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Lung cancer has been shown to be targetable by novel immunotherapies which reactivate the immune system and enable tumor cell killing. However, treatment failure and resistance to these therapies is common. Consideration of sex as a factor influencing therapy resistance is still rare. We hypothesize that the success of the treatment is impaired by the presence of the immunosuppressive pregnancy-associated glycoprotein glycodelin that is expressed in patients with non-small-cell lung cancer (NSCLC). We demonstrate that the glycan pattern of NSCLC-derived glycodelin detected by a lectin-based enrichment assay highly resembles amniotic fluid-derived glycodelin A, which is known to have immunosuppressive properties. NSCLC-derived glycodelin interacts with immune cells in vitro and regulates the expression of genes associated with inflammatory and tumor microenvironment pathways. In tumor microarray samples of patients, high glycodelin staining in tumor areas results in an impaired overall survival of female patients. Moreover, glycodelin colocalizes to tumor infiltrating CD8+ T cells and pro-tumorigenic M2 macrophages. High serum concentrations of glycodelin prior to immunotherapy are associated with a poor progression-free survival (p < 0.001) of female patients receiving PD-(L)1 inhibitors. In summary, our findings suggest that glycodelin not only is a promising immunological biomarker for early identification of female patients that do not benefit from the costly immunotherapy, but also represents a promising immunotherapeutic target in NSCLC to improve therapeutic options in lung cancer., Competing Interests: Declaration of competing interest The authors declare no conflict of interest, (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: Lung cancer is a major cause of cancer-related death worldwide and effective therapies, besides surgery, are available only for a small proportion of patients. Since cellular respiration is known to be broadly altered in malignant tumors, the cellular processes of respiration can be a potential therapeutic target. One important element of cellular respiration is creatine and its transport by the creatine transporter SLC6A8. Here we describe the expression of SLC6A8 at the RNA and protein level, epigenetic modifications as well as survival analysis in NSCLC tissues and matched controls., Materials and Methods: We analyzed epigenetic modifications of the SLC68A gene in 32 patients, of which 18 were additionally analyzed by transcriptome analysis. The expression of SLC6A8 at the protein level was assessed by immunohistochemistry using an independent cohort and correlated with clinicopathological data including survival. Kaplan-Meier analysis was performed to analyze the possible effects of the transcriptional levels of SLC6A8 in another separate cohort (n=1925)., Results: SLC6A8 loci are epigenetically modified in NSCLC compared with tumor-free controls. SLC6A8 is upregulated in NSCLC at the RNA and protein level. High mRNA expression of SLC6A8 was associated with an overall poor prognosis in lung adenocarcinoma patients and displayed the strongest adverse prognostic effect in male smokers with adenocarcinomas. Results of transcriptome analysis were partially confirmed at the protein level., Conclusions: Our results suggest an important role of creatine and its transport via SLC6A8 in NSCLC., (©The Author(s) 2024. Open Access. This article is licensed under a Creative Commons CC-BY International License.)

Background: Lung cancers arising in never smokers have been suggested to be substantially different from lung cancers in smokers at an epidemiological, genetic and molecular level. Focusing on non-small cell lung cancer (NSCLC), we characterized lung cancer patients in China looking for demographic and clinical differences between the smoking and never-smoking subgroups., Methods: In total, 891 patients with NSCLC, including 841 with adenocarcinoma and 50 with squamous cell carcinoma, were recruited in this study. Association of smoking status with demographic and clinical features of NSCLC was determined, and risk factors for lymph node metastasis and TNM stage were evaluated using Multivariate logistic regression analysis., Results: In patients with adenocarcinoma, never smokers showed a younger age at diagnosis (54.2 ± 12.7vs. 59.3 ± 9.4, p adjusted <0.001), a lower risk for lymph node metastasis than smokers (7,6% vs. 19.5%, p adjusted <0.001) and less severe disease as indicated by lower percentages of patients with TNM stage of III or IV (5.5% vs. 14.7%, p adjusted <0.001 ). By contrast, these associations were not observed in 50 patients with squamous cell carcinoma. Multivariate logistic regression analysis showed that smoking status was a risk factor for lymph node metastasis (OR = 2.70, 95% CI: 1.39-5.31, p = 0.004) but not for TNM stage (OR = 1.18, 95% CI: 0.09-14.43, p = 0.896) in adenocarcinoma., Conclusion: This study demonstrates that lung adenocarcinoma in never smokers significantly differ from those in smokers regarding both age at diagnosis and risk of lymph node metastasis, supporting the notion that they are distinct entries with different etiology and pathogenesis., (© 2022. The Author(s).)

Introduction: The advent of immune checkpoint blockade (ICB) has led to significantly improved disease outcome in lung adenocarcinoma (ADC), but response of ALK/EGFR-positive tumors to immune therapy is limited. The underlying immune biology is incompletely understood., Methods: We performed comparative mRNA expression profiling of 31 ALK-positive, 40 EGFR-positive and 43 ALK/EGFR-negative lung ADC focused on immune gene expression. The presence and levels of tumor infiltration lymphocytes (TILs) as well as fourteen specific immune cell populations were estimated from the gene expression profiles., Results: While total TILs were not lower in ALK-positive and EGFR-positive tumors compared to ALK/EGFR-negative tumors, specific immunosuppressive characteristics were detected in both subgroups: In ALK-positive tumors, regulatory T cells were significantly higher compared to EGFR-positive (fold change: FC = 1.9, p = 0.0013) and ALK/EGFR-negative tumors (FC = 2.1, p = 0.00047). In EGFR-positive tumors, cytotoxic cells were significantly lower compared to ALK-positive (FC =  - 1.7, p = 0.016) and to ALK/EGFR-negative tumors (FC =  - 2.1, p = 2.0E-05). A total number of 289 genes, 40 part of cytokine-cytokine receptor signaling, were differentially expressed between the three subgroups. Among the latter, five genes were differently expressed in both ALK-positive and EGFR-positive tumors, while twelve genes showed differential expression solely in ALK-positive tumors and eleven genes solely in EGFR-positive tumors., Conclusion: Targeted gene expression profiling is a promising tool to read out tumor microenvironment characteristics from routine diagnostic lung cancer biopsies. Significant immune reactivity including specific immunosuppressive characteristics in ALK- and EGFR-positive lung ADC, but not a total absence of immune infiltration supports further clinical evaluation of immune-modulators as partners of ICB in such tumors., (© 2021. The Author(s).)

