Seroepidemiology of tuberculosis in sheep in southern Spain.

Tuberculosis (TB) is a multi-host infectious disease caused by members of the Mycobacterium tuberculosis complex (MTC). In Mediterranean ecosystems, where multiple animal hosts of TB are present, identifying the role of the different species involved in the epidemiology of TB is a key point to be able to implement proper control measures. Sheep are susceptible to MTC infection but have traditionally been considered a spillover host. However, the occurrence of outbreaks involving sheep in recent years evidences the need to better understand the role of this small ruminant species in the epidemiology of the disease. Here, we aimed to determine the seroprevalence and risk factors associated with MTC seropositivity in sheep in Andalusia (southern Spain), a region with one of the highest prevalence of MTC infection in both cattle and wild ungulates. A total of 2266 sheep from 83 flocks were tested for antibodies against MTC using an in-house indirect ELISA. Anti-MTC antibodies were detected in 16 (0.7%) of the 2266 sheep (adjusted true prevalence 0.29%, 95% posterior probability interval 0.01-1.05). Seropositivity was found in 14.5% (12/83; 95%CI: 6.9-22.0) of the sheep farms analyzed. A semi-extensive management system was identified as a risk factor associated with MTC seropositivity in sheep farms (OR = 3.7; p < 0.038; 95%CI: 1.1-12.4) in the study area. To the best of the authors' knowledge, this is the first active TB surveillance study carried out to assess MTC exposure in sheep. Our results indicate MTC circulation in sheep farms in southern Spain. However, the low individual seroprevalence obtained suggests that sheep may play a limited role in the epidemiology of TB in this region. Serosurveillance programs could be a valuable tool to detect MTC circulation in sheep in risk scenarios or target farms, in order to optimize control measures on TB animal in multi-host Mediterranean ecosystems.

Epidemiological surveillance of Schmallenberg virus in small ruminants in southern Spain.

Schmallenberg virus (SBV) is an emerging Culicoides-borne Orthobunyavirus that affects ruminant species. Between 2011 and 2013, it was responsible for a large-scale epidemic in Europe. In the present study, we aimed to determine the seroprevalence, spatial distribution and risk factors associated with SBV exposure in sheep and goats in the region where the first Schmallenberg disease outbreak in Spain was reported. Blood samples from 1,796 small ruminants from 120 farms were collected in Andalusia (southern Spain) between 2015 and 2017. Antibodies against SBV were detected in 536 of 1,796 animals (29.8%; 95%CI: 27.7-32.0) using a commercial blocking ELISA. The individual seroprevalence according to species was 31.1% (280/900; 95%CI: 28.1-34.1) in sheep and 28.6% (256/896; 95%CI: 25.6-31.5) in goats. The farm prevalence was 76.7% (95%CI: 69.1-84.2). Seropositivity to SBV was confirmed in both sheep and goats in all provinces by virus neutralization test. Two significant (p < .001) spatial clusters of high seroprevalence were identified. The generalized estimating equation analysis showed that management system (extensive), temperature (>14ºC) and altitude (<400 metres above sea level) were risk factors associated with SBV exposure in small ruminants. Our results highlight widespread but not homogeneous circulation of SBV in small ruminant populations in Spain.

POT1 and Damage Response Malfunction Trigger Acquisition of Somatic Activating Mutations in the VEGF Pathway in Cardiac Angiosarcomas.

Background Mutations in the POT1 gene explain abnormally long telomeres and multiple tumors including cardiac angiosarcomas (CAS). However, the link between long telomeres and tumorigenesis is poorly understood. Methods and Results Here, we have studied the somatic landscape of 3 different angiosarcoma patients with mutations in the POT1 gene to further investigate this tumorigenesis process. In addition, the genetic landscape of 7 CAS patients without mutations in the POT1 gene has been studied. Patients with CAS and nonfunctional POT1 did not repress ATR (ataxia telangiectasia RAD3-related)-dependent DNA damage signaling and showed a constitutive increase of cell cycle arrest and somatic activating mutations in the VEGF (vascular endothelial growth factor)/angiogenesis pathway (KDR gene). The same observation was made in POT1 mutation carriers with tumors different from CAS and also in CAS patients without mutations in the POT1 gene but with mutations in other genes involved in DNA damage signaling. Conclusions Inhibition of POT1 function and damage-response malfunction activated DNA damage signaling and increased cell cycle arrest as well as interfered with apoptosis, which would permit acquisition of somatic mutations in the VEGF/angiogenesis pathway that drives tumor formation. Therapies based on the inhibition of damage signaling in asymptomatic carriers may diminish defects on cell cycle arrest and thus prevent the apoptosis deregulation that leads to the acquisition of driver mutations.

