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Contains fulltext : 252010.pdf (Publisher’s version ) (Open Access) OBJECTIVES: The number of targeted drugs in non-small cell lung cancer (NSCLC) is ever-expanding and requires testing of an increasing number of predictive biomarkers. We present a comprehensive real-world evaluation of molecular testing and treatment selection in stage IV NSCLC patients in the Netherlands from 2017 to 2019. MATERIALS AND METHODS: Molecular pathology reports of NSCLC patients were collected from the Dutch Pathology Registry in time intervals between Oct-2017 and April-2019 (N = 5,038 patients) to study diagnostic yield. Linkage between the Dutch Pathology Registry and the Netherlands Cancer Registry enabled studying molecular testing rates for stage IV NSCLC initially diagnosed in 2017-Q4 (N = 1,193) and application of targeted therapy in stage IV NSCLC patients with potentially druggable alterations reported between Oct-2017 and June-2018 (N = 401). RESULTS: Predictive molecular testing was performed in 85.0% of adenocarcinomas, 60.4% of NSCLC-not otherwise specified (NOS) and 17.4% of squamous cell carcinomas. Testing rates were highest for EGFR and ALK (adenocarcinoma: 82.7% and 80.7%, respectively). Incidence of molecular driver alterations (i.e. EGFR, KRAS, ALK, ROS1, BRAF, MET, ERBB2, FGFR1) was 61.1% for adenocarcinomas, 42.3% for NSCLC-NOS, and 24.7% for squamous cell carcinomas. Therapeutically relevant alterations were detected at a higher frequency by NGS- versus non-NGS-approaches (adenocarcinoma: 62.4% versus 56.5%, respectively (P = 0.004)) due to a lower failure rate, more comprehensive testing and higher sensitivity. Uptake of treatment with a registered targeted therapy in eligible patients varied per actionable target, i.e. EGFR: 85.8%, ALK: 74.7%, ROS1: 33.7%, BRAF: 51.5%. Treatment with agents in clinical studies/compassionate use was lower, i.e. MET: 22.8%, HER2: 18.9%, RET: 6.7%. CONCLUSION: Real-world data show NGS-based approaches to be superior to non-NGS. Uptake of molecular testing and the corresponding targeted treatments was less than expected based on guidelines and even more so for trials, off-label use and compassionate use, indicating less than optimal access to rational treatment options.

PURPOSE A large number of targeted treatment options for stage IV nonsquamous non–small-cell lung cancer with specific genetic aberrations in tumor DNA is available. It is therefore important to optimize diagnostic testing strategies, such that patients receive adequate personalized treatment that improves survival and quality of life. The aim of this study is to assess the efficacy (including diagnostic costs, turnaround time (TAT), unsuccessful tests, percentages of correct findings, therapeutic costs, and therapeutic effectiveness) of parallel next generation sequencing (NGS)–based versus sequential single-gene–based testing strategies routinely used in patients with metastasized non–small-cell lung cancer in the Netherlands. METHODS A diagnostic microsimulation model was developed to simulate 100,000 patients with prevalence of genetic aberrations, extracted from real-world data from the Dutch Pathology Registry. These simulated patients were modeled to undergo different testing strategies composed of multiple tests with different test characteristics including single-gene and panel tests, test accuracy, the probability of an unsuccessful test, and TAT. Diagnostic outcomes were linked to a previously developed treatment model, to predict average long-term survival, quality-adjusted life-years (QALYs), costs, and cost-effectiveness of parallel versus sequential testing. RESULTS NGS-based parallel testing for all actionable genetic aberrations is on average €266 cheaper than single-gene–based sequential testing, and detects additional relevant targetable genetic aberrations in 20.5% of the cases, given a TAT of maximally 2 weeks. Therapeutic costs increased by €8,358, and 0.12 QALYs were gained, leading to an incremental cost-effectiveness ratio of €69,614/QALY for parallel versus sequential testing. CONCLUSION NGS-based parallel testing is diagnostically superior over single-gene–based sequential testing, as it is cheaper and more effective than sequential testing. Parallel testing remains cost-effective with an incremental cost-effectiveness ratio of 69,614 €/QALY upon inclusion of therapeutic costs and long-term outcomes.

Introduction: Salivary gland cancer (SGC) is a rare cancer for which systemic treatment options are limited. Therefore, it is important to characterize its genetic landscape in search for actionable aberrations, such as NTRK gene fusions. This research aimed to identify these actionable aberrations by combining NGS-based analysis of RNA (gene fusions) and DNA (single and multiple nucleotide variants, copy number variants, microsatellite instability and tumor mutational burden) in a large cohort of SGC patients. Methods: RNA and DNA were extracted from archival tissue of 121 patients with various SGC subtypes. Gene fusion analysis was performed using a customized RNA-based targeted NGS panel. DNA was sequenced using a targeted NGS panel encompassing 523 cancer-related genes. Cross-validation of NGS-based NTRK fusion detection and pan-TRK immunohistochemistry (IHC) was performed. Results: Fusion transcripts were detected in 50% of the cases and included both known (MYB-NFIB, MYBL1-NFIB, CRTC1-MAML2) and previously unknown fusions (including transcripts involving RET, BRAF or RAD51B). Only one NTRK fusion transcript was detected, in a secretory carcinoma case. Pan-TRK IHC (clone EPR17341) was false positive in 74% of cases. The proportion of patients with targets for genetically matched therapies differed among subtypes (salivary duct carcinoma: 82%, adenoid cystic carcinoma 28%, mucoepidermoid carcinoma 50%, acinic cell carcinoma 33%). Actionable aberrations were most often located in PIK3CA (n = 18, 15%), ERBB2 (n = 15, 12%), HRAS and NOTCH1 (both n = 9, 7%). Conclusions: Actionable genetic aberrations were seen in 53.7% of all SGC cases on the RNA and DNA level, with varying percentages between subtypes.

PurposePredictive diagnostics play an increasingly important role in personalized medicine for cancer treatment. Whole genome sequencing (WGS) based treatment selection is expected to rapidly increase worldwide. Detailed and comparative cost analyses of diagnostic techniques are an essential element in decision-making. This study aimed to calculate and compare the total cost of currently used diagnostic techniques and of WGS in treatment of non-small cell lung carcinoma (NSCLC), melanoma, colorectal cancer (CRC) and gastrointestinal stromal tumor (GIST) in the Netherlands.MethodsThe activity-based costing (ABC) method was conducted to calculate the total cost of included diagnostic techniques based on data provided by Dutch pathology laboratories and the Dutch centralized cancer WGS facility. Costs were allocated to four categories: capital costs, maintenance costs, software costs and operational costs. Outcome measures were total cost per cancer patient per included technique, and the total cost per cancer patient per most commonly applied technique (combination) for each cancer type.ResultsThe total cost per cancer patient per technique varied from € 58 (Sanger sequencing, 3 amplicons) to € 4738 (paired tumor-normal WGS). The operational costs accounted for the vast majority over 90 % of the total per cancer patient technique costs. The most important operational cost drivers were consumables followed by personnel (for sample preparation and primary data analysis).ConclusionThis study outlined in detail all costing aspects and cost prices of current and new diagnostic modalities used in treatment of NSCLC, melanoma, CRC and GIST in the Netherlands. Detailed cost differences and value comparisons between these diagnostic techniques enable future economic evaluations to support decision-making on implementation of WGS and other diagnostic modalities in routine clinical practice.

