Background: Neoadjuvant chemotherapy (NACT) for non-metastatic breast cancer is often preferred over adjuvant chemotherapy to shrink tumours and facilitate surgical removal. NACT typically comprises 8 cycles over 20–24 weeks: 4 cycles of anthracycline with cyclophosphamide followed by 4 cycles of paclitaxel. After surgery, the therapeutic response is assessed histopathologically using the TNM classification, where ypT0 indicates pathological complete remission (PCR) and residual tumour cells (> ypT0) indicate non-complete remission (non-PCR). Currently, imaging techniques such as ultrasound are used during NACT to assess clinical response. Liquid biopsy-based methods may complement imaging by enabling early response monitoring. In this study, we used serum proteomics to identify marker candidates associated with PCR as early as after the second NACT cycle. Methods: Longitudinal, retrospective serum proteomic analyses were performed on 22 breast cancer patients (11 PCR, 11 non-PCR) and 21 age-matched healthy controls. Serum samples were collected pre-therapy and after two and six NACT cycles. Proteins were analysed using liquid chromatography–tandem mass spectrometry (LC–MS/MS) following immunoaffinity depletion, trypsin digestion, tandem mass tag labelling, and fractionation. Protein quantitation was performed with MaxQuant software, and abundance analysis utilised linear models of microarray analysis. Tumour-resident expression of a candidate marker was evaluated via immunohistochemistry in an independent cohort of 37 cases. Results: Across 84 samples, >390 proteins were consistently identified and quantified. Pre-therapy serum proteomes showed no significant differences between PCR and non-PCR groups. Longitudinal analysis revealed that serum levels of c-Met and N-cadherin could distinguish responders after the second NACT cycle with high predictive value (AUC 0.93). More pronounced changes were observed after the sixth cycle, including significant alterations in centrosomal protein, sex hormone-binding globulin, and cholinesterase levels. Additionally, N-cadherin expression was elevated in therapy-naïve tumour samples from patients achieving PCR. Conclusions: This study highlights the potential of serum proteomics for identifying markers to assess NACT efficacy in breast cancer. Soluble N-cadherin and c-Met may serve as promising serum markers for PCR, particularly when combined with (immune)histochemical tumour characterisation.

Breast cancer remains the most common cancer in women worldwide. Neoadjuvant chemotherapy (NACT) is often preferred to adjuvant chemotherapy to achieve tumour shrinkage, monitor response to therapy and facilitate surgical removal in the absence of metastases. In addition, there is strong evidence that pathological complete remission (pCR) is associated with prolonged survival. In this study, we sought to identify candidate markers that signal response or resistance to therapy. We present a retrospective longitudinal serum proteomic study of 22 breast cancer patients (11 with pCR and 11 with non-pCR) matched with 21 healthy controls. Serum was analysed by LC-MS/MS after depletion of abundant proteins by immunoaffinity, trypsinisation, isobaric labelling and fractionation by reversed-phase HPLC. We observed an inverse behaviour of the serum proteins c-Met and N-cadherin after the second cycle of chemotherapy with a high predictive value (AUC 0.93). More pronounced changes were observed after the 6th cycle of NACT, with significant changes in the intensity of the proteins contactin-1, centrosomal protein, sex hormone-binding globuline and cholinesterase. Our study highlights the possibility of monitoring response to NACT using serum as a liquid biopsy.

PURPOSE:Ovarian cancer is the seventh most frequent form of malignant diseases in women worldwide and over 150,000 women die from it every year. More than 70 percent of all ovarian cancer patients are diagnosed at a late-stage disease with poor prognosis necessitating the development of sufficient screening biomarkers. MicroRNAs displayed promising potential as early diagnostics in various malignant diseases including ovarian cancer. The presented study aimed at identifying single microRNAs and microRNA combinations detecting ovarian cancer in vitro and in vivo.METHODS:Intracellular, extracellular and urinary microRNA expression levels of twelve microRNAs (let-7a, let-7d, miR-10a, miR-15a, miR-15b, miR-19b, miR-20a, miR-21, miR-100, miR-125b, miR-155, miR-222) were quantified performing quantitative real-time-PCR. Therefore, the three ovarian cancer cell lines SK-OV-3, OAW-42, EFO-27 as well as urine samples of ovarian cancer patients and healthy controls were analyzed.RESULTS:MiR-15a, miR-20a and miR-222 showed expression level alterations extracellularly, whereas miR-125b did intracellularly across the analyzed cell lines. MicroRNA expression alterations in single cell lines suggest subtype specificity in both compartments. Hypoxia and acidosis showed scarce effects on single miRNA expression levels only. Furthermore, we were able to demonstrate the feasibility to clearly detect the 12 miRNAs in urine samples. In urine, miR-15a was upregulated whereas let-7a was down-regulated in ovarian cancer patients.CONCLUSION:Intracellular, extracellular and urinary microRNA expression alterations emphasize their great potential as biomarkers in liquid biopsies. Especially, miR-15a and let-7a qualify for possible circulating biomarkers in liquid biopsies of ovarian cancer patients.

