Genome-scale metabolic network of human carotid plaque reveals the pivotal role of glutamine/glutamate metabolism in macrophage modulating plaque inflammation and vulnerability.

BACKGROUND: Metabolism is increasingly recognized as a key regulator of the function and phenotype of the primary cellular constituents of the atherosclerotic vascular wall, including endothelial cells, smooth muscle cells, and inflammatory cells. However, a comprehensive analysis of metabolic changes associated with the transition of plaque from a stable to a hemorrhaged phenotype is lacking. METHODS: In this study, we integrated two large mRNA expression and protein abundance datasets (BIKE, n = 126; MaasHPS, n = 43) from human atherosclerotic carotid artery plaque to reconstruct a genome-scale metabolic network (GEM). Next, the GEM findings were linked to metabolomics data from MaasHPS, providing a comprehensive overview of metabolic changes in human plaque. RESULTS: Our study identified significant changes in lipid, cholesterol, and inositol metabolism, along with altered lysosomal lytic activity and increased inflammatory activity, in unstable plaques with intraplaque hemorrhage (IPH+) compared to non-hemorrhaged (IPH-) plaques. Moreover, topological analysis of this network model revealed that the conversion of glutamine to glutamate and their flux between the cytoplasm and mitochondria were notably compromised in hemorrhaged plaques, with a significant reduction in overall glutamate levels in IPH+ plaques. Additionally, reduced glutamate availability was associated with an increased presence of macrophages and a pro-inflammatory phenotype in IPH+ plaques, suggesting an inflammation-prone microenvironment. CONCLUSIONS: This study is the first to establish a robust and comprehensive GEM for atherosclerotic plaque, providing a valuable resource for understanding plaque metabolism. The utility of this GEM was illustrated by its ability to reliably predict dysregulation in the cholesterol hydroxylation, inositol metabolism, and the glutamine/glutamate pathway in rupture-prone hemorrhaged plaques, a finding that may pave the way to new diagnostic or therapeutic measures.

External Quality Assessment on Molecular Tumor Profiling with Circulating Tumor DNA-Based Methodologies Routinely Used in Clinical Pathology within the COIN Consortium.

BACKGROUND: Identification of tumor-derived variants in circulating tumor DNA (ctDNA) has potential as a sensitive and reliable surrogate for tumor tissue-based routine diagnostic testing. However, variations in pre(analytical) procedures affect the efficiency of ctDNA recovery. Here, an external quality assessment (EQA) was performed to determine the performance of ctDNA mutation detection work flows that are used in current diagnostic settings across laboratories within the Dutch COIN consortium (ctDNA on the road to implementation in The Netherlands). METHODS: Aliquots of 3 high-volume diagnostic leukapheresis (DLA) plasma samples and 3 artificial reference plasma samples with predetermined mutations were distributed among 16 Dutch laboratories. Participating laboratories were requested to perform ctDNA analysis for BRAF exon 15, EGFR exon 18-21, and KRAS exon 2-3 using their regular circulating cell-free DNA (ccfDNA) analysis work flow. Laboratories were assessed based on adherence to the study protocol, overall detection rate, and overall genotyping performance. RESULTS: A broad range of preanalytical conditions (e.g., plasma volume, elution volume, and extraction methods) and analytical methodologies (e.g., droplet digital PCR [ddPCR], small-panel PCR assays, and next-generation sequencing [NGS]) were used. Six laboratories (38%) had a performance score of >0.90; all other laboratories scored between 0.26 and 0.80. Although 13 laboratories (81%) reached a 100% overall detection rate, the therapeutically relevant EGFR p.(S752_I759del) (69%), EGFR p.(N771_H773dup) (50%), and KRAS p.(G12C) (48%) mutations were frequently not genotyped accurately. CONCLUSIONS: Divergent (pre)analytical protocols could lead to discrepant clinical outcomes when using the same plasma samples. Standardization of (pre)analytical work flows can facilitate the implementation of reproducible liquid biopsy testing in the clinical routine.

Dutch National Round Robin Trial on Plasma-Derived Circulating Cell-Free DNA Extraction Methods Routinely Used in Clinical Pathology for Molecular Tumor Profiling.