Background Molecular tumor boards (MTBs) provide rational, genomics‐driven, patient‐tailored treatment recommendations. Worldwide, MTBs differ in terms of scope, composition, methods, and recommendations. This study aimed to assess differences in methods and agreement in treatment recommendations among MTBs from tertiary cancer referral centers in The Netherlands. Materials and Methods MTBs from all tertiary cancer referral centers in The Netherlands were invited to participate. A survey assessing scope, value, logistics, composition, decision‐making method, reporting, and registration of the MTBs was completed through on‐site interviews with members from each MTB. Targeted therapy recommendations were compared using 10 anonymized cases. Participating MTBs were asked to provide a treatment recommendation in accordance with their own methods. Agreement was based on which molecular alteration(s) was considered actionable with the next line of targeted therapy. Results Interviews with 24 members of eight MTBs revealed that all participating MTBs focused on rare or complex mutational cancer profiles, operated independently of cancer type–specific multidisciplinary teams, and consisted of at least (thoracic and/or medical) oncologists, pathologists, and clinical scientists in molecular pathology. Differences were the types of cancer discussed and the methods used to achieve a recommendation. Nevertheless, agreement among MTB recommendations, based on identified actionable molecular alteration(s), was high for the 10 evaluated cases (86%). Conclusion MTBs associated with tertiary cancer referral centers in The Netherlands are similar in setup and reach a high agreement in recommendations for rare or complex mutational cancer profiles. We propose a “Dutch MTB model” for an optimal, collaborative, and nationally aligned MTB workflow. Implications for Practice Interpretation of genomic analyses for optimal choice of target therapy for patients with cancer is becoming increasingly complex. A molecular tumor board (MTB) supports oncologists in rationalizing therapy options. However, there is no consensus on the most optimal setup for an MTB, which can affect the quality of recommendations. This study reveals that the eight MTBs associated with tertiary cancer referral centers in The Netherlands are similar in setup and reach a high agreement in recommendations for rare or complex mutational profiles. The Dutch MTB model is based on a collaborative and nationally aligned workflow with interinstitutional collaboration and data sharing., Worldwide, molecular tumor boards (MTBs) differ in terms of scope, composition, methods, and recommendations. This article assesses differences in methods and agreement in treatment recommendations among MTBs from tertiary cancer referral centers in The Netherlands.

Background Molecular tumour boards (MTB) optimally match oncological therapies to patients with genetic aberrations. Prostate cancer (PCa) is underrepresented in these MTB discussions. This study describes the impact of routine genetic profiling and MTB referral on the outcome of PCa patients in a tertiary referral centre. Methods All PCa patients that received next-generation sequencing results and/or were discussed at an MTB between Jan 1, 2017 and Jan 1, 2020 were included. Genetically matched therapies (GMT) in clinical trials or compassionate use were linked to actionable alterations. Response to these agents was retrospectively evaluated. Results Out of the 277 genetically profiled PCa patients, 215 (78%) were discussed in at least one MTB meeting. A GMT was recommended to 102 patients (47%), of which 63 patients (62%) initiated the GMT. The most recommended therapies were PARP inhibitors (n = 74), programmed death-(ligand) 1 inhibitors (n = 21) and tyrosine kinase inhibitors (n = 19). Once started, 41.3% had a PFS of ≥6 months, 43.5% a PSA decline ≥50% and 38.5% an objective radiographic response. Conclusion Recommendation for a GMT is achieved in almost half of the patients with advanced prostate cancer, with GMT initiation leading to durable responses in over 40% of patients. These data justify routine referral of selected PCa patients to MTB’s.

BackgroundImmunotherapy is currently part of the standard of care for patients with advanced-stage non-small cell lung cancer (NSCLC). However, many patients do not respond to this treatment, therefore combination strategies are being explored to increase clinical benefit. The PEMBRO-RT trial combined the therapeutic programmed cell death 1 (PD-1) antibody pembrolizumab with stereotactic body radiation therapy (SBRT) to increase the overall response rate and study the effects on the tumor microenvironment (TME).MethodsHere, immune infiltrates in the TME of patients included in the PEMBRO-RT trial were investigated. Tumor biopsies of patients treated with pembrolizumab alone or combined with SBRT (a biopsy of the non-irradiated site) at baseline and during treatment were stained with multiplex immunofluorescence for CD3, CD8, CD20, CD103 and FoxP3 for lymphocytes, pan-cytokeratin for tumors, and HLA-ABC expression was determined.ResultsThe total number of lymphocytes increased significantly after 6 weeks of treatment in the anti-PD-1 group (fold change: 1.87, 95% CI: 1.06 to 3.29) and the anti-PD-1+SBRT group (fold change: 2.29, 95% CI: 1.46 to 3.60). The combination of SBRT and anti-PD-1 induced a 4.87-fold increase (95% CI: 2.45 to 9.68) in CD103+cytotoxic T-cells 6 weeks on treatment and a 2.56-fold increase (95% CI: 1.03 to 6.36) after anti-PD-1 therapy alone. Responders had a significantly higher number of lymphocytes at baseline than non-responders (fold difference 1.85, 95% CI: 1.04 to 3.29 for anti-PD-1 and fold change 1.93, 95% CI: 1.08 to 3.44 for anti-PD-1+SBRT).ConclusionThis explorative study shows that that lymphocyte infiltration in general, instead of the infiltration of a specific lymphocyte subset, is associated with response to therapy in patients with NSCLC.Furthermore, anti-PD-1+SBRT combination therapy induces an immunological abscopal effect in the TME represented by a superior infiltration of cytotoxic T cells as compared with anti-PD-1 monotherapy.

Contains fulltext : 235767.pdf (Publisher’s version ) (Open Access) To address the challenges posed by large-scale development, validation, and adoption of artificial intelligence (AI) in pathology, we have constituted a consortium of academics, small enterprises, and pharmaceutical companies and proposed the BIGPICTURE project to the Innovative Medicines Initiative. Our vision is to become the catalyst in the digital transformation of pathology by creating the first European, ethically compliant, and quality-controlled whole slide imaging platform, in which both large-scale data and AI algorithms will exist. Our mission is to develop this platform in a sustainable and inclusive way, by connecting the community of pathologists, researchers, AI developers, patients, and industry parties based on creating value and reciprocity in use based on a community model as the mechanism for ensuring sustainability of the platform.

