Non-neoplastic Lung Tissue Research Articles

Elevated expression of STIM1 is involved in lung tumorigenesis

This study aimed to address the potential role of STIM1 (stromal interaction molecule 1) in lung tumorigenesis. Colony formation in soft agar assay and tumorigenicity in nude mice assay were conducted. Western blot, immunohistochemistry and quantitative real-time polymerase chain reaction were used to measure the STIM1 expression. The distribution of cell cycle was detected by flow cytometry assay. Our results showed that the expression of STIM1 mRNA was significantly higher in human lung tumors than that in adjacent non-neoplastic lung tissues. Significantly increased expression of STIM1 mRNA and protein was observed in 16HBE-benzo(a)pyrene (BaP) cells and in BaP-treated mice lung tissues compared with 16HBE-control cells and the control group, respectively. Silencing STIM1 inhibited the proliferation and colony formation of A549 cells in in vitro experiments, attenuated the growth of tumor xenografts of A549 cells in in vivo experiments and induced the arrest of cell cycle in the G1 phase. The markedly decreased expression of cyclin D1 protein was observed in A549-shRNA-STIM1 cells as compared to A549-shRNA-control cells. The markedly increased expression of p21 protein was observed in A549-shRNA-STIM1 cells as compared to A549-shRNA-control cells. The expression levels of β-catenin and TGIF proteins were lower in A549-shRNA-STIM1 cells than those in A549-shRNA-control cells. In conclusion, this study indicated that the elevated expression of STIM1 might be involved in lung tumorigenesis.

Molecular heterogeneity of non-small cell lung carcinoma patient-derived xenografts closely reflect their primary tumors.

Availability of lung cancer models that closely mimic human tumors remains a significant gap in cancer research, as tumor cell lines and mouse models may not recapitulate the spectrum of lung cancer heterogeneity seen in patients. We aimed to establish a patient-derived tumor xenograft (PDX) resource from surgically resected non-small cell lung cancer (NSCLC). Fresh tumor tissue from surgical resection was implanted and grown in the subcutaneous pocket of non-obese severe combined immune deficient (NOD SCID) gamma mice. Subsequent passages were in NOD SCID mice. A subset of matched patient and PDX tumors and non-neoplastic lung tissues were profiled by whole exome sequencing, single nucleotide polymorphism (SNP) and methylation arrays, and phosphotyrosine (pY)-proteome by mass spectrometry. The data were compared to published NSCLC datasets of NSCLC primary and cell lines. 127 stable PDXs were established from 441 lung carcinomas representing all major histological subtypes: 52 adenocarcinomas, 62 squamous cell carcinomas, one adeno-squamous carcinoma, five sarcomatoid carcinomas, five large cell neuroendocrine carcinomas, and two small cell lung cancers. Somatic mutations, gene copy number and expression profiles, and pY-proteome landscape of 36 PDXs showed greater similarity with patient tumors than with established cell lines. Novel somatic mutations on cancer associated genes were identified but only in PDXs, likely due to selective clonal growth in the PDXs that allows detection of these low allelic frequency mutations. The results provide the strongest evidence yet that PDXs established from lung cancers closely mimic the characteristics of patient primary tumors.

Clinicopathological significance and a potential drugtarget of RARβ in non-small-cell lung carcinoma: a meta-analysis and a systematic review.

