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Neutrophils are prominent immune components of solid tumors, which can protect against the onset of cancer (N1) or have pro-tumor activity (N2). Circulating neutrophils, divided into high density neutrophils (HDN) and low density neutrophils (LDN), functionally mirror those N1 and N2 cells, respectively. LDN, a rare subset in non-pathological conditions, have been extensively studied in cancer, due to their frequency in this disease and their pro-tumor phenotype. However, this has been mainly demonstrated in animal models and proper validation in humans is an urgent need. Here, we further enlighten the clinical impact of LDN in a cohort of breast cancer (BC) patients. We observed that LDN were practically absent in healthy donors’ blood, while were significantly increased in the blood of BC patients, particularly with metastatic disease. Relevant for a clinical translation, within the population of non-metastatic patients, LDN were more prevalent in patients with poor response to neoadjuvant chemotherapy than in responders. The association of a higher incidence of circulating LDN and the worse prognosis of BC patients could be explained by the pro-tumor/immunosuppressive characteristics exhibited by these cells. Namely, there are more LDN expressing the immunosuppressive marker PD-L1, than HDN. Additionally, LDN also showed increased expression of activation markers; a robust formation of neutrophil extracellular traps; an augmented phagocytic activity; and a higher capacity to release reactive oxygen species, which may contribute for tumor development and metastization. Moreover, the percentage of LDN in BC patients’ blood was negatively correlated with activated cytotoxic T lymphocytes and positively correlated with the immunosuppressive CCR4+ regulatory T cells, corroborating their impairment on the anti-tumor immune responses, which was further demonstrated ex vivo. Hence, this study reveals the potential of LDN as a clinical meaningful biomarker of BC response to treatment and opens new avenues for developing targeted immunotherapies.

Breast cancer patients diagnosed with HR+/HER2– tumors face a persistent risk of distant recurrence long after completion of their treatment. Strategies to induce anti-tumor immune responses could complement standard-of-care therapies for these patients. The current study was performed to examine the feasibility, safety and immunogenicity of adding P10s-PADRE to standard-of-care chemotherapy in HR+/HER2− early-stage breast cancer patients. Twenty-five subjects were treated in a single-arm Phase Ib clinical trial. Five different immunization schedules were considered to evaluate the feasibility of eliciting an immune response. The primary immunogenicity endpoint was antibody titer. The expression of several activation markers on natural killer (NK) cells and serum concentrations of Th1/Th2 cytokines were also examined. The percentage of tumor-infiltrating lymphocytes (TILs) was determined. Antibody response was superior in schedule C where 3 weekly immunizations preceded the first dose of chemotherapy. A significant change in CD16, NKp46 and CD94 expression levels on NK cells and a rise in serum content of IFN-γ was observed after treatment. Schedule C showed an increase in TILs in residual lesions. The combination therapy is safe and immunogenic with treatment schedule C being immunologically promising. Randomized trials focused on long-term survival outcomes are needed to evaluate clinical benefits.

Background Tumor protein 53 (TP53) gene mutations are identified in up to 37% of breast tumors especially in HER-2 positive and basal-like subtype. Previous studies have indicated TP53 mutations as a prognostic biomarker in breast cancer. However, most of these studies performed immunohistochemistry (IHC) for the detection of TP53 mutations. Aim The purpose of our study is to evaluate the role of TP53 somatic mutations detected via next-generation sequencing (NGS) as a potential prognostic marker in patients with breast cancer. Materials and methods 82 female patients with Stage I-III breast cancer underwent NGS in paraffin blocks and blood samples during the period 25/09/2019 through 25/05/2021. 23 cases of somatic TP53 mutations and 23 cases of healthy controls were matched on age at diagnosis, menopausal status, histological subtype, histological grade, ki67 expression and disease stage. Results Mean age at diagnosis was 52.35 (SD; 11.47) years. The somatic TP53 mutation NM_000546.5:c.824G>A p.(Cys275Tyr) was most frequently detected. Co-existence of PIK3CA mutation was a common finding in somatic TP53-mutant tumors (4/23; 17.4%). Disease-free survival was shorter in TP53-mutated cases (16.3 months vs. 62.9 months). TP53 pathogenic somatic mutations were associated with a 8-fold risk of recurrence in the univariate Cox regression analysis (OR = 8.530, 95% CI: 1.81-40.117; p = 0.007). Conclusions Our case-control study suggests that TP53 somatic mutations detected by next-generation sequencing (NGS) are associated with an adverse prognosis in breast cancer.

Background: Radiomics involving quantitative analysis of imaging has shown promises in oncology to serve as non-invasive biomarkers. We investigated whether pre-treatment T2-weighted magnetic resonance imaging (MRI) can be used to predict response to neoadjuvant chemotherapy (NAC) in breast cancer. Materials and Methods: MRI scans were obtained for 102 patients with locally advanced breast cancer (LABC). All patients were treated with standard regimens of NAC as decided by the treating oncologist, followed by surgery and adjuvant treatment according to standard institutional practice. The primary tumor was segmented, and 11 texture features were extracted using the grey-level co-occurrence matrices analysis of the T2W-images from tumor cores and margins. Response assessment was done using clinical-pathological responses with patients classified into binary groups: responders and non-responders. Machine learning classifiers were used to develop a radiomics model, and a leave-one-out cross-validation technique was used to assess the performance. Results: 7 features were significantly (p < 0.05) different between the two response groups. The best classification accuracy was obtained using a k-nearest neighbor (kNN) model with sensitivity, specificity, accuracy, and area under curve of 63, 93, 87, and 0.78, respectively. Conclusions: Pre-treatment T2-weighted MRI texture features can predict NAC response with reasonable accuracy.

Altered expression of the translation factor eIF3e is associated with breast cancer occurrence. We have previously shown that eIF3e deficiency leads to an impaired DNA damage response with a marked decrease in DNA repair by homologous recombination. Here, we explored the possibility to exploit this DNA repair defect in targeted cancer therapy using PARP inhibitors. Surprisingly, eIF3e-deficient breast cancer cells are resistant to these drugs, in contrast to BRCA1-deficient cells. Studying this, we found that eIF3e-depleted cells synthesize lowered amounts of PARP1 protein, due to a weakened translation of the corresponding mRNA, associated with a strong decrease in cellular poly(ADP-ribosyl)ation. Additionally, we discovered that the mTORC1 signaling pathway is aberrantly activated in response to eIF3e suppression. Together, these PARP1 and mTORC1 dysfunctions upon eIF3e depletion are causally linked to induction of cellular senescence associated with a pro-inflammatory secretory phenotype. This study provides mechanistic insights into how eIF3e protects against breast cancer, with potential novel cancer therapeutic opportunities. While PARP inhibitors appear as inappropriate drugs for eIF3e-deficient breast tumors, our findings suggest that such cancers may benefit from senolytic drugs or mTORC1 inhibitors.

Purpose We develop a multi-centric response predictive model using QUS spectral parametric imaging and novel texture-derivate methods for determining tumour responses to neoadjuvant chemotherapy (NAC) prior to therapy initiation. Materials and methods QUS Spectroscopy provided parametric images of mid-band-fit (MBF), spectral-slope (SS), spectral-intercept (SI), average-scatterer-diameter (ASD), and average-acoustic-concentration (AAC) in 78 patients with locally advanced breast cancer (LABC) undergoing NAC. Ultrasound radiofrequency data were collected from Sunnybrook Health Sciences Center (SHSC), University of Texas MD Anderson Cancer Center (MD-ACC), and St. Michaels Hospital (SMH) using two different systems. Texture analysis was used to quantify heterogeneities of QUS parametric images. Further, a second-pass texture analysis was applied to obtain texture-derivate features. QUS, texture- and texture-derivate parameters were determined from both tumour core and a 5-mm tumour margin and were used in comparison to histopathological analysis for developing a response predictive model to classify responders versus non-responders. Model performance was assessed using leave-one-out cross-validation. Three standard classification algorithms including a linear discriminant analysis (LDA), k-nearest-neighbors (KNN), and support vector machines-radial basis function (SVM-RBF) were evaluated. Results A combination of tumour core and margin classification resulted in a peak response prediction performance of 88% sensitivity, 78% specificity, 84% accuracy, 0.86 AUC, 84% PPV, and 83% NPV, achieved using the SVM-RBF classification algorithm. Other parameters and classifiers performed less well running from 66% to 80% accuracy. Conclusions A QUS-based framework and novel texture-derivative method enabled accurate prediction of responses to NAC. Multi-centric response predictive model provides indications of the robustness of the approach to variations due to different ultrasound systems and acquisition parameters.

