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This study determined whether expression levels of a panel of biologically relevant microRNAs can be used as prognostic or predictive biomarkers in patients who participated in the International Adjuvant Lung Cancer Trial (IALT), the largest randomized study conducted to date of adjuvant chemotherapy in patients with radically resected non–small cell lung carcinoma (NSCLC). Expression of miR-21, miR-29b, miR-34a/b/c, miR-155, and let-7a was determined by quantitative real-time PCR in formalin-fixed paraffin-embedded tumor specimens from 639 IALT patients. The prognostic and predictive values of microRNA expression for survival were studied using a Cox model, which included every factor used in the stratified randomization, clinicopathologic prognostic factors, and other factors statistically related to microRNA expression. Investigation of the expression pattern of microRNAs in situ was performed. We also analyzed the association of TP53 mutation status and miR-34a/b/c expression, epidermal growth factor receptor and KRAS mutation status, and miR-21 and Let-7a expression. Finally, the association of p16 and miR-29b expression was assessed. Overall, no significant association was found between any of the tested microRNAs and survival, with the exception of miR-21 for which a deleterious prognostic effect of lowered expression was suggested. Otherwise, no single or combinatorial microRNA expression profile predicted response to adjuvant cisplatin-based chemotherapy. Together, our results indicate that the microRNA expression patterns examined were neither predictive nor prognostic in a large patient cohort with radically resected NSCLC, randomized to receive adjuvant cisplatin-based chemotherapy versus follow-up only. Cancer Res; 70(21); 8288–98. ©2010 AACR.

Supplementary Appendix 1 from MicroRNA Expression and Clinical Outcomes in Patients Treated with Adjuvant Chemotherapy after Complete Resection of Non–Small Cell Lung Carcinoma

Supplementary Figure 1 from MicroRNA Expression and Clinical Outcomes in Patients Treated with Adjuvant Chemotherapy after Complete Resection of Non–Small Cell Lung Carcinoma

Supplementary Tables 1-9 from MicroRNA Expression and Clinical Outcomes in Patients Treated with Adjuvant Chemotherapy after Complete Resection of Non–Small Cell Lung Carcinoma

Background: In 2D cell cultures, migration of tumor cells is characterized by epithelial-to-mesenchymal transition (EMT), whereby tumor cells loose epithelial and express mesenchymal markers and very often change into a spindle cell phenotype. In contrast to 2D cultures, in 3D cultures tumor cells do not lose their differentiation. Small cell lung cancer usually moves as single cells or in small clusters, squamous cell and adenocarcinomas preferentially move in large clusters of cells. In tissue specimen, all of these carcinomas migrate in a hybrid EMT, not loosing their cytokeratin and E-cadherin expression. In AC and SCC also polarity and specification do occur, as some tumor cells act as leaders, providing orientation for the followers. However, 3D culture system might better depict real migration and come closer to the in vivo system as 2D cell culture systems. Material and Method: A 3D bioprinter (TissueLabsR) was used to spot an alveolar-mimicking lung-specific matrix on cell culture wells. Microscopic channels out of pluronicsR are created within the matrix. Pluronics is removed by cooling, leaving empty channels within the protein matrix. These channels can be filled with different interleukins, such as IL23, which will direct migration of the tumor cells. Cultured cell lines from an adenocarcinoma (A549) and small cell carcinoma (NCI-H82) are layered on top of the matrix and migration into the matrix is studied using different time points. Results and Discussion: A matrix simulating an alveolar structure was created with MatrigelR. The A549 cells were seeded on the matrix and grown for 2,5, 10, and 14 days. The cells formed cell clusters on the matrix and after one week started to invade the matrix in cell complexes. By immunohistochemistry we could show, that the A549 cells retained their epithelial differentiation. The H82 cells showed a different behavior. They did not form large clusters, but started to invade in small groups. The use of the bioprinter enabled us not only to create a 3D tumor culture system to simulate the in vivo conditions but also to create matrix structures for studying invasion and migration and to stimulate carcinoma cells with migration-activating substances. Citation Format: Melina Hardt, Kurt Zatloukal, Helmut H. Popper. 3D model to study migration and invasion of lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3614.