Lung cancer is the leading cause of cancer-related mortality in men worldwide. Aberrant RARβ promoter methylation has been frequently investigated in non-small-cell lung carcinoma (NSCLC), the most common form of lung cancer. The aim of present study was to carry out a meta-analysis and a systematic review to evaluate clinicopathological significance of RARβ promoter hypermethylation in NSCLC. A systematic literature search was carried out. The data were extracted and assessed by two reviewers independently. The Cochrane software Review Manager 5.2 was used to conduct the review. Odds ratios (ORs) with 95% corresponding confidence intervals (CIs) were calculated. A total of 18 relevant articles were available for meta-analysis which included 1,871 participants. The frequency of RARβ hypermethylation was significantly increased in NSCLC than in nonmalignant lung tissue, and the pooled OR was 5.69 (P<0.00001). RARβ hypermethylation was significantly more frequently observed in adenocarcinoma (AC) than in squamous cell carcinoma (SCC), and the pooled OR was 1.47 (P=0.005). Hypermethylation of RARβ gene in NSCLC was 2.46 times higher in smoking than in nonsmoking individuals, and the pooled OR was 2.46 (P=0.0002). RARβ hypermethylation rate was not significantly correlated with stage of the disease and sex. RARβ gene methylation status was not associated with prognosis of patients with NSCLC. In conclusion, RARβ promoter hypermethylation significantly increased in NSCLC than in non-neoplastic lung tissue and is predominant in AC, suggesting that RARβ methylation contributes to the development of NSCLC, especially AC. RARβ gene is a potential novel target for development of personalized therapy in patients with NSCLC, and is promising in restoration of retinoic acid-target gene induction via demethylation of RARβ1′ promoter.

Identification of Reprogrammed Myeloid Cell Transcriptomes in NSCLC.

Lung cancer is the leading cause of cancer related mortality worldwide, with non-small cell lung cancer (NSCLC) as the most prevalent form. Despite advances in treatment options including minimally invasive surgery, CT-guided radiation, novel chemotherapeutic regimens, and targeted therapeutics, prognosis remains dismal. Therefore, further molecular analysis of NSCLC is necessary to identify novel molecular targets that impact prognosis and the design of new-targeted therapies. In recent years, tumor “activated/reprogrammed” stromal cells that promote carcinogenesis have emerged as potential therapeutic targets. However, the contribution of stromal cells to NSCLC is poorly understood. Here, we show increased numbers of bone marrow (BM)-derived hematopoietic cells in the tumor parenchyma of NSCLC patients compared with matched adjacent non-neoplastic lung tissue. By sorting specific cellular fractions from lung cancer patients, we compared the transcriptomes of intratumoral myeloid compartments within the tumor bed with their counterparts within adjacent non-neoplastic tissue from NSCLC patients. The RNA sequencing of specific myeloid compartments (immature monocytic myeloid cells and polymorphonuclear neutrophils) identified differentially regulated genes and mRNA isoforms, which were inconspicuous in whole tumor analysis. Genes encoding secreted factors, including osteopontin (OPN), chemokine (C-C motif) ligand 7 (CCL7) and thrombospondin 1 (TSP1) were identified, which enhanced tumorigenic properties of lung cancer cells indicative of their potential as targets for therapy. This study demonstrates that analysis of homogeneous stromal populations isolated directly from fresh clinical specimens can detect important stromal genes of therapeutic value.

High expression of kinesin light chain-2, a novel target of miR-125b, is associated with poor clinical outcome of elderly non-small-cell lung cancer patients.

Background:MiR-125b has critical role in non-small-cell lung cancer (NSCLC) cell migration, and its target genes have not been elucidated. Kinesin-1 light chain (KLC)-2 was predicted as one of miR-125b's targets by bioinformatics analysis. This study is to identify the function of KLC2 and its interaction with miR-125b in NSCLC.Methods:Kinesin-1 light chain-2 protein expression and its clinical relevance were analysed in 140 matched NSCLC and adjacent non-neoplastic lung tissues. Both KLC2 gain- and loss-of-function analyses were performed in NSCLC cell lines by transient transfection. The direct interaction between KLC2 and miR-125b was confirmed by a luciferase reporter assay and a transient co-transfection assay as well as an analysis of eight matched clinical samples.Results:KLC2 protein was upregulated in NSCLC cell lines and tissues, and was an independent predictor of poor prognosis for elderly NSCLC patients. Kinesin-1 light chain-2 remarkably enhanced the invasive and migratory ability of NSCLC cells. MiR-125b inhibited KLC2 3′-untranslated region luciferase activity and protein expression, and inversely correlated with KLC2 expression in clinical samples. Kinesin-1 light chain-2 almost completely reversed miR-125b-induced inhibition on migration and invasion.Conclusions:Kinesin-1 light chain-2 protein overexpression predicts poor survival in elderly NSCLC patients. Kinesin-1 light chain-2 acts as a proto-oncogene and a functional target of miR-125b in NSCLC cells.

