Development and Validation of a Novel Dual-Drop-off ddPCR Assay for the Simultaneous Detection of Ten Hotspots PIK3CA Mutations.

Breast cancer is the leading cause of cancer-related deaths in women worldwide. Approximately 40% of patients with hormone receptor-positive, human epidermal growth factor receptor-2-negative breast cancer have activating mutations in the PIK3CA gene. We developed a highly sensitive, specific, cost-effective, and reproducible dual-drop-off droplet digital polymerase chain reaction (PCR) assay for the simultaneous detection of ten hotspots of PIK3CA mutations in plasma cell-free (cf) DNA. We first evaluated the analytical specificity, sensitivity, limit of blank, repeatability, and reproducibility of the assay, which simultaneously detects seven mutations in exon9 and three in exon20. We further applied this assay in 11 gDNA and 18 plasma cfDNA samples from healthy donors and 35 plasma cfDNA samples from metastatic breast cancer patients. The assay is highly sensitive, specific, and applicable for clinical samples containing at least 1-5% mutant DNA. We detected PIK3CA mutations in 9/35(26%) plasma cfDNA samples in exon 9 and in 9/35(26%) in exon 20. Direct comparison of the developed assay with amplification refractory mutation system-based PCR (using plasma samples) and with the Food and Drug Administration-approved cobas PIK3CA mutation assay (using formalin fixed paraffin embedded samples) showed high concordance of our developed assay with the cobas PIK3CA assay. The developed assay is cost-effective and can reliably and simultaneously detect ten hotspot PIK3CA mutations in plasma cfDNA. The clinical performance of the assay will be further evaluated in liquid biopsy samples.

Effect of a new squalene synthase inhibitor on an ApoE-/- mouse model of atherosclerosis.

Ηypercholesterolemia/hyperlipidemia in conjunction with oxidative stress and inflammatory processes contribute synergistically to the pathogenesis of atherosclerosis. We hereby evaluated the antiatherosclerotic effect of the multi-target derivative 4-methyl-2-(10H-phenothiazin-3-yl)morpholin-2-ol hydrobromide 1 in apoE-/- mice; compound 1 is a potent antihyperlipidemic agent acting through Squalene Synthase inhibition, while it has exhibited an outstanding antioxidant and anti-inflammatory activity in various experimental animal models. The new analogue was evaluated in terms of its antiatherosclerotic/antioxidant effect in the ApoE-/- transgenic mouse model. Its toxicity profile was also assessed by measuring the levels of four sensitive indicators of liver toxicity. Prolonged administration of 1 in ApoE-/- mice fed with a western-type (wt) diet efficiently reduced the aortic atheromatic lesions, an effect that took place through a cholesterol lowering independent manner. In addition, 1 displayed a significant reduction not only of glucose but also of oxidative stress levels, while it did not cause any toxicity. To the best of our knowledge this is the first time that the antiatherosclerotic effect of a Squalene Synthase inhibitor is studied in this specific atherosclerosis mouse model. As a result, compound 1 may serve as a promising starting point towards developing new bioactive analogues against the onset and subsequent development of atherosclerosis.

A case report of recessive restrictive cardiomyopathy caused by a novel mutation in cardiac troponin I (TNNI3).

BACKGROUND: Restrictive cardiomyopathy is a rare cardiac disease, for which several genes including TNNT2, MYPN, FLNC and TNNI3 have been associated with its familial form. CASE PRESENTATION: Here we describe a female proband with a severely manifested restrictive phenotype leading to heart transplantation at the age of 41, who was found homozygous for the novel TNNI3 mutation: NM_000363.4:c.586G > C, p.(Asp196His). Her parents were third-degree cousins originating from a small village and although they were found heterozygous for the same variant they displayed no symptoms of the disease. Her older sister who was also found heterozygous was asymptomatic. Her twin sister and her brother who were homozygous for the same variant displayed a restrictive and a hypertrophic phenotype, respectively. Their children are all carriers of the mutation and remain asymptomatic until the age of 21. CONCLUSION: These observations point to a recessive mode of inheritance reported for the first time for this combination of gene/disease.

