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Supplementary Fig. S1 from Fibroblast growth factor receptor-mediated signals contribute to the malignant phenotype of non-small cell lung cancer cells: therapeutic implications and synergism with epidermal growth factor receptor inhibition

Supplementary Table S2 from Fibroblast growth factor receptor-mediated signals contribute to the malignant phenotype of non-small cell lung cancer cells: therapeutic implications and synergism with epidermal growth factor receptor inhibition

Fibroblast growth factors (FGF) and their high-affinity receptors (FGFR) represent an extensive cellular growth and survival system. Aim of this study was to evaluate the contribution of FGF/FGFR-mediated signals to the malignant growth of non-small cell lung cancer (NSCLC) and to assess their potential as targets for therapeutic interventions. Multiple FGFR mRNA splice variants were coexpressed in NSCLC cells (n = 16) with predominance of FGFR1. Accordingly, both expression of a dominant-negative FGFR1 (dnFGFR1) IIIc-green fluorescent protein fusion protein and application of FGFR small-molecule inhibitors (SU5402 and PD166866) significantly reduced growth, survival, clonogenicity, and migratory potential of the majority of NSCLC cell lines. Moreover, dnFGFR1 expression completely blocked or at least significantly attenuated s.c. tumor formation of NSCLC cells in severe combined immunodeficient mice. Xenograft tumors expressing dnFGFR1 exhibited significantly reduced size and mitosis rate, enhanced cell death, and decreased tissue invasion. When FGFR inhibitors were combined with chemotherapy, antagonistic to synergistic in vitro anticancer activities were obtained depending on the application schedule. In contrast, simultaneous blockage of FGFR- and epidermal growth factor receptor-mediated signals exerted synergistic effects. In summary, FGFR-mediated signals in cooperation with those transmitted by epidermal growth factor receptor are involved in growth and survival of human NSCLC cells and should be considered as targets for combined therapeutic approaches. [Mol Cancer Ther 2008;7(10):3408–19]

About 15% of patients with non-small cell lung cancer (NSCLC) harbor the Kirsten rat sarcoma homolog G12C mutation (KRASG12C). Selective KRASG12C inhibitors offer new treatment opportunities, but little is known about the prevalence, characteristics, and outcomes of standard-of-care treatment (SOC) in this population. We retrospectively assessed the clinicopathological features of patients with KRASG12C-mutated advanced NSCLC and responses to SOC at two high-volume centers in Austria. Out of 2495 NSCLC patients tested, we identified 174 patients with advanced-stage disease carrying a KRASG12C mutation. Most patients were ≥65 years old (55%), heavy smokers (55%), and presented with comorbidities. The most frequent co-alteration was TP53 (18%). PD-L1 expression was high (TPS ≥ 50%) in 31%, very high (TPS ≥ 90%) in 11%, and negative in 31% of patients. A total of 138 patients (79%) received oncologic systemic treatment. The most common first-line therapy (1 L) was anti-PD-1/PD-L1 plus platinum-based chemotherapy. Median overall survival measured from 1 L treatment was 15.3 months (95% CI, 8.6–21.9), 9.4 (95% CI, 5.3–13.5) from 2 L treatment, and 8.4 (95% CI, 1.7–15.1) from 3 L treatment. The time-to-next-treatment was 8.4 (95% CI, 5.2–11.6) from 1 L and 6.1 (95% CI, 2.7–9.7) months from 2 L to 3 L. These poor outcomes underscore the need for the implementation of new treatment options and for specific molecular testing.

The second-generation ALK tyrosine kinase inhibitor brigatinib has recently been approved in the European Union for use after crizotinib treatment in patients with EML4-ALK-rearranged lung cancer. In the current study, brigatinib was investigated as second-line or later-line treatment in 35 patients who had developed resistance to crizotinib, ceritinib, or alectinib. Most patients (68.6%) received brigatinib as second or third line (range: second to 12th line). In the total cohort, complete and partial responses were obtained for 9.1 and 75.8%, respectively. Overall median progression-free survival was 9.9 months, whereas the largest treatment cohort (brigatinib after crizotinib failure) showed a median progression-free survival of 8.4 months. Fifty-four percent of patients with baseline brain metastases responded to brigatinib treatment. Brigatinib was highly effective after crizotinib and ceritinib failure. Six patients had received alectinib as monotherapy, second-line, or third line before brigatinib; of these, four experienced partial responses and two progressed responses. Brigatinib treatment was well tolerated.

