composition and remodelling of the extracellular matrix (ECM) are important factors in the development and progression of cancers, and the ECM is implicated in promoting tumour growth and restricting anti-tumour therapies through multiple mechanisms. The characterisation of differences in ECM composition between normal and diseased tissues may aid in identifying novel diagnostic markers, prognostic indicators and therapeutic targets for drug development. Using tissue from non-small cell lung cancer (NSCLC) patients undergoing curative intent surgery, we characterised quantitative tumour-specific ECM proteome signatures by mass spectrometry. We identified 161 matrisome proteins differentially regulated between tumour tissue and nearby non-malignant lung tissue, and we defined a collagen hydroxylation functional protein network that is enriched in the lung tumour microenvironment. We validated two novel putative extracellular markers of NSCLC, the collagen cross-linking enzyme peroxidasin and a disintegrin and metalloproteinase with thrombospondin motifs 16 (ADAMTS16), for discrimination of malignant and non-malignant lung tissue. These proteins were up-regulated in lung tumour samples, and high PXDN and ADAMTS16 gene expression was associated with shorter survival of lung adenocarcinoma and squamous cell carcinoma patients, respectively. These data chart extensive remodelling of the lung extracellular niche and reveal tumour matrisome signatures in human NSCLC.

CAMSAP3 negatively regulates lung cancer cell invasion and angiogenesis through nucleolin/HIF-1α mRNA complex stabilization

AimsCancer metastasis is a major cause of lung cancer-related mortality, so identification of related molecular mechanisms is of interest. Calmodulin-regulated spectrin-associated protein 3 (CAMSAP3) has been implicated in lung cancer malignancies; however, its role in metastatic processes, including invasion and angiogenesis, is largely unknown. Main methodThe clinical relevance of CAMSAP3 expression in lung cancer was evaluated. The relevance of CAMSAP3 expression to in vitro cell invasion and angiogenesis was assessed in human lung cancer cells and endothelial cells, respectively. The molecular mechanism was identified by qRT–PCR, immunoprecipitation, mass spectrometry, and RNA immunoprecipitation. The in vivo metastatic and angiogenic activities of lung cancer cells were assessed. Key findingsLow CAMSAP3 expression was found in malignant lung tissues and strongly correlated with a poor prognosis in lung adenocarcinoma (LUAD). CAMSAP3-knockout NSCLC exhibited high invasive ability, and CAMSAP3 knockout induced HUVEC proliferation and tube formation; these effects were significantly attenuated by reintroduction of exogenous wild-type CAMSAP3. Mechanistically, in the absence of CAMSAP3, the expression of hypoxia-inducible factor-1α (HIF-1α) was upregulated, which increased the levels of downstream HIF-1α targets such as vascular endothelial growth factor A (VEGFA) and matrix metalloproteinases (MMPs) 2 and 9. Proteomic analysis revealed that nucleolin (NCL) bound to CAMSAP3 to regulate HIF-1α mRNA stabilization. In addition, CAMSAP3-knockout lung cancer cells displayed highly aggressive behavior in metastasis and angiogenesis in vivo. SignificanceThis study reveals that CAMSAP3 plays a negative regulatory role in lung cancer cell metastatic behavior both in vitro and in vivo through NCL/HIF-1α mRNA complex stabilization.

Overexpression of Human Aspartyl (Asparaginyl) β-hydroxylase in NSCLC: Its Diagnostic Value by Means of Exosomes of Bronchoalveolar Lavage.

