Molecular pathology testing for non-small cell lung cancer: an observational study of elements currently present in request forms and result reports and the opinion of different stakeholders.

BACKGROUND: For patients with non-small cell lung cancer (NSCLC), targeted therapies are becoming part of the standard treatment. It is of question which information the clinicians provide on test requests and how the laboratories adapt test conclusions to this knowledge and regulations. METHODS: This study consisted of two components; 1) checking the presence of pre-defined elements (administrative and key for therapy-choice) on completed requests and corresponding reports in Belgian laboratories, both for tissue- and liquid biopsy (LB)-testing and b) opinion analysis from Belgian pathologists/molecular biologists and clinicians during national pathology/oncology meetings. RESULTS: Data from 4 out of 6 Belgian laboratories with ISO-accreditation for LB-testing were analyzed, of which 75% were university hospitals. On the scored requests (N = 4), 12 out of 19 ISO-required elements were present for tissue and 11 for LB-testing. Especially relevant patient history, such as line of therapy (for LB), tumor histology and the reason for testing were lacking. Similarly, 11 and 9 out of 18 elements were present in the reports (N = 4) for tissue and LB, respectively. Elements that pathologists/molecular biologists (N = 18) were missing on the request were the initial activating mutation, previous therapies, a clinical question and testing-related information. For reporting, an item considered important by both groups is the clinical interpretation of the test result. In addition, clinicians (N = 28) indicated that they also wish to read the percentage of neoplastic cells. CONCLUSIONS: Communication flows between the laboratory and the clinician, together with possible pitfalls were identified. Based on the study results, templates for complete requesting and reporting were proposed.

Comparison of microsatellite instability detection by immunohistochemistry and molecular techniques in colorectal and endometrial cancer.

DNA mismatch repair deficiency (dMMR) testing is crucial for diagnosing Lynch syndrome and detection of microsatellite unstable (MSI) tumors eligible for immunotherapy. The aim of this study was to compare the relative diagnostic performance of three molecular MSI assays: polymerase chain reaction (PCR), MSI testing by Idylla and next-generation-sequencing (NGS) on 49 tumor samples (28 colorectal and 21 endometrial adenocarcinomas) versus immunohistochemistry (IHC). Discrepancies were investigated by MLH1 methylation analysis and integrated with germline results if available. Overall, the molecular assays achieved equivalent diagnostic performance for MSI detection with area under the ROC curves (AUC) of respectively 0.91 for Idylla and PCR, and 0.93 for NGS. In colorectal cancers with tumor cell percentages ≥ 30% all three molecular assays achieved 100% sensitivity and specificity (AUC = 1) versus IHC. Also, in endometrial cancers, all three molecular assays showed equivalent diagnostic performance, albeit at a clearly lower sensitivity ranging from 58% for Idylla to 75% for NGS, corresponding to negative predictive values from 78 to 86%. PCR, Idylla and NGS show similar diagnostic performance for dMMR detection in colorectal and endometrial cancers. Molecular MSI analysis has lower sensitivity for dMMR detection in endometrial cancer indicating that combined use of both IHC and molecular methods is recommended.Clinical Trial Number/IRB: B1172020000040, Ethical Committee, AZ Delta General Hospital.

Vascular and Microvascular Endothelial Function in Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Assessment of vascular endothelial function lacks consistency, and microvascular endothelial function has been only partly assessed in heart failure with preserved ejection fraction (HFpEF). METHODS: The study population consisted of 90 patients: 45 had well documented HFpEF, and 45 had hypertension and no history or evidence of heart failure. Patients with hypertension but no heart failure were matched with HFpEF patients for age, sex, and diabetes. They served as control subjects. All patients underwent 2-dimensional Doppler echocardiography and vascular function measurements, including assessment of arterial wave reflections and arterial stiffness, brachial artery flow-mediated dilation (FMD), and forearm cutaneous blood flow with the use of a laser Doppler flow probe at rest and after release of arterial occlusion for 5 minutes. RESULTS: Brachial artery FMD was lower in HFpEF than in control subjects (median (IQR) 3.6 (0.4-7.4) vs. 7.2 (3.2-17.2)%, P = .001). Forearm cutaneous blood flow at rest was similar in HFpEF and control subjects (P = .68). After release of arterial occlusion, forearm cutaneous peak blood flow was lower in HFpEF than in control subjects (P = .03). Estimated aortic systolic and mean blood pressures were similar in HFpEF and control subjects, whereas pulse pressure and pressure augmentation were greater in HFPEF than in control subjects (both P < .05). CONCLUSION: Compared with hypertensive control subjects, patients with HFpEF had a depressed endothelial function in the forearm vasculature and microvasculature.