Molecular tumour boards (MTB) optimally match oncological therapies to patients with genetic aberrations. Prostate cancer (PCa) is underrepresented in these MTB discussions. This study describes the impact of routine genetic profiling and MTB referral on the outcome of PCa patients in a tertiary referral centre.All PCa patients that received next-generation sequencing results and/or were discussed at an MTB between Jan 1, 2017 and Jan 1, 2020 were included. Genetically matched therapies (GMT) in clinical trials or compassionate use were linked to actionable alterations. Response to these agents was retrospectively evaluated.Out of the 277 genetically profiled PCa patients, 215 (78%) were discussed in at least one MTB meeting. A GMT was recommended to 102 patients (47%), of which 63 patients (62%) initiated the GMT. The most recommended therapies were PARP inhibitors (n = 74), programmed death-(ligand) 1 inhibitors (n = 21) and tyrosine kinase inhibitors (n = 19). Once started, 41.3% had a PFS of ≥6 months, 43.5% a PSA decline ≥50% and 38.5% an objective radiographic response.Recommendation for a GMT is achieved in almost half of the patients with advanced prostate cancer, with GMT initiation leading to durable responses in over 40% of patients. These data justify routine referral of selected PCa patients to MTB's.

Classification of non-small-cell lung cancer (NSCLC) into adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) via histopathology is a vital prerequisite to select the appropriate treatment for lung cancer patients. Most machine learning approaches rely on manually annotating large numbers of whole slide images (WSI) for training. However, manually delineating cancer areas or even single cancer cells on hundreds or thousands of slides is tedious, subjective and requires highly trained pathologists. We propose to use Neural Image Compression (NIC), which requires only slide-level labels, to classify NSCLC into LUSC and LUAD. NIC consists of two phases/networks. In the first phase the slides are compressed with a convolutional neural network (CNN) acting as an encoder. In the second phase the compressed slides are classified with a second CNN. We trained our classification model on >2,000 NIC-compressed slides from the TCGA and TCIA databases and evaluated the model performance additionally on several internal and external cohorts. We show that NIC approaches state of the art performance on lung cancer classification, with an average AUC of 0.94 on the TCGA and TCIA testdata, and AUCs between 0.84 and 0.98 on other independent datasets.

BackgroundLung cancer is the leading cause of cancer death worldwide. With the growing number of targeted therapies and the introduction of immuno-oncology (IO), personalized medicine has become standard of care in patients with metastatic disease. The development of predictive and prognostic biomarkers is of great importance. Mutational signatures harbor potential clinical value as predictors of therapy response in cancer. Here we set out to investigate particular mutational processes by assessing mutational signatures and associations with clinical features, tumor mutational burden (TMB) and targetable mutations.MethodsIn this retrospective study, we studied tumor DNA from patients with non-small cell lung cancer (NSCLC) irrespective of stage. The samples were sequenced using a 2 megabase (Mb) gene panel. On each sample TMB was determined and defined as the total number of single nucleotide mutations per Mb (mut/Mb) including non-synonymous mutations. Mutational signature profiling was performed on tumor samples in which at least 30 somatic single base substitutions (SBS) were detected.ResultsIn total 195 samples were sequenced. Median total TMB was 10.3 mut/Mb (range 0–109.3). Mutational signatures were evaluated in 76 tumor samples (39%; median TMB 15.2 mut/Mb). SBS signature 4 (SBS4), associated with tobacco smoking, was prominently present in 25 of 76 samples (33%). SBS2 and/or SBS13, both associated with activity of the AID/APOBEC family of cytidine deaminases, were observed in 11 of 76 samples (14%). SBS4 was significantly more present in early stages (I and II) versus advanced stages (III and IV;P = .005).ConclusionIn a large proportion of NSCLC patients tissue panel sequencing with a 2 Mb panel can be used to determine the mutational signatures. In general, mutational signature SBS4 was more often found in early versus advanced stages of NSCLC. Further studies are needed to determine the clinical utility of mutational signature analyses.

Objectives The pathogenesis of intestinal involvement in systemic sclerosis (SSc) is thought to be a sequential process (vascular, neuronal, and consecutive muscular impairment), but understanding of the underlying histological changes and how they translate to symptoms, is still lacking. Therefore, we systematically investigated histological characteristics of SSc in the intestines, compared to controls. Methods Autopsy material from the small bowel and colon was used for histological semiquantitative evaluation of the vasculature, enteric nervous system, interstitial cells of Cajal (ICC), and muscle layers, using a combination of histochemical and immunohistochemical stainings, according to guidelines of the Gastro 2009 International Working Group. Results Vascular changes were most frequently encountered, represented by intima fibrosis in both arteries and small vessels, and represented by venous dilatation. Second, generalized fibrosis of the circular muscle layer was significantly more found in SSc patients than in controls. Third, reduction of submucosal nerve fibers and myenteric neurons was shown in the colon of four SSc patients, which may explain severe symptoms of intestinal dysmotility. The density of myenteric ICC network was decreased in the small bowel of SSc patients. Conclusions The postulated sequential processes of intestinal involvement in SSc could not be supported by our histological evaluation. The interpatient diversity suggests that parallel processes occur, explaining the variety of histological features and clinical symptoms. Key Points• Histological analysis showed vascular changes, fibrosis in the muscularis propria, and reduction of the ENS and ICC network in the intestines of SSc patients.• Pathophysiological mechanisms leading to intestinal dysmotility in SSc may be parallel rather than sequential.• The interpatient diversity suggests parallel pathophysiological processes, explaining the variety of histological features and clinical symptoms.

