Pathological and imaging features of pulmonary invasive mucinous adenocarcinoma-a retrospective cohort study.

Background: Pulmonary invasive mucinous adenocarcinoma (IMA) is a rare subtype of lung cancer which is easily misdiagnosed as inflammatory nodules, tuberculosis, pulmonary diffuse lesions, or hamartomas due to the lack of clinical specificity. This study aims to identify the pathological and imaging characteristics of IMA, which will favor to improve the diagnostic and therapeutic efficacy. Methods: A retrospective study was conducted by enrolling patients histopathologically diagnosed with pulmonary IMA in the current study between January 2014 and December 2021. The clinical pathological and radiological data were collected for analysis to evaluate the radiological patterns and pathological and molecular characteristics of IMA. Results: A total of 136 patients were included in the study, of whom 58 were male and 78 were female. The patients had an average age of 63.0±9.7 years. The tumors were classified into the following three pathological types: pure mucinous (76 cases) featured by only mucinous cells observed under the microscope; mixed mucinous (23 cases) featured as an attached-wall, papillary, acinar, and solid tumor cells with more than 10% mucinous cells.; and mucinous-absent (29 cases) featured with the absence of mucous cells, but still can detect more than 10% of mucin expresses. In terms of the morphological classification based on the CT scans, 88 (64.7%) cases were identified as the nodular type, 31 (22.8%) as the inflammatory type, 15 (11.1%) as the mass-like type, and two (1.5%) as the diffuse type. For the molecular features, patients afflicted with IMA showed much lower levels of thyroid transcription factor-1 (15%) than those with usual adenocarcinoma (over 80%). However, cytokeratin 20 was more common in IMA (50%) than the usual adenocarcinoma (about 5%). The K-RAS mutation was prevalent in 75% of IMA, which contrasted sharply to its occurrence in a mere 15% of the usual adenocarcinoma. Epidermal growth factor receptor mutations were rarer in IMA (less than 5%) than the usual adenocarcinoma (about 50%). Conclusions: The pathological and imaging features enrich our understanding of the disease's heterogeneity, which will contribute to more personalized diagnostic and therapeutic strategies.

The cGAS-STING pathway drives inflammation in Usual Interstitial Pneumonia, phagocytosis could prevent inflammation but is inhibited by the don't eat me signal CD47.

BACKGROUND: Usual Interstitial Pneumonia (UIP) a fibrosing pneumonia is associated with idiopathic pulmonary fibrosis, chronic autoimmune disease (AID), or hypersensitivity pneumonia. Oxygen radicals, due to tobacco smoke, can damage DNA and might upregulate PARP1. Cytosolic DNA from dying pneumocytes activate cytosolic GMP-AMP-synthase-stimulator of interferon genes (cGAS-STING) pathway and TREX1. Prolonged inflammation induces senescence, which might be inhibited by phagocytosis, eliminating nuclear debris. We aimed to evaluate activation of cGAS-STING-TREX1 pathway in UIP, and if phagocytosis and anti-phagocytosis might counteract inflammation. METHODS: 44 cases of UIP with IPF or AID were studied for the expression of cGAS, pSTING, TREX1 and PARP1. LAMP1 and Rab7 expression served as phagocytosis markers. CD47 protecting phagocytosis and p16 to identify senescent cells were also studied. RESULTS: Epithelial cells in remodeled areas and macrophages expressed cGAS-pSTING, TREX1; epithelia but not macrophages stained for PARP1. Myofibroblasts, endothelia, and bronchial/bronchiolar epithelial cells were all negative except early myofibroblastic foci expressing cGAS. Type II pneumocytes expressed cGAS and PARP1, but less pSTING. TREX1 although expressed was not activated. Macrophages and many regenerating epithelial cells expressed LAMP1 and Rab7. CD47, the 'don't-eat-me-signal', was expressed by macrophages and epithelial cells including senescence cells within the remodeled areas. CONCLUSIONS: The cGAS-STING pathway is activated in macrophages and epithelial cells within remodeled areas. LikelyTREX1 because not activated cannot sufficiently degrade DNA fragments. PARP1 activation points to smoking-induced oxygen radical release, prolonging inflammation and leading to fibrosis. By expressing CD47 epithelial cells within remodeled areas protect themselves from being eliminated by phagocytosis.