Purpose In 2020, breast cancer still represents the most common type of cancer in women worldwide. Depending on the specific molecular subtype, clinical breast cancer management comprises surgery, radiotherapy, chemotherapy and targeted therapy. Furthermore, there are some therapeutic approaches from the field of complementary and alternative medicine. Current research focuses on the elucidation of new therapeutic targets for treatment development. Odorant substances affect apoptosis, proliferation and cell cycle in healthy and cancerous cells. Exact signalling pathways involved are not entirely clear. The present study aims to analyse their therapeutic potential in breast cancer. Methods This study focuses on the effect of commonly used odorant substances (citral, citrathal R, cyclovertal, para-cymol, hexylacetat, herbavert, dihydromyrcerol and limonen) on the breast cancer cell lines MDA-MB-231, T47-D and BT474. Methodologically, this study applied cell culturing, MTT assay for detection of IC50 of the odorant substance, RNA purification followed by qRT-PCR, protein isolation and Western Blot, as well as immunocytochemistry. Further, this study investigates the role of transient receptor potential channel V1 (TRPV1), involved in the mechanisms of action for some odorant substances. Therefore, capsazepine, a TRPV1 antagonist, was used. Results The odorant substances citral, citrathal R and cyclovertal have significant pro-apoptotic (p < 0.001), anti-proliferative (p < 0.001) and cell cycle-arresting effects measurable in RNA expression as well as in protein levels and immunocytochemical staining. The combination of citral and capsazepine no longer showed significant pro-apoptotic, antiproliferative, and cell cycle inhibitory effects compared to the compounds alone. This indicates that TRPV1 is necessary for the signal transduction of citral. Conclusion This present study reveals three odorant substances with effects on cell viability, indicating their potential use in breast cancer therapy.

Breast cancer (BC) represents the most common type of cancer in females worldwide. Endocrine therapy evolved as one of the main concepts in treatment of hormone-receptor positive BC. Current research focuses on the elucidation of tumour resistance mechanisms against endocrine therapy. In a translational in vitro approach, potential regulatory effects of clinically implemented BC anti-oestrogens on ERα, its coactivators DDX5, DDX17 and other DEADbox proteins as well as on the proliferation markers cyclin D1 and Ki67 were investigated on both the RNA and protein level. BC in vitro models for hormone-receptor positive (MCF-7, T-47D) and hormone-receptor negative cells (BT-20) were subjected to endocrine therapy. Anti-oestrogen-dependent expression regulation of target genes on the transcriptional and translational level was quantified and statistically assessed. Endocrine therapy decreases the expression levels of Ki67, cyclin D1 and ERα in hormone-receptor positive cells. In the hormone-receptor negative cells, the three parameters remained stable after endocrine therapy. Endoxifen triggers a downregulation of DDX5 and DDX23 in MCF-7 cells. Fulvestrant treatment downregulates the expression levels of all investigated DEADbox proteins in MCF-7 cells. In T-47D cells, endoxifen and fulvestrant lead to a decrease of all target gene expression levels. Interestingly, endocrine therapy affects DEADbox RNA expression levels in BT-20 cells, too. However, this result could only be confirmed for DDX1, immunocytologically. The investigated DEADbox proteins appear to correlate with the oestrogen-dependent tumourigenesis in hormone-receptor positive BC and show expression alterations after endocrine treatment.

Due to the positive association between neoadjuvant chemotherapy (NACT) and the promising early response rates of patients with triple negative breast cancer (TNBC), including probabilities of pathological complete response, NACT is increasingly used in TNBC management. Liquid biopsy-based biomarkers with the power to diagnose the early response to NACT may support established monitoring tools, which are to a certain extent imprecise and costly. Simple serum- or urine-based analyses of non-coding RNA (ncRNA) expression may allow for fast, minimally-invasive testing and timely adjustment of the therapy regimen. The present study investigated breast cancer-related ncRNAs [microRNA (miR)-7, -9, -15a, -17, -18a, -19b, -21, -30b, -222 and -320c, PIWI-interacting RNA-36743 and GlyCCC2] in triple positive BT-474 cells and three TNBC cell lines (BT-20, HS-578T and MDA-MB-231) treated with various chemotherapeutic agents using reverse transcription-quantitative PCR. Intracellular and secreted microvesicular ncRNA expression levels were analysed using a multivariable statistical regression analysis. Chemotherapy-driven effects were investigated by analysing cell cycle determinants at the mRNA and protein levels. Serum and urine specimens from 8 patients with TNBC were compared with 10 healthy females using two-sample t-tests. Samples from the patients with TNBC were compared at two time points. Chemotherapeutic treatments induced distinct changes in ncRNA expression in TNBC cell lines and the BT-474 cell line in intra- and extracellular compartments. Serum and urine-based ncRNA expression analysis was able to discriminate between patients with TNBC and controls. Time point comparisons in the urine samples of patients with TNBC revealed a general rise in the level of ncRNA. Serum data suggested a potential association between piR-36743, miR-17, -19b and -30b expression levels and an NACT-driven complete clinical response. The present study highlighted the potential of ncRNAs as liquid biopsy-based biomarkers in TNBC chemotherapy treatment. The ncRNAs tested in the present study have been previously investigated for their involvement in BC or TNBC chemotherapy responses; however, these previous studies were restricted to patient tissue or in vitro models. The data from the present study offer novel insight into ncRNA expression in liquid samples from patients with TNBC, and the study serves as an initial step in the evaluation of ncRNAs as diagnostic biomarkers in the monitoring of TNBC therapy.