Background Analysis of cell-free DNA from blood could provide an alternative method for identifying genomic changes in the tumors of patients with advanced lung adenocarcinoma. Objective We compared the performance of droplet digital PCR (ddPCR) and Cobas® EGFR Mutation Test v2 (Cobas) for detecting EGFR mutations in cell-free plasma DNA. Patients and Methods Plasma samples from patients with advanced EGFR-mutated lung adenocarcinoma were analyzed for EGFR T790M, exon 19 deletions, and L858R mutations by both ddPCR and Cobas. Results T790M testing was performed in 354 plasma samples collected from 129 patients. The concordance rate between ddPCR and Cobas for T790M, sensitivity, and specificity were 86, 100, and 85%, respectively. Exon 19 deletions were analyzed in 196 plasma samples obtained from 71 of the 129 patients using both platforms. The concordance rate between ddPCR and Cobas for exon 19 deletions, sensitivity, and specificity were 90, 92, and 89%, respectively. L858R mutations were studied in 124 plasma samples obtained from 44 of the 129 patients using both assays. The concordance rate between ddPCR and Cobas for L858R, sensitivity, and specificity were 90, 91, and 89%, respectively. In patients who progressed under treatment with an EGFR TKI (n = 50), the T790M positivity rate was 66% using ddPCR, but only 24% using Cobas. Conclusions We observed a high concordance between ddPCR and Cobas in detecting EGFR mutations in plasma samples of patients with advanced EGFR-mutated lung adenocarcinoma, but ddPCR was more sensitive than Cobas.

Malignant pleural mesothelioma (MPM) is a rare pleural cancer associated with asbestos exposure. According to current evidence, the combination of chemotherapy, surgery and radiotherapy improves patients’ survival. However, the optimal sequence and weighting of the respective treatment modalities is unclear. In anticipation of the upcoming results of the MARS-2 trial, we sought to determine the relative impact of the respective treatment modalities on complications and overall survival in our own consecutive institutional series of 112 patients. Fifty-seven patients (51%) underwent multimodality therapy with curative intent, while 55 patients (49%) were treated with palliative intent. The median overall survival (OS) of the entire cohort was 16.9 months (95% CI: 13.4–20.4) after diagnosis; 5-year survival was 29% for patients who underwent lung-preserving surgery. In univariate analysis, surgical treatment (p < 0.001), multimodality therapy (p < 0.001), epithelioid subtype (p < 0.001), early tumor stage (p = 0.02) and the absence of arterial hypertension (p = 0.034) were found to be prognostic factors for OS. In multivariate analysis, epithelioid subtype was associated with a survival benefit, whereas the occurrence of complications was associated with worse OS. Multimodality therapy including surgery significantly prolonged the OS of MPM patients compared with multimodal therapy without surgery.

In clinical practice, patients with anaplastic lymphoma kinase (ALK)-rearrangement&ndash, positive non&ndash, small-cell lung cancer commonly receive sequential treatment with ALK tyrosine kinase inhibitors. The third-generation agent lorlatinib has been shown to inhibit a wide range of ALK resistance mutations and thus offers potential benefit in later lines, although real-world data are lacking. This multicenter study retrospectively investigated later-line, real-world use of lorlatinib in patients with advanced ALK- or ROS1-positive lung cancer. Fifty-one patients registered in a compassionate use program in Austria, who received second- or later-line lorlatinib between January 2016 and May 2020, were included in this retrospective real-world data analysis. Median follow-up was 25.3 months. Median time of lorlatinib treatment was 4.4 months for ALK-positive and 12.2 months for ROS-positive patients. ALK-positive patients showed a response rate of 43.2%, while 85.7% percent of the ROS1-positive patients were considered responders. Median overall survival from lorlatinib initiation was 10.2 and 20.0 months for the ALK- and ROS1-positive groups, respectively. In the ALK-positive group, lorlatinib proved efficacy after both brigatinib and alectinib. Lorlatinib treatment was well tolerated. Later-line lorlatinib treatment can induce sustained responses in patients with advanced ALK- and ROS1-positive lung cancer.

Carcinoid tumours are rare neuroendocrine neoplasms that mostly occur in younger adults with low tendencies to metastasise. Based on their histological characteristics, they are divided into typical and atypical subtypes. The most common presenting symptoms are due to central airway obstruction. The first step in the diagnostic assessment should be a computed tomography (CT) scan, as it provides information both for local tumour extent and lymph node involvement. Bronchoscopy is the main tool for histological confirmation, evaluation of bronchial wall invasion and removal of endobronchial manifestation with subsequent resolution of atelectasis. Endobronchial ultrasound may be necessary to rule out lymph node metastasis. Somatostatin receptor scintigraphy in combination with CT can rule out further metastatic disease.Surgical resection using parenchyma-sparing techniques remains the gold standard for treatment. For selected patients, endobronchial therapy could be an alternative for minimal invasiveness. Long-term follow-up is suggested due to the high likelihood of recurrence.Here, we describe our clinical experience in a 35-year-old male patient who originally presented with haemoptysis and a central polypoid tumour in the left main bronchus revealed by a CT scan. The histological characteristics were indicative of a typical carcinoid. The patient was treated using an endobronchial approach only. No complications and no recurrences have been observed in a follow-up of 2 years.