The human aspartyl β-hydroxylase (ASPH) is overexpressed in tumor tissues. Bronchoalveolar lavage (BAL) is a diagnostic procedure for infections and malignancies. The aim of this study was to investigate whether tumor exosomes carrying ASPH gene marker were present in bronchoalveolar fluid of patients with non-small cell lung cancer (NSCLC). A tissue microarray analysis was applied to explore the expression of ASPH in different histologic NSCLC. The human NSCLC cell lines and normal bronchial cell lines were used to study exosomal ASPH exprerssion. A total of 27 NSCLC, 21 benign tumor, and 15 healthy controls underwent BAL. Immunohistochemistry was performed to study the ASPH expression in malignant and normal lung tissues. The expression characteristics of ASPH in different NSCLC and normal bronchial cells and pneumocytes were confirmed by cell blocks. A reverse transcription-quantitative polymerase chain reaction was carried out to study the levels of exosomal ASPH expression. Immunohistochemical staining of tissue microarray demonstrated that overexpression of ASPH was found in NSCLC tissues including adenocarcinoma, large cell carcinoma, and squamous cell carcinoma, but absent in adjacent normal tissues. All NSCLC specimens exhibited high levels of ASPH immunoreactivity, while nonmalignant and normal lung tissues exhibited a very low level of expression. Overexpression of ASPH was found in exosomes from NSCLC cell lines but absent from the normal bronchial cell line NL-20. ASPH level from BAL exosomes was significantly increased in NSCLC patients compared with that from nonmalignant or health group. Our method of isolation of BAL exosomes was easily performed in the clinical laboratory. BAL exosomal ASPH can be a potential biomarker for NSCLC diagnosis.

Single-Cell Expression Landscape of SARS-CoV-2 Receptor ACE2 and Host Proteases in Normal and Malignant Lung Tissues from Pulmonary Adenocarcinoma Patients.

Simple SummaryThe coronavirus disease 2019 (COVID-19) pandemic continues to spread rapidly on a global scale. When presenting with severe respiratory complications, COVID-19 results in markedly high death rates, particularly among patients with comorbidities such as cancer. Motivated by the ongoing global health crisis, we leveraged a growing in-house cohort of pulmonary tissues from lung cancer patients to analyze, at high resolution, the expression of host proteins implicated in the entryway of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into lung epithelial cells. Our results identify key pathways in lung pathobiology and inflammation that offer the potential to identify novel markers and therapeutic targets that can be repurposed for clinical management of COVID-19, particularly among lung cancer patients, a population that represents over half a million individuals in the United States alone. The novel coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Severely symptomatic COVID-19 is associated with lung inflammation, pneumonia, and respiratory failure, thereby raising concerns of elevated risk of COVID-19-associated mortality among lung cancer patients. Angiotensin-converting enzyme 2 (ACE2) is the major receptor for SARS-CoV-2 entry into lung cells. The single-cell expression landscape of ACE2 and other SARS-CoV-2-related genes in pulmonary tissues of lung cancer patients remains unknown. We sought to delineate single-cell expression profiles of ACE2 and other SARS-CoV-2-related genes in pulmonary tissues of lung adenocarcinoma (LUAD) patients. We examined the expression levels and cellular distribution of ACE2 and SARS-CoV-2-priming proteases TMPRSS2 and TMPRSS4 in 5 LUADs and 14 matched normal tissues by single-cell RNA-sequencing (scRNA-seq) analysis. scRNA-seq of 186,916 cells revealed epithelial-specific expression of ACE2, TMPRSS2, and TMPRSS4. Analysis of 70,030 LUAD- and normal-derived epithelial cells showed that ACE2 levels were highest in normal alveolar type 2 (AT2) cells and that TMPRSS2 was expressed in 65% of normal AT2 cells. Conversely, the expression of TMPRSS4 was highest and most frequently detected (75%) in lung cells with malignant features. ACE2-positive cells co-expressed genes implicated in lung pathobiology, including COPD-associated HHIP, and the scavengers CD36 and DMBT1. Notably, the viral scavenger DMBT1 was significantly positively correlated with ACE2 expression in AT2 cells. We describe normal and tumor lung epithelial populations that express SARS-CoV-2 receptor and proteases, as well as major host defense genes, thus comprising potential treatment targets for COVID-19 particularly among lung cancer patients.

ATR-FTIR spectroscopy as adjunct method to the microscopic examination of hematoxylin and eosin-stained tissues in diagnosing lung cancer.

