Comparison of TNF-α and IL-19 concentrations at different stages of breast cancer.

This study investigated the alteration of tumour necrosis factor (TNF-α) and interleukin-19 (IL-19) at different clinical disease stages, lymph node metastasis, and ductal carcinoma in women with breast cancer. Serum samples were collected from 90 individuals with an age range of 25-61 years. These individuals were divided into a control group (healthy people), consisting of 31 individuals, and a breast cancer patients (BCP) group, consisting of 59 individuals. The pathological data (tumour stage, lymph node metastasis, and ductal carcinoma) was obtained from the medical record of patients and confirmed by experienced histopathology. Enzyme-linked immunosorbent assay (ELISAs) technology was used to measure the serum concentrations of IL-19 and TNF-α. The results showed significant differences (P≤0.002) in the mean of BMI, interleukin-19, and TNF-α in BCP compared to controls, while the age factor did not play an important role. The stages of breast cancer caused clear differences in the levels of TNF-α and IL-19. According to the findings, BCPs had a greater level of TNF-α in lymph node metastases than healthy persons. The concentration of serum IL-19 in BCP with lymph node metastasis was significantly different in non-lymph node metastasis patients and healthy people. Additionally, BCP with ductal carcinoma showed significant differences in the mean levels of IL-19 and TNF-α in comparison with healthy people.

Breast invasive ductal carcinoma diagnosis with a three-miRNA panel in serum.

Aim: Breast cancer, especially invasive ductal carcinoma (IDC), is the cause of a great clinical burden. miRNA could be considered as a noninvasive biomarkers for IDC diagnosis. Materials & methods: Two hundred and sixty participants (135 IDC patients and 125 healthy controls) were enrolled in a three-cohort study. The expression of 28 miRNAs in serum were detected with quantitative reverse transcription-PCR. Bioinformatic analysis was used for predicting the target genes of three selected miRNAs. Results: The expression level of seven miRNAs (miR-9-5p, miR-34b-3p, miR-1-3p, miR-146a-5p, miR-20a-5p, miR-34a-5p, miR-125b-5p) was discrepant at the validation cohort. Through statistical test, a three-miRNA panel (miR-9-5p, miR-34b-3p, miR-146a-5p) was significant for IDC diagnosis (AUC = 0.880, sensitivity = 86.25%, specificity = 81.25%). Conclusion: The three-miRNA panel in serum could be used as a noninvasive biomarker in the diagnosis of IDC.

Platelet-Derived Procoagulant Microparticles as Blood-based Biomarker of Breast Cancer.

OBJECTIVE: Breast cancer is the main cause of cancer death in women worldwide. Elevated plasma levels of circulating cell-derived microparticles (MPs) have been reported in various types of cancer, including breast cancer, with the ability to mediate inflammation and thrombosis. Microparticles are bioactive agents, and it has been suggested that MPs can be used as a diagnostic, prognostic, or therapeutic biomarker in various diseases. The aim of this study was to investigate the levels of platelet-derived MPs (PMPs) in breast cancer patients. MATERIALS AND METHODS: In this case-control study, 30 patients with breast cancer and 20 normal subjects were sampled after obtaining written consent. MPs were isolated from blood samples by centrifugation technique. CD42b and annexin V markers were used respectively for counting PMPs and procoagulant MPs with flow cytometry. RESULTS: Flow cytometry results showed that the number of PMPs and procoagulant annexin V positive MPs was significantly higher in the breast cancer patients than normal subjects (p <0.001). The number of the annexin V MPs differed significantly in patients with high tumor size (T2) compared to the patients with low tumor size (T1) and controls (p <0.001). Significant and positive correlations were found between PMP levels and tissue-based biomarkers, tumor grading, and distant metastasis (p <0.05). Tumor histological type did not correlate with the numbers of PMPs (p=0.065). CONCLUSION: Increased levels of PMPs and activity in terms of hemostasis and having a positive and significant relationship with tumor grading and metastasis may indicate the effective role of PMPs in the pathogenesis and prognosis of breast cancer.

