A combination of urinary biomarker panel and PancRISK score for earlier detection of pancreatic cancer: A case–control study

Funder: Pancreatic Cancer Research Fund; funder-id: http://dx.doi.org/10.13039/100011704Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers, with around 9% of patients surviving >5 years. Asymptomatic in its initial stages, PDAC is mostly diagnosed late, when already a locally advanced or metastatic disease, as there are no useful biomarkers for detection in its early stages, when surgery can be curative. We have previously described a promising biomarker panel (LYVE1, REG1A, and TFF1) for earlier detection of PDAC in urine. Here, we aimed to establish the accuracy of an improved panel, including REG1B instead of REG1A, and an algorithm for data interpretation, the PancRISK score, in additional retrospectively collected urine specimens. We also assessed the complementarity of this panel with CA19-9 and explored the daily variation and stability of the biomarkers and their performance in common urinary tract cancers. Methods and findings: Clinical specimens were obtained from multiple centres: Barts Pancreas Tissue Bank, University College London, University of Liverpool, Spanish National Cancer Research Center, Cambridge University Hospital, and University of Belgrade. The biomarker panel was assayed on 590 urine specimens: 183 control samples, 208 benign hepatobiliary disease samples (of which 119 were chronic pancreatitis), and 199 PDAC samples (102 stage I–II and 97 stage III–IV); 50.7% were from female individuals. PDAC samples were collected from patients before treatment. The samples were assayed using commercially available ELISAs. Statistical analyses were performed using non-parametric Kruskal–Wallis tests adjusted for multiple comparisons, and multiple logistic regression. Training and validation datasets for controls and PDAC samples were obtained after random division of the whole available dataset in a 1:1 ratio. The substitution of REG1A with REG1B enhanced the performance of the panel to detect resectable PDAC. In a comparison of controls and PDAC stage I–II samples, the areas under the receiver operating characteristic curve (AUCs) increased from 0.900 (95% CI 0.843–0.957) and 0.926 (95% CI 0.843–1.000) in the training (50% of the dataset) and validation sets, respectively, to 0.936 in both the training (95% CI 0.903–0.969) and the validation (95% CI 0.888–0.984) datasets for the new panel including REG1B. This improved panel showed both sensitivity (SN) and specificity (SP) to be >85%. Plasma CA19-9 enhanced the performance of this panel in discriminating PDAC I–II patients from controls, with AUC = 0.992 (95% CI 0.983–1.000), SN = 0.963 (95% CI 0.913–1.000), and SP = 0.967 (95% CI 0.924–1.000). We demonstrate that the biomarkers do not show significant daily variation, and that they are stable for up to 5 days at room temperature. The main limitation of our study is the low number of stage I–IIA PDAC samples (n = 27) and lack of samples from individuals with hereditary predisposition to PDAC, for which specimens collected from control individuals were used as a proxy. Conclusions: We have successfully validated our urinary biomarker panel, which was improved by substituting REG1A with REG1B. At a pre-selected cutoff of >80% SN and SP for the affiliated PancRISK score, we demonstrate a clinically applicable risk stratification tool with a binary output for risk of developing PDAC (‘elevated’ or ‘normal’). PancRISK provides a step towards precision surveillance for PDAC patients, which we will test in a prospective clinical study, UroPanc

Clinical and molecular characterization of a cohort of patients with novel nucleotide alterations of the Dystrophin gene detected by direct sequencing

<p>Abstract</p> <p>Background</p> <p>Duchenne and Becker Muscular dystrophies (DMD/BMD) are allelic disorders caused by mutations in the dystrophin gene, which encodes a sarcolemmal protein responsible for muscle integrity. Deletions and duplications account for approximately 75% of mutations in DMD and 85% in BMD. The implementation of techniques allowing complete gene sequencing has focused attention on small point mutations and other mechanisms underlying complex rearrangements.</p> <p>Methods</p> <p>We selected 47 patients (41 families; 35 DMD, 6 BMD) without deletions and duplications in <it>DMD </it>gene (excluded by multiplex ligation-dependent probe amplification and multiplex polymerase chain reaction analysis). This cohort was investigated by systematic direct sequence analysis to study sequence variation. We focused our attention on rare mutational events which were further studied through transcript analysis.</p> <p>Results</p> <p>We identified 40 different nucleotide alterations in DMD gene and their clinical correlates; altogether, 16 mutations were novel. DMD probands carried 9 microinsertions/microdeletions, 19 nonsense mutations, and 7 splice-site mutations. BMD patients carried 2 nonsense mutations, 2 splice-site mutations, 1 missense substitution, and 1 single base insertion. The most frequent stop codon was TGA (n = 10 patients), followed by TAG (n = 7) and TAA (n = 4). We also analyzed the molecular mechanisms of five rare mutational events. They are two frame-shifting mutations in the <it>DMD </it>gene 3'end in BMD and three novel splicing defects: IVS42: c.6118-3C>A, which causes a leaky splice-site; c.9560A>G, which determines a cryptic splice-site activation and c.9564-426 T>G, which creates pseudoexon retention within IVS65.</p> <p>Conclusion</p> <p>The analysis of our patients' sample, carrying point mutations or complex rearrangements in <it>DMD </it>gene, contributes to the knowledge on phenotypic correlations in dystrophinopatic patients and can provide a better understanding of pre-mRNA maturation defects and dystrophin functional domains. These data can have a prognostic relevance and can be useful in directing new therapeutic approaches, which rely on a precise definition of the genetic defects as well as their molecular consequences.</p