Introduction Osimertinib is standard treatment for patients with advanced EGFR T790M-mutated non–small-cell lung cancer who have been pre-treated with EGFR–tyrosine kinase inhibitors (TKIs). We studied whether cell-free plasma DNA for T790M detection can be used to select patients for osimertinib treatment in the clinical routine. Methods From April 2015 to November 2016, we included 119 patients with advanced EGFR -mutated non–small-cell lung cancer who had progressed under treatment with an EGFR-TKI. The T790M mutation status was assessed in cell-free plasma DNA by droplet digital polymerase chain reaction in all patients and by tissue analyses in selected patients. Results T790M mutations were detected in 85 (93%) patients by analyses of cell-free plasma DNA and in 6 (7%) plasma-negative patients by tumor re-biopsy. Eighty-nine of 91 T790M-positive patients received osimertinib. Median progression-free survival (PFS) was 10.1 months (95% confidence interval [CI]: 8.1–12.1). Median survival was not reached and the 1-year survival was 64%. The response rate was 70% in T790M-positive patients (n = 91) in the intention-to-treat population. PFS trended to be shorter in patients with high T790M copy number (≥10 copies/mL) compared to those with low T790M copy number ( p = 0.09). A comparable trend was observed for overall survival (hazard ratio for overall survival = 2.16, 95% CI: 0.89–5.25, p = 0.09). No difference in response rate was observed based on T790M copy numbers. Conclusion Plasma genotyping using digital polymerase chain reaction is clinically useful for the selection of patients who had progressed during first-line EGFR-TKI therapy for treatment with osimertinib.

Molecular testing of EGFR is required to predict the response likelihood to targeted therapy in non–small cell lung cancer. Analysis of circulating tumor DNA in plasma may complement limitations of tumor tissue. This study evaluated the interlaboratory performance and reproducibility of a real-time PCR EGFR mutation test (cobas EGFR Mutation Test v2) to detect EGFR variants in plasma. Fourteen laboratories received two identical panels of 27 single-blinded plasma samples. Samples were wild type or spiked with plasmid DNA to contain seven common EGFR variants at six predefined concentrations from 50 to 5000 copies per milliliter. The circulating tumor DNA was extracted by a cell-free circulating DNA sample preparation kit (cobas cfDNA Sample Preparation Kit), followed by duplicate analysis with the real-time PCR EGFR mutation test (Roche Molecular Systems, Pleasanton, CA). Lowest sensitivities were obtained for the c.2156G>C p.(Gly719Ala) and c.2573T>G p.(Leu858Arg) variants for the lowest target copies. For all other variants, sensitivities varied between 96.3% and 100.0%. All specificities were 98.8% to 100.0%. Coefficients of variation indicated good intralaboratory and interlaboratory repeatability and reproducibility but increased for decreasing concentrations. Prediction models revealed a significant correlation for all variants between the predefined copy number and the observed semiquantitative index values, which reflect the samples' plasma mutation load. This study demonstrates an overall robust performance of the real-time PCR EGFR mutation test kit in plasma. Prediction models may be applied to estimate the plasma mutation load for diagnostic or research purposes.

Malignant pleural mesothelioma is an aggressive malignancy characterized by frequent resistance to chemo- and radiotherapy, poor outcome, and limited therapeutic options. Fibroblast growth factors (FGFs) and their receptors are potential targets for cancer therapy, but their significance in mesothelioma has remained largely undefined.To investigate the antimesothelioma potential of FGF receptor 1 (FGFR1) inhibition.Expression of FGFs and their receptors was analyzed in mesothelioma cell lines and tissue specimens. Several cell models were used to investigate FGFR1 inhibition in vitro and in combination with cisplatin and irradiation. Mouse intraperitoneal xenotransplant models were used for in vivo validation.FGFR1, FGF2, and FGF18 were overexpressed in mesothelioma. Stimulation with FGF2 led to increased cell proliferation, migration, and transition to a more sarcomatoid phenotype in subsets of mesothelioma cell lines. In contrast, inhibition of FGFR1 by a specific kinase inhibitor or a dominant-negative FGFR1 construct led to significantly decreased proliferation, clonogenicity, migration, spheroid formation, and G1 cell cycle arrest in several mesothelioma cell lines, accompanied by apoptosis induction and decreased mitogen-activated protein kinase pathway activity. Reduced tumor growth, proliferation, mitogenic signaling, and apoptosis induction were observed in vivo. Inhibition of FGFR1 synergistically enhanced the cytotoxic effects of ionizing radiation and cisplatin.Our data suggest that the malignant phenotype of mesothelioma cells depends on intact FGF signals, which should be considered as therapeutic targets with a promising chemo- and radiosensitizing potential.