Gene amplification is considered to be one responsible cause for upregulation of Programmed Death Ligand-1 (PD-L1) in non-small cell lung cancer (NSCLC) and to represent a specific molecular subgroup possibly associated with immunotherapy response. Our aim was to analyze the frequency of PD-L1 amplification, its relation to PD-L1 mRNA and protein expression, and to characterize the immune microenvironment of amplified cases. The study was based on two independent NSCLC cohorts, including 354 and 349 cases, respectively. Tissue microarrays were used to evaluate PD-L1 amplification by FISH and PD-L1 protein by immunohistochemistry. Immune infiltrates were characterized immunohistochemically by a panel of immune markers (CD3, CD4, CD8, PD-1, Foxp3, CD20, CD138, CD168, CD45RO, NKp46). Mutational status was determined by targeted sequencing. RNAseq data was available for 197 patients. PD-L1 amplification was detected in 4.5% of all evaluable cases. PD-L1 amplification correlated only weakly with mRNA and protein expression. About  37% of amplified cases were negative for PD-L1 protein. PD-L1 amplification did not show any association with the mutational status. In squamous cell cancer, PD-L1 amplified cases were enriched among patients with high tumoral immune cell infiltration and showed gene expression profiles related to immune exhaustion. In conclusion, PD-L1 amplification correlates with PD-L1 expression in squamous cell cancer and was associated with an immune cell rich tumor phenotype. The correlative findings help to understand the role of PD-L1 amplification as an important immune escape mechanism in NSCLC and suggest the need to further evaluate PD-L1 amplification as predictive biomarker for checkpoint inhibitor therapy., (© 2021. The Author(s).)

The only potentially curative treatment for lung adenocarcinoma patients remains complete resection of early-stage tumors. However, many patients develop recurrence and die of their disease despite curative surgery. Underlying mechanisms leading to establishment of systemic disease after complete resection are mostly unknown. We therefore aimed at identifying molecular signatures of resected lung adenocarcinomas associated with the risk of an early relapse. The study comprised 89 patients with totally resected stage IA-IIIA lung adenocarcinomas. Patients suffering from an early relapse within two years after surgery were compared to patients without a relapse in two years. Patients were clinically and molecular pathologically characterized. Tumor tissues were immunohistochemically analyzed for the expression of Ki67, CD45, CD4, CD8, PD1, PD-L1, PD-L2 and CD34, by Nanostring nCounter PanCancer Immune Profiling Panel as well as a comprehensive methylome profiling using the Infinium MethylationEPIC BeadChip. We detected differential DNA methylation patterns as well as significantly differentially expressed genes associated with an early relapse after complete resection. Especially, CD1A was identified as a potential biomarker, whose reduced expression is associated with an early relapse. These findings might help to develop biomarkers improving risk assessment and patient selection for adjuvant therapy as well as establish novel targeted therapeutic strategies.

Background: Lung cancer is the leading cause of cancer-related death in most western countries in both, males and females, accounting for roughly 20-25% of all cancer deaths. For choosing the most appropriate therapy regimen a definite diagnosis is a prerequisite. However, histological characterization of bronchoscopic biopsies particularly with low tumor cell content is often challenging. Therefore, this study aims at (a) determining the value of DNA methylation analysis applied to specimens obtained by bronchoscopic biopsy for the diagnosis of lung cancer and (b) at comparing aberrantly CpG loci identified in bronchoscopic biopsy with those identified by analyzing surgical specimens., Results: We report the HumanMethylation450-based DNA methylation analysis of paired samples of bronchoscopic biopsy specimens either from the tumor side or from the contralateral tumor-free bronchus in 37 patients with definite lung cancer diagnosis and 18 patients with suspicious diagnosis. A differential DNA methylation analysis between both biopsy sites of patients with definite diagnosis identified 1303 loci. Even those samples were separated by the set of 1303 loci in which histopathological analysis could not unambiguously define the dignity. Further differential DNA methylation analyses distinguished between SCLC and NSCLC. We validated our results in an independent cohort of 40 primary lung cancers obtained by open surgical resection and their corresponding controls from the same patient as well as in publically available DNA methylation data from a TCGA cohort which could also be classified with high accuracy., Conclusions: Considering that the prognosis correlates with tumor stage at time of diagnosis, early detection of lung cancer is vital and DNA methylation analysis might add valuable information to reliably characterize lung cancer even in histologically ambiguous sample material.

Background: The interplay of immune and cancer cells takes place in the tumor microenvironment where multiple signals are exchanged. The transforming growth factor beta (TGFB) pathway is known to be dysregulated in lung cancer and can impede an effective immune response. However, the exact mechanisms are yet to be determined. Especially which cells respond and where does this signaling take place with respect to the local microenvironment., Methods: Human non-small cell lung cancer samples were retrospectively analyzed by multiplexed immunohistochemistry for SMAD3 phosphorylation and programmed death ligand 1 expression in different immune cells with respect to their localization within the tumor tissue. Spatial relationships were studied to examine possible cell-cell interactions and analyzed in conjunction with clinical data., Results: TGFB pathway activation in CD3, CD8, Foxp3 and CD68 cells, as indicated by SMAD3 phosphorylation, negatively impacts overall and partially disease-free survival of patients with lung cancerindependent of histological subtype. A high frequency of Foxp3 regulatory T cells positive for SMAD3 phosphorylation in close vicinity of CD8 T cells within the tumor discriminate a rapidly progressing group of patients with lung cancer., Conclusions: TGFB pathway activation of local immune cells within the tumor microenvironment impacts survival of early stage lung cancer. This might benefit patients not eligible for targeted therapies or immune checkpoint therapy as a therapeutic option to re-activate the local immune response., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Aging affects the core processes of almost every organism, and the functional decline at the cellular and tissue levels influences disease development. Recently, it was shown that the methylation of certain CpG dinucleotides correlates with chronological age and that this epigenetic clock can be applied to study aging-related effects. We investigated these molecular age loci in non-small cell lung cancer (NSCLC) tissues from patients with adenocarcinomas (AC) and squamous cell carcinomas (SQC) as well as in matched tumor-free lung tissue. In both NSCLC subtypes, the calculated epigenetic age did not correlate with the chronological age. In particular, SQC exhibited rejuvenation compared to the corresponding normal lung tissue as well as with the chronological age of the donor. Moreover, the younger epigenetic pattern was associated with a trend toward stem cell-like gene expression patterns. These findings show deep phenotypic differences between the tumor entities AC and SQC, which might be useful for novel therapeutic and diagnostic approaches., (© 2018 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Background: ADAMs (a disintegrin and metalloproteinase) have long been associated with tumor progression. Recent findings indicate that members of the closely related ADAMTS (ADAMs with thrombospondin motifs) family are also critically involved in carcinogenesis. Gene silencing through DNA methylation at CpG loci around e.g. transcription start or enhancer sites is a major mechanism in cancer development. Here, we aimed at identifying genes of the ADAM and ADAMTS family showing altered DNA methylation in the development or colorectal cancer (CRC) and other epithelial tumors., Methods: We investigated potential changes of DNA methylation affecting ADAM and ADAMTS genes in 117 CRC, 40 lung cancer (LC) and 15 oral squamous-cell carcinoma (SCC) samples. Tumor tissue was analyzed in comparison to adjacent non-malignant tissue of the same patients. The methylation status of 1145 CpGs in 51 ADAM and ADAMTS genes was measured with the HumanMethylation450 BeadChip Array. ADAMTS16 protein expression was analyzed in CRC samples by immunohistochemistry., Results: In CRC, we identified 72 CpGs in 18 genes which were significantly affected by hyper- or hypomethylation in the tumor tissue compared to the adjacent non-malignant tissue. While notable/frequent alterations in methylation patterns within ADAM genes were not observed, conspicuous changes were found in ADAMTS16 and ADAMTS2. To figure out whether these differences would be CRC specific, additional LC and SCC tissue samples were analyzed. Overall, 78 differentially methylated CpGs were found in LC and 29 in SCC. Strikingly, 8 CpGs located in the ADAMTS16 gene were commonly differentially methylated in all three cancer entities. Six CpGs in the promoter region were hypermethylated, whereas 2 CpGs in the gene body were hypomethylated indicative of gene silencing. In line with these findings, ADAMTS16 protein was strongly expressed in globlet cells and colonocytes in control tissue but not in CRC samples. Functional in vitro studies using the colorectal carcinoma cell line HT29 revealed that ADAMTS16 expression restrained tumor cell proliferation., Conclusions: We identified ADAMTS16 as novel gene with cancer-specific promoter hypermethylation in CRC, LC and SCC patients implicating ADAMTS16 as potential biomarker for these tumors. Moreover, our results provide evidence that ADAMTS16 may have tumor suppressor properties.

