Differentiate Breast Cancer Patients Research Articles

CRISPR/Cas12a and aptamer-chemiluminescence based analysis for the relative abundance determination of tumor-related protein positive exosomes for breast cancer diagnosis

Exosomes, as novel biomarker for liquid biopsy, exhibit huge important potential value for cancer diagnosis. However, various proteins show different expression levels on exosomal membrane, and the absolute concentration of exosomes in clinical samples is easily influenced by a number of factors. Here, we developed a CRISPR/Cas12a and aptamer-chemiluminescence based analysis (CACBA) for the relative abundance determination of tumor-related protein positive exosomes in plasma for breast cancer diagnosis. The total concentration of exosomes was determined through captured CD63 using a CRISPR/Cas12a-based method with the LoD of 8.97 × 103 particles/μl. Meanwhile, EpCAM and MUC1 positive exosomes were quantitatively detected by aptamer-chemiluminescence (ACL) based method with the LoD of 1.45 × 102 and 3.73 × 102 particles/μl, respectively. It showed that the percentages of EpCAM and MUC1 positive exosomes offered an excellent capability to differentiate breast cancer patients and healthy donors. The high sensitivity, strong specificity, outstanding anti-interference capability, and steady recovery rate of this approach offered higher accuracy and robustness than the commercialized method in clinical trial. In addition with good stability, easy preparation and low cost, this method not only provides a new approach to rapid analysis of exosome proteins, it may be quickly extended to the diagnoses of various cancers.

Abstract 2436: In-depth analysis of microRNA signature in breast cancer derived extracellular vesicles: A potential biomarker repository for breast cancer diagnosis

Abstract Breast cancer remains one of the leading causes of cancer-related mortality among women worldwide. With the rising incidence of breast cancer, early and accurate diagnosis is pivotal in improving survival rates and patient outcomes. Our study addresses to identify vesicles associated with breast cancer in scope of miRNA signature. We focused on isolating breast cancer-derived extracellular vesicles (BEVs) from the bloodstream with immune affinity capture techniques. Then, we analyzed the miRNA profiles of BEVs in plasma samples from 120 breast cancer patients, 46 individuals with benign tumors, and 45 healthy controls. In this study, we analyzed to discover miRNA candidates for diagnosis of breast cancer with The Gene Expression Omnibus (GEO) database and found 379 down-regulated and 584 up-regulated differentially expressed miRNAs (DEMs) in tissue samples across 5 GEO datasets, and 28 down-regulated and 80 up-regulated DEMS in blood samples. Subsequently, we measured the expression levels of these 10 candidate miRNAs in BEVs from breast cancer cells and compared them with EVs from human mammary cells (MCF10a), using our BEV isolation method. The expression profiles of these miRNAs in BEVs were significantly elevated, supporting their potential as surrogate markers for breast cancer diagnosis. When comparing the AUC values of 10 candidate miRNAs through ROC analysis, 5 EV miRNAs (miR-21, miR-106b, miR-181a, miR-484, and miR-1260b) showed a high AUC values above 0.8. Subsequently, we verified a distinct signature of 5 EV miRNAs that effectively differentiate breast cancer patients from normal controls with 57.14% of sensitivity and 95% of specificity. Overall, our study not only complements existing diagnostic methods with high accuracy but also provides a deeper understanding of the molecular aspects of breast cancer, heralding a significant advancement in precision medicine and personalized cancer care. Citation Format: Jee Ye Kim, Min Woo Kim, Young Kim, Sol Moon, Suji Lee, Hyojung Lee, Joon Ye Kim, Seung Il Kim. In-depth analysis of microRNA signature in breast cancer derived extracellular vesicles: A potential biomarker repository for breast cancer diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2436.

Oxidative stress markers in breast cancer

Purpose: Oxidative stress is thought to play a role in the etiology of breast cancer. The aim of this study was to compare serum Total Antioxidant Status (TAS), Total Oxidant Status (TOS), Oxidative Stress Index (OSI) and Ischemia Modified Albumin (IMA) levels in breast cancer patients and healthy women and to determine whether there is a relationship between oxidative stress markers and breast cancer.
 Materials and methods: Newly diagnosed 106 breast cancer patients at the Bozyaka Training and Research Hospital General Surgery Clinic and 30 healthy women were included in the study. Serum levels of IMA, TAS, TOS were analyzed by spectrophotometric methods. 
 Results: IMA, TOS and OSI values were found significantly higher in the breast cancer group. Although the TAS values in patients with breast cancer were lower than the control group, the difference was not statistically significant. 
 Conclusions: In our study, it was seen that oxidative stress levels increased in breast cancer patients due to decreased TAS, significantly increased IMA, TOS and OSI results. Therefore, we think that increased oxidative stress may be related to breast cancer and that IMA, TOS and OSI can be used as markers of oxidative stress to differentiate breast cancer patients from healthy women.
 Keywords: breast cancer, cancer, biomarkers, ischemia-modified albumin, oxidative stress