Objectives: This study investigated harmonized pretreatment volume-based quantitative FDG-PET/CT parameters in breast cancer patients for prognostic value. Results: During a median overall follow-up period of 5.3 years, 91 patients had recurrence and 40 died. Multivariate analysis of ER-positive/HER2-negative patients showed high maximum standardized uptake value (SUVmax) (p = 0.018), high total lesion glycolysis (TLG) (p = 0.010), and clinical N-classification (p = 0.0027) as independent negative predictors of RFS, while high maximum SUVmax (p = 0.037), advanced clinical T-classification (p = 0.030), and advanced TNM stage (p = 0.0067) were independent negative predictors of OS. For recurrence and death in HER2-positive patients, high total TLG (p = 0.037, p = 0.0048, respectively) and advanced TNM stage (p = 0.048, p = 0.046, respectively) were independent prediction factors. In the triple-negative group, independent factors related to recurrence and death were high maximum SUVmax (p = 0.0014, p = 0.0003, respectively) and advanced TNM stage (p < 0.0001, p < 0.0001, respectively). Materials and Methods: Records of 546 stage I–III invasive breast cancer patients, including 344 estrogen receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative, 110 HER2-positive, and 92 triple-negative cases, treated at four institutions were reviewed retrospectively. Harmonized primary tumor and nodal maximum SUVmax, metabolic tumor volume (MTV), and TLG indicated in pretreatment FDG-PET/CT results were analyzed. Evaluations of relationships of clinicopathological factors, volume-based quantitative parameters, recurrence-free survival (RFS), and overall survival (OS) for each subtype were performed with a Cox proportional hazards model and log-rank test. Conclusions: The results indicated that potential surrogate markers for prognosis in patients with the three main subtypes of operable breast cancer include harmonized pretreatment quantitative volume-based FDG-PET/CT parameters, particularly whole-lesion SUVmax and TLG.

Introduction: Endocrine therapy has played an important role in the management of ER positive breast cancer over recent decades. Despite this, not all patients respond equally to endocrine intervention, which can lead to resistance, associated disease relapse and progression. Previous reports suggest that endocrine agents themselves may induce an invasive phenotype in ER positive breast cancers with low/aberrant expression of E-cadherin. Here we investigate this phenomenon further and provide data supporting a role for the ER co-receptor, PELP-1, in mediating an adverse response to endocrine agents.\ud\udMaterials and Methods: The effects of tamoxifen, fulvestrant and estrogen withdrawal (as a model for aromatase inhibitor therapy) on the invasive and migratory capacity of endocrine-sensitive MCF-7 and T47D cells, in the presence or absence of functional E-cadherin and/or PELP-1 (using siRNA knockdown), was assessed via Matrigel invasion and Boyden chamber migration assays. The effects of these endocrine therapies alongside E-cadherin/PELP-1 modulation on cell proliferation were further assessed by MTT assay. Western blotting using phospho-specific antibodies was performed to investigate signalling pathway changes associated with endocrine-induced changes in invasion and migration.\ud\udResults: Both tamoxifen and fulvestrant induced a pro-invasive and pro-migratory phenotype in ER positive breast cancer cells displaying a high basal expression of PELP-1, which was augmented in the context of poor cell-cell contact. This process occurred in a Src-dependent manner with Src inhibition reversing endocrine induced invasion/migration. While this adverse response was observed using both tamoxifen and fulvestrant therapy, it was not observed under conditions of estrogen withdrawal.\ud\udConclusions: Our data confirms previous reports that anti-estrogens induce an adverse cell phenotype in ER+ breast cancer, particularly in the absence of homotypic cell contact. These results implicate E-cadherin and PELP-1 as potential biomarkers when deciding upon optimum adjuvant endocrine therapy, whereby tumours with high PELP-1/low E-cadherin expression may benefit from estrogen withdrawal therapy via aromatase inhibition, as opposed to ER modulation/antagonism.

Centrosomes amplification is a hallmark of cancer. We hypothesize that 2-methoxyestradiol (2-ME) sensitizes breast cancer (BC) cells to taxanes by targeting amplified centrosomes. We assessed the extent by which 2-ME together with paclitaxel (PTX) induces centrosome alterations with subsequent mitotic catastrophe in different BC subtypes. 2-ME induced a significant reduction in PTX IC50 values in all cells tested ranging from 28–44% (P < 0.05). Treatment with both PTX and 2-ME significantly increased the number of misaligned metaphases compared to PTX alone (34%, 100% and 52% for MCF7, MDA-MB231 and SUM149, respectively; P < 0.05). The number of cells with multipolar spindle formation was significantly increased (81%, 220% and 285% for MCF7, MDA-MB231 and SUM 149, respectively; P < 0.05). PTX and 2-ME treatment significantly increased interphase declustering in cancer cells (56% for MCF7, 208% for MDA-MB231 and 218% for SUM149, respectively; P < 0.05) and metaphase declustering (1.4-fold, 1.56-fold and 2.48-fold increase for MCF7, MDA-MB231 and SUM149, respectively; P < 0.05). This report is the first to document centrosome declustering as a mechanism of action of 2-ME and provides a potential approach for reducing taxane toxicity in cancer treated patients.

Metastasis-associated in colon cancer-1 (MACC-1) is a newly identified tumor marker, found to express in various normal and cancerous tissue. This study is conducted to evaluate the serum MACC-1 level as a diagnostic marker for breast cancer (BC). Sixty new BC patients were included in this study. Patients who received neoadjuvant chemotherapy or with metastatic disease were excluded. Eighty patients of benign disease were taken as control group. All the patients were females with the mean age of 46.7 ± 10.6 years in study group and 40.2 ± 8.4 years in control group (p = 0.0001). The mean serum MACC-1 level in BC patients was 3.46 ± 1.3 ng/ml which was significantly higher than control mean serum MACC-1 level (1.90 ± 0.2 ng/ml) (p < 0.0001). On ROC analysis, the AUC was 0.98 (p ≤ 0.0001; 95% CI = 0.97-1.0) i.e., a good predictor for breast cancer. At the cut-off value of 2.12 ng/ml, the sensitivity and the specificity of serum MACC-1 were 96.7% and 92.5%, respectively. This study showed that serum MACC-1 can be a potential biomarker for diagnosis and tumor progression in patients with breast cancer.

The involvement of non-coding RNAs (ncRNAs) in cellular physiology and disease pathogenesis is becoming increasingly relevant in recent years specifically in cancer research. Breast cancer (BC) has become a health concern and accounts for most of the cancer-related incidences and mortalities reported amongst females. In spite of the presence of promising tools for BC therapy, the mortality rate of metastatic BC cases is still high. Therefore, the genomic exploration of the BC subtype and the use of ncRNAs for possible regulation is pivotal. The expression and prognostic values of AURKA gene were assessed by Oncomine, GEPIA, KM-plotter, and bc-GenExMiner v4.4, respectively. Associated proteins and functional enrichment were evaluated by Cytoscape and DAVID databases. Additionally, molecular docking approach was employed to investigate the regulatory role of hsa-miR-32-3p assisted argonaute (AGO) protein of AURKA gene in BC. AURKA gene was highly expressed in patients with BC relative to normal counterpart and significantly correlated with poor survival. The docking result suggested that AURKA could be regulated by hsa-miR-32-3p as confirmed by the reported binding energy and specific interactions. The study gives some insights into role of AURKA and its regulation by microRNAs through AGO protein. It also provides exciting opportunities for cancer therapeutic intervention.