Lung cancer remains the leading cause of cancer-related death worldwide. Since prognosis and treatment outcomes rely on fast and accurate diagnosis, there is a need for more cost-effective, sensitive, and specific method for lung cancer detection. Thus, this study aimed to determine the ability of ATR-FTIR in discriminating malignant from benign lung tissues and evaluate its concordance with H&E staining. Three (3) 5μm-thick sections were cut from formalin fixed paraffin embedded (FFPE) cell or tissue blocks from patients with lung lesions. The outer sections were H&E-stained and sent to two (2) pathologists to confirm the histopathologic diagnosis. The inner section was deparaffinized by standard xylene method and then subjected to ATR-FTIR analysis. Distinct spectral profiles that distinguished (p<0.05) one sample from another, called the “fingerprint region”, were observed in five (5) peak patterns representing the amides, lipids, and nucleic acids. Principal component analysis and hierarchical cluster analysis evidently clustered the benign from malignant tissues. ATR-FTIR showed 97.73% sensitivity, 92.45% specificity, 94.85% accuracy, 91.49% positive predictive value and 98.00% negative predictive value in discriminating benign from malignant lung tissue. Further, strong agreement was observed between histopathologic readings and ATR-FTIR analysis. This study shows the potential of ATR-FTIR spectroscopy as a potential adjunct method to the gold standard, the microscopic examination of hematoxylin and eosin (H&E)-stained tissues, in diagnosing lung cancer.

LMO7 and LIMCH1 interact with LRIG proteins in lung cancer, with prognostic implications for early-stage disease

ObjectivesThe human leucine-rich repeats and immunoglobulin-like domains (LRIG) protein family comprises the integral membrane proteins LRIG1, LRIG2 and LRIG3. LRIG1 is frequently down-regulated in human cancer, and high levels of LRIG1 in tumor tissue are associated with favorable clinical outcomes in several tumor types including non-small cell lung cancer (NSCLC). Mechanistically, LRIG1 negatively regulates receptor tyrosine kinases and functions as a tumor suppressor. However, the details of the molecular mechanisms involved are poorly understood, and even less is known about the functions of LRIG2 and LRIG3. The aim of this study was to further elucidate the functions and molecular interactions of the LRIG proteins. Materials and methodsA yeast two-hybrid screen was performed using a cytosolic LRIG3 peptide as bait. In transfected human cells, co-immunoprecipitation and co-localization experiments were performed. Proximity ligation assay was performed to investigate interactions between endogenously expressed proteins. Expression levels of LMO7 and LIMCH1 in normal and malignant lung tissue were investigated using qRT-PCR and through in silico analyses of public data sets. Finally, a clinical cohort comprising 355 surgically treated NSCLC cases was immunostained for LMO7. ResultsIn the yeast two-hybrid screen, the two paralogous proteins LMO7 and LIMCH1 were identified as interaction partners to LRIG3. LMO7 and LIMCH1 co-localized and co-immunoprecipitated with both LRIG1 and LRIG3. Endogenously expressed LMO7 was in close proximity of both LRIG1 and LRIG3. LMO7 and LIMCH1 were highly expressed in normal lung tissue and down-regulated in malignant lung tissue. LMO7 immunoreactivity was shown to be a negative prognostic factor in LRIG1 positive tumors, predicting poor patient survival. ConclusionThese findings suggest that LMO7 and LIMCH1 physically interact with LRIG proteins and that expression of LMO7 is of clinical importance in NSCLC.

Abstract 1948: Polo-like kinase 4 inhibition produces polyploidy and apoptotic death of lung cancers