Simultaneous detection of cancerous exosomal miRNA-21 and PD-L1 with a sensitive dual-cycling nanoprobe

Simultaneous detection of specific exosomal surface proteins and inner microRNAs are hampered by their heterogeneity, low abundance and spatial segregation in nanovesicles. Here, we design a dual-cycling nanoprobe (DCNP) to enable single-step simultaneous quantitation of cancerous exosomal surface programmed death-ligand 1 (PD-L1) (ExoPD-L1) and miRNA-21 (ExomiR-21) directly in exosome lysates, without resorting to either RNA extraction or time-consuming transmembrane penetration. In this design, DNA molecular machine-based dual-recognition probes co-assemble onto gold nanoparticle surface for engineering ‘silent’ DCNPs, which enable signal-amplified synchronous response to dual-targets as activated by ExomiR-21 and ExoPD-L1 within 20 min. Benefiting from cycling amplification of the molecular machine, DCNPs sensor achieves detection limits of tumor exosomes, ExoPD-L1 and ExomiR-21 down to 10 particles/μL, 0.17 pg/mL and 66 fM, respectively. Such a sensitive dual-response strategy allows simultaneous tracking the dynamic changes of ExoPD-L1 and ExomiR-21 expression regulated by signaling molecules or therapeutics. This approach further detects circulating ExoPD-L1 and ExomiR-21 in human plasma to differentiate breast cancer patients from healthy individuals with high accuracy, showing great potential of DCNPs for simultaneous profiling exosomal surface and inside biomarkers, and for clinical precision diagnosis.

Psychological factors associated with clinical fear of cancer recurrence in breast cancer patients in the early survivorship period.

Clinical fear of cancer recurrence (FCR) is highly prevalent among breast cancer patients and appears early in the disease trajectory. A better understanding of psychological factors associated with clinical FCR is essential to guide screening and intervention development. This cross-sectional study aimed to assess the contribution of attentional bias, intrusive thoughts, metacognitive beliefs, intolerance of uncertainty, thought suppression, and worry to clinical FCR in breast cancer patients in the early survivorship period. Seventy-four patients treated for non-metastatic breast cancer were enrolled at the end of their treatment. The FCR Inventory-Short Form (FCRI-SF) was used to discriminate between the patients with clinical versus nonclinical FCR. Attentional bias to negative and positive cancer-related and non-cancer-related emotional words was assessed with a dot-probe task. Words were presented for 17, 500, and 1500ms. Intrusive thoughts and thought suppression were assessed with the White Bear Suppression Inventory, metacognitive beliefs with the Metacognitions Questionnaire-30, intolerance of uncertainty with the Intolerance of Uncertainty Inventory-Part A, and worry with the Penn State Worry Questionnaire. According to univariate analyses, the patients with clinical FCR (FCRI-SF ≥13) significantly differed from those with nonclinical FCR in terms of intrusive thoughts (p=0.002), metacognitive beliefs (p=0.029), intolerance of uncertainty (p<0.001), and worry (p<0.001). Intolerance of uncertainty (odds ratio, OR=1.06; p=0.040) and worry (OR=1.09; p=0.013) remained in the final logistic regression models. All the patients showed vigilance to cancer-related words, whether with negative or positive valence, at automatic stages of processing (17ms). Intolerance of uncertainty and worry were the two psychological factors contributing directly to clinical FCR in our cross-sectional study. In addition, attentional bias did not differentiate breast cancer patients with clinical versus nonclinical FCR. Treatment approaches for clinical FCR in early survivorship care may need to integrate uncertainty and worry management intervention strategies.

