Prognostic significance of cyclins A2, B1, D1, and E1 and CCND1 numerical aberrations in oral squamous cell carcinomas

We analysed the expression of cyclins A2, B1, D1, and E1 by immunohistochemistry and numerical aberrations in CCND1 gene by fluorescence in situ hybridization technique in 67 primary oral squamous cell carcinomas (OSCC). Cyclin A2 expression was observed in 54 (83.1%) tumours, cyclin D1 in 58 (89.2%), cyclin B1 in 39 (60%), and cyclin E in 21 (32.8%). CCND1 region analysis revealed 26 (43.3%) tumours with the presence of numerical aberrations which were correlated with cyclin D1 high expression (Rho = 0.48; p < 0 001). Twenty-nine (45.3%) tumours were classified as high proliferative tumours assessed by Ki-67 protein expression and correlated with tumours with high expression of cyclin A2 (Rho = 0.30; p = 0 016) and cyclin B1 (Rho = 0.37; p = 0 003). In multivariate analysis for an overall five-year survival (OS), we found an adverse independent prognostic value for cyclin A2 high expression (p = 0 031) and for advanced tumour stage (p < 0 001). Our results confirm that several cyclins are commonly expressed in OSCC. CCND1 gene is abnormal in more than one-third of the cases and is frequently associated with cyclin D1 high expression. Moreover, cyclin A2 high expression is an independent indicator of worse OS suggesting that this protein may serve as a reliable biological marker to identify high-risk subgroups with poor prognosis.This study was supported by a grant from the Fundación de Investigación Médica Mutua Madrileña (Spain) and Instituto de Salud Carlos III (FIS PI 061902). The authors also thank the Biobanco del Complejo Hospitalario Universitario de Santiago and IINFACTS in CESPU (BubOral CESPU 2017; MacrOral CESPU 2017)S

From neural stem cells to glioblastoma: A natural history of GBM recapitulated in vitro

Due to its aggressive and invasive nature glioblastoma (GBM), the most common and aggressive primary brain tumour in adults, remains almost invariably lethal. Significant advances in the last several years have elucidated much of the molecular and genetic complexities of GBM. However, GBM exhibits a vast genetic variation and a wide diversity of phenotypes that have complicated the development of effective therapeutic strategies. This complex pathogenesis makes necessary the development of experimental models that could be used to further understand the disease, and also to provide a more realistic testing ground for potential therapies. In this report, we describe the process of transformation of primary mouse embryo astrocytes into immortalized cultures with neural stem cell characteristics, that are able to generate GBM when injected into the brain of C57BL/6 mice, or heterotopic tumours when injected IV. Overall, our results show that oncogenic transformation is the fate of NSC if cultured for long periods in vitro. In addition, as no additional hit is necessary to induce the oncogenic transformation, our model may be used to investigate the pathogenesis of gliomagenesis and to test the effectiveness of different drugs throughout the natural history of GBMThis study was supported in part by Xunta de Galicia (2014-PG029), University of Santiago de Compostela (2018-PU001), Ministerio de Economía y Competitividad (PI15/01129, José A. Costoya), the Centro Singular de Investigación de Galicia accreditation 2016–2019, ED431G/05), and the European Regional Development Fund (ERDF)S

AMPK activation by oncogenesis is required to maintain cancer cell proliferation in astrocytic tumors

5'-AMP-activated protein kinase (AMPK) is an energy sensor that controls cell metabolism, and it has been related to apoptosis and cell-cycle arrest. Although its role in metabolic homeostasis is well documented, its function in cancer is much less clear. In this study, we examined the role of AMPK in a mouse model of astrocytoma driven by oncogenic H-Ras(V12) and/or with PTEN deletion based on the common constitutive activation of the Raf/MEK/ERK and PI3K/AKT cascades in human astrocytomas. We also evaluated the activity and role of AMPK in human glioblastoma cells and xenografts. AMPK was constitutively activated in astrocytes expressing oncogenic H-Ras(V12) in parallel with high cell division rates. Genetic deletion of AMPK or attenuation of its activity in these cells was sufficient to reduce cell proliferation. The levels of pAMK were always related to the levels of phosphorylated retinoblastoma (Rb) at Ser804, which may indicate an AMPK-mediated phosphorylation of Rb. We confirmed this AMPK-Rb relationship in human glioblastoma cell lines and xenografts. In clinical specimens of human glioblastoma, elevated levels of activated AMPK appeared especially in areas of high proliferation surrounding the blood vessels. Together, our findings indicate that the initiation and progression of astrocytic tumors relies upon AMPK-dependent control of the cell cycle, thereby identifying AMPK as a candidate therapeutic target in this setting

