Glycomic Approaches for the Discovery of Targets in Gastrointestinal Cancer

Gastrointestinal (GI) cancer is the most common group of malignancies and many of its types are among the most deadly. Various glycoconjugates have been used in clinical practice as serum biomarker for several GI tumors, however, with limited diagnose application. Despite the good accessibility by endoscopy of many GI organs, the lack of reliable serum biomarkers often leads to late diagnosis of malignancy and consequently low 5-year survival rates. Recent advances in analytical techniques have provided novel glycoproteomic and glycomic data and generated functional information and putative biomarker targets in oncology. Glycosylation alterations have been demonstrated in a series of glycoconjugates (glycoproteins, proteoglycans, and glycosphingolipids) that are involved in cancer cell adhesion, signaling, invasion, and metastasis formation. In this review, we present an overview on the major glycosylation alterations in GI cancer and the current serological biomarkers used in the clinical oncology setting. We further describe recent glycomic studies in GI cancer, namely gastric, colorectal, and pancreatic cancer. Moreover, we discuss the role of glycosylation as a modulator of the function of several key players in cancer cell biology. Finally, we address several state-of-the-art techniques currently applied in this field, such as glycomic and glycoproteomic analyses, the application of glycoengineered cell line models, microarray and proximity ligation assay, and imaging mass spectrometry, and provide an outlook to future perspectives and clinical applications.We acknowledge the support from the European Union, Seventh Framework Programme, Gastric Glyco Explorer initial training network: grant number 316929. IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT, the Portuguese Foundation for Science and Technology. This work is funded by FEDER funds through the Operational Programme for Competitiveness Factors-COMPETE (FCOMP-01-0124- FEDER028188) and National Funds through the FCT-Foundation for Science and Technology, under the projects: PEst-C/SAU/ LA0003/2013, PTDC/BBB-EBI/0786/2012, and PTDC/BBBEBI/0567/2014. AM acknowledges the grant received from FCT, POPH (Programa Operacional Potencial Humano), and FSE (Fundo Social Europeu) (SFRH/BPD/75871/2011). MB acknowledges the University of Girona for pre-doctoral fellowship

Risk-Adjusted Cancer Screening and Prevention (RiskAP): Complementing Screening for Early Disease Detection by a Learning Screening Based on Risk Factors

Breast cancer; Evidence-generating care; Risk-adjusted preventionCáncer de mama; Atención generadora de evidencia; Prevención ajustada al riesgoCàncer de mama; Atenció generadora d'evidències; Prevenció ajustada al riscBackground: Risk-adjusted cancer screening and prevention is a promising and continuously emerging option for improving cancer prevention. It is driven by increasing knowledge of risk factors and the ability to determine them for individual risk prediction. However, there is a knowledge gap between evidence of increased risk and evidence of the effectiveness and efficiency of clinical preventive interventions based on increased risk. This gap is, in particular, aggravated by the extensive availability of genetic risk factor diagnostics, since the question of appropriate preventive measures immediately arises when an increased risk is identified. However, collecting proof of effective preventive measures, ideally by prospective randomized preventive studies, typically requires very long periods of time, while the knowledge about an increased risk immediately creates a high demand for action. Summary: Therefore, we propose a risk-adjusted prevention concept that is based on the best current evidence making needed and appropriate preventive measures available, and which is constantly evaluated through outcome evaluation, and continuously improved based on these results. We further discuss the structural and procedural requirements as well as legal and socioeconomical aspects relevant for the implementation of this concept.The project was funded by the German Federal Ministry of Health (grant No. 2515FSB401 to Rita Schmutzler and Christiane Woopen) for supporting the international expert meetings, and a grant of the EU Horizon 2020 program, BRIDGES (grant No. 634935, PI Peter Devilee, WP5-PI Rita Schmutzler), for the compilation of the most recent findings of genetic risk prediction

Identification of appropriate housekeeping genes for quantitative RT-PCR analysis in MDA-MB-231 and NCI-H460 human cancer cell lines under hypoxia and serum deprivation

