Usefulness of High-Sensitivity Cardiac Troponin T to Predict Long-Term Outcome in Patients with Hypertrophic Cardiomyopathy.

Since the first report of an association between cardiac troponin (cTn) and adverse outcome in hypertrophic cardiomyopathy (HD), there is a paucity in confirmative data. We performed a prospective, prespecified 5-year follow-up cohort study of 135 HC patients who participated in a national multicenter project and underwent clinical evaluation, MRI (cine, LGE and T2-weighted imaging) and biomarker assessment (high-sensitivity cTnT (hs-cTnT), N-terminal pro-B-type natriuretic peptide, soluble tumorgenicity suppressor-2, Galectin-3, Growth differentiation factor-15, C-terminal Propeptide of Type I Collagen (CICP)). An elevated hs-cTnT concentration was defined as ≥14ng/L. Follow-up was systematically performed for the primary endpoint: a composite of sudden cardiac death, heart failure related death, stroke-related death, heart failure hospitalization, hospitalization for stroke, spontaneous sustained ventricular tachycardia (VT) or appropriate ICD discharge, and progression to NYHA class III-IV. Elevated hs-cTnT was present in 33 of 135 (24%) HC patients. During a median follow-up of 5.0 years (IQR: 4.9-5.1) 18 patients reached the primary endpoint. Using Cox regression analysis, elevated hs-cTnT was univariately associated with the primary endpoint (HR: 3.4 (95%CI: 1.4-8.7, p=0.009). Also female sex, previous syncope, previous non-sustained VT, reduced LV ejection fraction (<50%) and CICP were associated with the primary endpoint. In multivariable analysis, elevated hs-cTnT remained independently associated with outcome (aHR: 4.7 (95%CI: 1.8-12.6, p = 0.002). In conclusion, this 5-year follow-up study is the first to prospectively confirm the association of elevated hs-cTnT and adverse outcomes. In addition to established clinical variables, cTn seems the biomarker of interest to further improve risk prediction in HC, which should be evaluated in larger prospective registries.

Exercise and myocardial injury in hypertrophic cardiomyopathy.

OBJECTIVE: Troponin and high signal intensity on T2-weighted (HighT2) cardiovascular magnetic resonance imaging (CMRi) are both markers of myocardial injury in hypertrophic cardiomyopathy (HCM). The interplay between exercise and disease development remains uncertain in HCM. We sought to assess the occurrence of postexercise troponin rises and its determinants. METHODS: Multicentre project on patients with HCM and mutation carriers without hypertrophy (controls). Participants performed a symptom limited bicycle test with hs-cTnT assessment pre-exercise and 6 hours postexercise. Pre-exercise CMRi was performed in patients with HCM to assess measures of hypertrophy and myocardial injury. Depending on baseline troponin (< or >13 ng/L), a rise was defined as a >50% or >20% increase, respectively. RESULTS: Troponin rises occurred in 18% (23/127) of patients with HCM and 4% (2/53) in mutation carriers (p=0.01). Comparing patients with HCM with and without a postexercise troponin rise, maximum heart rates (157±19 vs 143±23, p=0.004) and maximal wall thickness (20 mm vs 17 mm, p=0.023) were higher in the former, as was the presence of late gadolinium enhancement (85% vs 57%, p=0.02). HighT2 was seen in 65% (13/20) and 19% (15/79), respectively (p<0.001). HighT2 was the only independent predictor of troponin rise (adjusted odds ratio 7.9; 95% CI 2.7 to 23.3; p<0.001). CONCLUSIONS: Postexercise troponin rises were seen in about 20% of patients with HCM, almost five times more frequent than in mutation carriers. HighT2 on CMRi may identify a group of particularly vulnerable patients, supporting the concept that HighT2 reflects an active disease state, prone to additional injury after a short episode of high oxygen demand.

NT-proBNP correlates with LVEF decline in HER2-positive breast cancer patients treated with trastuzumab.