PARP1 Deficiency Reduces Tumour Growth by Decreasing E2F1 Hyperactivation: A Novel Mechanism in the Treatment of Cancer

In recent years, poly (ADP-ribose) polymerase (PARP) inhibitors have been evaluated for treating homologous recombination-deficient tumours, taking advantage of synthetic lethality. However, increasing evidence indicates that PARP1 exert several cellular functions unrelated with their role on DNA repair, including function as a co-activator of transcription through protein-protein interaction with E2F1. Since the RB/E2F1 pathway is among the most frequently mutated in many tumour types, we investigated whether the absence of PARP activity could counteract the consequences of E2F1 hyperactivation. Our results demonstrate that genetic ablation of Parp1 extends the survival of Rb-null embryos, while genetic inactivation of Parp1 results in reduced development of pRb-dependent tumours. Our results demonstrate that PARP1 plays a key role as a transcriptional co-activator of the transcription factor E2F1, an important component of the cell cycle regulation. Considering that most oncogenic processes are associated with cell cycle deregulation, the disruption of this PARP1-E2F1 interaction could provide a new therapeutic target of great interest and a wide spectrum of indicationsThis work received financial support from the Ministerio de Ciencia e Innovación (SAF2008-00543 and SAF2009-08629; J.A.C.) and ISCIII, Ministerio de Economía y Competitividad (PI15/01129), the Consellería de Cultura, Educación e Ordenación Universitaria (GPC2014/030, INCITE08PXIB208091PR and PXIB208091PR; J.A.C.), PRE/2011/131 (I.G.C.), the Centro Singular de Investigación de Galicia accreditation 2016–2019, ED431G/05), and the European Regional Development Fund (ERDF)S

Effects of Anti-IL-17 on Inflammation, Remodeling, and Oxidative Stress in an Experimental Model of Asthma Exacerbated by LPS

Inflammation plays a central role in the development of asthma, which is considered an allergic disease with a classic Th2 inflammatory profile. However, cytokine IL-17 has been examined to better understand the pathophysiology of this disease. Severe asthmatic patients experience frequent exacerbations, leading to infection, and subsequently show altered levels of inflammation that are unlikely to be due to the Th2 immune response alone. This study estimates the effects of anti-IL-17 therapy in the pulmonary parenchyma in a murine asthma model exacerbated by LPS. BALB/c mice were sensitized with intraperitoneal ovalbumin and repeatedly exposed to inhalation with ovalbumin, followed by treatment with or without anti-IL-17. Twenty-four hours prior to the end of the 29-day experimental protocol, the two groups received LPS (0.1 mg/ml intratracheal OVA-LPS and OVA-LPS IL-17). We subsequently evaluated bronchoalveolar lavage fluid, performed a lung tissue morphometric analysis, and measured IL-6 gene expression. OVA-LPS-treated animals treated with anti-IL-17 showed decreased pulmonary inflammation, edema, oxidative stress, and extracellular matrix remodeling compared to the non-treated OVA and OVA-LPS groups (p < 0.05). The anti-IL-17 treatment also decreased the numbers of dendritic cells, FOXP3, NF-kappa B, and Rho kinase 1-and 2-positive cells compared to the non-treated OVA and OVA-LPS groups (p < 0.05). In conclusion, these data suggest that inhibition of IL-17 is a promising therapeutic avenue, even in exacerbated asthmatic patients, and significantly contributes to the control of Th1/Th2/Th17 inflammation, chemokine expression, extracellular matrix remodeling, and oxidative stress in a murine experimental asthma model exacerbated by LPS.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)National Council of Scientific and the Technological Development (CNPq)Laboratory of Medical InvestigationsUniv Sao Paulo, Sch Med, Dept Med Sci, Sao Paulo, Brazil|Hosp Sirio Libanes, Sao Paulo, BrazilUniv Fed Sao Paulo, Inst Biomed Sci, Dept Biol Sci, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Biol Sci, Sao Paulo, BrazilUniv Fed Sao Paulo, Inst Biomed Sci, Dept Biol Sci, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Biol Sci, Sao Paulo, BrazilFAPESP: 2013/17944-1Laboratory of Medical Investigations: LIM-20 FMUSPWeb of Scienc

In search of an evidence-based strategy for quality assessment of human tissue samples: report of the tissue Biospecimen Research Working Group of the Spanish Biobank Network

