Cardiomyocyte Specific Deletion of ADAR1 Causes Severe Cardiac Dysfunction and Increased Lethality.

Background: Adenosine deaminase acting on RNA 1 (ADAR1) is a double-stranded RNA-editing enzyme that is involved in several functions including the deamination of adenosine to inosine, RNA interference (RNAi) mechanisms and microRNA (miRNA) processing, rendering ADAR1 essential for life. Methods and Results: To investigate whether maintenance of ADAR1 expression is required for normal myocardial homeostasis, we bypassed the early embryonic lethality of ADAR1-null mice through the use of a tamoxifen-inducible Cre recombinase under the control of the cardiac-specific α-myosin heavy chain promoter (αMHC). Targeted ADAR1 deletion in adult mice caused a significant increase in lethality accompanied by severe ventricular remodeling and quick and spontaneous cardiac dysfunction, induction of stress markers and overall reduced expression of miRNAs. Administration of a selective inhibitor of the unfolded protein response (UPR) stress significantly blunted the deleterious effects and improved cardiac function thereby prolonging animal survival. In vitro restoring miR-199a-5p levels in cardiomyocytes lacking ADAR1 diminished UPR activation and concomitant apoptosis. Conclusions: Our findings demonstrate an essential role for ADAR1 in cardiomyocyte survival and maintenance of cardiac function through a mechanism that integrates ADAR1 dependent miRNA processing and the suppression of UPR stress.

Modifying Graft-versus-Host Disease in a Humanized Mouse Model by Targeting Macrophages or B-Cells.

Humanized mouse models can well be modified to study specific aspects of Graft-versus-Host Disease (GvHD). This paper shows the results of both macrophage depletion and (early) B-cell depletion in a humanized mouse model using RAG2-/- γc-/- mice injected with HuPBMCs. Macrophage depletion showed a significant decrease in survival and also lead to a change in the histomorphology of the xenogeneic reaction. Higher levels of infiltrating B-cells were observed in various organs of mice depleted for macrophages. With (early) B-cell depletion using Rituximab, a clear improvement on clinical symptoms was observed, even when probably only inactivated B-cells were deleted. However, the histological examinations only showed a significant morphological effect on liver fibrosis. This may be related to a difference in the mRNA levels of TGF-ß. Also, lower mRNA levels of Tregs in some organs were observed after Rituximab treatment, which contradicts that a higher number of Tregs would always be related to less severe GvHD. Our data show that both macrophage depletion and (early) B-cell depletion in a xenogeneic mouse model can influence the clinical, histological, and cytokine production of a GvHD response.

One-fits-all pretreatment protocol facilitating Fluorescence In Situ Hybridization on formalin-fixed paraffin-embedded, fresh frozen and cytological slides.

BACKGROUND: The Fluorescence In Situ Hybridization (FISH) technique is a very useful tool for diagnostic and prognostic purposes in molecular pathology. However, clinical testing on patient tissue is challenging due to variables of tissue processing that can influence the quality of the results. This emphasizes the necessity of a standardized FISH protocol with a high hybridization efficiency. We present a pretreatment protocol that is easy, reproducible, cost-effective, and facilitates FISH on all types of patient material simultaneously with good quality results.During validation, FISH analysis was performed simultaneously on formalin-fixed paraffin-embedded, fresh frozen and cytological patient material in combination with commercial probes using our optimized one-fits-all pretreatment protocol. An optimally processed sample is characterized by strong specific signals, intact nuclear membranes, non-disturbing autofluorescence and a homogeneous DAPI staining. RESULTS: In our retrospective cohort of 3881 patient samples, overall 93% of the FISH samples displayed good quality results leading to a patient diagnosis. All FISH were assessed on quality aspects such as adequacy and consistency of signal strength (brightness), lack of background and / or cross-hybridization signals, and additionally the presence of appropriate control signals were evaluated to assure probe accuracy. In our analysis 38 different FISH probes from 3 commercial manufacturers were used (Cytocell, Vysis and ZytoLight). The majority of the patients in this cohort displayed good signal quality and barely non-specific background fluorescence on all tissue types independent of which commercial probe was used. CONCLUSION: The optimized one-fits-all FISH method is robust, reliable and reproducible to deliver an accurate result for patient diagnostics in a lean workflow and cost-effective manner. This protocol can be used for widespread application in cancer and non-cancer diagnostics and research.

