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Supplementary Fig. 1: Ectopic PIK3CAH1047R expression induces a modest activation of the EGFR downstream signaling pathway in BEAS-2B clones. Supplementary Fig. 2: the steady state level of miR-29b and the relative copy number of the miR-29b2 locus are significantly correlated in KRAS/EGFR wildtype tumors. Supplementary Fig. 3: miR-138 expression is not induced by mutant KRAS or mutant EGFR in adenocarcinoma tissues. Supplementary Fig. 4: Inhibitors of the EGFR signaling pathway downregulate expression of key downstream effectors. Supplementary Fig. 5: miR-29b confers resistance to TNFalpha+ActD-induced apoptosis. Supplementary Fig. 6: TNFAIP3 is a target of miR-29b. Supplementary Fig. 7: miR-29b sensitizes A549 cells to cisplatin-induced apoptosis.

Patient characteristics.

A global understanding of miRNA function in EGFR signaling pathways may provide insights into improving the management of KRAS-mutant lung cancers, which remain relatively recalcitrant to treatment. To identify miRNAs implicated in EGFR signaling, we transduced bronchial epithelial BEAS-2B cells with retroviral vectors expressing KRASG12V and monitored miRNA expression patterns by microarray analysis. Through this approach, we defined miR-29b as an important target for upregulation by mutant KRAS in non–small cell lung cancers. Cell biologic analyses showed that pharmacologic inhibition of EGFR or MEK was sufficient to reduce levels of miR-29b, while PI3K inhibition had no effect. In KRASG12V-transduced BEAS-2B cells, introduction of anti-miR-29b constructs increased the sensitivity to apoptosis, arguing that miR-29b mediated apoptotic resistance conferred by mutant KRAS. Mechanistic investigations traced this effect to the ability of miR-29b to target TNFAIP3/A20, a negative regulator of NF-κB signaling. Accordingly, overexpression of an miR-29b–refractory isoform of TNFAIP3 restored NF-κB and extrinsic apoptosis, confirming that TNFAIP3 is a functionally relevant target of miR-29b. We also noted that miR-29b could confer sensitivity to intrinsic apoptosis triggered by exposure to cisplatin, a drug used widely in lung cancer treatment. Thus, miR-29b expression may tilt cells from extrinsic to intrinsic mechanisms of apoptosis. Overall, our results reveal a complexity in cancer for miR-29b, which can act as either an oncogene or tumor suppressor gene depending on signaling context. Cancer Res; 76(14); 4160–9. ©2016 AACR.

Idiopathic pulmonary fibrosis (IPF) plays a special role within the group of interstitial lung diseases (ILDs) due to its inexorable progression and its specific medical treatment. With a median survival of only 2-3 years from the time of diagnosis, the prognosis is worse than many carcinomas.In contrast to other ILDs, IPF does not respond to anti-inflammatory treatment with corticosteroids but rather demands a specific medical therapy. Even though this cannot cure the disease, it can prolong survival. Lung transplantation is the only cure for progressive lung fibrosis. The clinical course is individual and difficult to predict. Acute exacerbations accelerate the clinical course and lead to high mortality.The underlying pathomechanisms of IPF, with its complex immunological and inflammatory processes and external impacts, have been the focus of recent research. Lifestyle and environmental influences are held responsible for much of its natural history. Smoking, pneumotoxic medications, and inhalation of dusts are well-known risk factors. Likewise, genetic and hereditary factors play a crucial role.This short review focuses on the peculiarities of IPF within the group of ILDs, especially in relation to its underlying mechanisms and clinical progression.

Interstitial lung diseases present clinically with unspecific respiratory symptoms and occur idiopathically or etiologically linked to various causes. The morphology of interstitial lung diseases (radiology or histopathology) may also be unspecific in the individual case, due to the limited arsenal of reaction patterns of the lungs. Only the combination of all findings assembled during multidisciplinary discussion (MDD) between pulmonologist, radiologist and pathologist, and if required other specialties, enables a highly reliable final diagnosis, permitting improved, personalized patient treatment. The necessity for histological evaluation and the means of tissue acquisition should also be decided on during MDD, considering clinical and radiological differential diagnoses, the risks involved in the procedures and patient-specific characteristics. In the current review, we discuss MDD as the diagnostic gold standard and exemplify its merit presenting a case of interstitial lung disease.

Background: Programmed cell death protein 1 (PD-1)/programmed cell death protein ligand 1 (PD-L1) immune checkpoint inhibitors have been approved for monotherapy of metastatic nonsmall cell lung cancer (mNSCLC) depending on tumour cells' PD-L1 expression. Pleural effusion is common in mNSCLC. The significance of immunocytochemistry PD-L1 analysis from pleural effusion samples is unclear. Aim: The aim of the study was to analyse the sensitivity regarding immunocytochemistry PD-L1 analysis of pleural effusion in NSCLC as compared to immunohistochemistry of pleural biopsies. Patients and Methods: Fifty consecutive subjects (17 female, median age 72.5 years, seven never-smokers) were enrolled in this prospective controlled two-centre study. Inclusion criteria were pleural effusion, suspected or known lung cancer, indication for pleural puncture and thoracoscopy, and written informed consent. Immunocytochemistry and immunohistochemistry PD-L1 analyses were performed with the Dako-PDL1-IHC-22C3pharmDx assay. Analysis for sensitivity, specificity, and positive and negative predictive value was performed for PD-L1 detection from pleural effusion. Results: 50 subjects underwent pleural puncture and thoracoscopy. Pathological diagnoses were lung cancer (48), lymphoma (1) and mesothelioma (1). Sensitivity, specificity, positive predictive value and negative predictive value of PD-L1-testing with expression ≥50% defined as positive were 100% (95% CI 46–100%), 63% (36–84%), 45% (18–75%) and 100% (66–100%), and with expression ≥1% defined as positive 86% (56–97%), 43% (12–80%), 75% (47–92%) and 60% (17–93%). Conclusion: PD-L1 analysis in tumour-positive pleural effusion samples shows a very high sensitivity and negative predictive value, especially regarding PD-L1 expression levels ≥50% (European Medicines Agency approval). Negative results are reliable and help in the decision against a first-line checkpoint inhibitor monotherapy. However, a 1% cut-off level (United States Food and Drug Administration approval) leads to a markedly lower negative predictive value, making other invasive procedures necessary (NCT02855281)., PD-L1 analysis in tumour-positive pleural effusion samples is characterised by a very high sensitivity and negative predictive value. Negative results seem very reliable and could thus help in the decision against a first-line checkpoint inhibitor therapy. https://bit.ly/2GPCLYS