Abstract Despite current treatments, lung cancers remain a major public health problem. Innovative ways are needed to treat or prevent these cancers. Centrosomes are critical for fidelity of mitosis. Abnormal centrosome numbers can cause aberrant mitosis and cell death. Polo-like kinase 4 (PLK4) is a serine/threonine kinase regulating centriole duplication and its deregulation alters centrosome number and mitosis. CFI-400945 is a highly selective PLK4 inhibitor that deregulates centriole duplication, causing mitotic defects and death of aneuploid cancers. Here, we explored CFI-400945 activity against lung cancer using in vitro and in vivo models. CFI-400945 caused polyploidy, growth inhibition and apoptotic death of murine and human lung cancer cells, despite expression of mutated KRAS or p53. Analysis of DNA content by propidium iodide (PI) staining revealed cells with &amp;gt; 4N DNA content (polyploidy) markedly increased after CFI-400945 treatment. Centrosome numbers and mitotic spindles were scored by individually staining treated and control cells with γ-tubulin, α-tubulin, and DAPI. CFI-400945 treatment produced supernumerary centrosomes and mitotic defects in the examined lung cancer cell lines. In vivo antineoplastic activity of CFI-400945 was established in mice with syngeneic lung cancer xenografts. Lung tumor growth was statistically significantly reduced at dosages that were well tolerated. Phospho-histone H3 staining of resected lung cancers following CFI-400945 treatment confirmed the presence of aberrant mitosis. PLK4 expression profiles in human lung cancers were explored using The Cancer Genome Atlas (TCGA) and RNA in situ hybridization (RNA ISH) of microarrays containing normal and malignant lung tissues. PLK4 expression was significantly higher in the malignant versus normal lung and conferred an unfavorable survival (P &amp;lt; 0.05) indicating the clinical relevance of PLK4 expression in lung cancer. Intriguingly, cyclin dependent kinase 2 (CDK2) antagonism (that confers multipolar anaphase catastrophe by inhibiting centrosome clustering) showed synergistic effects (CI &amp;lt; 1) on lung cancer cell growth when combined with CDI-400945. This is an appealing cooperating regimen since each agent increases multipolar cell division and cancer cell death. CFI-400945 is undergoing phase I clinical trial testing (NCT01954316). Taken together, targeting PLK4 for inhibition holds promise for lung cancer therapy either as a single agent or when combined with another agent that deregulates mitosis. Citation Format: Masanori Kawakami, Lisa Maria Mustachio, Lin Zheng, Yulong Chen, Jaime Rodriguez-Canales, Barbara Mino, Jonathan M. Kurie, Jason Roszik, Pamela Andrea Villalobos, Kelsie L. Thu, David W. Cescon, Jennifer Silvester, Ignacio Wistuba, Tak W. Mak, Xi Liu, Ethan Dmitrovsky. Polo-like kinase 4 inhibition produces polyploidy and apoptotic death of lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1948.

Abstract 5509: Early suppression of the TBX2 subfamily of transcription factors during in vivo tobacco carcinogen-mediated lung adenocarcinoma development

Abstract Targets for early treatment of lung adenocarcinoma (LUAD), the leading cause of cancer mortality among smokers, are far and few largely due to the paucity in our knowledge of the molecular pathogenesis of the malignancy. In our efforts to address this glaring gap in knowledge, we previously surveyed the expression of members of the T-box 2 subfamily of transcription factors (TBXs 2, 3, 4 and 5) in normal and malignant lung tissues owing to their evolutionarily conserved expression and function in normal lung organogenesis,. We found that all four members were profoundly suppressed in human premalignant lesions and malignant LUADs. While these findings showed that the TBX2 subfamily of transcription factors is suppressed in overt LUADs, their role in the molecular and temporal oncogenesis of the malignancy in smokers is still elusive. To fill this void, we first surveyed the expression of these genes in a mouse model of LUAD we had developed comprised of knockout of the airway-lineage specific gene Gprc5a (Gprc5a-/-) and exposure to the tobacco-specific carcinogen nicotine-derived nitrosamine ketone (NNK). We had previously found that NNK-exposed Gprc5a-/- mice developed adenomas and LUADs that harbored somatic activating mutations (p.G12D and p.Q61R) in the Kras oncogene – the same variants posited to function as drivers of LUAD in human smokers. In ongoing RNA-Seq efforts cataloging expression in frozen-fixed temporally collected (up to seven months post-NNK) Gprc5a-/- normal tissues, adenomas and LUADs, we found that all four TBX genes were markedly down-regulated (all P &amp;lt; 0.05) in all lesions and across all time points following exposure, relative to normal lung tissues. Concurrent analysis of canonical genes directly activated or repressed by the TBX2 subfamily were found to be consistently down-regulated or elevated, respectively, in the same NNK-exposed lung lesions relative to normal lung. We employed a mouse LUAD cell line (mF471 cells) we had previously derived from an NNK-exposed Gprc5a-/- mouse tumor to further functionally probe the role of TBX2 subfamily in LUAD oncogenesis. Transient over-expression of each of the four TBX2 subfamily members in the mF471 LUAD cells significantly inhibited cell proliferation and growth evidenced by trypan blue dye exclusion, MTT and cell cycle analyses. Over-expression of each of the four TBX2 subfamily members yielded similar anti-growth effects with significant increases in sub-G0 phase of the cell cycle indicating cell cycle arrest (up to 35%) 48 hours following transfection. These findings suggest that the TBX2 subfamily genes are suppressed at early stages in the development of smoking-associated Kras-mutant LUAD and play key tumor suppressor roles in the molecular pathogenesis of this common and fatal malignancy. Citation Format: Athar Khalil, Junya Fujimoto, Tina McDowell, Paul Scheet, Georges Nemer, Humam Kadara. Early suppression of the TBX2 subfamily of transcription factors during in vivo tobacco carcinogen-mediated lung adenocarcinoma development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5509.