Enhanced humoral immunity in breast cancer patients with high serum concentration of anti-HER2 autoantibody.

Humoral immunity plays a substantial role in the suppression of breast cancer. We have revealed that a high serum concentration of anti-HER2 autoantibody (HER2-AAb) is associated with favorable outcomes in patients with invasive breast cancer. Thus, we aimed to clarify the association between high serum concentration of HER2-AAb and humoral immune response in the tumor microenvironment. Out of 500 consecutive patients with invasive breast cancer, we selected those whose HER2-AAb values were high (n = 33) or low (n = 20) based on the distribution of HER2-AAb values of 100 healthy individuals. Tumor and regional lymph node formalin-fixed paraffin-embedded samples prepared from the surgical specimens were subjected to immunohistochemistry. We confirmed that the recurrence-free survival of the high HER2-AAb group was significantly longer than that of the low HER2-AAb group (p = 0.015). The numbers of tumor-infiltrating CD20+ immune cells (ICs) (p < 0.001), IGKC+ICs (p = 0.023), and CXCL13+ ICs (p = 0.044) were significantly higher in the high HER2-AAb group than in the low HER2-AAb group. The number of CD4+ ICs in the B-cell follicles of the regional lymph nodes was also significantly greater in the high HER2-AAb group than in the low HER2-AAb group (p = 0.026). Our findings indicate that a high level of HER2-AAb is associated with enhanced humoral immunity against breast cancer and thus may provide a rationale for the association of HER2-AAb with favorable prognosis.

Ultradeep targeted sequencing of circulating tumor DNA in plasma of early and advanced breast cancer.

We present a study to evaluate the feasibility and clinical utility of amplicon-based Oncomine Pan-Cancer cell-free assay to detect circulating tumor DNA (ctDNA) in patients with early or advanced breast cancer. In this study, 109 early and metastatic breast cancer patients were recruited before the initiation of treatment. ctDNA mutation profiles were assessed through unique molecular tagging (UMT) and ultradeep next generation sequencing (NGS). For patients with mutations, DNA from corresponding white blood cells (WBC) was sequenced to exclude variants of clonal-hematopoietic (CH) origin. UMT targeted sequencing from plasma of 109 patients achieved a median total coverage of 55 498X and a median molecular coverage of 4187X. Among 53 ctDNA positive samples, 38% were mutation positive by WBC sequencing, indicating potentially false-positive results contributed by CH origin. Prevalence of CH-related mutations was associated with age (P = 7.51 × 10-4 ). After exclusion of CH mutations, ctDNA detection rates were 37% for local or locally advanced breast cancer (stage I-III) and 81% for metastatic or recurrent breast cancer. The ctDNA detection rate correlated with disease stage (P = 2.60 × 10-4 ), nodal spread (P = 6.49 × 10-3 ) and the status of distant metastases (P = 5.00 × 10-4 ). ctDNA variants were detected mostly in TP53, PIK3CA and AKT1 genes, with variants showing therapeutic relevance. This pilot study endorses the use of targeted NGS for non-invasive molecular profiling of breast cancer. Paired sequencing of plasma ctDNA and WBC should be implemented to improve accurate interpretation of liquid biopsy.

Association study of miR-22 and miR-335 expression levels and G2 assay related inherent radiosensitivity in peripheral blood of ductal carcinoma breast cancer patients.