Central Exercise Action Increases the AMPK and mTOR Response to Leptin

AMP-activated protein kinase (AMPK) and mammalian Target of Rapamycin (mTOR) are key regulators of cellular energy balance and of the effects of leptin on food intake. Acute exercise is associated with increased sensitivity to the effects of leptin on food intake in an IL-6-dependent manner. To determine whether exercise ameliorates the AMPK and mTOR response to leptin in the hypothalamus in an IL-6-dependent manner, rats performed two 3-h exercise bouts, separated by one 45-min rest period. Intracerebroventricular IL-6 infusion reduced food intake and pretreatment with AMPK activators and mTOR inhibitor prevented IL-6-induced anorexia. Activators of AMPK and fasting increased food intake in control rats to a greater extent than that observed in exercised ones, whereas inhibitor of AMPK had the opposite effect. Furthermore, the reduction of AMPK and ACC phosphorylation and increase in phosphorylation of proteins involved in mTOR signal transduction, observed in the hypothalamus after leptin infusion, were more pronounced in both lean and diet-induced obesity rats after acute exercise. Treatment with leptin reduced food intake in exercised rats that were pretreated with vehicle, although no increase in responsiveness to leptin-induced anorexia after pretreatment with anti-IL6 antibody, AICAR or Rapamycin was detected. Thus, the effects of leptin on the AMPK/mTOR pathway, potentiated by acute exercise, may contribute to appetite suppressive actions in the hypothalamus

Patients' preferences for subcutaneous trastuzumab versus conventional intravenous infusion for the adjuvant treatment of HER2-positive early breast cancer: final analysis of 488 patients in the international, randomized, two-cohort PrefHer study

PrefHer revealed compelling and consistent patient preference for subcutaneous (s.c.) trastuzumab, regardless of delivery by single-use injection device or hand-held syringe. s.c. trastuzumab was well-tolerated and safety data, including immunogenicity, were consistent with previous reports. No new safety signals were identified compared with the known intravenous trastuzumab profile in early breast cance

Global disparities in surgeons’ workloads, academic engagement and rest periods: the on-calL shIft fOr geNEral SurgeonS (LIONESS) study

: The workload of general surgeons is multifaceted, encompassing not only surgical procedures but also a myriad of other responsibilities. From April to May 2023, we conducted a CHERRIES-compliant internet-based survey analyzing clinical practice, academic engagement, and post-on-call rest. The questionnaire featured six sections with 35 questions. Statistical analysis used Chi-square tests, ANOVA, and logistic regression (SPSS® v. 28). The survey received a total of 1.046 responses (65.4%). Over 78.0% of responders came from Europe, 65.1% came from a general surgery unit; 92.8% of European and 87.5% of North American respondents were involved in research, compared to 71.7% in Africa. Europe led in publishing research studies (6.6 ± 8.6 yearly). Teaching involvement was high in North America (100%) and Africa (91.7%). Surgeons reported an average of 6.7 ± 4.9 on-call shifts per month, with European and North American surgeons experiencing 6.5 ± 4.9 and 7.8 ± 4.1 on-calls monthly, respectively. African surgeons had the highest on-call frequency (8.7 ± 6.1). Post-on-call, only 35.1% of respondents received a day off. Europeans were most likely (40%) to have a day off, while African surgeons were least likely (6.7%). On the adjusted multivariable analysis HDI (Human Development Index) (aOR 1.993) hospital capacity &gt; 400 beds (aOR 2.423), working in a specialty surgery unit (aOR 2.087), and making the on-call in-house (aOR 5.446), significantly predicted the likelihood of having a day off after an on-call shift. Our study revealed critical insights into the disparities in workload, access to research, and professional opportunities for surgeons across different continents, underscored by the HDI