Differential expression patterns of Toll Like Receptors and Interleukin-37 between calcific aortic and mitral valve cusps in humans.

BACKGROUND: Significant differences are mentioned in the progress of calcification between aortic and mitral valve. Evidence of inflammation in calcific aortic and mitral valve disease suggests that pathways of Toll Like Receptors (TLR) and Interleukin (IL)-37 expression may contribute to this process. We sought to investigate the role of TLR-mediated inflammatory response and IL-37 pathway expression on aortic and mitral valve calcification. MATERIAL AND METHODS: One-hundred twenty stenotic valve cusps/leaflets (60 aortic, 60 mitral) were excised during surgery and were collected for histological, immunohistochemistry and morphometric analysis at our department. After total RNA isolation from a second part of valve cusps/leaflets, cDNA synthesis and quantitative reverse transcription polymerase chain reaction (qRT-PCR) protocols were performed and relative mRNA levels of target genes were assessed. RESULTS: By histological analysis, the anti-inflammatory IL-37 levels were increased in mitral valve leaflets (MVL) compared to aortic valve cusps (AVCu) while all other biomarkers, including TLR, presented a reverse pattern with decreased levels as compared to AVCu. In terms of calcification biomarkers, only osteopontin differed between AVCu and MVL. mRNA analysis confirmed increased expression of IL-37 and decreased levels of TLR in MVL compared to AVCu. CONCLUSIONS: Stenotic cusps of aortic valves express lower IL-37 and increased TLRs levels than stenotic mitral valve leaflets, suggesting a differential pro-calcification and pro-inflammatory profile between the two valves. This may explain the higher incidence of calcification of AVCu than MVL and offer therapeutic considerations.

Diagnosis and treatment complications of primary cardiac lymphoma in an immunocompetent 28-year old man: a case report.

BACKGROUND: Primary cardiac lymphomas (PCL) represent extremely rare cardiac tumors which are accompanied by poor prognosis, unless they are timely diagnosed and treated. CASE PRESENTATION: Herein we present a 28-year-old, immunocompetent man who presented to our hospital due to progressively worsening symptoms and signs of superior vena cava syndrome. Multi-modality imaging demonstrated a large intracardiac tumor, which was proven, by biopsy, to be a PCL. The patient received targeted chemotherapy which led to total remission of his disease, with no relapse over a 15-month follow-up period. CONCLUSIONS: Although PCLs are rare, they should always be kept in mind in the differential diagnosis of cardiac tumors. Timely diagnosis of PCLs and appropriate chemotherapy, alone or in combination with radiotherapy, seems to provide the best results.

ANCA-Negative Churg-Strauss Syndrome Presenting as Acute Multiple Cerebral Infarcts: A Case Report.

Eosinophilic granulomatosis with polyangiitis (EGPA, previously named Churg-Strauss syndrome) is a form of necrotizing vasculitis occurring in patients with asthma and eosinophilia. Ischemic stroke is a relatively rare complication of the disease. We report a case of a 63-year-old woman with multiple embolic infarcts, hypereosinophilia (for >7 years), and skin rash. Elevated cardiac enzymes and cardiac magnetic resonance imaging were consistent with endomyocarditis. The simultaneous presence of history of asthma, sinusitis, hypereosinophilia, and vasculitis led to the diagnosis of EGPA. This case contributes to the recent debate of the 2 possible presentations of the disease according to the ANCA (antineutrophil cytoplasmic antibodies) status. We furthermore underscore the need for careful differential diagnosis of the "ANCA negative" cases with persistent hypereosinophilia from the idiopathic hypereosinophilic syndrome.

Applying Broadband Dielectric Spectroscopy (BDS) for the Biophysical Characterization of Mammalian Tissues under a Variety of Cellular Stresses.

