Variations in the NBN/NBS1 gene and the risk of breast cancer in non-BRCA1/2 French Canadian families with high risk of breast cancer

<p>Abstract</p> <p>Background</p> <p>The Nijmegen Breakage Syndrome is a chromosomal instability disorder characterized by microcephaly, growth retardation, immunodeficiency, and increased frequency of cancers. Familial studies on relatives of these patients indicated that they also appear to be at increased risk of cancer.</p> <p>Methods</p> <p>In a candidate gene study aiming at identifying genetic determinants of breast cancer susceptibility, we undertook the full sequencing of the <it>NBN </it>gene in our cohort of 97 high-risk non-<it>BRCA1 </it>and -<it>BRCA2 </it>breast cancer families, along with 74 healthy unrelated controls, also from the French Canadian population. <it>In silico </it>programs (ESEfinder, NNSplice, Splice Site Finder and MatInspector) were used to assess the putative impact of the variants identified. The effect of the promoter variant was further studied by luciferase gene reporter assay in MCF-7, HEK293, HeLa and LNCaP cell lines.</p> <p>Results</p> <p>Twenty-four variants were identified in our case series and their frequency was further evaluated in healthy controls. The potentially deleterious p.Ile171Val variant was observed in one case only. The p.Arg215Trp variant, suggested to impair NBN binding to histone γ-H2AX, was observed in one breast cancer case and one healthy control. A promoter variant c.-242-110delAGTA displayed a significant variation in frequency between both sample sets. Luciferase reporter gene assay of the promoter construct bearing this variant did not suggest a variation of expression in the MCF-7 breast cancer cell line, but indicated a reduction of luciferase expression in both the HEK293 and LNCaP cell lines.</p> <p>Conclusion</p> <p>Our analysis of <it>NBN </it>sequence variations indicated that potential <it>NBN </it>alterations are present, albeit at a low frequency, in our cohort of high-risk breast cancer cases. Further analyses will be needed to fully ascertain the exact impact of those variants on breast cancer susceptibility, in particular for variants located in <it>NBN </it>promoter region.</p

Mutation analysis and characterization of ATR sequence variants in breast cancer cases from high-risk French Canadian breast/ovarian cancer families

BACKGROUND: Ataxia telangiectasia-mutated and Rad3-related (ATR) is a member of the PIK-related family which plays, along with ATM, a central role in cell-cycle regulation. ATR has been shown to phosphorylate several tumor suppressors like BRCA1, CHEK1 and TP53. ATR appears as a good candidate breast cancer susceptibility gene and the current study was designed to screen for ATR germline mutations potentially involved in breast cancer predisposition. METHODS: ATR direct sequencing was performed using a fluorescent method while widely available programs were used for linkage disequilibrium (LD), haplotype analyses, and tagging SNP (tSNP) identification. Expression analyses were carried out using real-time PCR. RESULTS: The complete sequence of all exons and flanking intronic sequences were analyzed in DNA samples from 54 individuals affected with breast cancer from non-BRCA1/2 high-risk French Canadian breast/ovarian families. Although no germline mutation has been identified in the coding region, we identified 41 sequence variants, including 16 coding variants, 3 of which are not reported in public databases. SNP haplotypes were established and tSNPs were identified in 73 healthy unrelated French Canadians, providing a valuable tool for further association studies involving the ATR gene, using large cohorts. Our analyses led to the identification of two novel alternative splice transcripts. In contrast to the transcript generated by an alternative splicing site in the intron 41, the one resulting from a deletion of 121 nucleotides in exon 33 is widely expressed, at significant but relatively low levels, in both normal and tumoral cells including normal breast and ovarian tissue. CONCLUSION: Although no deleterious mutations were identified in the ATR gene, the current study provides an haplotype analysis of the ATR gene polymorphisms, which allowed the identification of a set of SNPs that could be used as tSNPs for large-scale association studies. In addition, our study led to the characterization of a novel Δ33 splice form, which could generate a putative truncated protein lacking several functional domains. Additional studies in large cohorts and other populations will be needed to further evaluate if common and/or rare ATR sequence variants can be associated with a modest or intermediate breast cancer risk

Trastuzumab effects depend on HER2 phosphorylation in HER2-negative breast cancer cell lines.