BACKGROUND: Early identification of cardiac dysfunction by non-invasive imaging in HER2-positive breast cancer patients treated with trastuzumab is challenging. In particular multigated acquisition (MUGA) scan, which is most widely used, is unable to detect subclinical cardiac changes. The use of N-terminal pro-brain natriuretic peptide (NT-proBNP), a serum biomarker of myocardial stress, might improve timely diagnosis. METHODS: This prospective, single-center, cohort study included patients with HER2-positive breast cancer who started trastuzumab therapy. Echocardiography was scheduled at regular intervals every 3 months during one year follow-up for cardiac function monitoring. For research purposes, NT-proBNP was determined at the same time points. Trastuzumab-induced cardiotoxicity (TIC) was the primary study endpoint, defined as a left ventricular ejection fraction (LVEF) < 45%, and/or an absolute decline in LVEF > 10% since inclusion, and/or the incidence of a clinical cardiac event. RESULTS: A total of 135 patients were enrolled between April 2008 and June 2016, with a median age of 54 years (IQR: 47-61). By three-dimensional echocardiography (3DE), the median LVEF at baseline was 62% (IQR: 58-65). At a median of 6 months (IQR: 5-11), 45 patients (33%) reached the study endpoint of TIC. Patients with TIC had a mean change of - 9.5% in LVEF (95% CI -7.2 to - 11.7; p = 0.001) during 1 year of trastuzumab treatment. Both NT-proBNP at baseline (HR 1.04, 95% CI 1.02-1.07; p = 0.003) and LVEF decline during anthracycline treatment prior to the start of trastuzumab (HR 1.16, 95% CI 1.07-1.25; p < 0.001) were independently associated with development of TIC. The level of NT-proBNP during follow-up was associated too with development of TIC (HR 1.06 per 10 pmol/l difference, 95% CI 1.02-1.10; p = 0.008). No steadily or sudden increase in NT-proBNP prior to TIC was observed. CONCLUSIONS: NT-proBNP cannot be used as a surrogate monitoring tool for trastuzumab-induced cardiotoxicity in HER2-positive breast cancer patients during the first year of treatment. Patients showing an LVEF decline during anthracycline pre-treatment appeared vulnerable for trastuzumab-induced cardiotoxicity.

Prediction of Extensive Myocardial Fibrosis in Nonhigh Risk Patients With Hypertrophic Cardiomyopathy.

In nonhigh risk patients with hypertrophic cardiomyopathy (HC), the presence of extensive late gadolinium enhancement (LGEext) at cardiovascular magnetic resonance (CMR) imaging has been proposed as a risk modifier in the decision process for implantable cardioverter defibrillator implantation. With a pretest risk of about 10%, a strategy that alters the likelihood of LGEext could markedly affect efficacious CMR imaging. Our aim was to study the potential of clinical variables and biomarkers to predict LGEext. In 98 HC patients without any clear indication for implantable cardioverter defibrillator implantation, we determined the discriminative values of a set of clinical variables and a panel of biomarkers (hs-cTnT, NTproBNP, GDF-15, and Gal-3, CICP) for LGEext, that is, LGE ≥15% of the left ventricular mass. LGEext was present in 10% (10/98) of patients. The clinical prediction model contained a history of nonsustained ventricular tachycardia, maximal wall thickness and reduced systolic function (c-statistic: 0.868, p <0.001). Of all biomarkers, only hs-cTnT was associated with LGEext, in addition to the improved clinical model of diagnostic accuracy (p = 0.04). A biomarker-only strategy allowed the exclusion of LGEext in half of the cohort, in case of a hs-cTnT concentration less than the optimal cutoff (Youden index; 8 ng/L-sensitivity 100%, specificity 54%). In conclusion, in this nonhigh risk HC cohort, the pretest likelihood of LGEext can be altered using clinical variables and the addition of hs-cTnT. The promising findings with the use of hs-cTnT only call for new initiatives to study its impact on efficacious CMR imaging in a larger HC population, either with or without additional use of clinical variables.