The integrated stress response (ISR) is an essential stress-support pathway increasingly recognized as a determinant of tumorigenesis. Here we demonstrate that ISR is pivotal in lung adenocarcinoma (LUAD) development, the most common histological type of lung cancer and a leading cause of cancer death worldwide. Increased phosphorylation of the translation initiation factor eIF2 (p-eIF2α), the focal point of ISR, is related to invasiveness, increased growth, and poor outcome in 928 LUAD patients. Dissection of ISR mechanisms in KRAS-driven lung tumorigenesis in mice demonstrated that p-eIF2α causes the translational repression of dual specificity phosphatase 6 (DUSP6), resulting in increased phosphorylation of the extracellular signal-regulated kinase (p-ERK). Treatments with ISR inhibitors, including a memory-enhancing drug with limited toxicity, provides a suitable therapeutic option for KRAS-driven lung cancer insofar as they substantially reduce tumor growth and prolong mouse survival. Our data provide a rationale for the implementation of ISR-based regimens in LUAD treatment., The Integrated Stress Response (ISR) is a cytoprotective pathway upregulated in many cancers. Here the authors show that the activation of PERK/p-eIF2α arm of ISR enhances ERK phosphorylation through translation repression of DUSP6, thus resulting in KRAS-driven lung tumorigenesis.

Background Lung involvement in autoimmune disease (AID) is not uncommon, and may precede other organ manifestations. The histologic pattern is variable and difficult to interpret. Another problem was recently encountered: chronicity presenting with fibrosing pneumonia. This resulted that patients with chronic AID and usual interstitial pneumonia (UIP) were excluded from antifibrotic therapy. Creating a new category of UIP with autoimmune features (IPAF) for patients with AID enabled treatment for these patients. This however raised the opinion, that a pathological investigation for the underlying disease is not necessary. Results We retrospectively evaluated 66 cases of AID, 31 cases of hypersensitivity pneumonias (HP), and 10 clinically confirmed usual interstitial pneumonia/idiopathic pulmonary fibrosis (UIP/IPF) cases. 12 additional cases could not be assigned into any of these categories. Acute AID presented with lymphocytic interstitial pneumonia, immune complex deposition, cytotoxicity for specific cell compartments, and alveolar hemorrhage. Subacute patterns most often presented with organizing pneumonia, whereas chronic pattern most often presented as UIP. Granulomas were present in some patients. UIP pattern was the most common presentation in chronic AID and HP, whereas NSIP was rather rare. Conclusion The most important, statistically significant feature differentiating chronic AID or HP, from UIP/IPF are lymphocytic infiltrations into myofibroblastic foci. Other features such as granulomas, Langhans giant cells, and protein deposits were significantly associated with AID and HP, but were not encountered in all cases. This study demonstrated that the morphological analysis can uncover the possible etiology in many cases, and a more specific diagnosis can be provided to the clinicians.