The dielectric properties of biological tissues can contribute non-invasively to a better characterization and understanding of the structural properties and physiology of living organisms. The question we asked, is whether these induced changes are effected by an endogenous or exogenous cellular stress, and can they be detected non-invasively in the form of a dielectric response, e.g., an AC conductivity switch in the broadband frequency spectrum. This study constitutes the first methodological approach for the detection of environmental stress-induced damage in mammalian tissues by the means of broadband dielectric spectroscopy (BDS) at the frequencies of 1-106 Hz. Firstly, we used non-ionizing (NIR) and ionizing radiation (IR) as a typical environmental stress. Specifically, rats were exposed to either digital enhanced cordless telecommunication (DECT) radio frequency electromagnetic radiation or to γ-radiation, respectively. The other type of stress, characterized usually by high genomic instability, was the pathophysiological state of human cancer (lung and prostate). Analyzing the results of isothermal dielectric measurements provided information on the tissues' water fraction. In most cases, our methodology proved sufficient in detecting structural changes, especially in the case of IR and malignancy. Useful specific dielectric response patterns are detected and correlated with each type of stress. Our results point towards the development of a dielectric-based methodology for better understanding and, in a relatively invasive way, the biological and structural changes effected by radiation and developing lung or prostate cancer often associated with genomic instability.

Kisspeptin effect on endothelial monocyte activating polypeptide II (EMAP-II)-associated lymphocyte cell death and metastases in colorectal cancer patients.

Kisspeptin is an antimetastatic agent in some cancers that has also been associated with lymphoid cell apoptosis, a phenomenon favoring metastases. Our aim was to determine the association of kisspeptin with lymphocyte apoptosis and the presence of metastases in colorectal cancer patients. Blood was drawn from 69 colon cancer patients and 20 healthy volunteers. Tissue specimens from healthy and pathological tissue were immunohistochemically analyzed for kisspeptin and endothelial monocyte activating polypeptide II (EMAP-II) expression. Blood EMAP-II and soluble Fas ligand (sFasL) levels were examined by an enzyme-linked immunosorbent assay method. The kisspeptin and EMAP-II expression and secretion levels in the DLD-1 and HT-29 colon cancer cell lines were examined by quantitative real-time polymerase chain reaction, Western analysis and enzyme-linked immunosorbent assay, whereas lymphocyte viability was assessed by flow cytometry. The effect of kisspeptin on the viability of colon cancer cells was examined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. Exogenous, synthetic and naturally produced, kisspeptin induces through the G-protein-coupled receptor 54 (GPR54; also known as the kisspeptin receptor) the EMAP-II expression and secretion in colon cancer cell lines, inducing in vitro lymphocyte apoptosis, as verified by the use of an anti-EMAP-II antibody. These results were reversed with the use of kisspeptin inhibitors and by kisspeptin-silencing experiments. Tumor kisspeptin expression was associated with the tumor EMAP-II expression (p < 0.001). Elevated kisspeptin and EMAP-II expression in colon cancer tissues was associated with lack of metastases (p < 0.001) in colon cancer patients. These data indicate the antimetastatic effect of tumor-elevated kisspeptin in colon cancer patients that may be mediated by the effect of kisspeptin on EMAP-II expression in colon cancer tumors in patients with normal serum EMAP-II levels. These findings provide new insight into the role of kisspeptin in the context of metastases in colon cancer patients.

Nationwide Real-World Data of Microsatellite Instability and/or Mismatch Repair Deficiency in Cancer: Prevalence and Testing Patterns.