Epidemiological surveillance of Mycobacterium tuberculosis complex in extensively raised pigs in the south of Spain.

The role of domestic pigs in the epidemiology of Mycobacterium tuberculosis complex (MTC) is considered to be limited due to the characteristics of intensive production systems. However, in southwestern Spain, Iberian pigs are usually raised under extensive management systems, sharing their habitat with other domestic and wild species, some of which may act as reservoirs of MTC. Our objective was to determine the seroprevalence, risk factors, spatial distribution and spoligotypes of MTC circulating in extensively farmed pigs in Andalusia (southern Spain), a region with a high prevalence of tuberculosis in both cattle and wild boar populations. Serum samples from 3622 extensively-raised Iberian pigs from 129 randomly selected farms were tested for antibodies against MTC using an indirect (P22) ELISA. Antibodies to MTC were detected in 82 pigs (2.3%; 95%CI: 1.8-2.8%). Seropositivity was significantly higher in sows (3.7%) than in fattening pigs (1.7%) (P = 0.0001). Herd prevalence was 24.8% (95%CI: 17.4-32.3%). Two risk factors were associated with MTC seropositivity on farms: herd size (higher seroprevalence on larger farms) (OR=1.001; 95%CI: 1.000-1.002), and the presence of neighboring goat flocks (OR = 7.345; 95%CI: 1.464-36.848). Two statistically significant spatial clusters (P < 0.001) were identified in the north-west of Andalusia. A total of 25 different MTC spoligotypes were isolated in pigs bred extensively in the study area. Based on the serological and molecular results obtained in the current study, it is possible that extensively raised Iberian pigs may act as an MTC reservoir in Mediterranean ecosystems. The high herd prevalence, as well as the identification of significant spatial clusters, indicates widespread, but not homogenous MTC circulation among extensively-managed pig farms. Risk-based surveillance and control programs should be implemented on this type of farms in Spain.

In vivo phosphoproteomics reveals kinase activity profiles that predict treatment outcome in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) lacks prognostic and predictive markers. Here, we use high-throughput phosphoproteomics to build a functional TNBC taxonomy. A cluster of 159 phosphosites is upregulated in relapsed cases of a training set (n = 34 patients), with 11 hyperactive kinases accounting for this phosphoprofile. A mass-spectrometry-to-immunohistochemistry translation step, assessing 2 independent validation sets, reveals 6 kinases with preserved independent prognostic value. The kinases split the validation set into two patterns: one without hyperactive kinases being associated with a >90% relapse-free rate, and the other one showing ≥1 hyperactive kinase and being associated with an up to 9.5-fold higher relapse risk. Each kinase pattern encompasses different mutational patterns, simplifying mutation-based taxonomy. Drug regimens designed based on these 6 kinases show promising antitumour activity in TNBC cell lines and patient-derived xenografts. In summary, the present study elucidates phosphosites and kinases implicated in TNBC and suggests a target-based clinical classification system for TNBC.

Deep sequencing reveals microRNAs predictive of antiangiogenic drug response.

