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.

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.

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.

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.

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.

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.

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.

Glucocorticoids promote transition of ductal carcinoma in situ to invasive ductal carcinoma by inducing myoepithelial cell apoptosis.

BACKGROUND: The microenvironment and stress factors like glucocorticoids have a strong influence on breast cancer progression but their role in the first stages of breast cancer and, particularly, in myoepithelial cell regulation remains unclear. Consequently, we investigated the role of glucocorticoids in ductal carcinoma in situ (DCIS) in breast cancer, focusing specially on myoepithelial cells. METHODS: To clarify the role of glucocorticoids at breast cancer onset, we evaluated the effects of cortisol and corticosterone on epithelial and myoepithelial cells using 2D and 3D in vitro and in vivo approaches and human samples. RESULTS: Glucocorticoids induce a reduction in laminin levels and favour the disruption of the basement membrane by promotion of myoepithelial cell apoptosis in vitro. In an in vivo stress murine model, increased corticosterone levels fostered the transition from DCIS to invasive ductal carcinoma (IDC) via myoepithelial cell apoptosis and disappearance of the basement membrane. RU486 is able to partially block the effects of cortisol in vitro and in vivo. We found that myoepithelial cell apoptosis is more frequent in patients with DCIS+IDC than in patients with DCIS. CONCLUSIONS: Our findings show that physiological stress, through increased glucocorticoid blood levels, promotes the transition from DCIS to IDC, particularly by inducing myoepithelial cell apoptosis. Since this would be a prerequisite for invasive features in patients with DCIS breast cancer, its clinical management could help to prevent breast cancer progression to IDC.

Independent prognostic impact of preoperative serum carcinoembryonic antigen and cancer antigen 15-3 levels for early breast cancer subtypes.

BACKGROUND: Although the prognosis for operable breast cancers is reportedly worse if serum carcinoembryonic antigen (CEA) and cancer antigen 15-3 (CA15-3) levels are above normal, the usefulness of this prognosis is limited due to the low sensitivity and specificity; in addition, the optimal cutoff levels remain unknown. METHODS: A total of 1076 patients who were operated for breast cancers (test set = 608, validation set = 468) without evidence of metastasis were recruited, and their baseline and postoperative serum CEA and CA15-3 levels were analyzed. The optimal cutoff values of CEA and CA15-3 for disease-free survival (DFS) were 3.2 ng/mL and 13.3 U/mL, respectively, based on receiver operating characteristic curve and area under the curve analyses. RESULTS: The DFS of patients with high CEA levels (CEA-high: n = 191, 5-year DFS 70.6%) was significantly worse (p < 0.0001) than that of CEA-low patients (n = 885, 5-year DFS 87.2%). There was a significant difference in DFS (p < 0.0001) between CA15-3-high and CA15-3-low patients (n = 314 and n = 762, respectively; 5-year DFS 71.8 vs. 89.3%). Significant associations between DFS and CA15-3 levels were observed irrespective of the subtypes. Multivariable analysis indicated that tumor size, lymph node metastasis, tumor grade, and CEA (p = 0.0474) and CA15-3 (p < 0.0001) levels were independent prognostic factors (hazard ratio [HR] 1.520, 95% confidence interval [CI] 1.005-2.245 for CEA; HR 2.088, 95% CI 1.457-2.901 for CA15-3). CONCLUSIONS: These findings suggest that CEA and CA15-3 levels might be useful for predicting the prognosis of patients with operable early breast cancer irrespective of the subtype. Serum levels at baseline may reflect tumor characteristics for metastatic potential even when these levels are within the normal ranges.

LINE-1 METHYLATION IN BLOOD AND TISSUES OF PATIENTS WITH BREAST CANCER.