Polo-like kinase 4 inhibition produces polyploidy and apoptotic death of lung cancers

Polo-like kinase 4 (PLK4) is a serine/threonine kinase regulating centriole duplication. CFI-400945 is a highly selective PLK4 inhibitor that deregulates centriole duplication, causing mitotic defects and death of aneuploid cancers. Prior work was substantially extended by showing CFI-400945 causes polyploidy, growth inhibition, and apoptotic death of murine and human lung cancer cells, despite expression of mutated KRAS or p53. Analysis of DNA content by propidium iodide (PI) staining revealed cells with >4N DNA content (polyploidy) markedly increased after CFI-400945 treatment. Centrosome numbers and mitotic spindles were scored. CFI-400945 treatment produced supernumerary centrosomes and mitotic defects in lung cancer cells. In vivo antineoplastic activity of CFI-400945 was established in mice with syngeneic lung cancer xenografts. Lung tumor growth was significantly inhibited at well-tolerated dosages. Phosphohistone H3 staining of resected lung cancers following CFI-400945 treatment confirmed the presence of aberrant mitosis. PLK4 expression profiles in human lung cancers were explored using The Cancer Genome Atlas (TCGA) and RNA in situ hybridization (RNA ISH) of microarrays containing normal and malignant lung tissues. PLK4 expression was significantly higher in the malignant versus normal lung and conferred an unfavorable survival (P < 0.05). Intriguingly, cyclin dependent kinase 2 (CDK2) antagonism cooperated with PLK4 inhibition. Taken together, PLK4 inhibition alone or as part of a combination regimen is a promising way to combat lung cancer.

Abstract B37: Value of penetrated NIR spectroscopy in the diagnostic process of lung cancer

Abstract Background: Recently due to advances in biological therapy and immunotherapy there is an increased need for precise morphological and mutational verification of lung cancer. We have been testing the possibilities of NIR (near infrared) n spectroscopy in the process lung cancer detection. Methods: For the analyses, we used a custom-made endoscopic lensed microprobe consisting of one excitation and one collection optical fibers that were coupled to a NIR spectrometer. In 55 patients with lung cancer diagnosis undergoing bronchoscopy we carried on 110 measurements of NIR penetrated spectra from both normal and malignant lung tissues. Detected spectral curves of absolute transmittance for normal tissue and pathological tissue were further analyzed utilizing two chosen parameters which are corresponding to important characteristics of the curves. First parameter was statical moment of area under spectral curve referenced to vertical axis of maximal wavelength whose value reflected average intensity of detected signal as well as rate of shifting higher intensities to shorter wavelengths of spectral band (that means how much is investigated tissue permeable on shortest possible wavelengths). As the second parameter serve horizontal coordinates of the centrum of gravity of the area under spectral curve referenced to vertical axis of maximal wavelength whose value reflected just the rate of shifting signal to shorter wavelengths of spectral band regardless absolute intensity (this parameter compared shifting to shorter wavelengths for spectral curves as if were normalized to its maximal intensity value). Finally ratio of pathological to normal value for both parameters was calculated. Results: Resulting values of these ratios for both above mentioned parameters were higher than 1 for all types of pathologies. Moreover ratio for second parameter is significantly higher for adenocarcinoma than for any type of pathologies. Conclusion: NIR spectroscopy showed a potential for a reliable discrimination between normal and pathological lung tissue. We observed spectral differences between adenocarcinoma and all other types of lung pathologies. Acknowledgement: PROGRAM MPO-TRIO PROJECT: FV 10657, OPROX, a.s. Research and development of technologies and methods for optical penetrating spectroscopy of atypical lung tissue nodules. Note: This abstract was not presented at the conference. Citation Format: Tomas Bruha, Jiri Votruba, Vladimír Cech, Josef Skvarek. Value of penetrated NIR spectroscopy in the diagnostic process of lung cancer [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B37.