Richter’s transformation (RT) is an aggressive lymphoma that occurs upon progression from chronic lymphocytic leukemia (CLL). Transformation has been associated with genetic aberrations in the CLL phase involving TP53, CDKN2A, MYC, and NOTCH1; however, a significant proportion of RT cases lack CLL phase–associated events. Here, we report that high levels of AKT phosphorylation occur both in high-risk CLL patients harboring TP53 and NOTCH1 mutations as well as in patients with RT. Genetic overactivation of Akt in the murine Eµ-TCL1 CLL mouse model resulted in CLL transformation to RT with significantly reduced survival and an aggressive lymphoma phenotype. In the absence of recurrent mutations, we identified a profile of genomic aberrations intermediate between CLL and diffuse large B-cell lymphoma. Multiomics assessment by phosphoproteomic/proteomic and single-cell transcriptomic profiles of this Akt-induced murine RT revealed an S100 protein-defined subcluster of highly aggressive lymphoma cells that developed from CLL cells, through activation of Notch via Notch ligand expressed by T cells. Constitutively active Notch1 similarly induced RT of murine CLL. We identify Akt activation as an initiator of CLL transformation toward aggressive lymphoma by inducing Notch signaling between RT cells and microenvironmental T cells.

Adenocarcinoma (AC) is the number one pathological entity of lung cancer with approximately 30-40% of cases. It is known to be heterogeneous and has 5 histopathological growth patterns. We evaluated the long-term survival rates of patients with predominant subtypes.290 patients with AC underwent pulmonary resection between 2012 and 2017 at our institution. We excluded all patients with lymph node involvement and distant metastases. Hence, 163 patients were included for further analysis. Predominant growth pattern was defined if more than 10% of cells showed a growth pattern. 1, 3, and 5-year survival rates were evaluated. Survival was assessed by Kaplan-Meier curves and the Cox proportional hazards model was used to identify prognostic factors for overall survival.Predominant growth patterns10% were compared to10% growth patterns of the same subtype. 1-year, 3-year, and 5-year overall survival rates of patients with predominant solid tumor growth10% differed significantly from patients with10% (88.4% vs. 97.6%, p = 0.04; 65.8% vs. 87.4% p = 0.001, 36.4% vs. 65.9% p = 0.01). Survival rates did not differ between10% papillary and acinar growth compared to10%. Kaplan-Meier curves showed reduced overall survival for patients with solid tumor growth10% (log-rank 0.002). Solid tumor growth10% was an independent prognostic factor for worse long-term survival (Hazard ratio: 3.05, p = 0.01).Our study demonstrates that the presence of a predominant solid pattern in pulmonary adenocarcinoma is a factor for an unfavorable prognosis. This should be kept in mind in daily clinical practice.

Background: The detection of PD-L1 in non-small cell lung cancer (NSCLC) is of great relevance for individualized therapy and has so far been performed using immunohistochemical analysis (IHC). With accompanying pleural effusion, the immunocytochemical analysis (ICC) could be of additive diagnostic value. Methods: 50 patients (17 women, 70±10 years, 26±5 kg/m²) with highly suspected NSCLC and pleural effusion were examined. PD-L1 expression of IHC and ICC samples were compared using the PD-L1 IHC 22C3 pharmDx Kit (Dako). Sensitivity and specificity of tumour and PD-L1 detection of ICC was determined with IHC as reference. PD-L1 was quantified according to percent of tumour cells expressing PD-L1. Due to different approval statuses of available immune checkpoint inhibitors, we considered different cut-offs for PD-L1 positivity. Results: Regarding the detection of malignancy, 7 cases with inconclusive ICC results were defined as tumour negative. Failed PD-L1 analyses were defined as PD-L1-negative. Conclusion: The detection of tumour cells in pleural punctate shows good sensitivity. PD-L1 detection is less sensitive but may still contribute to the identification of PD-L1-positive lung carcinoma. In case when invasive diagnostics (bronchoscopy, thoracoscopy) are not feasible, ICC of pleural punctate may offer a clinically valuable alternative to detect PD-L1-positive lung carcinoma with the option for 1st-line immune checkpoint inhibitor monotherapy.

Molecular genetic testing (such as next-generation sequencing (NGS)) and programmed death ligand 1 (PD-L1) staining have become essential for the evaluation of lung cancer tissue [1, 2]. Endobronchial forceps biopsy (FB) is considered the gold standard for tissue sampling in central lung cancer [3]., In central exophytic lung cancer, the detection rate of oncogenic mutations and PDL1 positivity may be increased by combined sampling by forceps and EBUS-TBNA. The additional sampling of mediastinal lymph node and ctDNA may not be of additional benefit. https://bit.ly/2Ve41EF

Host-modulating therapies have become an important focus in the development of novel concepts for improved management of tuberculosis (TB). Previous in vitro studies revealed that the p38 MAP kinase signaling pathway coordinates several inflammatory and stress responses in Mycobacterium tuberculosis (Mtb)-infected host cells. Here we extend these findings and show that in vivo treatment of Mtb-infected C57BL/6 mice with doramapimod, a p38 MAP-kinase inhibitor, results in reduced inflammation, granuloma formation and lung pathology. Moreover, doramapimod, together with standard antibiotic treatment, significantly reduced lung and spleen mycobacterial loads compared to antibiotic treatment alone. Our in vivo data suggest the opportunity to repurpose p38 MAPK inhibitors for adjunct host directed therapies. We also provide first data on safety of p38 MAPK inhibition which is of relevance for future application of these substances in inflammatory diseases and concomitant TB.

Idiopathic pulmonary fibrosis (IPF) plays a special role within the group of interstitial lung diseases (ILDs) due to its inexorable progression and its specific medical treatment. With a median survival of only 2-3 years from the time of diagnosis, the prognosis is worse than many carcinomas.In contrast to other ILDs, IPF does not respond to anti-inflammatory treatment with corticosteroids but rather demands a specific medical therapy. Even though this cannot cure the disease, it can prolong survival. Lung transplantation is the only cure for progressive lung fibrosis. The clinical course is individual and difficult to predict. Acute exacerbations accelerate the clinical course and lead to high mortality.The underlying pathomechanisms of IPF, with its complex immunological and inflammatory processes and external impacts, have been the focus of recent research. Lifestyle and environmental influences are held responsible for much of its natural history. Smoking, pneumotoxic medications, and inhalation of dusts are well-known risk factors. Likewise, genetic and hereditary factors play a crucial role.This short review focuses on the peculiarities of IPF within the group of ILDs, especially in relation to its underlying mechanisms and clinical progression.