NPTX1 is a novel epigenetic regulation gene and associated with prognosis in lung cancer

BackgroundCpG island hypermethylation of gene promoters is a well-known mechanism of epigenetic regulation of tumor related-genes and is directly linked to lung carcinogenesis. Alterations in the pattern of methylation of the NPTX1 gene have not yet been studied in detail in human lung cancer. MethodsMethylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP) were used to analyze promoter methylation status, and real-time quantitative reverse transcription-PCR (qRT-PCR) examined mRNA levels. Subsequently, we compared the methylation profile of NPTX1 in samples of neoplastic and non-neoplastic lung tissue taken from the same patients by using quantitative methylation specific PCR (QMSP). ResultsCpG island hypermethylation in promoter of NPTX1 was confirmed in lung cancer cell lines. A significant increase in NPTX1 methylation was identified in lung cancer specimens compared to adjacent noncancerous tissues and that it was negatively correlated with its mRNA expression. The overall survival time among patients carrying methylated NPTX1 tumors was significantly shorter as compared to those with unmethylated NPTX1 tumors (P = 0.011). Moreover, methylation of NPTX1 gene was found to be an independent prognostic factor for poor overall survival based on multivariate analysis models (p = 0.021), as was age ≥60 years old (p = 0.012) and TNM stage (p < 0.001). ConclusionsThese results suggest that NPTX1 hypermethylation and consequent mRNA changes might be an important molecular mechanism in lung cancer. Epigenetic alterations in NPTX1 may serve as potential diagnostic and prognostic biomarkers in lung cancer.

Thyroid Transcription Factor-1 is a Specific Marker of Benign but Not Malignant Feline Lung Tumours

Feline lung tumours are currently subclassified according to the criteria of the World Health Organisation, but this scheme contains overlap in the tumour phenotype. The aims of the present study were to re-evaluate the histological features of feline lung tumours and to correlate these with expression of the markers thyroid transcription factor (TTF)-1 and Ki67. TTF-1 was found to be a highly specific marker for neoplastic and non-neoplastic lung tissue and thyroid tissue, but was expressed only weakly in invasive lung tumours. A combined semiquantitative score for Ki67 and TTF-1 expression correlated well with differentiation and invasive behaviour of the tumours and may thus be of potential value for evaluation of feline lung tumours.

Cellular localization of protein arginine methyltransferase-5 correlates with grade of lung tumors

BackgroundProtein arginine methyltransferase-5 (PRMT5) is a chromatin-modifying enzyme capable of methylating histone and non-histone proteins, and is involved in a wide range of cellular processes that range from transcriptional regulation to organelle biosynthesis. As such, its overexpression has been linked to tumor suppressor gene silencing, enhanced tumor cell growth and survival.Material and methodsQuantitative real-time polymerase chain reaction, Western immunoblot and immunohistochemistry were used to characterize PRMT5 expression in lung cancer cell lines and human tumors. Clinicopathological findings of tissue microarray based samples from 229 patients with non-small cell lung carcinomas (NSCLC) and 133 cases with pulmonary neuroendocrine tumors (NET) were analyzed with regard to nuclear and cytoplasmic PRMT5 expression.ResultsThere was statistically significant difference in PRMT5 messenger RNA expression between tumors and nonneoplastic lung tissues. Immunoblot experiments showed abundant expression of PRMT5 and its symmetric methylation mark H4R3 in lung carcinoma but not in non-neoplastic human pulmonary alveolar and bronchial epithelial cell lines. More than two thirds of lung tumors expressed PRMT5. High levels of cytoplasmic PRMT5 were detected in 20.5% of NSCLC and in 16.5% of NET; high levels of nuclear PRMT5 were detected in 38.0% of NSCLC and 24.0% of NET. Cytoplasmic PRMT5 was associated with high grade in both NSCLC and pulmonary NET while nuclear PRMT5 was more frequent in carcinoid tumors (p < 0.05).ConclusionThe observed findings support the role of PRMT5 in lung tumorigenesis and reflect its functional dichotomy in cellular compartments.Virtual slideThe virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1611895162102528