Although oncogenic activation of NFκB has been identified in various tumors, the NFκB-activating kinases (inhibitor of NFκB kinases, IKK) responsible for this are elusive. In this study, we determined the role of IKKα and IKKβ in KRAS -mutant lung adenocarcinomas induced by the carcinogen urethane and by respiratory epithelial expression of oncogenic KRAS G12D Using NFκB reporter mice and conditional deletions of IKKα and IKKβ, we identified two distinct early and late activation phases of NFκB during chemical and genetic lung adenocarcinoma development, which were characterized by nuclear translocation of Rel B, IκBβ, and IKKα in tumor-initiated cells. IKKα was a cardinal tumor promoter in chemical and genetic KRAS -mutant lung adenocarcinoma, and respiratory epithelial IKKα-deficient mice were markedly protected from the disease. IKKα specifically cooperated with mutant KRAS for tumor induction in a cell-autonomous fashion, providing mutant cells with a survival advantage in vitro and in vivo IKKα was highly expressed in human lung adenocarcinoma, and a heat shock protein 90 inhibitor that blocks IKK function delivered superior effects against KRAS -mutant lung adenocarcinoma compared with a specific IKKβ inhibitor. These results demonstrate an actionable requirement for IKKα in KRAS -mutant lung adenocarcinoma, marking the kinase as a therapeutic target against this disease. Significance: These findings report a novel requirement for IKKα in mutant KRAS lung tumor formation, with potential therapeutic applications. Cancer Res; 78(11); 2939-51. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Little is known about the human lung lipidome, its variability in different physiological states, its alterations during carcinogenesis and the development of pulmonary emphysema. We investigated how health status might be mirrored in the lung lipidome. Tissues were sampled for both lipidomic and histological analysis. Using a screening approach, we characterised lipidomes of lung cancer tissues and corresponding tumour-free alveolar tissues. We quantified 311 lipids from 11 classes in 43 tissue samples from 26 patients. Tumour tissues exhibited elevated levels of triacylglycerols and cholesteryl esters, as well as a significantly lower abundance of phosphatidylglycerols, which are typical lung surfactant components. Adenocarcinomas and squamous cell carcinomas were distinguished with high specificity based on lipid panels. Lipidomes of tumour biopsy samples showed clear changes depending on their histology and, in particular, their proportion of active tumour cells and stroma. Partial least squares regression showed correlations between lipid profiles of tumour-free alveolar tissues and the degree of emphysema, inflammation status, and the age of patients. Unsaturated long-chain phosphatidylserines and phosphatidylinositols showed a positive correlation with a worsened emphysema status and ageing. This work provides a resource for the human lung lipidome and a systematic data analysis strategy to link clinical characteristics and histology.

Targeting checkpoint inhibitors using monoclonal antibodies results in significantly better outcome of cancer patients compared to conventional chemotherapy. However, the current companion diagnostics to predict response is so far suboptimal, since they base on more or less reliable immunohistochemical approaches. In order to overcome these limitations, we analyzed epigenetic modifications of PDCD1 (PD1), CD274 (PD-L1), and CTLA4 in NSCLC tissues from 39 patients. Results were correlated with transcriptome data. Significant differences in the CpG-methylation patterns between tumor tissues and matched controls were observed for CTLA4 and PDCD1 (PD1) showing a decreased methylation of these genes compared to matched tumor-free tissues from the same patients. Results were confirmed by bisulfide sequencing in an independent validation cohort. Hypomethylation also resulted in increased expression of these genes as shown by transcriptome data. These epigenetic pathways as a hallmark of NSCLC might be useful to generate more precise diagnostic approaches in the future.

Targeting epidermal growth factor receptor (EGFR) in patients with non-small-cell lung cancer (NSCLC) having EGFR mutations is associated with an improved overall survival. The aim of this study is to verify, if EGFR mutations detected by immunohistochemistry (IHC) is a convincing way to preselect patients for DNA-sequencing and to figure out, the statistical association between EGFR mutation, wild-type EGFR overexpression, gene copy number gain, which are the main factors inducing EGFR tumorigenic activity and the clinicopathological data. Two hundred sixteen tumor tissue samples of primarily chemotherapeutic naïve NSCLC patients were analyzed for EGFR mutations E746-A750del and L858R and correlated with DNA-sequencing. Two hundred six of which were assessed by IHC, using 6B6 and 43B2 specific antibodies followed by DNA-sequencing of positive cases and 10 already genotyped tumor tissues were also included to investigate debugging accuracy of IHC. In addition, EGFR wild-type overexpression was IHC evaluated and EGFR gene copy number determination was performed by fluorescence in situ hybridization (FISH). Forty-one÷206 (19.9%) cases were positive for mutated EGFR by IHC. Eight of them had EGFR mutations of exons 18-21 by DNA-sequencing. Hit rate of 10 already genotyped NSCLC mutated cases was 90% by IHC. Positive association was found between EGFR mutations determined by IHC and both EGFR overexpression and increased gene copy number (p=0.002 and p<0.001, respectively). Additionally, positive association was detected between EGFR mutations, high tumor grade and clinical stage (p<0.001). IHC staining with mutation specific antibodies was demonstrated as a possible useful screening test to preselect patients for DNA-sequencing.