Human breast cancer which affects 1/8 women is rare at a cellular level. Even in the setting of germline BRCA1/BRCA2, which is present in all breast cells, solitary cancers or cancers arising at only several foci occur. The overwhelming majority of breast cells (109–1012 cells) resist transformation. Our hypothesis to explain this rareness of transformation is that mammary oncogenesis is regulated by the cell of origin’s critical window of differentiation so that target cells outside of this window cannot transform. Our novel hypothesis differs from both the multi-hit theory of carcinogenesis and the stem/progenitor cell compartmental theory of tumorigenesis and utilizes two well established murine transgenic models of breast oncogenesis, the FVB/N-Tg (MMTV-PyVT)634Mul/J and the FVB-Tg (MMTV-ErbB2) NK1Mul/J. Tail vein fibroblasts from each of these transgenics were used to generate iPSCs. When select clones were injected into cleared mammary fat pads, but not into non-orthotopic sites of background mice, they exhibited mammary ontogenesis and oncogenesis with the expression of their respective transgenes. iPSC clones, when differentiated along different non-mammary lineages in vitro, were also not able to exhibit either mammary ontogenesis or oncogenesis in vivo. Therefore, in vitro and in vivo regulation of differentiation is an important determinant of breast cancer oncogenesis.

Sacituzumab govitecan (SG) is an antibody-drug conjugate composed of a humanized anti-Trop-2 IgG antibody conjugated via a hydrolysable linker to SN-38, the topoisomerase I-inhibitory active component of irinotecan. We investigated whether Trop-2-expression and homologous recombination repair (HRR) of SN-38-mediated double-strand DNA (dsDNA) breaks play a role in the sensitivity of triple-negative breast cancer (TNBC) to SG. Activation of HRR pathways, as evidenced by Rad51 expression, was assessed in SG-sensitive cell lines with low and moderate Trop-2-expression (SK-MES-1 squamous cell lung carcinoma and HCC1806 TNBC, respectively), compared to a low Trop-2-expressing, less SG-sensitive TNBC cell line (MDA-MB-231). Further, two Trop-2-transfectants of MDA-MB-231, C13 and C39 (4- and 25-fold higher Trop-2, respectively), were treated in mice with SG to determine whether increasing Trop-2 expression improves SG efficacy. SG mediated >2-fold increase in Rad51 in MDA-MB-231 but had no effect in SK-MES-1 or HCC1806, resulting in lower levels of dsDNA breaks in MDA-MB-231. SG and saline produced similar effects in parental MDA-MB-231 tumor-bearing mice (median survival time (MST) = 21d and 19.5d, respectively). However, in mice bearing higher Trop-2-expressing C13 and C39 tumors after Trop-2 transfection, SG provided a significant survival benefit, even compared to irinotecan (MST = 97d vs. 35d for C13, and 81d vs. 28d for C39, respectively; P < 0.0007). These results suggest that SG could provide better clinical benefit than irinotecan in patients with HRR-proficient tumors expressing high levels of Trop-2, as well as to patients with HRR-deficient tumors expressing low/moderate levels of Trop-2.

Background To investigate quantitative ultrasound (QUS) based higher-order texture derivatives in predicting the response to neoadjuvant chemotherapy (NAC) in patients with locally advanced breast cancer (LABC). Materials and methods 100 Patients with LABC were scanned before starting NAC. Five QUS parametric image-types were generated from radio-frequency data over the tumor volume. From each QUS parametric-image, 4 grey level co-occurrence matrix-based texture images were derived (20 QUS-Tex1), which were further processed to create texture derivatives (80 QUS-Tex1-Tex2). Patients were classified into responders and non-responders based on clinical/pathological responses to treatment. Three machine learning algorithms based on linear discriminant (FLD), k-nearest-neighbors (KNN), and support vector machine (SVM) were used for developing radiomic models of response prediction. Results A KNN-model provided the best results with sensitivity, specificity, accuracy, and area under curve (AUC) of 87%, 81%, 82%, and 0.86, respectively. The most helpful features in separating the two response groups were QUS-Tex1-Tex2 features. The 5-year recurrence-free survival (RFS) calculated for KNN predicted responders and non-responders using QUS-Tex1-Tex2 model were comparable to RFS for the actual response groups. Conclusions We report the first study demonstrating QUS texture-derivative methods in predicting NAC responses in LABC, which leads to better results compared to using texture features alone.

Purpose High throughput panel sequencing to tailor therapy in precision oncology promises to improve outcome in patients with metastatic breast cancer. However, data that clearly show any benefit from such an approach is still pending. Materials and methods We performed a retrospective analysis of advanced breast cancer patients that underwent panel sequencing for suggestion of target related drugs. We aimed to (i) determine the frequency of actionable mutations per patient and to (ii) assess the clinical impact of results on treatment options. Results A total of 52 patients underwent panel sequencing of archived tumor tissue. Every sample showed at least one affected gene, accounting for actionable mutations in 45 of 52 patients (87%). New treatment options that would not have been used as indicated by standard predictive markers (such as hormonal receptor status or HER2-status) were found in 22 of 52 patients (42%). We detected therapeutic relevant pathogenic germline variants in 9,6% (5/52) of the patients. Conclusions Using a high throughput-panel sequencing approach to identify actionable mutations in patients with metastatic breast cancer, we identified potential target-related treatment options in a large proportion of our patients, some of which would not have been considered without this data. Prospective clinical trials with compounds targeting the identified actionable mutations are needed to determine which treatments can indeed improve survival or quality of life by limiting exposure to ineffective drugs in advanced breast cancer.

Interleukin-12 (IL-12) is a proinflammatory cytokine that links innate and adaptive immune responses against tumor cells. Single Nucleotide Polymorphisms (SNPs) in IL-12 genes have been associated with cancer risk. However, limited studies have assessed the role of IL-12 in breast cancer (BC) risk comprehensively, and these were done in European and Asian populations. Here, we evaluated the association of the IL-12 signaling pathway and BC risk in Puerto Rican women. A genetic association study was completed with 461 BC cases and 463 non-BC controls. By logistic regression, IL-12 signaling SNPs were associated with an increased BC risk, including rs2243123 (IL12A), rs3761041, rs401502 and rs404733 (IL12RB1), rs7849191 (JAK2), rs280500 (TYK2) and rs4274624 (STAT4). Conversely, other SNPs were associated with reduced BC risk including rs438421 (IL12RB1), rs6693065 (IL12RB2), rs10974947, and rs2274471 (JAK2), rs10168266 and rs925847 (STAT4), and rs2069718 (IFNG). Analyses based in hormone receptors such as estrogen (ER) and progesterone (PR) receptors also revealed protective (for SNPs rs3212227-IL12B; rs3024896 and rs3821236-STAT4) and predisposing (for rs2069705-IFNG SNP) BC associations. Haplotype analysis showed a decreased BC risk for IL12B and STAT4 SNPs, whereas increased risk for IL12RB1 SNPs. This study suggests a role of the IL-12 signaling axis and BC risk. SNPs in this pathway may alter IL-12 induced anti-tumor responses and modulate BC predisposition in a population-specific context. Functional studies will be necessary to confirm these findings, which potentially may benefit IL-12 related immunotherapeutic approaches towards BC.