Pulmonary veno-occlusive disease (PVOD) occurs in humans either as a heritable form (hPVOD) due to biallelic inactivating mutations of EIF2AK4 (encoding GCN2) or as a sporadic form in older age (sPVOD). The chemotherapeutic agent mitomycin C (MMC) is a potent inducer of PVOD in humans and in rats (MMC-PVOD). Here, we compared human hPVOD and sPVOD, and MMC-PVOD pathophysiology at the histological, cellular, and molecular levels to unravel common altered pathomechanisms. MMC exposure in rats was associated primarily with arterial and microvessel remodeling, and secondarily by venous remodeling, when PVOD became symptomatic. In all forms of PVOD tested, there was convergent GCN2-dependent but eIF2α-independent pulmonary protein overexpression of HO-1 (heme oxygenase 1) and CHOP (CCAAT-enhancer-binding protein [C/EBP] homologous protein), two downstream effectors of GCN2 signaling and endoplasmic reticulum stress. In human PVOD samples, CHOP immunohistochemical staining mainly labeled endothelial cells in remodeled veins and arteries. Strong HO-1 staining was observed only within capillary hemangiomatosis foci, where intense microvascular proliferation occurs. HO-1 and CHOP stainings were not observed in control and pulmonary arterial hypertension lung tissues, supporting the specificity for CHOP and HO-1 involvement in PVOD pathobiology. In vivo loss of GCN2 (EIF2AK4 mutations carriers and Eif2ak4-/- rats) or in vitro GCN2 inhibition in cultured pulmonary artery endothelial cells using pharmacological and siRNA approaches demonstrated that GCN2 loss of function negatively regulates BMP (bone morphogenetic protein)-dependent SMAD1/5/9 signaling. Exogenous BMP9 was still able to reverse GCN2 inhibition-induced proliferation of pulmonary artery endothelial cells. In conclusion, we identified CHOP and HO-1 inhibition, and BMP9, as potential therapeutic options for PVOD. ispartof: AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY vol:63 issue:1 pages:118-131 ispartof: location:United States status: published

When diagnosing and reporting lung adenocarcinoma (LAC), pathologists currently include an assessment of histologic tumor growth patterns because the predominant growth pattern has been reported to impact prognosis. However, the subjective nature of manual slide evaluation contributes to suboptimal inter-pathologist variability in tumor growth pattern assessment. We applied a deep learning approach to identify and automatically delineate areas of four tumor growth patterns (solid, acinar, micropapillary, and cribriform) and non-tumor areas in whole slide images (WSI) from resected LAC specimens. We trained a DenseNet model using patches from 109 slides collected at two institutions. The model was tested using 56 WSIs including 20 that were collected at a third institution. Using the same slide set, the concordance between the DenseNet model and an experienced pathologist (blinded to the DenseNet results) in determining the predominant tumor growth pattern was substantial (kappa score = 0.603). Using a subset of 36 test slides that were manually annotated for tumor growth patterns, we also measured the F1-score for each growth pattern: 0.95 (solid), 0.78 (acinar), 0.76 (micropapillary), 0.28 (cribriform) and 0.97 (non-tumor). Our results suggest that DenseNet assessment of WSIs with solid, acinar, and micropapillary predominant tumor growth is more robust than for the WSIs with predominant cribriform growth which are less frequently encountered.

Stratification of patients for targeted and immune-based therapies requires extensive genomic profiling that enables sensitive detection of clinically relevant variants and interrogation of biomarkers, such as tumor mutational burden (TMB) and microsatellite instability (MSI). Detection of single and multiple nucleotide variants, copy number variants, MSI, and TMB was evaluated using a commercially available next-generation sequencing panel containing 523 cancer-related genes (1.94 megabases). Analysis of formalin-fixed, paraffin-embedded tissue sections and cytologic material from 45 tumor samples showed that all previously known MSI-positive samples (n = 7), amplifications (n = 9), and pathogenic variants (n = 59) could be detected. TMB and MSI scores showed high intralaboratory and interlaboratory reproducibility (eight samples tested in 11 laboratories). For reliable TMB analysis, 20 ng DNA was shown to be sufficient, even for relatively poor-quality samples. A minimum of 20% neoplastic cells was required to minimize variations in TMB values induced by chromosomal instability or tumor heterogeneity. Subsequent analysis of 58 consecutive lung cancer samples in a diagnostic setting was successful and revealed sufficient somatic mutations to generate mutational signatures in 14 cases. In conclusion, the 523-gene assay can be applied for evaluation of multiple DNA-based biomarkers relevant for treatment selection.

Histopathology is a powerful tool to understand the COVID-19 pathogenesis and come up with rational treatment strategies in real time. We present lung histopathological features of biopsies of fatal COVID-19 cases together with anakinra (Kineret ®) Il-1α and IL-1β receptor blocking treatment of the disease. Lung biopsy of 8 cases were scored for alveolar, vascular and inflammatory features on a 4-point scale (none-severe) by 3 lung pathologists; consensus score was used. Anakinra was given in off-label setting to 3 COVID-19 cases receiving ICU treatment including mechanical ventilation. Lung pathology includes 1. Extensive epithelial damage with regenerative metaplasia with co-localization of neutrophils and macrophages, together with organizing pneumonia and scarring, 2. Alveolar oedema, haemorrhage, diffuse alveolar damage and a dominant pattern of acute and chronic arterial thrombosis in all cases as manifestations of vascular leakage and its sequelae, and 3. plasma cell or T cell endothelitis of the pulmonary arteries as a characteristic feature to COVID-19 in six out of eight cases, indicating a role for plasma cells or T cells in its vascular pathology. The temporal heterogeneity of both the epithelial damage and repair and the thrombosis and thrombotic arteriopathy within in all cases indicated ongoing disease. The anakinra-treated cases showed a rapid response with extubation in 2-4 days, drop in fever and inflammatory parameters. We propose that these distinctive features of COVID-19 are initiated through the IL-1 innate immunity pathway and operated by plasma cells. We further provide proof of concept that the IL-1 receptor antagonist anakinra was beneficial in the late and severe stage of COVID-19 disease. Funding: MGN was supported by an ERC Advanced Grant and a Spinoza Grant of the Netherlands Organization for Scientific Research (NWO). Declaration of Interest: None to declare. Ethical Approval: Only fully anonymized archival tissue was used in this study, for which ethical approval and informed consent is not required by law or by the institutional review board of Radboudumc.