Therapeutic Delivery of miR-148a Suppresses Ventricular Dilation in Heart Failure.

Heart failure is preceded by ventricular remodeling, changes in left ventricular mass, and myocardial volume after alterations in loading conditions. Concentric hypertrophy arises after pressure overload, involves wall thickening, and forms a substrate for diastolic dysfunction. Eccentric hypertrophy develops in volume overload conditions and leads wall thinning, chamber dilation, and reduced ejection fraction. The molecular events underlying these distinct forms of cardiac remodeling are poorly understood. Here, we demonstrate that miR-148a expression changes dynamically in distinct subtypes of heart failure: while it is elevated in concentric hypertrophy, it decreased in dilated cardiomyopathy. In line, antagomir-mediated silencing of miR-148a caused wall thinning, chamber dilation, increased left ventricle volume, and reduced ejection fraction. Additionally, adeno-associated viral delivery of miR-148a protected the mouse heart from pressure-overload-induced systolic dysfunction by preventing the transition of concentric hypertrophic remodeling toward dilation. Mechanistically, miR-148a targets the cytokine co-receptor glycoprotein 130 (gp130) and connects cardiomyocyte responsiveness to extracellular cytokines by modulating the Stat3 signaling. These findings show the ability of miR-148a to prevent the transition of pressure-overload induced concentric hypertrophic remodeling toward eccentric hypertrophy and dilated cardiomyopathy and provide evidence for the existence of separate molecular programs inducing distinct forms of myocardial remodeling.

Potential Diagnosis of Vitreoretinal Lymphoma by Detection of MYD88 Mutation in Aqueous Humor With Ultrasensitive Droplet Digital Polymerase Chain Reaction.

Importance: The diagnostic workup of patients suspected of having vitreoretinal lymphoma (VRL) is primarily based on vitreous fluid analysis, including the recently emerging myeloid differentiation primary response gene 88 (MYD88) mutation analysis. Aqueous humor paracentesis is a relatively less invasive and safer procedure than taking vitreous fluid specimens, and aqueous humor-based MYD88 mutation analysis would provide an additional liquid biopsy tool to diagnose and monitor patients with VRL. Objective: To investigate whether the detection of MYD88 L265P by highly sensitive droplet digital polymerase chain reaction (ddPCR) is feasible in the vitreous fluid and aqueous humor of patients with VRL. Design, Setting, and Participants: This cohort study includes aqueous humor and vitreous fluid samples from patients with VRL who were treated at the University Medical Center Utrecht, in Utrecht, the Netherlands, from August 2005 to August 2017. Ocular fluids were randomized and masked before MYD88 L265P analysis, which was performed using an in-house validated ddPCR platform. Patients with uveitis were included as a comparison group. Main Outcomes and Measures: The presence of MYD88 L265P mutation detected by ddPCR in AH and VF. Results: The study included 96 samples from 63 individuals, including 23 patients with VRL (of whom 10 were female and 13 male, with a mean [SD] age of 72 [7.3] years) and 40 individuals with uveitis (of whom 23 were female and 17 male, with a mean [SD] age of 58 [20.9] years). In 17 of 23 patients with VRL (74%), MYD88 L265P was detected; it was not detected in any of the patients with uveitis. It was detectable in both vitreous fluid and aqueous humor samples. In the paired samples, the mutation was detected in 8 of 9 aqueous humor samples (89%) of the MYD88 L265P-positive vitreous fluid samples. In vitreous fluid, the MYD88 ddPCR test showed a sensitivity of 75% (95% CI, 50%-92%) and a positive predictive value of 100%; in aqueous humor, sensitivity was 67% (95% CI, 42%-92%), and positive predictive value was 100%. Specificity was 100% in both fluids. After treatment, the mutation was no longer detectable in any ocular fluids. Conclusions and Relevance: The high concordance between aqueous humor and vitreous fluid samples suggests that use of the easily accessible aqueous humor is nearly as informative as vitreous fluid in the identification of key somatic mutations in patients with VRL. This approach may provide an additional minimally invasive tool for accurate diagnosis, detection of recurrence, and monitoring of treatment.