BACKGROUND: Efficient recovery of circulating tumor DNA (ctDNA) depends on the quantity and quality of circulating cell-free DNA (ccfDNA). Here, we evaluated whether various ccfDNA extraction methods routinely applied in Dutch laboratories affect ccfDNA yield, ccfDNA integrity, and mutant ctDNA detection, using identical lung cancer patient-derived plasma samples. METHODS: Aliquots of 4 high-volume diagnostic leukapheresis plasma samples and one artificial reference plasma sample with predetermined tumor-derived mutations were distributed among 14 Dutch laboratories. Extractions of ccfDNA were performed according to local routine standard operating procedures and were analyzed at a central reference laboratory for mutant detection and assessment of ccfDNA quantity and integrity. RESULTS: Mutant molecule levels in extracted ccfDNA samples varied considerably between laboratories, but there was no indication of consistent above or below average performance. Compared to silica membrane-based methods, samples extracted with magnetic beads-based kits revealed an overall lower total ccfDNA yield (-29%; P < 0.0001) and recovered fewer mutant molecules (-41%; P < 0.01). The variant allelic frequency and sample integrity were similar. In samples with a higher-than-average total ccfDNA yield, an augmented recovery of mutant molecules was observed. CONCLUSIONS: In the Netherlands, we encountered diversity in preanalytical workflows with potential consequences on mutant ctDNA detection in clinical practice. Silica membrane-based methodologies resulted in the highest total ccfDNA yield and are therefore preferred to detect low copy numbers of relevant mutations. Harmonization of the extraction workflow for accurate quantification and sensitive detection is required to prevent introduction of technical divergence in the preanalytical phase and reduce interlaboratory discrepancies.

Integrative multiomics analysis of human atherosclerosis reveals a serum response factor-driven network associated with intraplaque hemorrhage.

BACKGROUND: While single-omics analyses on human atherosclerotic plaque have been very useful to map stage- or disease-related differences in expression, they only partly capture the array of changes in this tissue and suffer from scale-intrinsic limitations. In order to better identify processes associated with intraplaque hemorrhage and plaque instability, we therefore combined multiple omics into an integrated model. METHODS: In this study, we compared protein and gene makeup of low- versus high-risk atherosclerotic lesion segments from carotid endarterectomy patients, as judged from the absence or presence of intraplaque hemorrhage, respectively. Transcriptomic, proteomic, and peptidomic data of this plaque cohort were aggregated and analyzed by DIABLO, an integrative multivariate classification and feature selection method. RESULTS: We identified a protein-gene associated multiomics model able to segregate stable, nonhemorrhaged from vulnerable, hemorrhaged lesions at high predictive performance (AUC >0.95). The dominant component of this model correlated with αSMA- PDGFRα+ fibroblast-like cell content (p = 2.4E-05) and Arg1+ macrophage content (p = 2.2E-04) and was driven by serum response factor (SRF), possibly in a megakaryoblastic leukemia-1/2 (MKL1/2) dependent manner. Gene set overrepresentation analysis on the selected key features of this model pointed to a clear cardiovascular disease signature, with overrepresentation of extracellular matrix synthesis and organization, focal adhesion, and cholesterol metabolism terms, suggestive of the model's relevance for the plaque vulnerability. Finally, we were able to corroborate the predictive power of the selected features in several independent mRNA and proteomic plaque cohorts. CONCLUSIONS: In conclusion, our integrative omics study has identified an intraplaque hemorrhage-associated cardiovascular signature that provides excellent stratification of low- from high-risk carotid artery plaques in several independent cohorts. Further study revealed suppression of an SRF-regulated disease network, controlling lesion stability, in vulnerable plaque, which can serve as a scaffold for the design of targeted intervention in plaque destabilization.

Performance analysis of high-throughput HPV testing on three automated workflows.

