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Objective: Systemic sclerosis-interstitial lung disease (SSc-ILD) is the leading cause of death in SSc, affecting around 50 % of the patients. Lung tissue of patients with early-stage SSc-ILD is characterized by a predominant inflammatory response with inconspicuous fibrosis, which may progress to honeycombing fibrosis. Hence, a better understanding of the molecular mechanisms underpinning SSc-ILD pathogenesis is needed to improve treatment options and progression prediction. This transcriptomic study aims to reveal the differential gene expression between control (ctrl) lung tissue and inflammatory, prefibrotic and fibrotic lung tissue to capture progression of early to late phase SSc-ILD., Methods: Twelve explanted lungs from patients with SSc-ILD were used to analyze gene expression from formalin-fixed paraffin-embedded lung tissues with varying stages of ILD (n = 18) and control lung tissue (n = 6). The SSc-ILD tissues were stratified into three ROIs: inflammatory, prefibrotic, and fibrotic using histological assessments to define a longitudinal simulation of early to late phases of SSc-ILD. The nanoString (nS) nCounter Human Fibrosis Panel was used to profile the transcriptome in the regions of interest. Validation of potential targetswas performed with immunohistochemistry in the same tissues that were used for transcriptome analysis., Results: To validate our simulation model, we performed subgroup analysis that showed an incremental increase in pathway scores related to the severity of fibrosis. Ctrl vs SSc-ILD comparison demonstrated 24 differentially expressed genes, two of which had the most pronounced p-values. Cyclin-dependent kinase inhibitor (cdkn2c) was overexpressed (P = 0.00052) in SSc-ILD compared to ctrl, while expression of Pellino E3 ubiquitin-protein ligase 1 (peli1) showed lower expression (P = 0.0012). Additionally, in all four groups, cdkn2c and peli1 gene expression showed an incremental increase and decrease, respectively. Immunohistochemistry of cdkn2c showed consistent results with the nS analysis., Conclusion: More cdkn2c and less peli1 expression were associated with more advanced stages of SSc-ILD on histologic assessment. We report the potential of the cell cycle inhibitor and senescence marker, cdkn2c (p18) to be associated with fibrosis progression., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The 2018 WHO edition on the classification of cutaneous melanocytic tumors recognizes eight evolutionary pathways of melanoma and describes tumors of uncertain malignant potential for each. When histology and immunohistochemistry do not support a confident conclusion about its malignant potential, a window of diagnostic uncertainty is created. Mutations in the telomerase reverse transcriptase gene promoter ( TERT p) are highly specific for melanoma and can be used as an ancillary technique to acquire a higher level of confidence in the diagnosis. However, little is known about the cost-effectiveness of testing for TERT p mutations. The aims of this study were to determine how often knowledge of the TERT p mutation status contributed to the final diagnosis and to develop a micro-costing framework to calculate cost-effectiveness. A retrospective analysis of all cutaneous melanocytic lesions that were discussed in the Noord-Nederland Melanoma Panel from January 2021 to October 2022 was performed to identify the cases in which the preliminary histopathological diagnosis was uncertain regarding malignancy (ambiguous, likely benign, or likely malignant). For cases in which a TERTp mutation analysis was performed, the final diagnoses were collected, and it was determined whether this impacted the overall conclusion. A micro-costing framework was established to model the financial impact of introducing TERTp mutation analyses and subsequent clinical procedures. The study included 367 cases, of which 175 diagnoses of uncertain malignant potential were initially reported. TERTp mutation analysis was performed for 151/175 (86%). In 38% of these cases, a higher level of confidence regarding malignant potential was obtained. The implementation of TERT p mutation analyses for cutaneous melanocytic proliferations with uncertain malignant potential can narrow the window of diagnostic uncertainty. For the patient group with an initial uncertain diagnosis, the increased cost for molecular testing (86.145 €) was compensated by a reduced overall treatment cost (-122.304 €). A microsimulation model to determine the cost-effectiveness of TERT p mutation analysis projected an overall saving for the healthcare system.

Introduction: Colorectal cancer (CRC) ranks as the third most prevalent malignancy globally, with a concerning rise in incidence among young adults. Despite progress in understanding genetic predispositions and lifestyle risk factors, the intricate molecular mechanisms of CRC demand exploration. MicroRNAs (miRNAs) emerge as key regulators of gene expression and their deregulation in tumor cells play pivotal roles in cancer progression., Methods: NanoString's nCounter technology was utilized to measure the expression of 827 cancer-related miRNAs in tumor tissue and adjacent non-involved normal colon tissue from five patients with locoregional CRC progression. These expression profiles were then compared to those from the primary colon adenocarcinoma (COAD) cohort in The Cancer Genome Atlas (TCGA)., Results and Discussion: Intriguingly, 156 miRNAs showed a contrasting dysregulation pattern in reccurent tumor compared to their expression in the TCGA COAD cohort. This observation implies dynamic alterations in miRNA expression patterns throughout disease progression. Our exploratory study contributes to understanding the regulatory landscape of recurrent CRC, emphasizing the role of miRNAs in disease relapse. Notable findings include the prominence of let-7 miRNA family, dysregulation of key target genes, and dynamic changes in miRNA expression patterns during progression. Univariate Cox proportional hazard models highlighted miRNAs associated with adverse outcomes and potential protective factors. The study underscores the need for more extensive investigations into miRNA dynamics during tumor progression and the value of stage specific biomarkers for prognosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Meyer, Kotnik, Noubissi Nzeteu, van Kempen, Mastik, Bockhorn and Troja.)