Molecular analysis in liquid biopsies for diagnostics of primary central nervous system lymphoma: Review of literature and future opportunities.

Primary central nervous system lymphoma (PCNSL) is an aggressive lymphoma with a poor prognosis, for which accurate and timely diagnosis is of utmost importance. Unfortunately, diagnosis of PCNSL can be challenging and a brain biopsy (gold standard for diagnosis) is an invasive procedure with the risk of major complications. Thus, there is an urgent need for an alternative strategy to diagnose and monitor these lymphomas. Currently, liquid biopsies from cerebrospinal fluid (CSF) are used for cytomorphologic and flow cytometric analysis. Recently, new biomarkers such as genetic mutations and interleukins have been identified in these liquid biopsies, further expanding the diagnostic armamentarium. In this review we present an overview of genetic aberrations (>70) reported in this unique lymphoma. Of these genes, we have selected those that are reported in ≥3 studies. Half of the selected genes are implicated in the NFκB pathway (CARD11, CD79B, MYD88, TBL1XR1 and TNFAIP3), while the other half are not related to this pathway (CDKN2A, ETV6, PIM1, PRDM1 and TOX). Although this underlines the crucial role of the NFκB pathway in PCNSL, CD79B and MYD88 are at present the only genes mentioned in liquid biopsy analysis. Finally, a stepwise approach is proposed for minimally invasive liquid biopsy analysis and work-up of PCNSL, incorporating molecular analysis. Prioritization and refinements of this approach can be constructed based upon multidisciplinary collaboration as well as novel scientific insights.

The use of droplet digital PCR in liquid biopsies: A highly sensitive technique for MYD88 p.(L265P) detection in cerebrospinal fluid.

The gold standard for diagnosis of central nervous system lymphomas still regards a stereotactic brain biopsy, with the risk of major complications for the patient. As tumor cells can be detected in cerebrospinal fluid (CSF), CSF analysis can be used as an alternative. In this respect, mutation analysis in CSF can be of added value to other diagnostic parameters such a cytomorphology and clonality analysis. A well-known example of targeted mutation analysis entails MYD88 p.(L265P) detection, which is present in the majority of Bing Neel syndrome and primary central nervous system lymphoma (PCNSL) patients. Unfortunately, tumor yield in CSF can be very low. Therefore, use of the highly sensitive droplet digital PCR (ddPCR) might be a suitable analysis strategy for targeted mutation detection. We analyzed 26 formalin fixed paraffin embedded (FFPE) samples (8 positive and 18 negative for MYD88 p.(L265P) mutation) by ddPCR, of which the results were compared with next generation sequencing (NGS). Subsequently, 32 CSF samples were analyzed by ddPCR. ddPCR and NGS results on FFPE material showed 100% concordance. Among the 32 CSF samples, 9 belonged to patients with lymphoplasmacytic lymphoma (LPL) and clinical suspicion of Bing Neel syndrome, and 3 belonged to patients with PCNSL. Nine of these samples tested positive for MYD88 p.(L265P) (8 LPL and 1 PCNSL). This study shows that sensitive MYD88 mutation analysis by ddPCR in CSF is highly reliable and can be applied even when DNA input is low. Therefore, ddPCR is of added value to current diagnostic parameters, especially when the available amount of DNA is limited.

The Interleukin-33/ST2 Pathway Is Expressed in the Failing Human Heart and Associated with Pro-fibrotic Remodeling of the Myocardium.