Non-small cell lung cancer (NSCLC) is characterized by early metastasis and has the highest mortality rate among all solid tumors, with the majority of patients diagnosed at an advanced stage where curative therapeutic options are lacking. In this study, we identify a targetable mechanism involving TGFβ elevation that orchestrates tumor progression in this disease. Substantial activation of this pathway was detected in human lung cancer tissues with concomitant downregulation of BAMBI, a negative regulator of the TGFβ signaling pathway. Alterations of epithelial-to-mesenchymal transition (EMT) marker expression were observed in lung cancer samples compared with tumor-free tissues. Distinct alterations in the DNA methylation of the gene regions encoding TGFβ pathway components were detected in NSCLC samples compared with tumor-free lung tissues. In particular, epigenetic silencing of BAMBI was identified as a hallmark of NSCLC. Reconstitution of BAMBI expression in NSCLC cells resulted in a marked reduction of TGFβ-induced EMT, migration, and invasion in vitro, along with reduced tumor burden and tumor growth in vivo In conclusion, our results demonstrate how BAMBI downregulation drives the invasiveness of NSCLC, highlighting TGFβ signaling as a candidate therapeutic target in this setting. Cancer Res; 76(13); 3785-801. ©2016 AACR., (©2016 American Association for Cancer Research.)

Background: Thoracic pathologists are frequently faced with tissue specimens from intrathoracic/mediastinal tumors. Specifically the differentiation between thymic and pulmonary squamous cell carcinomas (SqCC) can be challenging. In order to clarify the differential diagnostic value of CD5 and CD117 in this setting, we performed a large scale expression study of both markers in 1465 non-small cell lung cancer (NSCLC) cases., Methods: Tissue microarrays of formalin-fixed paraffin-embedded resection specimens of 1465 NSCLC were stained with antibodies against CD117 and CD5. Positivity of both markers was correlated with clinicopathological variables., Results: CD117 was positive in 145 out of 1457 evaluable cases (9.9 %) and CD5 was positive in 133 out of 1427 evaluable cases (9.3 %). 28 cases (1.9 %) showed coexpression of CD117 and CD5. Among the 145 cases that were positive for CD117, 97 (66.8 %) were adenocarcinomas (ADC), 34 (23.4 %) were SqCC, 5 (3.4 %) were adenosquamous carcinomas (ADSqCC), 8 (5.5 %) were large cell carcinomas (LC), and one (0.6 %) was a pleomorphic carcinoma (PC). In the CD5 positive group consisting of 133 cases, 123 (92.4 %) were ADC, 0 (0 %) were SqCC, 4 (3.0 %) were ADSqCC, 3 (2.2 %) LC and 3 (2.2 %) were PC. None of the 586 SqCC showed expression of CD5. No association of CD117- or CD5 positivity to patients' age, pathological stages or to T-, N-, or M- categories was observed., Conclusions: A substantial subset of NSCLC exhibit positivity of CD117 and CD5. Since CD5 expression was not observed in pulmonary SqCC, but is expressed in the majority of thymic squamous cell carcinomas, the application of this immunomarker is a valuable tool in the differential diagnosis of thoracic neoplasms.

Background: Several c-MET targeting inhibitory molecules have already shown promising results in the treatment of patients with Non-small Cell Lung Cancer (NSCLC). Combination of EGFR- and c-MET-specific molecules may overcome EGFR tyrosine kinase inhibitor (TKI) resistance. The aim of this study was to allow for the identification of patients who might benefit from TKI treatments targeting MET and to narrow in on the diagnostic assessment of MET., Methods: 222 tumor tissues of patients with NSCLC were analyzed concerning c-MET expression and activation in terms of phosphorylation (Y1234/1235 and Y1349) using a microarray format employing immunohistochemistry (IHC). Furthermore, protein expression and MET activation was correlated with the amplification status by Fluorescence in Situ Hybridization (FISH)., Results: Correlation was observed between phosphorylation of c-MET at Y1234/1235 and Y1349 (spearman correlation coefficient rs = 0.41; p < 0.0001). No significant correlation was shown between MET expression and phosphorylation (p > 0.05). c-MET gene amplification was detected in eight of 214 patients (3.7%). No significant association was observed between c-MET amplification, c-MET protein expression and phosphorylation., Conclusion: Our data indicate, that neither expression of c-MET nor the gene amplification status might be the best way to select patients for MET targeting therapies, since no correlation with the activation status of MET was observed. We propose to take into account analyzing the phosphorylation status of MET by IHC to select patients for MET targeting therapies. Signaling of the receptor and the activation of downstream molecules might be more crucial for the benefit of therapeutics targeting MET receptor tyrosine kinases than expression levels alone.

Background: Epidermal Growth Factor Receptor (EGFR) targeting therapies are currently of great relevance for the treatment of lung cancer. For this reason, in addition to mutational analysis immunohistochemistry (IHC) of EGFR in lung cancer has been discussed for the decision making of according therapeutic strategies. The aim of this study was to obtain standardization of EGFR-expression methods for the selection of patients who might benefit of EGFR targeting therapies., Methods: As a starting point of a broad investigation, aimed at elucidating the expression of EGFR on different biological levels, four EGFR specific antibodies were analyzed concerning potential differences in expression levels by Immunohistochemistry (IHC) and correlated with fluorescence in situ hybridization (FISH) analysis and clinicopathological data. 206 tumor tissues were analyzed in a tissue microarray format employing immunohistochemistry with four different antibodies including Dako PharmDx kit (clone 2-18C9), clone 31G7, clone 2.1E1 and clone SP84 using three different scoring methods. Protein expression was compared to FISH utilizing two different probes., Results: EGFR protein expression determined by IHC with Dako PharmDx kit, clone 31G7 and clone 2.1E1 (p ≤ 0.05) correlated significantly with both FISH probes independently of the three scoring methods; best correlation is shown for 31G7 using the scoring method that defined EGFR positivity when ≥ 10% of the tumor cells show membranous staining of moderate and severe intensity (p=0.001)., Conclusion: Overall, our data show differences in EGFR expression determined by IHC, due to the applied antibody. Highest concordance with FISH is shown for antibody clone 31G7, evaluated with score B (p=0.001). On this account, this antibody clone might by utilized for standard evaluation of EGFR expression by IHC., Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000&#95;2014&#95;165.