Multiphoton Microscopy: A Potential “Optical Biopsy” Tool for Real-Time Evaluation of Lung Tumors Without the Need for Exogenous Contrast Agents

Multiphoton microscopy (MPM) is an emerging, nonlinear, optical-biopsy technique, which can generate subcellular-resolution images from unprocessed and unstained tissue in real time. To assess the potential of MPM for lung tumor diagnosis. Fresh sections from tumor and adjacent nonneoplastic lung were imaged with MPM and then compared with corresponding hematoxylin-eosin slides. Alveoli, bronchi, blood vessels, pleura, smokers' macrophages, and lymphocytes were readily identified with MPM in nonneoplastic tissue. Atypical adenomatous hyperplasia (a preinvasive lesion) was identified in tissue adjacent to the tumor in one case. Of the 25 tumor specimens used for blinded pathologic diagnosis, 23 were diagnosable with MPM. Of these 23 cases, all but one adenocarcinoma (15 of 16; 94%) was correctly diagnosed on MPM, along with their histologic patterns. For squamous cell carcinoma, 4 of 7 specimens (57%) were correctly diagnosed. For the remaining 3 squamous cell carcinoma specimens, the solid pattern was correctly diagnosed in 2 additional cases (29%), but it was not possible to distinguish the squamous cell carcinoma from adenocarcinoma. The other squamous cell carcinoma specimen (1 of 7; 14%) was misdiagnosed as adenocarcinoma because of pseudogland formation. Invasive adenocarcinomas with acinar and solid pattern showed statistically significant increases in collagen. Interobserver agreement for collagen quantification (among 3 observers) was 80%. Our pilot study provides a proof of principle that MPM can differentiate neoplastic from nonneoplastic lung tissue and identify tumor subtypes. If confirmed in a future, larger study, we foresee real-time intraoperative applications of MPM, using miniaturized instruments for directing lung biopsies, assessing their adequacy for subsequent histopathologic analysis or banking, and evaluating surgical margins in limited lung resections.

Association of small foci of diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH) with adenocarcinoma of the lung

DIPNECH is regarded as a precursor lesion of neuroendocrine lung tumors, specifically carcinoids. A relationship with lung adenocarcinomas has not been clearly established so far. We present a series of four cases with a concomitant presence of adenocarcinoma and DIPNECH in the lung.The cases were retrieved from the archives of the Institutes of Pathology of the Jena University Hospital and the Charité, Berlin. The clinical data were collected from the hospital information system. The microscopic findings of adenocarcinoma and DIPNECH were reviewed. A panel of neuroendocrine and epithelial markers was analyzed immunohistochemically. In addition, the H&E slides of a series of 82 lung carcinomas were reevaluated for the presence of DIPNECH foci and the parameters of the IASLC/ATS/ERS classification for lung adenocarcinoma.DIPNECH foci were composed of small intramucosal nests of proliferating pulmonary neuroendocrine cells alongside or at the periphery of terminal airways. All detected foci measured less than 5mm in maximal diameter and showed a consistent reactivity against Synaptophysin. They did not express epithelial markers of squamous cell carcinoma and adenocarcinoma. In three cases, the DIPNECH foci were clearly associated with the adenocarcinoma, while in one case, they were observed in the non-neoplastic lung tissue. The adenocarcinoma with DIPNECH inside mainly showed low grade histology, while the fourth case was intermediate to high grade. The histologic evaluation of the HE slides of the other 82 lung cancer cases showed no suspected or definite DIPNECH foci. Within this series, we could confirm the prognostic significance of the IASLC/ATS/ERS classification of lung adenocarcinoma.Our series suggest that a subset of lung adenocarcinoma is characterized by the concomitant presence of DIPNECH within the tumor, suggesting a causal relationship. These adenocarcinomas seem to be low grade ones, and may have a particular tumorigenesis and clinical behavior. These observations need to be confirmed in larger tumor collectives. We could confirm the prognostic relevance of the new adenocarcinoma classification.