Lung adenocarcinoma (LUAD) is the most prevalent subtype of non‐small cell lung cancer. Despite the development of novel targeted and immune therapies, the 5‐year survival rate is still only 21%, indicating the need for more efficient treatment regimens. Lysine‐specific demethylase 1 (LSD1) is an epigenetic eraser that modifies histone 3 methylation status, and is highly overexpressed in LUAD. Using representative human cell culture systems and two autochthonous transgenic mouse models, we investigated inhibition of LSD1 as a novel therapeutic option for treating LUAD. The reversible LSD1 inhibitor HCI‐2509 significantly reduced cell growth with an IC 50 of 0.3–5 μm in vitro, which was linked to an enhancement of histone 3 lysine methylation. Most importantly, growth arrest, as well as inhibition of the invasion capacities, was independent of the underlying driver mutations. Subsequent expression profiling revealed that the cell cycle and replication machinery were prominently affected after LSD1 inhibition. In addition, our data provide evidence that LSD1 blockade significantly interferes with EGFR downstream signaling. Finally, our in vitro results were confirmed by preclinical therapeutic approaches, including the use of two autochthonous transgenic LUAD mouse models driven by either EGFR or KRAS mutations. Importantly, LSD1 inhibition resulted in significantly lower tumor formation and a strong reduction in tumor progression, which were independent of the underlying mutational background of the mouse models. Hence, our findings provide substantial evidence indicating that tumor growth of LUAD can be markedly decreased by HCI‐2509 treatment, suggesting its use as a single agent maintenance therapy or combined therapeutical application in novel concerted drug approaches.

Interstitial lung diseases present clinically with unspecific respiratory symptoms and occur idiopathically or etiologically linked to various causes. The morphology of interstitial lung diseases (radiology or histopathology) may also be unspecific in the individual case, due to the limited arsenal of reaction patterns of the lungs. Only the combination of all findings assembled during multidisciplinary discussion (MDD) between pulmonologist, radiologist and pathologist, and if required other specialties, enables a highly reliable final diagnosis, permitting improved, personalized patient treatment. The necessity for histological evaluation and the means of tissue acquisition should also be decided on during MDD, considering clinical and radiological differential diagnoses, the risks involved in the procedures and patient-specific characteristics. In the current review, we discuss MDD as the diagnostic gold standard and exemplify its merit presenting a case of interstitial lung disease.

The improvement in sensitive techniques has allowed the detection of tumor-specific aberrations in circulating tumor (ct) DNA. Amplification-refractory mutation system PCR has been used for the analysis of ctDNA to detect resistance-causing mutations in the epidermal growth factor receptor gene (eg, EGFR T790M) in lung cancer patients. So far, Streck tubes have been widely used as standard blood collection tubes. Here, we compared blood collection tubes manufactured by Streck with tubes from Roche and Qiagen regarding their utility in stabilizing ctDNA in plasma samples. Venous blood from healthy donors was collected in tubes from Streck, Roche, and Qiagen. Samples were spiked with artificially fragmented EGFR T790M-mutated DNA and stored for different periods of time or spiked with different DNA amounts before plasma preparation. Extracted ctDNA was analyzed by amplification-refractory mutation system PCR. Mutant DNA, spiked into blood samples from healthy donors at quantities of 1 or 3 ng, was still reliably detectable in all samples after 7 days. EGFR T790M could be detected when spiking was performed with an amount of artificial ctDNA of 0.5 ng when tubes from Roche and Qiagen were used. Blood collection tubes from Roche and Qiagen are highly suitable for ctDNA stabilization and subsequent liquid biopsy testing. Even low ctDNA concentrations allow the detection of somatic mutations.

Epithelial-myoepithelial carcinoma is a well differentiated malignant neoplasm, which originates from the salivary glands. The primary pulmonary manifestation is rare-about 30 cases have been reported worldwide. In the literature, anatomical resection has been described as the standard surgical approach. In the presented case, a wedge resection was performed, with no evidence for tumor relapse in the follow-up reevaluation after 24 months. This is the first case report of a primary pulmonary epithelial-myoepithelial carcinoma that has been treated with a non-anatomical wedge resection and lymph node dissection as a curative approach.

Immunotherapy that targets PD-1 or PD-L1 by monoclonal antibodies has emerged as effective and safe new treatment of malignant tumours. It is most beneficial in highly immunogenic tumours, in particular in malignant melanoma (1) and in non-small cell lung cancer (NSCLC) (2). Immunohistochemistry (IHC) of PD-L1 protein may serve as predictive biomarker for both PD-1 and PD-L1 inhibitors.

Next-generation-sequencing (NGS) from circulating (ctDNA) or tissue derived tumor DNA and PD-L1 analysis are essential for the evaluation of lung cancer. The objective was to evaluate the diagnostic value of endobronchial-ultrasound-guided transbronchial-needle-aspiration (EBUS-TBNA) from tumor and lymph nodes and ctDNA analysis compared to endobronchial forceps-biopsy (FB). 30 consecutive central lung cancer subjects (10 female, median age 66, 29 current/ex-smokers) with suspected exophytic lung cancer were included. The sequence of FB and EBUS-TBNA procedures for tissue sampling was randomized. Mediastinal lymph node tissue and blood plasma was sampled additionally. PD-L1 expression level was quantified by percent of tumor cells. Mutational analysis was performed by massively parallel sequencing, library generation by adapter-ligation and target-enrichment. NGS data could be generated in 21/23 subjects (91%), 7 small-cell-lung-cancer cases were not forwarded to NGS. Comparing primary tumor FB and EBUS-TBNA samples, the accordance regarding exon-mutation-detection was 1,371/1,380 exons (99%). However, 35% of the clinical cases showed discrepant NGS results, 24% showed different PD-L1 scores. Compared to the primary tumor findings, NGS analysis from lymph node samples and ctDNA lead to discrepant findings in only 3% and 0% of cases. FB and EBUS-TBNA samples deliver discrepant results from NGS analysis in about one third of cases and from PDL1 analysis in about a quarter of cases. There is no benefit from additional routine NGS analysis from lymph node samples and ctDNA.