Pathologic diagnosis of lung cancer - recent developments.

PURPOSE OF REVIEW: Diagnosis of lung cancer has previously been based on the evaluation of resection specimen. However, approximately 80% of lung cancers are diagnosed in stage IV. Targeted therapy has changed the practice of pathology. Diagnosis is usually based on small biopsies or even needle aspirations. Subtyping is important, as a molecular classification has to be added. RECENT FINDINGS: Molecular analysis has to be done in adenocarcinomas and on some of the rarer carcinoma types. Molecular analysis of squamous cell carcinomas should be done in never or former smokers, as they might present with targetable oncogenes. The same applies for adenosquamous carcinomas. Both high-grade neuroendocrine carcinomas should be subtyped. These subtypes might become relevant for new treatment options, currently investigated. Subtyping is done by immunohistochemistry with antibodies for ASCL1, NeuroD1, and POU2F3. In carcinoids, molecular investigation can better define cases with a higher risk of recurrence and metastasis. SUMMARY: Diagnosis of lung cancer is most often done on small biopsies or cytological preparations. Only a minimal number of tissues or cellular material is used for diagnosis. A considerable portion is reserved for molecular analysis. Molecular investigation is important in adenocarcinomas, but also for other rare tumor types.

Clinical insights into small cell lung cancer: Tumor heterogeneity, diagnosis, therapy, and future directions.

Small cell lung cancer (SCLC) is characterized by rapid growth and high metastatic capacity. It has strong epidemiologic and biologic links to tobacco carcinogens. Although the majority of SCLCs exhibit neuroendocrine features, an important subset of tumors lacks these properties. Genomic profiling of SCLC reveals genetic instability, almost universal inactivation of the tumor suppressor genes TP53 and RB1, and a high mutation burden. Because of early metastasis, only a small fraction of patients are amenable to curative-intent lung resection, and these individuals require adjuvant platinum-etoposide chemotherapy. Therefore, the vast majority of patients are currently being treated with chemoradiation with or without immunotherapy. In patients with disease confined to the chest, standard therapy includes thoracic radiotherapy and concurrent platinum-etoposide chemotherapy. Patients with metastatic (extensive-stage) disease are treated with a combination of platinum-etoposide chemotherapy plus immunotherapy with an anti-programmed death-ligand 1 monoclonal antibody. Although SCLC is initially very responsive to platinum-based chemotherapy, these responses are transient because of the development of drug resistance. In recent years, the authors have witnessed an accelerating pace of biologic insights into the disease, leading to the redefinition of the SCLC classification scheme. This emerging knowledge of SCLC molecular subtypes has the potential to define unique therapeutic vulnerabilities. Synthesizing these new discoveries with the current knowledge of SCLC biology and clinical management may lead to unprecedented advances in SCLC patient care. Here, the authors present an overview of multimodal clinical approaches in SCLC, with a special focus on illuminating how recent advancements in SCLC research could accelerate clinical development.

Does subtyping of high-grade pulmonary neuroendocrine carcinomas have an impact on therapy selection?

