Feasibility and Acceptability of Personalized Breast Cancer Screening (DECIDO Study): A Single-Arm Proof-of-Concept Trial.

The aim of this study was to assess the acceptability and feasibility of offering risk-based breast cancer screening and its integration into regular clinical practice. A single-arm proof-of-concept trial was conducted with a sample of 387 women aged 40-50 years residing in the city of Lleida (Spain). The study intervention consisted of breast cancer risk estimation, risk communication and screening recommendations, and a follow-up. A polygenic risk score with 83 single nucleotide polymorphisms was used to update the Breast Cancer Surveillance Consortium risk model and estimate the 5-year absolute risk of breast cancer. The women expressed a positive attitude towards varying the frequency of breast screening according to individual risk and, especially, more frequently inviting women at higher-than-average risk. A lower intensity screening for women at lower risk was not as welcome, although half of the participants would accept it. Knowledge of the benefits and harms of breast screening was low, especially with regard to false positives and overdiagnosis. The women expressed a high understanding of individual risk and screening recommendations. The participants' intention to participate in risk-based screening and satisfaction at 1-year were very high.

Feasibility and acceptability of personalised breast cancer screening (DECIDO study): protocol of a single-arm proof-of-concept trial.

INTRODUCTION: Personalised cancer screening aims to improve benefits, reduce harms and being more cost-effective than age-based screening. The objective of the DECIDO study is to assess the acceptability and feasibility of offering risk-based personalised breast cancer screening and its integration in regular clinical practice in a National Health System setting. METHODS AND ANALYSIS: The study is designed as a single-arm proof-of-concept trial. The study sample will include 385 women aged 40-50 years resident in a primary care health area in Spain. The study intervention consists of (1) a baseline visit; (2) breast cancer risk estimation; (3) a second visit for risk communication and screening recommendations based on breast cancer risk and (4) a follow-up to obtain the study outcomes.A polygenic risk score (PRS) will be constructed as a composite likelihood ratio of 83 single nucleotide polymorphisms. The Breast Cancer Surveillance Consortium risk model, including age, race/ethnicity, family history of breast cancer, benign breast disease and breast density will be used to estimate a preliminary 5-year absolute risk of breast cancer. A Bayesian approach will be used to update this risk with the PRS value.The primary outcome measures will be attitude towards, intention to participate in and satisfaction with personalised breast cancer screening. Secondary outcomes will include the proportions of women who accept to participate and who complete the different phases of the study. The exact binomial and the Student's t-test will be used to obtain 95% CIs. ETHICS AND DISSEMINATION: The study protocol was approved by the Drug Research Ethics Committee of the University Hospital Arnau de Vilanova. The trial will be conducted in compliance with this study protocol, the Declaration of Helsinki and Good Clinical Practice.The results will be published in peer-reviewed scientific journals and disseminated in scientific conferences and media. TRIAL REGISTRATION NUMBER: NCT03791008.

Genotyping of common SIRPB1 copy number variant using Paralogue Ratio Test coupled to MALDI-MS quantification.

Copy number variant (CNV) regions have been proven to have a significant impact on gene expression. Some of them have been also found to be associated to different human diseases. CNV genotyping is often prone to error and cross-validation with independent methods is frequently required. The platform of choice depends on whether it is a genome-wide discovery screening or a candidate CNV study, the cohort size and the number of CNVs included in the assay and, finally, the budget available. Here we illustrate a affordable approach to determine the CNV genotype using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and based on the quantitative determination of single nucleotide duplicated mismatches (SNDM) mapping the CNV region and a paralogue genomic region that is used as a two-copy reference. We have genotyped nsv436327, a common CNV mapping SIRPB1 intron 1 that has been associated to human personality behavior. SIRP cluster region was subjected to several ancestral duplication events what makes SIRPB1 CNV genotyping technically challenging. We designed three sets of primer pairs that amplified paralogue regions inside and outside the CNV, containing three SNDMs. Post-PCR extension analyses of sequencing oligonucleotides mapping immediately upstream each SNDM allowed us to quantify using MALDI-MS the proportion of PCR products derived from the CNV region versus the external reference. In contrast to other approaches, setting up this genotyping method requires an affordable investment.

Comparative analysis of hemolymph proteome maps in diapausing and non-diapausing larvae of Sesamia nonagrioides.