Primary high-risk human papillomavirus (hrHPV) DNA testing has been introduced in several countries worldwide, including The Netherlands. The objective of this study was to compare three automated workflow procedures for hrHPV testing of which the hrHPV detection assays meet the international guidelines for HPV testing. To mimic a realistic screening situation, we aimed to process 15 000 residual PreservCyt cervical samples in a period of 3 months. During a 3 months period, four technicians were involved in processing 5000 specimens per month on three automated platforms, (1) Qiagen Digene® HC2 HPV DNA test (HC2, signal amplification); (2) Roche Cobas® HPV test (DNA amplification), and (3) Hologic Aptima® HPV test (RNA amplification). We measured and scored general aspects (time-to-results, hands-on-time (HOT)), maintenance, pre-run, run and post-run aspects, inventory (orders, storage), and number of errors on a scale from 1 to 10. As determined for one complete workflow each, maximum processing capacity and HOT were 296 samples and 2 h:55 m, 282 samples and 3 h:20 m, and 264 samples and 4 h:15 m for Aptima, Cobas, and HC2, respectively. The mean throughput time per run was 5 h:51 m for Cobas in which 94 samples could be processed. For Aptima, the mean throughput time per run was 6 h:30 m for 60 samples. Mean throughput time for HC2 is longer since results were provided on day 2. In this study, the fully automated Aptima workflow scores best with a 7.2, followed by Cobas with a score of 7.1 and HC2 with a score of 5.8. Although all HPV tests used in this comparison meet the international test guidelines, the performance (workflow) characteristics of the assays vary widely. A specific choice of a laboratory for high-throughput testing can be different based on the laboratory's demands, but also hands-on-time, time-to-results/ # samples, maintenance, pre-run, run and post-run parameters, consumables, technical support, and number of errors are important operational factors for the selection of a fully automated workflow for hrHPV testing.

Recommendations for the clinical interpretation and reporting of copy number gains using gene panel NGS analysis in routine diagnostics.

Next-generation sequencing (NGS) panel analysis on DNA from formalin-fixed paraffin-embedded (FFPE) tissue is increasingly used to also identify actionable copy number gains (gene amplifications) in addition to sequence variants. While guidelines for the reporting of sequence variants are available, guidance with respect to reporting copy number gains from gene-panel NGS data is limited. Here, we report on Dutch consensus recommendations obtained in the context of the national Predictive Analysis for THerapy (PATH) project, which aims to optimize and harmonize routine diagnostics in molecular pathology. We briefly discuss two common approaches to detect gene copy number gains from NGS data, i.e., the relative coverage and B-allele frequencies. In addition, we provide recommendations for reporting gene copy gains for clinical purposes. In addition to general QC metrics associated with NGS in routine diagnostics, it is recommended to include clinically relevant quantitative parameters of copy number gains in the clinical report, such as (i) relative coverage and estimated copy numbers in neoplastic cells, (ii) statistical scores to show significance (e.g., z-scores), and (iii) the sensitivity of the assay and restrictions of NGS-based detection of copy number gains. Collectively, this information can guide clinical and analytical decisions such as the reliable detection of high-level gene amplifications and the requirement for additional in situ assays in case of borderline results or limited sensitivity.

Duplex-guided versus Conventional Percutaneous Transluminal Angioplasty of Iliac TASC II A and B Lesion: A Randomized Controlled Trial.

BACKGROUND: Percutaneous transluminal angioplasty (PTA) requires the use of nephrotoxic contrast. Patients with chronic kidney disease are more prone to develop contrast-induced nephropathy after utilization of contrast. Doppler ultrasound (duplex)-guided PTA (DuPTA) is a novel technique and has recently proven to be a successful alternative to conventional PTA in the treatment of iliac stenotic disease, without the use of contrast. In this randomized controlled trial, we evaluated whether DuPTA is as effective as conventional PTA in the treatment of iliac arterial stenotic disease. METHODS: From June 2013 till January 2017, 142 patients with symptomatic peripheral arterial disease (PAD), with significant (>70%) iliac stenotic lesions (both isolated and patients with multilevel disease without treatment of other lesions), were randomized to receive either conventional PTA or DuPTA of the iliac lesion, regardless of renal function. All patients received an ankle-brachial index, arterial mapping with duplex, and magnetic resonance angiography in the pre-operative work-up. Primary end point was procedural success, defined as passing the guidewire through the stenotic lesion and performing a PTA, with or without stenting. Reduction in peak systolic velocity (PSV) of ≥50% after successful PTA was required in the DuPTA group. Angiographic reduction of more than 50% was required in the interventional group. Decrease in PSV was evaluated in both groups 4 weeks post-procedure. RESULTS: Passing of the guidewire through the stenotic lesion was achieved in 96.5% of the DuPTA group and 98.8% of the PTA group (P = 0.34). Although PSV decreased significantly in both groups 4 weeks post-operative, PSV reduction ≥50% was significantly higher in the DuPTA group, respectively 78% vs. 58% in the PTA group (P < 0.01). The utilization of stents was significantly greater in the DuPTA group (52% vs. 18%, P < 0.01). After correction of potential confounders, significant difference in ≥50% PSV reduction remained; technical success did not differ significantly. CONCLUSIONS: DuPTA is a feasible alternative to conventional PTA in the treatment of PAD on the iliac anatomic level. Duplex examination before removal of the guidewire is recommended to evaluate adequate decrease in PSV and identify potential recoil.

