MicroRNAs derived from extracellular vesicles (EV-miRNAs) are circulating miRNAs considered as potential new diagnostic markers for cancer that can be easily detected in liquid biopsies. In this study, we performed RNA sequencing analysis as a screening strategy to identify EV-miRNAs derived from serum of clinically well-annotated breast cancer (BC) patients from the south of Brazil. EVs from three groups of samples (healthy controls (CT), luminal A (LA), and triple-negative (TNBC)) were isolated from serum using a precipitation method and analyzed by RNA-seq (screening phase). Subsequently, four EV-miRNAs (miR-142-5p, miR-150-5p, miR-320a, and miR-4433b-5p) were selected to be quantified by quantitative real-time PCR (RT-qPCR) in individual samples (test phase). A panel composed of miR-142-5p, miR-320a, and miR-4433b-5p distinguished BC patients from CT with an area under the curve (AUC) of 0.8387 (93.33% sensitivity, 68.75% specificity). The combination of miR-142-5p and miR-320a distinguished LA patients from CT with an AUC of 0.9410 (100% sensitivity, 93.80% specificity). Interestingly, decreased expression of miR-142-5p and miR-150-5p were significantly associated with more advanced tumor grades (grade III), while the decreased expression of miR-142-5p and miR-320a was associated with a larger tumor size. These results provide insights into the potential application of EVs-miRNAs from serum as novel specific markers for early diagnosis of BC.
Improvement of breast cancer (BC) patient's outcome is directly related to early detection. However, there is still a lack of reliable biomarkers for diagnosis, prognosis and, treatment follow up in BC, leading researchers to study the potential of liquid biopsy based on circulating microRNAs (c-miRNAs). These c-miRNAs can be cellfree or associated with extracellular vesicles (EVs), and have great advantages such as stability in biofluids, noninvasive accessibility compared to current techniques (core-biopsy and surgery), and expression associated with pathogenic conditions. Recently, a new promising field of EV-derived miRNAs (EV-miRNAs) as cancer biomarkers has emerged, receiving special attention due to their selective vesicle sorting which makes them accurate for disease detection. In this review, we discuss new findings about c-miRNA and their potential as biomarkers for BC diagnosis, prognosis, and therapy. Additionally, we address the impact of limitations associated with the standardization of analysis techniques and methods on the implementation of these biomarkers in the clinical setting. (Translational Research 2020; 223:40À60) Abbreviations: BC = breast cancer; c-miRNAs = circulating microRNAs; cf-miRNAs = cell-free microRNAs; EVs = extracellular vesicles; EV-miRNAs = EV-derived miRNAs; ER = estrogen receptor; PR = progesterone receptor; ERBB2 = human epidermal growth factor receptor 2; TNBC = triple negative breast cancer; CTCs = circulating tumor cells; CA15-3 = cancer antigen 15-3; CEA = carcinoembryonic antigen; RANTES/CCL5 = chemokine (CÀC motif) ligand 5; OPN = osteopontin; PAI-1 = plasminogen activator inhibitor-1; AUC = area under the curve; RT-qPCR = reverse transcription quantitative polymerase chain reaction; CT = controls; OS = overall survival; dPCR = digital PCR; NGS = next generation sequencing BACKGROUND Breast cancer (BC) is the most frequently diagnosed cancer in females and the leading cause of death by cancer worldwide, with the mortality rate predicted to reach around 626,679 deaths worldwide in 2018. 1 This cancer can be classified in several ways, one of the methods of clinical evaluation being the immunohistochemistry classification. 2 According to this classification, BC is stratified based on the expression of 4 main protein markers À estrogen receptor (ER), progesterone, receptor (PR), the proliferation marker and
Fanconi anemia is a rare hereditary disease showing genetic heterogeneity due to a variety of mutations in genes involved in DNA repair pathways, which may lead to different clinical manifestations. Phenotypic variability makes diagnosis difficult based only on clinical manifestations, therefore laboratory tests are necessary. New advances in molecular pathogenesis of this disease led researchers to develop a diagnostic test based on Western blot for FANCD2. The objective of the present study was to determine the efficacy of this method for the diagnosis of 84 Brazilian patients with Fanconi anemia, all of whom tested positive for the diepoxybutane test, and 98 healthy controls. The FANCD2 monoubiquitinated isoform (FANCDS+/FANCD2L-) was not detected in 77 patients (91.7%). In 2 patients (2.4%), there was an absence of both the monoubiquitinated and the non-ubiquitinated proteins (FANCD2S-/FANCD2L-) and 5 patients (5.9%) had both isoforms (FANCD2S+/FANCD2L+). This last phenotype suggests downstream subtypes or mosaicism. All controls were diepoxybutane negative and were also negative on the FANCD2 Western blot. The Western blot for FANCD2 presented a sensitivity of 94% (79/84) and specificity of 100% (98/98). This method was confirmed as an efficient approach to screen Brazilian patients with deleterious mutations on FANCD2 (FANCD2S-/FANCD2L-) or other upstream genes of the FA/BRCA pathway (FANCDS+/FANCD2L-), to confirm the chromosome breakage test and to classify patients according to the level of FA/BRCA pathway defects. However, patients showing both FANCD2 isoforms (FANCD2S+/FANCD2L+) require additional studies to confirm mutations on downstream Fanconi anemia genes or the presence of mosaicism.