The majority of metastatic renal cell carcinoma (RCC) patients are treated with tyrosine kinase inhibitors (TKI) in first-line treatment; however, a fraction are refractory to these antiangiogenic drugs. MicroRNAs (miRNAs) are regulatory molecules proven to be accurate biomarkers in cancer. Here, we identified miRNAs predictive of progressive disease under TKI treatment through deep sequencing of 74 metastatic clear cell RCC cases uniformly treated with these drugs. Twenty-nine miRNAs were differentially expressed in the tumors of patients who progressed under TKI therapy (P values from 6 × 10-9 to 3 × 10-3). Among 6 miRNAs selected for validation in an independent series, the most relevant associations corresponded to miR-1307-3p, miR-155-5p, and miR-221-3p (P = 4.6 × 10-3, 6.5 × 10-3, and 3.4 × 10-2, respectively). Furthermore, a 2 miRNA-based classifier discriminated individuals with progressive disease upon TKI treatment (AUC = 0.75, 95% CI, 0.64-0.85; P = 1.3 × 10-4) with better predictive value than clinicopathological risk factors commonly used. We also identified miRNAs significantly associated with progression-free survival and overall survival (P = 6.8 × 10-8 and 7.8 × 10-7 for top hits, respectively), and 7 overlapped with early progressive disease. In conclusion, this is the first miRNome comprehensive study, to our knowledge, that demonstrates a predictive value of miRNAs for TKI response and provides a new set of relevant markers that can help rationalize metastatic RCC treatment.

Analysis of Paired Primary-Metastatic Hormone-Receptor Positive Breast Tumors (HRPBC) Uncovers Potential Novel Drivers of Hormonal Resistance.

We sought to identify genetic variants associated with disease relapse and failure to hormonal treatment in hormone-receptor positive breast cancer (HRPBC). We analyzed a series of HRPBC with distant relapse, by sequencing pairs (n = 11) of tumors (primary and metastases) at >800X. Comparative genomic hybridization was performed as well. Top hits, based on the frequency of alteration and severity of the changes, were tested in the TCGA series. Genes determining the most parsimonious prognostic signature were studied for their functional role in vitro, by performing cell growth assays in hormonal-deprivation conditions, a setting that mimics treatment with aromatase inhibitors. Severe alterations were recurrently found in 18 genes in the pairs. However, only MYC, DNAH5, CSFR1, EPHA7, ARID1B, and KMT2C preserved an independent prognosis impact and/or showed a significantly different incidence of alterations between relapsed and non-relapsed cases in the TCGA series. The signature composed of MYC, KMT2C, and EPHA7 best discriminated the clinical course, (overall survival 90,7 vs. 144,5 months; p = 0.0001). Having an alteration in any of the genes of the signature implied a hazard ratio of death of 3.25 (p<0.0001), and early relapse during the adjuvant hormonal treatment. The presence of the D348N mutation in KMT2C and/or the T666I mutation in the kinase domain of EPHA7 conferred hormonal resistance in vitro. Novel inactivating mutations in KMT2C and EPHA7, which confer hormonal resistance, are linked to adverse clinical course in HRPBC.

Optimizing sharing of hospital biobank samples.

Implementing technical guidelines and standards as well as ways to boost cooperation should facilitate sharing of hospital biobank samples.

HIF1A expression in localized prostate cancer treated with dose escalation radiation therapy.

PURPOSE: To analyze the expression of hypoxia inducible factor 1 alpha (HIF1A) and its correlation with clinical outcome in men with localized prostate cancer (PC) treated with dose escalation radiotherapy (RT) and androgen deprivation (AD). METHODS: Between 1996 and 2004, 129 PC patients who had diagnostic biopsies pre-treatment and 24-36 months following RT were enrolled in this study. Median follow-up was 129 months. Suitable archival diagnostic tissue was obtained from 86 patients. Correlation analysis was done to assess association between HIF1A expression and clinical outcome. RESULTS: HIF1A expression was observed in 25/86 (29%) of diagnostic biopsies, and in 5/14 (36%) of post-RT biopsies. No significant association was noted between HIF1A expression and clinical and treatment parameters. We also failed to show a significant correlation between HIF1A overexpression and outcome. A borderline significant relationship was observed between expression of HIF1A and overall survival (OS) (HR 0.03, p=0.08). CONCLUSION: To our knowledge this is the first study assessing the pattern of change of HIF1A staining in biopsies of patients prior and following treatment. While we did not find significant variations in the expression of HIF1A following radio-hormone therapy, a high HIF1A expression was unexpectedly associated with a borderline improved OS.