Antitumoral activity of tyrosine kinase inhibitors in patients with non-small cell lung cancer harbouring rare epidermal growth factor receptor mutations.

INTRODUCTION: Mutations in the epidermal growth factor receptor (EGFR) have been reported as predictive markers of tumour response to tyrosine kinase inhibitors (TKIs) in patients with non-small cell lung cancer (NSCLC). Although the "common" EGFR mutations have been associated with response to EGFR-TKIs, the correlation with response to treatment for many other rare mutations is still unclear. The aim of this study was to investigate the clinical significance of rare and complex mutations, and the efficacy of EGFR-TKIs in this selected group of patients. METHODS: Three hundred and thirty patients with stage IIIB/IV NSCLC (106 females aged 62.5 ± 1.1 years; 224 males aged 68.0 ± 0.6 years) were enrolled in the study. Formalin fixed paraffin embedded tissue samples were screened for mutations using a high resolution melting technique, followed by Sanger sequencing of exons 18-21 of the EGFR-gene. Mutation status was also tested using the Roche Cobas(®) EGFR mutation test. RESULTS: EGFR mutations were detected in 31 tumours (9.4 %). Eleven cases carried novel mutations, six of these patients were treated with erlotinib or gefitinib. A response rate (RR) of 50.0 % was obtained in the group with rare EGFR mutations, the PFS was 3.0 months [standard deviation (STD) = 5.4 months]. The RR to EGFR-TKIs in patients with conventional EGFR mutations was 85 % with a median PFS of 10.5 months (STD = 3.6 months). CONCLUSION: We reported six patients with rare EGFR mutations of unknown clinical significance and their association with EGFR-TKIs. Report of cases harbouring rare mutations can support the decision making progress in this subset of patients.

Sepsis with an Atopobium-like species in a patient with Fournier's gangrene.

Atopobium species are Gram-positive, anaerobic, catalase-negative, fastidious bacteria belonging to the family Coriobacteriaceae. We report the isolation of an Atopobium-like species in a patient with Fournier's gangrene and highlight the role of 16S rRNA gene sequencing in the identification of fastidious organisms in the clinical laboratory.

First case of Pseudoclavibacter bifida bacteremia in an immunocompromised host with chronic obstructive pulmonary disease (COPD).

Pseudoclavibacter spp. are Gram-positive, aerobic, catalase-positive, coryneform bacteria belonging to the family of Microbacteriaceae. Identification of these species with conventional biochemical assays is difficult. This case report of a Pseudoclavibacter bifida bacteremia occurring in an immunocompromised host diagnosed with an acute exacerbation of chronic obstructive pulmonary disease, with a lethal outcome, confirms that this organism may be a human pathogen.

Animal models of organic heart valve disease.

Heart valve disease is a frequently encountered pathology, related to high morbidity and mortality rates in industrialized and developing countries. Animal models are interesting to investigate the causality, but also underlying mechanisms and potential treatments of human valvular diseases. Recently, animal models of heart valve disease have been developed, which allow to investigate the pathophysiology, and to follow the progression and the potential regression of disease with therapeutics over time. The present review provides an overview of animal models of primary, organic heart valve disease: myxoid age-related, infectious, drug-induced, degenerative calcified, and mechanically induced valvular heart disease.

Clinical and echocardiographic correlates of plasma B-type natriuretic peptide levels in patients with aortic valve stenosis and normal left ventricular ejection fraction.