Metabolic Alterations in Lung Cancer–Associated Fibroblasts Correlated with Increased Glycolytic Metabolism of the Tumor

Cancer cells undergo a metabolic reprogramming but little is known about metabolic alterations of other cells within tumors. We use mass spectrometry-based profiling and a metabolic pathway-based systems analysis to compare 21 primary human lung cancer-associated fibroblast lines (CAF) to "normal" fibroblast lines (NF) generated from adjacent nonneoplastic lung tissue. CAFs are protumorigenic, although the mechanisms by which CAFs support tumors have not been elucidated. We have identified several pathways whose metabolite abundance globally distinguished CAFs from NFs, suggesting that metabolic alterations are not limited to cancer cells. In addition, we found metabolic differences between CAFs from high and low glycolytic tumors that might reflect distinct roles of CAFs related to the tumor's glycolytic capacity. One such change was an increase of dipeptides in CAFs. Dipeptides primarily arise from the breakdown of proteins. We found in CAFs an increase in basal macroautophagy which likely accounts for the increase in dipeptides. Furthermore, we show a difference between CAFs and NFs in the induction of autophagy promoted by reduced glucose. In sum, our data suggest that increased autophagy may account for metabolic differences between CAFs and NFs and may play additional as yet undetermined roles in lung cancer.

The apoptosis of non-small cell lung cancer induced by cisplatin through modulation of STIM1

Cis-diamminedichloroplatinum (II) (cisplatin) is one of the most active antitumor agents used in human chemotherapy of non-small cell lung cancer. Cisplatin forms crosslinked DNA adducts and its cytotoxicity has been shown to be mediated by propagation of DNA damage recognition signals to downstream pathways prompting apoptosis. The steps involved in the process include changes in Ca(2+) signaling with dysregulated tumor cell turn-over. Stromal interaction molecules 1 (STIM1), as one of the most potent tumor suppressor genes, are identified as the endoplasmic-reticulum (ER) Ca(2+) sensor controlling store-operated Ca(2+) entry (SOCE) in non-excitable cells, which is main pathway to extracellular Ca(2+) influx. Its role in STIM1 cisplatin-induced apoptosis of non-small cell lung cancer was the focus of study with focus on SOCE inhibitors 2-APB- and SKF96365-cisplatin-induced apoptosis in the non-small cell lung cancer (NSCLC) cell lines A549 and H460. In this experimental model, cisplatin-induced apoptosis and decreased concentration of intracellular Ca(2+) was demonstrated. The expression of STIM1 was significantly higher in carcinoma tissue than in the adjacent non-neoplastic lung tissue. These findings support the conclusion that STIM1 may play an important role in the development of NSCLC which makes drugs that repress the expression of STIM1 to be a potential target for lung cancer therapy.

Abstract 1420: Mechanisms of stroma-derived CCL7 in promoting lung cancer.

Abstract Lung cancer is a leading cause of cancer related deaths worldwide, with non-small cell lung cancer (NSCLC) being the most prevalent form. Considerable attention has been directed to cancer cell intrinsic abnormalities (K-ras, p53, EGFR, AML-ALK4 fusions etc.), but little is known about the contribution of stromal cells to NSCLC progression. We have undertaken a comprehensive analysis to determine the contribution and biological function of the stromal cells in NSCLC. We show increased recruitment of BM hematopoietic cells in the tumor beds compared to matched adjacent non-neoplastic lung tissue in NSCLC patients. Genome-wide transcriptome analysis on specific cell populations from fresh samples of NSCLC patients identified differentially regulated genes and mRNA isoforms in NSCLC stromal cells. Genes that were successfully validated by independent RT-PCR on sorted stromal cells and tumor cells were used for further analysis. Many genes encoded secreted proteins, suggesting the activation of aberrant autocrine and paracrine regulatory loops likely to support stroma-mediated tumor progression. Among these genes, CCL7 was upregulated in myeloid progenitor cells and was selected for further investigation. Immunostaining of lung tumor and adjacent non-neoplastic tissue of patients shows highest expression of CCL7 in BM-derived cells (CD45+ cells) in the tumor. Cross species analysis showed that, consistent with the observation in human tissue, CCL7 was differentially regulated in BM derived cells in a mouse model of NSCLC generated in KrasG12D and p53-/- transgenic mice. We have genetically ablated CCL7 in the BM cells using lentiviral shRNA and BMT approaches and will discuss consequences of this ablation in CCL7-mediated crosstalk between tumor cells and stromal cells that regulate NSCLC progression. Citation Format: Hyejin Choi, Anna Durrans, Dingcheng Gao, Seongho Ryu, Nasser Altorki, Vivek Mittal. Mechanisms of stroma-derived CCL7 in promoting 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 1420. doi:10.1158/1538-7445.AM2013-1420

Abstract 4972: Analysis of the reprogrammed bone marrow-derived stroma in lung cancer unravels potential targets for prognosis and therapy.