Predictive value of PAK6 and PSMB4 expression in patients with localized prostate cancer treated with dose-escalation radiation therapy and androgen deprivation therapy.

PURPOSE: The present study analyzed the expression by immunochemistry of the novel markers P21-activated protein kinase 6 (PAK6) and proteasome beta-4 subunit (PSMB4) in men with localized prostate cancer (PC) who were treated with dose-escalation radiotherapy (RT) and androgen deprivation therapy. MATERIALS AND METHODS: Between 1996 and 2004, a cohort of 129 patients with PC who underwent diagnostic biopsies pretreatment and 24 to 36 months following RT were enrolled in this study. Suitable archival diagnostic tissue was obtained from 89 patients. Median follow-up was 129 months (48-198). Correlation analysis was done to assess association between PAK6 and PSMB4 expression and clinical outcome. RESULTS: PAK6 and PSMB4 were expressed in the cytoplasm in 62% and 96.7% of diagnostic biopsies, respectively. Increased staining for PAK6 was significantly (P = 0.04) correlated with higher Gleason scores. In the multivariate analysis, the intensity of PSMB4 staining was an independent predictor of local relapse (hazard ratio = 8.6, P = 0.04). CONCLUSIONS: To our knowledge, this is the first description of PAK6 and PSMB4 expression in the diagnostic specimens of men with PC who were treated with RT. If confirmed by further studies, increased expression of these genes could be used to identify patients at a high risk of developing local failure following high-dose RT, thus better tailoring treatments for the individual patient.

Surveillance of influenza viruses in waterfowl used as decoys in Andalusia, Spain.

A longitudinal study was carried out to determine the seroprevalence of avian influenza viruses (AIVs) in waterfowl used as decoys in Andalusia, southern Spain. A total of 2319 aquatic birds from 193 flocks were analyzed before and after the hunting season 2011-2012. In the first sampling, 403 out of 2319 (18.0%, CI95%: 15.8-19.0) decoys showed antibodies against AIVs by ELISA. The AI seroprevalence was significantly higher in geese (21.0%) than in ducks (11.7%) (P<0.001). Besides, the spatial distribution of AIVs was not homogeneous as significant differences among regions were observed. The prevalence of antibodies against AIVs subtypes H5 and H7 were 1.1% and 0.3%, respectively, using hemagglutination inhibition test (HI). The overall and H5 seroprevalences slightly increased after the hunting period (to 19.2% and 1.4%, respectively), while the H7 seroprevalence remained at the same level (0.3%). The proportion of flocks infected by AIVs was 65.3%, while 11.2% and 4.9% of flocks were positive for H5 and H7, respectively. Viral shedding was not detected in any of the 47 samples positive by both ELISA and HI, tested by RRT-PCR. The individual incidence after the hunting season was 3.4%. The fact that 57 animals seroconverted, 15 of which were confirmed by HI (12 H5 and 3 H7), was indication of contact with AIVs during the hunting period. The results indicate that waterfowl used as decoys are frequently exposed to AIVs and may be potentially useful as sentinels for AIVs monitoring. The seroprevalence detected and the seropositivity against AIVs H5 and H7, suggest that decoys can act as reservoirs of AIVs, which may be of animal and public health concern.

KiSS-1 methylation and protein expression patterns contribute to diagnostic and prognostic assessments in tissue specimens for colorectal cancer.