Pulmonary Arterial Hypertension and Hereditary Haemorrhagic Telangiectasia.

Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant inherited disease characterised by multisystemic vascular dysplasia. Heritable pulmonary arterial hypertension (HPAH) is a rare but severe complication of HHT. Both diseases can be the result of genetic mutations in ACVLR1 and ENG encoding for proteins involved in the transforming growth factor-beta (TGF-ß) superfamily, a signalling pathway that is essential for angiogenesis. Changes within this pathway can lead to both the proliferative vasculopathy of HPAH and arteriovenous malformations seen in HHT. Clinical signs of the disease combination may not be specific but early diagnosis is important for appropriate treatment. This review describes the molecular mechanism and management of HPAH and HHT.

Follow-up of pulmonary right-to-left shunt in hereditary haemorrhagic telangiectasia.

Pulmonary arteriovenous malformations (PAVMs) are associated with severe neurological complications in hereditary haemorrhagic telangiectasia (HHT). Transthoracic contrast echocardiography (TTCE) is recommended for screening of pulmonary right-to-left shunts (RLS). Although growth of PAVMs is shown in two small studies, no studies on follow-up with TTCE exist.All HHT patients underwent a second TTCE 5 years after initial screening. Patients with a history of PAVM embolisation were excluded. Pulmonary RLS grade on TTCE after 5 years was compared to the grade at screening.200 patients (53.5% female, mean±sd age at screening 44.7±14.1 years) were included. Increase in RLS grade occurred in 36 (18%) patients, of whom six (17%) underwent embolisation. The change in grade between screening and follow-up was not more than one grade. Of patients with nontreatable pulmonary RLS at screening (n=113), 14 (12.4%) underwent embolisation. In patients without pulmonary RLS at initial screening (n=87), no treatable PAVMs developed during follow-up.Within 5 years, no treatable PAVMs developed in HHT patients without pulmonary RLS at initial screening. Increase in pulmonary RLS grade occurred in 18% of patients, and never increased by more than one grade. Of patients with nontreatable pulmonary RLS at initial screening, 12% underwent embolisation.

Allelic imbalance at the HER2/TOP2A locus in breast cancer.

BACKGROUND: Breast cancer is a heterogeneous disease with various histological features and molecular markers. These are utilized for the prediction of clinical outcome and therapeutic decision making. In addition to well established markers such as HER2 overexpression and estrogen and progesterone receptor (ER and PR) status, chromosomal instability is evolving as an important hallmark of cancers. The HER2/TOP2A locus is of great importance in breast cancer. The copy number variability at this locus has been proposed to be a marker for the degree of chromosomal instability. We therefore developed a Single Nucleotide Polymorphism (SNP) assay to evaluate allelic imbalance at the HER2/TOP2A locus in three different entities of primary breast tumors. METHODS: Eleven SNPs were carefully selected and detected by real time PCR using DNA extracted from paired (histologically normal and tumor) paraffin-embedded tissues. Primary breast tumors of 44 patients were included, 15 tumors with HER2 overexpression, 16 triple negative tumors, defined by the absence of HER2 overexpression and a negative ER and PR status and 13 ER and PR positive tumors without HER2 overexpression. As controls, histologically normal breast tissues from 10 patients with no breast tumor were included. RESULTS: Allelic imbalance was observed in 13/15 (87 %) HER2 positive tumors, the remaining 2 being inconclusive. Of the 16 triple negative tumors, 12 (75 %) displayed instability, 3 (19 %) displayed no instability, and 1 was inconclusive. Of the 13 hormone receptor positive tumors, 5 (38 %) displayed allelic imbalance, while 8 did not. CONCLUSIONS: We conclude that the SNP assay is suitable for rapid testing of allelic (im)balance at the HER2/TOP2A locus using paraffin-embedded tissues. Based on allelic imbalance at this locus, both triple negative and ER and PR positive breast tumors can be subcategorized. The clinical relevance of the allelic (im)balance status at the HER2/TOP2A locus in breast cancer is subject of future study. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2086062232155220.

Pulmonary shunt fraction measurement compared to contrast echocardiography in hereditary haemorrhagic telangiectasia patients: time to abandon the 100% oxygen method?