Gene expression studies aimed at analyzing cancer cells under hypoxia and serum deprivation conditions show major potential for understanding molecular mechanisms associated with tumor progression as well as resistance to antitumor agents. To the best of our knowledge, a study for the identification of appropriate housekeeping genes in breast and lung cancer cells under hypoxia and serum deprivation conditions is currently missing. Given the relevance of a reliable and accurate normalization, we herein aimed to identify the appropriate housekeeping genes for breast and lung cancer cell lines cultured under hypoxia and/or serum deprivation. The stability of five commonly used housekeeping genes (ACTB, β 2M, GUSB, 18S rRNA, and PPIA) was assessed after reverse-transcription quantitative real-time PCR in MDA-MB-231 and NCI-H460 cancer cell lines using GeNorm, NormFinder and BestKeeper software. GeNorm and NormFinder ranking revealed ACTB, GUSB and PPIA as the most stable genes for both tumor cell lines. Our results support the use of ACTB/PPIA for MDA-MB-231 and GUSB/PPIA for NCI-H460 cells as the most stable combination for normalization of gene expression under hypoxic and serum deprivation conditions. Our results highlight the importance of the selection of the housekeeping genes in cancer cells subjected to different physiological stresses, such as hypoxia and serum deprivation.This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nıvel Superior (CAPES) [grant agreement No. 88881.132780/2016-01]; Fundac ão de Amparoa Ciência e Tecnologia de Pernambuco (FACEPE) [grant agreement No. 0883-2.08/13];and the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie [grant agreement No. 748880]. This work was also funded by the project NORTE-01-0145-FEDER-000029, supported by Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF); and by FEDER—Fundo Europeu de Desenvolvimento Regional funds through COMPETE 2020—Operational Programme for Competitiveness and Internationalization (POCI), Portugal 2020,and by Portuguese funds through FCT (Fundação para a Ciência e a Tecnologia)/Ministério da Ciência, Tecnologia e Inovação in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274), and the project POCI-01-0145-FEDER-016585 (PTDC/BBB EBI/0567/2014)

Different isolation approaches lead to diverse glycosylated extracellular vesicle populations

Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter-cellular communication and mediating a broad spectrum of biological functions. EVs cargo iscomposed of a large repertoire of molecules, including glycoconjugates. Herein, we report the firststudy on the impact of the isolation strategy on the EV populations’glycosylation profile. The use ofdifferent state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isola-tion (TEI), OptiPrepTMdensity gradient (ODG) and size exclusion chromatography (SEC) resulted in EVpopulations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODGand SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated themost distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EVglycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn(STn) were identified as packaged cargo into EVs independently of the isolation methodology. STncarrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-relatedproteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of usingdifferent isolation methodologies in the populations of EVs that are obtained, with consequences inthe glycosylation profile of the isolated population. Furthermore, our results highlight the importanceof selecting adequate EV isolation protocols and cell culture conditions to determine the structuraland functional complexity of the EV glycoconjugates.This work was funded by FEDER funds through theOperational Programme for Competitiveness Factors-COMPETE (POCI-01-0145-FEDER-016585; POCI-01-0145-FEDER-007274; POCI-01-0145-FEDER-028489) and NationalFunds through the Foundation for Science and Technology(FCT), under the projects: PTDC/BBB-EBI/0567/2014 (toCAR), PTDC/MED-ONC/28489/2017 (to AM) and UID/BIM/04293/2013; and the project NORTE-01-0145-FEDER-000029, supported by Norte Portugal Regional Programme(NORTE 2020), under the PORTUGAL 2020 PartnershipAgreement, through the European Regional DevelopmentFund (ERDF). DF acknowledges the FCT PhD Programmesand Programa Operacional Potencial Humano (POPH), speci-fically the Biotech Health Programme (Doctoral Programme onCellular and Molecular Biotechnology Applied to HealthSciences), with the reference PD/0016/2012 funded by FCTand the grant SFRH/BD/110636/2015 from FCT, POPH andFSE (Fundo Social Europeu); MB acknowledges the EuropeanUnion’s Horizon 2020 research and innovation programmeunder the Marie Sklodowska-Curie grant agreement No.748880; and JP acknowledges FCT (SFRH/BD/137319/2018).The authors acknowledge Rede Nacional de Espectrometria deMassa, ROTEIRO/0028/2013, ref. LISBOA-01-0145-FEDER-022125, supported by COMPETE and North PortugalRegional Operational Programme (Norte2020), under thePORTUGAL 2020 Partnership Agreement, through theEuropean Regional Development Fund (ERDF). SV acknowl-edges the Danish National Research Foundation (DNRF107)

The predictive ability of the 313 variant–based polygenic risk score for contralateral breast cancer risk prediction in women of European ancestry with a heterozygous BRCA1 or BRCA2 pathogenic variant