KISS1 is a metastasis suppressor lost in several solid malignancies. We evaluated the clinical relevance of KiSS-1 methylation and its protein expression in colorectal cancer. The epigenetic silencing of KiSS-1 by hypermethylation was tested in colon cancer cells (n = 5) before and after azacytidine treatment. KiSS-1 methylation was evaluated by methylation-specific PCR in colorectal cancer cells, and normal, benign, and tumor tissues (n = 352) were grouped in a training set (n = 62) and two independent validation cohorts (n = 100 and n = 190). KiSS-1 protein expression was analyzed by immunohistochemistry on tissue arrays. KiSS-1 hypermethylation correlated with transcript and protein expression loss, being increased in vitro by azacytidine. Methylation rates were 53.1, 70.0, and 80.0 % in the training and validation sets, respectively. In the training set, KiSS-1 methylation rendered a diagnostic accuracy of 72.7 % (p = 0.002). Combination of KiSS-1 methylation and serum CEA (p = 0.001) increased the prognostic utility of CEA alone (p = 0.022). In the first validation set, KiSS-1 methylation correlated with tumor grade (p = 0.011), predicted recurrence (p = 0.009), metastasis (p = 0.004), disease-free (p = 0.034), and overall survival (p = 0.015). In the second validation cohort, KiSS-1 methylation predicted disease-specific survival (p = 0.030). In the training set, cytoplasmic KiSS-1 expression was significantly higher in nonneoplastic biopsies as compared to colorectal tumors (p < 0.0005). In the validation set, loss of cytoplasmic expression correlated with tumor stage (p = 0.007), grade (p = 0.035), recurrence (p = 0.017), and disease-specific survival (p = 0.022). KiSS-1 was revealed epigenetically modified in colorectal cancer. The diagnostic and prognostic utility of KiSS-1 methylation and expression patterns suggests their assessment for the clinical management of colorectal cancer patients.

Cost model for biobanks.

Current economic conditions and budget constraints in publicly funded biomedical research have brought about a renewed interest in analyzing the cost and economic viability of research infrastructures. However, there are no proposals for specific cost accounting models for these types of organizations in the international scientific literature. The aim of this paper is to present the basis of a cost analysis model useful for any biobank regardless of the human biological samples that it stores for biomedical research. The development of a unique cost model for biobanks can be a complicated task due to the diversity of the biological samples they store. Different types of samples (DNA, tumor tissues, blood, serum, etc.) require different production processes. Nonetheless, the common basic steps of the production process can be identified. Thus, the costs incurred in each step can be analyzed in detail to provide cost information. Six stages and four cost objects were obtained by taking the production processes of biobanks belonging to the Spanish National Biobank Network as a starting point. Templates and examples are provided to help managers to identify and classify the costs involved in their own biobanks to implement the model. The application of this methodology will provide accurate information on cost objects, along with useful information to give an economic value to the stored samples, to analyze the efficiency of the production process and to evaluate the viability of some sample collections.

Preguntas y recomendaciones sobre integración de los archivos diagnósticos de los servicios de Anatomía Patológica en biobancos / Questions and recommendations on the integration of diagnostic files of the Anatomical-Pathology departments into biobanks

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Cyclooxygenase-2 expression in bladder cancer and patient prognosis: results from a large clinical cohort and meta-analysis.

Aberrant overexpression of cyclooxygenase-2 (COX2) is observed in urothelial carcinoma of the bladder (UCB). Studies evaluating COX2 as a prognostic marker in UCB report contradictory results. We determined the prognostic potential of COX2 expression in UCB and quantitatively summarize the results with those of the literature through a meta-analysis. Newly diagnosed UCB patients recruited between 1998-2001 in 18 Spanish hospitals were prospectively included in the study and followed-up (median, 70.7 months). Diagnostic slides were reviewed and uniformly classified by expert pathologists. Clinical data was retrieved from hospital charts. Tissue microarrays containing non-muscle invasive (n=557) and muscle invasive (n=216) tumours were analyzed by immunohistochemistry using quantitative image analysis. Expression was evaluated in Cox regression models to assess the risk of recurrence, progression and disease-specific mortality. Meta-hazard ratios were estimated using our results and those from 11 additional evaluable studies. COX2 expression was observed in 38% (211/557) of non-muscle invasive and 63% (137/216) of muscle invasive tumors. Expression was associated with advanced pathological stage and grade (p<0.0001). In the univariable analyses, COX2 expression - as a categorical variable - was not associated with any of the outcomes analyzed. As a continuous variable, a weak association with recurrence in non-muscle invasive tumors was observed (p-value=0.048). In the multivariable analyses, COX2 expression did not independently predict any of the considered outcomes. The meta-analysis confirmed these results. We did not find evidence that COX2 expression is an independent prognostic marker of recurrence, progression or survival in patients with UCB.