Integrin-linked kinase (ILK) is an intracellular signaling protein critically involved in cellular growth and motility. In non-small cell lung cancer (NSCLC), increased ILK expression has been associated with decreased recurrence-free and overall survival. Recently, ILK has also been detected in the serum of NSCLC patients.To assess the prognostic impact of preoperative serum ILK (sILK) concentration on overall survival in surgically amenable NSCLC.Preoperative sILK was quantified by ELISA in 50 newly diagnosed NSCLC patients. After surgery, patients were followed-up for a median interval of 2.5 years.Serum ILK concentrations ranged from 0 to 2.44 ng/ml. Mean sILK was around 2.3 times higher in the 16 patients who died as compared to the 34 patients who survived (1.04 vs. 0.45 ng/ml, p = 0.001). In univariate time-to-event analysis, increased sILK was associated with adverse survival [Hazard ratio (HR): 4.03, 95 % CI: 2.00-8.13, p0.001]. This association prevailed after multivariable adjustment for several clinical, demographic, and laboratory parameters (HR: 3.85, 95 % CI: 1.53-9.72, p = 0.004).Serum ILK shows potential as a novel strong and independent prognostic marker for postoperative survival in surgically amenable NSCLC.

The introduction of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) for non-small cell lung cancer (NSCLC) with activating mutations of the EGFR has opened a new area of lung cancer treatment strategies and led to an enthusiastic search for additional mutations. Since then, numerous driver mutations such as EML4-ALK and ROS1 have been detected and specific treatment options have already been developed. However, molecular tests have to follow specific rules if applied in daily practice. The Austrian Working Group on Pulmonary Pathology and Oncology (AWGPPO) is presenting an updated version of their previous recommendation published in 2011. Several practical questions raised during the last 2 years will be addressed, such as reflex testing, selection of tissues, order of molecular tests, and the issue of resistance mechanisms.

Integrin-linked kinase (ILK) is a cell membrane-bound molecule implicated in the metastatic progression of many tumour types. It phosphorylates the downstream target AKT (phosphorylated AKT, pAKT), and, by doing this, it activates anti-apoptotic pathways. We have recently shown ILK expression in malignant pleural mesothelioma (MPM). To determine whether ILK expression in MPM is connected with pAKT expression, and whether ILK and pAKT expression have any influence on the patient's prognosis, we correlated ILK and pAKT expression, as assessed by immunohistochemistry, with disease-related survival in a retrospective cohort of 80 MPM patients.The paraffin specimens of 80 MPM cases treated from 1990 to 2006 (52 surgical cases, 28 conservative cases) have been retrieved from the archive. The median (range) patients' age was 62 (28-83 years) years; the male-to-female ratio was 3:1. Fifty percent of the patients had an epitheloid subtype. The samples have been stained with anti-ILK as well as with anti-pAKT and scored by two independent pathologists. Intensity of ILK and pAKT expression has been correlated with disease-related survival.In total, 73 of 80 (91%) MPM samples expressed ILK; 65 of 74 (88%) MPM samples expressed pAKT. Comparing the 5-year disease-related survival according to ILK or pAKT expression, no statistically significant difference could be found between ILK and pAKT expressing or non-expressing patients. However, in the subgroup of conservatively treated MPM patients, those with strong ILK expression had a longer 5-year disease-related survival (p0.0001). In total, the only prognostic factor across all ILK, pAKT and therapy subgroups was the histological subtype (p = 0.01). The prognostic significance of the histological subtype has been confirmed in multivariate analysis (p = 0.005).The expression of ILK in MPM is connected with the expression of the downstream target pAKT, but neither ILK nor pAKT expression has a measurable influence on the patient's prognosis, except for certain subgroups of MPM. However, to shed light on the true prognostic impact of ILK and pAKT expression in MPM, prospective trials are needed.

Integrin-linked kinase (ILK) is a protein kinase that links integrins and growth factors to a range of signalling pathways. ILK expression and activity are increased in a variety of human cancers. However, little is known regarding the role of ILK in malignant pleural mesothelioma (MPM). In this study, we assessed the expression of ILK in samples of human MPM, and compared it with the expression of epidermal growth factor receptor (EGFR). Thirty-four samples of human malignant mesothelioma were stained with a polyclonal antibody against ILK. Two independent observers evaluated the morphological pattern and intensity of staining. The findings have been compared with the patient's characteristics. Most MPM and mesothelial cell proliferation samples (87.9%) showed cytoplasmic ILK staining of varying intensity. Normal mesothelial cells and normal lung parenchyma did not stain for ILK at all. Conversely, the percentage of positive EGFR staining was somewhat lower (75.8%). The ILK-positive patients were significantly older than the ILK-negative patients. Here we report for the first time that ILK is indeed expressed in malignant mesothelioma. For further validation of a causal association between ILK and neoplastic mesothelial transformation, these immunohistochemical results should be supplemented with clinical and molecular biological data.