Background: Predictive biomarker testing has a key role in the treatment decision-making for patients with non-small cell lung cancer (NSCLC) and is mandated by (inter)national guidelines. The aim of this study was to establish guideline-adherent biomarker testing rates in the Netherlands in 2019 and to examine associations of demographical, clinical, and environmental factors with guideline-adherent testing., Methods: This study involved the integration of clinical data of the Netherlands Cancer Registry with pathology reports of the Dutch Nationwide Pathology Databank. Data extracted from these reports included sample type, diagnosis, and molecular testing status of predictive biomarkers. The study population comprised all patients diagnosed with metastatic non-squamous NSCLC in the Netherlands in 2019., Results: In the cohort of 3877 patients with metastatic non-squamous NSCLC under investigation, overall molecular testing rates for non-fusion predictive biomarkers (EGFR, KRAS, BRAF, ERBB2, MET) ranged from 73.9 to 89.0 %, while molecular testing for fusion-drivers (ALK, ROS1, RET, NTRK) ranged from 12.6 % to 63.9 %. Guideline-adherent testing of EGFR, KRAS, and ALK was performed in 85.2 % of patients, with regional rates spanning from 76.0 % to 90.8 %. Demographical and clinical factors associated with guideline-adherent biomarker testing included lower age (OR = 1.05 per one year decrease; p < 0.001), female sex (OR = 1.36; p = 0.002), diagnosis of adenocarcinoma (OR = 2.48; p < 0.001), availability of histological tumor material (OR = 2.46; p < 0.001), and clinical stage of metastatic disease (p = 0.002). Other factors associated with guideline-adherent biomarker testing included diagnosis at academic center (OR = 1.87; p = 0.002) and patient's region of residence (p < 0∙001)., Conclusion: Optimization of the chain-of-care of predictive biomarker testing in patients with NSCLC in the Netherlands is needed to provide adequate care for these patients., Competing Interests: Declaration of Competing Interest WT has received consulting fees from Merck Sharp and Dohme, Bristol-Myers Squibb, and Altana (fees to institution), is board member of Dutch Society of Pathology and member of Council for Research and Innovation of the Federation of Medical Specialists (FMS). LvK has received institutional grants or contract from Amgen, AstraZeneca, Bayer, Janssen-Cilag, Merck, Roche, and Servier, has received institutional payments or honoraria from Amgen, AstraZeneca, Bayer, Bristol-Myers Squibb, Eli Lilly, Novartis, Pfizer, and Roche, has received institutional support for attending meeting and/or travel from Roche, has participated on a Data Safety Monitoring Board or Advisory Board from Janssen-Cilag, Merck, and Roche, has a leadership or fiduciary role in the Commission Personalized Medicine – Belgium (unpaid), has stock or stock options in Cyclomics (institutional/personal). AvdW has received grants or contracts from AstraZeneca, Boehringer-Ingelheim, Pfizer, Roche, and Takeda, has received consulting fees from AstraZeneca, Janssen, Lilly, Roche, and Takeda, has received payments or honoraria from AstraZeneca, BMS, Lilly, Pfizer, and Roche, has a leadership or fiduciary role in the oncology section NVALT, guideline committee NSCLC and CUP, dure geneesmiddelen committee NVALT and FMS. ES has received unrestricted grants (all paid to UMCG institution) from Abbott, Biocartis, AstraZeneca, Invitae/Archer, Bayer, Bio-Rad, Roche, Agena Bioscience, CC Diagnostics, MSD/MERCK, and Boehringer Ingelheim, has received consulting fees (all paid to UMCG institution) from MSD/Merck, AstraZeneca, Roche, Novartis, Bayer, BMS, Lilly, Amgen, Illumina, Agena Bioscience, CC Diagnostics, Janssen Cilag (Johnson & Johnson), Astellas Pharma, GSK, Sinnovisionlab, and Sysmex, has received payments or honoraria (all paid to UMCG institution) from Bio-Rad, Seracare, Roche, Biocartis, Lilly, Agena Bioscience, and Illumina, has received support for attending meetings and/or travel from BioRad, Biocartis, Ageno Sciences, and Illumina, is a board member for the Dutch Society of Pathology (unpaid), European Society of Pathology (unpaid), European Liquid Biopsy Society (unpaid), is a secretary/member of the advisory committee for assessment of molecular diagnostics (cieBOD) (honoraria paid to UMCG institution), is committee member of national guideline advisory (honoraria paid to UMCG institution). SW received unrestricted research grants and speaker’s fees from Bayer, Roche, Astra Zeneca, Pfizer, MSD, BMS, Novartis, Lilly, Amgen, Logex, all paid to the UMCG. He is chair of Palga foundation and vice chair of the scientific council from the Dutch Cancer Society (unpaid). All remaining authors have declared no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: Vaginal dryness is an important factor influencing sexual function in women with primary Sjögren syndrome (pSS). Previous studies showed a higher degree of inflammation in vaginal biopsies from patients with pSS compared to non-pSS controls. However, the molecular pathways that drive this inflammation remain unclear. Therefore, the aim of this study was to investigate inflammatory pathway activity in the vaginal tissue of patients with pSS., Methods: Vaginal biopsies of 8 premenopausal patients with pSS experiencing vaginal dryness and 7 age-matched non-pSS controls were included. Expression of genes involved in inflammation and tissue homeostasis was measured using NanoString technology and validated using TaqMan Real-Time PCR. Vaginal tissue sections were stained by immunohistochemistry for myxovirus resistance protein 1 (MxA) and CD123 (plasmacytoid dendritic cells [pDCs])., Results: The most enriched pathway in vaginal biopsies from patients with pSS compared to non-pSS controls was the interferon (IFN) signaling pathway ( P < 0.01). Pathway scores for Janus kinase and signal transducer and activator of transcription (JAK-STAT) and Notch signaling were also higher ( P < 0.01 for both pathways). Conversely, transforming growth factor-β signaling and angiogenesis pathway scores were lower in pSS ( P = 0.02 and P = 0.04, respectively). Differences in IFN signaling between patients with pSS and non-pSS controls were confirmed by PCR and MxA tissue staining. No CD123+ pDCs were detected in vaginal biopsies. IFN-stimulated gene expression levels correlated positively with CD45+ cell numbers in vaginal biopsies and serum anti-SSA/Ro positivity., Conclusion: Upregulation of IFN signaling in vaginal tissue of women with pSS, along with its association with tissue pathology, suggests that IFNs contribute to inflammation of the vaginal wall and potentially also to clinical symptomatology (ie, vaginal dryness)., (Copyright © 2024 by the Journal of Rheumatology.)

Background: Identification of tumor-derived variants in circulating tumor DNA (ctDNA) has potential as a sensitive and reliable surrogate for tumor tissue-based routine diagnostic testing. However, variations in pre(analytical) procedures affect the efficiency of ctDNA recovery. Here, an external quality assessment (EQA) was performed to determine the performance of ctDNA mutation detection work flows that are used in current diagnostic settings across laboratories within the Dutch COIN consortium (ctDNA on the road to implementation in The Netherlands)., Methods: Aliquots of 3 high-volume diagnostic leukapheresis (DLA) plasma samples and 3 artificial reference plasma samples with predetermined mutations were distributed among 16 Dutch laboratories. Participating laboratories were requested to perform ctDNA analysis for BRAF exon 15, EGFR exon 18-21, and KRAS exon 2-3 using their regular circulating cell-free DNA (ccfDNA) analysis work flow. Laboratories were assessed based on adherence to the study protocol, overall detection rate, and overall genotyping performance., Results: A broad range of preanalytical conditions (e.g., plasma volume, elution volume, and extraction methods) and analytical methodologies (e.g., droplet digital PCR [ddPCR], small-panel PCR assays, and next-generation sequencing [NGS]) were used. Six laboratories (38%) had a performance score of >0.90; all other laboratories scored between 0.26 and 0.80. Although 13 laboratories (81%) reached a 100% overall detection rate, the therapeutically relevant EGFR p.(S752&#95;I759del) (69%), EGFR p.(N771&#95;H773dup) (50%), and KRAS p.(G12C) (48%) mutations were frequently not genotyped accurately., Conclusions: Divergent (pre)analytical protocols could lead to discrepant clinical outcomes when using the same plasma samples. Standardization of (pre)analytical work flows can facilitate the implementation of reproducible liquid biopsy testing in the clinical routine., (© Association for Diagnostics & Laboratory Medicine 2024.)