Simple Summary This review presents and summarizes the key components and outcomes of a novel, unconventional radiation approach aimed to exploit immune-stimulatory radiation effects which, being added to direct radiation tumor cell killing, may improve the therapeutic ratio of radiotherapy. This technique, as a product of translational oncology research, was intentionally developed for the induction of immune-mediated bystander and abscopal effects in the treatment of unresectable bulky tumors which have much fewer therapeutic options and show poor prognoses after conventional treatments. This review offers insights into a unique unconventional radiotherapy technique which, due to its higher immunogenic potential, may improve the prognosis of patients affected by highly complex malignancies, providing additional opportunities for future research in terms of combining novel immuno-modulating agents with more modern radiotherapy approaches. Abstract Radiation-induced immune-mediated abscopal effects (AE) of conventional radiotherapy are very rare. Whole-tumor irradiation leads to lymphopenia due to killing of immune cells in the tumor microenvironment, resulting in immunosuppression and weak abscopal potential. This limitation may be overcome by partial tumor irradiation sparing the peritumoral immune-environment, and consequent shifting of immune-suppressive to immune-stimulatory effect. This would improve the radiation-directed tumor cell killing, adding to it a component of immune-mediated killing. Our preclinical findings showed that the high-single-dose irradiation of hypoxic tumor cells generates a stronger bystander effect (BE) and AE than the normoxic cells, suggesting their higher “immunogenic potential”. This led to the development of a novel Stereotactic Body RadioTherapy (SBRT)-based PArtial Tumor irradiation targeting HYpoxic segment (SBRT-PATHY) for induction of the immune-mediated BE and AE. Encouraging SBRT-PATHY-clinical outcomes, together with immunohistochemical and gene-expression analyses of surgically removed abscopal-tumor sites, suggested that delivery of the high-dose radiation to the partial (hypoxic) tumor volume, with optimal timing based on the homeostatic fluctuation of the immune response and sparing the peritumoral immune-environment, would significantly enhance the immune-mediated anti-tumor effects. This review discusses the current evidence on the safety and efficacy of SBRT-PATHY in the treatment of unresectable hypoxic bulky tumors and its bystander and abscopal immunomodulatory potential.

The worldwide incidence of pulmonary carcinoids is increasing, but little is known about their molecular characteristics. Through machine learning and multi-omics factor analysis, we compare and contrast the genomic profiles of 116 pulmonary carcinoids (including 35 atypical), 75 large-cell neuroendocrine carcinomas (LCNEC), and 66 small-cell lung cancers. Here we report that the integrative analyses on 257 lung neuroendocrine neoplasms stratify atypical carcinoids into two prognostic groups with a 10-year overall survival of 88% and 27%, respectively. We identify therapeutically relevant molecular groups of pulmonary carcinoids, suggesting DLL3 and the immune system as candidate therapeutic targets; we confirm the value of OTP expression levels for the prognosis and diagnosis of these diseases, and we unveil the group of supra-carcinoids. This group comprises samples with carcinoid-like morphology yet the molecular and clinical features of the deadly LCNEC, further supporting the previously proposed molecular link between the low- and high-grade lung neuroendocrine neoplasms., The worldwide incidence of pulmonary carcinoids is increasing, but little is known about their molecular characteristics. Here, Alcala and colleagues present a multi-omics analysis of these tumours, revealing distinct molecular and prognostic subgroups.

Interstitial processes in the lungs of children can be due to several underlying diseases. Knowledge of the child's age is important as genetic aberrations play a major role in diseases in the first 2 years, whereas immunological diseases are more common starting in kindergarden age. In general lung diseases are rare in children, which makes the diagnostics difficult and results in a delayed diagnosis. In addition, pediatric pulmonologists are often very reluctant to perform lung biopsies due to a lack of a specialized pathologist. In order to make a contribution to the diagnostics of pediatric pulmonary diseases, pathologists should be specialized in pulmonary pathology, have a good knowledge of genetic methods and fetal lung development, which includes the genetic factors involved in lung growth and differentiation. A close cooperation with the pediatric pulmonologist is necessary and each patient should be discussed jointly on an interstitial lung disease board to promote the quality of diagnostics. The pathologist should be aware that the developing lungs of children are not just a smaller form of adult lungs and often react very differently. In this article, we mainly focus on diffuse infiltration patterns, such as ground glass and reticulonodular infiltrations as described in high-resolution computed tomography (HRCT). Localized interstitial processes, which can sometimes be tumor-like and malformations are not dealt with; however, vascular malformations are included as these often manifest as diffuse interstitial infiltrations and must therefore be taken into consideration for the differential diagnostics.