BACKGROUND: Several studies suggest that BNP testing may help define the timing of aortic valve surgery in patients with aortic valve stenosis (AVS) prior onset of overt LV systolic dysfunction. The aim of this study was to identify clinical and echocardiographic correlates of plasma BNP levels in a large cohort of patients with AVS and preserved LV ejection fraction. METHOD AND RESULTS: One hundred thirty-five consecutive patients were prospectively included in the present study (Mean age 73 ± 13 years old, 66 (49%) male). Eighty-nine patients (66%) had severe AVS (aortic valve area <0.6 cm(2) /m(2) BSA). Plasma BNP levels, clinical and comprehensive Doppler echocardiography evaluation was performed in all patients. Independent clinical correlates of plasma BNP levels (R(2) = 0.19) were older age (P < 0.0001) and presence of AVS symptoms (P = 0.004). Independent echocardiographic correlates of plasma BNP levels (R(2) = 0.38) were E/Ea ratio (P = 0.01), LV mass index (P = 0.018), left atrial surface (P < 0.0001) and systolic pulmonary artery pressure (sPAP; P = 0.004). Overall, independent correlates of plasma BNP levels (R(2) = 0.47) were older age (P = 0.001), known coronary artery disease (P = 0.047), increased LV mass index (P = 0.001), left atrial enlargement (P = 0.002), and increased sPAP (P = 0.003). CONCLUSIONS: In patients with AVS and normal LV ejection fraction, plasma BNP predominantly reflects the clinical and echocardiographic consequences of afterload burden imposed on the left ventricle rather than the severity of valve stenosis, per se.

Atherosclerotic-like process in aortic stenosis: activation of the tissue factor-thrombin pathway and potential role through osteopontin alteration.

BACKGROUND: We recently demonstrated in an experimental model the expression of tissue factor (TF) in aortic valves. Thrombin, generated at the end of the TF-initiated coagulation cascade, has been shown to cleave the anti-calcific osteopontin (OSP) liberating the pro-inflammatory OSP N-half. OBJECTIVES: We hypothesized that TF might play an important role in calcific aortic valve stenosis (AS) through thrombin generation and hence evaluated the valvular expression of TF and its inhibitor (TF pathway inhibitor), α-thrombin, OSP and its thrombin-cleaved form (OSP N-half). METHODS: Calcified aortic valves were obtained from patients undergoing valve replacement. Protein expression was evaluated by immunostaining and measured using ELISA kits. Transcripts were analyzed by RT-PCR. RESULTS: We included 52 patients (31 men; age 70 ± 10 years; aortic valve area index 0.35 ± 0.13 cm(2)/m(2)). Immunohistochemistry revealed that TF, OSP and α-thrombin expressions were associated with calcifications at the aortic side of the leaflets. There was an overexpression in calcified regions as compared to non-calcified zones of TF (733.3 ± 70.5 pg/mg vs. 429.4 ± 73.2 pg/mg; p<0.0001), OSP (88.9 ± 12.7 ng/mg vs. 15.0 ± 3.3 ng/mg; p<0.0001) and OSP N-half (0.41 ± 0.06 pmol/mg vs. 0.056 ± 0.011 pmol/mg; p<0.0001). Additionally, both TF and α-thrombin expressions were associated with OSP N-half (r=0.52; p<0.0001 and r=0.33; p=0.019, respectively). CONCLUSIONS: Aortic leaflet TF and α-thrombin expressions and their association with the thrombin-cleaved form of OSP, are a new and important feature of AS. We hypothesize that TF may be involved in the mineralization process of aortic valves by enhancing the generation of the pro-inflammatory OSP N-half through thrombin induction. This pathway deserves further studies to address its implication in the aortic valve calcification process.

Frequency of abdominal aortic aneurysm in patients undergoing coronary artery bypass grafting.

The aims of this study were to clarify the prevalence and the risk factors for unsuspected abdominal aortic aneurysm (AAA) in patients who underwent coronary artery bypass grafting for severe coronary artery disease and to identify the most at risk patients for AAA. Among 217 patients (189 men, mean age 64 +/- 11 years), asymptomatic AAAs, as prospectively identified by echocardiography, were found in 15 patients (6.9%). All patients with AAAs were men and smokers or past smokers. Factors significantly associated by univariate analysis with asymptomatic AAA presence were smoking (p = 0.003), symptomatic peripheral artery disease (p = 0.006), significant carotid artery stenosis (p = 0.007), and larger femoral and popliteal diameters (p = 0.008 and p = 0.0012, respectively). The other classic demographic, clinical, and biologic features were equally distributed among patients. In conclusion, in patients who underwent coronary artery bypass grafting who were men and aged <75 years with smoking histories, the prevalence of AAA was as high as 24% when they had concomitant peripheral arterial disease and/or carotid artery stenosis (vs 4.4% in the absence of either condition, p = 0.007), justifying consideration of AAA screening in this subgroup of in-hospital patients.