Chromosomal rearrangements are important drivers in cancer, and their robust detection is essential for diagnosis, prognosis, and treatment selection, particularly for bone and soft tissue tumors. Current diagnostic methods are hindered by limitations, including difficulties with multiplexing targets and poor quality of RNA. A novel targeted DNA-based next-generation sequencing method, formalin-fixed, paraffin-embedded-targeted locus capture (FFPE-TLC), has shown advantages over current diagnostic methods when applied on FFPE lymphomas, including the ability to detect novel rearrangements. We evaluated the utility of FFPE-TLC in bone and soft tissue tumor diagnostics. FFPE-TLC sequencing was successfully applied on noncalcified and decalcified FFPE samples (n = 44) and control samples (n = 19). In total, 58 rearrangements were identified in 40 FFPE tumor samples, including three previously negative samples, and none was identified in the FFPE control samples. In all five discordant cases, FFPE-TLC could identify gene fusions where other methods had failed due to either detection limits or poor sample quality. FFPE-TLC achieved a high specificity and sensitivity (no false positives and negatives). These results indicate that FFPE-TLC is applicable in cancer diagnostics to simultaneously analyze many genes for their involvement in gene fusions. Similar to the observation in lymphomas, FFPE-TLC is a good DNA-based alternative to the conventional methods for detection of rearrangements in bone and soft tissue tumors., (Copyright © 2023 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Purpose: Dedifferentiated melanoma (DedM) poses significant diagnostic challenges. We aimed to investigate the clinical, histopathological and molecular features of DedM. Methylation signature (MS) and copy number profiling (CNP) were carried out in a subgroup of cases., Patients and Methods: A retrospective series of 78 DedM tissue samples from 61 patients retrieved from EORTC (European Organisation for Research and Treatment of Cancer) Melanoma Group centres were centrally reviewed. Clinical and histopathological features were retrieved. In a subgroup of patients, genotyping through Infinium Methylation microarray and CNP analysis was carried out., Results: Most patients (60/61) had a metastatic DedM showing most frequently an unclassified pleomorphic, spindle cell, or small round cell morphology akin to undifferentiated soft tissue sarcoma, rarely associated with heterologous elements. Overall, among 20 successfully analysed tissue samples from 16 patients, we found retained melanoma-like MS in only 7 tissue samples while a non-melanoma-like MS was observed in 13 tissue samples. In two patients from whom multiple specimens were analysed, some of the samples had a preserved cutaneous melanoma MS while other specimens exhibited an epigenetic shift towards a mesenchymal/sarcoma-like profile, matching the histological features. In these two patients, CNP was largely identical across all analysed specimens, in line with their common clonal origin, despite significant modification of their epigenome., Conclusions: Our study further highlights that DedM represents a real diagnostic challenge. While MS and genomic CNP may help pathologists to diagnose DedM, we provide proof-of-concept that dedifferentiation in melanoma is frequently associated with epigenetic modifications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Cribriform adenocarcinoma of salivary gland (CASG) is a rare, salivary gland tumor. In this report, we describe a case of CASG harboring a novel PPP2R2A::PRKD1 fusion. A 58-year-old female presented with an intraoral mass adjacent to the lower left third molar region. Morphological features at histological examination, immunohistochemical staining (p63+, p40-), and tumor location were indicative of CASG. However, due to the potential focal presence of a biphasic component within the tumor, RNA sequencing was performed to confirm the diagnosis. The subsequently found novel PPP2R2A::PRKD1 fusion adds to the rapidly evolving molecular landscape of salivary gland tumors. Additionally, we report that CASG may show some entrapment of pre-existent salivary gland ducts, which may be misinterpreted as tumor cells with myoepithelial differentiation., (© 2023 The Authors. Genes, Chromosomes and Cancer published by Wiley Periodicals LLC.)

Circulating tumor DNA (ctDNA) is a promising new biomarker with multiple potential applications in cancer care. Estimating total cost of ctDNA testing is necessary for reimbursement and implementation, but challenging because of variations in workflow. We aimed to develop a micro-costing framework for consistent cost calculation of ctDNA testing. First, the foundation of the framework was built, based on the complete step-wise diagnostic workflow of ctDNA testing. Second, the costing method was set up, including costs for personnel, materials, equipment, overhead, and failures. Third, the framework was evaluated by experts and applied to six case studies, including PCR-, mass spectrometry-, and next-generation sequencing-based platforms, from three Dutch hospitals. The developed ctDNA micro-costing framework includes the diagnostic workflow from blood sample collection to diagnostic test result. The framework was developed from a Dutch perspective and takes testing volume into account. An open access tool is provided to allow for laboratory-specific calculations to explore the total costs of ctDNA testing specific workflow parameters matching the setting of interest. It also allows to straightforwardly assess the impact of alternative prices or assumptions on the cost per sample by simply varying the input parameters. The case studies showed a wide range of costs, from €168 to €7638 ($199 to $9124) per sample, and generated information. These costs are sensitive to the (coverage of) platform, setting, and testing volume., (Copyright © 2023 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Targeted therapy in lung cancer requires the assessment of multiple oncogenic driver alterations, including fusion genes. This retrospective study evaluated the Idylla GeneFusion prototype, an automated and ease-of-use (<2 minutes) test, with a short turnaround time (3 hours) to detect fusions involving ALK, ROS1, RET, and NTRK1/2/3 genes and MET exon 14 skipping. This multicenter study (18 centers) included 313 tissue samples from lung cancer patients with 97 ALK, 44 ROS1, 20 RET, and 5 NTRKs fusions, 32 MET exon 14 skipping, and 115 wild-type samples, previously identified with reference methods (RNA-based next-generation sequencing/fluorescence in situ hybridization/quantitative PCR). Valid results were obtained for 306 cases (98%), overall concordance between Idylla and the reference methods was 89% (273/306); overall sensitivity and specificity were 85% (165/193) and 96% (108/113), respectively. Discordances were observed in 28 samples, where Idylla did not detect the alteration identified by the reference methods; and 5 samples where Idylla identified an alteration not detected by the reference methods. All of the ALK-, ROS1-, and RET-specific fusions and MET exon 14 skipping identified by Idylla GeneFusion were confirmed by reference method. To conclude, Idylla GeneFusion is a clinically valuable test that does not require a specific infrastructure, allowing a rapid result. The absence of alteration or the detection of expression imbalance only requires additional testing by orthogonal methods., (Copyright © 2022 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Efficient recovery of circulating tumor DNA (ctDNA) depends on the quantity and quality of circulating cell-free DNA (ccfDNA). Here, we evaluated whether various ccfDNA extraction methods routinely applied in Dutch laboratories affect ccfDNA yield, ccfDNA integrity, and mutant ctDNA detection, using identical lung cancer patient-derived plasma samples., Methods: Aliquots of 4 high-volume diagnostic leukapheresis plasma samples and one artificial reference plasma sample with predetermined tumor-derived mutations were distributed among 14 Dutch laboratories. Extractions of ccfDNA were performed according to local routine standard operating procedures and were analyzed at a central reference laboratory for mutant detection and assessment of ccfDNA quantity and integrity., Results: Mutant molecule levels in extracted ccfDNA samples varied considerably between laboratories, but there was no indication of consistent above or below average performance. Compared to silica membrane-based methods, samples extracted with magnetic beads-based kits revealed an overall lower total ccfDNA yield (-29%; P < 0.0001) and recovered fewer mutant molecules (-41%; P < 0.01). The variant allelic frequency and sample integrity were similar. In samples with a higher-than-average total ccfDNA yield, an augmented recovery of mutant molecules was observed., Conclusions: In the Netherlands, we encountered diversity in preanalytical workflows with potential consequences on mutant ctDNA detection in clinical practice. Silica membrane-based methodologies resulted in the highest total ccfDNA yield and are therefore preferred to detect low copy numbers of relevant mutations. Harmonization of the extraction workflow for accurate quantification and sensitive detection is required to prevent introduction of technical divergence in the preanalytical phase and reduce interlaboratory discrepancies., (© American Association for Clinical Chemistry 2022.)