Predicció de risc de càncer de mama; Dones europees; Variant patògena heterozigotaPredicción del riesgo de cáncer de mama; Mujeres europeas; Variante patógena heterocigotaBreast cancer risk prediction; European women; Heterozygous pathogenic variantPurpose To evaluate the association between a previously published 313 variant–based breast cancer (BC) polygenic risk score (PRS313) and contralateral breast cancer (CBC) risk, in BRCA1 and BRCA2 pathogenic variant heterozygotes. Methods We included women of European ancestry with a prevalent first primary invasive BC (BRCA1 = 6,591 with 1,402 prevalent CBC cases; BRCA2 = 4,208 with 647 prevalent CBC cases) from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA), a large international retrospective series. Cox regression analysis was performed to assess the association between overall and ER-specific PRS313 and CBC risk. Results For BRCA1 heterozygotes the estrogen receptor (ER)-negative PRS313 showed the largest association with CBC risk, hazard ratio (HR) per SD = 1.12, 95% confidence interval (CI) (1.06–1.18), C-index = 0.53; for BRCA2 heterozygotes, this was the ER-positive PRS313, HR = 1.15, 95% CI (1.07–1.25), C-index = 0.57. Adjusting for family history, age at diagnosis, treatment, or pathological characteristics for the first BC did not change association effect sizes. For women developing first BC < age 40 years, the cumulative PRS313 5th and 95th percentile 10-year CBC risks were 22% and 32% for BRCA1 and 13% and 23% for BRCA2 heterozygotes, respectively. Conclusion The PRS313 can be used to refine individual CBC risks for BRCA1/2 heterozygotes of European ancestry, however the PRS313 needs to be considered in the context of a multifactorial risk model to evaluate whether it might influence clinical decision-making

Overall survival in the OlympiA phase III trial of adjuvant olaparib in patients with germline pathogenic variants in BRCA1/2 and high-risk, early breast cancer

Adjuvant therapy; Breast cancer; OlaparibTerapia adyuvante; Cáncer de mama; OlaparibTeràpia adjuvant; Càncer de mama; OlaparibBackground The randomized, double-blind OlympiA trial compared 1 year of the oral poly(adenosine diphosphate-ribose) polymerase inhibitor, olaparib, to matching placebo as adjuvant therapy for patients with pathogenic or likely pathogenic variants in germline BRCA1 or BRCA2 (gBRCA1/2pv) and high-risk, human epidermal growth factor receptor 2-negative, early breast cancer (EBC). The first pre-specified interim analysis (IA) previously demonstrated statistically significant improvement in invasive disease-free survival (IDFS) and distant disease-free survival (DDFS). The olaparib group had fewer deaths than the placebo group, but the difference did not reach statistical significance for overall survival (OS). We now report the pre-specified second IA of OS with updates of IDFS, DDFS, and safety. Patients and methods One thousand eight hundred and thirty-six patients were randomly assigned to olaparib or placebo following (neo)adjuvant chemotherapy, surgery, and radiation therapy if indicated. Endocrine therapy was given concurrently with study medication for hormone receptor-positive cancers. Statistical significance for OS at this IA required P < 0.015. Results With a median follow-up of 3.5 years, the second IA of OS demonstrated significant improvement in the olaparib group relative to the placebo group [hazard ratio 0.68; 98.5% confidence interval (CI) 0.47-0.97; P = 0.009]. Four-year OS was 89.8% in the olaparib group and 86.4% in the placebo group (Δ 3.4%, 95% CI −0.1% to 6.8%). Four-year IDFS for the olaparib group versus placebo group was 82.7% versus 75.4% (Δ 7.3%, 95% CI 3.0% to 11.5%) and 4-year DDFS was 86.5% versus 79.1% (Δ 7.4%, 95% CI 3.6% to 11.3%), respectively. Subset analyses for OS, IDFS, and DDFS demonstrated benefit across major subgroups. No new safety signals were identified including no new cases of acute myeloid leukemia or myelodysplastic syndrome. Conclusion With 3.5 years of median follow-up, OlympiA demonstrates statistically significant improvement in OS with adjuvant olaparib compared with placebo for gBRCA1/2pv-associated EBC and maintained improvements in the previously reported, statistically significant endpoints of IDFS and DDFS with no new safety signals.Funding for this work, which was conducted as a collaborative partnership among the Breast International Group, NRG Oncology, Frontier Science, AstraZeneca, and Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, U.S.A. (MSD), was provided by the National Institutes of Health (grant numbers: U10CA 180868, UG1CA 189867, and U10CA 180822) and by AstraZeneca as part of an alliance between AstraZeneca and MSD. Provision of olaparib and placebo was from AstraZeneca