Background: Small cell lung cancer (SCLC) and large cell neuroendocrine carcinomas (LCNEC) are characterized by a rapid progressive course. Therapy for SCLC has not much changed for decades, and in LCNEC controversies exist, favoring either SCLC-like or non-small cell lung cancer (NSCLC)-like therapy. Three subtypes of SCLC identified in cell cultures, namely ASCL1, NeuroD1, and POU2F3 have been confirmed by immunohistochemistry. The fourth type based on the expression of YAP1 was questioned, and another type, inflamed SCLC, was proposed. Methods: SCLC and LCNEC samples were investigated by immunohistochemistry for different subtypes. Additionally, immunohistochemical markers as potential tools to identify patients who might respond to targeted treatment were investigated. For validation a biopsy set was added. Results: ASCL1, NeuroD1, and POU2F3 were expressed in different percentages in SCLC and LCNEC. Similar percentages of expression were found in biopsies. ATOH was expressed in combination with one of the subtypes. YAP1 and TAZ were expressed in some SCLC and LCNEC cases. HES1 expression was seen in few cases. Predominantly stroma cells expressed programmed cell death ligand 1 (PD-L1). The dominant MYC protein was N-MYC. Aurora kinase A (AURKA) was expressed in the majority of both carcinomas, whereas fibroblast growth factor receptor 2 (FGFR2) in few. Conclusions: SCLC and LCNEC can be subtyped into ASCL1-, NeuroD1-, and POU2F3-positive types. AURKA expression and positivity for N-MYC protein was not associated with subtypes. AURKA and FGFR2 are both possible targets for inhibition in SCLC and LCNEC, but patients' selection should be based on expression of the enzyme. Combined chemo- and immunotherapy might be decided by PD-L1 staining of stroma cells.

Expression patterns and prognostic relevance of subtype-specific transcription factors in surgically resected small-cell lung cancer: an international multicenter study.

The tissue distribution and prognostic relevance of subtype-specific proteins (ASCL1, NEUROD1, POU2F3, YAP1) present an evolving area of research in small-cell lung cancer (SCLC). The expression of subtype-specific transcription factors and P53 and RB1 proteins were measured by immunohistochemistry (IHC) in 386 surgically resected SCLC samples. Correlations between subtype-specific proteins and in vitro efficacy of various therapeutic agents were investigated by proteomics and cell viability assays in 26 human SCLC cell lines. Besides SCLC-A (ASCL1-dominant), SCLC-AN (combined ASCL1/NEUROD1), SCLC-N (NEUROD1-dominant), and SCLC-P (POU2F3-dominant), IHC and cluster analyses identified a quadruple-negative SCLC subtype (SCLC-QN). No unique YAP1-subtype was found. The highest overall survival rates were associated with non-neuroendocrine subtypes (SCLC-P and SCLC-QN) and the lowest with neuroendocrine subtypes (SCLC-A, SCLC-N, SCLC-AN). In univariate analyses, high ASCL1 expression was associated with poor prognosis and high POU2F3 expression with good prognosis. Notably, high ASCL1 expression influenced survival outcomes independently of other variables in a multivariate model. High POU2F3 and YAP1 protein abundances correlated with sensitivity and resistance to standard-of-care chemotherapeutics, respectively. Specific correlation patterns were also found between the efficacy of targeted agents and subtype-specific protein abundances. In conclusion, we investigated the clinicopathological relevance of SCLC molecular subtypes in a large cohort of surgically resected specimens. Differential IHC expression of ASCL1, NEUROD1, and POU2F3 defines SCLC subtypes. No YAP1-subtype can be distinguished by IHC. High POU2F3 expression is associated with improved survival in a univariate analysis, whereas elevated ASCL1 expression is an independent negative prognosticator. Proteomic and cell viability assays of human SCLC cell lines revealed distinct vulnerability profiles defined by transcription regulators. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Lung fibrosis in autoimmune diseases and hypersensitivity: how to separate these from idiopathic pulmonary fibrosis.

Lung involvement in autoimmune diseases (AID) is uncommon, but may precede other organ manifestations. A diagnostic problem is chronicity presenting with lung fibrosis. A new category of interstitial pneumonia with autoimmune features for patients with clinical symptoms of AID and presenting with usual interstitial pneumonia (UIP) enables antifibrotic treatment for these patients. Hypersensitivity pneumonia (HP) and other forms of lung fibrosis were not included into this category. As these diseases based on adverse immune reactions often present with unspecific clinical symptoms, a specified pathological diagnosis will assist the clinical evaluation. We aimed to establish etiology-relevant differences of patterns associated with AID or HP combined with lung fibrosis. We retrospectively evaluated 51 cases of AID, and 29 cases of HP with lung fibrosis, and compared these to 24 cases of idiopathic pulmonary fibrosis (UIP/IPF). Subacute AID and HP most often presented with organizing pneumonia (OP), whereas chronicity was associated with UIP. Unspecified fibrosis was seen in a few cases, whereas NSIP pattern was rare. In 9 cases, the underlying etiology could not be defined. Statistically significant features differentiating chronic AID or HP from UIP/IPF are lymphocytic infiltrations into myofibroblastic/fibroblastic foci. Other features significantly associated with AID and HP were granulomas, isolated Langhans giant cells, and protein deposits, but seen in only a minority of cases. A combination of UIP with one of these features enabled a specific etiology-based diagnosis. Besides the antifibrotic drug regimen, additional therapies might be considered.