BACKGROUND: Sesamia nonagrioides is a noctuid that feeds on maize, sugar cane and sorghum in North Africa and Southern Europe. Larvae reared under long day conditions pupate after 5 or 6 larval instars, whereas larvae reared under short day conditions enter diapause and undergo up to 12 molts before dying or pupating. To better understand the mechanism of larval development and diapause, we identified proteins with different expressions in the sixth instar of diapausing and non-diapausing larvae. RESULTS: A total of 52 differentially regulated proteins were detected in the hemolymph of the diapausing or non-diapausing larvae at the beginning or end of the sixth instar. From these proteins, 11 were identified by mass spectrometry (MALDI-TOF MS or MALDI-TOF/TOF MS/MS): 5 were upregulated in the hemolymph of non-diapausing larvae and 6 in the hemolymph of the diapausing larvae. Interestingly, some proteins were expressed only in non-diapausing larvae but none was expressed only in the hemolymph of diapausing larvae. The possible functions of some of these proteins related to diapause maintenance or to larval-pupal metamorphosis are discussed. CONCLUSIONS: The 2-DE proteomic map of S. nonagrioides hemolymph shows differential protein expression in diapausing and non-diapausing larvae. Some proteins that showed higher expression in the diapausing larvae at the end of the sixth instar could be involved in JH level maintenance thus in the diapause status maintenance. On the contrary, other proteins that showed the highest expression or that were expressed only in the non-diapausing larvae could be involved in larval-pupal metamorphosis.

The contribution of apoptosis-inducing factor, caspase-activated DNase, and inhibitor of caspase-activated DNase to the nuclear phenotype and DNA degradation during apoptosis.

We have assessed the contribution of apoptosis-inducing factor (AIF) and inhibitor of caspase-activated DNase (ICAD) to the nuclear morphology and DNA degradation pattern in staurosporine-induced apoptosis. Expression of D117E ICAD, a mutant that is resistant to caspase cleavage at residue 117, prevented low molecular weight (LMW) DNA fragmentation, stage II nuclear morphology, and detection of terminal deoxynucleotidyl transferase staining. However, high molecular weight (HMW) DNA fragmentation and stage I nuclear morphology remained unaffected. On the other hand, expression of either D224E or wild type ICAD had no effect on DNA fragmentation or nuclear morphology. In addition, both HMW and LMW DNA degradation required functional executor caspases. Interestingly, silencing of endogenous AIF abolished type I nuclear morphology without any effect on HMW or LMW DNA fragmentation. Together, these results demonstrate that AIF is responsible for stage I nuclear morphology and suggest that HMW DNA degradation is a caspase-activated DNase and AIF-independent process.

Characterization of splice variants of human caspase-activated DNase with CIDE-N structure and function.

Internucleosomal DNA fragmentation is an apoptotic event that depends on the activity of different nucleases. Among them, the DNA fragmentation factor B, better known as caspase-activated DNase (CAD), is mainly responsible for this DNA fragmentation in dying cells. CAD is an endonuclease that is chaperoned and inhibited by inhibitor of CAD (ICAD). Activation of CAD needs the cleavage of ICAD by activated caspase-3. During the characterization of the staurosporine-induced apoptotic process in human neuroblastoma cell lines, we have found three novel splice variants of CAD. In all three messengers, the open reading frame is truncated after the second exon of the CAD gene. This truncated open reading frame codifies the CAD protein amino terminal part corresponding to the cell death-inducing DFF45-like effector-N (CIDE-N) domain. We have detected these splicing variants in human tissues and in peripheral white blood cells from 10 unrelated individuals, and their products have been showed to be expressed in certain mouse tissues. We demonstrate that these truncated forms of CAD are soluble proteins that interact with ICAD. We also provided evidences that these CIDE-N forms of CAD promote apoptosis in a caspase-dependent manner.

The prevention of the staurosporine-induced apoptosis by Bcl-X(L), but not by Bcl-2 or caspase inhibitors, allows the extensive differentiation of human neuroblastoma cells.

Staurosporine is one of the best apoptotic inducers in different cell types including neuroblastomas. In this study we have compared the efficiency and final outcome of three different anti-apoptotic strategies in staurosporine-treated SH-SY5Y human neuroblastoma cells. At staurosporine concentrations up to 500 nm, z-VAD.fmk a broad-spectrum, noncompetitive inhibitor of caspases, reduced apoptosis in SH-SY5Y cells. At higher concentrations, z-VAD.fmk continued to inhibit caspases and the apoptotic phenotype but not cell death which seems to result from oxidative damage. Stable over-expression of Bcl-2 in SH-SY5Y protected cells from death at doses of staurosporine up to 1 microm. At higher doses, cytochrome c release from mitochondria occurred, caspases were activated and cells died by apoptosis. Therefore, we conclude that Bcl-2 increased the threshold for apoptotic cell death commitment. Over-expression of Bcl-X(L) was far more effective than Bcl-2. Bcl-X(L) transfected cells showed a remarkable resistance staurosporine-induced cytochrome c release and associated apoptotic changes and survived for up to 15 days in 1 microm staurosporine. In these conditions, SH-SY5Y displayed a remarkable phenotype of neuronal differentiation as assessed by neurite outgrowth and expression of neurofilament, Tau and MAP-2 neuronal specific proteins.