40 Background: Castration-resistant prostate cancer (CRPC) comprises distinct molecular actionable subtypes. Radboudumc, a tertiary referral center for PCa, hosts a Molecular Tumor Board (MTB), and frequently sees CRPC patients (pts) for referral for genetic testing. In the period 2017-2020, almost 50% of heavily pre-treated CRPC pts received a recommendation for a genetically matched therapy (GMT). In 2020 we initiated the PROMPT trial (NCT04746300) to prospectively study the impact of routine early molecular characterization in CRPC. Increased GMT utilization of targeted- and immunotherapy may improve outcome and quality of life of pts with metastatic CRPC, and lead to lower medical resource utilization (MRU). Here we report on first results from our observational trial. Methods: Within the PROMPT trial, prior to receiving first- or second line standard of care CRPC therapy, up to 300 pts were offered molecular tumor characterization with the TruSightOncology 500 panel (TSO500). Formalin-fixed paraffin-embedded prostate or metastatic tissue biopsies were used, preferably newly obtained. To assess both mutations and copy number alterations, the TSO500 required a minimal tumor cell percentage (TC%) of 30%. Results were discussed within the Radboudumc MTB for GMT recommendation. Actionable targets were defined per Precision Medicine Working group criteria and, when druggable, within the Drug Rediscovery Protocol trial (NCT02925234). All pts with tumor mutations carrying with a risk for cancer predisposition were referred for genetic counselling. Follow-up with patient-reported outcomes (EORTC QLQ-C30, EQ5D, BPI & EPIC-26 questionnaires) and MRU was conducted every 3 months until withdrawal or death. Results: From February 2020 until October 2021 we included 284 consecutive CRPC pts with a median age of 70 years (range 46-86) with a median follow-up of 6.8 months. Newly obtained biopsies and archival material was used in 131 and 126 pts, respectively. Median TC% was 60% (range: 20-90%). TSO500 results could be reported in 254 (89%) cases, with at least one putative clinically relevant aberration in 188 evaluable pts (74%). In 100 pts (39,4%) ≥ 1 druggable target was found. Most common actionable alterations were in PTEN (19%), BRCA2 (9%) or in mismatch-repair genes or resulting in high tumor mutational burden (5%). Out of the 100 druggable pts, 31 pts (31%) initiated a form of GMT, 4 pts (4%; 2 BRCA2, 2 PTEN) died prior to receiving GMT, in 65 pts (65%) GMT is pending as they receive standard of care. Conclusions: Routine molecular profiling early in the CRPC setting is feasible in a tertiary referral center with a MTB and high volume of CRPC pts. Almost 40% CRPC pts harbored an actionable target with 31% of these pts already allocated to a GMT. Failure rates of NGS were low at 11%.

Kidney failure is frequently observed during and after COVID-19, but it remains elusive whether this is a direct effect of the virus. Here, we report that SARS-CoV-2 directly infects kidney cells and is associated with increased tubule-interstitial kidney fibrosis in patient autopsy samples. To study direct effects of the virus on the kidney independent of systemic effects of COVID-19, we infected human induced pluripotent stem cell-derived kidney organoids with SARS-CoV-2. Single cell RNA-sequencing indicated injury and dedifferentiation of infected cells with activation of pro-fibrotic signaling pathways. Importantly, SARS-CoV-2 infection also led to increased collagen 1 protein expression in organoids. A SARS-CoV-2 protease inhibitor was able to ameliorate the infection of kidney cells by SARS-CoV-2. Our results suggest that SARS-CoV-2 can directly infect kidney cells and induce cell injury with subsequent fibrosis. These data could explain both acute kidney injury in COVID-19 patients and the development of chronic kidney disease in Long-COVID., Graphical Abstract, Jansen, Reimer, Nagai et al report that SARS-CoV-2 infects kidney cells and is associated with kidney fibrosis in patients. Using single cell transcriptomics of infected kidney organoids, they show that SARS-CoV-2 causes kidney injury and stimulates pro-fibrotic signaling. Viral infection in organoids was inhibited by a recently developed protease blocker.

Contains fulltext : 207194.pdf (Publisher’s version ) (Open Access) Advances in molecular tumour diagnostics and the number of targeted therapies increase rapidly. Molecular tumour boards (MTBs) are designated to interpret these data and provide clinical recommendations. Not all patients with cancer have access to advice of an MTB. We aimed to determine the current status, opportunities, and challenges of the organisation of MTBs in the Netherlands. We interviewed several stakeholders about their experiences with an MTB, using template analysis. Most clinicians and patient representatives underscore the significance of an MTB, because it can stimulate rational treatment options, enrolment in clinical trials, and interdisciplinary knowledge transfer. Health insurance companies and financial managers are concerned about increasing costs. Registries to assess the clinical benefit of MTBs, guidelines on quality control, financial agreements, and logistical resources are lacking. The national organisation of MTBs and a registry of molecular and clinical data are important issues to address.

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Contains fulltext : 208535.pdf (Publisher’s version ) (Open Access) Among the many uses of digital pathology, remote consultation, remote revision, and virtual slide panels may be the most important ones. This requires basic slide scanner infrastructure in participating laboratories to produce whole-slide images. More importantly, a software platform is needed for exchange of these images and functionality to support the processes around discussing and reporting on these images without breaching patient privacy. This poses high demands on the setup of such a platform, given the inherent complexity of the handling of digital pathology images. In this article, we describe the setup and validation of the Pathology Image Exchange project, which aimed to create a vendor-independent platform for exchange of whole-slide images between Dutch pathology laboratories to facilitate efficient teleconsultation, telerevision, and virtual slide panels. Pathology Image Exchange was released in April 2018 after technical validation, and a first successful validation in real life has been performed for hematopathology cases.

Contains fulltext : 204299.pdf (Publisher’s version ) (Open Access) Vascular malformations are part of overgrowth syndromes characterized by somatic mosaic mutations or rarely by germline mutations. Due to their similarities and diversity, clinicopathological classification can be challenging. A comprehensive targeted Next Generation Sequencing screen using Unique Molecular Identifiers with a technical sensitivity of 1% mutant alleles was performed for frequently mutated positions in >/=21 genes on 319 formalin-fixed paraffin-embedded samples. In 132 out of 319 cases pathogenic mosaic mutations were detected affecting genes previously linked to vascular malformations e.g. PIK3CA (n=80), TEK (TIE2) (n=11), AKT1 (n=1), GNAQ (n=7), GNA11 (n=4), IDH1 (n=3), KRAS (n=9), and NRAS (n=1). Six cases harbored a combination of mutations in PIK3CA and in GNA11 (n=2), GNAQ (n=2), or IDH1 (n=2). Aberrations in PTEN and RASA1 with a variant allele frequency approaching 50% suggestive of germline origin were identified in six out of 102 cases tested; four contained a potential second hit at a lower allele frequency. Ninety-one of the total 142 pathogenic mutations were present at a variant allele frequency

Aims: Variation in health-care is undesirable, as this is potentially harmful for patients. In the Netherlands, an e-learning module was developed to standardise pathological evaluation of colorectal adenomas. We studied the effect of e-learning on interlaboratory variability in grading of dysplasia in screened conventional colorectal adenomas. Methods and results: A cross-sectional retrospective study was performed, including all colorectal adenomas from the Dutch population-based colorectal cancer screening programme, retrieved from the Dutch Pathology Registry (PALGA) from January 2014 to July 2015. The e-learning tool, commissioned by the National Institute for Public Health, was implemented among screening pathologists from October 2014. Proportions of high-grade dysplasia (HGD) were compared before (January–July 2014) and after implementation (October 2014–July 2015) of the e-learning module. Interlaboratory variation was assessed by multilevel mixed-effects analysis. In total, 20 713 colonoscopies (20 546 patients) were performed after a positive faecal immunochemical screening test, resulting in the inclusion of 56 355 conventional adenomas from 37 pathology laboratories. Before implementation, 12 614 adenomas were diagnosed, including 4.3% with HGD. After implementation, 43 741 adenomas were diagnosed, and the HGD proportion decreased to 3.9%. Univariable analysis showed less deviant proportions of HGD after implementation in 62% of the laboratories (P = 0.019). Multilevel analysis confirmed decreased variation in the risk of diagnosing HGD (P = 0.021). Conclusions: Interlaboratory variability in grading HGD in colorectal adenomas after a positive screening test decreased after implementation of an e-learning module for pathologists. We therefore conclude that e-learning has a favourable influence on decreasing diagnostic variability, making this a relevant strategy for health-care standardisation.