Diagnosis and Molecular Profiles of Large Cell Neuroendocrine Carcinoma With Potential Targets for Therapy.

Large cell neuroendocrine carcinoma (LCNEC) together with small cell carcinoma (SCLC) and typical and atypical carcinoids form the group of pulmonary neuroendocrine tumors. LCNEC and SCLC are high-grade carcinomas. Although both can be found outside the thoracic cavity, they are most common in the lung. LCNEC differs from SCLC by morphologic pattern, and by cytological features such as nuclear size, nucleoli, chromatin pattern, but also by genetic differences. Originally thought to represent a single entity, it became evident, that three subgroups of LCNEC can be identified at the molecular level: a SCLC-like type with loss of retinoblastoma 1 gene (RB1) and TP53 mutations; a non-small cell lung carcinoma (NSCLC)-like type with wildtype RB1, TP53 mutation, and activating mutations of the phosphoinositol-3 kinase (PI3K-CA), or loss of PTEN; and a carcinoid-like type with MEN1 gene mutation. These subtypes can be identified by immunohistochemical staining for RB1, p53, and molecular analysis for PI3K and MEN1 mutations. These subtypes might also respond differently to chemotherapy. Immuno-oncologic treatment has also been applied to LCNEC, however, in addition to the evaluation of tumor cells the stroma evaluation seems to be important. Based on personal experiences with these tumors and available references this review will try to encompass our present knowledge in this rare entity and provoke new studies for better treatment of this carcinoma.

Genomic and transcriptional alterations in first-line chemotherapy exert a potentially unfavorable influence on subsequent immunotherapy in NSCLC.

Background: Recent studies in non-small cell lung cancer (NSCLC) patients have demonstrated that first-line immunotherapy is associated with better therapeutic response than second-line treatment. So far, the mechanisms need to be explored. It prompted us to evaluate the association between first-line chemotherapy and subsequent immunotherapy in NSCLC as well as its underlying mechanisms at the genomic and transcriptomic level. Methods: We launched a prospective, observational clinical study, paired tumor biopsies before and after chemotherapy were collected from NSCLC patients without tyrosine kinase inhibitor (TKI)-related driver gene mutations. The analyses included genomic and transcriptional changes performed by next-generation sequencing (NGS)-based whole-exome sequencing (WES) and messager ribonucleic acid (mRNA) sequencing. Characteristic mutational alterations in 1574 genes were investigated based on mutational status, clinicopathological factors, and chemotherapy responses. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, neoantigen prediction and intratumoral heterogeneity evaluation were also performed. Results: Samples and information from 32 NSCLC patients without TKI-related driver gene mutations were obtained. We found that the total number of single nucleotide variants (SNV)/insertion-deletion (INDEL) mutations did not change significantly after chemotherapy. The tumor mutation burden (TMB) decreased significantly after chemotherapy in smoking patients and the decreased TMB correlated with a better survival of smoking patients. The change in copy number variations (CNVs) exhibited a decreasing trend during chemotherapy. Subsequent analysis at mRNA level revealed a significant decrease in the expression levels of genes related to antigen processing and presentation as well as other factors relevant for response to immunotherapy. Pathway enrichment analysis confirmed that the immune-related signaling pathways or biological processes were decreased after first-line chemotherapy. Conclusions: Our study presents an explanation for the unsatisfactory results of immunotherapy when given after chemotherapy, and suggests that first-line chemotherapy is able to influence the tumor microenvironment and decrease the efficacy of subsequent immunotherapy. The study was registered at ClinicalTrials.gov, number NCT03764917, and has completed enrolment; patients are still in follow-up.