BACKGROUND: The presence of pulmonary right-to-left shunting (RLS) is associated with severe neurological complications from paradoxical embolisation in patients with hereditary haemorrhagic telangiectasia (HHT) and screening is warranted. Pulmonary shunt fraction measurement with the 100% oxygen method can be used for the detection and quantification of functional pulmonary RLS, although transthoracic contrast echocardiography (TTCE) has emerged as the gold standard over the last few years. OBJECTIVE: The aim of this study was to determine the true diagnostic accuracy of the established 100% oxygen method in detecting pulmonary RLS, as compared to TTCE. METHODS: We analysed 628 persons screened for HHT between 2004 and 2010, all of whom underwent TTCE. A quantitative 3-point grading scale was used to differentiate between minimal, moderate or extensive pulmonary RLS on TTCE (grade 1-3, respectively). Additional shunt fraction measurement with the 100% oxygen method was pursued in cases of pO2 <13 or <12 kPa in patients younger or older than 30 years, respectively. A shunt fraction >5% was considered pathological. RESULTS: Both TTCE and the 100% oxygen method were performed in 210 subjects. Although the presence of a pathological shunt fraction correlated with an increased pulmonary shunt grade on TTCE, the 100% oxygen method confirmed a >5% shunt fraction in only 51% of patients with pulmonary RLS on TTCE (14, 20 and 72% for grade 1, 2 and 3, respectively). CONCLUSION: Pulmonary shunt fraction measurement with the 100% oxygen method is not a useful screening technique for the detection of pulmonary RLS in HHT as its sensitivity is too low and large pulmonary shunts on TTCE may remain undetected using this method.

Predicting the size of pulmonary arteriovenous malformations on chest computed tomography: a role for transthoracic contrast echocardiography.

This study aimed to investigate whether pulmonary shunt grade on transthoracic contrast echocardiography (TTCE) predicts the size of pulmonary arteriovenous malformations (PAVMs) on chest computed tomography (CT) and subsequent feasibility for transcatheter embolotherapy. We prospectively included 772 persons with possible or definite hereditary haemorrhagic telangiectasia, who underwent both TTCE and chest CT for screening of PAVMs. A quantitative three-point grading scale was used to classify the pulmonary shunt size on TTCE (grade 1-3). Transcatheter embolotherapy was performed for PAVMs deemed large enough for endovascular closure on chest CT. TTCE documented pulmonary shunting in 510 (66.1%) patients. The positive predictive value of a pulmonary shunt grade 1, 2 and 3 on TTCE for presence of PAVMs on chest CT was 13.4%, 45.3% and 92.5%, respectively (p<0.001). None of the 201 persons with a pulmonary shunt grade 1 on TTCE had PAVMs on chest CT large enough for transcatheter embolotherapy, while 38 (25.3%) and 123 (77.4%) individuals with a pulmonary shunt grade 2 and 3 on TTCE, respectively, underwent endovascular closure of PAVMs. Pulmonary shunt grade on TTCE predicts the size of PAVMs on chest CT and their feasibility for subsequent transcatheter embolotherapy. Chest CT can be safely withheld from all persons with a pulmonary shunt grade 1 on TTCE, as any PAVM found in these subjects will be too small for transcatheter embolotherapy.

Role of compression stockings after endovenous laser therapy for primary varicosis.

OBJECTIVE: The objective of this prospective, randomized trial was to evaluate the effect of compression stockings after endovenous laser therapy (EVLT) for insufficiency of the great saphenous vein. Effect evaluation was focused on differences in postoperative pain within 6 weeks. METHODS: The study randomized 111 patients undergoing EVLT for duplex ultrasound-confirmed great saphenous vein insufficiency (C2-4EPASPR) to receive either 2 weeks of elastic stockings (class II, thigh length) or no elastic stockings after an initial 24-hour period of wearing bandages. Patients registered pain scores (primary outcome) and use of analgesics in a diary on a visual analog scale. At 2 days, 14 days, and 6 weeks after the procedure, leg circumferences, Aberdeen Varicose Vein Questionnaire scores, RAND 36-Item Health Survey scores, patient satisfaction (5-point scale), time before returning to work, and adverse events (secondary outcomes) were evaluated. RESULTS: In the 79 patients (15 men and 64 women; mean age, 52 years) who completed the treatment and follow-up, small but significant differences in pain scores were observed during the first week after laser surgery, with more favorable scores in the group wearing stockings. Patients not wearing stockings used more analgesics than did patients wearing stockings (P < .05). Patients wearing stockings reported a statistically significantly higher score of satisfaction at 2 days (4.44 vs 4.15) and 6 weeks (4.59 vs 4.18), although the absolute differences were small. No significant differences were found regarding time to return to work, Aberdeen Varicose Vein Questionnaire scores, RAND 36-Item Health Survey scores, leg circumference measurements, and risk of complications. CONCLUSIONS: Wearing of postoperative stockings for 2 weeks after an initial 24-hour period of wearing bandages results in a small but significant reduction of postoperative pain and use of analgesics compared with not wearing compression stockings after EVLT for great saphenous vein insufficiency.