Objectives: The recent accelerated FDA approval of sotorasib, a highly selective KRAS G12C inhibitor, offers new opportunities for the treatment of KRAS p.(G12C)-mutated non-squamous non-small cell lung cancer (NSCLC). The objective of the current study was to the determine the prevalence of KRAS mutations in stage IV non-squamous NSCLC in The Netherlands to reveal the potential impact of upcoming KRAS targeted therapy., Materials and Methods: All patients diagnosed with stage IV non-squamous NSCLC in 2013, 2015 and 2017 in the Netherlands were selected by linking the nation-wide Netherlands Cancer Registry (NCR) and the Dutch Pathology Registry (PALGA). Demographic and pathological variables were retrieved from the pathology reports including sex, age, KRAS mutation status, molecular test method used, and the mutation status of other genes., Results: Prevalence for any KRAS mutations in codon 12/13/61/146 was 39.1%. KRAS p.(G12C) was detected in 15.5% of all non-squamous NSCLC cases representing 39.6% of all KRAS-mutant cases. National testing rate for KRAS mutations increased from 70% in 2013 to 82% in 2017. Testing techniques changed significantly over time with next generation sequencing as the main used method in 2017 (71.6%) but did not affect prevalence of KRAS mutations over time. When KRAS was tested as part of a larger panel, the KRAS p.(G12C) mutation was frequently reported with a concurrent mutation in TP53 (47.7%) or STK11 (10.3%)., Conclusion: The high prevalence for KRAS p.(G12C) offers a promising new specific treatment option for 15% of all stage IV non-squamous NSCLC patients., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Oral potentially malignant disorders (OPMD) may precede oral squamous cell carcinoma (OSCC). Reported rates of malignant transformation of OPMD range from 3 to 50%. While some clinical, histological, and molecular factors have been associated with a high-risk OPMD, they are, to date, insufficiently accurate for treatment decision-making. Moreover, this range highlights differences in the clinical definition of OPMD, variation in follow-up periods, and molecular and biological heterogeneity of OPMD. Finally, while treatment of OPMD may improve outcome, standard therapy has been shown to be ineffective to prevent OSCC development in patients with OPMD. In this perspective paper, several experts discuss the main challenges in oral cancer prevention, in particular the need to (i) to define an OPMD classification system by integrating new pathological and molecular characteristics, aiming (ii) to better identify OPMD at high risk of malignant transformation, and (iii) to develop treatment strategies to eradicate OPMD or prevent malignant transformation.

Background and Aims: Nonanastomotic biliary strictures (NAS) are a major cause of morbidity after orthotopic liver transplantation (OLT). Although ischemic injury of peribiliary glands (PBGs) and peribiliary vascular plexus during OLT has been associated with the later development of NAS, the exact underlying mechanisms remain unclear. We hypothesized that bile ducts of patients with NAS suffer from ongoing biliary hypoxia and lack of regeneration from PBG stem/progenitor cells., Approach and Results: Forty-two patients, requiring retransplantation for either NAS (n = 18), hepatic artery thrombosis (HAT; n = 13), or nonbiliary graft failure (controls; n = 11), were included in this study. Histomorphological analysis of perihilar bile ducts was performed to assess differences in markers of cell proliferation and differentiation in PBGs, microvascular density (MVD), and hypoxia. In addition, isolated human biliary tree stem cells (hBTSCs) were used to examine exo-metabolomics during in vitro differentiation toward mature cholangiocytes. Bile ducts of patients with NAS or HAT had significantly reduced indices of PBG mass, cellular proliferation and differentiation (mucus production, secretin receptor expression, and primary cilia), reduced MVD, and increased PBG apoptosis and hypoxia marker expression, compared to controls. Metabolomics of hBTSCs during in vitro differentiation toward cholangiocytes revealed a switch from a glycolytic to oxidative metabolism, indicating the need for oxygen., Conclusions: NAS are characterized by a microscopic phenotype of chronic biliary hypoxia attributed to loss of microvasculature, resulting in reduced proliferation and differentiation of PBG stem/progenitor cells into mature cholangiocytes. These findings suggest that persistent biliary hypoxia is a key mechanism underlying the development of NAS after OLT., (© 2021 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Gene fusions involving NTRK1 , NTRK2, and NTRK3 are rare drivers of cancer that can be targeted with histology-agnostic inhibitors. This study aimed to determine the nationwide landscape of NTRK /TRK testing in the Netherlands and the usage of pan-TRK immunohistochemistry (IHC) as a preselection tool to detect NTRK fusions. All pathology reports in 2017-2020 containing the search term 'TRK' were retrieved from the Dutch Pathology Registry (PALGA). Patient characteristics, tumor histology, NTRK /TRK testing methods, and reported results were extracted. NTRK /TRK testing was reported for 7457 tumors. Absolute testing rates increased from 815 (2017) to 3380 (2020). Tumors were tested with DNA/RNA-based molecular assay(s) (48%), IHC (47%), or in combination (5%). A total of 69 fusions involving NTRK1 ( n = 22), NTRK2 ( n = 6) and NTRK3 ( n = 41) were identified in tumors from adult ( n = 51) and pediatric ( n = 18) patients. In patients tested with both IHC and a molecular assay ( n = 327, of which 29 NTRK fusion-positive), pan-TRK IHC had a sensitivity of 77% (95% confidence interval (CI), 56-91) and a specificity of 84% (95% CI, 78-88%). These results showed that pan-TRK IHC has a low sensitivity in current routine practice and warrants the introduction of quality guidelines regarding the implementation and interpretation of pan-TRK IHC.

Introduction: Non-small cell lung cancer (NSCLC) patients with Anaplastic Lymphoma Kinase (ALK) gene fusions respond well to ALK inhibitors but commonly develop on-target resistance mutations. The objective of this study is to collect clinical evidence for subsequent treatment with ALK inhibitors., Patients and Methods: Local experience with on-target ALK resistance mutations and review of the literature identified 387 patients with ALK inhibitor resistance mutations. Clinical benefit of mutation-inhibitor combinations was assessed based on reported response, progression-free survival and duration of treatment. Furthermore, this clinical evidence was compared to previously reported in vitro sensitivity of mutations to the inhibitors., Results: Of the pooled population of 387 patients in this analysis, 239 (62%) received at least 1 additional line of ALK inhibition after developing on-target resistance to ALK inhibitor therapy. Clinical benefit was reported for 177 (68%) patients, but differed for each mutation-inhibitor combination. Agreement between in vitro predicted sensitivity of 6 published models and observed clinical benefit ranged from 69% to 89%. The observed clinical evidence for highest probability of response in the context of specific on-target ALK inhibitor resistance mutations is presented., Conclusion: Molecular diagnostics performed on tissue samples that are refractive to ALK inhibitor therapy can reveal new options for targeted therapy for NSCLC patients. Our comprehensive overview of clinical evidence of drug actionability of ALK on-target resistance mechanisms may serve as a practical guide to select the most optimal drug for individual patients., (Copyright © 2021. Published by Elsevier Inc.)

In classical Hodgkin lymphoma (cHL), the highly abundant CD4+ T cells in the vicinity of tumor cells are considered essential for tumor cell survival, but are ill-defined. Although they are activated, they consistently lack expression of activation marker CD26. In this study, we compared sorted CD4+CD26- and CD4+CD26+ T cells from cHL lymph node cell suspensions by RNA sequencing and T cell receptor variable gene segment usage analysis. This revealed that although CD4+CD26- T cells are antigen experienced, they have not clonally expanded. This may well be explained by the expression of exhaustion associated transcription factors TOX and TOX2 , immune checkpoints PDCD1 and CD200 , and chemokine CXCL13 , which were amongst the 100 significantly enriched genes in comparison with the CD4+CD26+ T cells. Findings were validated in single-cell RNA sequencing data from an independent cohort. Interestingly, immunohistochemistry revealed predominant and high frequency of staining for TOX and TOX2 in the T cells attached to the tumor cells. In conclusion, the dominant CD4+CD26- T cell population in cHL is antigen experienced, polyclonal, and exhausted. This population is likely a main contributor to the very high response rates to immune checkpoint inhibitors in cHL., Competing Interests: A.D. receives research funding from Millenium/Takeda. C.S. is an advisory board member for Curis Inc., AbbVie, Seattle Genetics, and Roche, reports receiving commercial research grants from Bristol-Myers Squibb and Trillium Therapeutics, and remuneration from Bayer and Juno Therapeutics. No potential conflicts of interest were disclosed by the other authors., (© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Immunotherapy for metastasized non-small-cell lung cancer (NSCLC) can show long-lasting clinical responses. Selection of patients based on programmed death-ligand 1 (PD-L1) expression shows limited predictive value for durable clinical benefit (DCB). We investigated whether early treatment effects as measured by a change in circulating tumor DNA (ctDNA) level is a proxy of early tumor response to immunotherapy according to response evaluation criteria in solid tumors v1.1 criteria, progression-free survival (PFS), DCB, and overall survival (OS). To this aim, blood tubes were collected from advanced-stage lung adenocarcinoma patients (n = 100) receiving immune checkpoint inhibitors (ICI) at baseline (t 0 ) and prior to first treatment evaluation (4-6 weeks; t 1 ). Nontargetable (driver) mutations detected in the pretreatment tumor biopsy were used to quantify tumor-specific ctDNA levels using droplet digital PCR. We found that changes in ctDNA levels were strongly associated with tumor response. A > 30% decrease in ctDNA at t 1 correlated with a longer PFS and OS. In total, 80% of patients with a DCB of ≥ 26 weeks displayed a > 30% decrease in ctDNA levels. For patients with a PD-L1 tumor proportion score of ≥ 1%, decreasing ctDNA levels were associated with a higher frequency a DCB (80%) and a prolonged median PFS (85 weeks) and OS (101 weeks) compared with patients with no decrease in ctDNA (34%; 11 and 39 weeks, respectively). This study shows that monitoring of ctDNA dynamics is an easy-to-use and promising tool for assessing PFS, DCB, and OS for ICI-treated NSCLC patients., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