Abstract Background: Lung cancer is the leading cause of cancer related mortality, with NSCLC the most prevalent form. Despite advances in treatment options (minimally invasive surgery, CT-guided radiation, novel chemotherapy regimens, targeted therapeutics) prognosis remains dismal. Most research to date has focused on cancer cell intrinsic mutagenesis (K-ras, p53, EGFR, EML4-ALK fusion), however recent studies have begun to recognize that cross talk between tumor epithelial cells and intratumoral stromal cells contributes to tumor progression. Unfortunately, little is known about the contribution or role of the stroma in NSCLC. Here, we present the first comprehensive analysis of specific individual stromal components of human NSCLC, which has led to the identification of stromal-specific activated pro-tumorigenic genes of prognostic and therapeutic potential. Materials and Methods: Surgically resected Stage I/II adenocarcinoma and matched adjacent non-neoplastic lung tissue were mechanically dissociated within 1hour of resection. Cell populations marked with specific antibodies were sorted to homogeneity by flow cytometry. RNA was isolated to construct cDNA libraries, which were deep sequenced using the Illumina HiSeq 2000. Validation of differentially regulated genes was carried out by RT-PCR and immunostaining. Mouse models of K-rasG12D and p53flox/flox derived NSCLC together with a bone marrow transplantation approach were used to determine the functional role of activated genes, and a NSCLC tissue microarray was used to ascertain their prognostic value. Results: Flow cytometry analysis revealed a 4-fold increase in the number of intratumoral hematopoietic cells, compared with adjacent non-neoplastic lung tissue. Deep RNA sequencing of two predominant myeloid specific subpopulations, CD11b+CD33+ monocytic myeloid cells and CD11b+CD33- polymorphonuclear neutrophils, revealed 783 and 938 differentially regulated genes respectively when comparing tumor vs. adjacent lung-derived cells. Many genes encoded secreted proteins such as OPN, TSP1, CCL7, and MMP12, suggesting activation of aberrant autocrine and paracrine regulatory pathways likely to support stromal-mediated tumor progression. We will present data showing the prognostic and therapeutic potential of these genes in NSCLC. Conclusions: This study is based on an emerging paradigm that activated intratumoral stromal cells play a fundamental role in promoting tumor growth. Using a unique and novel approach of quantitative analysis of individual stromal components from freshly procured NSCLC tumor tissue, we have identified differentially regulated genes previously undiscovered in traditional whole tumor profiling approaches. The activated stromal genes constitute novel therapeutic targets against lung cancer, either alone or in combination with treatments that target cancer cells. Citation Format: Anna Durrans, Dingcheng Gao, Ravi Gupta, Seongho Ryu, Navneet Narula, Kari R. Fischer, Hyejin Choi, Brendon M. Stiles, Abu Nasar, Ramana V. Davuluri, Nasser K. Altorki, Vivek Mittal. Analysis of the reprogrammed bone marrow-derived stroma in lung cancer unravels potential targets for prognosis and therapy. [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 4972. doi:10.1158/1538-7445.AM2013-4972

Abstract 2373: The LCBRN: a biospecimen resource for lung cancer biomarker and discovery science.

