Platelet hyperaggregability and venous thrombosis risk: results from the RETROVE project.

The aim of the study was to determine whether platelet hyperaggregability correlates with short closure times (PFA-100) and if hyperaggregability is associated with the risk of venous thrombosis in a Spanish population. Case--control study (RETROVE project) involving 400 patients with venous thrombosis and 400 healthy controls. We determined platelet aggregation in platelet-rich plasma (PRP) by light transmission aggregometry. Various concentrations of two aggregation agonists [ADP and epinephrine (EPI)] were tested to determine the percentage of maximal aggregation and the percentage area under the curve (AUC). Venous thrombosis risk associated with platelet hyperaggregability was calculated by logistic regression. We estimated the crude and adjusted (by sex and age) odds ratios (OR) for venous thrombosis risk. An agonist concentration of 0.5 µmol/l differentiated between hypo-responders and hyper-responders at the following AUC cut-off values: EPI: the 50th percentile for aggregation with 0.5 µmol/l of EPI (EPI_AUC) was 22.53% (>22.53% = hyper-EPI); the crude risk for venous thrombosis was statistically significant (OR = 1.37; 95% CI 1.03-1.82); ADP: the 75th percentile for aggregation with 0.5 µmol/l of ADP (ADP_AUC) was 29.6% (>29.6% = hyper-ADP), with a significant crude risk for venous thrombosis (OR = 1.44; 95% CI 1.05-1.98). However, after adjustment for confounders (age), the ORs for EPI or ADP aggregation were no longer significant. EPI_AUC and PFA-100 values with the EPI agonist were significantly correlated (R = -0.342, P < 0.01). Only 12% of the PFA-100 values were explained by platelet aggregation. In this case--control study, platelet hyperaggregability was not associated with the risk of developing venous thrombosis.

VAMP8 and serotonin transporter levels are associated with venous thrombosis risk in a Spanish female population. Results from the RETROVE Project.

INTRODUCTION: Platelet hyper-reactivity has been associated with thrombosis and high levels of human vesicle-associated membrane protein 8 (VAMP8) and serotonin transporter (SERT). Two polymorphisms (rs1010 of VAMP8 gene and in SERT gene (SLC6A4)) are associated with arterial thrombosis. AIM: To determine if levels of serotonin, SERT and/or VAMP8 and these polymorphisms are associated with the risk of venous thrombosis. MATERIAL AND METHODS: A total of 324 individuals were included in the RETROVE Study (Riesgo de Enfermedad TROmboembólica VEnosa). VAMP8, SERT and serotonin were determined by ELISA; polymorphisms of SLC6A4 and VAMP8 by polymerase chain reaction (PCR) and real time PCR. The venous thrombotic risk was calculated by a logistic regression method to estimate the crude and adjusted OR (adjusted for sex, age, body mass index and venous thrombosis risk co-factors). RESULTS: Statistically significant high levels of VAMP8 and SERT were found in patients, but not in controls. In contrast, serotonin showed lower levels in patients than in controls. When individuals were studied by gender, only women exhibited a statistically significant difference: the OR for VAMP8 was 3.25 (1.61-6.56 95% CI). The adjusted OR did not change. The OR for SERT was 2.76 (1.36-5.60 95% CI), the adjusted OR was maintained also. For serotonin with OR of 2.62 (1.40-4.92 95% CI), the adjusted OR was not significant. In contrast males did not show significant differences. No statistically differences between patients and controls were found for both polymorphisms. CONCLUSIONS: VAMP8 and SERT levels are associated with venous thrombosis in a female Spanish population.

Recomendaciones del Grupo Catalán de Trombosis (Tromboc@t Working Group) para el tratamiento de los pacientes que reciben anticoagulantes orales directos / Tromboc@t Working Group recommendations for management in patients receiving direct oral anticoagulants