Applicability of pan-TRK immunohistochemistry for identification of NTRK fusions in lung carcinoma.

In the last two decades, various therapies have been introduced for lung carcinoma patients, including tyrosine-kinase inhibitors for different mutations. While some of them are specific to specific tumor types, others, like NTRK1-3 fusions, are found in various solid tumors. The occurrence of an NTRK1,2 or 3 fusion acts as a biomarker for efficient treatment with NTRK inhibitors, irrespectively of the tumor type. However, the occurrence of the NTRK1-3 fusions in lung carcinomas is extremely rare. We performed a retrospective analysis to evaluate the applicability of immunohistochemistry with the pan-TRK antibody in the detection of NTRK fusions in lung carcinomas. The study cohort included 176 adenocarcinomas (AC), 161 squamous cell carcinomas (SCC), 31 large-cell neuroendocrine carcinomas (LCNEC), and 19 small cell lung carcinomas (SCLC). Immunohistochemistry (IHC) was performed using the pan-TRK antibody (clone EPR17341, Ventana) on tissue microarrays, while confirmation for all positive cases was done using RNA-based Archer FusionPlex MUG Lung Panel. On IHC staining, 12/387 samples (3.1%) demonstrated a positive reaction. Ten SCC cases (10/161, 6.2%), and two LCNEC cases (2/31, 6.5%) were positive. Positive cases demonstrated heterogeneous staining of tumor cells, mostly membranous with some cytoplasmic and in one case nuclear pattern. RNA-based sequencing did not demonstrate any NTRK1-3 fusion in our patients' collective. Our study demonstrates that pan-TRK expression in lung carcinoma is very low across different histologic types. NTRK1-3 fusions using an RNA-based sequencing approached could not be detected. This stresses the importance of confirmation of immunohistochemistry results by molecular methods.

Triptolide inhibits epithelial-mesenchymal transition phenotype through the p70S6k/GSK3/ß-catenin signaling pathway in taxol-resistant human lung adenocarcinoma.

BACKGROUND: Chemotherapy is one of the primary treatments for both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), however, chemoresistance develops over time and is a bottleneck to effective chemotherapy worldwide. Therefore, the development of new potent therapeutic agents to overcome chemoresistance is of utmost importance. Triptolide is a natural component extracted from Tripterygium Wilfordii, a Chinese plant; our study aimed to evaluate its anti-tumor effects in taxol-resistant human lung adenocarcinoma and investigate its molecular mechanisms of chemoresistance. METHODS: Triptolide's inhibition of cell viability was detected by sulforhodamine B (SRB) assay. Cell cycle was measured by flow cytometry and cell apoptosis was assessed by flow cytometry and western blot. Expression of ß-catenin was analyzed by western blot and immunofluorescence (IF). The anti-tumor effects of triptolide were determined using a subcutaneous in-vivo model. Cell proliferation and apoptosis were evaluated by immunohistochemistry (IHC) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, respectively. The expression level of p-p70S6K and p-GSK-3α/ß was evaluated by western blot and IHC. RESULTS: Triptolide inhibited cell proliferation, induced S-phase cell cycle arrest and apoptosis in taxol-resistant A549 (A549/TaxR) cells. Moreover, intraperitoneal injection of triptolide resulted in a significant delay of tumor growth without obvious systemic toxicity in mice. Additionally, triptolide reversed epithelial-mesenchymal transition (EMT) through repression of the p70S6K/GSK3/ß-catenin signaling pathway. CONCLUSIONS: Our study provides evidence that triptolide can reverse EMT in taxol-resistant lung adenocarcinoma cells and impairs tumor growth by inhibiting the p70S6K/GSK3/ß-catenin pathway, indicating that triptolide has potential to be used as a new therapeutic agent for taxol-resistant lung adenocarcinoma.