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., (© 2020 The Authors. The Oncologist published by Wiley Periodicals LLC on behalf of AlphaMed Press.)

EGFR mutation analysis in non-small-cell lung cancer (NSCLC) patients is currently standard-of-care. We determined the uptake of EGFR testing, test results and survival of EGFR -mutant NSCLC patients in the Netherlands, with the overall objective to characterize the landscape of clinically actionable EGFR mutations and determine the role and clinical relevance of uncommon and composite EGFR mutations. Non-squamous NSCLC patients diagnosed in 2013, 2015 and 2017 were identified in the Netherlands Cancer Registry (NCR) and matched to the Dutch Pathology Registry (PALGA). Overall, 10,254 patients were included. Between 2013-2017, the uptake of EGFR testing gradually increased from 72.7% to 80.9% ( p < 0.001). Multi-gene testing via next-generation sequencing (increased from 7.8% to 78.7% ( p < 0.001), but did not affect the number of detected EGFR mutations ( n = 925; 11.7%; 95% confidence interval (CI), 11.0-12.4) nor the distribution of variants. For patients treated with first-line EGFR inhibitors ( n = 651), exon 19 deletions were associated with longer OS than L858R (HR 1.58; 95% CI, 1.30-1.92; p < 0.001) or uncommon, actionable variants (HR 2.13; 95% CI, 1.60-2.84; p < 0.001). Interestingly, OS for patients with L858R was similar to those with uncommon, actionable variants (HR 1.31; 95% CI, 0.98-1.75; p = 0.069). Our analysis indicates that grouping exon 19 deletions and L858R into one class of 'common' EGFR mutations in a clinical trial may mask the true activity of an EGFR inhibitor towards specific mutations.

The recent WHO classification of skin tumors has underscored the importance of acknowledging intermediate grade melanocytic proliferations. A multistep acquisition of oncogenic events drives the progressive transformation of nevi into melanomas. The various pathways described are modulated by the initial oncogenic drivers that define the common, blue, and Spitz nevi groups. Intermediate lesions are most often the result of a clonal evolution within such nevi. Based on this established classification, we have suggested for each pathway a practical diagnostic approach, benefiting from the recently developed molecular tools, both in the setting of general pathology labs and expert centers. Moreover, recommendations regarding the re-excision and clinical follow-up are given to support decision-making in multidisciplinary tumor boards., (© 2021. Crown.)

In routine diagnostic pathology, cancer biopsies are preserved by formalin-fixed, paraffin-embedding (FFPE) procedures for examination of (intra-) cellular morphology. Such procedures inadvertently induce DNA fragmentation, which compromises sequencing-based analyses of chromosomal rearrangements. Yet, rearrangements drive many types of hematolymphoid malignancies and solid tumors, and their manifestation is instructive for diagnosis, prognosis, and treatment. Here, we present FFPE-targeted locus capture (FFPE-TLC) for targeted sequencing of proximity-ligation products formed in FFPE tissue blocks, and PLIER, a computational framework that allows automated identification and characterization of rearrangements involving selected, clinically relevant, loci. FFPE-TLC, blindly applied to 149 lymphoma and control FFPE samples, identifies the known and previously uncharacterized rearrangement partners. It outperforms fluorescence in situ hybridization (FISH) in sensitivity and specificity, and shows clear advantages over standard capture-NGS methods, finding rearrangements involving repetitive sequences which they typically miss. FFPE-TLC is therefore a powerful clinical diagnostics tool for accurate targeted rearrangement detection in FFPE specimens.

Early-life adversity (ELA) is a major predictor of psychopathology, and is thought to increase lifetime risk by epigenetically regulating the genome. Here, focusing on the lateral amygdala, a major brain site for emotional homeostasis, we describe molecular cross-talk among multiple mechanisms of genomic regulation, including 6 histone marks and DNA methylation, and the transcriptome, in subjects with a history of ELA and controls. In the healthy brain tissue, we first uncover interactions between different histone marks and non-CG methylation in the CAC context. Additionally, we find that ELA associates with methylomic changes that are as frequent in the CAC as in the canonical CG context, while these two forms of plasticity occur in sharply distinct genomic regions, features, and chromatin states. Combining these multiple data indicates that immune-related and small GTPase signaling pathways are most consistently impaired in the amygdala of ELA individuals. Overall, this work provides insights into genomic brain regulation as a function of early-life experience.

Background: The purpose of this study was to build radiogenomics models from texture signatures derived from computed tomography (CT) and 18 F-FDG PET-CT (FDG PET-CT) images of non-small cell lung cancer (NSCLC) with and without epidermal growth factor receptor ( EGFR ) mutations., Methods: Fifty patients diagnosed with NSCLC between 2011 and 2015 and with known EGFR mutation status were retrospectively identified. Texture features extracted from pretreatment CT and FDG PET-CT images by manual contouring of the primary tumor were used to develop multivariate logistic regression (LR) models to predict EGFR mutations in exon 19 and exon 20., Results: An LR model evaluating FDG PET-texture features was able to differentiate EGFR mutant from wild type with an area under the curve (AUC), sensitivity, specificity, and accuracy of 0.87, 0.76, 0.66, and 0.71, respectively. The model derived from CT texture features had an AUC, sensitivity, specificity, and accuracy of 0.83, 0.84, 0.73, and 0.78, respectively. FDG PET-texture features that could discriminate between mutations in EGFR exon 19 and 21 demonstrated AUC, sensitivity, specificity, and accuracy of 0.86, 0.84, 0.73, and 0.78, respectively. Based on CT texture features, the AUC, sensitivity, specificity, and accuracy were 0.75, 0.81, 0.69, and 0.75, respectively., Conclusion: Non-small cell lung cancer texture analysis using FGD-PET and CT images can identify tumors with mutations in EGFR . Imaging signatures could be valuable for pretreatment assessment and prognosis in precision therapy.