The interleukin-33 (IL-33)/suppression of tumorigenicity 2 (ST2) pathway is a potential pathophysiological mediator of cardiac fibrosis. Soluble ST2 (sST2) is one of the main isoforms of ST2 with strong prognostic value in cardiac disease. The exact role of sST2 in cardiac fibrosis is unknown. The aim of this study was (1) to investigate myocardial expression of the IL-33/ST2 pathway in relation to myocardial fibrosis in end-stage heart failure patients and (2) to study whether plasma sST2 is associated with histologically determined cardiac fibrosis. In 38 patients undergoing left ventricular assist device implantation, mRNA expression of sST2, total ST2, and IL-33 was measured in cardiac tissue obtained during the implantation. In the same tissue, histological fibrosis was digitally quantified and mRNA expression of pro-fibrotic signaling molecules, connective tissue growth factor (CTGF) and transforming growth factor beta 1 (TGFß1), was measured. In addition, plasma levels of sST2 were determined. Expression levels of IL-33/ST2 pathway factors in myocardial tissue were significantly associated with cardiac fibrosis and the expression levels of CTGF and TGFß1. Plasma levels of sST2 did not correlate with tissue expression of ST2, the amount of fibrosis or myocardial expression of pro-fibrotic signaling proteins. The interleukin-33/ST2 pathway is expressed in the failing human heart and its expression is associated with cardiac fibrosis and pro-fibrotic signaling proteins, suggesting a role in pro-fibrotic myocardial remodeling. Soluble ST2 levels in the circulation did not correlate with the amount of cardiac fibrosis or myocardial ST2 expression, however. Therefore, other pathophysiological processes such as inflammation might also substantially affect sST2 plasma levels.

Preanalytical blood sample workup for cell-free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics.

In current molecular cancer diagnostics, using blood samples of cancer patients for the detection of genetic alterations in plasma (cell-free) circulating tumor DNA (ctDNA) is an emerging practice. Since ctDNA levels in blood are low, highly sensitive Droplet Digital PCR (ddPCR) can be used for detecting rare mutational targets. In order to perform ddPCR on blood samples, a standardized procedure for processing and analyzing blood samples is necessary to facilitate implementation into clinical practice. Therefore, we assessed the technical sample workup procedure for ddPCR on blood plasma samples. Blood samples from healthy individuals, as well as lung cancer patients were analyzed. We compared different methods and protocols for sample collection, storage, centrifugation, isolation, and quantification. Cell-free DNA (cfDNA) concentrations of several wild-type targets and BRAF and EGFR-mutant ctDNA concentrations quantified by ddPCR were primary outcome measurements. Highest cfDNA concentrations were measured in blood collected in serum tubes. No significant differences in cfDNA concentrations were detected between various time points of up to 24 h until centrifugation. Highest cfDNA concentrations were detected after DNA isolation with the Quick cfDNA Serum & Plasma Kit, while plasma isolation using the QIAamp Circulating Nucleic Acid Kit yielded the most consistent results. DdPCR results on cfDNA are highly dependent on multiple factors during preanalytical sample workup, which need to be addressed during the development of this diagnostic tool for cancer diagnostics in the future.

Distinct fibrosis pattern in desmosomal and phospholamban mutation carriers in hereditary cardiomyopathies.

BACKGROUND: Desmosomal and phospholamban (PLN) mutations are associated with arrhythmogenic cardiomyopathy. Ultimately, most cardiomyopathic hearts develop significant cardiac fibrosis. OBJECTIVE: To compare the fibrosis patterns of desmosomal and p. Arg14del PLN-associated cardiomyopathies with the pattern in hearts with other hereditary cardiomyopathies. METHODS: A midventricular transversal slice was obtained from hearts of 30 patients with a cardiomyopathy with a known underlying mutation and from 8 controls. Fibrosis and fatty changes were quantitatively analyzed using digital microscopy. RESULTS: Hearts from patients with desmosomal mutations (n = 6) showed fibrosis and fibrofatty replacement in the left ventricular (LV) outer myocardium, mainly in the posterolateral wall, and in the right ventricle. A similar phenotype, but with significantly more severe fibrotic changes in the LV, was found in the PLN mutation group (n = 8). Cardiomyopathies associated with lamin A/C (n = 5), sarcomeric (n = 8), and desmin (n = 3) mutations all showed a different pattern from that of the desmosomal and PLN mutation carriers. The posterolateral LV wall appeared to be the most discriminative area with fibrosis and fatty changes predominantly at the outer compact myocardium in 13 of 14 hearts with desmosomal and PLN mutations (93%), in 0 of 13 hearts with lamin A/C and sarcomeric mutations (0%), and in 1 of 3 desminopathic hearts (33%) (P < .001). CONCLUSION: Desmosomal- and PLN-associated cardiomyopathies have a fibrosis pattern distinct from the patterns in other hereditary cardiomyopathies. The posterolateral LV wall appeared to be the most discriminative region between mutation groups. These results may provide a roadmap for cardiac imaging interpretation and may help in further unraveling disease mechanisms.

