Comparing deep learning and pathologist quantification of cell-level PD-L1 expression in non-small cell lung cancer whole-slide images.

Programmed death-ligand 1 (PD-L1) expression is currently used in the clinic to assess eligibility for immune-checkpoint inhibitors via the tumor proportion score (TPS), but its efficacy is limited by high interobserver variability. Multiple papers have presented systems for the automatic quantification of TPS, but none report on the task of determining cell-level PD-L1 expression and often reserve their evaluation to a single PD-L1 monoclonal antibody or clinical center. In this paper, we report on a deep learning algorithm for detecting PD-L1 negative and positive tumor cells at a cellular level and evaluate it on a cell-level reference standard established by six readers on a multi-centric, multi PD-L1 assay dataset. This reference standard also provides for the first time a benchmark for computer vision algorithms. In addition, in line with other papers, we also evaluate our algorithm at slide-level by measuring the agreement between the algorithm and six pathologists on TPS quantification. We find a moderately low interobserver agreement at cell-level level (mean reader-reader F1 score = 0.68) which our algorithm sits slightly under (mean reader-AI F1 score = 0.55), especially for cases from the clinical center not included in the training set. Despite this, we find good AI-pathologist agreement on quantifying TPS compared to the interobserver agreement (mean reader-reader Cohen's kappa = 0.54, 95% CI 0.26-0.81, mean reader-AI kappa = 0.49, 95% CI 0.27-0.72). In conclusion, our deep learning algorithm demonstrates promise in detecting PD-L1 expression at a cellular level and exhibits favorable agreement with pathologists in quantifying the tumor proportion score (TPS). We publicly release our models for use via the Grand-Challenge platform.

Tumor microenvironment shows an immunological abscopal effect in patients with NSCLC treated with pembrolizumab-radiotherapy combination.

BACKGROUND: Immunotherapy 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). METHODS: Here, 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. RESULTS: The 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). CONCLUSION: This 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.

Identification of Fusion Genes and Targets for Genetically Matched Therapies in a Large Cohort of Salivary Gland Cancer Patients.

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.

Cost-Effectiveness of Parallel Versus Sequential Testing of Genetic Aberrations for Stage IV Non-Small-Cell Lung Cancer in the Netherlands.

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.

Mutation-tailored treatment selection in non-small cell lung cancer patients in daily clinical practice.

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.

Impact of molecular tumour board discussion on targeted therapy allocation in advanced prostate cancer.

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.

Mutational signature analysis in non-small cell lung cancer patients with a high tumor mutational burden.

BACKGROUND: Lung 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. METHODS: In 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. RESULTS: In 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). CONCLUSION: In 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.

Nationwide evaluation of mutation-tailored anti-EGFR therapy selection in patients with colorectal cancer in daily clinical practice.

For a nationwide real-word data study on the application of predictive mutation testing of patients with colorectal cancer (CRC) for anti-epidermal growth factor receptor (EGFR) therapy stratification, pathology data were collected from the Dutch Pathology Registry from October 2017 until June 2019 (N=4060) and linked with the Netherlands Cancer Registry. Mutation testing rates increased from 24% at diagnosis of stage IV disease to 60% after 20-23 months of follow-up (p<0.001). Application of anti-EGFR therapy in KRAS/NRAS wild-type patients was mainly observed from the third treatment line onwards (65% vs 17% in first/second treatment line (p<0.001)). The national average KRAS/NRAS/BRAF mutation rate was 63.9%, being similar for next-generation sequencing (NGS)-based approaches and single gene tests (64.4% vs 61.2%, p=ns). NGS-based approaches detected more additional potential biomarkers, for example, ERBB2 amplifications (p<0.05). Therefore, single gene tests are suitable to stratify patients with mCRC for anti-EGFR therapy, but NGS is superior enabling upfront identification of therapy resistance or facilitate enrolment into clinical trials.

Nationwide evaluation of mutation-tailored treatment of gastrointestinal stromal tumors in daily clinical practice.

BACKGROUND: Molecular analysis of KIT and PDGFRA is critical for tyrosine kinase inhibitor treatment selection of gastrointestinal stromal tumors (GISTs) and hence recommended by international guidelines. We performed a nationwide study into the application of predictive mutation testing in GIST patients and its impact on targeted treatment decisions in clinical practice. METHODS: Real-world clinical and pathology information was obtained from GIST patients with initial diagnosis in 2017-2018 through database linkage between the Netherlands Cancer Registry and the nationwide Dutch Pathology Registry. RESULTS: Predictive mutation analysis was performed in 89% of the patients with high risk or metastatic disease. Molecular testing rates were higher for patients treated in expertise centers (96%) compared to non-expertise centers (75%, P < 0.01). Imatinib therapy was applied in 81% of the patients with high risk or metastatic disease without patient's refusal or adverse characteristics, e.g., comorbidities or resistance mutations. Mutation analysis that was performed in 97% of these imatinib-treated cases, did not guarantee mutation-tailored treatment: 2% of these patients had the PDGFRA p.D842V resistance mutation and 7% initiated imatinib therapy at the normal instead of high dose despite of having a KIT exon 9 mutation. CONCLUSION: In conclusion, nationwide real-world data show that over 81% of the eligible high risk or metastatic disease patients receive targeted therapy, which was tailored to the mutation status as recommended in guidelines in 88% of cases. Therefore, still 27% of these GIST patients misses out on mutation-tailored treatment. The reasons for suboptimal uptake of testing and treatment require further study.

Micro-costing diagnostics in oncology: from single-gene testing to whole- genome sequencing.

Purpose: Predictive 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. 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.Methods: The activity-based costing (ABC) method was conducted to calculate 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.Results: The total cost per cancer patient per technique varied from € 58 (Sanger sequencing, three amplicons) to € 2925 (paired tumor-normal WGS). The operational costs accounted for the vast majority (over 90%) of the total per cancer patient technique costs.Conclusion: This 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.

Multicenter Comparison of Molecular Tumor Boards in The Netherlands: Definition, Composition, Methods, and Targeted Therapy Recommendations.

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.

Evaluation of a Hybrid Capture-Based Pan-Cancer Panel for Analysis of Treatment Stratifying Oncogenic Aberrations and Processes.

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.

Comparison of Human and Experimental Pulmonary Veno-Occlusive Disease.

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.

Pathology Image Exchange: The Dutch Digital Pathology Platform for Exchange of Whole-Slide Images for Efficient Teleconsultation, Telerevision, and Virtual Expert Panels.

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.

Molecular tumour boards and molecular diagnostics for patients with cancer in the Netherlands: experiences, challenges, and aspirations.

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.

Decrease of variation in the grading of dysplasia in colorectal adenomas with a national e-learning module.

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.

Comprehensive molecular and clinicopathological analysis of vascular malformations: A study of 319 cases.

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 <10% illustrating the importance of sensitive molecular analysis. Clinicopathological characteristics showed a broad spectrum and overlap when correlated with molecular data. Sensitive screening of recurrently mutated genes in vascular malformations may help to confirm the diagnosis and reveals potential therapeutic options with a significant contribution of PIK3CA/mTOR and RAS-MAPK pathway mutations. The co-existence of two activating pathogenic mutations in parallel pathways illustrates potential treatment challenges and underlines the importance of multigene testing. Detected germline mutations have major clinical impact.

Myositis ossificans - Another condition with USP6 rearrangement, providing evidence of a relationship with nodular fasciitis and aneurysmal bone cyst.

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 56 years (mean 27 years). 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.