Breast cancer (BC) is the most frequent malignant disease in women worldwide and the leading cause of cancer associated death as well. As especially advanced stages are challenging for the healthcare system, early BC detection is crucial striving for improved overall outcome and disease management. Established screening procedures show limited success in the early diagnosis of the disease and inherit several limitations. Aiming at a faster, easier and non-invasive BC screening, liquid biopsy biomarkers bear great potential to complete the current state-of-the-art diagnostics. This study explores the diagnostic potential of BC specific urinary microRNAs as a potential non-invasive BC screening method. Based on a case-control-study (69 BC patients vs. 40 healthy controls), expression level quantification and subsequent biostatistical computation of thirteen urine-derived microRNAs was performed to evaluate their diagnostic relevance in BC. Multilateral statistical assessment determined and repeatedly confirmed a specific panel of four urinary microRNAs (miR-424/miR-423/mir-660/let7-i) as highly specific combinatory biomarker tool. Herewith, it is possible to discriminate BC patients from healthy controls with 98.6 % sensitivity and 100 % specificity. Smaller feasibility studies have proven this method as highly potent already years ago. The given study finally provides large scale observations, sufficient statistic analysis, more refined methods and highly satisfying sensitivity and specificity. Therefore, it serves the implementation of urinary BC detection in the routine screening and offers a promising non-invasive alternative.

BACKGROUNDDeregulated microRNAs (miRNAs) in breast and gynecological cancer might contribute to improve early detection of female malignancies.OBJECTIVESpecification of miRNA types in serum and urine as minimally-invasive biomarkers for breast (BC), endometrial (EC) and ovarian cancer (OC).METHODSIn a discovery phase, serum and urine samples from 17 BC, five EC and five OC patients vs. ten healthy controls (CTRL) were analyzed with Agilent human miRNA microarray chip. Selected miRNA types were further investigated by RT-qPCR in serum (31 BC, 13 EC, 15 OC patients, 32 CTRL) and urine (25 BC, 10 EC, 10 OC patients, 30 CTRL) applying two-sample t-tests.RESULTSSeveral miRNA biomarker candidates exhibited diagnostic features due to distinctive expression levels (serum: 26; urine: 22). Among these, miR-518b, -4719 and -6757-3p were found specifically deregulated in BC serum. Four, non-entity-specific, novel biomarker candidates with unknown functional roles were identified in urine (miR-3973; -4426; -5089-5p and -6841). RT-qPCR identified miR-484/-23a (all p⩽ 0.001) in serum as potential diagnostic markers for EC and OC while miR-23a may also serve as an endogenous control in BC diagnosis.CONCLUSIONSPromising miRNAs as liquid biopsy-based tools in the detection of BC, EC and OC qualified for external validation in larger cohorts.

Due to their post-transcriptional regulatory impact on gene expression, microRNAs (miRNA, miRs) influence decisively cellular processes of differentiation, proliferation and apoptosis. In oncogenic pathways various miRNAs exert either oncogenic or tumor suppressor activities in a stage-specific manner. Dysregulation of miRNA expression pattern has been associated with several human cancers including endometrial cancer (EC). In the present study, expression profile alterations of EC associated secreted miRNAs were determined under the microenvironmental stress situations hypoxia and acidosis occurring in tumor progression and metastasis. The potential influence of hypoxia and acidosis vs. control conditions on the expression levels of 24 EC-relevant miRNA types was quantitatively accessed via real-time PCR in three established EC in vitro models. Expression data were analyzed statistically. In vitro application of hypoxia resulted in downregulation of miR-15a, miR-20a, miR-20b and miR-128-1 in Ishikawa cells (type I EC) and upregulation of miR-21 in EFE-184 cells (type I EC). Acidosis triggered upregulation of tumor promoting miR-125b in AN3-CA cell (type II EC), whereas in Ishikawa cells (type I EC) miRNAs with tumor suppressive function were found altered in divergent directions, both up- (let-7a) and down- (miR-22) regulated. Our current findings emphasize the functional importance of secreted miRNAs in the immediate response of EC cells to exogenic stress situations such as the typical tumor epiphenomena hypoxia and acidosis. Focusing on the specific potential of secreted, thus circulating miRNA molecules, alterations in expression levels not only influence intracellular gene expression and signaling cascades, but also transfer the induction of (tumor)biological cellular changes to adjacent cells.