Determination of microsatellite instability (MSI)/mismatch repair (MMR) status in cancer has several clinical implications. Our aim was to integrate MSI/MMR status from patients tested in Greece to assess the prevalence of MSI-high (MSI-H)/deficient MMR (dMMR) per tumor type, testing patterns over time and concordance between MSI and MMR status. We retrospectively recorded MSI/MMR testing data of patients with diverse tumor types performed in pathology and molecular diagnostics laboratories across Greece. Overall, 18 of 22 pathology and/or molecular diagnostics laboratories accepted our invitation to participate. In the 18 laboratories located across the country, 7916 tumor samples were evaluated for MSI/MMR status. MSI/MMR testing significantly increased in patients with colorectal cancer (CRC) and other tumor types overtime (p < 0.05). The highest prevalence was reported in endometrial cancer (47 of 225 patients, 20.9%). MSI-H/dMMR was observed in most tumor types, even in low proportions. Among 904 tumors assessed both for MSI and MMR status, 21 had discordant results (overall discordance rate, 2.3%). We reported MSI-H/dMMR prevalence rates in patients with diverse cancers, while demonstrating increasing referral patterns from medical oncologists in the country overtime. The anticipated high rate of concordance between MSI and MMR status in paired analysis was confirmed.

Tissue and Serum Biomarkers in Degenerative Aortic Stenosis-Insights into Pathogenesis, Prevention and Therapy.

BACKGROUND AND AIM: Degenerative Aortic Stenosis (DAS) is a common disease that causes substantial morbidity and mortality worldwide, especially in the older population. Our aim was to further investigate novel serum and tissue biomarkers to elucidate biological processes involved in this entity. MATERIAL AND METHODS: We evaluated the expression of six biomarkers significantly involved in cardiovascular pathology, i.e., irisin, periostin, osteoglycin, interleukin 18, high mobility group box 1 and proprotein convertase subtilisin/kexin type 9 in the serum at the protein level, and in the tissue at both the protein and mRNA levels of patients with AS (N = 60). Five normal valves obtained after transplantation from hearts of patients with idiopathic dilated cardiomyopathy were also studied. Serum measurements were also performed in 22 individuals without valvular disease who served as controls (C). RESULTS: Higher levels of all factors were found in DAS patients' serum than in normal C. IHC and PCR mRNA tissue analysis showed the presence of all biomarkers in the aortic valve cusps with DAS, but no trace of PCR mRNA was found in the five transplantation valves. Moreover, periostin serum levels correlated significantly with IHC and mRNA tissue levels in AS patients. CONCLUSION: We showed that six widely prevalent biomarkers affecting the atherosclerotic process were also involved in DAS, suggesting a strong osteogenic and pro-inflammatory profile, indicating that aortic valve calcification is a multifactorial biological process.

CSF2RB mutation-related hereditary pulmonary alveolar proteinosis: the "long and winding road" into adulthood.

Genetic analysis pre-lung transplantation diagnosed a case of hereditary pulmonary alveolar proteinosis (PAP) complicated by fibrosis in adulthood. The need for genetic testing in GM-CSF autoantibody negative and unclassifiable PAP is highlighted. https://bit.ly/3QcsYwM.

Deferoxamine attenuates lipid peroxidation, blocks interleukin-6 production, ameliorates sepsis inflammatory response syndrome, and confers renoprotection after acute hepatic ischemia in pigs.