Identifying patient's cellular radiosensitivity before radiotherapy (RT) in breast cancer (BC) patients allows proper alternations in routinely used treatment programs and reduces the adverse side effects in exposed patients. This study was conducted on blood samples taken from 60 women diagnosed with Invasive Ductal Carcinoma (IDC) BC (mean age: 47±9.93) and 30 healthy women (mean age: 44.43±6.7). The standard G2 assay was performed to predict cellular radiosensitivity. To investigate miR-22 and miR-335 expression levels in peripheral blood mononuclear cells (PBMCs), qPCR was performed. The sensitivity and specificity of the mentioned miRNAs were assessed by plotting the Receiver Operating Characteristic (ROC) curve. Binary logistic regression was performed to identify the miRNA involvement in BC and cellular radiosensitivity (CR) of BC patients. The frequency of spontaneous and radiation-induced chromatid breaks (CBs) was significantly different between control and patient groups (p<0.05). A cut-off value was determined to differentiate the patients with and without cellular radiosensitivity. miR-22 and miR-335 were significantly downregulated in BC patients. miRNAs expression levels were directly associated with CR. ROC curve assessment identified that both miRNAs had acceptable specificity and sensitivity in the prediction of BC and CR of BC patients. Binary logistic regression showed that both miRNAs could also predict BC successfully. Although only miR-22 was shown potent to predict CR of BC patients, both miR-22 and miR-335 might act as tumor suppressor miRNAs in BC. miR-22 and miR-335 may be promising potential biomarkers in BC prediction along with other important biomarkers. Moreover, mirR-22 might be a potential biomarker for the prediction of CR in BC patients.

Shotgun proteomics coupled to nanoparticle-based biomarker enrichment reveals a novel panel of extracellular matrix proteins as candidate serum protein biomarkers for early-stage breast cancer detection.

BACKGROUND: The lack of specificity and high degree of false positive and false negative rates when using mammographic screening for detecting early-stage breast cancer is a critical issue. Blood-based molecular assays that could be used in adjunct with mammography for increased specificity and sensitivity could have profound clinical impact. Our objective was to discover and independently verify a panel of candidate blood-based biomarkers that could identify the earliest stages of breast cancer and complement current mammographic screening approaches. METHODS: We used affinity hydrogel nanoparticles coupled with LC-MS/MS analysis to enrich and analyze low-abundance proteins in serum samples from 20 patients with invasive ductal carcinoma (IDC) breast cancer and 20 female control individuals with positive mammograms and benign pathology at biopsy. We compared these results to those obtained from five cohorts of individuals diagnosed with cancer in organs other than breast (ovarian, lung, prostate, and colon cancer, as well as melanoma) to establish IDC-specific protein signatures. Twenty-four IDC candidate biomarkers were then verified by multiple reaction monitoring (LC-MRM) in an independent validation cohort of 60 serum samples specifically including earliest-stage breast cancer and benign controls (19 early-stage (T1a) IDC and 41 controls). RESULTS: In our discovery set, 56 proteins were increased in the serum samples from IDC patients, and 32 of these proteins were specific to IDC. Verification of a subset of these proteins in an independent cohort of early-stage T1a breast cancer yielded a panel of 4 proteins, ITGA2B (integrin subunit alpha IIb), FLNA (Filamin A), RAP1A (Ras-associated protein-1A), and TLN-1 (Talin-1), which classified breast cancer patients with 100% sensitivity and 85% specificity (AUC of 0.93). CONCLUSIONS: Using a nanoparticle-based protein enrichment technology, we identified and verified a highly specific and sensitive protein signature indicative of early-stage breast cancer with no false positives when assessing benign and inflammatory controls. These markers have been previously reported in cell-ECM interaction and tumor microenvironment biology. Further studies with larger cohorts are needed to evaluate whether this biomarker panel improves the positive predictive value of mammography for breast cancer detection.

HCK can serve as novel prognostic biomarker and therapeutic target for Breast Cancer patients.

The role of HCK expression in the prognosis of breast cancer patients is unclear. Thus, this study aimed to explore the clinical implications of HCK expression in breast cancer. We assessed HCK expression and genetic variations in breast cancer using Oncomine, GEPIA, UALCAN, and cBioPortal databases. Then, immunochemistry was used to analyze HCK expression in breast cancer specimens, non-cancer tissues and metastatic cancer tissues. Consequently, we evaluated the effect of HCK expression on survival outcomes set as disease-free survival (DFS) and overall survival (OS). Finally, STRING, Coexpedia, and TISIDB database were explored to identify the molecular functions and regulation pathways of HCK. We found that breast cancer tissues have more HCK mRNA transcripts than non-cancer tissues. Patients with HCK expression had significantly shorter DFS and OS. The ratio of HCK expression was higher in cancer tissues than in non-cancer tissues. These results from STRING database, FunRich software, and TISIDB database showed that HCK was involved in mediating multiple biological processes including immune response-regulating signaling pathway, cell growth and maintenance through multiple signaling pathways including epithelial to mesenchymal transition, PI3K/AKT signaling pathway, and focal adhesion. Overall, HCK may be an oncogene in the development of breast cancer and thus may as a novel biomarker and therapeutic target for breast cancer.