Background: Patients treated with anti-EGFR tyrosine kinase inhibitors (TKI) may receive dose reduction due to toxicity; however, the impact on treatment efficacy and patient outcome remains unknown., Patients and Methods: Patients with stage IV NSCLC harbouring EGFR mutations and treated at our institution were retrospectively analyzed for treatment with TKI in 2012 or later., Results: Thirty patients with EGFR mutated adenocarcinoma were identified meeting the inclusion criteria. Eleven patients received cytotoxic therapy as first line treatment yielding in a response rate of 36 %. All patients were treated with TKI as first or second-line therapy which resulted in disease stabilization (23 %) or response (77 %) in all patients. The median progression-free survival was 21.4 months with 21 patients still on treatment with TKI. For 7 patients, the dose of TKI treatment had to be reduced due to co-morbidities (n = 2) or skin toxicity (rash ≥ grade 3; n = 5). These patients demonstrated a similar response and outcome as compared to patients receiving full dose TKI treatment., Conclusion: Patients with EGFR mutated NSCLC and the need for TKI dose reduction seem to benefit in a similar manner from TKI therapy., (© Georg Thieme Verlag KG Stuttgart · New York.)

Alterations in the DNA methylome are characteristic for numerous diseases and a typical hallmark of cancer. Therefore, DNA methylation is currently under investigation in research labs and has also entered diagnostics. Recently, protocols like the BeadChip technology have become commercially available to study DNA methylation in an array format and semiquantitative fashion. However, it is known that fixation of the sample material with formalin prior to BeadChip analysis can affect the results. In this study we compared the influence of fixation on the outcome of BeadChip analysis. From six patients each a lung cancer tissue sample and a corresponding tumor-free lung tissue sample were collected. The samples were separated into three pieces. One piece of each sample was fixed with formalin, another one by the non-cross-linking HOPE technique (Hepes-glutamic acid buffer mediated Organic solvent Protection Effect). Subsequently, both became paraffin embedded. As a reference, the remaining third piece was cryopreserved. In addition we used three adenocarcinoma cell lines (H838, A549, and H1650) to validate the results from patient tissues. We show that using the HOPE technique instead of formalin largely prevents the introduction of formalin-fixation related artifacts. An ANOVA analysis significantly separated HOPE- and cryopreserved from formalin-fixed samples (FDR<0.05), while differences in the methylation data obtained from HOPE-fixed and cryopreserved material were minor. Consequently, HOPE fixation is superior to formalin fixation if a subsequent BeadChip analysis of paraffin-embedded sample material is intended.

Lung cancer is frequently complicated by pulmonary infections which may impair prognosis of this disease. Therefore, we investigated the effect of bacterial lipopolysaccharides (LPS) on tumor proliferation in vitro in the non-small cell lung cancer (NSCLC) cell line A549, ex vivo in a tissue culture model using human NSCLC specimens and in vivo in the A549 adenocarcinoma mouse model. LPS induced a time- and dose-dependent increase in proliferation of A549 cells as quantified by MTS activity and cell counting. In parallel, an increased expression of the proliferation marker Ki-67 and cyclooxygenase (COX)-2 was detected both in A549 cells and in ex vivo human NSCLC tissue. Large amounts of COX-2-derived prostaglandin (PG)E(2) were secreted from LPS-stimulated A549 cells. Pharmacological interventions revealed that the proliferative effect of LPS was dependent on CD14 and Toll-like receptor (TLR)4. Moreover, blocking of the epidermal growth factor receptor (EGFR) also decreased LPS-induced proliferation of A549 cells. Inhibition of COX-2 activity in A549 cells severely attenuated both PGE(2) release and proliferation in response to LPS. Synthesis of PGE(2) was also reduced by inhibiting CD14, TLR4 and EGFR in A549 cells. The proliferative effect of LPS on A549 cells could be reproduced in the A549 adenocarcinoma mouse model with enhancement of tumor growth and Ki-67 expression in implanted tumors. In summary, LPS induces proliferation of NSCLC cells in vitro, ex vivo in human NSCLC specimen and in vivo in a mouse model of NSCLC. Pulmonary infection may thus directly induce tumor progression in NSCLC.

Background: Different therapy regimens in non-small-cell lung cancer (NSCLC) are of rising clinical importance, and therefore a clear-cut subdifferentiation is mandatory. The common immunohistochemical markers available today are well applicable for subdifferentiation, but a fraction of indistinct cases still remains, demanding upgrades of the panel by new markers., Methods: We report here the generation and evaluation of a new monoclonal antibody carrying the MAdL designation, which was raised against primary isolated human alveolar epithelial cells type 2., Results: Upon screening, one clone (MAdL) was identified as a marker for alveolar epithelial cell type II, alveolar macrophages and adenocarcinomas of the lung. In a large-scale study, this antibody, with an optimised staining procedure for formalin-fixed tissues, was then evaluated together with the established markers thyroid transcription factor-1, surfactant protein-A, pro-surfactant protein-B and napsin A in a series of 362 lung cancer specimens. The MAdL displays a high specificity (>99%) for adenocarcinomas of the lung, together with a sensitivity of 76.5%, and is capable of delivering independent additional diagnostic information to the established markers., Conclusion: We conclude that MAdL is a new specific marker for adenocarcinomas of the lung, which helps to clarify subdifferentiation in a considerable portion of NSCLCs.

Proteome analyses provide diagnostic information which can be essential for therapeutic predictions. The application of such techniques for analyzing paraffin-embedded tissue samples is widely hampered by the use of formalin fixation requiring antigen retrieval procedures in molecular pathology. In prior studies, the HEPES-glutamic acid buffer-mediated organic solvent protection effect (HOPE) technique of tissue fixation has been shown to provide a broad array of biochemical investigations with excellent preservation of morphological structures, DNA, RNA, and proteins, thus supporting the multimethod analysis of archived specimens. Here we show that HOPE fixation is also useful in proteomic investigations by allowing two-dimensional electrophoresis (2DE) and mass spectrometry, using lung cancer tissues. Two-dimensional gels of two-protein extraction protocols derived from HOPE-fixed material displayed characteristic spot patterns with high reproducibility. For comparison, 2DE analysis of ethanol-fixed, formalin-fixed, and frozen samples from the same tissues was performed. Western blotting confirmed immunoreactivity of 2DE-separated proteins from HOPE-fixed tissue samples. Additionally, distinct spots were excised from HOPE-derived 2D gels and successfully subjected to peptide mass fingerprinting. In conclusion, paraffin archives containing HOPE-fixed tissues are applicable to a wide spectrum of molecular investigations including common biochemical methods for proteome analyses and therefore represent a unique source for molecular investigations in the rapidly growing field of molecular pathology. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Despite considerable progress in the development of individualized targeted therapies of tumor diseases, identification of additional reliable target molecules is still mandatory. One of the most recent targets is microtubule-associated human EML4 generating a fusion-type oncogene with ALK demonstrating marked transforming activity in lung cancer. Since EML4 is a poorly characterized protein with regard to expression, function and regulation in human tissue, specimens of human tumor and tumor-free tissues obtained from patients with NSCLC were analyzed to determine the cellular localization. All tissue samples have been previously fixed with the novel HOPE-technique and paraffin embedded. Determination of both gene expression and protein levels of EML4 were performed using RT-PCR, in situ hybridization as well as immunohistochemistry, respectively. In human NSCLC tissue samples, possible regulation of EML4 transcription upon chemotherapy with combinations of most established cytotoxic drugs for NSCLC treatment was also studied employing the recently established ex vivo tissue culture model STST. In normal lung, both marked mRNA and protein levels of EML4 were localized in alveolar macrophages. In contrast, lung tumor tissues always showed consistent transcriptional expression in situ and by RT-PCR. Stimulation of NSCLC tissues with chemotherapeutics revealed heterogeneous effects on EML4 mRNA levels. Based on its expression patterns in both tumor-free lung and NSCLC tissues, human EML4 is likely to be closely associated with processes involved in local inflammation of the lung as well as with tumor behavior. Thus, our results suggest that EML4 may have the potential as a therapeutic target molecule in NSCLC chemotherapy.