Seroprevalence and risk factors associated to West Nile virus in horses from Andalusia, Southern Spain.

West Nile virus (WNV) is recognized as an emerging zoonotic pathogen, whose incidence in horses, humans and birds has increased significantly in different European countries in the last decade. A serosurvey study was carried out in non vaccinated horses to determine the geographical distribution of WNV in Andalusia (Southern Spain), and to assess the factors that influence the risk of WNV infection in horses. Antibodies to WNV were detected in 54 out of 510 horses analyzed by a blocking ELISA, of which 36 were confirmed by micro virus neutralization test (7.1%; CI(95%): 4.9-9.3). A total of 28 out of the 348 equine herds (8.3%; CI(95%): 5.4-11.2) had at least one seropositive animal. A generalized estimating equations model showed that the main risk factors associated to WNV seroprevalence were: number of horses within the holding (low), transport of the horse within the last six months (Yes) and presence of mosquitoes in the holding (Yes). The results demonstrated that WNV circulation in Andalusia was more widespread than previously reported. Besides, the distribution of WNV infections was not homogeneous as significant differences among provinces were observed. The results show the need to improve the active surveillance in Spain, so that the early detection of WNV circulation allows the establishment control measures such as vaccination and implementation of vector control programs during the risk period.

Use of sentinel serosurveillance of mules and donkeys in the monitoring of West Nile virus infection.

A cross-sectional study was carried out on clinically normal mules and donkeys in a region of southern Spain to assess the seroprevalence of West Nile virus (WNV) following detection of infection in contiguous horse and human populations. Antibodies against WNV were detected by a blocking ELISA and micro-virus neutralisation test in 12/165 (7.3%; CI(95%) 4.3-11.3) of the animals sampled. Even though the individual seroprevalence was higher in mules (9.6%; 8/83) than in donkeys (4.9%; 4/82), the difference was not statistically significant. Nine of 90 herds (10.0%; CI(95%) 3.8-16.2) contained at least one seropositive animal. Antibodies against WNV were also detected in 1/4 (25%) donkeys tested on three farms where WNV cases had been confirmed in horses. None of 26 potential explanatory variables was identified as a risk factor for seropositivity. Such serosurveillance of sentinel mules or donkeys may be a useful tool in the epidemiological monitoring of WNV in regions where horses are vaccinated.

Stage IV and age over 45 years are the only prognostic factors of the International Prognostic Score for the outcome of advanced Hodgkin lymphoma in the Spanish Hodgkin Lymphoma Study Group series.

The International Prognostic Score (IPS) is the most widely used system to date for identifying risk groups for the outcome of patients with advanced Hodgkin lymphoma, although important limitations have been recognized. We analyzed the value of the IPS in a series of 311 patients with advanced classical Hodgkin lymphoma (cHL) (Ann Arbor stage III, IV or stage II with B symptoms and/or bulky masses) treated with first-line chemotherapy including adriamycin (adriamycin, bleomycin, vinblastine, dacarbazine [ABVD] or equivalent variants). In univariate and multivariate analyses, stage IV disease and age ≥ 45 years were the only factors with independent predictive significance for overall survival (OS) (p = 0.002 and p < 0.001, respectively). Stage IV was still significant for freedom from progression (FFP) (p = 0.001) and age ≥ 45 years was borderline significant (p = 0.058). IPS separates prognostic groups, as in the original publication, but this is mainly due to the high statistical significance of stage IV and age ≥ 45 years. Moreover, the combination of these two factors enables a simpler system to be constructed that separates groups with different FFP and OS. In conclusion, in our series, stage IV and age ≥ 45 years are the key prognostic factors for the outcome of advanced cHL.

West Nile fever outbreak in horses and humans, Spain, 2010.

TO THE EDITOR: West Nile virus (WNV) is a member of the genus Flavivirus within the Japanese encephalitis antigenic complex. The enzootic virus cycle involves transmission between avian hosts and ornithophilic mosquitoes, whereas humans and horses are considered dead-end hosts. Given the recent increase of WNV infection in humans and horses in Europe, concern has been raised regarding public and animal health.