C-C motif chemokine ligand 5 and C-C chemokine receptor type 5: possible diagnostic application in breast cancer patients.

The chemokine CCL5 and its receptor CCR5 play important roles in cancer invasion and metastasis. Based on our knowledge, our results were the first that presented the diagnostic usefulness of CCL5 and CCR5 in breast cancer (BC) patients, based on receiver operating characteristic (ROC) curve analysis. We wished to examine further if CCL5 and CCR5 are appropriate to be applied as BC markers for early screening. Values of tested parameters in patients' plasma were determined by CMIA method (Chemiluminescent Microparticle Immunoassay, CA 15-3) as well as by ELISA method (Enzyme-Linked Immunosorbent Assay, CCL5 and CCR5). Levels of CCL5 in the plasma were markedly increased, while those of CCR5 were remarkably lower in BC patients when compared to the control groups. Moreover, higher levels of CCL5 in BC corresponded to advanced tumor stage, while the levels of CCR5 decreased with increasing the disease stage. CCL5 concentration was characterized by high sensitivity (SE) (68.04%) and high specificity (SP) (100.00%) in the BC patients. Results indicated that area under the curve (AUC) corresponding to CCL5 (0.8116) had a higher value than this corresponding to CA 15-3. The AUC value of CCL5 was significantly increased in the early phase of BC (stage I - 0.7089; stage II - 0.8313). The maximum range in the BC patients was observed for the combined analysis of the tested measurands with CA 15-3 (0.8335). In conclusion, our research indicates that examination of plasma CCL5 and CCR5 may be useful in BC diagnosis at the early stage of the disorder, especially when combined with CA 15-3.

Pre-surgical level of von Willebrand factor as an evident indicator of breast cancer recurrence.

BACKGROUND: Endothelial and platelet activation as well as a disruption of haemostatic balance are crucial in cancer-dependent venous thromboembolism development. OBJECTIVE: The aim of this study was to investigate the influence of von Willebrand factor (VWF), sE-selectin, sP-selectin as well as VWF/sE-selectin and sP-selectin/sE-selectin ratios on the probability of disease relapse in invasive breast carcinoma (IBrC) cases. METHODS: Eighty-four patients with IA-IIB stage of IBrC who passed a comprehensive clinicopathologic evaluation were included in the study. Follow-up was completed in all patients with a 15.48 % recurrence rate. An immunoassay of VWF antigen, sE-selectin, sP-selectin, as well as an immunohistochemistry of oestrogen and progesterone receptors, human epidermal growth factor receptor 2 (HER2) and Ki67 was performed in all cases. RESULTS: The VWF/sE-selectin ratio was significantly higher in patients with poorly differentiated tumours than in those with high-differentiated tumours. A positive correlation between VWF concentration and tumour grade was noted. Eleven of 13 events happened in patients with VWF value below 600 mU/mL with recurrence rate of 25%, but only two events occurred in subject with VWF values above the 600 mU/mL (5%; P= 0.0028). CONCLUSIONS: Our study show that VWF could be considered as a suitable biomarker of breast cancer relapse.

Prognostic significance of preoperative albumin to fibrinogen ratio associated nomograms in patients with breast invasive ductal carcinoma.