Contains fulltext : 171056.pdf (Publisher’s version ) (Open Access) BACKGROUND: Data on non-small-cell lung cancer (NSCLC) patients with non-classic epidermal growth factor receptor (EGFR) mutations are scarce, especially in non-Asian populations. The purpose of this study was to evaluate prevalence, clinical characteristics and outcome on EGFR-TKI treatment according to type of EGFR mutation in a Dutch cohort of NSCLC patients. METHODS: We retrospectively evaluated a cohort of 240 EGFR-mutated NSCLC patients. Data on demographics, clinical and tumour-related features, EGFR-TKI treatment and clinical outcome were collected and compared between patients with classic EGFR mutations, EGFR exon 20 insertions and other uncommon EGFR mutations. RESULTS: Classic EGFR mutations were detected in 186 patients (77.5%) and non-classic EGFR mutations in 54 patients (22.5%); 23 patients with an exon 20 insertion (9.6%) and 31 patients with an uncommon EGFR mutation (12.9%). Median progression-free survival (PFS) and overall survival (OS) on EGFR-TKI treatment were 2.9 and 9.7 months, respectively, for patients with an EGFR exon 20 insertion, and 6.4 and 20.2 months, respectively, for patients with an uncommon EGFR mutation. Patients with a double uncommon EGFR mutation that included G719X/L861Q/S768I had longer PFS and OS on EGFR-TKI treatment compared with patients with a single G719X/L861Q/S768I EGFR mutation (both P=0.02). CONCLUSIONS: In our Dutch cohort, prevalence and genotype distribution of non-classic EGFR mutations were in accordance with previously reported data. The PFS and OS on EGFR-TKI treatment in patients with an uncommon EGFR mutation were shorter compared with patients with classic EGFR mutations, but varied among different uncommon EGFR mutations. 9 p.

Differentiation grade of colorectal adenocarcinoma (CRC) is a prognostic factor and important for therapy selection. In patients with stage II colon cancer, poor differentiation is an indication for adjuvant chemotherapy. The variability in daily practice in the grading of CRC was assessed in a nationwide cohort. Using the Dutch Pathology Registry (PALGA), all synoptically reported CRC resections from 2010 to 2013 were identified. Proportions of poorly differentiated (PD) adenocarcinomas were determined and compared between 35 laboratories by univariable and multivariable logistic regression analyses. In total, 11,719 resections of 11,681 patients were included, of which 1427 (12.2%) were PD (range between 35 laboratories: 5.0% to 33.2%). After adjustment for case mix, 4 (11%) laboratories still reported a significantly lower (n=2) or higher (n=2) proportion of PD adenocarcinoma compared with the reference laboratory. Seven of 8 investigated laboratories showed considerable intralaboratory variation between pathologists as well. In a subgroup of 2812 patients (2813 tumors) who could have been eligible for adjuvant chemotherapy solely on the basis of the differentiation grade (stage II colon cancer patients without other high-risk factors [ie, T4

Contains fulltext : 172088.pdf (Publisher’s version ) (Open Access) With around 500 cases published worldwide, pulmonary alveolar microlithiasis is a rare disorder with an autosomal recessive pattern of inheritance. We show for the first time that homozygous deletions encompassing the entire SCL34A2 can be associated with this rare genetic pulmonary disease.

Contains fulltext : 193424.pdf (Publisher’s version ) (Open Access) Myositis ossificans is defined as a self-limiting pseudotumor composed of reactive hypercellular fibrous tissue and bone. USP6 rearrangements have been identified as a consistent genetic driving event in aneurysmal bone cyst and nodular fasciitis. It is therefore an integral part of the diagnostic workup when dealing with (myo)fibroblastic lesions of soft tissue and bone. Two cases of myositis ossificans with USP6 rearrangement were published so far. We determine herein the incidence of USP6 rearrangement in myositis ossificans using USP6 fluorescence in situ hybridization analysis (FISH). Of the 11 cases included, seven patients were female and four were male. Age ranged from 6 to 56years (mean 27years). Lesions were located in the thigh (n=5), knee (n=1), lower leg (n=1), lower arm (n=1), perineum (n=1), gluteal (n=1) and thoracic wall (n=1). All assessable cases except one (8/9) showed rearrangement of USP6 providing evidence that myositis ossificans is genetically related to nodular fasciitis and aneurysmal bone cyst.

Older asthmatic patients may develop fixed airway obstruction and clinical signs of chronic obstructive pulmonary disease (COPD). We investigated the added value of pathological evaluation of bronchial biopsies to help differentiate asthma from COPD, taking into account smoking, age, and inhaled corticosteroid (ICS) use. Asthma and COPD patients (24 of each category) were matched for ICS use, age, FEV1, and smoking habits. Five pulmonary and five general pathologists examined bronchial biopsies using an interactive website, without knowing patient information. They were asked to diagnose asthma or COPD on biopsy findings in both a pairwise and randomly mixed order of cases during four different phases, with intervals of 4–6 weeks, covering a maximal period of 36 weeks. Clinically concordant diagnoses of asthma or COPD varied between 63 %-73 %, without important differences between pairwise vs randomly mixed examination or between general vs pulmonary pathologists. The highest percentage of concordant diagnoses was in young asthmatic patients without ICS use and in COPD patients with ICS use. In non ICS users with fixed airway obstruction, a COPD diagnosis was favored if abnormal presence of glands, squamous metaplasia, and submucosal infiltrate was present and an asthma diagnosis in case of abnormal presence of goblet cells. In ICS users with fixed airway obstruction, abnormal presence of submucosal infiltrates, basement membrane thickening, eosinophils, and glands was associated with asthma. Histological characteristics in bronchial biopsies are reproducibly recognized by pathologists, yet the differentiation by histopathology between asthma and COPD is difficult without information about ICS use. Electronic supplementary material The online version of this article (doi:10.1007/s00428-015-1824-6) contains supplementary material, which is available to authorized users.