Antecedentes y objetivos: En los últimos años los anticoagulantes orales directos (ACOD) se han convertido en una alternativa a los antagonistas de la vitamina K (AVK) para la prevención del ictus y embolia sistémica en pacientes con fibrilación auricular no valvular (FANV), así como para la prevención y tratamiento de la trombosis venosa profunda. Los ensayos clínicos han demostrado la no inferioridad y la potencial superioridad en comparación con la warfarina, lo cual permite ampliar las opciones de anticoagulación. En nuestro medio, las Unidades de Tratamiento Anticoagulante (UTA) y los Centros de Atención Primaria (CAP) son los encargados de la educación, seguimiento, control de adherencia y del manejo en situaciones especiales de los pacientes anticoagulados. Estas consideraciones han motivado la preparación del presente documento de consenso, que tiene como objetivo establecer recomendaciones que incorporen los hallazgos de la investigación científica a la práctica clínica para mejorar la calidad asistencial en el ámbito de la anticoagulación. Material y métodos: Un grupo de expertos del Grupo Catalán de Trombosis (TROMBOC@T) ha revisado la bibliografía publicada entre 2007 y 2016 para poder establecer recomendaciones basadas en la evidencia clínica. Resultados: Como resultado del proyecto se han establecido un conjunto de recomendaciones de carácter práctico que facilitarán el tratamiento, educación, seguimiento y manejo en situaciones especiales de los pacientes anticoagulados con ACOD. Conclusiones: El aumento progresivo del uso de los ACOD requiere establecer y homogeneizar las directrices de actuación clínica en el paciente anticoagulado con estos antitrombóticos tanto en las UTA como en los CAP

Background and objectives: In recent years, direct oral anticoagulants (DOACs) have become an alternative to vitamin K antagonists (VKA) for the prevention of stroke and systemic embolism in patients with non-valvular atrial fibrillation (NVAF) as well as for prevention and treatment of deep venous thrombosis. Pivotal trials have demonstrated non-inferiority and potential superiority compared to warfarin, which increases the options of anticoagulant treatment. In our setting, the Anticoagulant Treatment Units (ATUs) and Primary Care Centres (PCCs) play an important role in the education, follow-up, adherence control and management in special situations of anticoagulated patients. These considerations have motivated us to elaborate the present consensus document that aims to establish clear recommendations that incorporate the findings of scientific research into clinical practice to improve the quality of care in the field of anticoagulation. Material and methods: A group of experts from the Catalan Thrombosis Group (TROMBOC@T) reviewed all published literature from 2009 to 2016, in order to provide recommendations based on clinical evidence. Results: As a result of the project, a set of practical recommendations have been established that will facilitate treatment, education, follow-up and management in special situations of anticoagulated patients with ACODs. Conclusions: Progressive increase in the use of DOACs calls for measures to establish and homogenise clinical management guidelines for patients anticoagulated with DOACs in ATUs and PCCs

Tromboc@t Working Group recommendations for management in patients receiving direct oral anticoagulants. / Recomendaciones del Grupo Catalán de Trombosis (Tromboc@t Working Group) para el tratamiento de los pacientes que reciben anticoagulantes orales directos.

BACKGROUND AND OBJECTIVES: In recent years, direct oral anticoagulants (DOACs) have become an alternative to vitamin K antagonists (VKA) for the prevention of stroke and systemic embolism in patients with non-valvular atrial fibrillation (NVAF) as well as for prevention and treatment of deep venous thrombosis. Pivotal trials have demonstrated non-inferiority and potential superiority compared to warfarin, which increases the options of anticoagulant treatment. In our setting, the Anticoagulant Treatment Units (ATUs) and Primary Care Centres (PCCs) play an important role in the education, follow-up, adherence control and management in special situations of anticoagulated patients. These considerations have motivated us to elaborate the present consensus document that aims to establish clear recommendations that incorporate the findings of scientific research into clinical practice to improve the quality of care in the field of anticoagulation. MATERIAL AND METHODS: A group of experts from the Catalan Thrombosis Group (TROMBOC@T) reviewed all published literature from 2009 to 2016, in order to provide recommendations based on clinical evidence. RESULTS: As a result of the project, a set of practical recommendations have been established that will facilitate treatment, education, follow-up and management in special situations of anticoagulated patients with ACODs. CONCLUSIONS: Progressive increase in the use of DOACs calls for measures to establish and homogenise clinical management guidelines for patients anticoagulated with DOACs in ATUs and PCCs.

HLA-DQA1 and PLA2R1 polymorphisms and risk of idiopathic membranous nephropathy.