Abstract The Lung Cancer Biospecimen Resource Network (LCBRN) is an open access biorepository that obtains specimens from lung cancer patients (tissue, blood, urine, bronchial lavage fluid and saliva) and makes them available to biomedical researchers. Specimen collection is supported at 3 collection sites (Medical University of South Carolina, The University of Virginia, and Washington University at St. Louis) in compliance with uniform standard operating procedures. The specimens are annotated with clinical, radiologic and pathologic data. Histologic and molecular quality control procedures are in place for tissue samples. Lung cancer and non-neoplastic lung tissue aliquots are processed into DNA, RNA and protein fractions, which are also available. Fluid biosamples are available from subjects prior to, and after surgery to remove the cancer. A cohort of subjects are followed for recurrence with serial biofluid sampling. Specimens are currently available from over 300 research subjects. The application form for biospecimens and additional information is available at http://LCBRN.org. Citation Format: Christopher A. Moskaluk. The LCBRN: a biospecimen resource for lung cancer biomarker and discovery science. [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 2373. doi:10.1158/1538-7445.AM2013-2373

Expression of Adiponectin Receptor 1 Is Indicative of Favorable Prognosis in Non-small Cell Lung Carcinoma

Lung cancer is a major cause of cancer-related death worldwide. It is believed that obesity-related malignancies such as breast, endometrial, colorectal, and kidney carcinomas have lower plasma level and/or tissue expression of adiponectin receptors. However, the association between adiponectin receptors and lung cancer, a non obesity-related malignancy, is still unknown. We evaluated the tissue expression of adiponectin receptor (AdipoR) 1 and AdipoR2 in 83 cases of non-small cell lung carcinoma (NSCLC) and matched non-neoplastic lung tissues by immunohistochemistry and real-time polymerase chain reaction (PCR). Clinicopathological data, including smoking history, smoker's bronchiolitis, emphysema, lymph node metastasis, and T-stage were collected and evaluated. Expression of immunohistochemically stained AdipoR1 and AdipoR2 was observed in all samples of non-neoplastic lung tissues. Both receptors showed higher mRNA expression in non-neoplastic than neoplastic tissues (p < 0.05). In NSCLC tissues, AdipoR1 immunohistochemical expression was not observed in most of patients with squamous cell carcinoma and current smoking history (31/42, p = 0.04 and 25/29, p = 0.003, respectively). Additionally, AdipoR1 mRNA expression was significantly lower in patients with lymph node metastasis (p = 0.05). Meanwhile, AdipoR2 immunohistochemical stain expression was inversely correlated with T-stage (p = 0.05) and AdipoR2 mRNA expression was significantly lower in patients with smoker's bronchiolitis (p = 0.01) and emphysema (p = 0.03). Patients with expression of AdipoR1 had longer overall survival. AdipoR2 expression was not correlated with patients' survival. In conclusion, we suggest that expression of AdipoR1 is indicative of favorable prognosis and may be used as prognostic marker in NSCLC.

Full-field optical coherence tomography for the analysis of fresh unstained human lobectomy specimens

Background: Full-field optical coherence tomography (FFOCT) is a real-time imaging technique that generates high-resolution three-dimensional tomographic images from unprocessed and unstained tissues. Lack of tissue processing and associated artifacts, along with the ability to generate large-field images quickly, warrants its exploration as an alternative diagnostic tool. Materials and Methods: One section each from the tumor and from adjacent non-neoplastic tissue was collected from 13 human lobectomy specimens. They were imaged fresh with FFOCT and then submitted for routine histopathology. Two blinded pathologists independently rendered diagnoses based on FFOCT images. Results: Normal lung architecture (alveoli, bronchi, pleura and blood vessels) was readily identified with FFOCT. Using FFOCT images alone, the study pathologists were able to correctly identify all tumor specimens and in many cases, the histological subtype of tumor (e.g., adenocarcinomas with various patterns). However, benign diagnosis was provided with high confidence in roughly half the tumor-free specimens (others were diagnosed as equivocal or false positive). Further analysis of these images revealed two major confounding features: (a) Extensive lung collapse and (b) presence of smoker’s macrophages. On a closer inspection, however, the smoker’s macrophages could often be identified as distinct from tumor cells based on their relative location in the alveoli, size and presence of anthracosis. We posit that greater pathologist experience, complemented with morphometric analysis and color-coding of image components, may help minimize the contribution of these confounders in the future. Conclusion: Our study provides evidence for the potential utility of FFOCT in identifying and differentiating lung tumors from non-neoplastic lung tissue. We foresee its potential as an adjunct to intra-surgical frozen section analysis for margin assessment, especially in limited lung resections.