NT-proBNP is associated with mortality and adverse cardiac events in patients with atrial fibrillation presenting to the emergency department.

BACKGROUND: Atrial fibrillation (AF) is the most common cardiac arrhythmia in the emergency department. The CHA2 DS2 -VASc score helps to predict thromboembolic risk; however, the rate of other adverse cardiac events is more difficult to predict. HYPOTHESIS: The biomarker N-terminal pro-brain natriuretic peptide (NT-proBNP) has prognostic value in patients presenting to the emergency department with AF. METHODS: During a 1.5-year period, a prospective study was performed in consecutive patients presenting to the emergency department with AF on the presenting electrocardiogram. At baseline, NT-proBNP was measured. The primary endpoints were all-cause death and major adverse cardiac events (MACE: all-cause mortality, myocardial infarction, or revascularization). RESULTS: A total of 355 patients were included (mean age, 71 years; 55% male). The median duration of follow-up was 2 years. After adjustment for baseline variables, the logNT-proBNP was independently correlated with death (hazard ratio [HR]: 1.54, 95% confidence interval [CI]: 1.18-1.99) and MACE (HR: 1.27, 95% CI: 1.03-1.58). After adjustment for baseline variables, a high NT-proBNP value (>500 pmol/L) was independently correlated with death (HR: 2.26, 95% CI: 1.19-4.28), and for MACE a trend was seen (HR: 1.67, 95% CI: 0.96-2.91) compared with a low value (<250 pmol/L). CONCLUSIONS: In patients presenting to the emergency department with AF, higher NT-proBNP values are independently associated with an increased mortality and MACE. Therefore, this biomarker may be a useful prognostic marker in the management and treatment of these patients.

High T2-weighted signal intensity for risk prediction of sudden cardiac death in hypertrophic cardiomyopathy.

In search of improved risk stratification in hypertrophic cardiomyopathy (HCM), CMR imaging has been implicated as a potential tool for prediction of sudden cardiac death (SCD). In follow-up of the promising results with extensive late gadolinium enhancement (LGE), high signal-intensity on T2-weighted imaging (HighT2) has become subject of interest given its association with markers of adverse disease progression, such as LGE, elevated troponin and non-sustained ventricular tachycardia. In lack of follow-up cohorts, we initiated an exploratory study on the association between HighT2 and the internationally defined risk categories of SCD. In a cohort of 109 HCM patients from a multicenter study on CMR imaging and biomarkers, we estimated the 5-year SCD risk (HCM Risk-SCD model). Patients were categorized as low (< 4%), intermediate (≥ 4-<6%) or high (≥ 6%) risk. In addition, risk categorization according to the ACC/AHA guidelines was performed. HighT2 was present in 27% (29/109). Patients with HighT2 were more often at an intermediate-high risk of SCD according to the European (28 vs. 10%, p = .032) and American guidelines (41 vs. 18%, p = .010) compared to those without HighT2. The estimated 5-year SCD risk of our cohort was 1.9% (IQR 1.3-2.9%), and projected SCD rates were higher in patients with than without HighT2 (2.8 vs. 1.8%, p = .002). In conclusion, HCM patients with HighT2 were more likely to be intermediate-high risk, with projected SCD rates that were 1.5 fold higher than in patients without HighT2. These pilot findings call for corroborative studies with more intermediate-high risk HCM patients and clinical follow-up to assess whether HighT2 may have additional value to current risk stratification.

High T2-weighted signal intensity is associated with elevated troponin T in hypertrophic cardiomyopathy.