Allogeneic Mesenchymal Stem Cells Stimulate Cartilage Regeneration and Are Safe for Single-Stage Cartilage Repair in Humans upon Mixture with Recycled Autologous Chondrons.

Traditionally, mesenchymal stem cells (MSCs) isolated from adult bone marrow were described as being capable of differentiating to various lineages including cartilage. Despite increasing interest in these MSCs, concerns regarding their safety, in vivo behavior and clinical effectiveness have restrained their clinical application. We hypothesized that MSCs have trophic effects that stimulate recycled chondrons (chondrocytes with their native pericellular matrix) to regenerate cartilage. Searching for a proof of principle, this phase I (first-in-man) clinical trial applied allogeneic MSCs mixed with either 10% or 20% recycled autologous cartilage-derived cells (chondrons) for treatment of cartilage defects in the knee in symptomatic cartilage defect patients. This unique first in man series demonstrated no treatment-related adverse events up to one year postoperatively. At 12 months, all patients showed statistically significant improvement in clinical outcome compared to baseline. Magnetic resonance imaging and second-look arthroscopies showed completely filled defects with regenerative cartilage tissue. Histological analysis on biopsies of the grafts indicated hyaline-like regeneration with a high concentration of proteoglycans and type II collagen. Short tandem repeat analysis showed the regenerative tissue only contained patient-own DNA. These findings support the novel insight that the use of allogeneic MSCs is safe and opens opportunities for other applications. Stem cell-induced paracrine mechanisms may play an important role in the chondrogenesis and successful tissue regeneration found. Stem Cells 2017;35:256-264.

Lymphoblastic lymphoma with a triple-hit profile: a rare but distinct and relevant entity.

Follicular lymphoma with progression to a high-grade lymphoma bears a poor prognosis. We describe a case of a 60-year-old man who presented in 2012 with an epidural mass, diagnosed as a diffuse large B-cell lymphoma (DLBCL) with concurrent low-grade follicular lymphoma. Three years later, the patient presented with a cervical mass, diagnosed as a lymphoblastic lymphoma (LBL). Both the DLBCL and LBL contained a "triple hit" with BCL2, BCL6, and cMYC translocations demonstrated by fluorescence in situ hybridization analysis and a complex karyotype by single-nucleotide polymorphism array analysis. Furthermore, the 2 lymphomas were shown to be clonally related by clonality analysis and single-nucleotide polymorphism array analysis. This case report presents a highly unusual case of an LBL with a triple hit, originating from a DLBCL, which has rarely been described in the literature and deserves further exploration.

Targeted Next Generation Sequencing as a Reliable Diagnostic Assay for the Detection of Somatic Mutations in Tumours Using Minimal DNA Amounts from Formalin Fixed Paraffin Embedded Material.

BACKGROUND: Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making. METHOD: We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering. RESULTS: Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data. CONCLUSION: Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA.

Predicted Indirectly ReCognizable HLA Epitopes Class I Promote Antileukemia Responses after Cord Blood Transplantation: Indications for a Potential Novel Donor Selection Tool.

Unrelated cord blood transplantation (UCBT) provides a curative therapy for patients with hematological malignancies. The effect of HLA mismatches in UCBT is currently the subject of debate. HLA-mismatched UCBT may lead to improved leukemia control but also to graft-versus-host disease (GVHD), resulting in nonrelapse mortality (NRM). The aim of this study was to investigate whether indirect recognition of mismatched HLA provides an explanation for the graft-versus-tumor effect and risk of GVHD. The probability of indirect recognition was predicted by the Predicted Indirectly ReCognizable HLA Epitopes (PIRCHE) model. The effect of the numbers of PIRCHE presented on HLA class I and II (PIRCHE-I and -II) was studied in 134 pediatric patients. To study the effects of higher numbers of PIRCHE, patients were divided in 2 equally sized groups, using the median number of PIRCHE as cutoff values. Proportional hazard models and competing risk analyses were performed to study the effect of PIRCHE on the clinical outcomes relapse, acute and chronic GVHD, NRM, and disease-free and overall survival. Above median PIRCHE-I were associated with reduced relapse risk (HR, .26; 95% CI, .07 to .94; P = .04), evaluating the 50 patients transplanted for a malignancy. Both PIRCHE-I and -II were not associated with other clinical outcomes, including GVHD and NRM. These data suggest that high PIRCHE-I may lead to improved graft-versus-tumor effects after UCBT, without an accompanying GVHD risk. Inclusion of PIRCHE in UCB selection criteria may enhance UCBT outcome, which needs to be tested in prospective studies.