In the last decade, pathologic approaches concerning diagnosis and treatment of lung carcinomas have increasingly moved towards the implementation of molecular methods into the process of decision. In this study, an overview is given referring to the variety of tumors in the lung including common primary lung neoplasms and secondary tumors, and a modus operandi is presented which integrates immunology as well as molecular pathology within the process of finding correct diagnoses. Besides the conventional and approved methods and techniques leading to appropriate treatment including so-called targeted therapies, pathologist's work meanwhile depends on both histologic and molecular results. Since molecular techniques have increasingly entered the field of routine diagnostics, challenges and possibilities have changed and are still rapidly developing. The proceeding integration of molecular-biologic investigations into the process of diagnosing has changed the nature of diagnostics and will continuously grow in the near future. Only by obtaining a proper diagnosis, the optimal treatment of a patient can be assured, whereupon the knowledge of gene mutations and/or altered protein expression is crucial. By identifying those novel molecular target structures, the therapeutic spectrum is tremendously enlarged and will finally improve the patient's prognosis by personalized targeted therapies.

Besides its main function, i.e., the binding of free hemoglobin and prevention of oxidative stress, the acute phase protein haptoglobin acts as a potent immunoreactive modulator. As part of an investigation that aimed at illuminating the role of acute phase proteins in the local defense of the lungs, this study is the first to describe the expression and synthesis of haptoglobin in human lung tissues and lung tumors. Prompted by the results obtained from a transcription array study, we analyzed 115 lung (cancer) specimens using immunohistochemistry. Thirty-seven specimens were subjected to mRNA-in situ hybridization. 40.4% of the adenocarcinomas showed distinct granular and perinuclear staining of the tumor cells. By contrast, only 4.8% of the squamous cell carcinomas showed haptoglobin within tumor cells, but 19% displayed haptoglobin expressing alveolar epithelial cells type II surrounding the tumor. One small cell lung cancer displayed haptoglobin expression. In tumor-free lungs, we located haptoglobin in alveolar macrophages, alveolar epithelial cells type II, and bronchiolar cells. In situ hybridization verified the results of immunohistochemistry. The results were further verified by RT-PCR and Western blot compared to liver tissues, which both showed comparable amounts of haptoglobin mRNA and protein in NSCLC and in liver, while tumor-free lung tissues showed lower expression. Due to the known immunomodulatory effects of haptoglobin, its broad expression and synthesis within human lung tissues strongly suggests a function as a fundamental pulmonary local defense element.

Even though the lung represents a special immune compartment with the capacity of a high inflammatory response, ineffective anti-tumour immunity is common in lung-associated malignancies. We asked whether a differential composition of the immune cell infiltrate in malignant (MLTAs) and non-malignant lung tissue areas (N-MLTAs) exists and might potentially contribute to this effect. We performed a comparative analysis of immune cells residing in MLTAs and N-MLTAs of non-small cell lung cancer (NSCLC) patients. To this end, we used immunophenotyping and functional analyses on directly isolated immune cells and tissue arrays on archived paraffin-embedded specimens. A strong T cell infiltration was prominent in both tissue compartments whereas CD4(+)CD25(+)CD127(-) T regulatory cells were present in MLTAs only. Nonetheless, concurrent functional ex vivo T cell analyses revealed no significant difference between T cells of MLTA and N-MLTA, suggesting that tumour-infiltrating T cells were not functionally impaired. Interestingly, T cell infiltration was less pronounced in specimens with a high neutrophilic infiltrate. NK cell infiltration was strikingly heterogenous between MLTA and N-MLTA. While NK cells were almost absent in the malignant tissue regions, non-malignant counterparts were selectively populated by NK cells and those NK cells showed strong cytotoxic activity ex vivo. We report that malignant and non-malignant tissue areas in NSCLC are selectively infiltrated by certain immune cell types with NK cells being displaced from the tumour tissue. These phenomena have important implications for tumour immunology of NSCLC and should be considered for the development of future immunologic intervention therapies.

Lung cancer is still the leading cause of death from cancer worldwide primarily because of the fact that most lung cancers are diagnosed at advanced stages. Overexpression of the high mobility group protein HMGA2 has been observed in a variety of malignant tumors and often correlates with poor prognosis. Herein, HMGA2 expression levels were analyzed in matching cancerous and non-cancerous lung samples of 17 patients with adenocarcinoma (AC) and 17 patients with squamous cell carcinoma (SCC) with real-time quantitative RT-PCR (qRT-PCR). Transcript levels were compared to results obtained by immunohistochemistry (IHC). HMGA2 expression was detectable by qRT-PCR in all samples tested and varied from 5422 to 16 991 545 copies per 250 ng total RNA in the carcinoma samples and from 289 to 525 947 copies in the non-cancerous tissue samples. In 33/34 non-small cell lung cancer (NSCLC) samples tested, an overexpression of HMGA2 was revealed with statistically highly significant differences between non-neoplastic and tumor samples for both AC (P < 0.0001) as well as for SCC (P < 0.0001). Expression varies strongly and is increased up to 911-fold for AC and up to 2504-fold for SCC, respectively, with statistically significant higher increase in SCC (P < 0.05). The results presented herein indicate that HMGA2 overexpression is a common event in NSCLC and could serve as molecular marker for lung cancer., ((c) 2007 Wiley-Liss, Inc.)