Senescence and autophagy in usual interstitial pneumonia of different etiology.

Idiopathic pulmonary fibrosis (IPF) is a disease with a dismal prognosis. Currently, the causing agent(s) are poorly understood. Recent data suggest that senescence and autophagy might play a role in its development, as well as changes in metabolism due to hypoxic conditions. In this study, the expression of senescence markers in 23 cases of usual interstitial pneumonia (UIP)/IPF and UIP/chronic autoimmune diseases (UIP/AuD) was investigated. The status of autophagy was evaluated with respect to either antiinflammatory or antihypoxia function. Formalin-fixed paraffin-embedded tissues of UIP were selected for immunohistochemistry with antibodies for p21, p16, and ß-galactosidase (senescence); for LC3, SIRT1, MAP1S, and pAMKα (autophagy); and for LDH and GLUT1 (metabolism). Epithelial cells in cystic remodeled areas of UIP stained for p16 and p21, p16 being more specific compared with p21. Myofibroblasts were negative in all cases. An upregulation of all four autophagy markers was seen not only in epithelia within remodeled areas and proliferating myofibroblasts, but also in bronchial epithelia and pneumocytes. Upregulated autophagy points to a compensatory mechanism for hypoxia; therefore, LDH and GLUT1 were investigated. Their expression was present in epithelia within cystic remodeling and in myofibroblasts. The cells within the remodeled areas stained for cytokeratin 5, but coexpressed TTF1, confirming their origin from basal cells of bronchioles. Within this population, senescent cells arise. Our results indicated that autophagy in UIP very likely helps cells to survive in hypoxic condition. By phagocytosis of cellular debris, they supplement their need for nutrition, and by upregulating LDH and GLUT1, they compensate for local hypoxia.

Myopericytoma arising from myopericytosis-a hitherto unrecognized entity within the lung.

Two cases of myopericytosis combined with pericytoma originating within the lung are reported. These are rare pulmonary tumors. The differential diagnosis for hemangiopericytoma and pericytic tumors with glomus elements is discussed. Both myopericytic lesions mimic other lesions, which are more commonly seen in the lung. Based on the expression of vascular growth factor receptors 2 and 3, an antiangiogenic therapy was suggested for the patient with the myopericytoma. A treatment with an angiogenesis inhibitor resulted in a regression of the tumor, but not the precursor lesion. Probably a more specific therapy using tyrosine kinase inhibitors for VEGFR2/3 might better control these myopericytic proliferations.

Primary tumor and metastasis-sectioning the different steps of the metastatic cascade.

Patients with lung cancer in the majority die of metastases. Treatment options include surgery, chemo- and radiotherapy, targeted therapy by tyrosine kinase inhibitors (TKIs), and immuno-oncologic treatment. Despite the success with these treatment options, cure of lung cancer is achieved in only a very small proportion of patients. In most patients' recurrence and metastasis will occur, and finally kill the patient. Metastasis is a multistep procedure. It requires a change in adhesion of tumor cells for detachment from their neighboring cells. The next step is migration either as single cells [epithelial-mesenchymal transition (EMT)], or as cell clusters (hybrid-EMT or bulk migration). A combination of genetic changes is required to facilitate migration. Then tumor cells have to orient themselves along matrix proteins, detect oxygen concentrations, prevent attacks by immune cells, and induce a tumor-friendly switch of stroma cells (macrophages, myofibroblasts, etc.). Having entered the blood stream tumor cells need to adapt to shear stress, avoid being trapped by coagulation, but also use coagulation in small veins for adherence to endothelia, and express homing molecules for extravasation. Within a metastatic site, tumor cells need a well-prepared niche to establish a metastatic focus. Tumor cells again have to establish a vascular net for maintaining nutrition and oxygen supply, communicate with stroma cells, grow out and set further metastases. In this review the different steps will be discussed with a focus on pulmonary carcinomas. The vast amount of research manuscripts published so far are not easy to analyze: in most reports' single steps of the metastatic cascade are interpreted as evidence for the whole process; for example, migration is interpreted as evidence for metastasis. In lung cancer most often latency periods are shorter, in between 1-5 years. In other cases, despite widespread migration occurs, tumor cells die within the circulation and do not reach a metastatic site. Therefore, migration is a requisite, but does not necessarily predict metastasis. The intention of this review is to point to these different aspects and hopefully provoke research directed into a more functional analysis of the metastatic process.