Primary neuroendocrine tumors (NET) in the mediastinum are very rare and among them thymic NETs are the most common. They represent 5 % of all thymic and mediastinal tumors. The WHO classification from 2015 subdivides thymic NETs into three groups; low grade (typical carcinoid), intermediate grade (atypical carcinoid) and high grade (large cell neuroendocrine carcinoma and small cell carcinoma). Through this change of mediastinal/thymic NET classification into three groups of malignancy, the nomenclature was adapted to that of the lungs, while the histological criteria for each entity remained the same. Thymic NETs typically occur in middle-aged adults and predominantly in males. Approximately 30 % are asymptomatic and the rest present with symptoms caused by local tumor growth, distant metastases and/or endocrine manifestations. Carcinoids can also occur as a part of multiple endocrine neoplasia type 1 (MEN1) and at the time of diagnosis commonly present with regional lymph node or distant metastases, which most often affect the lungs and bones. For the correct diagnosis tumor cell morphology, mitotic count and/or necrosis are crucial. Patients with typical carcinoids have the best prognosis, whereas the prognosis is slightly worse for atypical carcinoids but very poor for large cell neuroendocrine carcinomas. Small cell carcinomas have the worst prognosis and the shortest median survival time of approximately 14 months.

Pulmonary Langerhans cell histiocytosis is regarded as a reactive proliferation of the dendritic Langerhans cell population stimulated by chronic tobacco-derived plant proteins due to incomplete combustion but can also occur in childhood as a tumor-like systemic disease. Currently, both these forms cannot be morphologically distinguished. In the lungs a nodular proliferation of Langerhans cells occurs in the bronchial mucosa and also peripherally in the alveolar septa with an accompanying infiltration by eosinophilic granulocytes and destruction of the bronchial wall. Langerhans cells can be selectively detected with antibodies against CD1a and langerin. In the reactive isolated pulmonary form, abstinence from tobacco smoking in most patients leads to regression of infiltration and improvement of symptoms. In high-resolution computed tomography (HRCT) the small star-like scars can still be detected even after complete cessation of tobacco smoking.

CCX CKR is a decoy chemokine receptor that specifically binds the chemokines CCL19, CCL25 and CCL21. CCL19 was previously found to be upregulated in pulmonary sarcoidosis. We have, therefore, investigated CCX CKR expression in this inflammatory disease.CCX CKR mRNA was semiquantitated using RT-PCR in unseparated bronchoalveolar (BAL) cells from sarcoidosis patients (S, n = 29) and healthy control subjects (C, n = 9). CCX CKR transcripts were upregulated in patients (mean +/- SEM); S, 0.82 +/- 0.10; C, 0.44 +/- 0.04; p = 0.01; this upregulation paralleled the disease course as assessed by the chest radiographic stage (p0.02). Immunocytochemistry localised the CCX CKR protein to ciliated bronchial cells. Flow cytometric fluorescent ligand uptake assay showed that these cells are able to internalize its ligand.These observations implicate CCX CKR in the modulation of the inflammatory response in sarcoidosis.

A few human tumor types have been modeled in mice using genetic or chemical tools. The final goal of these efforts is to establish models that mimic not only the location and cellular origin of human cancers but also their genetic aberrations and morphologic appearances. The latter has been neglected by most investigators, and comparative histopathology of human versus mouse cancers is not readily available. This issue is exacerbated by the fact that some human malignancies comprise a whole spectrum of cancer subtypes that differ molecularly and morphologically. Lung cancer is a paradigm that appears not only as non-small cell and small-cell lung cancer but comprises a plethora of subtypes with distinct morphologic features. This review discusses species-specific and common morphological features of non-small cell lung cancer in mice and humans. Potential inconsistencies and the need for refined genetic tools are discussed in the context of a comparative analysis between commonly employed RAS-induced mouse tumors and human lung cancers.

From the aspect of the contemporary pathologic diagnostics of lung cancer, it is a key issue of the tissue obtained since small biopsies and cytology still play a major role. In the non-small cell lung cancer era, cytology considered equal to biopsy. However, in recent years it is unable to provide quality diagnosis and must be replaced by biopsy. Various molecular techniques can handle various different tissue samples which must be considered during molecular pathology diagnosis. Besides, tumor cell-normal cell ratio in the obtained tissue as well as the absolute tumor cell number have great significance whose information must be provided in the primary lung cancer diagnosis. Last but not least, for continuous sustainable molecular diagnostics of lung cancer rational algorythms, affordable technology and appropriate reimbursement are equally necessary.