Plasma albumin to fibrinogen ratio is involved in human cancer, but its prognostic significance in breast cancer is controversy. In the context of breast invasive ductal carcinoma, this research aims to retrospectively evaluate by preoperative plasma albumin to fibrinogen ratio (AFR) and forecast oncological outcome and recurrence.This retrospective study comprised 230 patients with non-metastatic breast invasive ductal carcinoma who underwent surgery between January 2009 and April 2012 in Fourth Hospital of Hebei Medical University. Patients were categorized base on an optimal value of preoperative plasma fibrinogen (Fib) and albumin. Progression-free and cancer-specific survival were assessed using Kaplan-Meier method. The associations between albumin to fibrinogen ratio and clinical outcomes were assessed with univariate and multivariate analysis. A number of risk factors were used to form nomograms to evaluate survival, and Harrell concordance index (C-index) was used to evaluate the predictive accuracy.Plasma AFR was significantly associated with diminished disease-free survival (DFS) and overall survival (OS). Multivariate analysis revealed that plasma AFR was an independent prognostic indicator for DFS (HR = 1.346; 95% CI: 1.107-1.636; P = .03) and overall survival (OS) (HR = 1.485; 95% CI: 1.106-1.993; P = .008). Two prediction model of 3-, 5-years OS and DFS based on the AFR was developed.Elevated preoperative plasma AFR is an independent prognostic factor for oncological outcomes in patients with breast invasive ductal carcinoma. The formulated nomogram showed superior predictive accuracy for DFS and OS.

Factors predictive of invasive ductal carcinoma in cases preoperatively diagnosed as ductal carcinoma in situ.

BACKGROUND: Invasion is often found during postoperative pathological examination of cases diagnosed as ductal carcinoma in situ (DCIS) by histological examinations such as core needle biopsy (CNB) or vacuum-assisted biopsy (VAB). A meta-analysis reported that 25.9% of invasive ductal carcinoma (IDC) cases are preoperatively diagnosed by CNB as DCIS. Risk factors for invasion have been studied by postoperative examination, but no factors have been found that could be obtained preoperatively from blood tests. In this study, we investigated factors predictive of invasion based on preoperative blood tests in patients diagnosed with DCIS by preoperative biopsy. METHODS: In this study, 118 patients who were diagnosed with DCIS by preoperative biopsy were included. Biopsies were performed with 16-gauge CNB or VAB. Peripheral blood was obtained at the time of diagnosis. This study evaluated absolute platelet count, absolute lymphocyte count, lactate dehydrogenase, carcinoembryonic antigen, and cancer antigen 15-3 (CA15-3). The platelet-lymphocyte ratio (PLR) was calculated by dividing the absolute platelet count by the absolute lymphocyte count, and patients were grouped into high PLR (≥160.0) and low PLR (< 160.0) groups. RESULTS: Invasion was found more frequently after surgery in pathologically high-grade cases than in pathologically not-high-grade cases (p = 0.015). The median PLR was 138.9 and 48 patients (40.7%) were classified into the high PLR group. The high PLR group was significantly more likely to have invasion detected by the postoperative pathology than the low PLR group (p = 0.018). In multivariate analysis of factors predictive of invasion in postoperative pathology, a high PLR (p = 0.006, odds ratio [OR] = 3.526) and biopsy method (VAB vs. CNB, p = 0.001, OR = 0.201) was an independent risk factor. CONCLUSIONS: The PLR may be a predictor of invasion in the postoperative pathology for patients diagnosed with DCIS by preoperative biopsy.

Neoadjuvant chemotherapy modifies serum pyrrolidone carboxypeptidase specific activity in women with breast cancer and influences circulating levels of GnRH and gonadotropins.