We have previously shown that deferoxamine (DFO) infusion protected myocardium against reperfusion injury in patients undergoing open heart surgery, and reduced brain edema, intracranial pressure, and lung injury in pigs with acute hepatic ischemia (AHI). The purpose of this research was to study if DFO could attenuate sepsis inflammatory response syndrome (SIRS) and confer renoprotection in the same model of AHI in anesthetized pigs. Fourteen animals were randomly allocated to two groups. In the Group DFO (n=7), 150mg/kg of DFO dissolved in normal saline was continuously infused in animals undergoing hepatic devascularization and portacaval anastomosis. The control group (Group C, n=7) underwent the same surgical procedure and received the same volume of normal saline infusion. Animals were euthanized after 24h. Hematological, biochemical parameters, malondialdehyde (MDA), and cytokines (interleukin [IL]-1ß, IL-6, IL-8, IL-10, and tumor necrosis factor-α) were determined from sera obtained at baseline, at 12h, and after euthanasia. Hematoxylin-eosin and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling were used to evaluate necrosis and apoptosis, respectively, in kidney sections obtained after euthanasia. A rapid and substantial elevation (more than 100-fold) of serum IL-6 levels was observed in Group C reaching peak at the end of the experiment, associated with increased production of oxygen free radicals and lipid peroxidation (MDA 3.2±0.1nmol/mL at baseline and 5.5±0.9nmol/mL at the end of the experiment, P<0.05) and various manifestations of SIRS and multiple organ dysfunction (MOD), including elevation of high-sensitivity C-reactive protein, severe hypotension, leukocytosis, thrombocytopenia, hypoproteinemia, and increased serum levels of lactate dehydrogenase (fourfold), alkaline phosphatase (fourfold), alanine aminotransferase (14-fold), and ammonia (sevenfold). In sharp contrast, IL-6 production and lipid peroxidation were completely blocked in DFO-treated animals offering remarkable resistance to the development of SIRS and MOD. Profound proteinuria, strips of extensive necrosis of tubular epithelial cells, and occasional apoptotic tubular epithelial cells were already present in Group C, but not in Group DFO animals at the time of euthanasia. DFO infusion attenuated lipid peroxidation, blocked IL-6 production, and substantially diminished SIRS and MOD, including tubulointerstitial damage in pigs after acute ischemic hepatic failure. This finding shows that iron, IL-6, and lipid peroxidation are important participants in the pathophysiology of renal injury in the course of generalized inflammation and provides novel pathways of therapeutic interventions for renal protection.

Phenotype Characterization and Course of Patients With Arrhythmogenic Right Ventricular Cardiomyopathy and Biventricular Advanced Heart Failure: A Report of 3 Cases.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) may be complicated by heart failure. Management of advanced heart failure in this context is challenging. METHODS: We reviewed our center's experience with advanced heart failure therapies in patients with ARVC. Three rapidly deteriorating patients with ARVC with biventricular heart failure were found. Their management and outcomes are presented. Data on ventricular fibrosis were available in 2 of them and are also included. RESULTS: The first patient underwent initially successful paracorporeal pulsatile biventricular assist device (BiVAD) implantation. However, a large ischemic stroke occurred 2 weeks later, and the patient died after 2 months. The second patient underwent urgent BiVAD implantation after extracorporeal membrane oxygenation support because of cardiogenic shock, but his course was complicated by multiorgan failure due to systemic infection and the patient died. The last patient, being at Interagency Registry for Mechanically Assisted Circulatory Support 3-4 profile, underwent heart transplant with uneventful recovery. Extensive fibrosis was present in both ventricles of 2 patients undergoing pathology examination. CONCLUSIONS: Patients with ARVC and advanced biventricular heart failure are characterized by extensive ventricular fibrosis and considerable risk, but data on their management are limited. Biventricular circulatory support is associated with suboptimal outcomes, and prioritization for heart transplant seems preferable.

The learning experience for endoscopic submucosal dissection in a non-academic western hospital: a single operator's untutored, prevalence-based approach.

BACKGROUND: Studies of learning experience in endoscopic submucosal dissection (ESD) commonly originate from the East. Little is known about the performance of ESD in low-volume western centers. Furthermore, it is unclear whether ESD can be self-taught without a tutored approach. METHODS: We performed a retrospective analysis of consecutive ESDs, performed in an untutored prevalence-based fashion by a single operator at a private Greek hospital from 2016-2020. Out of 60 lesions, standard ESD was applied for 54 and enucleation for 6; 41 were mucosal and 19 submucosal; 3 esophageal, 24 gastric, one duodenal, 12 colonic, and 20 rectal. RESULTS: Pathology revealed carcinoma (n=14), neuroendocrine tumor (n=7), precancerous lesion (n=27), or other submucosal tumors (n=12). The rates of en bloc and R0 resection were 98% and 91%, respectively. The median resection speed was <3 cm2/h for the first 20 cases, but improved progressively to ≥9 cm2/h after 40 cases. Two patients underwent laparoscopic surgery for colonic perforation, and one received a blood transfusion because of delayed bleeding (serious adverse event rate: 5%). No deaths occurred. The median hospital stay was 1.3 days. Variables associated with improvement in ESD speed during the second period of the study were the application of countertraction and the experience acquired through other endosurgical techniques. CONCLUSIONS: ESD was safe and effective in a low-volume center, with an acceptable adverse events rate. At least 40 mixed cases were needed to achieve a high resection speed. Additive experience gained through other endosurgical procedures probably contributed to the improvement in performance.