Polymorphisms in ACVRL1 and endoglin genes are not associated with sporadic and HHT-related brain AVMs in Dutch patients.

We aimed to replicate the association of the IVS3-35A>G polymorphism in the activin receptor-like kinase (ACVRL) 1 gene and the 207G>A polymorphism in the endoglin (ENG) gene with sporadic brain arteriovenous malformations (BAVM) in Dutch BAVM patients. In addition, we assessed whether these polymorphisms contribute to the risk of BAVM in patients with hereditary haemorrhagic telangiectasia type 1 (HHT1). We genotyped 143 Dutch sporadic BAVM patients and 360 healthy volunteers for four variants in the ACVRL1 gene including IVS3-35A>G and two variants in the ENG gene including 207G>A. Differences in allele and genotype frequencies between sporadic BAVM patients and controls and their combined effect were analysed with a likelihood ratio test. Furthermore, we compared the allele and genotype frequencies between 24 HHT1 patients with a BAVM with those of a relative with HHT1 without a BAVM in a matched pair analysis using Wilcoxon signed rank test. No significant differences in allele frequency were found between sporadic BAVM cases and controls or between HHT1 patients with and without BAVM for any of the polymorphisms or the combination of ACVRL1 and ENG polymorphisms. Meta-analysis of the current and the two previous studies for the ACVRL1 IVS3-35A polymorphism showed a persisting association between the ACVRL1 IVS3-35A polymorphism and risk of sporadic BAVM (odds ratio, 1.86; 95% CI: 1.32-2.61, p<0.001). We did not replicate the previously found association between a polymorphism in ACVRL1 IVS3-35A>G and BAVM in Dutch patients. However, meta-analysis did not rule out a possible effect.

Role of transthoracic contrast echocardiography in the clinical diagnosis of hereditary hemorrhagic telangiectasia.

BACKGROUND: Hereditary hemorrhagic telangiectasia (HHT) can be diagnosed according to the four clinical Curaçao criteria, including the presence of pulmonary arteriovenous malformations (PAVMs). In the past few years, transthoracic contrast echocardiography (TTCE) replaced chest high-resolution CT (HRCT) imaging for the screening of PAVMs. The objective of this study was to determine whether the presence of any pulmonary shunt on TTCE can be accepted as a new clinical Curaçao criterion in diagnosing HHT. METHODS: Between 2004 and 2012, we included 487 first-degree relatives of known HHT-causing mutation carriers who underwent both TTCE and chest HRCT imaging to screen for PAVMs. A quantitative three-point grading scale was used to differentiate among minimal, moderate, or extensive pulmonary shunt on TTCE (grade 1-3). Genetic testing was performed in all people and considered the gold standard for the diagnosis of HHT. RESULTS: Chest HRCT imaging demonstrated PAVMs in 114 of 218 patients (52.3%) with a pulmonary shunt on TTCE. The addition of any pulmonary shunt on TTCE to the current clinical Curaçao criteria increased the number of positive criteria in 92 of 487 individuals (18.9%), which increased the sensitivity in diagnosing HHT from 88% to 94% at the expense of a decreased specificity from 74% to 70%. Accepting only pulmonary shunt grades ≥ 2 on TTCE as a diagnostic criterion for HHT enhanced the number of positive criteria in 30 (6.2%) individuals, which led to an increased sensitivity of 90% with no decrease in specificity (74%). CONCLUSIONS: The addition of only pulmonary shunt grades ≥ 2 on TTCE to the current clinical Curaçao criteria increases its sensitivity without affecting specificity in the diagnosis of HHT.

Hereditary hemorrhagic telangiectasia: how accurate are the clinical criteria?