A novel method for quantification of beam's-eye-view tumor tracking performance.

In-treatment imaging using an electronic portal imaging device (EPID) can be used to confirm patient and tumor positioning. Real-time tumor tracking performance using current digital megavolt (MV) imagers is hindered by poor image quality. Novel EPID designs may help to improve quantum noise response, while also preserving the high spatial resolution of the current clinical detector. Recently investigated EPID design improvements include but are not limited to multi-layer imager (MLI) architecture, thick crystalline and amorphous scintillators, and phosphor pixilation and focusing. The goal of the present study was to provide a method of quantitating improvement in tracking performance as well as to reveal the physical underpinnings of detector design that impact tracking quality. The study employs a generalizable ideal observer methodology for the quantification of tumor tracking performance. The analysis is applied to study both the effect of increasing scintillator thickness on a standard, single-layer imager (SLI) design as well as the effect of MLI architecture on tracking performance. The present study uses the ideal observer signal-to-noise ratio (d') as a surrogate for tracking performance. We employ functions which model clinically relevant tasks and generalized frequency-domain imaging metrics to connect image quality with tumor tracking. A detection task for relevant Cartesian shapes (i.e., spheres and cylinders) was used to quantitate trackability of cases employing fiducial markers. Automated lung tumor tracking algorithms often leverage the differences in benign and malignant lung tissue textures. These types of algorithms (e.g., soft-tissue localization - STiL) were simulated by designing a discrimination task, which quantifies the differentiation of tissue textures, measured experimentally and fit as a power-law in trend (with exponent β) using a cohort of MV images of patient lungs. The modeled MTF and NPS were used to investigate the effect of scintillator thickness and MLI architecture on tumor tracking performance. Quantification of MV images of lung tissue as an inverse power-law with respect to frequency yields exponent values of β=3.11 and 3.29 for benign and malignant tissues, respectively. Tracking performance with and without fiducials was found to be generally limited by quantum noise, a factor dominated by quantum detective efficiency (QDE). For generic SLI construction, increasing the scintillator thickness (gadolinium oxysulfide - GOS) from a standard 290μm to 1720μm reduces noise to about 10%. However, 81% of this reduction is appreciated between 290 and 1000μm. In comparing MLI and SLI detectors of equivalent individual GOS layer thickness, the improvement in noise is equal to the number of layers in the detector (i.e., 4) with almost no difference in MTF. Further, improvement in tracking performance was slightly less than the square-root of the reduction in noise, approximately 84-90%. In comparing an MLI detector with an SLI with a GOS scintillator of equivalent total thickness, improvement in object detectability is approximately 34-39%. We have presented a novel method for quantification of tumor tracking quality and have applied this model to evaluate the performance of SLI and MLI EPID designs. We showed that improved tracking quality is primarily limited by improvements in NPS. When compared to very thick scintillator SLI, employing MLI architecture exhibits the same gains in QDE, but by mitigating the effect of optical Swank noise, results in more dramatic improvements in tracking performance.

Midkine and pleiotrophin concentrations in needle biopsies of breast and lung masses.

Midkine (MDK) and pleiotrophin (PTN) are two closely related heparin-binding growth factors which are overexpressed in a wide variety of human cancers. We hypothesized that the concentrations of these factors in washout of biopsy needles would be higher in breast and lung cancer than in benign lesions. Seventy subjects underwent pre-operative core needle biopsies of 78 breast masses (16 malignancies). In 11 subjects, fine needle aspiration was performed ex vivo on 7 non-small cell lung cancers and 11 normal lung specimens within surgically excised lung tissue. The biopsy needle was washed with buffer for immunoassay. The MDK/DNA and the PTN/DNA ratio in most of the malignant breast masses were similar to the ratios in benign masses except one lobular carcinoma in situ (24-fold higher PTN/DNA ratio than the average benign mass). The MDK/DNA and PTN/DNA ratio were similar in most malignant and normal lung tissue except one squamous cell carcinoma (38-fold higher MDK/DNA ratio than the average of normal lung tissue). Both MDK and PTN are readily measurable in washout of needle biopsy samples from breast and lung masses and levels are highly elevated only in a specific subset of these malignancies.