Expression level of miR-155, miR-15a and miR-19a in peripheral blood of ductal carcinoma breast cancer patients: Possible bioindicators for cellular inherent radiosensitivity

Examination of cellular radiosensitivity (RS) helps prevent the adverse side-effects of radiotherapy in radioresistant tumors. We aim to study whether miRNA-155 (miR-155), miR-19a and miR-15a can predict inherent RS according to cellular RS in breast cancer (BC) patients.This study was done on the blood samples of 40 invasive ductal carcinoma (IDC) BC patients and 15 healthy women. G2 assay was performed to evaluate cellular RS. To study the expression level of these miRNAs in blood, qRT-PCR was used. The sensitivity and specificity of the studied miRNAs were assessed by the receiver operating characteristic (ROC) curve.The yield of spontaneous (SY) and radiation-induced (RIY) chromatid breaks (CBs) was significantly different between control and patient groups (p < 0.0001). A cut-off value was specified to recognize the patients with cellular RS from those without. Expression of miR-15a was significantly downregulated (p < 0.0001) in BC patients. However, miR-19a showed upregulation in the blood of BC patients. It was also found the expression level of miR-155 and miR-19a were significantly associated with frequency of CBs (FCB) (p < 0.05). ROC curve analysis manifested that the miR-15a and miR-19a differentiate BC patients and healthy women with 0.91 and 0.68 yielding an area under the ROC curve, respectively. miR-155 and miR-19a discriminate between BC patients with and without cellular RS with area under the ROC curve 0.98 and 0.68.Our findings uncovered miR-155 and miR-19a could be applied as a bioindicator to predict cellular radiosensitivity of BC patients.

Salivary biomarkers in breast cancer diagnosis: A systematic review and diagnostic meta-analysis.

BackgroundSalivary diagnostics and their utility as a nonaggressive approach for breast cancer diagnosis have been extensively studied in recent years. This meta‐analysis assesses the diagnostic value of salivary biomarkers in differentiating between patients with breast cancer and controls.MethodsWe conducted a meta‐analysis and systematic review of studies related to salivary diagnostics published in PubMed, EMBASE, Scopus, Ovid, Science Direct, Web of Science (WOS), and Google Scholar. The articles were chosen utilizing inclusion and exclusion criteria, as well as assessing their quality. Specificity and sensitivity, along with negative and positive likelihood ratios (NLR and PLR) and diagnostic odds ratio (DOR), were calculated based on random‐ or fixed‐effects model. Area under the curve (AUC) and summary receiver‐operating characteristic (SROC) were plotted and evaluated, and Fagan's Nomogram was evaluated for clinical utility.ResultsOur systematic review and meta‐analysis included 14 papers containing 121 study units with 8639 adult subjects (4149 breast cancer patients and 4490 controls without cancer). The pooled specificity and sensitivity were 0.727 (95% CI: 0.713–0.740) and 0.717 (95% CI: 0.703–0.730), respectively. The pooled NLR and PLR were 0.396 (95% CI: 0.364–0.432) and 2.597 (95% CI: 2.389–2.824), respectively. The pooled DOR was 7.837 (95% CI: 6.624–9.277), with the AUC equal to 0.801. The Fagan's nomogram showed post‐test probabilities of 28% and 72% for negative and positive outcomes, respectively. We also conducted subgroup analyses to determine specificity, sensitivity, DOR, PLR, and NLR based on the mean age of patients (≤52 or >52 years old), saliva type (stimulated and unstimulated saliva), biomarker measurement method (mass spectrometry [MS] and non‐MS measurement methods), sample size (≤55 or >55), biomarker type (proteomics, metabolomics, transcriptomics and proteomics, and reagent‐free biophotonic), and nations.ConclusionSaliva, as a noninvasive biomarker, has the potential to accurately differentiate breast cancer patients from healthy controls.

Association between Bone Mineral Density and Bone Turnover Markers in Breast Cancer Patients and Bone-Only Metastasis.