BACKGROUND AND OBJECTIVES: Single nucleotide polymorphisms (SNPs) within HLA complex class II HLA-DQ α-chain 1 (HLA-DQA1) and M-type phospholipase A2 receptor (PLA2R1) genes were identified as strong risk factors for idiopathic membranous nephropathy (IMN) development in a recent genome-wide association study. Copy number variants (CNVs) within the Fc gamma receptor III (FCGR3) locus have been associated with several autoimmune diseases, but their role in IMN has not been studied. This study aimed to validate the association of HLA-DQA1 and PLA2R1 risk alleles with IMN in a Spanish cohort, test the putative association of FCGR3A and FCGR3B CNVs with IMN, and assess the use of these genetic factors to predict the clinical outcome of the disease. DESIGN, SETTINGS, PARTICIPANTS, & MEASUREMENTS: A Spanish cohort of 89 IMN patients and 286 matched controls without nephropathy was recruited between October of 2009 and July of 2012. Case-control studies for SNPs within HLA-DQA1 (rs2187668) and PLA2R1 (rs4664308) genes and CNVs for FCGR3A and FCGR3B genes were performed. The contribution of these polymorphisms to predict clinical outcome and renal function decline was analyzed. RESULTS: This study validated the association of these HLA-DQA1 and PLA2R1 SNPs with IMN in a Spanish cohort and its increased risk when combining both risk genotypes. No significant association was found between FCGR3 CNVs and IMN. These results revealed that HLA-DQA1 and PLA2R1 genotype combination adjusted for baseline proteinuria strongly predicted response to immunosuppressive therapy. HLA-DQA1 genotype adjusted for proteinuria was also linked with renal function decline. CONCLUSION: This study confirms that HLA-DQA1 and PLA2R1 genotypes are risk factors for IMN, whereas no association was identified for FCGR3 CNVs. This study provides, for the first time, evidence of the contribution of these HLA-DQA1 and PLA2R1 polymorphisms in predicting IMN response to immunosuppressors and disease progression. Future studies are needed to validate and identify prognostic markers.

Cost-effective PKHD1 genetic testing for autosomal recessive polycystic kidney disease.

BACKGROUND: Genetic diagnosis of autosomal recessive polycystic kidney disease (ARPKD) is challenging due to the length and allelic heterogeneity of the PKHD1 gene. Mutations appear to be clustered at specific exons, depending on the geographic origin of the patient. We aimed to identify the PKHD1 exons most likely mutated in Spanish ARPKD patients. METHODS: Mutation analysis was performed in 50 ARPKD probands and nine ARPKD-suspicious patients by sequencing PKHD1 exons arranged by their reported mutation frequency. Haplotypes containing the most frequent mutations were analyzed. Other PKD genes (HNF1B, PKD1, PKD2) were sequenced in PKHD1-negative cases. RESULTS: Thirty-six different mutations (concentrated in 24 PKHD1 exons) were detected, giving a mutation detection rate of 86%. The screening of five exons (58, 32, 34, 36, 37) yielded a 54% chance of detecting one mutation; the screening of nine additional exons (3, 9, 39, 61, 5, 22, 26, 41, 57) increased the chance to 76%. The c.9689delA mutation was present in 17 (34%) patients, all of whom shared the same haplotype. Two HNF1B mutations and one PKD1 variant were detected in negative cases. CONCLUSIONS: Establishing a PKHD1 exon mutation profile in a specific population and starting the analysis with the most likely mutated exons might significantly enhance the efficacy of genetic testing in ARPKD. Analysis of other PKD genes might be considered, especially in suspicious cases.

Molecular lymph node staging in bladder urothelial carcinoma: impact on survival.

BACKGROUND: Routine histologic analysis of lymph nodes (LN) for detecting disseminated bladder urothelial carcinoma (BUC) lacks sensitivity. OBJECTIVE: To identify and test potential mRNA markers of BUC dissemination in LN that has been missed by histological analysis, and to compare the performance of selected markers with patients' clinical outcome. DESIGN, SETTING, AND PARTICIPANTS: Microarray data and a literature search were used to identify potential markers expressed in BUC but absent in LN. Five genes were finally selected to be studied by quantitative real-time RT-PCR (qRT-PCR) in 181 and 29 LN from 102 BUC patients and 29 controls, respectively, collected from 2002 to 2004 (median follow-up of 35 mo). MEASUREMENTS: The three most expressed genes plus two additional markers selected from the literature were finally evaluated by qRT-PCR. Gene expression values were statistically compared with histologic results and clinical outcome. RESULTS AND LIMITATIONS: A discriminant analysis showed that the combination of FXYD3 and KRT20 genes yielded a 100% sensitivity and specificity differentiating LN with BUC dissemination from controls. Combined, the expression of both genes allowed the identification of urothelial cells in LN in 20.5% of patients with previous histologically negative LN. These patients did not have a significantly worse survival than those who were negative by qRT-PCR. CONCLUSIONS: Using molecular markers it was possible to improve the sensitivity of LN histologic analysis. However, since 20.5% of patients that reclassified as positive by qRT-PCR did not have a significantly worse survival, we assume lymphadenectomy was important to remove residual disease.