Objectives Resistance to tyrosine kinase inhibitor (TKI) therapy occurs inevitably in lung cancer patients with targetable genetic alterations. MET amplification has found to be an oncogenic driver in lung cancer with several reports showing response to MET TKI especially in cases with high-level amplification. Materials and methods We report the case of a patient with lung adenocarcinoma harbouring low-level MET amplification and strong MET expression who was treated with crizotinib. Results The patient developed a durable response to crizotinib. A KRAS mutation and loss of MET amplification was found in a new lesion at time of progression as a potential mechanism of acquired resistance. Conclusion MET amplification is a continuous biomarker with responses to MET TKI observed even in patients with low-level amplification. KRAS mutations may act as a resistance mechanism to MET inhibition in MET dependent lung cancer.

A global understanding of miRNA function in EGFR signaling pathways may provide insights into improving the management of KRAS-mutant lung cancers, which remain relatively recalcitrant to treatment. To identify miRNAs implicated in EGFR signaling, we transduced bronchial epithelial BEAS-2B cells with retroviral vectors expressing KRASG12V and monitored miRNA expression patterns by microarray analysis. Through this approach, we defined miR-29b as an important target for upregulation by mutant KRAS in non–small cell lung cancers. Cell biologic analyses showed that pharmacologic inhibition of EGFR or MEK was sufficient to reduce levels of miR-29b, while PI3K inhibition had no effect. In KRASG12V-transduced BEAS-2B cells, introduction of anti-miR-29b constructs increased the sensitivity to apoptosis, arguing that miR-29b mediated apoptotic resistance conferred by mutant KRAS. Mechanistic investigations traced this effect to the ability of miR-29b to target TNFAIP3/A20, a negative regulator of NF-κB signaling. Accordingly, overexpression of an miR-29b–refractory isoform of TNFAIP3 restored NF-κB and extrinsic apoptosis, confirming that TNFAIP3 is a functionally relevant target of miR-29b. We also noted that miR-29b could confer sensitivity to intrinsic apoptosis triggered by exposure to cisplatin, a drug used widely in lung cancer treatment. Thus, miR-29b expression may tilt cells from extrinsic to intrinsic mechanisms of apoptosis. Overall, our results reveal a complexity in cancer for miR-29b, which can act as either an oncogene or tumor suppressor gene depending on signaling context. Cancer Res; 76(14); 4160–9. ©2016 AACR.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

The epigenetic writer lysine-specific demethylase 1 (LSD1) is aberrantly upregulated in many cancer types and its overexpression correlates with poor survival and tumor progression. In this study, we analysed LSD1 function in non-small cell lung cancer adenocarcinomas. Expression profiling of 182 cases of lung adenocarcinoma proved a significant correlation of LSD1 overexpression with lung adenocarcinoma progression and metastasis. KRAS-mutated lung cancer cell clones were stably silenced for LSD1 expression. RNA-seq and comprehensive pathway analysis revealed, that genes related to a recently described non-canonical integrin β3 pathway, were significantly downregulated by LSD1 silencing. Hence, invasion and self-renewal capabilities were strongly decreased. Notably, this novel defined LSD1/integrin β3 axis, was also detected in human lung adenocarcinoma specimens. Furthermore, the linkage of LSD1 to an altered expression pattern of lung-lineage specific transcription factors and genes, which are involved in alveolar epithelial differentiation, was demonstrated. Thus, our findings point to a LSD1-integrin β3 axis, conferring attributes of invasiveness and tumor progression to lung adenocarcinoma.

Wir berichten über eine 72-jährige Patientin mit B-Symptomatik und einem persistierenden pulmonalen Infiltrat trotz antibiotischer Behandlung. In der transbronchialen Biopsie fand sich intraalveoläres Granulationsgewebe vereinbar mit einer organisierenden Pneumonie. Eine Steroid-Therapie führte zu einer prompten Besserung der B-Symptomatik sowie zu einer Regredienz des Infiltrates. Auch wenn aufgrund einer chronisch-lymphatischen Leukämie (CLL) sowie einer vier Monate früher behandelten Pneumonie eine mögliche Ursache für eine sekundär organisierende Pneumonie vorlag, handelte es sich am ehesten trotzdem um eine kryptogen organisierende Pneumonie (COP).

57 Background: Although relapses after radiotherapy are common in prostate cancer (PCa) patients, there are no clinical models or markers to identify patients at high risk for radioresistance. So far, only in vitro studies and xenograft models have been performed to identify gene expression patterns associated with radioresistance. However, studies which address the protein pattern to predict radioresistance in humans are completely missing. In order to determine potential biomarkers for radioresistance, we compared protein expression profiles of radioresistant PCa patients with PCa of primary prostatectomized patients. Methods: Two study groups consisting of: I) 30 patients who were treated by salvage prostatectomy and II) 94 patients treated by primary prostatectomy were formed. Tissue microarrays were constructed and immunostained for 15 proteins which are suggested to be associated with radioresistance by in vitro findings. Kruskal-Wallis test was used for multiple group comparison and followed by Dunn-Bonferroni-Test to detect intergroup differences. Cohen’s d was used to calculate the intergroup effect size. Results: Most proteins studied did not show any relevant differences between radioresistant PCa and primary PCa, except for two (AR and AKR1C3). On comparing immunostaining patterns between radioresistant PCa and primary PCa separated by Gleason risk groups, we observed only AR (androgen receptor) to be most expressed in radioresistant PCa (89.7%) and, in 87.8% of primary PCa of the high-risk group ( > 7a) (p = 0.851, Cohen’s d = 0.05), while only 67.3% PCa of the low-risk group (≤7a) (p = 0.017, Cohen’s d = 0.55) were positive. Considering the highest Gleason pattern per patient, only AKR1C3 (Aldo-Keto Reductase Family 1 Member C3) was seen to be similarly expressed in radiation-resistant PCa and patients with Gleason patterns 4 and 5 (p = 0.827, Cohen’s d = 0.05 and p = 0.893, Cohen’s d = 0.10) as compared to Gleason pattern 3 (p = 0.20, Cohen’s d = 0.69) in primary PCa. Conclusions: This is the first study evaluating protein expression profiles to predict radioresistance in PCa, where AR and AKR1C3 were identified to be the most promising protein markers.

Retroperitoneal location of bronchogenic cysts is extremely rare. Most commonly they are encountered in the posterior mediastinum. Bronchogenic cysts arise from developmental aberrations of the tracheobronchial tree in the early embryologic period. We report a 42-year-old female patient with a retroperitoneal bronchogenic cyst in the left adrenal region. She was admitted to our hospital with epigastric pain and subsequently underwent CT of the abdomen. The examination revealed a mass related to the left adrenal gland. Endocrine tests for adrenal hypersecretion were negative. Because of the uncertain entity, laparoscopic adrenalectomy was performed. Pathological examination revealed a bronchogenic cyst in proximity to an inconspicuous left adrenal gland. Although very rare, bronchogenic cysts should be considered in the differential diagnosis of retroperitoneal cystic lesions and surgical resection pursued for symptom resolution and to establish a definitive histology.