OBJECTIVE: Areas of high signal intensity (HighT2) on T2-weighted cardiovascular magnetic resonance (CMR) imaging have been demonstrated in hypertrophic cardiomyopathy (HCM). It has been hypothesised that HighT2 may indicate active tissue injury in HCM. In this context, we studied HighT2 in relation to cardiac troponin. METHODS: Outpatient HCM patients without a history of coronary artery disease underwent CMR imaging at 1.5 T using T2-weighted, cine and late gadolinium enhancement (LGE) imaging to assess HighT2, left ventricular (LV) function, LV mass and the presence and extent of LGE. Highly sensitive cardiac troponin T (hs-cTnT) was assessed as a marker of injury, with hs-cTnT ≥14 and >3 ng/L defined as an elevated and detectable troponin. RESULTS: HighT2 was present in 28% of patients (28/101). An elevated hs-cTnT was present in 54% of patients with HighT2 (15/28) compared with 14% of patients without HighT2 (10/73) (p<0.001). Hs-cTnT was detectable in 96% of patients with HighT2 (27/28) compared with 66% of patients without HighT2 (48/73) (p=0.002). In case of an undetectable hs-cTnT, HighT2 was only seen in 4% (1/26). In addition, the extent of HighT2 was related with increasing hs-cTnT concentrations (Spearman's ρ: 0.42, p<0.001). CONCLUSIONS: In this CMR study of patients with HCM, we observed HighT2 in a quarter of patients, and demonstrated that HighT2 was associated with an elevated hs-cTnT. This observation, combined with the very high negative predictive value of an undetectable hs-cTnT for HighT2, provides supportive evidence for the hypothesis that HighT2 is indicative of recently sustained myocyte injury.

Introduction of a new cobalamin (vitamin B12) assay: lessons from a flawed validation study.

BACKGROUND: Plasma vitamin B12 [cobalamin (Cbl)] concentrations are usually measured as a screening marker for vitamin B12 deficiency. Siemens Healthcare Diagnostics has introduced Cbl assays for various platforms, i.e., the immulite (IML) 2000 and 2500. In our laboratories, regular validation studies for the IML 2500 were conducted and showed acceptable quality specifications. After the introduction of the IML 2500 Cbl assay, clinicians in the department of internal medicine reported an increased frequency of patients with Cbl-concentrations less than 148 pmol/L. METHODS: In order to investigate this claim from the clinicians, we retrospectively analyzed the internal and external quality control (QC) of the Cbl assay. In addition, the monthly patient means for the Cbl assay were analyzed both before and after the introduction of the new Cbl assay. RESULTS: No abnormalities were found in the internal and external QCs. However, the monthly patient means for the Cbl assay showed a statistically significant decrease in cobalamin concentrations. Siemens acknowledged the problems and formulated a new Cbl assay, which was subsequently validated in our laboratories and showed equivocal Cbl results when compared to the IML 2000 Cbl assay. CONCLUSIONS: We report a flawed validation study conducted by the manufacturer that resulted in an undetected analytical problem in the IML 2500 Cbl assay, its subsequent introduction on the market, the final recognition of the poor performance of the assay by our clinicians, and the eventual resolution by the manufacturer. Hence, it emphasizes the utmost importance for thorough comparison between assays over the entire measurement range, even when both assays are produced by the same manufacturer.

Rabip4' is an effector of rab5 and rab4 and regulates transport through early endosomes.

We describe the characterization of an 80-kDa protein cross-reacting with a monoclonal antibody against the human La autoantigen. The 80-kDa protein is a variant of rabip4 with an N-terminal extension of 108 amino acids and is expressed in the same cells. For this reason, we named it rabip4'. rabip4' is a peripheral membrane protein, which colocalized with internalized transferrin and EEA1 on early endosomes. Membrane association required the presence of the FYVE domain and was perturbed by the phosphatidylinositol 3-kinase inhibitor wortmannin. Expression of a dominant negative rabip4' mutant reduced internalization and recycling of transferrin from early endosomes, suggesting that it may be functionally linked to rab4 and rab5. In agreement with this, we found that rabip4' colocalized with the two GTPases on early endosomes and bound specifically and simultaneously to the GTP form of both rab4 and rab5. We conclude that rabip4' may coordinate the activities of rab4 and rab5, regulating membrane dynamics in the early endosomal system.