The effect of paracorporeal pulsatile biventricular assist devices on allosensitization in adults: A comparison with left ventricular assist devices.

Ventricular assist devices (VADs) have been associated with the development of anti-HLA antibodies ('allosensitization'), but data on devices providing biventricular support in adults are limited. We sought to characterize differences in anti-HLA antibody formation in adult patients receiving left- (LVAD) versus biventricular- (BiVAD) assist devices as bridge to transplantation (BTT) by retrospectively reviewing the records of adult patients who have undergone VAD implantation at our institution. We assessed 82 patients supported with a pulsatile-flow paracorporeal BiVAD and compared them with 40 patients receiving LVAD till 2018. Forty-eight (58.5%) of the BiVAD and 23 (57.5%) of the LVAD patients were eventually transplanted (p = 0.91) with an average time to transplantation 559 and 598 days, respectively (p = 0.73). Evidence of sensitization pre-VAD was found in 11.0% of the BiVAD patients and 15.0% of the LVAD ones (p = 0.53); these percentages rose to 43.9% (p < 0.001) and 40.0% (p = 0.01), respectively. The post-VAD sensitization status was not significantly different between the BiVAD and the LVAD group (p = 0.68). De novo sensitization was comparable between the two groups (p = 0.55). Post-transplantation outcomes regarding rejections and cardiac allograft vasculopathy were also similar. Conclusively, BiVAD- and LVAD- induced allosensitization do not appear to differ significantly.

Myocardial inflammation in Duchenne Muscular Dystrophy as a precipitating factor for heart failure: a prospective study.

BACKGROUND: In patients with Duchenne Muscular Dystrophy (DMD), the absent or diminished dystrophin leads to progressive skeletal muscle and heart failure. We evaluated the role of myocardial inflammation as a precipitating factor in the development of heart failure in DMD. METHODS: 20 DMD patients (aged 15-18 yrs) and 20 age-matched healthy volunteers were studied and followed-up for 2 years. Evaluation of myocarditis with cardiovascular magnetic resonance imaging (CMR) was performed using STIR T2-weighted (T2W), T1-weighted (T1W) before and after contrast media and late enhanced images (LGE). Left ventricular volumes and ejection fraction were also calculated. Myocardial biopsy was performed in patients with positive CMR and immunohistologic and polymerase chain reaction (PCR) analysis was employed. RESULTS: In DMD patients, left ventricular end-diastolic volume (LVEDV) was not different compared to controls. Left ventricular end-systolic volume (LVESV) was higher (45.1 +/- 6.6 vs. 37.3 +/- 3.8 ml, p < 0.001) and left ventricular ejection fraction (LVEF) was lower (53.9 +/- 2.1 vs. 63 +/- 2.4%, p < 0.001). T2 heart/skeletal muscle ratio and early T1 ratio values in DMD patients presented no difference compared to controls. LGE areas were identified in six DMD patients. In four of them with CMR evidence of myocarditis, myocardial biopsy was performed. Active myocarditis was identified in one and healing myocarditis in three using immunohistology. All six patients with CMR evidence of myocarditis had a rapid deterioration of left ventricular function during the next year. CONCLUSIONS: DMD patients with myocardial inflammation documented by CMR had a rigorous progression to heart failure.