Microabstract: Integration of Next Generation Sequencing (NGS) information for use in distinguishing between Multiple Primary Lung Cancer and intrapulmonary metastasis was evaluated. We used a probabilistic model, comprehensive histologic assessment and NGS to classify patients. Integrating NGS data confirmed initial diagnosis (n = 41), revised the diagnosis (n = 12), while resulted in non-informative data (n = 8). Accuracy of diagnosis can be significantly improved with integration of NGS data., Background: Distinguishing between multiple primary lung cancers (MPLC) and intrapulmonary metastases (IPM) is challenging. The goal of this study was to evaluate how Next Generation Sequencing (NGS) information may be integrated in the diagnostic strategy., Patients and Methods: Patients with multiple lung adenocarcinomas were classified using both the comprehensive histologic assessment and NGS. We computed the joint probability of each pair having independent mutations by chance (thus being classified as MPLC). These probabilities were computed using the marginal mutation rates of each mutation, and the known negative dependencies between driver genes and different gene loci. With these NGS-driven data, cases were re-classified as MPLC or IPM., Results: We analyzed 61 patients with a total of 131 tumors. The most frequent mutation was KRAS (57.3%) which occured at a rate higher than expected (p < 0.001) in lung cancer. No mutation was detected in 25/131 tumors (19.1%). Discordant molecular findings between tumor sites were found in 46 patients (75.4%); 11 patients (18.0%) had concordant molecular findings, and 4 patients (6.6%) had concordant molecular findings at 2 of the 3 sites. After integration of the NGS data, the initial diagnosis was confirmed for 41 patients (67.2%), the diagnosis was revised for 12 patients (19.7%) or was considered as non-informative for 8 patients (13.1%)., Conclusion: Integrating the information of NGS data may significantly improve accuracy of diagnosis and staging., (Copyright © 2021. Published by Elsevier Ltd.)

Purpose: Plasma detection of EGFR T790M mutations is an emerging alternative to tumor rebiopsy in acquired epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor resistance. Validation of analytical sensitivity and clinical utility is required before routine diagnostic use in clinical laboratories., Patients and Methods: Sixty-three patients with advanced EGFR -mutant lung cancer at 7 Canadian centers, who were being screened for the ASTRIS trial (ClinicalTrials.gov identifier: NCT02474355), participated in this companion study. Plasma T790M mutation was detected using droplet digital polymerase chain reaction, Cobas (Roche Diagnostics, Indianapolis, IN), or next-generation sequencing in 4 laboratories. T790M concordance was assessed between plasma and tumor samples., Results: Assessment of T790M in tumor biopsy tissue was successful in 81% of patients; 49% had confirmed T790M results (tumor or plasma) for ASTRIS. Plasma testing in this companion study yielded T790M results in 97% of patients; 62% had T790M-positive results, 36% had negative results, and 2% had indeterminate results. Of 38 patients with negative or indeterminate biopsy results, 55% had positive plasma T790M results, increasing the proportion with T790M-positive results to 73%. Sensitivity of plasma T790M testing was 75%. Overall concordance between tissue and plasma was 64%, and concordance among laboratories was 90.3%. Response to osimertinib and duration of therapy were similar irrespective of testing method (overall response rate, 62.5% for tissue, 66.7% for plasma, and 70.6% for both)., Conclusion: This multicenter validation study demonstrates that plasma EGFR T790M testing can identify significantly more patients than biopsy alone who may benefit from targeted therapy.

Purpose: Molecular tumor boards (MTBs) provide physicians with a treatment recommendation for complex tumor-specific genomic alterations. National and international consensus to reach a recommendation is lacking. In this article, we analyze the effectiveness of an MTB decision-making methodology for patients with non-small-cell lung cancer (NSCLC) with rare or complex mutational profiles as implemented in the University Medical Center Groningen (UMCG)., Methods: The UMCG-MTB comprises (pulmonary) oncologists, pathologists, clinical scientists in molecular pathology, and structural biologists. Recommendations are based on reported actionability of variants and molecular interpretation of pathways affected by the variant and supported by molecular modeling. A retrospective analysis of 110 NSCLC cases (representing 106 patients) with suggested treatment of complex genomic alterations and corresponding treatment outcomes for targeted therapy was performed., Results: The MTB recommended targeted therapy for 59 of 110 NSCLC cases with complex molecular profiles: 24 within a clinical trial, 15 in accordance with guidelines (on label) and 20 off label. All but 16 recommendations involved patients with an EGFR or ALK mutation. Treatment outcome was analyzed for patients with available follow-up (10 on label and 16 off label). Adherence to the MTB recommendation (21 of 26; 81%) resulted in an objective response rate of 67% (14 of 21), with a median progression-free survival of 6.3 months (interquartile range, 3.2-10.6 months) and an overall survival of 10.4 months (interquartile range, 6.3-14.6 months)., Conclusion: Targeted therapy recommendations resulting from the UMCG-MTB workflow for complex molecular profiles were highly adhered to and resulted in a positive clinical response in the majority of patients with metastatic NSCLC.

Background: Sentinel lymph node (SLN) biopsy remains crucial for melanoma staging. The European Organisation for Research and Treatment of Cancer Melanoma Group recommends performing immunohistochemical stainings for reproducible identification of melanoma metastases. S100 protein (pS100) is a commonly used melanocytic antigen because of its high sensitivity in spite of relatively low specificity. SRY-related HMG-box 10 protein (SOX10) is a transcription factor characterising neural crest-derived cells. It is uniformly expressed mostly in the nuclei of melanocytes, neural, and myoepithelial cells. Pathologists sometimes prefer SOX10 as a melanoma marker, but it has not yet been investigated on a large-scale to confirm that it is reliable and recommendable for routine SLN evaluation., Methods: Four hundred one treatment-naïve lymph node (LN) metastatic melanomas were included in high-density tissue microarrays and were assessed for the presence of SOX10 and pS100 by immunohistochemistry. The slides were digitalised, shared and evaluated by a panel of experienced melanoma pathologists., Results: The vast majority of melanomas were double-positive for pS100 and SOX10 (93.2%); a small percentage of the cases (3.9%) were double-negative melanomas. Discordance between the two markers was observed: 1.9% pS100(-)/SOX10(+) and 0.75% pS100(+)/SOX10(-). SOX10 was not expressed by immune cell types in the LN, resulting in a less controversial interpretation of the staining., Conclusions: SOX10 is as equally specific as pS100 for the detection of melanoma metastases in LNs. The interpretation of SOX10 staining is highly reproducible among different centres and different pathologists because of the absence of staining of immune cells., Competing Interests: Conflict of interest statement None declared., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Purpose: To identify computed tomography (CT) features of epidermal growth factor receptor (EGFR) mutation-positive lung adenocarcinoma in Canadian population and whether imaging-based surrogate markers of EGFR mutation in our population were similar to those found in the Asian population., Materials and Methods: Pretreatment CT scans of 223 patients with adenocarcinoma of the lung (112 with EGFR mutation and 111 without mutation) were retrospectively assessed for 20 specific CT features by 2 radiologists, who were blinded to the status of EGFR mutation. Univariate and multivariate logistic regression analyses as well as areas under the receiver operating characteristic curve were performed to discriminate characteristics of EGFR-activating mutation features., Results: Epidermal growth factor receptor mutation-positive adenocarcinomas were more frequently found in female ( P < .03), less than 20 pack-year smoking history ( P < .001), smaller tumor ( P < .01), spiculated margins ( P < .05), without centrilobular emphysema ( P < .001), and without lymphadenopathy ( P < .05), similarly to the Asian population. Multivariate logistic regression analyses of combined clinical and radiological features identified less than 20 pack-year smoking history, smaller tumor diameter, fine or coarse spiculations, noncentral location of the tumor, and lack of centrilobular emphysema and pleural attachment as the strongest independent prognostic factors for the presence of an EGFR mutation. These combined features improved prognostic ability area under the curve to 0.879, compared to 0.788 for clinical features only., Conclusion: Several CT findings may help predict the presence of an activating mutation in EGFR in lung adenocarcinomas in our Canadian population. Combining clinical and radiological features improves prognostic ability to determine the EGFR mutation status compared to clinical features alone.