The human La (SS-B) autoantigen interacts with DDX15/hPrp43, a putative DEAH-box RNA helicase.

The human La (SS-B) autoantigen is an abundantly expressed putative RNA chaperone, functioning in various intracellular processes involving RNA. To further explore the molecular mechanisms by which La functions in these processes, we performed large-scale immunoprecipitations of La from HeLa S100 extracts using the anti-La monoclonal antibody SW5. La-associated proteins were subsequently identified by sequence analysis. This approach allowed the identification of DDX15 as a protein interacting with La. DDX15, the human ortholog of yeast Prp43, is a member of the superfamily of DEAH-box RNA helicases that appeared to interact with La both in vivo and in vitro. The region needed for the interaction with La partly overlaps the DEAH-box domain of DDX15. Immunofluorescence data indicated that endogenous DDX15 accumulates in U snRNP containing nuclear speckles in HEp-2 cells. Surprisingly DDX15 also accumulates in the nucleoli of HEp-2 cells. Moreover, DDX15 and La seem to colocalize in the nucleoli. Regions of DDX15 involved in nuclear, nuclear speckle, and nucleolar localization are located within the N- and C-terminal regions flanking the DEAH-box. RNA coprecipitation experiments indicated that DDX15 is associated with spliceosomal U small nuclear RNAs in HeLa cell extracts. The possible functional implications of the interaction between La and DDX15 are discussed.

Cross-reactivity of the anti-La monoclonal antibody SW5 with early endosome antigen 2.

Coimmunoprecipitation studies with SW5, a frequently used and specific mouse monoclonal antibody (mAb) directed against the human La autoantigen, led to the identification of a functionally unrelated 80 000 MW protein, designated early endosome antigen 2 (EEA2). EEA2 appeared to be directly targeted by mAb SW5. Because an RNA-binding domain, a structural element of La containing the SW5-epitope, was not discernable in the primary structure of EEA2, the SW5-epitope on EEA2 was determined. Coiled-coil region 3 of EEA2 appeared to contain the epitope recognized by SW5. The SW5 epitope regions of La and EEA2 share a limited sequence homology and probably share a higher degree of structural similarity at the tertiary level. Most likely, the most critical determinants for recognition by SW5 reside in elements adopting alpha-helical conformations. These data indicate that the application of specific mAbs to purify and characterize (functionally) interacting proteins can be severely obscured by the cross-reactivity of mAbs with structurally, but not functionally, similar proteins.

Nucleolin associates with a subset of the human Ro ribonucleoprotein complexes.

Ro RNPs are evolutionarily conserved, small cytoplasmic RNA-protein complexes with an unknown function. In human cells, Ro RNPs consist of one of the four hY RNAs and two core proteins: Ro60 and La. Recently, the association of hnRNP I and hnRNP K with particles containing hY1 and hY3 RNAs has been described. The association of three other proteins, namely calreticulin, Ro52 and RoBPI, with (subsets of) the Ro RNPs is still controversial. To gain more insight into the composition and function of the Ro RNPs, we have immunopurified these particles from HeLa cell extracts using monoclonal antibodies against Ro60 and La. Using this approach, we have identified the RNA-binding protein nucleolin as a novel subunit of Ro RNP particles containing hY1 or hY3 RNA, but not hY4 and hY5 RNA. Using an in vitro hY RNA-binding assay we established that the internal pyrimidine-rich loop of hY1 and hY3 RNA is essential for the association of nucleolin. The binding is critically dependent on the presence of all four RNP motifs of nucleolin, but not of the C-terminal RGG-box. Moreover, we demonstrate that, in contrast to nucleolin and hnRNP K, nucleolin and hnRNP I can bind simultaneously to the internal pyrimidine-rich loop of hY1 RNA. We postulate that nucleolin functions in the biogenesis and/or trafficking of hY1 and hY3 RNPs through the nucleolus and subsequent transport to the cytoplasm.