Background Pulmonary tumorlets are usually an incidental pathologic curiosity of no clinical importance, but may be mistaken for epithelial and nonepithelial neoplasms. Fine needle aspiration (FNA) of this cell proliferation has rarely been reported. We describe a pulmonary tumorlet associated with branch acentric granulomatosis presenting as a tumorous consolidation on chest radiograph. Case In a hitherto healthy 70-year-old man admitted for acute respiratory infection, a solid consolidation was found on chest radiograph. Medical history was uneventful except right-sided pleurisy in 1949. Computed tomography-guided FNA sample was composed of loose clusters of small columnar cells with cyanophilic cytoplasm and centrally located round to oval nuclei. With a tentative diagnosis of well-differentiated adenocarcinoma, lumpectomy was performed. Intraoperative cytology demonstrated lymphocytes, epithelioid cells, giant cells of Longerbang type and clusters of columnar cells. Definitive histologic examination confirmed the intraoperative diagnosis of necrotizing granulomatosis and tumorlet. Neuroendocrine origin of the cells was confirmed by immunocytochemical and immunohistochemical studies resulting in strong reactivity of the cells to synaptophysin, NSE, chromogranin A and N-Cam. Conclusion Knowledge of the cytomorphologic presentation oftumorlets in FNA and consideration of the appropriate differential diagnoses combined with ancillary studies might have prevented lung resection.

Sprouty (Spry) proteins function as inhibitors of receptor tyrosine kinase signaling mainly by interfering with the Ras/Raf/mitogen-activated protein kinase cascade, a pathway known to be frequently deregulated in human non–small cell lung cancer (NSCLC). In this study, we show a consistently lowered Spry2 expression in NSCLC when compared with the corresponding normal lung epithelium. Based on these findings, we investigated the influence of Spry2 expression on the malignant phenotype of NSCLC cells. Ectopic expression of Spry2 antagonized mitogen-activated protein kinase activity and inhibited cell migration in cell lines homozygous for K-Ras wild type, whereas in NSCLC cells expressing mutated K-Ras, Spry2 failed to diminish extracellular signal-regulated kinase (ERK) phosphorylation. Nonetheless, Spry2 significantly reduced cell proliferation in all investigated cell lines and blocked tumor formation in mice. Accordingly, a Spry2 mutant unable to inhibit ERK phosphorylation reduced cell proliferation significantly but less pronounced compared with the wild-type protein. Therefore, we conclude that Spry2 interferes with ERK phosphorylation and another yet unidentified pathway. Our results suggest that Spry2 plays a role as tumor suppressor in NSCLC by antagonizing receptor tyrosine kinase–induced signaling at different levels, indicating feasibility for the usage of Spry in targeted gene therapy of NSCLC. (Mol Cancer Res 2007;5(5):509–20)

Sprouty2 is a modulator of receptor tyrosine kinase-mediated signalling with an important role during lung carcinogenesis. Here, we characterize a Sprouty2 variant harbouring a substitution of proline 106 with serine. Serine substitution fails to influence expression, but accumulation of slower migrating phosphatase-sensitive forms indicates that its presence facilitates phosphorylation. In normal lung cells the serine variant is slightly more potent in inhibiting proliferation and migration. Additionally non-malignant cells expressing the major Sprouty2 variant attach more effective to fibronectin, while the serine variant only weakly stimulates cell adhesion. Mechanistically, the serine variant interferes less effectively with mitogen-activated protein kinase induction in response to serum. Concerning the positive Sprouty2 effect on epidermal growth factor receptor activation the serine variant is more potent. In all lung cancer-derived cell lines proliferation is more effectively inhibited if the Sprouty2 protein harbours the serine. In contrast, an increased interference of the serine Sprouty2 variant is only observed in cells with unaltered K-Ras. In cells harbouring a K-Ras mutation the serine conversion weakens the reduction of migration velocity indicating that dependent on the status of K-Ras the serine influences Sprouty2 functions differently. Accordingly, cell adhesion in cells with unaffected K-Ras is only stimulated by a Sprouty2 protein harbouring proline, while a serine conversion improves the attachment of the cells with constitutive active Ras. In summary our studies demonstrate that substitution of proline by serine at position 106 has biological significance and that the observed effects of this conversion depend on the activation status of endogenous K-Ras. J. Cell. Biochem. 117: 1822-1832, 2016. © 2016 Wiley Periodicals, Inc.

Basic fibroblast growth factor (FGF-2) has been implicated in the progression of human tumours via both autocrine and paracrine (angiogenic) activities. We investigated the expression of FGF-2 and FGF receptors (FGFR-1 to -4) in NSCLC cell lines (N = 16), NSCLC surgical specimens (N = 21) and 2 control cell lines. Our data show that almost all NSCLC cells produce elevated levels of FGF-2 and FGFR in vitro and in vivo. FGF-2 expression did correlate with a short doubling time as well as with potent anchorage-independent growth of NSCLC cell lines. In contrast with control cells, NSCLC cells did not secrete considerable amounts of FGF-2 into the extracellular space. Expression levels of FGFR-1 and -2 in NSCLC cell lines correlated with FGF-2 production. FGFR were located at the plasma membranes in some low FGF-2-producing NSCLC and control cell lines. These cells were sensitive to the proliferative effect of recombinant FGF-2 (rFGF-2). In NSCLC cell lines with an enhanced FGF-2 production, representing the majority studied, FGFR localisation was predominantly intracellular. These cells were insensitive to both the proliferative effect of rFGF-2 and growth inhibition by FGF-2-neutralising antibodies. In contrast, several agents antagonised FGF-2 intracellularly impaired growth of almost all NSCLC cell lines. Our data suggest a role of FGF-2 and FGFR in the growth stimulation of NSCLC cells possibly via an intracrine mechanism.