The purpose of the present work is to underline the importance of obtaining a standardized procedure to ensure and evaluate both clinical and research usability of human tissue samples. The study, which was carried out by the Biospecimen Science Working Group of the Spanish Biobank Network, is based on a general overview of the current situation about quality assurance in human tissue biospecimens. It was conducted an exhaustive review of the analytical techniques used to evaluate the quality of human tissue samples over the past 30 years, as well as their reference values if they were published, and classified them according to the biomolecules evaluated: (i) DNA, (ii) RNA, and (iii) soluble or/and fixed proteins for immunochemistry. More than 130 publications released between 1989 and 2019 were analysed, most of them reporting results focused on the analysis of tumour and biopsy samples. A quality assessment proposal with an algorithm has been developed for both frozen tissue samples and formalin-fixed paraffin-embedded (FFPE) samples, according to the expected quality of sample based on the available pre-analytical information and the experience of the participants in the Working Group. The high heterogeneity of human tissue samples and the wide number of pre-analytic factors associated to quality of samples makes it very difficult to harmonize the quality criteria. However, the proposed method to assess human tissue sample integrity and antigenicity will not only help to evaluate whether stored human tissue samples fit for the purpose of biomarker development, but will also allow to perform further studies, such as assessing the impact of different pre-analytical factors on very well characterized samples or evaluating the readjustment of tissue sample collection, processing and storing procedures. By ensuring the quality of the samples used on research, the reproducibility of scientific results will be guaranteed.This work was funded by the Ministerio de Ciencia, Innovacion y Universidades of Spain and Instituto de Salud Carlos III (PI16/00528, PI16/00946, PI16/01207 and PI16/01276), co-funded by the Spanish Biobank Network (PT13/0010/0030, PT17/0015/0001, PT17/0015/0021, PT17/0015/0049, PT17/0015/0018, PT17/0015/0002, PT17/0015/0016, PT17/0015/0038, PT17/0015/0027, PT17/0015/0004, PT17/0015/0047, PT17/0015/0014, PT17/0015/0041, and PT17/0015/0006), European Regional Development Fund (FEDER) "A way to make Europe" and granted by Conselleria d'Innovacio, Recerca i Turisme del Govern de les Illes Balears (TEC/002/2017).S

Breast feeding, parity and breast cancer subtypes in a Spanish cohort

Background: Differences in the incidence and outcome of breast cancer among Hispanic women compared with white women are well documented and are likely explained by ethnic differences in genetic composition, lifestyle, or environmental exposures. METHODOLGY/PRINCIPAL FINDINGS: A population-based study was conducted in Galicia, Spain. A total of 510 women diagnosed with operable invasive breast cancer between 1997 and 2010 participated in the study. Data on demographics, breast cancer risk factors, and clinico-pathological characteristics were collected. The different breast cancer tumor subtypes were compared on their clinico-pathological characteristics and risk factor profiles, particularly reproductive variables and breastfeeding. Among the 501 breast cancer patients (with known ER and PR receptors), 85% were ER+/PR+ and 15% were ER-&PR-. Among the 405 breast cancer with known ER, PR and HER2 status, 71% were ER+/PR+/HER2- (luminal A), 14% were ER+/PR+/HER2+ (luminal B), 10% were ER-/PR-/HER2- (triple negative breast cancer, TNBC), and 5% were ER-/PR-/HER2+ (non-luminal). A lifetime breastfeeding period equal to or longer than 7 months was less frequent in case patients with TNBC (OR = 0.25, 95% CI = 0.08-0.68) compared to luminal A breast cancers. Both a low (2 or fewer pregnancies) and a high (3-4 pregnancies) number of pregnancies combined with a long breastfeeding period were associated with reduced odds of TNBC compared with luminal A breast cancer, although the association seemed to be slightly more pronounced among women with a low number of pregnancies (OR = 0.09, 95% CI = 0.005-0.54). Conclusions/significance: In case-case analyses with the luminal A cases as the reference group, we observed a lower proportion of TNBC among women who breastfed 7 or more months. The combination of longer breastfeeding duration and lower parity seemed to further reduce the odds of having a TNBC compared to a luminal A breast cancer