Direct Cell-Cell Contact with Chondrocytes Is a Key Mechanism in Multipotent Mesenchymal Stromal Cell-Mediated Chondrogenesis.

Using a combination of articular chondrocytes (ACs) and mesenchymal stromal cells (MSCs) has shown to be a viable option for a single-stage cell-based treatment of focal cartilage defects. However, there is still considerable debate whether MSCs differentiate or have a chondroinductive role through trophic factors. In addition, it remains unclear whether direct cell-cell contact is necessary for chondrogenesis. Therefore, the aim of this study was to investigate whether direct or indirect cell-cell contact between ACs and MSCs is essential for increased cartilage production in different cellular environments and elucidate the mechanisms behind these cellular interactions. Human ACs and MSCs were cultured in a 10:90 ratio in alginate beads, fibrin scaffolds, and pellets. Cells were mixed in direct cocultures, separated by a Transwell filter (indirect cocultures), or cultured with conditioned medium. Short tandem repeat analysis revealed that the percentages of ACs increased during culture, while those of MSCs decreased, with the biggest change in fibrin glue scaffolds. For alginate, where the lack of cell-cell contact could be confirmed by histological analysis, no difference was found in matrix production between direct and indirect cocultures. For fibrin scaffolds and pellet cultures, an increased glycosaminoglycan production and type II collagen deposition were found in direct cocultures compared with indirect cocultures and conditioned medium. Positive connexin 43 staining and transfer of cytosolic calcein indicated communication through gap junctions in direct cocultures. Taken together, these results suggest that MSCs stimulate cartilage formation when placed in close proximity to chondrocytes and that direct cell-cell contact and communication through gap junctions are essential in this chondroinductive interplay.

Comparison of next-generation sequencing and mutation-specific platforms in clinical practice.

OBJECTIVES: To compare next-generation sequencing (NGS) platforms with mutation-specific analysis platforms in a clinical setting, in terms of sensitivity, mutation specificity, costs, capacity, and ease of use. METHODS: We analyzed 25 formalin-fixed, paraffin-embedded lung cancer samples of different size and tumor percentage for known KRAS and EGFR hotspot mutations with two dedicated genotyping platforms (cobas [Roche Diagnostics, Almere, The Netherlands] and Rotor-Gene [QIAGEN, Venlo, The Netherlands]) and two NGS platforms (454 Genome Sequencer [GS] junior [Roche Diagnostics] and Ion Torrent Personal Genome Machine [Life Technologies, Bleiswijk, The Netherlands]). RESULTS: All platforms, except the 454 GS junior, detected the mutations originally detected by Sanger sequencing and high-resolution melting prescreening and detected an additional KRAS mutation. The dedicated genotyping platforms outperformed the NGS platforms in speed and ease of use. The large sequencing capacity of the NGS platforms enabled them to deliver all mutation information for all samples at once. CONCLUSIONS: Sensitivity for detecting mutations was highly comparable among all platforms. The choice for either a dedicated genotyping platform or an NGS platform is basically a trade-off between speed and genetic information.

TP53 Y220C Is a Hotspot Mutation in Oropharyngeal Squamous Cell Carcinoma.

OBJECTIVES: Although TP53 mutations in head and neck squamous cell carcinoma (HNSCC) have been extensively studied, their association with the different subsites in the head and neck region has never been described. METHODS: Sanger sequence analysis evaluating exons 4-9 in the TP53 gene was performed on 116 HNSCC patients. The exon location, exact codon and corresponding substitution in relation to the anatomical site (subsite) of the HNSCC were evaluated. RESULTS: We found nonsynonymous TP53 mutations in 70% (81/116) of the patients. In oral cavity carcinomas, most mutations occurred in exon 7 (37%). In oropharyngeal and laryngeal tumors, mutations were mainly found in exons 6 and 7. The most common mutation was located in codon 220, and all of these were an Y220C mutation. Five out of nine (56%) Y220C mutations occurred in oropharyngeal tumors. Additionally, 22% of all mutations observed in oropharyngeal squamous cell carcinoma (OPSCC) consisted of Y220C mutations. CONCLUSION: In this study, the subsite-related distribution of TP53 mutations underlines the biological diversity between tumors arising from different anatomical regions in the head and neck region. Moreover, the Y220C mutation was by far the most prevalent TP53 mutation in HNSCC and a relative hotspot mutation in the oropharynx. © 2015 S. Karger AG, Basel.