Methylation is an epigenetic alteration proved to be involved in many disease processes including cancer. This change affects mainly gene promoters and repetitive sequences in genome. Long Interspersed Nuclear Element-1 (LINE-1) is a family of retrotransposons - repetitive elements that modify gene activity and can themselves be targeted by epigenetic mechanisms. LINE-1 methylation level is a surrogate marker for global methylation. In many conditions this parameter is found to be altered not only in affected cell groups, but also throughout other tissues. The aim of our study was to compare LINE-1 methylation pattern in DNA extracted from blood of the patients with benign and malignant breast tissue. In addition, we investigated correlation of LINE-1 methylation in blood and tissues of same patients and relationship of all variables with histopathologic and phenotypic characteristics of tumors. Patients with biopsy-proved ductal invasive carcinoma of breast and no preoperative chemo/radiotherapy were chosen for the study group. Another pool of patients with various benign breast lesions represented controls. Blood samples from both group members were collected preoperatively. Tumor tissue sections were processed for pathology report and part of remaining tissue was used for methylation study. LINE-1 methylation level was quantified using ELISA-based assay. It was analyzed in combination with histologic and phenotypic tumor parameters and compared between different tissues and different study groups. LINE-1 was found to be significantly hypomethylated in breast cancer tissue compared to blood. Blood samples of patients with malignant tumors showed slightly lower methylation level, than samples obtained from control group members. Lymphovascular invasion was the only aggressiveness-determining factor that was found to be at least weakly correlated with LINE-1 hypomethylation in blood. We can conclude, that global hypomethylation measured by LINE-1 methylation level is significant in tumor tissue. But there is no significant difference between LINE-1 methylation levels in blood of patients with benign and malignant breast tumors; therefor LINE-1 hypomethylation in blood cannot be used as a marker for early tumor detection. Neither is it valid for determination of tumor behavior.

Detection of ESR1 mutations in plasma and tumors from metastatic breast cancer patients using next-generation sequencing.

PURPOSE: Liquid biopsy using digital PCR (dPCR) has been widely used for the screening of ESR1 mutations, since they are frequently identified in the hotspot. However, dPCR is limited to the known mutations. Therefore, we aimed to analyze the utility of next-generation sequencing (NGS) to discover novel ESR1 mutations. METHODS: Whole exon sequencing of the ESR1 gene using NGS was performed in 16 primary and 47 recurrent tumor samples and 38 plasma samples from hormone receptor-positive metastatic breast cancer patients. Functional analyses were then performed for the novel mutations we detected. RESULTS: We identified no mutations in primary tumors and six mutations in five recurrent tumors, including three types of known mutations (Y537C, Y537N, and D538G) and two novel mutations (E279V and G557R). We also identified seven mutations in five plasma samples, including three types of known mutations (S463P, Y537S, and D538G) and one mutation not reported in COSMIC database (L536H). All nine patients with ESR1 mutations were treated with aromatase inhibitors (AIs) prior to sampling, and the mutations were frequently detected in patients who received AI treatments in the metastatic setting. Among the three novel mutations (E279V, L536H, and G557R), L536H, but not E279V and G557R, showed ligand-independent activity. All three mutant proteins showed nuclear localization and had no relation with non-genomic ER pathways. CONCLUSIONS: Although the molecular mechanisms of the E279V and G557R mutations remain unclear, our data suggest the utility of NGS as a liquid biopsy for metastatic breast cancer patients and the potential to identify novel ESR1 mutations.

Criteria derived from serum markers can precisely evaluate axillary status in breast cancer patients.

BACKGROUND: A noninvasive method to confirm the presence of lymph node metastases (LNM) in breast cancer patients is lacking. This study aimed to identify markers from peripheral blood that have diagnostic value in evaluating axillary LNM. METHODS: We tested 26 factors in serum from 57 patients with resectable breast cancer by the Luminex assay. Differences between node-negative and node-positive patients were assessed. The diagnostic value of the factors was determined by further analyses and a validation test. RESULTS: Matrix metalloproteinase-1, hepatocyte growth factor, and chemokine ligand 5 were independent risk factors for LNM. However, receiver operating characteristic analysis showed that these factors alone were not ideal predictors. The LNM score (LNMS), derived from combining these markers, correlated significantly with numbers of positive lymph nodes. Patients with LNMS of 0 had few LNM, axillary lymph node dissection (ALND) could be avoided, and sentinel lymph node biopsy (SLNB) was unnecessary. Very high accuracy was achieved for patients with LNMS of 1 with SLNB using only methylene blue, patients with LNMS of 3 required ALND, and patients with LNMS of 2 needed SLNB using both a radioactive isotope and methylene blue, and ALND. CONCLUSIONS: The LNMS derived from matrix metalloproteinase-1, hepatocyte growth factor, and chemokine ligand 5 serum levels identified the axillary lymph node status with high accuracy. Patients with higher LNMS had a greater probability of LNM.

MiR-16: A novel hereditary marker in breast cancer and their offspring.