Exosomes are extracellular vesicles that are present in body fluids and known to play key roles in intercellular signaling communication. Exosomes carry several types of molecules of their tumor of origin, including miRNAs, and therefore present potential as biomarkers for cancer prognosis and treatment. Triple-negative breast cancer (TNBC) is a clinically aggressive subtype of breast cancer, associated with treatment resistance, recurrence and high mortality rates. These tumors are molecularly heterogeneous, which impairs the identification of effective prognostic molecular markers and target therapies. The main objective of this study was to characterize the miRNA expression profile of exosomes derived from TNBC cells in comparison to normal controls and to the luminal A (LA) breast cancer subtype. The exosomal miRNAs were isolated from 19 serum samples of breast cancer patients (10 with the LA and 9 with the TNBC subtype) and 10 serum samples of healthy women (control group) using the exosome precipitation method. Exosome characterization and quantification was assessed by Western blot using the exosomal surface markers, CD6 and CD9, and Nanotracking analysis. The tumor and normal exosomal samples were combined into 6 different pools (of at least 4 samples in each group) and sequenced in the Ion Torrent Proton platform (Applied Biosystems). RNA-seq analysis revealed differentially expressed (DE) levels of exosomal miRNAs, with log2FC ranging from 1.2 to 3.6, among the four groups studied (control, cancer (TNBC+LA), TNBC and LA groups): 22 DE miRNAs were observed in the cancer vs. control, 19 in the LA vs control, 30 in the TNBC vs. control and 7 in the LA vs. TNBC groups. Eleven miRNAs were commonly DE in the cancer, LA and TNBC groups when compared to the control group, which included miRNAs, such as miR-103a-3p, miR-107 and miR-423-5p, involved in the biosynthesis of unsaturated fatty acids and neutrophin signaling pathways. Nine miRNAs were only observed DE in the TNBC group (vs. control), with the majority of them being downregulated. The top 10 significant functional enriched pathways affected by these miRNAs included proteoglycans, pathways in cancer and TGF-beta signaling pathways. Only three miRNAs were observed DE in the TNBC vs. LA groups, all with downregulated expression in the TNBC group. These miRNAs were observed mostly involved in fatty acid biosynthesis, proteoglycans in cancer and TGF-beta signaling pathways. In conclusion, our results indicate that expression alterations of exosomal miRNAs affect critical cancer-associated signaling pathways, suggesting their potential use as noninvasive cancer biomarkers. Additional studies should be performed to determine whether the expression patterns of these exosomal miRNAs reflect the ones observed in the corresponding patients' TNBC tissue. Funding: CAPES, PPSUS-Fundação Araucária, Brazil. Citation Format: Patricia M. Ozawa, Débora S. Lemos, Evelyn Vieira, Ingrid L. Souza, Silvio M. Zanata, Vânia C. Pankievicz, Thalita R. Tuleski, Emanuel M. Souza, Rosiane V. Silva, Pryscilla F. Wowk, Rodrigo C. Almeida, Iglenir J. Cavalli, Danielle M. Ferreira, Luciane R. Cavalli, Enilze M. Ribeiro. Exosomal miRNA expression profiling in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5392.
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