Breast feeding, parity and breast cancer subtypes in a Spanish cohort

Background: Differences in the incidence and outcome of breast cancer among Hispanic women compared with white women are well documented and are likely explained by ethnic differences in genetic composition, lifestyle, or environmental exposures. METHODOLGY/PRINCIPAL FINDINGS: A population-based study was conducted in Galicia, Spain. A total of 510 women diagnosed with operable invasive breast cancer between 1997 and 2010 participated in the study. Data on demographics, breast cancer risk factors, and clinico-pathological characteristics were collected. The different breast cancer tumor subtypes were compared on their clinico-pathological characteristics and risk factor profiles, particularly reproductive variables and breastfeeding. Among the 501 breast cancer patients (with known ER and PR receptors), 85% were ER+/PR+ and 15% were ER-&PR-. Among the 405 breast cancer with known ER, PR and HER2 status, 71% were ER+/PR+/HER2- (luminal A), 14% were ER+/PR+/HER2+ (luminal B), 10% were ER-/PR-/HER2- (triple negative breast cancer, TNBC), and 5% were ER-/PR-/HER2+ (non-luminal). A lifetime breastfeeding period equal to or longer than 7 months was less frequent in case patients with TNBC (OR = 0.25, 95% CI = 0.08-0.68) compared to luminal A breast cancers. Both a low (2 or fewer pregnancies) and a high (3-4 pregnancies) number of pregnancies combined with a long breastfeeding period were associated with reduced odds of TNBC compared with luminal A breast cancer, although the association seemed to be slightly more pronounced among women with a low number of pregnancies (OR = 0.09, 95% CI = 0.005-0.54). Conclusions/significance: In case-case analyses with the luminal A cases as the reference group, we observed a lower proportion of TNBC among women who breastfed 7 or more months. The combination of longer breastfeeding duration and lower parity seemed to further reduce the odds of having a TNBC compared to a luminal A breast cancer

Usefulness of haptoglobin and serum amyloid A proteins as biomarkers for atherothrombotic ischemic stroke diagnosis confirmation

Objective To identify protein biomarkers in order to classify ischemic stroke subtypes using proteomic analysis and immunoenzymatic tools for clinical validation. Methods and results We performed a proteomic analysis in serum samples of 24 patients with ischemic stroke (12 atherothrombotic patients and 12 cardioembolic patients). In this study, based on two-dimensional electrophoresis and mass spectrometry we found four spots whose expression intensity was at least four times stronger in atherothrombotic patients than in cardioembolic patients. These spots were identified as haptoglobin related protein, serum amyloid A (two spots) and haptoglobin α chain. We validated the possible value of haptoglobin and serum amyloid A in a larger series of patients (n = 262) with ischemic stroke using ELISA techniques. Haptoglobin levels >1040 μg/mL identified atherothrombotic patients with 95% sensitivity and 88% specificity whereas serum amyloid A levels >160 μg/mL identified atherothrombotic patients with 91% sensitivity and 83% specificity. Conclusions Haptoglobin and serum amyloid A are useful biomarkers for atherothrombotic ischemic stroke diagnosis confirmation.This project has been partially supported by grants from the Spanish Ministry of Health (Instituto de Salud Carlos III, RETICS-RD06/0026); and Xunta de Galicia (Consellería de Sanidade, Código PS07/29; and Consellería de Educación e Ordenación Universitaria of Xunta de Galicia, Axudas para a Consolidación e Estruturación de Unidades de Investigación Competitivas. Expediente: 80/2006); and Fundación de Investigación Médica Mútua Madrileña.Peer reviewe

Chronic lymphocytic leukemia cells in lymph nodes show frequent NOTCH1 activation

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western world. Pathogenic mechanisms involve multiple external events (such as microenvironmental and antigenic stimuli) and internal events (genetic and epigenetic alterations) that are associated with the transformation, progression and evolution of CLL. CLL is characterized by an accumulation of mature B cells in peripheral blood, bone marrow and lymphoid tissues. Extracellular stimuli play an important role in the development and maintenance of neoplastic cells. B-CLL cells proliferate and activate pathogenic signaling pathways in anatomical structures known as proliferation centers, which are usually more conspicuous in involved lymph nodes.1 Its clinical course is quite heterogeneous, whereby some patients progress rapidly and have short survival, whereas others have a more stable clinical course that may not need treatment for years.This work was supported by grants from the Ministerio de Economía y Competitividad (MINECO) (SAF2013-47416-R) Instituto de Salud Carlos III (ISCIII)- FEDER – MINECO- AES (CP11/00018, PI10/00621, RD012/0036/0060), and Asociación Española contra el Cancer (AECC). MS-B is supported by a Miguel Servet contract from ISCIII-FEDER (CP11/00018). Salary support to SG is provided by CP11/00018, from ISCIII-FEDER. JG-R is supported by a predoctoral grant from the Fundación Investigación Puerta de Hierro.S