The influence of breast cancer cells on normal cells of the microenvironment, such as fibroblasts and macrophages, has been heavily studied but the influence of normal epithelial cells on breast cancer cells has not. Here using in vivo and in vitro models we demonstrate the impact epithelial cells and the mammary microenvironment can exert on breast cancer cells. Under specific conditions, signals that originate in epithelial cells can induce phenotypic and genotypic changes in cancer cells. We have termed this phenomenon “cancer cell redirection.” Once breast cancer cells are redirected, either in vivo or in vitro, they lose their tumor forming capacity and undergo a genetic expression profile shift away from one that supports a cancer profile towards one that supports a non-tumorigenic epithelial profile. These findings indicate that epithelial cells and the normal microenvironment influence breast cancer cells and that under certain circumstances restrict proliferation of tumorigenic cells.

TGF-β1 is an epithelial-mesenchymal transition (EMT)-inducing factor that is critical in tumor progression. However, whether the effect of TGF-β1 on breast cancer is through the EMT pathway remains to be determined, and drug development based on this mechanism needs to be improved. Results of this study showed that TGF-β1 dysregulation significantly correlated with the expression levels of EMT-associated markers and transcriptional factors. Exogenous expression of TGF-β1 promoted breast cancer cell metastasis and EMT progression. In addition, direct binding of baicalin to TGF-β1 caused its inactivation, which subsequently blocked signal transduction and inhibited breast cancer cell metastasis. In vivo experiment results further invalidated the inhibitory effect of baicalin on TGF-β1-induced tumor metastasis. These results suggest that baicalin, an active ingredient used in traditional Chinese medicine, exhibits a potential therapeutic effect on breast cancer metastasis by regulating TGF-β1-dependent EMT progression.

Targeted drug approaches have been a major focus for developing new anticancer therapies. Although many such agents approved in the last 20 years have improved outcomes, almost all have underperformed expectations. The full potential of such agents may yet be obtained through novel combinations. Previously, we showed that anti-estrogen drugs combined with a dendritic cell-based anti-HER-2 vaccine known to induce strong Th1-polarized immunity dramatically improved clinical response rates in patients with HER-2pos/ERpos early breast cancer. Here, we show that the small molecule Akt antagonist MK-2206, when combined with the Th1 cytokines IFN-gamma and TNF-alpha, maximize indicators of apoptotic cell death in a panel of phenotypically-diverse human breast cancer lines. These findings were mirrored by other, structurally-unrelated Akt-targeting drugs that work through different mechanisms. Interestingly, we found that MK-2206, as well as the other Akt antagonist drugs, also had a tendency to suppress Th1 cytokine expression in stimulated human and murine lymphocytes, potentially complicating their use in conjunction with active immunotherapy. After verifying that MK-2206 plus IFN-gamma could show similar combined effects against breast cancer lines, even in the absence of TNF-alpha, we tested in a rodent HER-2pos breast cancer model either a HER-2-based DC vaccine, or recombinant IFN-gamma with or without MK-2206 administration. We found that for MK-2206, co-administration of recombinant IFN-gamma outperformed co-administration of DC vaccination for slowing tumor growth kinetics. These findings suggest a combined therapy approach for Akt-targeting drugs that incorporates recombinant Interferon-gamma and is potentially translatable to humans.

1,25-Dihydroxyvitamin D3 (1,25D3) induces growth arrest and apoptosis in breast cancer cells in vivo and in vitro, however the exact mechanisms are unclear. Although the vitamin D receptor (VDR), a ligand dependent transcription factor, is required for growth regulation by vitamin D, the specific target genes that trigger these effects are unknown. Genomic profiling of murine mammary tumor cells with differential VDR expression identified 35 transcripts that were altered by the 1,25D3-VDR complex including Hyaluronan Synthase-2 (Has2). Here we confirmed that 1,25D3 reduces both HAS2 gene expression and hyaluronic acid (HA) synthesis in multiple models of breast cancer. Furthermore, we show that the growth inhibitory effects of 1,25D3 are partially reversed in the presence of high molecular weight HA. HAS2 expression and HA production are elevated in immortalized human mammary epithelial cells induced to undergo epithelial-mesenchymal transition (EMT) through stable expression of TGFβ, SNAIL or TWIST and in those expressing oncogenic H-RASV12, indicating that deregulation of HA production may be an early and frequent event in breast tumorigenesis. 1,25D3 also reduces HA secretion and acts additively with an HA synthesis inhibitor to slow growth of cells expressing TGFβ, SNAIL and TWIST. Analysis of mammary gland and tumors from Vdr knockout mice suggest that loss of VDR is associated with enhanced HAS2 expression and HA production in vivo. These data define a novel role for 1,25D3 and the VDR in control of HA synthesis in epithelial tissues that likely contributes to its anti-cancer actions.

Objective: To study GRB7 protein expression in normal human tissues and breast and ovarian cancers, and determine its clinical significance. Results: GRB7 protein was expressed in multiple tissues, including myoepithelial cells of normal breast and fibroadenoma. Cytoplasmic GRB7 expression was seen predominantly in HER-2 positive and, to a lesser extent, triple negative breast cancer. Membrane localization of GRB7 was present in a subset of breast cancers with high cytoplasmic GRB7 expression. Univariate and multivariate analysis found that cytoplasmic GRB7 expression was associated with a negative progesterone receptor status, while membrane GRB7 expression was associated with a negative axillary nodal status. Membrane associated GRB7 expression was present in a subset of ovarian cancers with high cytoplasmic GRB7 expression. Membrane GRB7 expression displayed a trend towards improved recurrence free survival (RFS). Landmark analysis suggested an RFS advantage for ovarian cancers that had GRB7 membrane expression and survived beyond 27 months; GRB7 membrane expression in two or more cores (out of three) predicted an improved RFS. Membrane expression of GRB7 protein was observed in breast cancer cell lines with high GRB7 protein expression in vitro. Conclusion: GRB7 protein membrane expression may be associated with a better prognosis in breast and ovarian cancers. The favorable prognostic value of GRB7 protein membrane expression and its underlying mechanism is worthy of further investigation. Methods: Immunohistochemistry of normal human tissues, breast tissues of various pathologies, and clinically annotated ovarian cancers was performed to correlate the patterns of GRB7 expression with biomarkers or clinical outcome.

Despite relevant medical advancements, metastatic breast cancer remains an uncurable disease. HER2 signaling conditions tumor behavior and treatment strategies of HER2 expressing breast cancer. Cancer treatment guidelines uniformly identify dual blockade with pertuzumab and trastuzumab plus a taxane as best first line and trastuzumab emtansine as preferred second line choice. However, there is no prospectively designed available study focusing on the sequence and outcomes of patients treated with T-DM1 following the triplet. In the following report, data concerning a wide series of patients treated in a real-life setting are presented. Results obtained in terms of response and median progression free survival suggests a significant role for T-DM1 in disease control of metastatic HER2 expressing breast cancer.

Epidemiological studies provide evidence that physical activity reduces the risk of cancer, particularly of breast cancer. However, little is known about the underlying molecular mechanisms as related to microRNAs. The goal of the herein presented study is to explore the involvement of miRNAs in beneficial effects exerted by physical activity in breast cancer prevention. Thirty subjects (mean age: 57.1 ± 14.7 years) underwent 45 minutes of treadmill walking under standardized conditions. The levels of extracellular miRNAs were evaluated in blood plasma before and after structured exercise by means of microarray analysis of 1,900 miRNAs identifying mostly modulated miRNAs. Structured exercise has been found to modulate the expression of 14 miRNAs involved in pathways relevant to cancer. The different expression of two miRNAs involved in breast cancer progression, i. e. up-regulation of miR-206 and down-regulation of anti-miR-30c, were the most striking effects induced by exercise. The biological effects of these miRNAs were investigated in MCF-7 human breast cancer cells. miR-206 transfection and anti-miR-30c silencing, inhibited cell growth and increased apoptosis of MCF-7 cells. Moreover, the combined use of the two miRNAs further enhanced apoptosis and induced growth arrest in the G1/S phase of cell cycle. Our results support that physical activity effectively change the expression of extracellular miRNAs. Specifically, miR-206 up-regulation and anti-miR-30c down-regulation act as suppressors in breast cancer cells. The evaluation of these miRNAs in blood can be used as non-invasive biomarkers for breast cancer prevention.