Myocardial fibrosis and pro-fibrotic markers in end-stage heart failure patients during continuous-flow left ventricular assist device support.

OBJECTIVES: During support with a left ventricular assist device (LVAD), partial reverse remodelling takes place in which fibrosis plays an important role. In this study, we analysed the histological changes and expression of fibrotic markers in patients with advanced heart failure (HF) during continuous-flow LVAD (cf-LVAD) support. METHODS: In 25 patients, myocardial tissue at the time of LVAD implantation (pre-LVAD) was compared with tissue from the explanted left ventricle (post-LVAD). Interstitial fibrosis and cardiomyocyte size were analysed pre- and post-LVAD. Plasma was obtained from all patients before and during LVAD support. Plasma levels, cardiac mRNA and protein expression of brain natriuretic peptide (BNP), galectin-3 (Gal-3), connective tissue growth factor (CTGF), osteopontin (OPN) and transforming growth factor ß-1 were determined. RESULTS: Fibrosis increased during cf-LVAD unloading (P < 0.05). Cardiomyocytes elongated (P < 0.05), whereas cross-sectional area did not change. BNP, Gal-3, CTGF and OPN were significantly elevated pre-LVAD in comparison with controls. BNP decreased significantly after 1 month of cf-LVAD support (P < 0.001) to near-normal levels. Pro-fibrotic markers remained elevated in comparison with controls. CONCLUSIONS: cf-LVAD support is associated with lengthening of cardiomyocytes, without alterations in diameter size. Remarkably, myocardial fibrosis increased as well as circulating pro-fibrotic markers. Whether the morphological changes are a direct effect of reduced pulsatility during cf-LVAD support or due to HF progression requires further investigation.

Simultaneous detection of clinically relevant mutations and amplifications for routine cancer pathology.

In routine cancer molecular pathology, various independent experiments are required to determine mutation and amplification status of clinically relevant genes. Most of these tests are designed to identify a limited number of genetic aberrations, most likely in a given tumor type. We present a modified version of a multiplexed PCR and IonTorrent-based sequencing approach that can replace a large number of existing assays. The test allows for the simultaneous detection of point mutations and gene amplifications in 40 genes, including known hotspot regions in oncogenes (KRAS, BRAF), inactivating mutations in tumor suppressors (TP53, PTEN), and oncogene amplifications (ERBB2, EGFR). All point mutations were confirmed using certified diagnostic assays, and a sensitivity and specificity of 100% (95% CI, 0.875-1.0) and 99% (95% CI, 0.960-0.999), respectively, were determined for amplifications in FFPE material. Implementation of a single assay to effectively detect mutations and amplifications in clinically relevant genes not only improves the efficiency of the workflow within diagnostic laboratories but also increases the chance of detecting (rare) actionable variants for a given tumor type that are typically missed in routine pathology. The ability to obtain comprehensive and rapid mutational overviews is key for improving the efficiency of cancer patient care through tailoring treatments based on the genetic characteristics of individual tumors.

Humanized mouse models in transplantation research.

The interest in the use of humanized mouse models for research topics like Graft versus Host Disease (GvHD), allograft studies and other studies to the human immune system is growing. The design of these models is still improving and enables even more complicated studies to these topics. For researchers it can be difficult to choose the best option from the current pool of available models. The decision will depend on which hypothesis needs to be tested, in which field of interest, and therefore 'the best model' will differ from one to another. In this review, we provide a guide to the most common available humanized mouse models, with regards to different mouse strains, transplantation material, transplantation techniques, pre- and post-conditioning and references to advantages and disadvantages. Also, an evaluation of experiences with humanized mouse models in studies on GvHD and allograft rejection is provided.