PURPOSE: Functional studies have demonstrated that gonadotropin-releasing hormone (GnRH) regulates cell proliferation, apoptosis, and tissue remodeling. GnRH is metabolized by the proteolytic regulatory enzyme pyrrolidone carboxypeptidase (Pcp) (E.C. 3.4.19.3), which is an omega peptidase widely distributed in fluids and tissues. We previously reported a decrease in both rat and human Pcp activity in breast cancer, suggesting that GnRH may be an important local hormonal factor in the pathogenesis of breast cancer. Recently, we have described that postmenopausal women with breast cancer show lower levels of serum Pcp activity than control postmenopausal women. To determine the effect of neoadjuvant chemotherapy (NACT) on serum Pcp specific activity and circulating levels of GnRH, luteinizing hormone (LH), follicle-stimulating hormone (FSH) and steroid hormones 17-ß-estradiol and progesterone in pre- and postmenopausal women diagnosed with infiltrating ductal carcinoma. METHODS: Serum Pcp activity was measured fluorometrically using pyroglutamyl-ß-naphthylamide. Circulating GnRH levels were dosed using a commercial RIA kit. Circulating LH and FSH levels were measured by enzyme immunoassays. Levels of steroid hormones were measured in serum samples by dissociation-enhanced lanthanide fluorescence immunoassay. RESULTS AND CONCLUSION: Our results show the effect of NACT on the hypothalamic-pituitary axis, with the consequent alteration of circulating gonadotropins in premenopausal women with breast cancer. However, the results obtained in postmenopausal women with breast cancer treated with NACT, that is, the significant decrease in the concentration of GnRH and FSH compared to control postmenopausal women, differ from those obtained for premenopausal women. The only difference between pre- and postmenopausal women is their hormonal profile at the beginning of the study, that is, the presence of menopause and the consequent alteration of the hypothalamic-pituitary-gonadal axis.

Breast cancer diagnosis by analysis of serum N-glycans using MALDI-TOF mass spectroscopy.

Blood and serum N-glycans can be used as markers for cancer diagnosis, as alterations in protein glycosylation are associated with cancer pathogenesis and progression. We aimed to develop a platform for breast cancer (BrC) diagnosis based on serum N-glycan profiles using MALDI-TOF mass spectroscopy. Serum N-glycans from BrC patients and healthy volunteers were evaluated using NosQuest's software "NosIDsys." BrC-associated "NosID" N-glycan biomarkers were selected based on abundance and NosIDsys analysis, and their diagnostic potential was determined using NosIDsys and receiver operating characteristic curves. Results showed an efficient pattern recognition of invasive ductal carcinoma patients, with very high diagnostic performance [area under the curve (AUC): 0.93 and 95% confidence interval (CI): 0.917-0.947]. We achieved effective stage-specific differentiation of BrC patients from healthy controls with 82.3% specificity, 84.1% sensitivity, and 82.8% accuracy for stage 1 BrC and recognized hormone receptor-2 and lymph node invasion subtypes based on N-glycan profiles. Our novel technique supplements conventional diagnostic strategies for BrC detection and can be developed as an independent platform for BrC screening.

Expression and Circulating Levels of Perlecan in Breast Cancer - Implications for Oestrogen Dependent Stromal Remodeling.

Localised breast cancer can be cured by surgery and adjuvant treatments, but mortality remains high as some tumours metastasize early. Perlecan is a basement membrane (BM) protein involved in tumour development and progression. Here, mRNA and protein expression of perlecan, and mRNA expression of matrix degrading enzymes were studied in normal breast and invasive breast cancer, and correlated to prognostic risk factors, in particular oestrogen status. Moreover, plasma levels of perlecan were measured in patients with breast cancer and compared with controls. mRNA data was extracted from the Cancer Genome Atlas database. Perlecan protein expression was visualized using immunofluorescence and plasma levels measured by ELISA assay. Perlecan mRNA levels were twice as high in normal breast compared with breast cancer tissue. A strong correlation was found between mRNA expression of perlecan and several matrix-degrading enzymes in oestrogen receptor positive (ER+) tumours. Perlecan protein was localized to both epithelial and vascular BMs, but absent in the stroma in normal breast. In breast cancer, the expression of perlecan in epithelial BM was fragmented or completely lost, with a marked upregulation of perlecan expression in the stroma. Significantly higher levels of perlecan were found in plasma of ER+ patients when compared with ER- patients. This study shows that perlecan expression and degradation in breast cancer may be linked to the ER status of the tumour.

Possible Prognostic Potential of RANKL and OPG in Metastatic Breast Cancer Egyptian Females.