Objectives Adenocarcinoma (ADC) of the lung may harbor EGFR - or KRAS -mutations, which are relevant for treatment decisions. There is no consensus on the percentages of EGFR - and KRAS -mutations that are allowed to be missed by a diagnostic algorithm, although a percentage of less than 1% for EGFR -mutations has been suggested. The current guidelines do not advise to perform EGFR -mutation analysis in unequivocal squamous cell carcinoma (SqCC). For KRAS -mutations no threshold for missing cases is suggested yet. To improve segregation between ADC and SqCC in small samples, the classification of lung cancer was updated in 2011, adding immunohistochemistry (IHC) for p63 and TTF-1 to the diagnostic algorithm. In this study we examined how many cases with an EGFR - or KRAS -mutation in our database would have been missed, if the current guideline for selecting cases for mutation analysis would have been applied. Materials and methods From an institutional lung cancer database of specimens analyzed for EGFR - and KRAS -mutations ( n = 816), cases harboring a mutation without being treated prior with an EGFR-TKI were selected ( n = 336). Corresponding original histological diagnoses and IHC for TTF-1, p63 and PAS-D were collected. Cases with SqCC on HE or with an IHC pattern favoring SqCC were reassessed according to the criteria of the 2011-classification. Results From the 336 cases 70% had a KRAS -mutation and 30% an EGFR -mutation. The number of cases with SqCC on HE and/or an IHC-profile favoring SqCC was 12. After the reassessment six specimens (1.8%) would not have been tested for EGFR-/KRAS-mutations, if the current diagnostic algorithm had been used: 2.0% of EGFR -mutations and 1.7% KRAS -mutations. All six cases were NSCLC with an IHC-profile favoring SqCC. Conclusion Most NSCLC-cases with EGFR- and KRAS -mutations are selected by the current diagnostic algorithm. As a small but relevant fraction is missed, there is room for improvement.

Lung resection is a standard treatment in patients with clinical stages I and II and selected patients with stage IIIA nonsmall cell lung cancer [1]. Major lung resection (MLR), such as (bi)lobectomy or pneumonectomy, occasionally lead to pulmonary hypertension (PH). Several studies report an increase in pulmonary artery pressures in about one-third of these patients up to 5 years postoperatively [2–4]. The development of PH after MLR may be explained simply by the fact that total cardiac output flows through a smaller vascular bed. Because there are no reports in the literature of histological studies performed after MLR, it remains unknown whether flow-induced structural changes in the remaining lung vasculature lead to progressive increases in pulmonary vascular resistance., Major lung resection results in mild pulmonary vascular remodelling http://ow.ly/JRvW30doTC0

Contains fulltext : 174854.pdf (Publisher’s version ) (Open Access) In idiopathic pulmonary arterial hypertension (PAH), increased pulmonary vascular resistance is associated with structural narrowing of small (resistance) vessels and increased vascular tone. Current information on pulmonary vascular remodeling is mostly limited to averaged increases in wall thickness, but information on number of vessels affected and internal diameter decreases for vessels of different sizes is limited. Our aim was to quantify numbers of affected vessels and their internal diameter decrease for differently sized vessels in PAH in comparison with non-PAH patients. Internal and external diameters of transversally cut vessels were measured in five control subjects and six PAH patients. Resistance vessels were classified in Strahler orders, internal diameters 13 mum (order 1) to 500 mum (order 8). The number fraction, that is, percentage of affected vessels, and the internal diameter fraction, that is, percentage diameter of normal diameter, were calculated. In PAH, not all resistance vessels are affected. The number fraction is about 30%, that is, 70% of vessels have diameters not different from vessels of control subjects. Within each order, the decrease in diameter of affected vessels is variable with an averaged diameter fraction of 50-70%. Narrowing of resistance vessels is heterogeneous: not all vessels are narrowed, and the decrease in internal diameters, even within a single order, vary largely. This heterogeneous narrowing alone cannot explain the large resistance increase in PAH We suggest that rarefaction could be an important contributor to the hemodynamic changes.

Contains fulltext : 174831.pdf (Publisher’s version ) (Open Access) Pulmonary hypertension (PH) with complicating chronic lung diseases and/or hypoxia falls into group 3 of the updated classification of PH. Patients with chronic obstructive lung disease (COPD), diffuse lung disease (such as idiopathic pulmonary fibrosis (IPF)) and with sleep disordered breathing are particularly exposed to the risk of developing PH. Although PH in such a context is usually mild, a minority of patients exhibit severe haemodynamic impairment, defined by a mean pulmonary arterial pressure (mPAP) of >/=35 mmHg or mPAP values ranging between 25 mmHg and 35 mmHg with a low cardiac index (

TPS3149 Background: With the emergence of large-scale genetic tumor profiling and the increasing availability of approved targeted therapies, precision medicine has become crucial in cancer treatment. However, for many cancers the relative contribution of either tumor type or genetic aberration to drug sensitivity often remains unknown. Since drug access is generally limited to the on-label indication and outcome of off-label use is not systematically collected in clinical practice, innovative trials facilitating drug access, whilst systematically analyzing treatment outcomes, are urgently needed. Methods: The Drug Rediscovery Protocol (DRUP) is an ongoing, prospective, non-randomized, multi-drug, and pan-cancer trial, in which patients with advanced cancer, who have exhausted all standard of care treatment options, are treated with either targeted or immunotherapy matched to their genetic tumor profile. All submitted patients are reviewed and enrolled in multiple parallel cohorts, preceded by a baseline tumor biopsy for whole genome sequencing to confirm previously identified variants and for exploratory biomarker analyses. Each cohort is defined by a study drug, histologic tumor type, and molecular tumor profile. Efficacy is analyzed per cohort: 8 patients in stage I and 16 more in stage II if ≥ 1 response is observed in the first stage. Primary endpoints include objective response rate, stable disease at 16 weeks, and grade ≥3 adverse events. Since the start of recruitment in September 2016, 870 patients have been submitted for review and 365 patients (42%) have started treatment in one of 101 opened cohorts. Eight cohorts have graduated to the second stage, two cohorts completed accrual in either their first or second stage, and one cohort was closed due to a registered indication. Twenty-two different study treatments (i.e. immunotherapy, monoclonal antibodies, and PARP/small molecule inhibitors), provided by 11 different pharmaceutical companies, are currently available in DRUP. Data sharing with similar trials such as TAPUR and CAPTUR enables to achieve completion of slow accruing cohorts and affirm conclusions. Clinical trial information: NCT02925234.