The breast cancer (BC) biomarker HER2 (Human Epidermal Receptor 2) is overexpressed in 25% of BC. Only patients with HER2-positive tumors receive HER2-targeting therapies, like trastuzumab (Herceptin). However, some women with a HER2-negative BC could benefit from trastuzumab. This could be explained by the activation/phosphorylation of HER2 that can be recognized by trastuzumab. The aim of this study is to examine trastuzumab effects on HER2 phosphorylation at tyrosine Y877 (pHER2Y877). HER2 and pHER2Y877 status were evaluated in a cohort of BC patients representative of molecular subtypes distribution (n = 497) and in a series of BC cell lines (n = 7). Immunohistochemistry against pHER2Y877 was performed on tissue micro arrays. Cellular proliferation assays were performed on BC cell lines presenting different combinations of HER2 and pHER2Y877 status and treated with increasing doses of trastuzumab (0-150 μg/ml). The prevalence of pHER2Y877 in this cohort was 6%. Nearly 5% of patients with HER2-negative tumors (n = 406, 82%) overexpressed pHER2Y877. Among triple negative BC patients (n = 39, 8%), 7.7% expressed pHER2Y877. Trastuzumab treatment decreased cell proliferation in HER2-/pHER2Y877+ BC cell lines, to an extent comparable to what occurs in HER2+ cell lines, but did not affect HER2-/pHER2Y877- cell lines. Trastuzumab sensitivity in HER2-/pHER2Y877+ cell line is specific to HER2 tyrosine 877 phosphorylation. Hence, with further confirmation in a bigger cohort, trastuzumab treatment could be envisaged as a treatment option to women presenting with HER2-/pHER2+ tumors, representing more than 1000 BC women in Canada in 2019

An isoform of AIF1 involved in breast cancer

Abstract Background Inflammation is a major player in breast cancer (BC) progression. Allograft-inflammatory factor-1 (AIF1) is a crucial mediator in the inflammatory response. AIF1 reportedly plays a role in BC, but the mechanism remains to be elucidated. We identified two AIF1 isoforms, AIF1v1 and AIF1v3, which were differentially expressed between affected and unaffected sisters from families with high risk of BC with no deleterious BRCA1/BRCA2 mutations (BRCAX). We investigated potential functions of AIFv1/v3 in BC of varying severity and breast adipose tissue by evaluating their expression, and association with metabolic and clinical parameters of BC patients. Methods AIF1v1/v3 expression was determined in BC tissues and cell lines using quantitative real-time PCR. Potential roles and mechanisms were examined in the microenvironment (fibroblasts, adipose tissue, monocytes and macrophages), inflammatory response (cell reaction in BC subgroups), and metabolism [treatment with docosahexaenoic acid (DHA)]. Association of AIF1 transcript expression with clinical factors was determined by Spearman’s rank correlation. Bioinformatics analyses were performed to characterize transcripts. Results AIF1v1/v3 were mostly expressed in the less severe BC samples, and their expression appeared to originate from the tumor microenvironment. AIF1 isoforms had different expression rates and sources in breast adipose tissue; lymphocytes mostly expressed AIF1v1 while activated macrophages mainly expressed AIF1v3. Bioinformatics analysis revealed major structural differences suggesting distinct functions in BC progression. Lymphocytes were the most infiltrating cells in breast tumors and their number correlated with AIF1v1 adipose expression. Furthermore, DHA supplementation significantly lowered the expression of AIF1 isoforms in BRCAX cell lines. Finally, the expression of AIF1 isoforms in BC and breast adipose tissue correlated with clinical parameters of BC patients. Conclusions Results strongly suggest that AIF1v1 as much as AIF1v3 play a major role in the crosstalk between BC and infiltrating immune cells mediating tumor progression, implying their high potential as target molecules for BC diagnostic, prognostication and treatment

Role of Secreted Frizzled-Related Protein 1 in Early Breast Carcinogenesis and Breast Cancer Aggressiveness

A human transcriptome array on ERα-positive breast cancer continuum of risk identified Secreted Frizzled-Related Protein 1 (SFRP1) as decreased during breast cancer progression. In addition, SFRP1 was inversely associated with breast tissue age-related lobular involution, and differentially regulated in women with regard to their parity status and the presence of microcalcifications. The causal role of SFRP1 in breast carcinogenesis remains, nevertheless, not well understood. In this study, we characterized mammary epithelial cells from both nulliparous and multiparous mice in organoid culture ex vivo, in the presence of estradiol (E2) and/or hydroxyapatite microcalcifications (HA). Furthermore, we have modulated SFRP1 expression in breast cancer cell lines, including the MCF10A series, and investigated their tumoral properties. We observed that organoids obtained from multiparous mice were resistant to E2 treatment, while organoids obtained from nulliparous mice developed the luminal phenotype associated with a lower ratio between Sfrp1 and Esr1 expression. The decrease in SFRP1 expression in MCF10A and MCF10AT1 cell lines increased their tumorigenic properties in vitro. On the other hand, the overexpression of SFRP1 in MCF10DCIS, MCF10CA1a, and MCF7 reduced their aggressiveness. Our results support the hypothesis that a lack of SFRP1 could have a causal role in early breast carcinogenesis