Rosette-forming glioneuronal tumor (RGNT) is a recently introduced, indolent neoplasm composed of diminutive circular aggregates of neurocytic-like cells on a noninfiltrative astrocytic background, typically located in the cerebellar midline The traded concept of RGNT being derived from site-specific periventricular precursors may be questioned in the face of extracerebellar examples as well as ones occurring in combination with other representatives of the glioneuronal family. We describe a hitherto not documented example of asymptomatic RGNT discovered during autopsy of a 74-year-old male. Located in the tuberal vermis, this lesion of 6 mm diameter consisted of several microscopic nests of what were felt to represent nascent stages of RGNT, all of them centered on the internal granular layer, and ranging from mucoid dehiscences thereof to fully evolved - if small - tumor foci. Molecular genetic analysis revealed a missense mutation in Exon 20 of the PIK3CA gene involving an A→G transition at Nucleotide 3140. On the other hand, neither codeletion of chromosomes 1p/19q nor pathogenic mutations of IDH1/2 were detected. By analogy with in situ paradigms in other organs, we propose that this tumor is likely to have arisen from the internal granular layer, rather than the plate of the 4th ventricle. A suggestive departure from the wholesale argument of "undifferentiated precursors", this finding also indirectly indicates that a subset of non-classical RGNTs - in particular extracerebellar examples, whose origin cannot be mechanistically accounted for by either of the above structures - may possibly reflect an instance of phenotypic convergence, rather than a lineage-restricted entity.

OBJECTIVES: The pre-treatment of tumour neovessels by low-level photodynamic therapy (PDT) improves the distribution of concomitantly administered systemic chemotherapy. The mechanism by which PDT permeabilizes the tumour vessel wall is only partially known. We have recently shown that leukocyte-endothelial cell interaction is essential for photodynamic drug delivery to normal tissue. The present study investigates whether PDT enhances drug delivery in malignant mesothelioma and whether it involves comparable mechanisms of actions. METHODS: Human mesothelioma xenografts (H-meso-1) were grown in the dorsal skinfold chambers of 28 nude mice. By intravital microscopy, the rolling and recruitment of leukocytes were assessed in tumour vessels following PDT (Visudyne(®) 400 μg/kg, fluence rate 200 mW/cm(2) and fluence 60 J/cm(2)) using intravital microscopy. Likewise, the distribution of fluorescently labelled macromolecular dextran (FITC-dextran, MW 2000 kDa) was determined after PDT. Study groups included no PDT, PDT, PDT plus a functionally blocking anti-pan-selectin antibody cocktail and PDT plus isotype control antibody. RESULTS: PDT significantly enhanced the extravascular accumulation of FITC-dextran in mesothelioma xenografts, but not in normal tissue. PDT significantly increased leukocyte-endothelial cell interaction in tumour. While PDT-induced leukocyte recruitment was significantly blunted by the anti-pan-selectin antibodies in the tumour xenograft, this manipulation did not affect the PDT-induced extravasation of FITC-dextran. CONCLUSIONS: Low-level PDT pre-treatment selectively enhances the uptake of systemically circulating macromolecular drugs in malignant mesothelioma, but not in normal tissue. Leukocyte-endothelial cell interaction is not required for PDT-induced drug delivery to malignant mesothelioma.

BACKGROUND Most molecular techniques currently require fresh frozen tumor tissue, which in the case of prostatectomy specimen is a challenge to obtain for a variety of intrinsic reasons. Prostate cancers are usually located in the organ periphery and hence meticulous attention has to be paid to the relation between the tumor and the surgical margin. In this article we describe a new technique that allows to obtain fresh frozen tumor material in rather large quantities and without jeopardizing diagnostic accuracy. METHOD An inner triangle, representing roughly 50% of the entire prostate tissue, is removed from native prostatectomy specimen and cryopreserved, leaving the periphery of the organ for routine histomorphological analysis. We have validated the technique using a series of 180 archived radical prostatectomy specimen that had been studied by histology in their entirety, as well as 42 prostatectomy specimen worked-up by the new technique. RESULTS The described technique is effective, yielding frozen tumor tissue in 84.2% of unilateral (≤pT2b) and in 91.8% of bilateral (pT2c) carcinomas. The number of tumor containing tissue blocks ranged from 1 to 7 blocks per carcinoma (mean 5.6 ± 3.2). The remaining peripheral tissue portion subjected to histological analysis allowed to estimate total tumor volume with a high degree to confidentiality (r2 ≥ 0.90 by linear regression analysis). The number of blocks to be routinely examined for each prostatectomy specimen is significantly reduced, thus saving costs without loss of diagnostic accuracy. CONCLUSION The described technique for prostate cancer cryoconservtion is feasible, highly standardized, effective, safe, and economic. Prostate 69: 191–197, 2009. © 2008 Wiley–Liss, Inc.

Any academic pathologist will sooner or later be confronted with the need to use human tissues for quality control purposes or for research projects or else with the demand for human tissue samples for research projects of external researchers. Over the last 10 years, the use of human tissues for such non-diagnostic purposes has been the subject of extensive debate and wide-reaching regulations. In particular, questions of medical secrecy, safety, autonomy and anonymization have been addressed, and the role of ethical review boards defined. However, these guidelines are not uniform in all countries and unfortunately tend to suffer from a certain lack of precision, which may in part be due to the fact that they are usually edited by multiple authors with quite diverse backgrounds (physicians, lawyers, ethicists, philosophers, anthropologists, theologists or lay persons, for example). The wide spectrum of interpretations of such regulations may be embarrassing to such an extent that a continuation of academic activities with human tissues appears to many pathologists as almost impossible or even outright illegal. This paper describes a set of characteristic, recurrent situations to which an academic pathologist may be confronted and proposes simple, realistic solutions that are nevertheless in line with most current regulations.