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Item does not contain fulltext RATIONALE: Altered pulmonary hemodynamics and fluid flow-induced high shear stress (HSS) are characteristic hallmarks in the pathogenesis of pulmonary arterial hypertension (PAH). However, the contribution of HSS to cellular and vascular alterations in PAH is unclear. OBJECTIVES: We hypothesize that failing shear adaptation is an essential part of the endothelial dysfunction in all forms of PAH and tested whether microvascular endothelial cells (MVECs) or pulmonary arterial endothelial cells (PAECs) from lungs of patients with PAH adapt to HSS and if the shear defect partakes in vascular remodeling in vivo. METHODS: PAH MVEC (n = 7) and PAH PAEC (n = 3) morphology, function, protein, and gene expressions were compared with control MVEC (n = 8) under static culture conditions and after 24, 72, and 120 hours of HSS. MEASUREMENTS AND MAIN RESULTS: PAH MVEC showed a significantly delayed morphological shear adaptation (P = 0.03) and evidence of cell injury at sites of nonuniform shear profiles that are critical loci for vascular remodeling in PAH. In clear contrast, PAEC isolated from the same PAH lungs showed no impairments. PAH MVEC gene expression and transcriptional shear activation were not altered but showed significant decreased protein levels (P = 0.02) and disturbed interendothelial localization of the shear sensor platelet endothelial cell adhesion molecule-1 (PECAM-1). The decreased PECAM-1 levels were caused by caspase-mediated cytoplasmic cleavage but not increased cell apoptosis. Caspase blockade stabilized PECAM-1 levels, restored endothelial shear responsiveness in vitro, and attenuated occlusive vascular remodeling in chronically hypoxic Sugen5416-treated rats modeling severe PAH. CONCLUSIONS: Delayed shear adaptation, which promotes shear-induced endothelial injury, is a newly identified dysfunction specific to the microvascular endothelium in PAH. The shear response is normalized on stabilization of PECAM-1, which reverses intimal remodeling in vivo.

Contains fulltext : 172841pub.pdf (Publisher’s version ) (Closed access) AIMS: Although high-grade dysplasia (HGD) is a risk factor for malignant transformation and the future development of adenomas/carcinomas, grade is not incorporated in the Dutch guidelines for colonoscopy surveillance, partly because of presumed interobserver variability. The aim of this study was to analyse, in a nationwide cohort of colorectal adenomas, the interlaboratory variability in the grading of dysplasia in daily practice. METHODS AND RESULTS: From the Dutch Pathology Registry, all synoptically reported classic adenomas in The Netherlands in 2013 were identified. The proportion of adenomas with HGD was determined for biopsies and polypectomies, and compared between 37 laboratories by the use of multivariable logistic regression analyses. In total, 21 030 colonoscopies of 20 270 patients were included. HGD was reported in 530 (3.6%) of 14 866 adenomas diagnosed on biopsies (range between laboratories: 0-13.6%) and in 983 (11.8%) of 8346 adenomas diagnosed on polypectomies (range: 3.1-42.9%). After adjustment for case mix, 13 (35%) laboratories reported a significantly lower or higher frequency of HGD than average. CONCLUSIONS: We observed considerable interlaboratory variation in the grading of dysplasia in colorectal adenomas, which could be only partly explained by differences in case mix. Therefore, better standardization of grading criteria is needed before grade of dysplasia can usefully be incorporated in colonoscopy surveillance guidelines.

5036Background: Predictive biomarkers are needed to improve the proportion of pts with mCRPC that may benefit from anti-PD-1/PD-L1 ICB. A comprehensive characterization of both genomic alterations ...

Introduction: Idiopathic Pulmonary Arterial Hypertension (iPAH) is a fatal lung disease characterized by remodeling of the lung vasculature leading to a 5-fold increase of the pulmonary vascular resistance (PVR). Quantitative information on vascular remodeling is limited to assessments of increased wall thickness, and the extent of internal diameter decrease and thus the contribution to PVR increase, is unknown. Therefore, we quantified the number of affected arteries and their internal diameter to estimate the PVR increase using an anatomy based model. Methods: Internal and external diameters of resistance arteries (external diameter of 13 to 500 μm) were measured in 5 controls and 6 iPAH subjects. The vessels were classified in Strahler orders, and number fraction of narrowed vessels and their relative internal diameter decrease in iPAH were determined. To predict arterial resistance increase as a result of diameter decrease, the arterial bed was modeled as a tree, using Poiseuille9s Law, and data on diameters, lengths, and numbers. Results: Only 30 to 50% of the resistance arteries in iPAH patients were narrowed. Their internal diameters decreased to approximately 50 to 70%. This remodeling resulted in an arterial resistance increase of only 3 times in iPAH. Conclusions: In iPAH, narrowing of resistance arteries is heterogeneous: not all arteries are narrowed, and internal diameters vary largely. Diameter changes cannot fully explain the increased PVR seen in iPAH. Therefore, future experiments should determine numbers and sizes of vessels in control and iPAH subjects to gain further insight into the possible role of arterial rarefaction and venous changes in iPAH.

Introduction: Recent reports have shown that mutations in the eukaryotic initiation factor 2 alpha kinase 4 (EIF2AK4) gene cause autosomal recessively inheritable pulmonary veno-occlusive disease (PVOD) in familial and a subset of sporadic cases. The pathophysiology evolving from EIF2AK4 mutations remains still to be elucidated. Aim and objective: We sought to differentiate histopathological characteristics between EIF2AK4 mutation-carriers and non-carriers with PVOD. Methods: Histology of lung samples obtained after transplantation of EIF2AK4 mutation-carriers (n=9) and non-carriers (n=9) displaying clinical PVOD were systematically analyzed and scored in a blinded fashion, using quantitative semi-automated morphometry. Results: EIF2AK4 mutation-carriers were significantly distinctive from non-carriers regarding several patterns: a) intimal fibrosis (more severe) and media hypertrophy (less severe) of pulmonary arteries; b) diffuse interstitial fibrosis (less frequent) and patchy hemangiomatosis-like foci (more frequent) within the parenchyma. A trend to heavier remodeling of interlobular septal veins was observed. Conclusion: Histology of bi-allelic EIF2AK4 mutation-carriers can be differentiated from non-carriers due to arterial and interstitial remodeling patterns. Whether the severe arterial remodeling is of primary or secondary nature in the setting of heritable PVOD requires further insight into EIF2AK4 biology.

A polarization sensitive endoscopic optical frequency domain imaging (PS-OFDI) system with a motorized distal scanning catheter is demonstrated. It employs a passive polarization delay unit to multiplex two orthogonal probing polarization states in depth, and a polarization diverse detection unit to detect interference signal in two orthogonal polarization channels. Per depth location four electro-magnetic field components are measured that can be represented in a complex 2x2 field matrix. A Jones matrix of the sample is derived and the sample birefringence is extracted by eigenvalue decomposition. The condition of balanced detection and the polarization mode dispersion are quantified. A complex field averaging method based on the alignment of randomly pointing field phasors is developed to reduce speckle noise. The variation of the polarization states incident on the tissue due to the circular scanning and catheter sheath birefringence is investigated. With this system we demonstrated imaging of ex vivo chicken muscle, in vivo pig lung and ex vivo human lung specimens. (C)2015 Optical Society of America