MicroRNAs can govern up to hundred different mRNAs and are important regulators of gene expression programs in development and disease. We analyzed the expression of microRNA-21, one of the most common oncomirs, in non-small cell lung cancer (NSCLC). Using northern blots the microRNA-21 expression levels of NSCLC-derived tissue and cell lines were measured. In line with earlier observations we show that mature microRNA-21 expression levels are highly increased in NSCLC-derived tissue compared to normal lung tissue. Additionally, we demonstrate that microRNA-21 levels correlate with malignancy since its expression in higher staged tumors is significantly more elevated compared to stage 1A. Interestingly, microRNA-21 levels in cultured NSCLC-derived cells are comparable to the expression detected in non-malignant lung tissue. Since microRNA-21 levels showed no fluctuation during the cell cycle, accelerated proliferation of tumor cells is not responsible for microRNA-21 upregulation in the tumor compartment. Similarly to NSCLC-derived cancer cells, the tumor-associated fibroblasts show low expression levels of microRNA-21. Together, these data indicate that rather microenviromental and growth conditional changes than intrinsic features of the cancer cells are responsible for the observed increase of microRNA-21 levels in tumor tissues. Subsequently culturing conditions were changed to assess the impact of co-cultivation with fibroblasts, hypoxia and anchorage-independent growth on microRNA-21 expression. While co-cultivation with tumor-associated fibroblasts had no effect on microRNA-21 expression, both hypoxia and anchorage-independent growth cause a microRNA-21 elevation. In summary, our data demonstrate that growth conditions especially expected in more malignant tumors result in microRNA-21 upregulation explaining the observed increase in higher staged lung cancer tissue, but not in lung cancer-derived cells.

We report on a 55-year-old patient who was admitted to hospital because of recurrent pneumonia. CT imaging provided airway narrowing and mural thickening of the distal trachea and mainstem bronchi, compatible with endobronchial polypoid, toric-shaped changes of the distal tracheal wall spreading into both the left and right bronchial system. Bronchoscopy was performed and biopsies revealed the diagnosis of tracheobronchial amyloidosis. We performed a combination of bronchoscopic debulking and consecutive external beam radiation therapy with the result of no further progression of the disease, stable endobronchial situation, and functional improvements at a follow up at 6 months.

e20572Background: The EGFR T790M resistance mutation located on Exon 20 is the most common mechanism of drug resistance to EGFR tyrosine kinase inhibitors (TKI). The mutation can be detected by re-...

Einleitung: Das maligne Pleuramesotheliom (MPM) ist eine aggressive, asbest-assoziierte Krebserkrankung mit begrenzten Therapieoptionen und schlechter Prognose. Fibroblasten Wachstumsfaktoren (FGF) und deren Rezeptoren (FGFR) regulieren Wachstum, Wanderungsverhalten und Uberleben von Zellen und sind an wichtigen physiologischen Prozessen aber auch an der Entstehung und dem Fortschreiten von Krebs beteiligt. Der FGF Signalweg wurde bereits als potentielles Ziel fur neuartige Therapien in diverse Krebsarten untersucht, aber bisher noch nicht fur das maligne Pleuramesotheliom. Ziel dieser Studie war die Evaluierung der FGF/FGFR Achse als mogliches „Therapietarget“ auch fur MPM. Methoden: Die Expression aller vier FGFRs samt Isoformen und der 22 FGFs wurde mittels konventioneller PCR, qRT-PCR, Immunfluoreszenz, genetischen Reporterkonstrukten in MPM Zellinien (n=9) und immunhistochemischer Farbungen in Tumorgewebeproben (N=60) untersucht. Zur Stimulierung beziehungsweise Blockierung des FGF Signalwegs wurden rekombinantes FGF2, die synthetischen Inhibitoren SU5402 und PD166866 sowie ein dominant-negativer FGFR1-Adenovirus verwendet. Zellwachstum und Wanderung wurde durch MTT, Clonogenic, Platypus und Transwell Assays ermittelt beziehungsweise durch Farbung mit Hoechst/PI und mikroskopische Betrachtung. Anderungen in intrazellularen Signalwegen wurden mittels Western Blot Analyse dargestellt. Ergebnisse: Expressionsanalysen ergaben eine starke Uberexpression des FGFR1 und eine hohe Expression der Liganden FGF2 und FGF18, sowohl in den Zelllinien als auch Tumorgewebeproben. Die Blockierung des FGFR1 durch spezifische Inhibitoren sowie ein Adenoviruskonstrukt fuhrte in allen Zelllinien zu signifikant vermindertem Zellwachstum, Stimulation mit FGF2 hingegen zu einer Erhohung sowie zu drastischen morphologischen Veranderungen. Ahnliche Effekte wurden auch in Bezug auf das Wanderungsverhalten von MPM Zellen beobachtet, begleitet von Anderungen in intrazellularen Signaltransduktionskaskaden. Des Weiteren resultierte die Inhibierung des FGFR1 in stark verminderter Spheroidbildung, Kombination von FGFR-Inhibierung mit herkommlicher Chemo- und Radiotherapie fuhrte in den meisten Fallen zu einer Verstarkung der Wirkung. Diskussion: Unsere Daten zeigen, dass der FGF Signalweg eine wichtige Rolle fur Wachstum, Uberleben, Wanderungsverhalten sowie Chemo- und Strahlenresistenz in MPM Zellen spielt und eine weitere Exploration als potenziell neuer Therapieansatz im MPM sinnvoll ist.