OBJECTIVES: Searching for sensitive, minimally invasive biomarkers that represent tumor-associated changes in the peripheral blood might enable the early diagnosis of breast cancer (BC) and monitoring of tumor progression. METHODS: Herein, we investigated the association of some circulating biomarkers with the risk of metastasis. In the current study, 115 BC patients which were subdivided into two groups: nonmetastatic breast cancer patients (NMBC) (n=83) and metastatic breast cancer patients (MBC) (n=32), and 79 apparently healthy controls were recruited. Serum protein levels of lysosomal protein transmembrane 4 beta (LAPTM4B), receptor activator of nuclear factor-kappa b (NF-Kb) ligand (RANKL), osteoprotegerin (OPG), vitamin D (VIT D), chitinase-3-like protein 1 (also known as YKL-40), and sirtuin 1 (SIRT1) were assessed in blood samples using ELISA technique. RESULTS: The results showed that RANKL and OPG had the highest diagnostic potential for MBC detection, with area under the curve values of 0.97 and 0.94, respectively. Moreover, logistic regression analysis showed that RANKL had the highest differentiation power in the discrimination of MBC from NMBC. CONCLUSION: The study highlighted that measuring RANKL and OPG may be helpful in the early detection of metastasis in Egyptian patients with BC.

Plasma oxysterol levels in luminal subtype breast cancer patients are associated with clinical data.

Oxygenated metabolites of cholesterol (oxysterols) have been previously demonstrated to contribute to progression of various cancers and to modulate resistance to breast cancer endocrine therapy in vitro. We measured prognostic roles of circulating levels of seven major oxysterols in the progression of luminal subtype breast carcinoma. Liquid chromatography coupled with tandem mass spectrometry was used for determination of levels of non-esterified 25-hydroxycholesterol, 27-hydroxycholesterol, 7α-hydroxycholesterol, 7-ketocholesterol, cholesterol-5α,6α-epoxide, cholesterol-5ß,6ß-epoxide, and cholestane-3ß,5α,6ß-triol in plasma samples collected from patients (n = 58) before surgical removal of tumors. Oxysterol levels were then associated with clinical data of patients. All oxysterols except cholesterol-5α,6α-epoxide were detected in patient plasma samples. Circulating levels of 7α-hydroxycholesterol and 27-hydroxycholesterol were significantly lower in patients with small tumors (pT1) and cholesterol-5ß,6ß-epoxide and cholestane-3ß,5α,6ß-triol were lower in patients with stage IA disease compared to larger tumors or more advanced stages. Patients with higher than median cholestane-3ß,5α,6ß-triol levels had significantly worse disease-free survival than patients with lower levels (p = 0.037 for all patients and p = 0.015 for subgroup treated only with tamoxifen). In conclusion, this study shows, for the first time, that circulating levels of oxysterols, especially cholestane-3ß,5α,6ß-triol, may have prognostic roles in patients with luminal subtype breast cancer.

Circulating miR-34a and miR-125b as Promising non Invasive Biomarkers in Egyptian Locally Advanced Breast Cancer Patients.

Background: Breast cancer (BC) is the second most common cancer worldwide. MicroRNAs are a group of non-coding, single stranded RNAs of ~ 22 nucleotides, which regulate gene expression at the post-transcriptional level. Circulating miRNAs have been found as potential blood based predictive biomarkers. Purpose: we aim to evaluate miR-34a and miR-125b to predict outcome from neoadjuvant chemotherapy in Egyptian BC patients. Methodology: Quantitative assessment of plasma miR-34a and miR-125b expression was performed by qRT-PCR. Thirty nine newly diagnosed locally advanced BC female patients with 10 age and sex matched healthy volunteers were included in the study. Results: We performed ROC curve analysis to evaluate the diagnostic value for the miR-34a with AUCs = 0.995, cutoff point of 2.57 sensitivity 97.4%, specificity 100%, PPV 100%, NPV 83.3% and accuracy 97.7%. miR-125b had AUC = 0.68 and a cutoff point of 8.69 with sensitivity 66.7%, specificity 70.0%, PPV 90.6%, NPV 41.2% and accuracy 73.5%. miR-34a expression were significantly higher in BC patients compared to controls with p value <0.001*. Also, miR-34a expression level was significantly higher in patients with progressive disease with P value =0.03*. However, miR-125b expression levels were insignificantly higher in responsive patients with p value = 0.2. Conclusion: miRNAs are crucial candidates for novel molecular targeted therapies due to their capability to regulate numerous genes in molecular pathways. Our data suggest that circulating miR-34a and miR-125b expression levels could be promising highly accurate non-invasive biomarkers in diagnosing BCs. miR-34a can predict chemotherapeutic resistance associated with higher expression levels in non-responsive patients.