The clinical diagnosis of hereditary hemorrhagic telangiectasia (HHT) is based on the Curaçao criteria. Three out of four criteria are required for a definite clinical diagnosis HHT, two criteria are considered "possible" HHT, and 0 or 1 criterion makes the diagnosis unlikely. However, these consensus diagnostic criteria have not been validated. We report on the diagnostic accuracy of the clinical criteria. A total of 450 consecutive persons ≥16 years of age were screened for HHT between May 2004 and September 2009, including a chest CT to screen for pulmonary arteriovenous malformations (AVMs). We selected 263 first-degree relatives of disease-causing mutation carriers who underwent mutation analysis. Genetic test results were considered the gold standard. The family mutation was present in 186 patients (mean age 42.9 ± 14.6 yr; 54.8% female). A clinical diagnosis was definite, "possible", and unlikely in 168 (90.3%), 17 (9.1%), and 1 (0.5%) patient, respectively. In 77 persons the family mutation was absent (mean age 37.1 ± 12.3 yr, 59.7% female). In this group a clinical diagnosis was definite, possible, and unlikely in 0, 35 (45.5%), and 42 (54.5%) persons, respectively. The positive predictive value of a definite clinical diagnosis was 100% (95% CI 97.8-100), the negative predictive value of an unlikely diagnosis 97.7% (95% CI 87.9-99.6). Of 52 patients with "possible" HHT, 17 (32.7%) displayed an HHT-causing mutation. The Curaçao clinical criteria have a good diagnostic performance. Genetic testing is particularly helpful in patients with a "possible" clinical diagnosis HHT.

Grade of pulmonary right-to-left shunt on contrast echocardiography and cerebral complications: a striking association.

BACKGROUND: A pulmonary right-to-left shunt (RLS) carries the risk of cerebral paradoxical embolization and severe neurologic complications. Recognizing patients at risk is important to facilitate appropriate management strategies, but a direct relation between pulmonary shunt size and risk of complications remains controversial. This study evaluated the potential relation between pulmonary shunt grade on transthoracic contrast echocardiography (TTCE) and prevalence of cerebral manifestations in patients screened for hereditary hemorrhagic telangiectasia (HHT). METHODS: We conducted a two-center, cross-sectional study of all consecutive patients screened for HHT between 2004 and 2011. Pulmonary shunt grading on TTCE (grade 0, no microbubbles; grade 1, < 30 microbubbles; grade 2, 30-100 microbubbles; grade 3, > 100 microbubbles) was performed according to contrast opacification of the left ventricle. Cerebral complications were defined as ischemic stroke, transient ischemic attack, or brain abscess diagnosed by a neurologist and confirmed by appropriate imaging techniques. RESULTS: A pulmonary RLS was present in 530 out of 1,038 patients (51.1%; mean age, 44.3 ± 15.6 years; 58.6% women). The presence of a cerebral manifestation (n = 51) differed significantly among pulmonary shunt grades on TTCE: 1.4%, 0.4%, 6.5%, and 20.9% for grades 0, 1, 2 and 3, respectively. A pulmonary shunt grade 1 was not associated with an increased prevalence of cerebral manifestations (OR, 0.44; 95% CI, 0.05-4.13; P = .47), whereas pulmonary shunt grade 2 (OR, 4.78; 95% CI, 1.14-20.0; P = .03) and grade 3 (OR, 10.4; 95% CI, 2.4-45.3; P = .002) were both independent predictors for the prevalence of a cerebral ischemic event or brain abscess. CONCLUSIONS: The pulmonary RLS grade on TTCE is strongly associated with the prevalence of cerebral complications in patients screened for HHT.

Single nucleotide polymorphism (SNP)-based loss of heterozygosity (LOH) testing by real time PCR in patients suspect of myeloproliferative disease.