OBJECTIVE: To analyse micro ribonucleic acid-16 in sera of invasive intraductal breast carcinoma in stage III and compare its expression in their daughters and healthy women. METHODS: The study took place from January 2013 till December 2015. This case-control study was conducted at the Ziauddin Cancer Hospital, Karachi, and comprised breast cancer patients and healthy individuals. Stage III invasive intraductal breast cancer patients (cases), their age-matched healthy individuals (control group A) and patients' daughters (control group B) were included. Subjects with stage I cancer and their daughters and subjects with stage IV and their daughters were also included. Serum tests were run on real-time quantitative reverse transcription polymerase chain reaction. Threshold cycle was determined and fold change was calculated. Fold change was applied between the groups. SPSS 20 was used for data analysis. RESULTS: Of the 194 participants, there were 50(25.8%) cases, 50(25.8%) group A controls, 35(18%) group B controls, 20(10.3%) stage I patients, 11(5.7%) daughters of stage 1 patients, 20(10.3%) patients of stage IV and 8(4.1%) daughters of stage IV patients. Micro ribonucleic acid-16 was higher in cases than controls (p=0.001). Group B showed significant gene expression than group A (p=0.001). Stage IV patients and daughters showed expression of micro ribonucleic acid-16 (p=0.001). Triple negative receptor cases showed a greater expression of gene (p=0.001). CONCLUSIONS: Micro ribonucleic acid-16 can be used as a prognostic, diagnostic as well as a predictive marker in breast cancer patients and their offspring.

Protective effect of naturally occurring anti-HER2 autoantibodies on breast cancer.

Anti-HER2-autoantibodies (HER2-AAbs) are found in breast cancer patients as well as healthy individuals. However, the clinical relevance of the antibodies is unknown. We established an enzyme-linked immunosorbent assay with high sensitivity and quantified serum HER2-AAbs in 100 healthy women, 100 untreated patients with ductal carcinoma in situ (DCIS), and 500 untreated patients with invasive breast carcinoma (IBC). The associations between the levels of HER2-AAbs and breast cancer risk, and recurrence-free survival, were examined. High levels of HER2-AAbs were significantly associated with a reduced risk of DCIS (odds ratio [OR] 0.19, P = 4.6 × 10(-7)) or IBC (OR 0.31, P = 3.7 × 10(-7)). Subgroup analysis of IBC revealed a stronger association of HER2-AAbs with a reduced risk of the hormone receptor (HR)(-)/HER2(+) subtype (OR 0.12) than the other subtypes (HR(+)/HER2(-) [OR = 0.32], HR(+)/HER2(+) [OR 0.38], and HR(-)/HER2(-) [OR 0.29]). When we set the cutoff of HER2-AAbs at 20 ng/mL, recurrence-free survival of HER2-AAb-positive patients (N = 74) was significantly better than that of HER2-AAb-negative patients (N = 426) (P = 0.015). Univariate and multivariate analyses demonstrated that HER2-AAbs, as well as histological grade, were independently and significantly (P = 0.0065 and 0.049, respectively) associated with recurrence-free survival. Our exploratory study suggests a protective effect of naturally occurring HER2-AAbs on the development of primary and recurrent breast cancer. Further studies on HER2-AAbs are warranted.

Plasma DNA integrity index as a potential molecular diagnostic marker for breast cancer.

Plasma DNA integrity index is increased in various malignancies including breast cancer, the most common cancer in women worldwide; early detection is crucial for successful treatment. Current screening methods fail to detect many cases of breast cancer at an early stage. In this study, we evaluated the level of plasma DNA integrity index in 260 females (95 with breast cancer, 95 with benign breast lesions, and 70 healthy controls) to verify its potential value in discriminating malignant from benign breast lesions. The criteria of the American Joint Committee on Cancer were used for staging of breast cancer patients. DNA integrity index was measured by real-time PCR. DNA integrity index was significantly higher in breast cancer than in benign breast patients and healthy subjects (P = <0.001). DNA integrity index is correlated with TNM stage. Given 100 % specificity, the highest sensitivity achieved in detecting cancer group was 85.3 % at 0.55 DNA integrity index cutoff. In conclusion, the plasma DNA integrity index may be a promising molecular diagnostic marker of malignancy in breast lesions.

AKT1 (E17K) mutation profiling in breast cancer: prevalence, concurrent oncogenic alterations, and blood-based detection.