OLFM4 Expression in Ductal Carcinoma In Situ and in Invasive Breast Cancer Cohorts by a SWATH-Based Proteomic Approach.

Human olfactomedin-4 (OLFM4) is a secreted protein involved in a variety of cellular functions including proliferation, differentiation, apoptosis, and cell adhesion. OLFM4 expression has been studied in several tumor types including gastric, colorectal, lung, and endometrioid cancers where it has been suggested to be an independent favorable or unfavorable prognostic marker. For breast cancer, the clinical significance of OLFM4 is still unclear. In the present study, SWATH-MS is used as a tool for the robust identification and quantification of breast tissue proteins. SWATH-MS data show that OLFM4 expression is higher in DCIS than in invasive breast cancer. In-depth analysis of the breast tumor proteome show that OLFM4 is a favorable pronostic marker. Serum OLFM4 levels in peripheral blood are also analyzed by ELISA in 825 cases, including 94 cases of healthy individuals, 61 cases of non-invasive breast tumor (DCIS) and 670 cases of breast cancer (BC). It is found that serum OLFM4 levels are significantly higher in the DCIS cohort and in the breast cancer cohort compared with the healthy controls. This result suggests that circulating OLFM4 could be an interesting biomarker of early breast cancer. Data are available via ProteomeXchange with identifier PXD014194.

Overexpression of serum exosomal HOTAIR is correlated with poor survival and poor response to chemotherapy in breast cancer patients.

To investigate the source of serum exosomal HOTAIR, to uncover the diagnostic and prognostic values of serum exosomal HOTAIR, and to discern the expression of serum exosomal HOTAIR between neoadjuvant chemotherapy and response to tamoxifen therapy. Samples were collected from the Third Affiliated Hospital of Kunming Medical University, Tumor Hospital of Yunnan. Exosomes were isolated from serum, cell culture medium and tumor tissues. We used transmission electron microscopy and western immunoblotting assay to characterize exosomes, and real-time PCR (qPCR) to assess HOTAIR expression. Neoadjuvant chemotherapy and tamoxifen therapy were carried out according to established guidelines. Breast cancer patients expressed higher serum exosomal HOTAIR than did healthy individuals (P\0.001). Serum exosomal HOTAIR levels 3 months after surgery were markedly decreased compared with levels before surgery (P\0.001), and the expression level of exosomal HOTAIR in cell culture medium increased with time in both breast cancer cell lines (72 h greater than 48 h greater than 24 h, 48 h vs 24 h [ [P less than 0.05]; 72 h vs 24 h [P less than 0.01]. Expression of serum exosomal HOTAIR in nude mice was notably greater than in the mock control group (P less than 0.001). The results of the ROC analysis revealed an AUC for serum exosomal HOTAIR of 0.9178 with a 95% CI of 0.8407-1.017 (P less than 0.01). The AUC for the CA15-3 cell line was 0.7378 (95% CI, 0.5585-0.9170; P = 0.03). High expression of exosomal HOTAIR led to a worse disease-free survival (P = 0.0481) and overall survival (P = 0.0463). In the high-expression chemotherapy group, six patients achieved a partial response (PR) and eight demonstrated stable disease (SD) and nine patients achieved PR and two SD in the low-expression group (P = 0.048). In the low-expression tamoxifen group, one patient had a recurrence of breast cancer and another 10 patients exhibited no recurrence, while six showed recurrence, and seven had none in the highexpression group (P = 0.035). We isolated exosomes successfully, and demonstrated that serum exosomal HOTAIR originated from primary breast cancer tissue. We conclude that serum exosomal HOTAIR exhibits the potential to be a diagnostic and prognostic biomarker. High expression of serum exosomal HOTAIR was also correlated with poor neoadjuvant chemotherapy and response to tamoxifen therapy.