Objectives: Epidermal growth factor receptor (EGFR)-targeted therapies are a valid therapeutic option for advanced non-small-cell lung cancer (NSCLC), but unequivocally recognised predictive factors for therapeutic response are lacking. However, intrinsic resistance might occur due to loss of EGFR expression during the course of the disease or its treatment. Methods: Paraffin-embedded tissue from cases with metastatic NSCLC obtained at autopsy was retrieved from our archive. Specimens of primary tumour (n = 39; 64% adenocarcinoma) and of all corresponding metastases (n = 70) were immunohistochemically stained for EGFR expression. Two observers independently scored staining intensity and evaluated the percentage of positively stained cells. Identical staining intensity and 10% difference in number of stained cells were defined as perfect concordance; and one-increment difference in staining intensity and less than 30% difference in number of stained cells were defined as good concordance. Results: Twenty-seven out of 39 primary tumours (69%) were EGFR-positive on immunohistochemistry (IHC), with 12/27 (44%) of positive tumours exhibiting intense or moderate EGFR expression. The median number of EGFR-expressing cells in primary tumours was 50% (range 0—100%). Overall Spearman’s rank correlations for staining intensity and percentage of positively stained cells between primary tumours and paired metastases were 0.78 (p < 0.001) and 0.60 (p < 0.001), respectively. Perfect concordance was observed in 51% (20/39) and good concordance in 18% (7/39) of corresponding pairs, respectively, whereas 9/12 metastases showing discordant staining with their corresponding primary tumours had lacked EGFR expression. Conclusions: In most NSCLCs, EGFR status of primary tumours correlates with EGFR status of correspondingmetastases. Hence,lossof EGFRexpressionis unlikelyduringdiseaseprogression,localornon-EGFR-targetingsystemictreatment. # 2010 European Association for Cardio-Thoracic Surgery. Published by Elsevier B.V. All rights reserved.

Synovial sarcoma of the lung (SSL) is a very rare but aggressive primary lung tumor. Due to its unusual histological features, it can easily be misdiagnosed, if only small biopsies of the tumor are investigated. Here, we review two recent cases of SSL diagnosed and treated in our institution. The first case is a 37-year-old male with a round nodule in the right lower lobe; he underwent a lobectomy. Histologically, the nodule resembled a biphasic tumor. Cytogenetic analysis revealed a translocation t (X; 18), and the diagnosis of primary SSL could be established. The patient is alive and disease-free since 45 months following surgery. The second case is a 41-year-old male with a cystic lesion in the right lower lobe, removed by video-assisted thoracic surgery (VATS) segmentectomy. In the tumor tissue, spindle cell-rich and cystic structures could be found, together with epithelial elements. Because the tumor contained also a translocation t (X; 18), it could be diagnosed as monophasic SSL. The patient is alive and disease-free since 11 months. Since rare diseases of the lung may present as subtle and focal changes, complete removal of suspect pulmonary lesions is always advisable.

Fibroblast growth factors (FGF) and their high-affinity receptors (FGFR) represent an extensive cellular growth and survival system. Aim of this study was to evaluate the contribution of FGF/FGFR-mediated signals to the malignant growth of non-small cell lung cancer (NSCLC) and to assess their potential as targets for therapeutic interventions. Multiple FGFR mRNA splice variants were coexpressed in NSCLC cells (n = 16) with predominance of FGFR1. Accordingly, both expression of a dominant-negative FGFR1 (dnFGFR1) IIIc-green fluorescent protein fusion protein and application of FGFR small-molecule inhibitors (SU5402 and PD166866) significantly reduced growth, survival, clonogenicity, and migratory potential of the majority of NSCLC cell lines. Moreover, dnFGFR1 expression completely blocked or at least significantly attenuated s.c. tumor formation of NSCLC cells in severe combined immunodeficient mice. Xenograft tumors expressing dnFGFR1 exhibited significantly reduced size and mitosis rate, enhanced cell death, and decreased tissue invasion. When FGFR inhibitors were combined with chemotherapy, antagonistic to synergistic in vitro anticancer activities were obtained depending on the application schedule. In contrast, simultaneous blockage of FGFR- and epidermal growth factor receptor-mediated signals exerted synergistic effects. In summary, FGFR-mediated signals in cooperation with those transmitted by epidermal growth factor receptor are involved in growth and survival of human NSCLC cells and should be considered as targets for combined therapeutic approaches. [Mol Cancer Ther 2008;7(10):3408–19]