Abstract A14: Proteomic analysis of paired malignant and non-malignant tissues from patients with NSCLC adenocarcinoma identified changes in translation initiation factors potentially important in oncogenesis

Abstract An important aspect of the control of mRNA translation into protein in mammalian cells is the initiation process, which involves the activities of multiple eukaryotic translation initiation factors or eIFs. Genetic analyses of eIFs in yeast (S. cerevisiae) have defined the eIFs necessary for the basic translation of mRNAs into proteins, but additional subunits found in mammalian cells have not been as well defined in normal cells so how the activities and functions of eIFs may be dysregulated in cancers such as lung cancer are still being determined. Lung cancer causes the highest cancer mortality in men and women in the United States. Lung cancer, mostly non-small cell (NSCLC) adenocarcinoma is responsible for about 13% of all new cancer cases and 26% of all cancer deaths. We undertook a proteomic analysis of lung cancer by analyzing 39 pairs of malignant and non-malignant lung tissues obtained from patients with early stage NSCLC adenocarcinoma using trypsin digestion and tandem MS/MS. Examination of eIFs in these tissues showed significant proteomic differences with significant increases in eIFs, especially in eIF2A, eIF2B, 1 alpha, and eIF2B, 5 episilon (p-values &amp;lt; 0.05). Smaller, but significant increased fold changes were also observed in eIF3B, eIF3G, eIF3J, eIF4G1, eIF4E eIF4A1 and eIF4H. Increased protein levels of EIF2AK2, the interferon induced eIF2A protein kinase was also detected. The eIF2alpha exchange rate may be an important aspect of tumorigenesis in lung cancer tissues with the increased exchange of GTP for GDP in eIF2A, hence the large increases in eIF2B subunits. Increases in eIF3 subunits, especially eIF3j, as well as increases in eIF4E and eIF4G1 correspond to increased protein synthesis rates and cellular proliferation. These early results need further investigation and related functional analysis to determine if the changes in eIFs correspond to important activities in the tumor cells or surrounding microenvironment and have mechanistic consequences in the growth and progression of lung cancer tumors. Citation Format: Suzanne Miyamoto, Johannes F. Fahrmann, Dmitry Grapov, Brett Phinney, Harvey Pass. Proteomic analysis of paired malignant and non-malignant tissues from patients with NSCLC adenocarcinoma identified changes in translation initiation factors potentially important in oncogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A14.

Matrix Metalloproteinase 14 promotes lung cancer by cleavage of Heparin-Binding EGF-like Growth Factor

Molecularly targeted therapies benefit approximately 15–20% of non-small cell lung cancer (NSCLC) patients carrying specific drug-sensitive mutations. Thus, there is a clinically unmet need for the identification of novel targets for drug development. Here, we performed RNA-deep sequencing to identify altered gene expression between malignant and non-malignant lung tissue. Matrix Metalloproteinase 14 (MMP14), a membrane-bound proteinase, was significantly up-regulated in the tumor epithelial cells and intratumoral myeloid compartments in both mouse and human NSCLC. Overexpression of a soluble dominant negative MMP14 (DN-MMP14) or pharmacological inhibition of MMP14 blocked invasion of lung cancer cells through a collagen I matrix in vitro and reduced tumor incidence in an orthotopic K-RasG12D/+p53−/− mouse model of lung cancer. Additionally, MMP14 activity mediated proteolytic processing and activation of Heparin-Binding EGF-like Growth Factor (HB-EGF), stimulating the EGFR signaling pathway to increase proliferation and tumor growth. This study highlights the potential for development of therapeutic strategies that target MMP14 in NSCLC with particular focus on MMP14-HB-EGF axis.

Sprouty4 mRNA variants and protein expressions in breast and lung-derived cells.