Background and Objectives: The aim of this study was to determine the influence of bone turnover markers, namely the N-terminal cross-linking telopeptide (NTx) and alpha C-terminal cross-linking telopeptide of type I collagen (α-CTx), in detecting bone metastasis (bone-only) in breast cancer (BC) patients, as well as to determine whether this effect is related to changes in bone mineral density (BMD). Materials and Methods: The participants in this study comprised 30 postmenopausal BC patients with bone metastases (age range: 59.56 ± 9.02), 20 postmenopausal BC patients without bone metastases (age range: 55.30 ± 11.55), and 20 healthy postmenopausal female controls (age range: 55.55 ± 5.85). Bone turnover markers (serum NTx and urine α-CTx) were measured using the ELISA method. A densitometer using dual-energy X-ray absorptiometry (DEXA) was used to analyze the BMD, and tumor markers were measured using the chemiluminescent immunometric assay. Results: The corresponding levels of serum NTx (p = 0.004), parathyroid hormone (PTH) (p = 0.001), and urine α-CTx (p < 0.001) of BC patients were found to be higher than the standard levels. After the BC patients were divided into subgroups on the basis of the presence of metastasis, the urine α-CTx levels (p = 0.001) were seen to be at critically high levels in those patients suffering from BC with metastasis. Though the BMD values in the lumbar spine (p < 0.001) and femoral neck (p = 0.001) were found to be significantly low in BC patients, no statistically substantial difference in the BMD levels of BC patients suffering from metastasis was observed. It was observed that urine α-CTx (specificity: 70%; sensitivity: 85%) values are critical factors that differentiate BC patients with metastasis from BC patients without metastasis. Conclusions: We found that alterations in bone turnover could be detected by using the values of urine α-CTx while differentiating BC patients with metastasis from BC patients without metastasis. Using the biochemical markers of bone turnover and BMD together would be pertinent for determining the level of metastasis present and examining the efficiency of bone density preservation therapy. Ideally, BMD measurement would be evaluated together with biochemical markers.

Hematological Parameters Changes in Patients with Breast Cancer.

Breast cancer is the most common cancer diagnosis among women worldwide. It accounts for 25% of all women's cancers. This cancer is invasive with a high mortality rate. Evaluation of hematological factors is one of the reliable paraclinical methods to diagnose diseases. Hematological parameters can predict severity, mortality, and follow-up treatment in patients with breast cancer. The aim of this study was to evaluate hematological parameters as useful markers to distinguish between patients with breast cancer and healthy individuals. This study was conducted on 160 women with breast cancer as case groups and 160 healthy women as controls. Hematological parameters were analyzed using the Sysmex KX-21N™ automated hematology analyzer system. The difference between the breast cancer patients and the control group was significant in Hb, HCT, MCV, RDW parameters, and MPV/PLT ratio (p < 0.05) (effect size > 0.8). Also, there was a moderate difference between the two groups in MPV and MCH parameters (p < 0.05) (0.5 < effect size < 0.8). Meanwhile, two groups had a minimal difference in NLR, PLR, and MCHC parameters and WBC and RBC count (p < 0.05) (0.2 < effect size < 0.5). The routine blood test is the most accessible and essential examination tool for disease diagnosis. Our results showed that hematological parameters, like MCV, RDW, MPV, MPV/PLT count, NLR, and PLR, can differentiate breast cancer patients from healthy individuals.

ПЛАЗМЕННЫЕ УРОВНИ НЕКОТОРЫХ микроРНК ПРИ РАКЕ МОЛОЧНОЙ ЖЕЛЕЗЫ В КАЗАХСКОЙ ПОПУЛЯЦИИ

Breast cancer is the most common cancer among women worldwide. The use of mammography screening for women, in the age range the most at risk to breast cancer, has led to a significant reduction in mortality from this disease. However, mammography shows a significant number of false positives in women at a younger age. This problem indicates the need to find new reliable, minimally invasive and cheap biomarkers of breast cancer. The relevance of the research is determined by the fact that breast cancer in Kazakh women often develops at a young age. The aim of this study was to test the diagnostic value of six plasma miRNAs in Kazakh women. For this, using the quantitative PCR technique, we compared the levels of the miRNAs in the plasma of breast cancer patients (n = 27) and healthy controls (n = 33), as well as in the breast tumor and adjacent normal tissue (n = 28). Quantitative data were normalized relative to endogenous control miR-16-5p. Plasma concentrations of miR-145-5p, miR-191-5p, and miR-21-5p were significantly increased in patients compared to controls (P = 6.58e-7, 2.70e-5, and 0.049, respectively). The levels of miR-191-5p and miR-210-3p were significantly increased, while the level of miR-145-5p was significantly reduced in the tumor compared to normal tissue (1.88e-6, 6.56e-7 and 9.66e-4, respectively). To test the hypothesis of the secretory origin of the studied plasma miRNAs, we analyzed the correlation of miRNA levels in plasma, tumor, and healthy breast tissue. Correlation analysis showed that there may be a relationship between plasma and tumor levels of miR-145-5p. Plasma level of miR-210-3p correlated with tissue ∆∆Ct, plasma level of miR-222-3p correlated with its expression in healthy tissue; however, the concentrations of these miRNAs did not differ in plasma of breast cancer patients and healthy controls. To test whether our circulating miRNAs can be used to differentiate breast cancer patients from healthy individuals, we performed a ROC analysis. The largest area under the ROC-curve (AUC) was obtained for miR-145-5p (0.854), slightly less for miR-191-5p (0.818), and miR-21-5p was significantly inferior in this indicator (0.649). Using three microRNAs together, it was possible to separate patients from healthy women with 85 % accuracy, high specificity (94 %) and medium sensitivity (74 %). When assessing the potential of the studied miRNAs as markers of tumorigenesis in breast tissue, we found that using miR-191-5p or miR-210-3p, it is possible to distinguish cancer from healthy tissue with equally high accuracy (84% each). Although individually miR-145-5p showed medium separation accuracy (71 %), it complemented two other miRNAs in both paired and triple models. Thus, miR-145-5p and miR-191-5p can be considered potential plasma biomarkers, while miR-191-5p, miR-210-3p, and miR-145-5p can be considered potential tissue biomarkers for the diagnosis of breast cancer. The findings need to be confirmed on a more representative cohort of samples.