Distribution and prognostic significance of gluconeogenesis and glycolysis in lung cancer.

Inhibition of glycolysis has been considered as a therapeutic approach in aggressive cancers including lung cancer. Abbreviated gluconeogenesis, mediated by phosphoenolpyruvate carboxykinase (PEPCK), was recently discovered to partially circumvent the need for glycolysis in lung cancer cells. However, the interplay of glycolysis and gluconeogenesis in lung cancer is still poorly understood. Here, we analyzed the expression of GLUT1, the prime glucose transporter, and of PCK1 and PCK2, the cytoplasmic and mitochondrial isoforms of PEPCK, in 450 samples of non-small cell lung cancer (NSCLC) and in 54 NSCLC metastases using tissue microarrays and whole tumor sections. Spatial distribution was assessed by automated image analysis. Additionally, glycolytic and gluconeogenic gene expression was inferred from The Cancer Genome Atlas (TCGA) datasets. We found that PCK2 was preferentially expressed in the lung adenocarcinoma subtype, while GLUT1 expression was higher in squamous cell carcinoma. GLUT1 and PCK2 were inversely correlated, GLUT1 showing elevated expression in larger tumors while PCK2 was highest in smaller tumors. However, a mixed phenotype showing the presence of both, glycolytic and gluconeogenic cancer cells was frequent. In lung adenocarcinoma, PCK2 expression was associated with significantly improved overall survival, while the opposite was found for GLUT1. The metabolic tumor microenvironment and the 3-dimensional context play an important role in modulating both pathways, since PCK2 expression preferentially occurred at the tumor margin and hypoxia regulated both, glycolysis and gluconeogenesis, in NSCLC cells in vitro, albeit in opposite directions. PCK1/2 expression was enhanced in metastases compared to primary tumors, possibly related to the different environment. The results of this study show that glycolysis and gluconeogenesis are activated in NSCLC in a tumor size and oxygenation modulated manner and differentially correlate with outcome. The frequent co-activation of gluconeogenesis and glycolysis in NSCLC should be considered in potential future therapeutic strategies targeting cancer cell metabolism.

Pleuropulmonary blastoma type I might arise in congenital pulmonary airway malformation type 4 by acquiring a Dicer 1 mutation.

Congenital pulmonary airway malformation (CPAM) occurs most commonly in infants. It is divided into 5 types. The most common types 1 and 2 are cystic, type 0 presents as bronchial buds without alveolar tissue, most likely corresponding to alveolar dysgenesis, while type 3 is composed of branching bronchioles and appears as a solid lesion. A defect in the epithelial-mesenchymal crosstalk might be the underlying mechanism for all. Type 4 is a peripheral cystic lesion with a thin cyst wall covered by pneumocytes. CPAM 4 has been mixed up with pleuropulmonary blastoma (PPB) type I and some authors question its existence. We investigated five cases of CPAM type 4 for the presence or absence of rhabdomyoblasts, and for markers associated with CPAM development. In addition, all cases were evaluated for mutations within the Dicer gene and for mutations of the RAS family of oncogenes. All five cases showed smooth muscle actin and desmin-positive cells; however, only one case showed a few cells positive for MyoD. The same case showed a mutation of Dicer 1. All cases were negative for mutations of the RAS family of genes. Fibroblast growth factor 10 was similarly expressed in all cases, and thus cannot be used to differentiate CPAM4 from PPB-I. Low expression of the proliferation marker Ki67 was seen in our CPAM 4 cases and the probable PPB-I case. YingYang-1 protein seems to play an active role in the development of PPB-I. CPAM 4 can be separated from PPB-I based on the presence of rhabdomyoblasts and mutations in Dicer 1 gene. These cells might not be numerous; therefore, all available tissue has to be evaluated. As CPAM 4 morphologically looks very similar to PPB-I, it might be speculated, that there exists a potential for progression from CPAM 4 to PPB-I, by acquiring somatic mutations in Dicer 1.