Pulmonary tumorlets are usually an incidental pathologic curiosity of no clinical importance, but may be mistaken for epithelial and nonepithelial neoplasms. Fine needle aspiration (FNA) of this cell proliferation has rarely been reported. We describe a pulmonary tumorlet associated with bronchocentric granulomatosis presenting as a tumorous consolidation on chest radiograph.In a hitherto healthy 70-year-old man admitted for acute respiratory infection, a solid consolidation was found on chest radiograph. Medical history was uneventful except right-sided pleurisy in 1949. Computed tomography-guided FNA sample was composed of loose clusters of small columnar cells with cyanophilic cytoplasm and centrally located round to oval nuclei. With a tentative diagnosis of well-differentiated adenocarcinoma, lumpectomy was performed. Intraoperative cytology demonstrated lymphocytes, epithelioid cells, giant cells of Langerhans type and clusters of columnar cells. Definitive histologic examination confirmed the intraoperative diagnosis of necrotizing granulomatosis and tumorlet. Neuroendocrine origin of the cells was confirmed by immunocytochemical and immunohistochemical studies resulting in strong reactivity of the cells to synaptophysin, NSE, chromogranin A and N-Cam.Knowledge of the cytomorphologic presentation of tumorlets in FNA and consideration of the appropriate differential diagnoses combined with ancillary studies might have prevented lung resection.

Fibroblast growth factor 5 (FGF5) is widely expressed in embryonic but scarcely in adult tissues. Here we report simultaneous overexpression of FGF5 and its predominant high-affinity receptor (FGFR1 IIIc) in astrocytic brain tumour specimens (N=49) and cell cultures (N=49). The levels of both ligand and receptor increased with enhanced malignancy in vivo and in vitro. Furthermore, secreted FGF5 protein was generally present in the supernatants of glioblastoma (GBM) cells. siRNA-mediated FGF5 down-modulation reduced moderately but significantly GBM cell proliferation while recombinant FGF5 (rFGF5) increased this parameter preferentially in cell lines with low endogenous expression levels. Apoptosis induction by prolonged serum starvation was significantly prevented by rFGF5. Moreover, tumour cell migration was distinctly stimulated by rFGF5 but attenuated by FGF5 siRNA. Blockade of FGFR1-mediated signals by pharmacological FGFR inhibitors or a dominant-negative FGFR1 IIIc protein inhibited GBM cell proliferation and/or induced apoptotic cell death. Moreover, rFGF5 and supernatants of highly FGF5-positive GBM cell lines specifically stimulated proliferation, migration and tube formation of human umbilical vein endothelial cells. In summary, we demonstrate for the first time that FGF5 contributes to the malignant progression of human astrocytic brain tumours by both autocrine and paracrine effects.

We report on two Turkish children who presented with progressive airway obstruction. Open lung biopsy revealed follicular bronchiolitis. The children were treated with systemic steroids and various topical medications. Whereas the respiratory situation of patient 1 required immunosuppressive therapy, the condition of patient 2 stabilised without systemic medication. Conclusion Diagnosis of follicular bronchiolitis should be considered when children present with recurrent respiratory tract infections, progressive dyspnoea, and chronic bronchial obstruction. Children in whom follicular bronchiolitis is suspected should undergo open lung biopsy for confirmation of diagnosis.

Aims: Malignant pleural mesothelioma is a devastating cancer with increasing incidence. Despite the fact that for establishing the diagnosis a number of immunohistochemical markers are needed, there is only one pathological prognosticator accepted and utilized, namely the histological subtype. Accordingly, we have investigated the prognostic significance of the Ki76 labeling index in malignant pleural mesothelioma. Patients and Methods: Ki67 index has been determined by assessing the labeled nuclei per high power field and correlated in a retrospective manner with patients’ clinical history and outcome. The prognostic power of the routine proliferation marker Ki67 was first determined in a test cohort (TC) of paraffin-embedded tissue samples from 55 patients. The independence of prognostic power against basic clinicopathological characteristics has been determined by multivariate analysis. Next, we measured the prognostic power in an independent validation cohort (VC) of 42 patients from another center. Results: Median Ki67 index was found to be 10 for both the test and validation. Patients whose tumor sample was categorized by low Ki67 index (≥ 10) had significantly longer survival times than those with higher Ki76 indices (in the test cohort: hazard ratio [HR] 2.49, 95% confidence interval [CI] 1.30-4.79, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1712. doi:1538-7445.AM2012-1712

a Division of Thoracic Surgery, Otto Wagner Hospital, Vienna, Austria b Karl Landsteiner Institute for Thoracic Oncology, Otto Wagner Hospital, Vienna, Austria c Division of Pathology, Otto Wagner Hospital, Vienna, Austria d Division of Cytology, Medical University of Graz, Graz, Austria e Division of Pathology, Vienna Medical University, Vienna, Austria f Department of Cardiothoracic Surgery, Vienna Medical University, Vienna, Austria