© Gomes et al. Copyright: Gomes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited., The role of RANKL-RANK pathway in progesterone-driven mammary carcinogenesis and triple negative breast cancer tumorigenesis has been well characterized. However, and despite evidences of the existence of RANK-positive hormone receptor (HR)-positive breast tumors, the implication of RANK expression in HR-positive breast cancers has not been addressed before. Here, we report that RANK pathway affects the expression of cell cycle regulators and decreases sensitivity to fulvestrant of estrogen receptor (ER)-positive (ER+)/HER2- breast cancer cells, MCF-7 and T47D. Moreover, RANK overexpressing cells had a staminal and mesenchymal phenotype, with decreased proliferation rate and decreased susceptibility to chemotherapy, but were more invasive in vivo. In silico analysis of the transcriptome of human breast tumors, confirmed the association between RANK expression and stem cell and mesenchymal markers in ER+HER2- tumors. Importantly, exposure of ER+HER2- cells to continuous RANK pathway activation by exogenous RANKL, in vitro and in vivo, induced a negative feedback effect, independent of RANK levels, leading to the downregulation of HR and increased resistance to hormone therapy. These results suggest that ER+HER2- RANK-positive cells may constitute an important reservoir of slow cycling, therapy-resistance cancer cells; and that RANK pathway activation is deleterious in all ER+HER2- breast cancer cells, independently of RANK levels., This project was funded by the research project PTDC/MED-ONC/28636/2017 from Fundação para a Ciência e a Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Ensino Superior (MCTES) through Fundos do Orçamento de Estado. IG is supported by the FCT PhD grant SFRH/BD/139178/2018

Assay for transposase-accessible chromatin using sequencing (ATAC-seq) has not yet been widely used in cancer research. Clinical implications of chromatin accessibility assessed by ATAC-seq profiling in human cancers especially in a large patient cohort is largely unknown. In this study, we analyzed ATAC-seq data in 404 cancer patients from the Cancer Genome Atlas, representing the largest cancer patient cohort with ATAC-seq data, and correlated chromatin accessibility with patient demographics, tumor histology, molecular subtypes, and survival. Our results showed that chromatin accessibility varies from chromosome to chromosome, and is different in different genomic regions along the same chromosome. Chromatin accessibility especially on the X chromosome is strongly dependent on patient sex, but not much on patient age or tumor stage. Striking difference in chromatin accessibility is observed between lung adenocarcinoma and lung squamous cell carcinoma, the two most common histological subgroups in lung cancer. Furthermore, chromatin accessibility was different between basal and non-basal breast cancer. Finally, we identified prognostic peaks in the promoter regions that were significantly correlated with survival. In particular, we identified six peaks in the ESR1 gene promoter region in the ATAC-seq profiling and found that the peak about 247 bp away from the transcription start site was significantly associated with better survival. In conclusion, our study provides an alternative mechanism underlying tumor prognosis.

Background: Cancer and fibroadenoma are the most common breast tumors in women of reproductive age. Nuclear factor erythroid 2-related factor 2 (Nrf2) and the nuclear factor kappaB (NF-κB) transcription factor play an important role in the inflammatory process and in cell proliferation. However, few studies have analyzed these markers in breast cancer and fibroadenoma in women of reproductive age. Results: Light microscopy showed a higher concentration of anti-Nrf2 and anti-NF-κB-stained nuclei in breast cancer than in fibroadenoma. The mean percentage of stained nuclei for Nrf2 was 7.12 ± 5.2 and 43.21 ± 19.83 in the control and study groups, respectively (p < 0.0001). The mean percentage of anti-NF-κB was 10.75 ± 7.09 and 56.14 ± 21.19 (mean ± standard deviation) in the control and study groups, respectively (p < 0.0001). Histological grade 3 tumors showed a significantly higher expression of Nrf2 and NF-κB than grade 1 tumors (p < 0.05). Material and methods: This study was approved by the Institutional Review Board of Federal University of Piaui and all patients assigned an inform consent term prior to the study initiation. Nrf2 and NF-κB expression was evaluated by immunohistochemistry in 66 patients, divided into two groups, control (fibroadenoma, n = 36) and study (cancer, n = 30). The data were analyzed using ANOVA test and the statistical significance was established at p < 0.05. Conclusion: Nrf2 and NF-κB expression was significantly higher in breast cancer than in fibroadenoma, in addition to having a greater association with more aggressive tumors.

// Tali Sinai-Livne 1 , Metsada Pasmanik-Chor 2 , Zoya Cohen 3 , Ilan Tsarfaty 4 , Haim Werner 1 , 5 and Raanan Berger 3 1 Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel 2 Bioinformatics Unit, George Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel 3 Institute of Oncology, Chaim Sheba Medical Center, Tel Hashomer 52620, Israel 4 Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel 5 Yoran Institute for Human Genome Research, Tel Aviv University, Tel Aviv 69978, Israel Correspondence to: Haim Werner, email: hwerner@post.tau.ac.il Keywords: insulin-like growth factor-1 (IGF1); IGF1 receptor (IGF1R); targeted therapy; breast cancer; proteomic analysis Received: March 01, 2020 Accepted: April 03, 2020 Published: April 28, 2020 ABSTRACT Clinical, epidemiological and experimental data identified the insulin-like growth factor-1 receptor (IGF1R) as a candidate therapeutic target in oncology. While this paradigm is based on well-established biological facts, including the potent anti-apoptotic and cell survival capabilities of the receptor, most Phase III clinical trials designed to target the IGF1R led to disappointing results. The present study was aimed at evaluating the hypothesis that combined treatment composed of selective IGF1R inhibitor along with classical chemotherapy might be more effective than individual monotherapies in breast cancer treatment. Analyses included comprehensive measurements of the synergism achieved by various combination regimens using the CompuSyn software. In addition, proteomic analyses were conducted to identify the proteins involved in the synergistic killing effect at a global level. Data presented here demonstrates that co-treatment of IGF1R inhibitor along with chemotherapeutic drugs markedly improves the therapeutic efficiency in breast cancer cells. Of clinical relevance, our analyses indicate that high IGF1R baseline expression may serve as a predictive biomarker for IGF1R targeted therapy. In addition, we identified a ten-genes signature with potential predictive value. In conclusion, the use of a series of bioinformatics tools shed light on some of the biological pathways that might be responsible for synergysm in cancer therapy.

SYK has been reported to possess both tumour promotor and repressor activities and deletion has been linked to a pro-proliferative / pro-invasive phenotype in breast tumours. It is unclear whether this is a consequence of protein deletion or loss of kinase activity. The SYK inhibitor, BI 1002494, caused no increase in proliferation in breast cancer cells or primary mammary epithelial cells in 2D or 3D cultures, nor changes in proliferation (CD1/2, CDK4, PCNA, Ki67) or invadopodia markers (MMP14, PARP, phospho-vimentin Ser56). BI 1002494 did not alter SYK protein expression. There was no change in phenotype observed in 3D cultures after addition of BI 1002494. Thirteen weeks of treatment with BI 1002494 resulted in no ductal branching or cellular proliferation in the mammary glands of mice. An in silico genetic analysis in breast tumour samples revealed no evidence that SYK has a typical tumour suppressor gene profile such as focal deletion, inactivating mutations or lower expression levels. Furthermore, SYK mutations were not associated with reduction in survival and disease-free period in breast cancer patients. In conclusion, small molecule inhibition of the kinase function of SYK does not contribute to a typical tumour suppressor profile.