Five microRNAs in Serum Are Able to Differentiate Breast Cancer Patients From Healthy Individuals.

Breast cancer is the cancer with the most incidence and mortality in women. microRNAs are emerging as novel prognosis/diagnostic tools. Our aim was to identify a serum microRNA signature useful to predict cancer development. We focused on studying the expression levels of 30 microRNAs in the serum of 96 breast cancer patients vs. 92 control individuals. Bioinformatic studies provide a microRNA signature, designated as a predictor, based on the expression levels of five microRNAs. Then, we tested the predictor in a group of 60 randomly chosen women. Lastly, a proteomic study unveiled the overexpression and downregulation of proteins differently expressed in the serum of breast cancer patients vs. that of control individuals. Twenty-six microRNAs differentiate cancer tissue from healthy tissue, and 16 microRNAs differentiate the serum of cancer patients from that of the control group. The tissue expression of miR-99a, miR-497, miR-362, and miR-1274, and the serum levels of miR-141 correlated with patient survival. Moreover, the predictor consisting of miR-125b, miR-29c, miR-16, miR-1260, and miR-451 was able to differentiate breast cancer patients from controls. The predictor was validated in 20 new cases of breast cancer patients and tested in 60 volunteer women, assigning 11 out of 60 women to the cancer group. An association of low levels of miR-16 with a high content of CD44 protein in serum was found. Circulating microRNAs in serum can represent biomarkers for cancer prediction. Their clinical relevance and the potential use of the predictor here described are discussed.

Identification of a Profile of Neutrophil-Derived Granule Proteins in the Surface of Gold Nanoparticles after Their Interaction with Human Breast Cancer Sera.

It is well known that the interaction of a nanomaterial with a biological fluid leads to the formation of a protein corona (PC) surrounding the nanomaterial. Using standard blood analyses, alterations in protein patterns are difficult to detect. PC acts as a “nano-concentrator” of serum proteins with affinity for nanoparticles’ surface. Consequently, characterization of PC could allow detection of otherwise undetectable changes in protein concentration at an early stage of a disease, such as breast cancer (BC). Here, we employed gold nanoparticles (AuNPsdiameter: 10.02 ± 0.91 nm) as an enrichment platform to analyze the human serum proteome of BC patients (n = 42) and healthy controls (n = 42). Importantly, the analysis of the PC formed around AuNPs after their interaction with serum samples of BC patients showed a profile of proteins that could differentiate breast cancer patients from healthy controls. These proteins developed a significant role in the immune and/or innate immune system, some of them being neutrophil-derived granule proteins. The analysis of the PC also revealed serum proteome alterations at the subtype level.

Evaluation of the diagnostic value of circulating tumor cells with CytoSorter® CTC capture system in patients with breast cancer.