The adaptor protein MYD88 is critical for relaying activation of Toll-like receptor signaling to NF-κB activation. MYD88 mutations, particularly the p.L265P mutation, have been described in numerous distinct B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL). Twenty-nine percent of activated B-cell-type DLBCL (ABC-DLBCL), which is characterized by constitutive activation of the NF-κB pathway, carry the p.L265P mutation. In addition, ABC-DLBCL frequently displays focal copy number gains affecting BCL2 Here, we generated a novel mouse model in which Cre-mediated recombination, specifically in B cells, leads to the conditional expression of Myd88(p.L252P) (the orthologous position of the human MYD88(p.L265P) mutation) from the endogenous locus. These mice develop a lymphoproliferative disease and occasional transformation into clonal lymphomas. The clonal disease displays the morphologic and immunophenotypical characteristics of ABC-DLBCL. Lymphomagenesis can be accelerated by crossing in a further novel allele, which mediates conditional overexpression of BCL2 Cross-validation experiments in human DLBCL samples revealed that both MYD88 and CD79B mutations are substantially enriched in ABC-DLBCL compared with germinal center B-cell DLBCL. Furthermore, analyses of human DLBCL genome sequencing data confirmed that BCL2 amplifications frequently co-occurred with MYD88 mutations, further validating our approach. Finally, in silico experiments revealed that MYD88-mutant ABC-DLBCL cells in particular display an actionable addiction to BCL2. Altogether, we generated a novel autochthonous mouse model of ABC-DLBCL that could be used as a preclinical platform for the development and validation of novel therapeutic approaches for the treatment of ABC-DLBCL.

Lung adenosquamous carcinoma is a particular subtype of non-small cell lung carcinoma that is defined by the coexistence of adenocarcinoma and squamous cell carcinoma components. The aim of this study was to assess the mutational profile in each component of 16 adenosquamous carcinoma samples from a Caucasian population by a combination of next generation sequencing using the cancer hotspot panel as well as the colon and lung cancer panel and FISH. Identified mutations were confirmed by Sanger sequencing of DNA from cancer cells of each component collected by Laser Capture microdissection. Mutations typical for adenocarcinoma as well as squamous cell carcinoma were identified. Driver mutations were predominantly in the trunk suggesting a monoclonal origin of adenosquamous carcinoma. Most remarkably, EGFR mutations and mutations in the PI3K signaling pathway, which accounted for 30% and 25% of tumors respectively, were more prevalent while KRAS mutations were less prevalent than expected for a Caucasian population. Surprisingly, expression of classifier miR-205 was intermediate between that of classical adenocarcinoma and squamous cell carcinoma suggesting that adenosquamous carcinoma is a transitional stage between these tumor types. The high prevalence of therapy-relevant targets opens new options of therapeutic intervention for adenosquamous carcinoma patients.

Introduction: IPF is a devastating lung disease with a median survival of 3 years. The etiology is unknown. IPF deteriorates upon viral or bacterial lung infection although pulmonary infection (pneumonia) in healthy lungs rarely induces fibrosis. Bacterial lipopolysaccharide (LPS) activates TLR4 initiating pro-inflammatory pathways. Since TLR4 has been linked to hepatic fibrosis and scleroderma, we investigate the role of TLR4 in IPF. Methods: Lung sections of IPF patients were analysed for TLR4 expression (IHC, IF, qPCR). Primary human lung fibroblasts (HLF LONZA®), isolated normal lung fibroblasts (NLFB), as well as fibroblasts from IPF lungs (IPF-FB) were exposed to LPS and TGF-β and evaluated by qPCR, flow cytometry for pro-fibrotic mediator expression. Results: TLR4 is expressed in fibroblast foci of IPF lungs as well as in HLF, NLFB, IPF-FB. LPS had no direct pro-fibrotic effect on HLF in vitro . As a model for pneumonia in normal lung, NLFB were exposed simultaneously to LPS and TGF-β, which reduced pro-fibrotic connective tissue growth factor (CTGF) significantly compared to TGF-β alone (p To model pneumonia in IPF lung, HLF were differentiated into myofibroblasts by TGF-β. In myofibroblasts CTGF expression was not reduced after LPS. Our in vitro model was confirmed in preliminary experiments using IPF-FB. Conclusion: TLR4 is present in fibroblast foci of IPF lungs. LPS stimulation had no direct pro-fibrotic effect on HLF in vitro , but can influence the pro-fibrotic effect of TGF-β dependent on time of exposure. These findings might explain why pneumonia in healthy individuals does not lead to fibrosis opposed to IPF, where infection often induces disease progression.

Long-term treatment with glucocorticoids is associated with mild to moderate hypertension. We reported previously that downregulation of endothelial NO synthase (eNOS) expression and activity is likely to contribute to this increase in blood pressure. In the present study, we tested the effects of dexamethasone on the vasodilation of microvascular arterioles using implanted dorsal skin-fold chambers in anesthetized C57BL/6J mice. Experiments were performed on control mice or on mice treated with dexamethasone (0.1–3 mg/kg of body wt). Endothelium-dependent vasodilation in response to ACh (0.1–10 μM) was reduced by dexamethasone in a dose-dependent fashion. Comparable inhibition was seen in tissues superfused with 30 μM NG-nitro-l-arginine methyl ester. In contrast, endothelium-independent vasodilation in response to S-nitroso- N-acetyl-d,l-penicillamine (10 μM) was not influenced by either dexamethasone or NG-nitro-l-arginine methyl ester. Levels of eNOS mRNA in murine hearts and NO2−/NO3−in serum were suppressed by dexamethasone (down to 63 and 50% of control values, respectively, at 3 mg/kg of body wt) along with a reduction in eNOS protein to 85.6%. Dexamethasone also concentration dependently reduced the expression of the cationic amino acid transporter-1 in murine hearts and cultured endothelial cells. The suppression by dexamethasone of the ACh-induced vasodilation could be partially reversed by dietary l-arginine (50 mg/kg of body wt) and by dietary vitamin C (10 g/kg of diet). We conclude that suppression by dexamethasone of the endothelium-mediated microvascular vasodilation involves several mechanisms including 1) downregulation of eNOS, 2) downregulation of cationic amino acid transporter-1, and 3) generation of reactive oxygen species. The demonstration that l-arginine and vitamin C can partially offset the effects of dexamethasone on microvascular arterioles suggests the potential clinical usefulness of these agents for the reduction of glucocorticoid-induced hypertension.