Hb Köln [a2b298(FG5) val-met] identified by DNA analysis in a Brazilian family

Hb Köln was identified by DNA analysis in a Brazilian patient. A four-year old Brazilian female, with jaundice since birth, presented an abnormal band, between A2 and S, in hemoglobin electrophoresis on a cellulose acetate membrane, and a band with electrophoretic migration similar to Hb C on agar gel. Thermic instability and isopropanol precipitation tests were positive. Heinz bodies were observed in the patient?s peripheral blood. Sequencing of the three exons of the b globin gene detected a transition from G to A in the first position of codon 98. This alteration does not create or abolish any known restriction site. In this case, confirmation of the mutation was accomplished by allele-specific oligonucleotide hybridization, which is a simple and fast identification method when the clinical data and hematological and electrophoretic patterns are suggestive of Hb Köln.Hb Köln resulta da substituição do aminoácido valina por metionina na posição 98 da cadeia da hemoglobina. Embora seja a mais freqüente das hemoglobinas instáveis, possui poucas características que permitem distinguíla sem a realização de análise estrutural da molécula. Com o objetivo de desenvolver um procedimento alternativo à análise protéica, este trabalho descreve a identificação da Hb Köln através de análise de DNA. Uma paciente branca, sexo feminino, 4 anos de idade, com icterícia desde o nascimento, foi analisada, apresentando uma banda anômala entre A2 e S em eletroforese em acetato de celulose, e migração eletroforética semelhante a Hb C em gel de agar. Os testes de instabilidade térmica e de precipitação pelo isopropanol foram positivos e foram observados corpúsculos de Heinz no sangue periférico. Os três exons do gene da globina foram seqüenciados e uma transição de G ® A foi identificada na primeira posição do codon 98. Essa alteração não cria ou abole nenhum sítio conhecido de enzima de restrição. Desta forma, a confirmação da mutação foi levada a efeito através de hibridização com oligonucleotídeos aleloespecíficos, o que permitiu o estabelecimento de um método simples e rápido de identificação de novos casos quando os dados clínicos e o padrão hematológico e eletroforético sugerem Hb Köln.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Dnaase I Hypersensitive Site 3' To The Beta-globin Gene Cluster Contains A Taa Insertion Specific For Beta(s)-benin Haplotype.

Analysis of DNA polymorphic sites is a powerful tool for detection of gene flow in human evolutionary studies and to trace genetic background associated with abnormal genes. The beta-globin locus contains more than 20 single-base restriction fragment length polymorphism (RFLP) sites spanning over 80 kb on chromosome 11. Far downstream of the expressed genes, there is a hypersensitive site (HS). The function of the 3'-HS remains unknown. As an approach to the understanding of the 3'-HS region in sickle cell anemia we searched for sequence polymorphism in the AT-rich region, using a non-radioactive polymerase chain reaction (PCR)-single strand conformational polymorphism (SSCP) technique. A 460 bp fragment located at the 3' of the b globin gene was amplified from patients (with sickle cell anemia and HbSC disease), and from AS individuals. Standard RFLP-haplotyping was performed and compared with the PCR-SSCP screening strategy. Two distinct band patterns were revealed by SSCP testing, each one in strict linkage disequilibrium with either Benin or Bantu haplotypes. Direct sequencing of the amplified segment revealed a TAA insertion in the AT-rich region, in all 121 beta(S) Benin chromosomes tested, but not in other beta(S) haplotypes from the total of 380 beta(S) chromosomes typed. SSCP analysis could easily distinguish sequence variations in the 3'AT-rich region of the beta-globin cluster, and a TAA insertion in this region seems to be specific for the Benin-beta(S) chromosome.87246-

Mutation analysis of the HFE gene in brazilian populations

Universidade de Campinas. Faculdade de Ciências Médicas. Hemocentro and Departamento de Clínica Médica. São Paulo, SP, Brasil.Universidade de Campinas. Faculdade de Ciências Médicas. Hemocentro and Departamento de Clínica Médica. São Paulo, SP, Brasil.Universidade de Campinas. Faculdade de Ciências Médicas. Hemocentro and Departamento de Clínica Médica. São Paulo, SP, Brasil.Ministério da Saúde. Fundação Nacional de Saúde. Instituto Evandro Chagas. Belém, PA, Brasil.Ministério da Saúde. Fundação Nacional de Saúde. Instituto Evandro Chagas. Belém, PA, Brasil.Universidade de Campinas. Faculdade de Ciências Médicas. Hemocentro and Departamento de Clínica Médica. São Paulo, SP, Brasil.Universidade de Campinas. Faculdade de Ciências Médicas. Hemocentro and Departamento de Clínica Médica. São Paulo, SP, Brasil.Universidade de Campinas. Faculdade de Ciências Médicas. Hemocentro and Departamento de Clínica Médica. São Paulo, SP, Brasil.We analyzed the frequency of the C282Y and H63D mutations in the HFE gene in 227 individuals from Brazil comprising 71 Caucasians, 91 racially mixed Caucasian African-derived Amerindians (both populations from Southeast Brazil), 85 African-derived subjects (from Northeast Brazil) and 75 Parakanã Indians. Allelic frequency of the mutation C. 845G6A (C282Y) was 1.4% in the Caucasian population, 1.1% in the African-derived population, 1.1% in the racially mixed normal controls and 0% in the Parakanã Indians. In the African-derived population, the C282Y mutation was present on chromosomes bearing the haplotype 6/1h according to Beutler and West (1997). Allelic frequency of the mutation C. 187C6G (H63D) was 16.3% in the Caucasian population, 7.5% in the African-derived population, 9.8% in the racially mixed controls and 0% in the Amerindians. The presence of these mutations in the African-derived population reflects the fact that these subjects may have undergone a non-identified racial admixture in their past history. The absence of both defects in the Amerindians suggests that these mutations have emerged after the migration of Polynesians to America, or that they may not have reached the Polynesian population until after the migration to America had occurred