Sprouty proteins are modulators of mitogen-induced signalling processes and are therefore hypothesized to affect malignant diseases. As a member of the Sprouty family, Sprouty4 has been previously shown to function as a tumour suppressor in lung and breast cancer. The present study analysed the expression of two known Sprouty4 splice variants in cells established from malignant and normal lung and breast tissues using semi-quantitative reverse transcription-polymerase chain reaction and immunoblotting. The results indicated that the expression of the two messenger RNA (mRNA) variants was reduced in the cells derived from malignant tissue in comparison to the normal counterparts. Although the expression of the two splice variants were associated in both tissue types, on average, the relative expression of the longer variant was slightly increased in malignant cells compared with normal tissues. Notably, the protein levels reflected the expression observed at the mRNA level only in breast-derived cells. Contrarily, with regards to the measured mRNA levels, Sprouty4 protein was disproportional augmented in lung cells known to harbour the mutated K-Ras gene.

RNA Activation of the Vascular Endothelial Growth Factor Gene (VEGF) Promoter by Double-Stranded RNA and Hypoxia: Role of Noncoding VEGF Promoter Transcripts.

RNA activation (RNAa) is a gene regulation process in which promoter-targeted short double-stranded RNAs (dsRNAs) or microRNAs (miRs) induce target gene expression at the transcriptional level. Here, we investigate the presence of cryptic promoter transcripts within the VEGF promoter. Single-strand sense and antisense noncoding vascular endothelial growth factor (NcVEGF) promoter transcripts are identified, and their respective expression is studied in cells transfected with a VEGF promoter targeted dsRNA, namely, dsVEGF706, in hypoxic cells and in human malignant lung tissues. Interestingly, in dsVEGF706-transfected, as well as in hypoxic cells, NcVEGF expression levels increase coordinately with coding VEGF expression. Ago2 interaction with both sense and antisense NcVEGFs is increased in hypoxic cells, whereas in dsVEGF706-transfected cells, Ago2 and the antisense strand of the dsRNA interact specifically with the sense NcVEGF transcript. Furthermore, both dsVEGF706 and ectopic NcVEGF transcripts are able to activate the VEGF promoter endogenously present or in a reporter construct. Finally, using small interfering RNA targeting Ago2, we show that RNAa plays a role in the maintenance of increased VEGF and NcVEGF expression after hypoxia. Given the central role of VEGF in major human diseases, including cancer, this novel molecular mechanism is poised to reveal promising possibilities for therapeutic interventions.

Analysis of the acetylcholine‐signaling pathway reveals differences between normal and malignant lung tissue

NSCLC is characterized by aggressive clinical course, rapid doubling time and a dismal survival rate. The development of NSCLC is closely correlated with smoking habits. A small percentage of NSCLC cases also occur in non‐smokers. Targeted therapies like EGFR‐inhibitors, EML‐ALK4 inhibitors, and B‐Raf inhibitors show excellent therapeutic response in NSCLC patients who are non‐smokers. However, they do not work well in NSCLC patients who are smokers. Therefore, the identification of novel molecular targets in NSCLC patients who are exposed to cigarette smoke is urgently needed. Tobacco components like nicotine and NNK have been known to accelerate the progression of lung cancer. One of the major mechanisms underlying the proliferative actions of nicotine and NNK is that they upregulate the levels of the growth factor acetylcholine (ACh). This ACh binds back to its target receptors (on NSCLC cells) and stimulates the proliferation of NSCLC cells in an autocrine manner. The ACh signaling loop is amplified by exposure to tobacco components and cigarette smoke. With this background in mind, we aim to compare the ACh‐signaling machinery between normal lung cells and NSCLC cells. We find that both normal lung epithelial cells and NSCLC cells express all the components of the ACh‐signaling pathway, namely choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), acetylcholinesterase (AChE) and choline transporter (ChT). Some of the ACh‐signaling proteins like ChAT, VAChT and AChE show differences in expression and activity between normal lung tissue and NSCLC cells. We believe that such differences may form the basis of targeted therapies in NSCLC patients who are exposed to cigarette smoke.Support or Funding InformationFunding for our study was supported by the an NIH R15‐AREA Grant (1R15HL113681‐01A1), ACTSI Grant from Marshall University, the Flight Attendant Medical Research Institute YCSA Grant (82115),and the West Virginia IDeA Network of Biomedical Research Excellence (P20RR016477 and P20GM103434).