During tumor development, loss of heterozygosity (LOH) often occurs. When LOH is preceded by an oncogene activating mutation, the mutant allele may be further potentiated if the wild-type allele is lost or inactivated. In myeloproliferative neoplasms (MPN) somatic acquisition of JAK2V617F may be followed by LOH resulting in loss of the wild type allele. The occurrence of LOH in MPN and other proliferative diseases may lead to a further potentiating the mutant allele and thereby increasing morbidity. A real time PCR based SNP profiling assay was developed and validated for LOH detection of the JAK2 region (JAK2LOH). Blood of a cohort of 12 JAK2V617F-positive patients (n=6 25-50% and n=6>50% JAK2V617F) and a cohort of 81 patients suspected of MPN was stored with EDTA and subsequently used for validation. To generate germ-line profiles, non-neoplastic formalin-fixed paraffin-embedded tissue from each patient was analyzed. Results of the SNP assay were compared to those of an established Short Tandem Repeat (STR) assay. Both assays revealed JAK2LOH in 1/6 patients with 25-50% JAK2V617F. In patients with >50% JAK2V617F, JAK2LOH was detected in 6/6 by the SNP assay and 5/6 patients by the STR assay. Of the 81 patients suspected of MPN, 18 patients carried JAK2V617F. Both the SNP and STR assay demonstrated the occurrence of JAK2LOH in 5 of them. In the 63 JAK2V617F-negative patients, no JAK2LOH was observed by SNP and STR analyses. The presented SNP assay reliably detects JAK2LOH and is a fast and easy to perform alternative for STR analyses. We therefore anticipate the SNP approach as a proof of principle for the development of LOH SNP-assays for other clinically relevant LOH loci.

Sensitive detection and quantification of the JAK2V617F allele by real-time PCR blocking wild-type amplification by using a peptide nucleic acid oligonucleotide.

A single G-to-T missense mutation in the gene for the JAK2 tyrosine kinase, leading to a V617F amino acid substitution, is commonly found in several myeloproliferative neoplasms. Reliable quantification of this mutant allele is of increasing clinical and therapeutic interest in predicting and diagnosing this group of neoplasms. Because JAK2V617F is somatically acquired and may be followed by loss of heterozygosity, the percentage of mutant versus wild-type DNA in blood can vary between 0% and almost 100%. Therefore, we developed a real-time PCR assay for detection and quantification of the low-to-high range of the JAK2V617F allele burden. To allow the assay to meet these criteria, amplification of the wild-type JAK2 was blocked with a peptide nucleic acid oligonucleotide. JAK2V617F patient DNA diluted in JAK2 wild-type DNA could be amplified linearly from 0.05% to 100%, with acceptable reproducibility of quantification. The sensitivity of the assay was 0.05% (n = 3 of 3). In 9 of 100 healthy blood donors, a weak positive/background signal was observed in DNA isolated from blood, corresponding to approximately 0.01% JAK2V617F allele. In one healthy individual, we observed this signal in duplicate. The clinical relevance of this finding is not clear. By inhibiting amplification of the wild-type allele, we developed a sensitive and linear real-time PCR assay to detect and quantify JAK2V617F.

Relation between migraine and size of echocardiographic intrapulmonary right-to-left shunt.

An increased prevalence of intrapulmonary right-to-left shunt (RLS) has been shown in patients with migraine. The aim of this study was to determine whether the size of intrapulmonary RLS was associated with migraine with aura (MA+) and migraine without aura (MA-) in subjects screened for hereditary hemorrhagic telangiectasia. A total of 462 consecutive subjects were screened for hereditary hemorrhagic telangiectasia and underwent transthoracic contrast echocardiography. A pulmonary shunt was established when contrast appeared in the left atrium after 4 cardiac cycles. Shunt size was assessed semiquantitatively as small (<30 microbubbles), moderate (30 to 100 microbubbles), or large (>100 microbubbles). A headache questionnaire was completed by 420 subjects (91%). Two independent neurologists diagnosed migraine according to the International Headache Society criteria. Of 420 screened subjects (mean age 43.4 ± 15.4 years, 61.4% women), 44 (10.5%) had MA+ and 45 (10.7%) had MA-. MA+ was an independent predictor for an intrapulmonary RLS (odds ratio [OR] 2.96, 95% confidence interval [CI] 1.36 to 6.47, p=0.006) in multivariate analysis. MA- was not correlated with RLS (OR 1.21, 95% CI 0.56 to 2.64, p=0.60). When comparing patients with MA+ to those without migraine in a multivariate analysis, the presence of an intrapulmonary shunt predicted MA+ (OR 2.5, 95% CI 1.2 to 5.2, p=0.01), as did female gender (OR 3.15, 95% CI 1.29 to 7.65, p<0.01). The correlation of MA+ and RLS could be entirely attributed to large intrapulmonary shunts (OR 7.61, 95% CI 3.11 to 18.61, p<0.001), as small (OR 0.6, 95% CI 0.13 to 2.78, p=0.52) and moderate (OR 1.33, 95% CI 0.35 to 5.02, p=0.68) shunts did not appear to be risk factors for MA+. In conclusion, patients with large intrapulmonary RLS have an increased risk for MA+.