Richter's syndrome (RS) is an aggressive transformation of Chronic Lymphocytic Leukemia (CLL) to Diffuse Large B Cell Lymphoma (DLBCL) refractory to current therapies with dismal prognosis. Richter Syndrome arises from CLL cells independent of common DLBCL mutations. Frequently, mutations in p53, CDKN2 or cMyc genes are involved, but a significant proportion displays no specifically acquired driver mutation. We could observe activation of AKT in 6 out 48 Richter syndrome biopsies by positive staining for active phosphorylated AKT while in CLL lymph nodes, DLBCL and Burkitt´s Lymphoma no phospho-AKT by IHC could be observed. However in primary patient CLL cases we could detect varying levels of pAKT by Western blot, elevated levels were identified predominantly in patients harboring high-risk mutations such p53, ATM, NOTCH1 and XPO1. Furthermore, B-cell receptor mediated stimulation of the PI3K/AKT axis provided protection towards genotoxic stress induced apoptosis via post-translation stabilization of MCL1. This provides subsequently a synergistic induction of apoptosis by combining idelalisib and bendamustin. Thus we analyzed the functional impact of AKT signaling using a conditional constitutive allele for AKT (AKT-C) specifically activated using CD19-Cre and Cγ1-Cre fro post-GC-activation. AKT activation alone could not induce a malignant phenotype, however we could demonstrate that Eµ-Tcl-1 mice with AKT-C develop Richter Syndrome. Both in EµTCL1:CD19-CreAKT-C (TCA) and EµTCL1:Cγ1- CreAKT-C (TCγ1A) mice developed a high-grade lymphoma phenotype leading to decreased survival. Transformed cells displayed blastoid characteristics with significantly increased cellular size and the histomorphological features of DLBCL. Large transformed cells show high percentage of KI67-positive staining (>90%) and frequent mitotic figures. Here, AKT-mediated GSK-3b inhibition and subsequent cMyc and Mcl-1 stabilization might transform CLL to RS cells and combinatory treatments with DNA-damaging and PI3K-inhibiting compounds revealed promising therapeutic results. Collectively, we have identified AKT signaling as an oncogenic signaling pathway in progression of CLL towards Richter´s syndrome and generated the first murine Richter Syndrome model (TCA and TCγ1A) providing novel mechanistic insights into the molecular understanding of Richter's transformation that is amenable to model therapeutic strategies and to address the efficacy of synergistic treatment combinations. Disclosures Klapper: Roche, Novartis, Amgen, Takeda: Research Funding. Hallek:Amgen: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Mundipharma: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; F. Hoffmann-LaRoche: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Janssen-Cilag: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau.

Background: The understanding of molecular mechanisms leading to poor prognosis in pancreatic cancer may help develop treatment options. N-myc downstream-regulated gene-1 (NDRG1) has been correlated to better prognosis in pancreatic cancer. Therefore, we thought to analyze how the loss of NDRG1 affects progression in an orthotopic xenograft animal model of recurrence. Methods: Capan-1 cells were silenced for NDRG1 (Csil) or transfected with scrambled shRNA (Cscr) and compared for anchorage-dependent and anchorage-independent growth, invasion and tube formation in vitro. In an orthotopic xenograft model of recurrence tumors were grown in the pancreatic tail. The effect of NDRG1 silencing was evaluated on tumor size and metastasis. Results: The silencing of NDRG1 in Capan-1 cells leads to more aggressive tumor growth and metastasis. We found faster cell growth, double count of invaded cells and 1.8-fold increase in tube formation in vitro. In vivo local tumors were 5.9-fold larger (p = 0.006) and the number of metastases was higher in animals with tumors silenced for NDRG1 primarily (3 vs. 1.1; p = 0.005) and at recurrence (3.3 vs. 0.9; p = 0.015). Conclusion: NDRG1 may be an interesting therapeutic target as its silencing in human pancreatic cancer cells leads to a phenotype with more aggressive tumor growth and metastasis.

Myoepithelioma is a dimorphic neoplasm with contractile-epithelial phenotype, originally interpreted as deriving from, but not actually restricted to the salivary glands. As a novel addition to the list of exquisitely rare intracranial salivary gland-type tumors and tumor-like lesions, we report on an example of myoepithelioma encountered in the left cerebellopontine angle of a 32-year-old male. Clinically presenting with ataxia and dizziness, this extraaxial mass of 4 × 3.5 × 3 cm was surgically resected, and the patient is alive 6 years postoperatively. Histologically, the tumor exhibited a continuum ranging from compact fascicles of spindle cells to epithelial nests and trabeculae partitioned by hyalinized septa, while lacking tubular differentiation. Regardless of architectural variations, there was robust immunoexpression of S100 protein, smooth muscle actin, GFAP, cytokeratin, and vimentin. Cytologic atypia tended to be modest throughout, and the MIB1 labeling index averaged less than 1%. Fluorescent in situ hybridization indicated no rearrangement of the EWSR1 locus. We interpret these results to suggest that myoepithelioma of the posterior fossa - along with related salivary epithelial tumors in this ostensibly incongruous locale - may possibly represent analogous neoplasms to their orthotopic counterparts, ones arising within aberrant salivary anlagen. The presence of the latter lends itself to being mechanistically accounted for by either postulating placodal remnants in the wake of branchial arch development, or linking them to exocrine glandular nests within endodermal cysts. Alternatively, myoepithelioma at this site could be regarded as a non tissue-specific lesion similar to its relatives ubiquitously occurring in the soft parts.

It is known that hypertension is associated with endothelial dysfunction and that Angiotensin II (Ang II) is a key player in the pathogenesis of hypertension. We aimed to elucidate whether endothelial dysfunction is a specific feature of Ang II-mediated hypertension or a common finding of hypertension, independently of underlying etiology. We studied endothelial-dependent vasorelaxation in precapillary resistance arterioles and in various large-caliber conductance arteries in wild-type mice with Ang II-dependent hypertension (2-kidney 1-clip (2K1C) model) or Ang II-independent (volume overload) hypertension (1-kidney 1-clip model (1K1C)). Normotensive sham mice were used as controls. Aortic mechanical properties were also evaluated. Intravital microscopy of precapillary arterioles revealed a significantly impaired endothelium-dependent vasorelaxation in 2K1C mice compared with sham mice, as quantified by the ratio of acetylcholine (ACh)-induced over S-nitroso-N-acetyl-D,L-penicillamine (SNAP)-induced vasorelaxation (2K1C: 0.49±0.12 vs. sham: 0.87±0.11, P=0.018). In contrast, the ACh/SNAP ratio in volume-overload hypertension 1K1C mice was not significantly different from sham mice, indicating no specific endothelial dysfunction (1K1C: 0.77±0.27 vs. sham: 0.87±0.11, P=0.138). Mechanical aortic wall properties and endothelium-dependent vasorelaxation, assessed ex vivo in rings of large-caliber conductance (abdominal and thoracic aorta, carotid and femoral arteries), were not different between 2K1C, 1K1C and sham mice. Endothelial dysfunction is an early feature of Ang II- but not volume-overload-mediated hypertension. This occurs exclusively at the level of precapillary arterioles and not in conduit arteries. Our findings, if confirmed in clinical studies, will provide a better understanding of the pathophysiological mechanisms of hypertension.