Clinical utility of fluorescent in situ hybridization for the surveillance of bladder cancer patients treated with bacillus Calmette-Guérin therapy.

OBJECTIVE: To evaluate the use of a multiprobe fluorescent in situ hybridization (FISH) assay for determining the response of patients with high-risk superficial bladder tumour (HRSBT) to bacillus Calmette-Guérin (BCG) therapy. METHODS: Bladder washing specimens from 65 HRSBT patients collected before and after BCG therapy were analyzed by FISH. Labelled probes for chromosomes 3, 7, 9, and 17 were used to assess chromosomal abnormalities indicative of malignancy. RESULTS: Fifty-five of 65 (85%) patients had a positive pre-BCG FISH result; 29 (45%) patients had a positive and 36 (55%) had a negative post-BCG FISH result. Patients with a positive post-BCG FISH status had a 2.7 times higher risk for tumour recurrence than patients with a negative post-BCG FISH status (p = 0.017; 95% CI: 1.18-6.15). In addition, patients who maintained a positive FISH status before and after BCG therapy had a risk for tumour recurrence 2.96 times higher than patients whose FISH result changed from positive to negative after BCG (p = 0.02; 95% CI: 1.17-7.54). On the other hand, there was no significant difference between the risk for tumour progression in patients with a positive versus a negative post-BCG FISH result (p = 0.49). CONCLUSIONS: The high percentage of positive pre-BCG FISH results suggests the need for adjuvant therapy in patients with HRSBT after the initial transurethral resection. In addition, patients with a positive post-BCG FISH result were more likely to relapse after therapy. Thus, FISH appears to be useful for the surveillance of patients with HRSBT following BCG therapy.

Utility of a multiprobe fluorescence in situ hybridization assay in the detection of superficial urothelial bladder cancer.

We evaluated the performance of a multiprobe FISH (fluorescence in situ hybridization) assay for noninvasive detection of superficial urothelial carcinoma (UC) in the bladder, in comparison to urinary cytology. Voided urine samples from 74 patients with superficial UC were analyzed by both techniques. Urine samples from 19 patients with muscle-invasive tumors and from 19 healthy control subjects were also studied. For FISH analysis, labeled probes for chromosomes 3, 7, 9, and 17 were used to assess chromosomal abnormalities indicative of malignancy. We found a significant difference between the overall sensitivity of FISH and cytology in superficial UC detection (70.3 versus 35.1%, respectively; P < 0.0001). This significant difference was maintained when superficial UCs were broken down into low grade (52.8 versus 13.9%, respectively; P < 0.0005) and high grade (86.8 versus 55.3%, respectively; P < 0.0015) tumors. Overall specificity was 100% for cytology and 94.7% for FISH (difference not significant). Of patients with suspicious cytology, 69% were positive by FISH. Together, these findings suggest that FISH assay for chromosomes 3, 7, 9, and 17 has a higher sensitivity than cytology and a similar specificity in the detection of superficial UC--which could be useful for reducing some cystoscopies in the accurate follow-up usually performed in these patients.

Association after linkage analysis indicates that homozygosity for the 46C-->T polymorphism in the F12 gene is a genetic risk factor for venous thrombosis.

In a family-based study called GAIT (Genetic Analysis of Idiopathic Thrombophilia) that included a genome-wide scan we demonstrated that a polymorphism (46C-->T) in the F12 locus jointly influences variability of plasma (Factor XII) FXII levels and susceptibility to thrombotic disease. It then became germane to determine the prevalence of the 46C-->T polymorphism and its relative risk of thrombotic disease. We followed up evidence for genetic linkage with a case-control study, including 250 unrelated consecutive Spanish patients suffering from venous thrombotic disease and 250 Spanish subjects matched for sex and age as a controls. We measured FXII levels and genotyped the 46C-->T polymorphism, as well as a number of classical risk factors for thrombotic disease. We confirmed that individuals with different genotypes for this polymorphism showed significant differences in their FXII levels. Most importantly, the mutated T allele in the homozygous state (genotype T/T) was associated with an increased risk of thrombosis (adjusted OR of 4.82; 95% CI 1.5-15.6), suggesting that the polymorphism itself is an independent risk factor for venous thromboembolism. This study confirms that the 46C-->T polymorphism is a genetic risk factor for venous thrombosis in the Spanish population. In addition, our results confirm that a genome-wide scan coupled with a classical case-control association study is an extremely valuable approach to identify DNA variants that affect complex diseases.