Tissue factor pathway inhibitor-2 gene methylation is associated with low expression in carotid atherosclerotic plaques.

BACKGROUND: The tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine-protease inhibitor which is expressed in atherosclerotic plaques. Epigenetic regulation of the TFPI-2 gene, through methylation of CpG islands, has been advocated in cancer. We hypothesized that TFPI-2 gene methylation could regulate TFPI-2 expression in atherosclerosis. METHODS: We used Methylation Specific PCR (MSP) and pyrosequencing in order to identify 18 CpG of the TFPI-2 promoter, in 59 carotid atherosclerotic plaques and 26 control mammary arteries. RESULTS: MSP showed methylation of the TFPI-2 gene (MSP+) in 16 plaques (27%), while no methylation (MSP-) was found in control arteries. Pyrosequencing confirmed that MSP+ plaques presented higher methylation levels than MSP- ones and arteries (p=0.03 and 0.01). Moreover, the TFPI-2 mRNA levels were lower in methylated plaques than in unmethylated ones and than in arteries (p=0.04 and <0.0001). The methylated plaques contained less lipids and macrophage infiltration than unmethylated ones. Their TFPI-2 immunoreactivity was mainly detected in the macrophages located in the media on the adventitial side, rather than in the lipid-rich core. CONCLUSION: Methylation of the TFPI-2 gene takes place in atherosclerotic plaques and is associated with decreased TFPI-2 expression. The place of this process in atherosclerosis progression remains to be investigated.

The endothelium-derived hyperpolarising factor (EDHF) in isolated bovine choroidal arteries.

The present study reports of an endothelium-dependent and NO- and prostanoid-independent relaxation in isolated choroidal arteries, and evaluates the hypothesis of an endothelium-derived hyperpolarising factor (EDHF) playing a role in the choroidal circulation. Choroidal arteries were isolated from bovine eyes and mounted in a small vessel wire-myograph for isometric tension recording. Concentration-response curves for acetylcholine (0.1nM-10microM) were constructed in isolated choroidal arteries contracted with 10microM norepinephrine. Acetylcholine induced a concentration-dependent relaxation in the choroidal arteries. The presence of the NO-synthase inhibitor L-NA and the cyclo-oxygenase inhibitor indomethacin only had a limited effect on this relaxation. All further experiments were performed in the presence of L-NA and indomethacin, in order to study the NO- and prostanoid-independent part of the acetylcholine-relaxations. Both removal of the vascular endothelium or the presence of an increased K(+) concentration in the organ bath abolished the NO- and prostanoid-independent part of the acetylcholine-relaxations. The presence of TEA, a rather non-specific K(+) channel blocker, significantly reduced the acetylcholine-relaxations. Simultaneous application of apamin (an inhibitor of small-conductance Ca(2+)-activated K(+) channels) and charybdotoxin (an inhibitor of intermediate- and large-conductance Ca(2+)-activated K(+) channels) abolished the acetylcholine-induced relaxation and even resulted in a concentration-dependent contraction. Transmembrane potential recordings in isolated choroidal arteries revealed a clear membrane hyperpolarisation in the vascular smooth muscle cells of isolated choroidal arteries. It was therefore concluded that the acetylcholine-induced relaxation of choroidal arteries in the presence of NO-synthase and cyclo-oxygenase inhibitors is mediated by an endothelium-derived hyperpolarising factor. This EDHF seems to be of more importance than endothelium-derived NO or prostanoids.

Methanandamide hyperpolarizes gastric arteries by stimulation of TRPV1 receptors on perivascular CGRP containing nerves.

