Desmosomal and phospholamban (PLN) mutations are associated with arrhythmogenic cardiomyopathy. Ultimately, most cardiomyopathic hearts develop significant cardiac fibrosis.
To compare the fibrosis ...patterns of desmosomal and p. Arg14del PLN-associated cardiomyopathies with the pattern in hearts with other hereditary cardiomyopathies.
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.
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).
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.
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.
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.
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.
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.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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
Overview of the IMPACT therapy. During a mini‐arthrotomy cartilage is acquired form the debrided rim of the treated cartilage (a). The previously deemed waste cartilage is minced into small pieces of approximately 1 × 1 × 1 mm and transferred to a tube containing the digestion medium (b). During 40 minutes the cubical cartilage pieces are digested, releasing the chondrons (c). After washing the chondrons they are combined with the human allogeneic MSCs (d). The cell product containing the defect derived autologous chondrons and allogeneic MSCs is resuspended in the fibrin glue (e). The surgeon will apply the cell loaded fibrin glue to the debrided isolated cartilage defect (f). See http://youtu.be/CbZQ18Bvvtw for a video demonstration. Patients were followed for 18 months after treatment and were subjected to a rehabilitation program. No treatment‐related adverse events were found and all patients showed statistically significant clinical improvement.
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.
Raso et al. show that miR-148a is elevated in concentric hypertrophy and decreased in dilated cardiomyopathy. Adeno-associated viral delivery of miR-148a protects the mouse heart from cardiac dilation. Mechanistically, miR-148a regulates the sensitivity of the heart to extracellular cytokines.
Aims
Growth differentiation factor‐15 (GDF‐15) is a stress‐responsive cytokine and is emerging as a biomarker of cardiac remodelling. Left ventricular assist devices (LVADs) provide unloading of the ...left ventricle, resulting in partial reverse remodelling. Our aim was to study GDF‐15 in patients with a non‐ischaemic dilated cardiomyopathy (DCM) during LVAD support.
Methods and results
We analysed circulating GDF‐15 in 30 patients before and 1, 3, and 6 months after LVAD implantation and before heart transplantation or explantation. In addition, mRNA and protein expression of GDF‐15 were evaluated in myocardial tissue obtained prior to and after LVAD support. Circulating GDF‐15 was significantly higher before LVAD implantation as compared with healthy controls (P < 0.001). After 1 month of mechanical support, GDF‐15 levels were significantly decreased compared with pre‐implantation levels (P < 0.001) and remained stable thereafter. Circulating GDF‐15 was significantly correlated with kidney function and the severity of myocardial fibrosis. Interestingly, GDF‐15 mRNA and protein expression in the myocardium were hardly detectable.
Conclusions
High circulating levels of GDF‐15 in patients with end‐stage non‐ischaemic DCM correlate with myocardial fibrosis and kidney function and decline strongly after 1 month of mechanical unloading, remaining stable thereafter. However, cardiac mRNA and protein expression of GDF‐15 are very low, suggesting that the heart is not an important source of GDF‐15 production in these patients.
Cardiac allograft vasculopathy (CAV) is a multifactorial pathology limiting the survival of cardiac transplants. The etiology of CAV is unclear, but antibody-mediated and cellular-mediated responses ...have been implicated. We, and others, have observed ectopic lymphoid structures (ELS) surrounding epicardial coronary arteries with CAV. The potential contribution of these ELS to CAV has not been elucidated.
Epicardial coronary arteries were collected from 59 transplant patients at 2 centers and studied for ELS presence and composition using immunohistochemistry. The intima and ELS were isolated, and the expression of the genes involved in tertiary lymphoid organ (TLO) formation was measured by quantitative polymerase chain reaction.
ELS presence was related to survival after transplantation (p = 0.013) and histologic composition of CAV (p < 0.001). ELS contain B and T lymphocytes, macrophages, and antibody-producing (immunoglobulin Ig M and/or IgG) plasma cells. A sub-population of B lymphocytes appeared to be cluster of differentiation (CD)20(+)CD27(+) memory B lymphocytes. The messenger RNA expression of TLO markers (lymphotoxin-β, and chemokine C-C motif ligand 19 and 21) was significantly higher in ELS than in the neointimal lesions. The ELS observed in this study exhibited some TLO markers but did not exhibit the distinct areas rich in B and T lymphocytes that are normally found in classic TLOs.
The cellular composition of the ELS differs from the cellular infiltrate in CAV intimal lesions. The presence of memory B lymphocytes and plasma producing IgM and IgG cells suggests that ELS are related to local antibody production, potentially contributing to antibody-mediated CAV. ELS associated with coronary vessels containing CAV show features of underdeveloped TLOs; classic TLOs may not develop due to patient immunosuppression.
•Many genetic aberrations reported in PCNSL are implicated in the NFkB pathway.•Only few targets for liquid biopsy analysis in PCNSL are currently reported.•Proper liquid biopsy analysis of PCNSL ...requires a multivariable analysis approach.
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.
Molecular pathology is becoming more and more important in present day pathology. A major challenge for any molecular test is its ability to reliably detect mutations in samples consisting of ...mixtures of tumor cells and normal cells, especially when the tumor content is low. The minimum percentage of tumor cells required to detect genetic abnormalities is a major variable. Information on tumor cell percentage is essential for a correct interpretation of the result. In daily practice, the percentage of tumor cells is estimated by pathologists on hematoxylin and eosin (H&E)-stained slides, the reliability of which has been questioned. This study aimed to determine the reliability of estimated tumor cell percentages in tissue samples by pathologists. On 47 H&E-stained slides of lung tumors a tumor area was marked. The percentage of tumor cells within this area was estimated independently by nine pathologists, using categories of 0–5%, 6–10%, 11–20%, 21–30%, and so on, until 91–100%. As gold standard, the percentage of tumor cells was counted manually. On average, the range between the lowest and the highest estimate per sample was 6.3 categories. In 33% of estimates, the deviation from the gold standard was at least three categories. The mean absolute deviation was 2.0 categories (range between observers 1.5–3.1 categories). There was a significant difference between the observers (P<0.001). If 20% of tumor cells were considered the lower limit to detect a mutation, samples with an insufficient tumor cell percentage (<20%) would have been estimated to contain enough tumor cells in 27/72 (38%) observations, possibly causing false negative results. In conclusion, estimates of tumor cell percentages on H&E-stained slides are not accurate, which could result in misinterpretation of test results. Reliability could possibly be improved by using a training set with feedback.
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.
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