Triple-negative breast cancer (TNBC) shows limited therapeutic efficacy. PARP inhibitor has been approved to treat advanced BRCA-mutant breast cancer but shows high resistance. Therefore, the development of new therapeutics that sensitize TNBC irrespective of BRCA status is urgently needed. The neddylation pathway plays a critical role in many physiological processes by regulating the degradation of proteins. MLN4924, a selective inhibitor of the key neddylation enzyme NEDD8 Activation Enzyme (NAE1), shows higher sensitivity to both BRCA1-wild type and -mutant TNBCs compared to other breast cancer subtypes. MLN4924 induced re-replication with >4N DNA content leading to robust DNA damage. Accumulation of unrepaired DNA damage resulted in S and G2/M arrest causing apoptosis and senescence, due to the stabilization of the replication initiation protein CDT1 and the accumulation of cell cycle proteins upon MLN4924 treatment. Moreover, adding MLN4924 to the standard TNBC chemotherapeutic agent cisplatin increased the DNA damage level, further enhancing the sensitivity. In vivo, MLN4924 reduced tumor growth in a NOD-SCID mouse xenograft model by inducing DNA damage which was further augmented with the MLN4924 and cisplatin cotreatment. NAE1 is overexpressed in TNBC cell lines and in patients compared to other breast cancer subtypes suggesting that NAE1 status is prognostic of MLN4924 treatment response and outcome. Taken together, we demonstrated the mechanism of TNBC sensitization by the MLN4924 and MLN4924/cisplatin treatments irrespective of BRCA1 status, provided a strong justification for using MLN4924 alone or in combination with cisplatin, and identified a genetic background in which this combination will be particularly effective.

The effect of bevacizumab plus paclitaxel therapy on progression-free survival (PFS) is prominent; however, no overall survival (OS) benefit has been demonstrated. Our aim was to study the predictive efficacy of peripheral immune-related parameters, neutrophil-to-lymphocyte ratio (NLR), absolute lymphocyte count (ALC), and c-reactive protein (CRP) in locally advanced and metastatic breast cancers. A total of 179 patients treated with bevacizumab plus paclitaxel were recruited from three institutes in the test cohort. The cut-off values of NLR, ALC, and CRP were set at 3, 1500/μL, and 1.0 mg/dL, respectively, and baseline values of these factors were measured. The PFS of patients with NLR-low was significantly longer than that of patients with -high (median, 12.6 vs. 7.2 months; hazard ratio (HR), 0.48, 95% confidence interval (95% CI), 0.31–0.73; p = 0.0004). OS of patients with NLR-low was significantly better than those with-high (22.2 vs. 13.5 months; HR, 0.57, 95% CI, 0.39–0.83; p = 0.0032). Similarly, improved PFS and OS were recognized in patients with CRP-low as compared with patients with -high (HR, 0.44, 95% CI, 0.28–0.68; p = 0.0001 and HR, 0.39, 95% CI, 0.26–0.61, p < 0.0001, respectively). In the validation cohort from two institutes (n = 57), similar significant improvements in PFS and OS were confirmed for patients with NLR-low (p = 0.0344 and p = 0.0233, respectively) and CRP-low groups (p < 0.0001 and p = 0.0001, respectively). Low levels of NLR and CRP at baseline were significantly associated with improved prognosis in patients treated with bevacizumab plus paclitaxel.

Triple-negative breast cancers are often characterized by aggressive behavior and short clinical course once they become chemotherapy-resistant. We describe below a patient who has shown a response to combination of chemotherapy with Elenagen, a plasmid encoding p62. Elenagen was tested in a previous phase I/II study in patients with refractory solid tumors and shown to be safe. Also, plasmid ability to halt tumor progression and restore sensitivity to chemotherapy was found. Preclinical data supports effects on tumor grade and change the tumor's microenvironment in spontaneous canine breast cancers. We describe here a 48-year old female with triple-negative and BRCA1/2-negative breast cancer who had a primary resistance to chemotherapy and negative dynamics despite the use of multiple lines of treatments. Elenagen was applied intramuscularly at a dose of 1 mg weekly in combination with standard chemotherapy scheme CMF (cyclophosphamide, methotrexate, fluorouracil). In this patient we observed partial tumor regression (by 33%) and 19 weeks of progression-free survival. This first observed objective response to a combination of Elenagen with chemotherapy demonstrates that even in heavily pretreated chemo-resistant triple-negative tumor, the addition of Elenagen to a chemotherapy regimen can cause an objective response and increase in progression-free survival. Such a regimen is worthy of further study in a larger number of patients.

Dysregulated adenosine signaling pathway has been evidenced in the pathogenesis of breast cancer. However, the role of adenosine kinase (ADK) in tumorigenesis remains unclear while it crucially regulates the removal and availability of adenosine. ADK has two isoforms that localize to discrete subcellular spaces: i.e., nuclear, long-isoform (ADK-L) and cytosolic, short-isoform (ADK-S). We hypothesized that these two ADK isoforms would be differentially expressed in breast cancer and may contribute to divergent cellular actions in cancer. In this study, we examined the expression profiles of ADK isoforms in breast cancer tissues from 46 patient and followed up with an in vitro investigation by knocking down the expression of ADK-L or ADK-S using CRISPR gene editing to evaluate the role of ADK isoform in cancer progression and metastasis of cultured triple-negative breast cancer cell line MDA-MB-231. We demonstrated that (i) ADK-L expression level was significantly increased in breast cancer tissues versus paired normal tissues adjacent to tumor, whereas the ADK-S expression levels were not significantly different between cancerous and normal tissues; (ii) CRISPR/Cas9-mediated downregulation of ADK isoforms, led to suppressed cellular proliferation, division, and migration of cultured breast cancer cells; (iii) ADK-L knockdown significantly upregulated gene expression of matrix metalloproteinase (ADAM23, 9.93-fold; MMP9, 24.58-fold) and downregulated expression of cyclin D2 (CCND2, -30.76-fold), adhesive glycoprotein THBS1 (-8.28-fold), and cystatin E/M (CST6, -16.32-fold). Our findings suggest a potential role of ADK-L in mitogenesis, tumorigenesis, and tumor-associated tissue remodeling and invasion; and the manipulation of ADK-L holds promise as a therapeutic strategy for aggressive breast cancer.

// Luana Mota Martins 1 , Carla Solange de Melo Escorcio Dourado 1 , Larysse Maira Campos-Verdes 1 , Fabiane Araujo Sampaio 1 , Camila Maria Simplicio Revoredo 1 , Danylo Rafhael Costa-Silva 1 , Maria da Conceicao Barros-Oliveira 1 , Elmo de Jesus Nery Junior 1 , Lucia Maria do Rego-Medeiros 2 , Luiz Henrique Gebrim 1 , Francisco Adelton Alves-Ribeiro 1 , Gilmara Peres Rodrigues 1 , Diego Cipriano Chagas 1 , Dilina do Nascimento Marreiro 1 and Benedito Borges da Silva 1 , 2 1 Postgraduate Program, Northeast Biotechnology Network (RENORBIO), Biotechnology in Health, Federal University of Piaui, Teresina, Piaui 64000-020, Brazil 2 Facid / Wyden Differential Integral Medicine Faculty, Department of Mastology, Teresina, Piaui 64052-810, Brazill Correspondence to: Benedito Borges da Silva, email: beneditoborges@globo.com Keywords: breast neoplasms; fibroadenoma; matrix metalloproteinase-2; matrix metalloproteinase-9; immunohistochemistry Received: September 17, 2019 Accepted: November 06, 2019 Published: December 03, 2019 ABSTRACT Background Matrix metalloproteinases (MMPs) 2 and 9 may play an important role in cell proliferation and dissemination of cancer. However, few studies have compared the expression of these proteins between breast cancer and fibroadenoma. Material and methods A randomized, double-blind study was carried out in 66 premenopausal women, aged 20-49 years, who had been diagnosed with fibroadenoma or breast cancer. The patients were divided into two groups: Group A, control (fibroadenoma, n=36) and Group B, study (cancer, n=30). Immunohistochemical analysis was performed using tissue samples of fibroadenoma and breast cancer to assess MMP-2 and MMP-9 antigen expression. Cells were considered positive if exhibiting brown cytoplasmic staining. Fisher’s exact test was used to compare the percentage of cases with cells expressing MMP-2 and MMP-9 in control and study groups (p < 0.05). Results Light microscopy showed a higher concentration of cells with positive cytoplasmic staining for MMP-2 and MMP-9 expression in breast cancer than in fibroadenoma. The percentage of cases with cells expressing MMP-2 in the control and study groups was 41.67% and 86.11%, respectively (p < 0.0009), whereas the percentage of cases with cells expressing MMP-9 in groups A and B was 66.67% and 93.33%, respectively (p