Surfactant proteins play important roles in lung surfactant function and innate immunity. The DNA methylation state of 11 CpG sites of surfactant protein (SP)-A1, -B, -C, and -D was determined using universal bead arrays. A total of 90 cancerous and non-cancerous tissues from 23 patients with adenocarcinoma and 22 with squamous cell carcinoma were studied. These were divided into a training set and a testing set. The results indicate that DNA methylation profiling of these CpGs is associated with lung cancer. Four CpG sites, SP-A1&#95;370, SP-A1&#95;1080, SP-D&#95;1170, and SP-D&#95;1370, were hypomethylated in cancer and were significantly associated with both adenocarcinoma and squamous cell carcinoma, indicating that they have the potential to be used as biomarkers for lung cancer diagnosis and treatment. Normal lung tissues with a higher level of unmethylated SP-A1&#95;1468 and SP-D&#95;1170 CpG exhibited a higher level of SP-A1 and SP-D gene transcripts indicating that CpG methylation may play a role in gene expression. When the non-cancerous tissues were compared to cancerous tissues in patients with adenocarcinoma, the methylation profile results of these 46 samples (23 cancerous and 23 non-cancerous) could be clustered into 4 groups by agglomerative nesting. The percentage of tumor samples in each group was 0, 58, 91, and 100, respectively. A similar pattern was observed in squamous cell carcinoma patients. We speculate that SP-A1 and SP-D are subject to methylation/demethylation regulatory mechanisms and are involved in lung cancer pathogenesis by virtue of their function in innate host defense and/or regulation of inflammation.

Background: Non-small cell lung cancer (NSCLC) causes most of cancer related deaths in humans and is characterized by poor prognosis regarding efficiency of chemotherapeutical treatment and long-term survival of the patients. The purpose of the present study was the development of a human ex vivo tissue culture model and the analysis of the effects of conventional chemotherapy, which then can serve as a tool to test new chemotherapeutical regimens in NSCLC., Methods: In a short-term tissue culture model designated STST (Short-Term Stimulation of Tissues) in combination with the novel *HOPE-fixation and paraffin embedding method we examined the responsiveness of 41 human NSCLC tissue specimens to the individual cytotoxic drugs carboplatin, vinorelbine or gemcitabine. Viability was analyzed by LIFE/DEAD assay, TUNEL-staining and colorimetric MTT assay. Expression of Ki-67 protein and of BrdU (bromodeoxyuridine) uptake as markers for proliferation and of cleaved (activated) effector caspase-3 as indicator of late phase apoptosis were assessed by immunohistochemistry. Transcription of caspase-3 was analyzed by RT-PCR. Flow cytometry was utilized to determine caspase-3 in human cancer cell lines., Results: Viability, proliferation and apoptosis of the tissues were moderately affected by cultivation. In human breast cancer, small-cell lung cancer (SCLC) and human cell lines (CPC-N, HEK) proliferative capacity was clearly reduced by all 3 chemotherapeutic agents in a very similar manner. Cleavage of caspase-3 was induced in the chemo-sensitive types of cancer (breast cancer, SCLC). Drug-induced effects in human NSCLC tissues were less evident than in the chemo-sensitive tumors with more pronounced effects in adenocarcinomas as compared to squamous cell carcinomas., Conclusion: Although there was high heterogeneity among the individual tumor tissue responses as expected, we clearly demonstrate specific multiple drug-induced effects simultaneously. Thus, STST provides a useful human model to study numerous aspects of mechanisms underlying tumor responsiveness towards improved anticancer treatment. The results presented here shall serve as a base for multiple functional tests of novel chemotherapeutic approaches to NSCLC in the future.

Studies have highlighted molecular and cell biological mechanisms of the NKG2D-NKG2D ligand system in the activation of natural killer (NK) cell and T cell functions. In this study we explore the potential of genetic overexpression of human major histocompatibility complex class I chain-related gene A (MICA), a powerful NKG2D ligand, for the induction of NK cell-mediated antitumor immunity in a humanized murine model of non-small cell lung cancer. Using tissue microarray technology we detected expression of MICA in only 30% of the samples from patients with lung cancer. Staining was always weak and focal, indicating that expression of MICA is detectable but limited in human lung cancer. Genetic overexpression of MICA by means of adenoviral transduction or transfection of expression plasmids was feasible in cell lines in vitro, primary human cancer tissue ex vivo, and in experimental human cancers in vivo. The presence of MICA on the surface of largely NK cell-resistant human lung cancer cells reestablished NK cell susceptibility and provoked NK cell-mediated antitumor immunity by murine and human NK cells in two different experimental therapy models. In this study we analyze the interaction of human NK cells with MICA-positive human cancer cells in an in vivo setting. Our data demonstrate that MICA overexpression can function as NK cell-mediated immunotherapy in experimental lung cancer.

This article analyzes phenotype and genotype alterations of the lung in association with lung cancer. The frequency of phenotype preneoplastic lesions (atypical adenomatoid hyperplasia (AAH) and squamous cell dysplasia (SCD)) was analyzed at distinct distances from the tumor boundary in 150 lung carcinomas. AAH was noted in 19/150 (13%) cases and more frequently seen in adeno carcinomas, squamous cell dysplasia was noted in 46/150 (31%) cases and more frequently seen in squamous cell carcinomas. The degree of cellular atypia decreased with increasing distance from tumor boundary in both AAH and SCD. At similar distances, genotype (chromosome) alterations of surrounding bronchial mucosa were studied in additional 55 primary and secondary lung tumors by karyotype analysis. Numerical chromosome aberrations occur frequently in primary lung carcinomas and adjacent bronchial mucosa, and affect at average 4.5/10 metaphases in primary lung cancer and 2/10 metaphases in metastases. Most abnormal metaphases were induced by chromosome losses, only few by additional copies, i.e. trisomy, etc. Losses of y chromosome were seen in both malignancy and adjacent bronchial mucosa, and interpreted as "tumor related", losses of chromosome 21 in adjacent bronchial mucosa were non-tumor related in adenocarcinoma and metastases, losses of chromosome 19 in adjacent bronchial mucosa occurred independently in squamous cell and large cell carcinomas. The data suggest the hypothesis that preneoplastic lesions in the lung might be partly induced by the tumor itself.

Background: CpG-oligonucleotides (CpG-ODN), which induce signaling through Toll-like receptor 9 (TLR9), are currently under investigation as adjuvants in therapy against infections and cancer. CpG-ODN function as Th-1 adjuvants and are able to activate dendritic cells. In humans TLR9 has been described to be strongly expressed in B-lymphocytes, monocytes, plasmacytoid dendritic cells and at low levels in human respiratory cells. We determined whether a direct interaction of bacterial DNA with the tumor cells themselves is possible and investigated the expression and function of TLR9 in human malignant solid tumors and cell lines. TLR9 expression by malignant tumor cells, would affect treatment approaches using CpG-ODN on the one hand, and, on the other hand, provide additional novel information about the role of tumor cells in tumor-immunology., Methods: The expression of TLR9 in HOPE-fixed non-small lung cancer, non-malignant tissue and tumor cell lines was assessed using immunohistochemistry, confocal microscopy, in situ hybridization, RT-PCR and DNA-sequencing. Apoptosis and chemokine expression was detected by FACS analysis and the Bio-Plex system., Results: We found high TLR9 signal intensities in the cytoplasm of tumor cells in the majority of lung cancer specimens as well as in all tested tumor cell lines. In contrast to this non-malignant lung tissues showed only sporadically weak expression. Stimulation of HeLa and A549 cells with CpG-ODN induced secretion of monocyte chemoattractant protein-1 and reduction of spontaneous and tumor necrosis factor-alpha induced apoptosis., Conclusions: Here we show that TLR9 is expressed in a selection of human lung cancer tissues and various tumor cell lines. The expression of functionally active TLR9 in human malignant tumors might affect treatment approaches using CpG-ODN and shows that malignant cells can be regarded as active players in tumor-immunology.