Endogenous as well as synthetic cannabinoids have potent vasodilatory actions in a variety of vascular preparations. Their precise mechanism of action is as yet unclear, but several studies point to the activation of type 1 vanilloid (TRPV1) receptors on primary afferent perivascular nerves, stimulating the release of calcitonin gene related peptide (CGRP). Given the documented gastroprotective function of these nerves, and the various gastrointestinal effects reported for cannabinoids, we explored a possible link between these systems in the gastric circulation by comparing responses of small gastric arteries to cannabinoids and to calcitonin gene related peptide using conventional microelectrode techniques. Exposure of small gastric arteries to the stable endocannabinoid analogue methanandamide caused a hyperpolarization of the vascular smooth muscle cells, which was completely abolished by the vanilloid receptor antagonist capsazepine (P < 0.01). Exposure to exogenous calcitonin gene related peptide evoked fully reproducible (P > 0.05) hyperpolarizations with similar time course, unaffected by capsazepine. Preincubation with glibenclamide, an inhibitor of ATP-sensitive potassium (KATP) channels, reversed both responses to methanandamide (P < 0.01) and calcitonin gene related peptide (P < 0.05). Similar results were found in rat mesenteric arteries. These findings show that cannabinoids stimulate TRPV1 receptors, presumably causing the release of calcitonin gene related peptide, which hyperpolarizes the smooth muscle cells by activation of KATP channels. Because membrane hyperpolarization is a powerful mediator of vasorelaxation, this novel pathway might prove to be an important mechanism affording gastroprotection.

Characterization of the vasorelaxation to methanandamide in rat gastric arteries.

In the present study, the relaxant effect of the cannabinoid methanandamide was explored in rat gastric arteries. Since in some vessels cannabinoids have been shown to release calcitonin gene-related peptide (CGRP) from perivascular nerves, the influence of methanandamide was compared with that of exogenous CGRP. Methanandamide and CGRP elicited concentration-dependent, endothelium-independent relaxations. Methanandamide-induced relaxations were unaffected by the CB1 receptor antagonist AM251, the CB2 receptor antagonists AM630 and SR144528, and combined pre-exposure to AM251 and SR144528. Pre-exposure to O-1918, an antagonist of a novel nonCB1/nonCB2 cannabinoid receptor, did not influence the relaxations to methanandamide. Capsaicin or capsazepine treatment slightly inhibited methanandamide-induced relaxations. Preincubation with 30 mmol/L extracellular K+ or 3 mmol/L TEA had no significant effect on the responses elicited by methanandamide, but reduced CGRP-induced relaxations. Relaxation to 10(-5) mol/L methanandamide was significantly blunted by Bay K8644 and by preincubation with nifedipine. Furthermore, 10(-5) mol/L methanandamide significantly inhibited CaCl2-induced contractions in norepinephrine-stimulated vessels previously depleted of intra- and extracellular Ca2+. Finally, preincubation with 10(-5) mol/L methanandamide almost completely abolished high K+-induced contractions. These findings suggest that the vasorelaxant action of methanandamide in rat gastric arteries is not mediated by stimulation of known cannabinoid receptors and only partly related to stimulation of TRPV1 receptors on perivascular nerves. At high concentrations, methanandamide might induce relaxation by reducing calcium entry into the smooth muscle cells.

Role of Ba2+-resistant K+ channels in endothelium-dependent hyperpolarization of rat small mesenteric arteries.

In rat small mesenteric arteries, the influence of modulation of basal smooth muscle K+ efflux on the mechanism of endothelium-dependent hyperpolarization was investigated. The membrane potentials of the vascular smooth muscle cells were measured using conventional microelectrode techniques. Incubation of resting arteries with the gap junction uncoupler carbenoxolone (20 micro M) decreased the endothelium-dependent hyperpolarization elicited by a submaximal concentration of acetylcholine (3 micro M) to about 65% of the control. In the presence of Ba2+ (200 micro M), which depolarized the membrane potential by 10 mV, the acetylcholine-induced membrane potential response was doubled in magnitude, reaching values not different from control. Moreover, the hyperpolarization was more resistant to carbenoxolone in these conditions. Finally, both in the absence and in the presence of carbenoxolone, the combined application of Ba2+ and ouabain (0.5 mM) did not abolish the acetylcholine response. These results suggest that gap junctional coupling plays a role in endothelium-dependent hyperpolarization of smooth muscle cells of resting rat small mesenteric arteries. Additionally, these findings show that the hyperpolarization does not rely on activation of inward rectifying K+ channels. Although a minor contribution of Na-K pumping cannot be excluded, the Ba2+ experiments show that the membrane electrical response is mediated by activation of a Ba2+-resistant K+ conductance.