The human transcriptional coactivator PC4 has numerous roles to play in the cell. Other than its transcriptional coactivation function, it facilitates chromatin organization, DNA damage repair, viral DNA replication, etc. Although it was found to be an essential protein in vivo, the importance of this multifunctional protein in the regulation of different cellular pathways has not been investigated in details, particularly in oncogenesis. In this study, PC4 downregulation was observed in a significant proportion of mammary tissues obtained from Breast cancer patient samples as well as in a subset of highly invasive and metastatic Breast cancer patient-derived cell lines. We have identified a miRNA, miR-29a which potentially reduce the expression of PC4 both in RNA and protein level. This miR-29a was found to be indeed overexpressed in a substantial number of Breast cancer patient samples and cell lines as well, suggesting one of the key mechanisms of PC4 downregulation. Stable Knockdown of PC4 in MCF7 cells induced its migratory as well as invasive properties. Furthermore, in an orthotopic breast cancer mice model system; we have shown that reduced expression of PC4 enhances the tumorigenic potential substantially. Absence of PC4 led to the upregulation of several genes involved in Epithelial to Mesenchymal Transition (EMT), indicating the possible mechanism of uniform tumour progression in the orthotropic mice. Collectively these data establish the role of PC4 in tumour suppression.

// Maria Jose Godoy-Calderon 1 , 2 , Eglys Gonzalez-Marcano 1 , 2 , Jeismar Carballo 2 and Ana Federica Convit 1 , 2 1 Unidad Experimental de Inmunoterapia, Fundacion Jacinto Convit, Caracas, Venezuela 2 Jacinto Convit World Organization, Inc., Palo Alto, CA, USA Correspondence to: Maria Jose Godoy-Calderon, email: publicaciones@jacintoconvit.org Keywords: combination immunotherapies; cancer immunotherapy; breast cancer; autologous tumor cells vaccine; anti-PD-1 Received: May 29, 2019 Accepted: September 24, 2019 Published: November 12, 2019 ABSTRACT Breast cancer therapies using checkpoints alone have not been highly effective. Based on previous experiences using the ConvitVax, an autologous tumor cells/bacillus Calmette-Guerin (BCG)/formalin-based vaccine, in breast cancer and the potential success of combined therapies, we sought to ascertain whether the ConvitVax combined with anti-PD-1 enhances the antitumor effect in a 4T1 breast cancer model. Animals received four weekly injections of either PBS (G1), ConvitVax (200 μg cell homogenate, 0.0625 mg BCG, 0.02% formalin) (G2), 50 μg anti-PD-1 (G3), or ConvitVax plus anti-PD-1 (200 μg cell homogenate, 0.0625 mg BCG, 0.02% formalin, 50 μg anti-PD-1) (G4). Five weeks post tumor induction all mice were euthanized, tumors extracted and evaluated pathologically and by immunohistochemistry. The combination group (G4) showed 10% more tumor necrosis, greater infiltration of PD-1 + cells and lower infiltration of TAMs, evidencing that the combination of ConvitVax and anti-PD-1 can improve the antitumor effect of the vaccine. Using a higher anti-PD-1 dose and administering each treatment at different times could further potentiate the effect of our therapy. Given the vaccine’s low cost and simple preparation, its use in combination with checkpoints or other target-specific compounds may lead to a highly effective personalized breast cancer immunotherapy.

Introduction Mammography is the gold standard for early breast cancer detection, but shows important limitations. Blood-based approaches on basis of cell-free DNA (cfDNA) provide minimally invasive screening tools to characterize epigenetic alterations of tumor suppressor genes and could serve as a liquid biopsy, complementing mammography. Methods Potential biomarkers were identified from The Cancer Genome Atlas (TCGA), using HumanMethylation450-BeadChip data. Promoter methylation status was evaluated quantitatively by pyrosequencing in a serum test cohort (n = 103), a serum validation cohort (n = 368) and a plasma cohort (n = 125). Results SPAG6, NKX2-6 and PER1 were identified as novel biomarker candidates. ITIH5 was included on basis of our previous work. In the serum test cohort, a panel of SPAG6 and ITIH5 showed 63% sensitivity for DCIS and 51% sensitivity for early invasive tumor (pT1, pN0) detection at 80% specificity. The serum validation cohort revealed 50% sensitivity for DCIS detection on basis of NKX2-6 and ITIH5. Furthermore, an inverse correlation between methylation frequency and cfDNA concentration was uncovered. Therefore, markers were tested in a plasma cohort, achieving a 64% sensitivity for breast cancer detection using SPAG6, PER1 and ITIH5. Conclusions Although liquid biopsy remains challenging, a combination of SPAG6, NKX2-6, ITIH5 and PER1 (SNiPER) provides a promising tool for blood-based breast cancer detection.

Importance: Metastatic breast cancer with central nervous system (CNS) metastases carries a poor prognosis. Recently, CDK4/6 inhibitors have demonstrated a progression free survival (PFS) and overall survival benefit when combined with standard endocrine therapy in advanced hormone receptor (HR)+/HER2- breast cancer. Pre-clinical data suggests possible activity of CDK4/6 inhibitors in the brain, but their CNS-specific benefit has not been explored in clinical practice. Methods: We reviewed clinical trials investigating the efficacy of CDK4/6 inhibitors for advanced or metastatic HR+/HER2- breast cancer. We also reviewed pre-clinical studies that demonstrated the ability of CDK4/6 inhibitors to cross the blood-brain barrier (BBB) and halt the growth of brain metastases in animal models. Findings: An ongoing phase II trial (NCT02308020) was designed to investigate the safety and tolerability of abemaciclib for treatment of patients with CNS metastases, with preliminary data showing partial response in some patients. Review of key randomized phase III trials revealed a scarcity of data pertaining to the development of new CNS metastases. Pre-clinical models demonstrate that CDK4/6 inhibitors are able to cross the BBB and can delay the growth of brain metastases. Conclusions: Despite encouraging pre-clinical evidence, there is a lack of clinical data to inform CNS-specific response rates to CDK4/6 inhibitors among patients with metastatic breast cancer. Given that the treatment of patients with breast cancer brain metastases represents an area of unmet medical need, enrollment of patients with CNS metastases in ongoing clinical trials should be encouraged; innovative trials that examine response of CNS metastases to CDK4/6 inhibitors are also of interest.

GZ17-6.02 is presently undergoing clinical evaluation in solid tumors and lymphoma. The present studies were performed to define its biology in estrogen receptor positive breast cancer cells and to determine whether it interacted with palbociclib to enhance tumor cell killing. GZ17-6.02 interacted in an additive fashion with palbociclib to kill ER+ breast cancer cells. GZ17-6.02 and palbociclib cooperated to inactivate mTOR and AKT and to activate ULK1 and PERK. The drugs interacted to increase the expression of FAS-L and BAX, and to decrease the levels of MCL1, the estrogen receptor, and HDACs 1–3. Palbociclib activated ERBB3, an effect blocked by GZ17-6.02. GZ17-6.02 and palbociclib interacted to increase the expression of multiple toxic BH3 domain proteins and to reduce MCL1 and BCL-XL expression. Knock down of FAS-L reduced the lethality of [GZ17-6.02 + palbociclib]. GZ17-6.02 and palbociclib interacted to enhance autophagosome formation and autophagic flux. Knock down of Beclin1, ATG5, BAG3, eIF2α, toxic BH3 domain proteins or CD95 significantly reduced drug combination lethality. GZ17-6.02 and palbociclib increased the expression of Beclin1 and ATG5, effects blocked by knock down of eIF2α. The drugs also increased the phosphorylation of the AMPK and ATG13, effects blocked by knock down of ATM. Knock down of ATM or the AMPK, or expression of activated mTOR significantly reduced the abilities of GZ17-6.02 and palbociclib to enhance autophagosome formation and autophagic flux.