The frequency of preneoplastic lesions of the lung and bronchial mucosa as well as potential genotype alterations in spatial relationship to pulmonary malignancies still need intensive investigations in order to understand the occurrence and manifestation of lung cancer in detail. To investigate the contemporary manifestation of lung cancer precursor lesions, peripheral (non-neoplastic) lung parenchyma and bronchial mucosa of operated lung carcinomas were analyzed at distinct distances (1, 2, 3, and 4 cm) from the tumor boundary for pre-neoplastic lesions--atypical adenomatoid hyperplasia (AAH) and squamous cell dysplasia (SCD), in 150 surgical specimens. Short-term tissue cultures of additional 55 primary and secondary lung tumors and their surrounding non-neoplastic bronchial mucosa were performed at the same distances in order to search for chromosome alterations, i.e. genotype aberrations. In phenotype observations, atypical adenomatoid hyperplasia was noted in 19/150 (13%) cases, and squamous cell dysplasia in 46/150 (31%) cases. The degree of cellular atypia decreased with increasing distance from the tumor boundary in both AAH and SCM. AAH was observed more frequently in adenocarcinomas, SCQ more frequently in squamous cell carcinomas. In genotype observations, the average number of abnormal metaphases measured 4.5/10 high power fields (HPF) in primary lung carcinomas, and only 2/10 in metastases. Data indicate that the so-called preneoplastic lesions in the lung are not completely tumor-precursor lesions, but, in addition, induced by the tumor itself.

Unlabelled: Objective of this study was to investigate the balance of surfactant-apoprotein A1 and A2 (SP-A1 and SP-A2) at transcriptional level in tumor and tumor-free lung-tissues of patients with non-small-cell lung-carcinomas., Materials and Methods: Nonfixed snap-frozen tumor and tumor-free sections of lungs, excised from 21 patients with adenocarcinomas of the lung and 6 patients with squamous cell carcinomas (control group) were analyzed. This was accomplished by RT-PCR using a SP-A1/SP-A2 consensus sequence, followed by enzymatic restriction., Results: Tumor-containing tissues of adenocarcinomas showed higher SP-A1/SP-A2 ratios than the tumor free tissues. There was a significant difference in expression of SP-A1/SP-A2-mRNA in intra-individual tumor and tumor-free lung-tissues of adenocarcinoma patients (p = 0,05). In cases of squamous cell carcinomas this was not observed., Conclusions: A novel method to investigate the transcriptional activity of surfactant-apoproteins A1 and A2 in snap-frozen lung tissues was established. It was shown, that adenocarcinoma-tissues display higher SP-A1/SP-A2 than the corresponding tumor-free tissues and that the variation of SP-A-mRNA expression rises in cases of higher tumor-grading.

Background: Adenomatous hyperplasia of the peripheral lung has been suggested to be a preneoplastic lesion leading to peripherally localized lung carcinomas. The paucity of data about cellular and vascular characteristics of this lesion in comparison to normal lung prompted this investigation., Material and Methods: We describe results of two investigations comprising 75 cases and 70 cases, respectively, with atypical adenomatous hyperplasia (AAH) of the lung, respectively: (a) a prospective study part with thorough analysis of surgical lung specimens (lobes and lungs) for light microscopical detection of the lesion; and (b) a retrospective study part with immuno- and lectin histochemical analysis of AAH and non-neoplastic lung parenchyma monitoring expression of growth-related markers and changes in vascularization patterns. Sections of the individual cases were examined by an image-analyzing system including automated measurement of staining intensities and structure analysis., Results: The prospective study part revealed an incidence of AAH in 2/31 cases with squamous cell carcinoma and in 5/32 cases with adenocarcinomas. No relation to pT- or pN stages was detectable, high grade AAHs were seen to be close to the tumor lesions (<2 cm distance) and those with low grade at greater distances. Statistically significantly increased levels of expression of anti-apoptotic bcl-2, macrophage migration inhibitory factor (MIF) capable to suppress p53 activities, heparin-binding lectin, interleukin-2, galectin-1 and of binding sites for the endogenous lectins galectins-1, -3 and -7 were determined. In addition, alveolar-lining cells, which express these markers, formed spatial clusters, which harbor different levels of structural entropy. AAH displayed an increased level of vascularization characterized by regular size and increased number of newly formed vessels., Interpretation: The prospective and retrospective study parts point to a close association of AAH with peripherally localized adenocarcinoma of the lung. AAH is characterized by pronounced alteration of expression of several growth-related markers and probably non-reversible changes in vascularization.

We report two cases of patients suffering from multiple tumors in the lungs with questionable origin. In these cases, the expression of Surfactant-apoprotein-A (SP-A) by the tumor cells has been a helpful feature in the process of diagnosis by indicating primary carcinomas of the lungs. Surfactant-apoprotein-A is expressed by the pneumocytes II in lung tissue and a portion of non-small cell lung carcinomas and has not yet been found to be expressed by other tumors when detected immunohistochemically by use of the monoclonal antibody PE-10. We analyzed formalin-fixed, paraffin-embedded specimens of different malignancies with pulmonary location for the expression of SP-A by the use of PE-10 with other antibodies against additional epitopes such as the thyroid transcription factor-1. In normal lung areas a distinct staining of the pneumocytes II has been observed. All the control sections of primary and metastatic nonlung-carcinoma specimen in our study remained negative. The monoclonal antibody PE-10 used in this study provides high specificity when compared to the results obtained with polyclonal antibodies. One of the two cases reported, a mucinous carcinoma of the lungs, had been negative for thyroid transcription factor-1 but positive for SP-A. Thus, SP-A detected by immunohistochemistry using the monoclonal antibody PE-10, together with other parameters such as thyroid transcription factor-1, may be a useful tool for individual diagnosis of malignomas located in the lungs with a questionable primary origin. Ann Diagn Pathol 5:84-90, 2001., (Copyright 2001 by W.B. Saunders Company)