PurposeIn this study, we aimed to investigate the viability of utilizing CytoSorter® system to detect circulating tumor cells (CTCs) and to evaluate the diagnostic value of CTCs in breast cancer (BC).MethodsA total of 366 females patients suspected of having BC and 30 healthy female volunteers were enrolled in this study. CTCs were enriched by CytoSorter®, a microfluidic‐based CTCs capturing platform. CTC detection was performed before operation or biopsy. Based on the biopsy results, patients were divided into two groups, namely patients with BC and patients with benign breast diseases (BBD). Patients with BBD and healthy volunteers were serving as controls. The correlation between CTC enumeration and patients' clinicopathological characteristics was evaluated. The receiver operating characteristic (ROC) curve was plotted to assess the diagnostic potency of CytoSorter® system in BC.ResultsBased on the biopsy results, 130 BC patients at different cancer stages and 236 patients with BBD were enrolled in the study. Seven subjects were dropped out from the study. CTCs were detected in 109 of 128 BC patients, in one of 29 healthy volunteers, and in 37 of 232 patients with BBD. Maximum CTC counts detected in BC patients, healthy volunteers, and patients with BBD were 8, 1, and 4, respectively. Statistical analysis showed CTCs could be used to distinguish BC patients from healthy volunteers and patients with BBD (P < .0001). Circulating tumor cells were statistically associated with patients' cancer stage (P = .0126), tumor size (tumor node metastasis [TNM] T stage, P = .0253), cancer type (invasive vs noninvasive, P = .0141), and lymph node metastasis (P = .0436). More CTCs were found in patients at advanced cancer stage or TNM T stage and in patients with invasive tumor or lymph node metastasis. Furthermore, CTC detection rates in BC patients at Tis and T1‐4 stages were 50%, 81.67%, 91.07%, 100%, and 100%, respectively. When the CTC cut‐off value was set to 2, the ROC curve gave an area under the curve (AUC) of 0.86 with a specificity and sensitivity of 95.4% and 76.56%, respectively. Taken together, CTCs could be used as a diagnostic aid in assistance of cancer screening and staging.ConclusionCirculating tumor cells were successfully isolated in BC patients using CytoSorter® system. CTCs can be used to differentiate BC patients from the patients with BBD or healthy volunteers, and as a diagnostic aid for early cancer diagnosis and cancer staging.

Implication of Soluble HLA-G and HLA-G +3142G/C Polymorphism in Breast Cancer Patients Receiving Adjuvant Therapy in Tanzania.

Background:During cancer growth, immunosuppressive microenvironment is created that enables tumour cells to evade an eliminative immune response and hence manage to grow into malignancy. HLA-G, existing as either membrane-bound (mHLA-G) or soluble (sHLA-G) molecule is thought to be immunosuppressive and produced more by tumor cells. The +3142G/C polymorphism in HLA-G gene affects its expression, and G allele is considered to be a protective mutant allele associated with less expression of HLA-G. The implication of HLA-G in cancer development has been reported in different cancers and populations. But, its implication in most African populations has not yet been investigated. The aim of this study was to determine the possible associations of soluble HLA-G and HLA-G +3142G/C SNP with breast cancer. Materials and Methods:75 breast cancer patients and 84 normal controls were recruited in this study. The genotyping of HLA-G +3142G/C polymorphism was determined by LightSNiP typing assay using quantitative Real-Time PCR and sHLA-G levels were determined by ELISA. Results:The sHLA-G levels were significantly lower in breast cancer patients than in controls (p<0.001). Also, they were significantly lower in mastectomized patients compared to non-mastectomized patients (p=0.018). The ROC analysis revealed a significant ability of sHLA-G to differentiate breast cancer patients versus normal controls (AUC=0.697, 95% CI= 0.619-0.767, p<0.001) and identify mastectomized patients (AUC=0.667, 95% CI= 0.549 to 0.772, p=0.041). The assessment of +3142G/C polymorphism revealed a relatively similar distribution of frequencies of genotypes and alleles between breast cancer patients and normal controls (p>0.05) and was neither associated with sHLA-G levels. Conclusion:While the +3142G/C SNP was found not to be relevant to breast cancer, the changes of sHLA-G levels in response to medical interventions such as mastectomy may be translated into its potential prognostic utility for breast cancer. More studies are needed to provide clear evidence of sHLA-G as a diagnostic and prognostic marker of breast cancer in Tanzania.

Tryptophan catabolism increases in breast cancer patients compared to healthy controls without affecting the cancer outcome or response to chemotherapy