Diagnostic histopathology of soft tissue tumors can be troublesome as many entities are quite rare and have overlapping morphologic features. Many soft tissue tumors harbor tumor-defining gene translocations, which may provide an important ancillary tool for tumor diagnosis. The NanoString nCounter platform enables multiplex detection of pre-defined gene fusion transcripts in formalin-fixed and paraffin-embedded tissue. A cohort of 104 soft tissue tumors representing 20 different histological types was analyzed for the expression of 174 unique gene fusion transcripts. A tumor-defining gene fusion transcript was detected in 60 cases (58%). Sensitivity and specificity of the NanoString assay calculated against the result of an alternative molecular method were 85% and 100%, respectively. Highest diagnostic coverage was obtained for Ewing sarcoma, synovial sarcoma, myxoid liposarcoma, alveolar rhabdomyosarcoma, and desmoplastic small round cell tumor. For these tumor types, the NanoString assay is a rapid, cost-effective, sensitive, and specific ancillary screening tool for molecular diagnosis. For other sarcomas, additional molecular testing may be required when a translocation transcript is not identified with the current 174 gene fusion panel., (© 2020 The Authors. Genes, Chromosomes & Cancer published by Wiley Periodicals, Inc.)

Background: Sensitive and reliable molecular diagnostics is needed to guide therapeutic decisions for cancer patients. Although less material becomes available for testing, genetic markers are rapidly expanding. Simultaneous detection of predictive markers, including mutations, gene amplifications and MSI, will save valuable material, time and costs., Methods: Using a single-molecule molecular inversion probe (smMIP)-based targeted next-generation sequencing (NGS) approach, we developed an NGS panel allowing detection of predictive mutations in 33 genes, gene amplifications of 13 genes and microsatellite instability (MSI) by the evaluation of 55 microsatellite markers. The panel was designed to target all clinically relevant single and multiple nucleotide mutations in routinely available lung cancer, colorectal cancer, melanoma, and gastro-intestinal stromal tumor samples, but is useful for a broader set of tumor types., Results: The smMIP-based NGS panel was successfully validated and cut-off values were established for reliable gene amplification analysis (i.e. relative coverage ≥3) and MSI detection (≥30% unstable loci). After validation, 728 routine diagnostic tumor samples including a broad range of tumor types were sequenced with sufficient sensitivity (2.4% drop-out), including samples with low DNA input (< 10 ng; 88% successful), low tumor purity (5-10%; 77% successful), and cytological material (90% successful). 75% of these tumor samples showed ≥1 (likely) pathogenic mutation, including targetable mutations (e.g. EGFR, BRAF, MET, ERBB2, KIT, PDGFRA). Amplifications were observed in 5.5% of the samples, comprising clinically relevant amplifications (e.g. MET, ERBB2, FGFR1). 1.5% of the tumor samples were classified as MSI-high, including both MSI-prone and non-MSI-prone tumors., Conclusions: We developed a comprehensive workflow for predictive analysis of diagnostic tumor samples. The smMIP-based NGS analysis was shown suitable for limited amounts of histological and cytological material. As smMIP technology allows easy adaptation of panels, this approach can comply with the rapidly expanding molecular markers.

Melanoma is among the most aggressive cancers due to its tendency to metastasize early. Phenotype switching between a proliferative and an invasive state has been suggested as a critical process for metastasis, though the mechanisms that regulate state transitions are complex and remain poorly understood. Brother of Regulator of Imprinted Sites (BORIS), also known as CCCTC binding factor-Like (CTCFL), is a transcriptional modulator that becomes aberrantly expressed in melanoma. Yet, the role of BORIS in melanoma remains elusive. Here, we show that BORIS is involved in melanoma phenotype switching. Genetic modification of BORIS expression in melanoma cells combined with whole-transcriptome analysis indicated that BORIS expression contributes to an invasion-associated transcriptome. In line with these findings, inducible BORIS overexpression in melanoma cells reduced proliferation and increased migration and invasion, demonstrating that the transcriptional switch is accompanied by a phenotypic switch. Mechanistically, we reveal that BORIS binds near the promoter of transforming growth factor-beta 1 ( TFGB1 ), a well-recognized factor involved in the transition towards an invasive state, which coincided with increased expression of TGFB1 . Overall, our study indicates a pro-invasive role for BORIS in melanoma via transcriptional reprogramming., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© The Author(s) 2020.)

Large-scale genomic studies have detailed the molecular landscape of tumors from patients with high-grade serous ovarian cancers (HGSC) who underwent primary debulking surgery and correlated the identified subgroups to survival. In recent years, there is increased use of neoadjuvant chemotherapy (NACT) for patients with HGSC and while abundant data exist for patients who underwent primary debulking, little data are available on the cancer cells remaining after NACT that could lead to recurrences. We aimed to analyze gene expression profiles of NACT-treated HGSC tumor samples, and correlate them to treatment response and outcome. Tumor samples were collected from patients with stage III or IV HGSC (NACT cohort, N = 57) at the time of surgery and diagnosis (biopsy samples N = 8). Tumor content was validated by histologic examination and bioinformatics. Gene expression analysis was performed using a tailored NanoString-based assay, while sequencing was performed using MiSeq. A cross-validated survival classifier revealed patient clusters with either a "Better" or "Worse" prognostic outcome. The association with overall survival remained significant after controlling for clinical variables, and differential gene expression, gene set enrichment analyses, and the appropriate survival models were used to assess the associations between alterations in gene expression in cancer cells remaining after NACT and outcome. Pathway-based analysis of the differentially expressed genes revealed comparatively high levels of cell cycle and DNA repair gene expression in the poor outcome group. IMPLICATIONS: Our work suggests mRNA expression patterns in key genes following NACT may reflect response to treatment and outcome in patient with HGSC., (©2019 American Association for Cancer Research.)

The sentinel lymph node (SLN) biopsy is a highly accurate staging procedure and the most important prognostic factor in melanoma patients. The European Organisation for Research and Treatment of Cancer (EORTC) Melanoma Group aimed to design an updated evolved SLN protocol for the histopathological workup and reporting. We herein recommend extending the distance between steps according to the short axis dimension of the lymph node and optimise both conventional sectioning and staining procedures including immunohistochemistry. We also provide guidance on the description of the spatial localisation of melanoma deposits in a SLN. The histopathological features to be reported include the following: presence or absence of the metastasis, the intranodal location of the metastasis (subcapsular, parenchymal, combined, extensive confluent and extensive multifocal), the number of the metastatic deposits (1, 2-5, 6-10, 11-20 and >20), the maximum dimension of the largest metastasis (indicating its site) and the presence of extracapsular extension and of naevus cells. This updated EORTC protocol is expected to clarify and simplify the existing procedures, ensuring a reasonable workload for the laboratory and for the pathologists resulting in cost saving with no loss, and possible increase, in accuracy., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Next-generation sequencing (NGS) panel analysis on DNA from formalin-fixed paraffin-embedded (FFPE) tissue is increasingly used to also identify actionable copy number gains (gene amplifications) in addition to sequence variants. While guidelines for the reporting of sequence variants are available, guidance with respect to reporting copy number gains from gene-panel NGS data is limited. Here, we report on Dutch consensus recommendations obtained in the context of the national Predictive Analysis for THerapy (PATH) project, which aims to optimize and harmonize routine diagnostics in molecular pathology. We briefly discuss two common approaches to detect gene copy number gains from NGS data, i.e., the relative coverage and B-allele frequencies. In addition, we provide recommendations for reporting gene copy gains for clinical purposes. In addition to general QC metrics associated with NGS in routine diagnostics, it is recommended to include clinically relevant quantitative parameters of copy number gains in the clinical report, such as (i) relative coverage and estimated copy numbers in neoplastic cells, (ii) statistical scores to show significance (e.g., z-scores), and (iii) the sensitivity of the assay and restrictions of NGS-based detection of copy number gains. Collectively, this information can guide clinical and analytical decisions such as the reliable detection of high-level gene amplifications and the requirement for additional in situ assays in case of borderline results or limited sensitivity.