We previously reported that excess of deoxycorticosterone-acetate (DOCA)/salt-induced cardiac hypertrophy in the absence of hypertension in one-renin gene mice. This model allows us to study molecular mechanisms of high-salt intake in the development of cardiovascular remodeling, independently of blood pressure in a high mineralocorticoid state. In this study, we compared the effect of 5-wk low- and high-salt intake on cardiovascular remodeling and cardiac differential gene expression in mice receiving the same amount of DOCA. Differential gene and protein expression was measured by high-density cDNA microarray assays, real-time PCR and Western blot analysis in DOCA-high salt (HS) vs. DOCA-low salt (LS) mice. DOCA-HS mice developed cardiac hypertrophy, coronary perivascular fibrosis, and left ventricular dysfunction. Differential gene and protein expression demonstrated that high-salt intake upregulated a subset of genes encoding for proteins involved in inflammation and extracellular matrix remodeling (e.g., Col3a1, Col1a2, Hmox1, and Lcn2). A major subset of downregulated genes encoded for transcription factors, including myeloid differentiation primary response (MyD) genes. Our data provide some evidence that vascular remodeling, fibrosis, and inflammation are important consequences of a high-salt intake in DOCA mice. Our study suggests that among the different pathogenic factors of cardiac and vascular remodeling, such as hypertension and mineralocorticoid excess and sodium intake, the latter is critical for the development of the profibrotic and proinflammatory phenotype observed in the heart of normotensive DOCA-treated mice.

The uncommon simultaneous occurrence of an exuberant, angioma-like proliferation of superficial cerebral microvessels along with absence of the kidneys has been proposed to constitute a syndromic complex for which the term "meningocerebral angiodysplasia (or angiomatosis) with renal agenesis" (MCA-RA) is being descriptively used. We observed this constellation in one of a pair of dichorionic male twins following postpartal death in the 38th week of pregnancy. General autopsy revealed rudimentary metanephric anlagen made up of few residual glomeruli, cysts lined by flattened tubular epithelium, and islands of cartilage - corresponding to renal aplastic dysplasia. Largely inconspicuous with respect to its gyral pattern, as well as the configuration of the ventricular system, the brain microscopically showed extensive replacement of the cortex by a lattice of proliferating capillaries with necrosis of the intervening parenchyma. Minute foci of calcified necrosis were scattered in the deep subcortical white matter as well, while the ventricular ependyma and the subventricular germ cell layer remained remarkably intact. The cerebellum and brain stem appeared unaffected as well. Karyotyping of skin fibroblasts indicated a normal chromosome set of 46XY without gross structural anomalies. We interpret these findings as ones apt to being reasonably accommodated within the spectrum of MCA-RA. Although exceedingly rare, accurate identification of individual cases of MCA-RA is relevant both to differential diagnosis from its prognostically different look-alike "proliferative vasculopathy and hydranencephaly-hydrocephaly" (PVHH), and to refine the nosology of unconventional pediatric vascular malformations, for which the rather nonspecific label "angiodysgenetic necrotizing encephalopathy" is still commonly used.

We report a 26-year-old female patient who was diagnosed within 4 years with chest sarcoma, lung adenocarcinoma, and breast cancer. While her family history was unremarkable, DNA sequencing of TP53 revealed a germline de novo non-sense mutation in exon 6 p.Arg213X. One year later, she further developed a contralateral ductal carcinoma in situ, and 18 months later a jaw osteosarcoma. This case illustrates the therapeutic pitfalls in the care of a young cancer patient with TP53 de novo germline mutations and the complications related to her first-line therapy. Suggestion is made to use the less stringent Chompret criteria for germline TP53 mutation screening. Our observation underlines the possibly negative effect of radiotherapy in generating second tumors in patients with a TP53 mutation. We also present a review of six previously reported cases, comparing their cancer phenotypes with those generally produced by TP53 mutations.

We report herein the case of a 57-year-old lady who had two concomittant lesions, an inflammatory myofibroblastic tumor in the trachea, and severe granulomatous lesions in the adjacent hilar lymph nodes. While these two lesions shared histological and some immunohistochemical features lesions. They differed in terms of ALK-1 expression, which was positive in the tracheal tumor and negative in the lymph nodes. The discussion of the case circles around putative pathophysiological links between the lesions. The authors favor the idea that the lymph nodes present a sarcoid-like granulomatous reaction to the inflammatory myofibroblastic tumor in the trachea over a coexistence of two independent entities. However, no conclusive evidence for this interpretation can be presented based on the existing literature.

Atrial natriuretic peptide (ANP) regulates arterial blood pressure and volume. Its guanylyl cyclase-A (GC-A) receptor is expressed in vascular endothelium and mediates increases in cGMP, but the functional relevance is controversial. Notably, mice with endothelial-restricted GC-A deletion [EC GC-A knockout (KO) mice] exhibit significant chronic hypervolemic hypertension. The present study aimed to characterize the endothelial effects of ANP and their relevance for the acute regulation of intravascular fluid volume. We studied the effect of ANP on microvascular permeability to fluorescein isothiocyanate-labeled albumin (BSA) using intravital microscopy on mouse dorsal skinfold chambers. Local superfusion of ANP (100 nm) increased microvascular fluorescein isothiocyanate-BSA extravasation in control but not EC GC-A KO mice. Intravenous infusion of synthetic ANP (500 ng/kg·min) caused immediate increases in hematocrit in control mice, indicating intravascular volume contraction. In EC GC-A KO mice, the hematocrit responses were not only abolished but even reversed. Furthermore, acute vascular volume expansion, which caused release of endogenous cardiac ANP, did not affect resting central venous pressure of control mice but rapidly and significantly increased central venous pressure of EC GC-A KO mice. In cultured lung endothelial cells, ANP provoked cGMP-dependent protein kinase I-mediated phosphorylation of vasodilator-stimulated phosphoprotein. We conclude that ANP, via GC-A, enhances microvascular endothelial macromolecule permeability in vivo. This effect might be mediated by cGMP-dependent protein kinase I-dependent phosphorylation of vasodilator-stimulated phosphoprotein. Modulation of transcapillary protein and fluid transport may represent one of the most important hypovolemic actions of ANP.