BackgroundIndoleamine 2,3-dioxygenase catalyzes the conversion of tryptophan to kynurenine, an immunosuppressive metabolite involved in T regulatory cell differentiation. Indoleamine 2,3-dioxygenase is expressed in many cancer types, including breast cancer. Here, we analyze kynurenine and tryptophan and their ratio in breast cancer patients and healthy controls.MethodsBreast cancer patients and healthy controls were prospectively enrolled in our study. All subjects underwent blood sample withdrawal at diagnosis or on the day of screening mammography for the healthy controls. Plasmatic kynurenine and tryptophan were determined on a TQ5500 tandem mass spectrometer after chromatographic separation.ResultsWe enrolled 146 healthy controls and 202 women with stages I–III breast cancer of all subtypes. All patients underwent surgery, 126 underwent neoadjuvant chemotherapy with 43 showing a pathological complete response, and 43 underwent adjuvant chemotherapy. We observed significantly higher plasmatic kynurenine, tryptophan and their ratio for the healthy controls compared to patients with breast cancer. We observed a lower plasmatic tryptophan and a higher kynurenine/tryptophan ratio in hormone receptor-negative patients compared to hormone receptor-positive cancers. Lobular cancers showed a lower ratio than any other histologies. Advanced cancers were associated with a lower tryptophan level and higher grades with an increased kynurenine/tryptophan ratio. Pathological complete response was associated with higher kynurenine values. The plasmatic kynurenine, tryptophan and kynurenine/tryptophan ratios were not predictive of survival.ConclusionsThe plasmatic kynurenine, tryptophan and kynurenine/tryptophan ratio could differentiate breast cancer patients from healthy controls. The Kyn/Trp ratio and Trp also showed different values according to hormone receptor status, TNM stage, T grade and histology. These results suggest a rapid metabolism in breast cancer, but no associations with outcome or sensitivity to chemotherapy were observed.

Panel of SEREX-defined antigens for breast cancer autoantibodies profile detection

Content: Identification of panel of SEREX-defined antigens for breast cancer autoantibodies profile detection.Objective: To create panel of antigens that can differentiate breast cancer patients and healthy individuals.Methods: SEREX (serological analysis of cDNA expression libraries) method, ELISA (enzyme-linked immunosorbent assay), qPCR (quantitative polymerase chain reaction).Results: In large-scale screening of 16 SEREX-antigens by sera of breast cancer patients and healthy donors, a combination of six antigens (RAD50, PARD3, SPP1, SAP30BP, NY-BR-62 and NY-CO-58) was identified, which can differentiate breast cancer patients and healthy donors with 70% sensitivity and 91% specificity. Elevated mRNA expression of SPP1 gene was revealed in breast tumors (2–7-fold) that correlated with SPP1 antigen immunoreactivity in autologous patients’ sera.Conclusions: The new panel of six SEREX-antigens was proposed, which enables creation of serological assay for breast cancer diagnostics and/or prognosis.

Abstract P2-08-09: Prognostic values of circulating tumor cell (CTC) enumeration and their clusters in advanced breast cancer

Abstract Background The enumeration of circulating tumor cells (CTCs) has been proven to have prognostic values in several solid tumors including breast cancer. It has been established that a cut-off of 5 CTCs in 7.5 ml of blood may significantly differentiate breast cancer patients with favorable and unfavorable survival. However, CTC enumeration has not been shown to further predict the prognosis in those patients with more than 5 CTCs in 7.5 ml of blood. There are several recent in vitro and in vivo studies suggesting that clusters of CTC can be identified in blood and those clusters may play an important role in tumor progression and metastasis. Few clinical studies have been reported to enumerate CTC clusters and evaluate their prognostic values. In the current study, we hypothesize that the enumeration of CTC clusters play an important role in the prognostication of advanced breast cancer patients by providing additional predictive performance independent of CTC enumeration. Methods In an ongoing study of blood-based breast cancer biomarkers, we enrolled 114 patients with stages III and IV breast cancer. Among them, 68 patients had inflammatory breast cancer (IBC), an extremely aggressive form of breast cancer with a much lower survival rate than non-IBC breast cancer patients. The number of single CTCs and CTC clusters (two or more CTCs bound together) in 7.5 ml blood sample were counted using the CellSearch™ system (Janssen Diagnostic) at baseline study entry, and their associations with the progression-free survival (PFS) of patients were evaluated using Kaplan Meier curves and Cox proportional hazards modeling. Results Baseline CTCs were detected in 67 (58.77%) patients. Thirty-five (30.70%) and 19 patients (16.67%) had elevated CTCs (≥5 CTCs/7.5 mL) and clusters, respectively. IBC patients had a slightly higher percentage of cluster (17.65%) compared to non-IBC patients (15.22%). Patients with elevated baseline CTC and cluster had worse PFS (log rank P, 0.0009 and 0.0035, respectively). Compared to patients with &amp;lt; 5 CTC and without cluster, those patients with elevated CTC without cluster, and those with elevated CTC with cluster had an increasingly higher risk of disease progression with an hazard ratio [HR] of 1.93 (95% confidence interval [CI] 1.01-3.67) and 2.91 (1.54-5.50), respectively (P for trend = 0.001). Moreover, the combined analysis of baseline CTC and cluster enumerations showed similar effect when the analysis was restricted to IBC patients (HR 3.03, 95% CI 1.34-6.86). Conclusion Baseline enumerations of both individual CTCs and CTC clusters predict PFS in advanced stage breast cancer patients. CTC clusters provide further prognostic value in patients with elevated CTC and their molecular characterizations may provide novel insights into the metastasis process. Citation Format: Ye Z, Mu Z, Wang C, Palazzo JP, Biederman L, Li B, Jaslow R, Avery T, Austin L, Yang H, Cristofanilli M. Prognostic values of circulating tumor cell (CTC) enumeration and their clusters in advanced breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-08-09.