Déploiement d’un algorithme de soins en réponse à la crise des opioïdes

Contexte : Entre janvier 2016 et juin 2020, le Canada a enregistré plus de 17 602 décès attribuables à des cas de surdose aux opioïdes ; une hausse inquiétante qui incite les professionnels de la santé à réfléchir à des interventions dans les salles d’urgence (SU) qui sont souvent la première ligne de soins pour les personnes à risque de surdose aux opioïdes (RSO).En avril 2018, un groupe pluridisciplinaire de cliniciens a développé le projet SuboxED pour établir un processus de mise en oeuvre d’un algorithme clinique pour la distribution de naloxone intranasale pour les patients à RSO et la prescription de buprénorphine-naloxone (B/n) dans trois SU du Québec pour les patients ayant un trouble de l’usage d’opioïdes (TUO).Méthodologie : Le projet SuboxED se présente en deux phases, la phase pré et mise en oeuvre et la phase d’évaluation clinique. Nous traiterons ici de la première phase qui s’est déroulée du 1er avril 2018 au 30 avril 2019 par un processus conçu en plusieurs étapes progressives : 1) constituer un groupe pluridisciplinaire de cliniciens ; 2) identifier trois SU, des cliniques TAO et les pharmacies partenaires au Québec ; 3) établir les critères d’éligibilité à la naloxone intranasale et la B/n basée sur les recommandations scientifiques pour créer l’algorithme ; 4) former le personnel des SU ; 5) mettre en oeuvre l’algorithme pour les patients RSO.Conclusion : Le projet SuboxED a développé un algorithme clinique en réponse à la crise des opioïdes au Québec et a contribué à la gratuité de la naloxone, tout en relevant de nombreux défis. La mise en oeuvre de tel algorithme est faisable et devrait être déployée largement surtout en temps de pandémie. Une phase de l’évaluation clinique suivra.Between January 2016 and June 2020, Canada recorded more than 17,602 deaths attributable to opioid overdose, an increase that prompts healthcare professionals to develop clinical interventions aimed at decreasing population overdose. As emergency departments (EDs) are often the first point of care for people at risk of opioid overdose, the current intervention focused on dispensing take-home naloxone (THN) and initiating opioid agonist treatment (OAT) among ED patients who are at risk of opioid overdose (ROO). In 2018, the SuboxED project convened a multidisciplinary group of clinical experts to implement a clinical algorithm for dispensing THN and prescribing buprenorphine/naloxone (B/n) for at ROO patients in 3 Québec EDs.Methodology: This project had two phases: 1) planning and implementation, and 2) evaluation. This article will describe the first phase, from April 1, 2018 to April 30, 2019, which included several progressive stages: 1) convening a multidisciplinary group of clinicians ; 2) identifying the EDs, OAT clinics, and pharmacy partnerships in Quebec ; 3) establishing the eligibility criteria for intranasal naloxone and B/n based on scientific recommendations ; 4) developing training tools and the ED algorithm ; and 5) implementing the algorithm for patients at ROO.Conclusion: The SuboxED project developed a clinical algorithm in response to the opioid crisis in Quebec and contributed to improving naloxone access in EDs, despite many challenges. The implementation of such an algorithm is feasible and should be deployed widely, especially during health crises such as the COVID-19 pandemic. The clinical evaluation of the implementation process will follow.Contexto: entre enero de 2016 y junio de 2020 Canadá registró más de 17 602 casos de muertes atribuibles a casos de sobredosis de opioides, un aumento inquietante que incita a los profesionales de la salud a considerar las intervenciones que se pueden realizar en las salas de emergencia para disminuir los riesgos de sobredosis, puesto que las emergencias están a menudo en la primera línea de la atención médica en los casos de personas a riesgo de sobredosis de opioides.En abril de 2018, un grupo multidisciplinario de personal médico desarrolló el proyecto SuboxED para establecer un proceso de puesta en práctica de un algoritmo clínico para la distribución de naloxona intranasal para los pacientes con riesgo de sobredosis de opioides y de la prescripción de buprenorfina-naloxona (B-n) en tres salas de emergencia de Quebec destinada a los pacientes que presentan un problema de consumo de opioides.Metodología: el proyecto SuboxED se presenta en dos etapas, la etapa previa a la implementación y la implementación y la etapa evaluación clínica de la implementación. Trataremos aquí la primera etapa que se ha aplicado del 1 de abril de 22018 al 30 de abril de 2019 por medio de un proceso concebido en varias etapas progresivas: 1) constituir un grupo multidisciplinario de personal médico ; 2) identificar tres salas de emergencia, las clínicas para pacientes que presentan problemas de consumo de drogas y las farmacias asociadas de Quebec ; 3) establecer los criterios de elegibilidad para la naloxona intranasal y la B-n sobre la base de las recomendaciones científicas para crear el algoritmo ; 4) formar el personal de las salas de emergencia ; 5) implementar el algoritmo para los pacientes a riesgo de una sobredosis por opioides.Conclusión: el proyecto SuboxED ha desarrollado un algoritmo clínico en respuesta a la crisis de los opioides en Quebec y ha contribuido a la gratuidad de la naloxona, debiendo enfrentar al mismo tiempo una cantidad de desafíos. La aplicación del algoritmo es factible y debería implementarse ampliamente, sobre todo en época de pandemia. Se realizará posteriormente una etapa de evaluación clínica