Manipulation of the immune system by non-small cell lung cancer and possible therapeutic interference.

Pulmonary carcinomas have developed mechanisms by which they escape the attack of immune cells. Immune checkpoint molecules programmed death 1 - programmed death ligand 1 (PD1-PDL1) and the cytotoxic T-lymphocyte antigen 4 system have gained attention. The expression of PDL1 by tumor cells causes immune tolerance, and further influences the microenvironment via orchestration by cytokines. Therapy with PDL1 antibodies could restore the cytotoxicity of T-lymphocytes towards tumor cells. Many patients will respond to this treatment. However, resistance mechanisms will counteract this therapy. New investigations have identified additional immune checkpoint inhibitors such as lymphocyte activation gene 3 and T cell immunoglobulin and mucin-domain containing-3. Tumor cells also induce tolerance by manipulating cells of the innate immune system. Macrophages are polarized to tumor-friendly M2, neutrophils into N2 types, and dendritic cells and myeloid suppressor cells are switched to assist tumor cells. Regulatory T cells enter the tumor microenvironment and signal tolerance to cytotoxic cells, inhibiting the influx of NK cells. Soluble mediators either released by tumor cells or cells of the tumor stroma induce immune tolerance, examples including tryptophan and indolamine dioxygenases, arginine and adenosine. Treatment options to counteract these molecules are currently being tested. The tumor stroma has been classified as immune-inflamed, immune-excluded, and immune-desert types. The latter might be switched to an inflamed type by induction of tertiary lymph follicles. Dendritic cells and macrophages normally phagocytose tumor antigens, but inhibitors of phagocytosis can block this. Interference with these molecules is another option for re-establishing the cytotoxic action of the immune system against tumor cells. In this review we will discuss these aspects with a special emphasis on non-small cell lung cancer.

Atypical goblet cell hyperplasia occurs in CPAM 1, 2, and 3, and is a probable precursor lesion for childhood adenocarcinoma.

Congenital pulmonary airway malformation (CPAM) is a developmental disorder. Types 1-2-3 are the more common ones. Atypical goblet cell hyperplasia (AGCH) in CPAM might be a precursor lesion for pulmonary adenocarcinomas. In nine out of 33 CPAM cases, types 1-3 showed foci of goblet cell proliferations. As these cells completely replace normal epithelium, we prefer to name these proliferations AGCH. In 5 cases, adenocarcinomas were seen (AC). All cases were analyzed for proteins possibly being associated with CPAM development: fibroblast growth factor 10 (FGF10) and receptor 2 (FGFR2), forkhead box A1 (FOXA1) and A2 (FOXA2), MUC protein 5AC (MUC5AC), human epidermal growth factor receptor 2 (erbB2, HER2/neu), hepatocyte nuclear factor 4α (HNF4α), SOX2, and Ying Yang protein 1 (YY1). By next generation sequencing, AGCH and adenocarcinomas were evaluated for driver mutations. Expression for FGF10, FGFR2, FOXA1, and FOXA2 was seen in CPAM epithelium and stroma, but not differently in AGCH and AC. SOX2 was positive in CPAM epithelium and AGCH, however weakly in AC. YY1 and MUC5AC showed more intense staining in AGCH and AC than in CPAM epithelium. HER2 was intensely expressed in AC and less intensely in AGCH, but not in CPAM epithelium. KRAS mutation in exon 2 was detected in all AGCH and AC, but was absent in CPAM epithelia. AGCH can arise in CPAM types 1-3. Oncogenic KRAS mutation seems to be the oncogenic driver already in AGCH, proving its role as a precursor lesion for adenocarcinoma. It might upregulate HER2 at the protein level. YY1 seems to be involved in carcinogenesis.