Safety of assisted reproductive techniques in young women harboring germline pathogenic variants in BRCA1/2 with a pregnancy after prior history of breast cancer

Breast cancer fertility; Pregnancy; SurvivalFertilidad y cáncer de mama; Embarazo; SupervivenciaFertilitat i càncer de mama; Embaràs; SupervivènciaBackground Knowledge is growing on the safety of assisted reproductive techniques (ART) in cancer survivors. No data exist, however, for the specific population of breast cancer patients harboring germline BRCA1/2 pathogenic variants. Patients and methods This is a multicenter retrospective cohort study across 30 centers worldwide including women diagnosed at ≤40 years with stage I-III breast cancer, between January 2000 and December 2012, harboring known germline BRCA1/2 pathogenic variants. Patients included in this analysis had a post-treatment pregnancy either achieved through use of ART (ART group) or naturally (non-ART group). ART procedures included ovulation induction, ovarian stimulation for in vitro fertilization or intracytoplasmic sperm injection, and embryo transfer under hormonal replacement therapy. Results Among the 1424 patients registered in the study, 168 were eligible for inclusion in the present analysis, of whom 22 were in the ART group and 146 in the non-ART group. Survivors in the ART group conceived at an older age compared with those in the non-ART group (median age: 39.7 versus 35.4 years, respectively). Women in the ART group experienced more delivery complications compared with those in the non-ART group (22.1% versus 4.1%, respectively). No other apparent differences in obstetrical outcomes were observed between cohorts. The median follow-up from pregnancy was 3.4 years (range: 0.8-8.6 years) in the ART group and 5.0 years (range: 0.8-17.6 years) in the non-ART group. Two patients (9.1%) in the ART group experienced a disease-free survival event (specifically, a locoregional recurrence) compared with 40 patients (27.4%) in the non-ART group. In the ART group, no patients deceased compared with 10 patients (6.9%) in the non-ART group. Conclusion This study provides encouraging safety data on the use of ART in breast cancer survivors harboring germline pathogenic variants in BRCA1/2, when natural conception fails or when they opt for ART in order to carry out preimplantation genetic testing.The present work was supported by the Italian Association for Cancer Research (‘Associazione Italiana per la Ricerca sul Cancro’, AIRC; MFAG 2020 ID 24698) and the Italian Ministry of Health (5 × 1000 funds 2017). MC and ID acknowledge the Fonds National de la Recherche Scientifique (FNRS and Télévie 7.6508.20) and Fonds Erasme for their financial support

Molecular features of the basal-like breast cancer subtype based on BRCA1 mutation status

BRCA1-mutated breast cancer is associated with basal-like disease; however, it is currently unclear if the presence of a BRCA1 mutation depicts a different entity within this subgroup. In this study, we compared the molecular features among basal-like tumors with and without BRCA1 mutations. Fourteen patients with BRCA1-mutated (nine germline and five somatic) tumors and basal-like disease, and 79 patients with BRCA1 non-mutated tumors and basal-like disease, were identified from the cancer genome atlas dataset. The following molecular data types were evaluated: global gene expression, selected protein and phospho-protein expression, global miRNA expression, global DNA methylation, total number of somatic mutations, TP53 and PIK3CA somatic mutations, and global DNA copy-number aberrations. For intrinsic subtype identification, we used the PAM50 subtype predictor. Within the basal-like disease, we observed minor molecular differences in terms of gene, protein, and miRNA expression, and DNA methylation variation, according to BRCA1 status (either germinal or somatic). However, there were significant differences according to average number of mutations and DNA copy-number aberrations, and four amplified regions (2q32.2, 3q29, 6p22.3, and 22q12.2), which are characteristic in high-grade serous ovarian carcinomas, were observed in both germline and somatic BRCA1-mutated breast tumors. These results suggest that minor, but potentially relevant, baseline molecular features exist among basal-like tumors according to BRCA1 status. Additional studies are needed to better clarify if BRCA1 genetic status is an independent prognostic feature, and more importantly, if BRCA1 mutation status is a predictive biomarker of benefit from DNA-damaging agents among basal-like disease.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-014-3056-x) contains supplementary material, which is available to authorized users