The clinical expectations how pathologists should submit lung cancer diagnosis have changed dramatically. Until mid 90-ties a clear separation between small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) was mostly sufficient. With the invention of antiangiogenic treatment a differentiation between squamous and non-squamous NSCLC was requested. When epidermal growth factor receptor (EGFR) mutation was detected in patients with pulmonary adenocarcinomas and subsequent specific treatment with tyrosine kinase inhibitors (TKIs) was invented, sub-classification of NSCLC and molecular analysis of the tumor tissue for mutations was asked for. Pathologists no longer submit just a diagnosis, but instead are involved in a multidisciplinary team for lung cancer patient management. After EGFR several other driver genes such as echinoderm microtubule associated protein like 4-AL-Kinase 1 (EML4-ALK1), c-ros oncogene 1, receptor tyrosine kinase (ROS1), discoidin domain receptor tyrosine kinase 2 (DDR2), fibroblast growth factor receptor 1 (FGFR1) were discovered, and more to come. Due to new developments in bronchology (EUS, EBUS) the amount of tissue submitted for diagnosis and molecular analysis is decreasing, however, the genes to be analyzed are increasing. Many of these driver gene aberrations are inversions or translocations and thus require FISH analysis. Each of these analyses requires a certain amount of tumor cells or one to two tissue sections from an already limited amount of tissues or cells. In this respect new genetic test systems have been introduced such as next generation sequencing, which enables not only to detect multiple mutations in different genes, but also amplifications and fusion genes. As soon as these methods have been validated for routine molecular analysis this will enable the analysis of multiple genetic changes simultaneously. In this review we will focus on genetic aberrations in NSCLC, resistance to new target therapies, and also to methodological requirements for a meaningful evaluation of lung cancer tissue and cells.

Lymphoepithelioma-like carcinoma (LELC) is a rare form of lung cancer, usually encountered in Chinese patients. Similar to nasopharyngeal carcinoma, LELC of the lung is strongly associated with Epstein-Barr virus (EBV) infection in Asian patients, but there is controversy over whether an association exists in patients from Western countries. To determine whether such a relationship exists, we retrospectively studied 6 cases of primary LELC of the lung, all of which were in Western patients. There were 4 men and 2 women, ranging in age from 49 to 75 years. The tumors ranged from 1 to 4.5 cm in diameter. Four patients had stage I disease, 1 had stage IIb disease, and 1 had stage IIIa disease. All patients are alive without evidence of disease with a follow-up of 18 to 30 months. Formalin-fixed, paraffin-embedded tissue was stained with hematoxylin-eosin for routine evaluation and immunostained for keratin and leukocyte common antigen (LCA). LCA staining was performed to exclude large-cell lymphoma. Immunoperoxidase staining (1:500 clone CS1-4; Dako, Carpinteria, CA) and in situ hybridization were performed to detect EBV. Tumors consisted of solid nests of undifferentiated tumor cells in a syncytial arrangement surrounded by heavy lymphoplasmacytic infiltrate. Tumor cells stained positively for keratin but negative for LCA. All 6 cases were negative for EBV, suggesting no association between EBV and LELC in the Western population.

