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Introduction: Phosphatase and tensin homolog (PTEN) loss is frequently observed in NSCLC and associated with both phosphoinositide 3-kinase activation and tumoral immunosuppression. PTEN immunohistochemistry is a valuable readout, but lacks standardized staining protocol and cutoff value. Methods: After an external quality assessment using SP218, 138G6 and 6H2.1 anti-PTEN antibodies, scored on webbook and tissue microarray, the European Thoracic Oncology Platform cohort samples (n = 2245 NSCLC patients, 8980 tissue microarray cores) were stained with SP218. All cores were H-scored by pathologists and by computerized pixel-based intensity measurements calibrated by pathologists. Results: All three antibodies differentiated six PTEN+ versus six PTEN- cases on external quality assessment. For 138G6 and SP218, high sensitivity and specificity was found for all H-score threshold values including prospectively defined 0, calculated 8 (pathologists), and calculated 5 (computer). High concordance among pathologists in setting computer-based intensities and between pathologists and computer in H-scoring was observed. Because of over-integration of the human eye, pixel-based computer H-scores were overall 54% lower. For all cutoff values, PTEN-was associated with smoking history, squamous cell histology, and higher tumor stage (p < 0.001). In adenocarcinomas, PTEN-was associated with poor survival. Conclusion: Calibration of immunoreactivity intensities by pathologists following computerized H-score measurements has the potential to improve reproducibility and homogeneity of biomarker detection regarding epitope validation in multicenter studies. (C) 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Clear cell papillary renal cell carcinoma (ccpRCC) and renal angiomyoadenomatous tumor (RAT) share morphologic similarities with clear cell (ccRCC) and papillary renal cell carcinoma (pRCC). It is a matter of controversy whether their morphologic, immunophenotypic and molecular features allow the definition of a separate renal carcinoma entity. The aim of our project was to investigate specific renal immunohistochemical biomarkers involved in the hypoxia-inducible factor pathway and mutations in the VHL gene to clarify the relationship between ccpRCC and RAT. We investigated 28 ccpRCC and 9 RAT samples by immunohistochemistry using 25 markers. VHL gene mutations and allele losses were investigated by Sanger sequencing and fluorescence in situ hybridization (FISH). Clinical follow-up data were obtained for a subset of the patients. No tumor recurrence or tumor-related death was observed in any of the patients. Immunohistochemistry and molecular analyses led to the reclassification of three tumors as ccRCC and TFE3 translocation carcinomas. The immunohistochemical profile of ccpRCC and RAT samples was very similar but not identical, differing from both ccRCC and pRCC. Especially, the parafibromin and hKIM-1 expression exhibited differences in ccpRCC/RAT compared with ccRCC and pRCC. Genetic analysis revealed VHL mutations in 2/27 (7%) and 1/7 (14%) ccpRCC and RAT samples, respectively. FISH analysis disclosed a 3p loss in 2/20 (10 %) ccpRCC samples. ccpRCC and RAT have a specific morphologic and immunohistochemical profile but they share similarities with the more aggressive renal tumors. Based on our results, we regard ccpRCC/RAT as a distinct entity of renal cell carcinomas.

Reliable detection of anaplastic lymphoma kinase (ALK) rearrangements is a prerequisite for personalized treatment of lung cancer patients, as ALK rearrangements represent a predictive biomarker for the therapy with specific tyrosine kinase inhibitors. Currently, fluorescent in situ hybridization (FISH) is considered to be the standard method for assessing formalin-fixed and paraffin-embedded tissue for ALK inversions and translocations. However, FISH requires a specialized equipment, the signals fade rapidly and it is difficult to detect overall morphology and tumor heterogeneity. Chromogenic in situ hybridization (CISH) has been successfully introduced as an alternative test for the detection of several genetic aberrations. This study validates a newly developed ALK CISH assay by comparing FISH and CISH signal patterns in lung cancer samples with and without ALK rearrangements. One hundred adenocarcinomas of the lung were included in this study, among them 17 with known ALK rearrangement. FISH and CISH were carried out and evaluated according to the manufacturers' recommendations. For both assays, tumors were considered positive if ≥15% of tumor cells showed either isolated 3' signals or break-apart patterns or a combination of both. A subset of tumors was exemplarily examined by using a novel EML4 (echinoderm microtubule-associated protein-like 4) CISH probe. Red, green and fusion CISH signals were clearcut and different signal patterns were easily recognized. The percentage of aberrant tumor cells was statistically highly correlated (P0.001) between FISH and CISH. On the basis of 86 samples that were evaluable by ALK CISH, we found a 100% sensitivity and 100% specificity of this assay. Furthermore, EML4 rearrangements could be recognized by CISH. CISH is a highly reliable, sensitive and specific method for the detection of ALK gene rearrangements in pulmonary adenocarcinomas. Our results suggest that CISH might serve as a suitable alternative to FISH, which is the current gold standard.

The human histamine H(4)-receptor (hH(4)R) possesses high constitutive activity and, like the human H(1)-receptor (hH(1)R), is involved in the pathogenesis of type-I allergic reactions. The study aims were to explore the value of dual H(1)/H(4)R antagonists as antiallergy drugs and to address the question of whether H(1)R ligands bind to hH(4)R. In an acute murine asthma model, the H(1)R antagonist mepyramine and the H(4)R antagonist 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methyl-piperazine (JNJ 7777120) exhibited synergistic inhibitory effects on eosinophil accumulation in the bronchoalveolar lavage fluid. At the hH(4)R expressed in Sf9 insect cells, 18 H(1)R antagonists and 22 H(1)R agonists showed lower affinity to hH(4)R than to hH(1)R as assessed in competition binding experiments. For a small number of H(1)R antagonists, hH(4)R partial agonism was observed in the steady-state GTPase assay. Most compounds were neutral antagonists or inverse agonists. Twelve phenylhistamine-type hH(1)R partial agonists were also hH(4)R partial agonists. Four histaprodifen-type hH(1)R partial agonists were hH(4)R inverse agonists. Dimeric histaprodifen was a more efficacious hH(4)R inverse agonist than the reference compound thioperamide. Suprahistaprodifen was the only histaprodifen acting as hH(4)R partial agonist. Suprahistaprodifen was docked into the binding pocket of inactive and active hH(4)R models in two different orientations, predominantly stabilizing the active state of hH(4)R. Collectively, the synergistic effects of H(1)R and H(4)R antagonists in an acute asthma model and the overlapping interaction of structurally diverse H(1)R ligands with hH(1)R and hH(4)R indicate that the development of dual H(1)R/H(4)R antagonists is a worthwhile and technically feasible goal for the treatment of type-I allergic reactions.

[Figure][1] [![Graphic][3] ][3][![Graphic][4] ][4] A 9-year-old boy was admitted to our emergency department with acute chest pain and elevated troponin I. His mother had died of a ruptured aortic aneurysm. Computed tomography angiography of the chest and upper abdomen was