BACKGROUND: The single hotspot mutation AKT1 [G49A:E17K] has been described in several cancers, with the highest incidence observed in breast cancer. However, its precise role in disease etiology remains unknown. METHODS: We analyzed more than 600 breast cancer tumor samples and circulating tumor DNA for AKT1 (E17K) and alterations in other cancer-associated genes using Beads, Emulsions, Amplification, and Magnetics digital polymerase chain reaction technology and targeted exome sequencing. RESULTS: Overall AKT1 (E17K) mutation prevalence was 6.3 % and not correlated with age or menopausal stage. AKT1 (E17K) mutation frequency tended to be lower in patients with grade 3 disease (1.9 %) compared with those with grade 1 (11.1 %) or grade 2 (6 %) disease. In two cohorts of patients with advanced metastatic disease, 98.0 % (n = 50) and 97.1 % (n = 35) concordance was obtained between tissue and blood samples for the AKT1 (E17K) mutation, and mutation capture rates of 66.7 % (2/3) and 85.7 % (6/7) in blood versus tissue samples were observed. Although AKT1-mutant tumor specimens were often found to harbor concurrent alterations in other driver genes, a subset of specimens harboring AKT1 (E17K) as the only known driver alteration was also identified. Initial follow-up survival data suggest that AKT1 (E17K) could be associated with increased mortality. These findings warrant additional long-term follow-up. CONCLUSIONS: The data suggest that AKT1 (E17K) is the most likely disease driver in certain breast cancer patients. Blood-based mutation detection is achievable in advanced-stage disease. These findings underpin the need for a further enhanced-precision medicine paradigm in the treatment of breast cancer.

Modelling circulating tumour cells for personalised survival prediction in metastatic breast cancer.

Ductal carcinoma is one of the most common cancers among women, and the main cause of death is the formation of metastases. The development of metastases is caused by cancer cells that migrate from the primary tumour site (the mammary duct) through the blood vessels and extravasating they initiate metastasis. Here, we propose a multi-compartment model which mimics the dynamics of tumoural cells in the mammary duct, in the circulatory system and in the bone. Through a branching process model, we describe the relation between the survival times and the four markers mainly involved in metastatic breast cancer (EPCAM, CD47, CD44 and MET). In particular, the model takes into account the gene expression profile of circulating tumour cells to predict personalised survival probability. We also include the administration of drugs as bisphosphonates, which reduce the formation of circulating tumour cells and their survival in the blood vessels, in order to analyse the dynamic changes induced by the therapy. We analyse the effects of circulating tumour cells on the progression of the disease providing a quantitative measure of the cell driver mutations needed for invading the bone tissue. Our model allows to design intervention scenarios that alter the patient-specific survival probability by modifying the populations of circulating tumour cells and it could be extended to other cancer metastasis dynamics.

Biological markers of invasive breast cancer.

Biological markers for breast cancer are biomolecules that result from cancer-related processes and are associated with particular clinical outcomes; they thus help predict responses to therapy. In recent years, gene expression profiling has made the molecular classification of breast cancer possible. Classification of breast cancer by immunohistochemical expression of estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2 and Ki-67 is standard practice for clinical decision-making. Assessments of hormone receptor expression and human epidermal growth factor receptor 2 overexpression help estimate benefits from targeted therapies and have greatly improved prognoses for women with these breast cancer types. Although Ki-67 positivity is associated with an adverse outcome, its clear identification is an aid to optimal disease management. Standardization of testing methodology to minimize inter-laboratory measurement variations is a remaining issue. Multi-gene assays provide prognostic information and identify those most likely to benefit from systemic chemotherapy. Incorporating molecular profiles with conventional pathological classification would be more precise, and could enhance the clinical development of personalized therapy in breast cancer.

Effect of polyclonal activators on cytokine production by blood cells and by malignant breast cancer cells.

The production of cytokines by peripheral blood cells and biopsy specimens of tumors stimulated by polyclonal activators (PAs) was evaluated in 34 patients with invasive ductal breast carcinoma using enzyme-linked immunosorbent assay (ELISA). Positive correlation between the stimulation index of polyclonal activators (SIPA) for IL-18 production by the tumor and the relative content of poorly differentiated cells was revealed. The latter, in turn, was positively correlated with the numbers of normal and pathologic mitoses and the degree of malignancy. Cancer cells can produce IL-18, which is involved in the process of angiogenesis, stimulates invasion and metastasis. Decrease in SIPA for the production of IL-6 and GCSF by peripheral blood cells could serve as an indicator of malignant progression in invasive ductal breast carcinoma.