Overexpression of aldolase A and cytokeratin 19 in ovine pulmonary adenocarcinoma

Ovine pulmonary adenocarcinoma (OPA) is a transmissible lung cancer of sheep caused by jaagsiekte sheep retrovirus (JSRV). In the present study the protein profiles of five neoplastic and three non-neoplastic sheep lung tissues were examined for the identification of proteins overexpressed in ovine pulmonary adenocarcinoma. Lung sections of the experimental group of sheep were collected during necropsies for proteomic and immunohistochemical examination. Two dimensional electrophoresis (2DE) was performed using gel strips with immobilized pH gradient 3-10. As a result of 2DE gel analysis 14 spots characterized by over 2-fold higher expression in tumour proteomes were selected for mass spectrometry. In eleven spots more than one polypeptide was identified indicating overlapping of proteins in gels. In two spots demonstrating over 3-fold higher expression in OPA proteomes, single proteins: cytokerarin 19 (CK19) and aldolase A were identified. Immunohistochemical studies revealed that CK19 and aldolase A were expressed in the cytoplasm of epithelial cells of bronchioles in non-neoplastic lung sections, as well as epithelial cells of bronchioles and neoplastic cells in lung sections of OPA affected sheep. The results indicate that the overexpression of the two proteins reflects the presence of neoplastic cells in the lungs of OPA affected sheep.

CK2 Enzyme Affinity Against c-myc424-434Substrate in Human Lung Cancer Tissue

CK2 is a serine threonine kinase that participates in a variety of cellular processes with more than 300 defined substrates. This critical enzyme is known to be upregulated in cancers, but the role of this upregulation in carcinogenesis is not yet fully understood but c-myc, one of the defined CK2 substrates, is a well-known proto- oncogene that is normally essential in developmental process but is also involved in tumor development. We evaluated the optimal enzyme and substrate concentrations for CK2 activity in both neoplastic and non-neoplastic human lung tissues using the c-myc 424-434 peptide (EQKLISEEDL) as a substrate. The activities measured for the neoplastic tissue were 600-750 U/mg protein while those for the control tissue was in the range of 650-800 U/ mg. Km value for c-myc peptide was determined as 0.33 μM in non-neoplastic tissue and 0.18 μM in neoplastic tissue. In this study, we did not observe an increased activity in the neoplastic tissue when compared with the non-neoplastic lung tissue, but we recorded two times higher affinity for c-myc 424-434 in cancer tissue. Considering the metabolic position of c-myc 424-434, our results suggest that phosphorylation by CK2 may be important in dimerization and thus it might affect the regulation of c-myc in cancer tissues.

WT1 CpG islands methylation in human lung cancer: A pilot study

BackgroundCpG island hypermethylation of gene promoters and regulatory regions is a well-known mechanism of epigenetic silencing of tumor suppressors and is directly linked to carcinogenesis. Wilm’s tumor gene (WT1) is a tumor suppressor protein involved in the regulation of human cell growth and differentiation and a modulator of oncogenic K Ras signaling in lung cancer. Changes in the pattern of methylation of the WT1 gene have not yet been studied in detail in human lung cancer. In this study we compared the methylation profile of WT1 gene in samples of neoplastic and non-neoplastic lung tissue taken from the same patients. MethodsDNA was extracted from neoplastic and normal lung tissue obtained from 16 patients with non small cell lung cancer (NSCLC). The methylation status of 29 CpG islands in the 5′ region of WT1 was determined by pyrosequencing. Statistical analysis was carried out by T test and Mann Whitney test. ResultsThe mean percentage of methylation, considering all CpG islands of WT1 in the neoplastic tissues of the 16 NSCLC patients, was 16.2±3.4, whereas in the normal lung tissue from the same patients it was 5.6±1.7 (p<0.001). Adenocarcinomas presented higher methylation levels than squamous cell carcinomas (p<0,001). ConclusionsMethylation of WT1 gene is significantly increased in NSCLC. Both histotype and exposure to cigarette smoke heavily influence the pattern of CpG islands which undergo hypermethylation.