Replication Timing Aberrations and Aneuploidy in Peripheral Blood Lymphocytes of Breast Cancer Patients

Peripheral blood lymphocytes of patients with hematological malignancies or solid tumors, such as renal cell carcinoma or prostate cancer, display epigenetic aberrations (loss of synchronous replication of allelic counterparts) and genetic changes (aneuploidy) characteristic of the cancerous phenotype. This study sought to determine whether such alterations could differentiate breast cancer patients from cancer-free subjects. The HER2 locus-an oncogene assigned to chromosome 17 whose amplification is associated with breast cancer (BCA)-and the pericentromeric satellite sequence of chromosome 17 (CEN17) were used for replication timing assessments. Aneuploidy was monitored by enumerating the copy numbers of chromosome 17. Replication timing and aneuploidy were detected cytogenetically using fluorescence in situ hybridization technology applied to phytohemagglutinin-stimulated lymphocytes of 20 women with BCA and 10 control subjects. We showed that both the HER2 and CEN17 loci in the stimulated BCA lymphocytes altered their characteristic pattern of synchronous replication and exhibited asynchronicity. In addition, there was an increase in chromosome 17 aneuploidy. The frequency of cells displaying asynchronous replication in the patients' samples was significantly higher (P < 10(-12) for HER2 and P < 10(-6) for CEN17) than the corresponding values in the control samples. Similarly, aneuploidy in patients' cells was significantly higher (P < 10(-9)) than that in the controls. The HER2 and CEN17 aberrant replication differentiated clearly between BCA patients and control subjects. Thus, monitoring the replication of these genes offers potential blood markers for the detection and monitoring of breast cancer.

Abstract 814: A systemic miRNA signature predictive of early breast cancer

Abstract Background: The potential of miRNAs as novel tumor markers has been the focus of much recent attention due to their tissue specificity and unique ability to predict clinicopathological parameters with superior accuracy to mRNA expression profiling. Recent reports have documented altered serum or plasma miRNAs in a variety of cancers including prostate, colon, lung and breast. However it is unknown whether the specific miRNAs reported to be altered in these studies are tumor specific or a general cancer phenomenon. We wished to determine whether a panel of 9 circulating ‘oncomirs’ were tumor specific or similarly expressed in all cancers, and thus identify systemic miRNA signatures predictive of specific cancers. Methods: Using a real-time quantitative PCR approach, expression levels of a panel of 9 oncogenic miRNAs were quantified in blood specimens from 163 cancer patients (breast, colon, renal, prostate and melanoma) and 44 age-matched disease free control individuals. Advanced QBase Plus software and SPSS were used for biostatistical analysis of the data, and correlation with clinicopathological variables. Results: Our panel of circulating miRNAs were detectable at various levels in the circulation of all cancer patients. Expression of general oncomirs such as let 7a and miR-21 were similarly expressed in all cancers. However other specific miRNAs were remarkably different between cancer types. MiR-10b and miR-145 were significantly decreased in the circulation of colon cancer patients compared to other cancers (p=0.000 and p=0.017 respectively). MiR-195, miR-342 and miR-181c were found to be breast cancer specific; elevated levels of these three miRNAs could reliably differentiate breast cancer patients from healthy controls and from patients with other malignancies with high sensitivities (area under curve by ROC analysis: 0.941, 0.963, and 0.926 respectively). Furthermore, systemic miR-195 was detectable in patients with in-situ disease, levels increased progressively as stage of disease advanced, and decreased significantly post curative tumor resection to levels comparable with controls (p=0.001). Conclusion: These findings suggest that individual malignancies, in particular breast cancers, display specific systemic miRNAs profiles, which could aid in early diagnosis and in discriminating one cancer from another. This is of major clinical importance as it illustrates the potential for systemic miRNAs as sensitive and specific yet non-invasive cancer biomarkers. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 814.