Lung cancer accounts for approximately 14% of all newly diagnosed cancers and is the leading cause of cancer-related deaths. Chimeric RNA resulting from gene fusions (RNA fusions) and other RNA splicing errors are driver events and clinically addressable targets for non-small cell lung cancer (NSCLC). The reliable assessment of these RNA markers by next-generation sequencing requires integrated reagents, protocols, and interpretive software that can harmonize procedures and ensure consistent results across laboratories. We describe the development and verification of a system for targeted RNA sequencing for the analysis of challenging, low-input solid tumor biopsies that includes reagents for nucleic acid quantification and library preparation, run controls, and companion bioinformatics software. Assay development reconciled sequence discrepancies in public databases, created predictive formalin-fixed, paraffin-embedded RNA qualification metrics, and eliminated read misidentification attributable to index hopping events on the next-generation sequencing flow cell. The optimized and standardized system was analytically verified internally and in a multiphase study conducted at five independent laboratories. The results show accurate, reproducible, and sensitive detection of RNA fusions, alternative splicing events, and other expression markers of NSCLC. This comprehensive approach, combining sample quantification, quality control, library preparation, and interpretive bioinformatics software, may accelerate the routine implementation of targeted RNA sequencing of formalin-fixed, paraffin-embedded samples relevant to NSCLC., (Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Current genomic models in diffuse large B-cell lymphoma (DLBCL) are based on single tumor biopsies, which might underestimate heterogeneity. Data on mutational evolution largely remains unknown. An exploratory study using whole exome sequencing on paired (primary and relapse) formalin fixed paraffin embedded DLBCL biopsies ( n = 14) of 6 patients was performed to globally assess the mutational evolution and to identify gene mutations specific for relapse samples from patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone. A minority of the mutations detected in the primary sample (median 7.6%, range 4.8⁻66.2%) could not be detected in the matching relapse sample. Relapsed DLBCL samples showed a mild increase of mutations (median 12.5%, range 9.4⁻87.6%) as compared to primary tumor biopsies. We identified 264 genes possibly related to therapy resistance, including tyrosine kinases ( n = 18), (transmembrane) glycoproteins ( n = 73), and genes involved in the JAK-STAT pathway ( n = 7). Among the potentially resistance related genes were PIM1 , SOCS1 , and MYC , which have been reported to convey a risk for treatment failure. In conclusion, we show modest temporal heterogeneity between paired tumor samples with the acquisition of new mutations and identification of genes possibly related to therapy resistance. The mutational evolution could have implications for treatment decisions and development of novel targeted drugs.

The intracellular parasite Toxoplasma gondii promotes infection by targeting multiple host cell processes; however, whether it modulates mRNA translation is currently unknown. Here, we show that infection of primary murine macrophages with type I or II T. gondii strains causes a profound perturbation of the host cell translatome. Notably, translation of transcripts encoding proteins involved in metabolic activity and components of the translation machinery was activated upon infection. In contrast, the translational efficiency of mRNAs related to immune cell activation and cytoskeleton/cytoplasm organization was largely suppressed. Mechanistically, T. gondii bolstered mechanistic target of rapamycin (mTOR) signaling to selectively activate the translation of mTOR-sensitive mRNAs, including those with a 5'-terminal oligopyrimidine (5' TOP) motif and those encoding mitochondrion-related proteins. Consistent with parasite modulation of host mTOR-sensitive translation to promote infection, inhibition of mTOR activity suppressed T. gondii replication. Thus, selective reprogramming of host mRNA translation represents an important subversion strategy during T. gondii infection., (Copyright © 2018 American Society for Microbiology.)

Orphan G-protein-coupled receptors (oGPCRs) possess untapped potential for drug discovery. In the brain, oGPCRs are generally expressed at low abundance and their function is understudied. Expression profiling is an essential step to position oGPCRs in brain function and disease, however public databases provide only partial information. Here, we fine-map expression of 78 brain-oGPCRs in the mouse, using customized probes in both standard and supersensitive in situ hybridization. Images are available at http://ogpcr-neuromap.douglas.qc.ca. This searchable database contains over 8000 coronal brain sections across 1350 slides, providing the first public mapping resource dedicated to oGPCRs. Analysis with public mouse (60 oGPCRs) and human (56 oGPCRs) genome-wide datasets identifies 25 oGPCRs with potential to address emotional and/or cognitive dimensions of psychiatric conditions. We probe their expression in postmortem human brains using nanoString, and included data in the resource. Correlating human with mouse datasets reveals excellent suitability of mouse models for oGPCRs in neuropsychiatric research., Competing Interests: The authors declare no competing interests.

Macrophages represent one of the first lines of defense during infections and are essential for resolution of inflammation following pathogen clearance. Rapid activation or suppression of protein synthesis via changes in translational efficiency allows cells of the immune system, including macrophages, to quickly respond to external triggers or cues without de novo mRNA synthesis. The translational repressors eIF4E-binding proteins 4E-BP1 and 4E-BP2 (4E-BP1/2) are central regulators of proinflammatory cytokine synthesis during viral and parasitic infections. However, it remains to be established whether 4E-BP1/2 play a role in translational control of anti-inflammatory responses. By comparing translational efficiencies of immune-related transcripts in macrophages from wild-type and 4E-BP1/2 double-knockout mice, we found that translation of mRNAs encoding two major regulators of inflammation, IL-10 and PG-endoperoxide synthase 2/cyclooxygenase-2, is controlled by 4E-BP1/2. Genetic deletion of 4E-BP1/2 in macrophages increased endogenous IL-10 and PGE 2 protein synthesis in response to TLR4 stimulation and reduced their bactericidal capacity. The molecular mechanism involves enhanced anti-inflammatory gene expression (s Il1ra , Nfil3 , Arg1 , Serpinb2 ) owing to upregulation of IL-10-STAT3 and PGE 2 -C/EBPβ signaling. These data provide evidence that 4E-BP1/2 limit anti-inflammatory responses in macrophages and suggest that dysregulated activity of 4E-BP1/2 might be involved in reprogramming of the translational and downstream transcriptional landscape of macrophages during pathological conditions, such as infections and cancer., (Copyright © 2018 by The American Association of Immunologists, Inc.)

The brain cancer medulloblastoma consists of different transcriptional subgroups. To characterize medulloblastoma at the phosphoprotein-signaling level, we performed high-throughput peptide phosphorylation profiling on a large cohort of SHH (Sonic Hedgehog), group 3, and group 4 medulloblastomas. We identified two major protein-signaling profiles. One profile was associated with rapid death post-recurrence and resembled MYC-like signaling for which MYC lesions are sufficient but not necessary. The second profile showed enrichment for DNA damage, as well as apoptotic and neuronal signaling. Integrative analysis demonstrated that heterogeneous transcriptional input converges on these protein-signaling profiles: all SHH and a subset of group 3 patients exhibited the MYC-like protein-signaling profile; the majority of the other group 3 subset and group 4 patients displayed the DNA damage/apoptotic/neuronal signaling profile. Functional analysis of enriched pathways highlighted cell-cycle progression and protein synthesis as therapeutic targets for MYC-like medulloblastoma., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)