Established clinicopathologic characteristics of non-small cell lung cancer patients define a subgroup responding better to EGFR-TK inhibitors: adenocarcinoma histology, ethnicity, sex, smoking status, presence of activating EGFR mutation, and/or K-RAS wild type. However, EGFR mutation does not automatically lead to increased activity of the protein influenced by several factors. As adenocarcinoma can be further divided into histologic subclasses, we compared adenocarcinomas without lepidic growth pattern (NLAC) to those characterized by pure or predominant lepidic growth (LAC) for EGFR protein expression and autophosphorylation activity (Y1173), as determined by immunohistochemistry. This pretarget therapy cohort comprised a total of 110 surgically operated patients of stage I non-small cell lung cancer: 49 NLAC and 61 LAC variants. The LAC group had a significantly better prognosis and the incidence of phospho-EGFR-positive tumors was significantly higher compared with NLAC. Patient sex did not influence EGFR activity, but the incidence of pEGFR-positive tumors was significantly lower among smoker patients. There was no statistically significant difference in EGFR or KRAS mutation frequencies between the 2 groups. In NLAC, pEGFR-positive tumors occurred exclusively among EGFR-mutant/K-RAS wild-type tumors. On the contrary, in LAC tumors, pEGFR-positive tumors were similarly frequent in the EGFR or K-RAS mutant groups indicating an interesting feedback activation of EGFR signaling in K-RAS mutant tumors. Our data also indicate that EGFR mutation leads to EGFR autophosphorylation only in a small fraction of adenocarcinoma patients, which might have clinical significance.

We retrospectively evaluated 107 fiberoptic bronchoscopies with and without transbronchial lung biopsy (TBLB) in 98 consecutive patients with haematologic malignancies and pulmonary infiltrates. Bronchoalveolar lavage (BAL) was performed in 45 and BAL plus TBLB in 62 procedures. There was no procedure-related severe haemorrhage, pneumothorax or death. Infectious aetiology was identified in 26 of 107 (24%), toxic pneumonitis in 17 of 107 (16%) and neoplastic infiltration in 9 of 107 (8.5%) episodes. Combined BAL and TBLB was significantly superior to BAL alone with respect to the diagnosis of neoplastic infiltrates (p=0.008) and toxic pneumonitis (p

Fluoroscopically guided transthoracic fine needle biopsies were performed in 890 patients (380 female, 582 male, mean age: 56 years). In 795 patients (89.4%) the first biopsy was successful. The success rate of biopsy correlated closely with the size of the lesion and increased with larger size. Sensitivity in the diagnosis of malignant lesions was 94.6%, specificity 99.5%. Due to cytological, histological, and clinical confirmation of benign lung lesions in 300 patients (33.7% of all patients), the number of unnecessary diagnostic thoracotomies for benign disease could be reduced significantly. In 9 of 113 cases (8%) histological reclassification of the cytologic results was necessary. Reclassification was not necessary in the differentiation between benign and malignant lesions and between small cell and non-small cell carcinomas. The overall complication rate was 24.7% (220 patients). Only 36 patients (4% of all patients) required chest-tube insertion. There were no fatal complications. Percutaneous fluoroscopically guided transthoracic fine needle biopsy of the lung, performed with the Rotex needle, is a time-effective, safe, and efficient method for diagnosing focal pulmonary lesions.

Low-grade flat ductal intraepithelial neoplasia (DIN1a, flat epithelial atypia) is one of the earliest morphologically recognizable neoplastic lesions of the breast. Frequently, it occurs concomitantly with lobular intraepithelial neoplasia (LIN). We aimed to elucidate chromosomal aberrations in these early neoplastic breast lesions with the use of array comparative genomic hybridization analysis.Laser capture microdissection of 12 archival formalin-fixed, paraffin-embedded specimens harbouring foci of both DIN1a and LIN was performed. All analysed cases of DIN1a and LIN showed chromosomal gains and losses. The aberration encountered most often was loss of 16q, noted in seven DIN1a (70% of those successfully examined) and 10 LIN (91%) cases. The next most common alteration was a gain on 1q, noted in four DIN1a (40%) and seven LIN (64%) cases.The results show concurrent chromosomal aberrations of 1q gains and 16q losses in several cases with coexisting LIN and DIN1a. These aberrations are known to be common in low-grade invasive (ductal and lobular) carcinomas as well as in more advanced (conventional) types of low-grade ductal intraepithelial neoplasia (DIN) (low-grade ductal carcinoma in situ). Our results raise the possibility of similar molecular-genetic pathways in coexisting LIN and low-grade flat DIN.