Breast cancer (BC) and colorectal cancer (CRC) are common and show poor survival in advanced stages. Using The Cancer Genome Atlas (TCGA) computational tool cBioPortal, we evaluated overall patient survival in BRCA1 mRNA-low versus -high cohorts (1.05 SD from mean BRCA1 expression, respectively). Analysis included 1082 BC patients with mRNA data (PanCancer Atlas), 382 CRCs (Firehose Legacy) and 592 CRCs (PanCancer Atlas). As previously reported, BRCA1 mRNA-low tumor expression positively correlated with BC patient survival but was negatively associated in CRC. We observed a correlation between BRCA1 mRNA-high and age 0.6) with BRCA1 in BC and CRC, whereas LMNB2 correlates with BRCA1 in CRC, suggesting tissue-specific BRCA1 interactions. Our results indicate potential for BRCA1 mRNA expression levels as a prognostic biomarker in BC and CRC, suggest tissue-specificity in BRCA1 molecular interactions, and point to BRCA1 mRNA-high levels as a characteristic of CRC tumors in younger versus older individuals.

Background: The purpose of the study was to investigate the role of pre-treatment quantitative ultrasound (QUS)-radiomics in predicting recurrence for patients with locally advanced breast cancer (LABC). Materials and Methods: A prospective study was conducted in patients with LABC (n = 83). Primary tumours were scanned using a clinical ultrasound device before starting treatment. Ninety-five imaging features were extracted-spectral features, texture, and texture-derivatives. Patients were determined to have recurrence or no recurrence based on clinical outcomes. Machine learning classifiers with k-nearest neighbour (KNN) and support vector machine (SVM) were evaluated for model development using a maximum of 3 features and leave-one-out cross-validation. Results: With a median follow up of 69 months (range 7–118 months), 28 patients had disease recurrence (local or distant). The best classification results were obtained using an SVM classifier with a sensitivity, specificity, accuracy and area under curve of 71%, 87%, 82%, and 0.76, respectively. Using the SVM model for the predicted non-recurrence and recurrence groups, the estimated 5-year recurrence-free survival was 83% and 54% (p = 0.003), and the predicted 5-year overall survival was 85% and 74% (p = 0.083), respectively. Conclusions: A QUS-radiomics model using higher-order texture derivatives can identify patients with LABC at higher risk of disease recurrence before starting treatment.

Background In 2014 the standardised incidence rate for breast cancer in Poland reached 51.6/100,000, while the mortality rate reached 14.8/100,000. The incidence rate for breast cancer in the EU was 106.6/100,000, the mortality rate - 22.4/100,000. In 2014 the incidence rate for cervical cancer in Poland was 8.8/100,000, the mortality rate - 4.5/100,000. The incidence rate in the EU was 11.3/100,000 and the mortality rate - 3.7/100,000. Objective The aim of the paper was to establish the number of health policy programmes concerned with breast cancer and cervical cancer in women carried out in 2009-2014 by local government units, with specification of the type of programme, type of local government units that carried out the programmes and the costs of implementation of the programmes. Methods The study was based on a desk research. The analysis covered data included in annual reports submitted by voivodes to Minister of Health, concerning health policy programmes implemented by local government units in 2009-2014. Results The greatest number of programmes concerned with prevention of breast cancer and cervical cancer were implemented in municipalities, followed by counties and finally - self-governed voivodeships. The number of programmes concerned with primary prevention was three times smaller (656) than the number of programmes concerned with secondary prevention (2,229). The greatest number of primary prevention programmes were implemented in Dolnośląskie, Wielkopolskie and Mazowieckie Voivodeships, and the greatest number of secondary prevention programmes - in Wielkopolskie, Mazowieckie and Zachodniopomorskie Voivodeships. Conclusion It was found that the number of programmes implemented by particular local government units and the financial resources employed in the implementation of the programmes were different. It is probable that some of the initiatives of local government units related to secondary prevention coincide with the actions undertaken under the National Programme for Fighting Cancer. The entities that carry out breast cancer and cervical cancer prevention programmes need to coordinate their actions.

Breast cancer (BC) in Nigeria is characterized by disproportionately aggressive molecular subtypes. C-reactive protein (CRP) is associated with risk and aggressiveness for several types of cancer. We examined the association of high-sensitivity CRP (hsCRP) with odds of BC by molecular subtype among Nigerian women. Among 296 newly diagnosed BC cases and 259 healthy controls, multivariable logistic regression models were used to estimate adjusted odds ratios (aOR) and 95% confidence intervals (CI) for the association between hsCRP and odds of BC overall and by molecular subtype (luminal A, luminal B, HER2-enriched and triple-negative or TNBC). High hsCRP (> 3 mg/L) was observed in 57% of cases and 31% of controls and was associated with 4 times the odds of BC (aOR: 4.43; 95% CI: 2.56, 7.66) after adjusting for socio-demographic, reproductive, and clinical variables. This association persisted regardless of menopausal status and body mass index (BMI) category. High hsCRP was associated with increased odds of TNBC (aOR: 3.32; 95% CI: 1.07, 10.35), luminal A BC (aOR: 4.03; 95% CI: 1.29, 12.64), and HER2-enriched BC (aOR: 6.27; 95% CI: 1.69, 23.25). Future studies are necessary in this population to further evaluate a potential role for CRP as a predictive biomarker for BC.

The presence of macrophages within breast tumors correlates with metastatic potential. These tumor-associated macrophages often take on a pro-tumorigenic (M2-like) phenotype resulting in the secretion of growth factors and proteases, including the lysosomal protease cathepsin L. Since cathepsin L also is frequently secreted by breast cancer cells and contributes to tumor invasion, metastasis, and angiogenesis, we hypothesized that secretion of cathepsin L by both tumor-associated macrophages and neoplastic cells would facilitate the metastatic phenotype. Our results showed that the novel cathepsin L/K inhibitors KGP94 and KGP207 could inhibit in vitro M2 macrophage invasion and reduce the macrophage-stimulated invasion of 4T1 murine breast cancer cells. KGP94 and KGP207 treatment also reduced the expression of several M2-associated markers, suggesting that cathepsin L activity may be important for IL-4-driven M0 to M2 differentiation. In addition, cathepsin L shRNA knockdown studies revealed that cathepsin L from both the tumor cell and the macrophage population is important for tumor cell invasion. Thus our data suggest that tumor cells and macrophages may both contribute to the cathepsin L-driven metastatic phenotype of breast cancer. Taken together, these studies highlight the importance of cathepsin L in macrophage functions and suggest that cathepsin inhibition strategies may be therapeutically beneficial by impairing the progression of tumors with high infiltration of M2 macrophages.

// Ming-Bo Huang 1 , * , Jennifer Y. Wu 2 , * , James Lillard 1 and Vincent C. Bond 1 1 Department of Microbiology, Biochemistry, and Immunology, Morehouse School of Medicine, Atlanta, GA 30310, USA 2 Columbia College, Columbia University, New York, NY 10027, USA * These authors contributed equally to this work Correspondence to: Ming-Bo Huang, email: mhuang@msm.edu Keywords: extracellular vesicles; mortalin; complement; SMR peptides; breast cancer Received: May 14, 2019 Accepted: July 24, 2019 Published: September 10, 2019 ABSTRACT Background: Mortalin/GRP-75/mt-hsp70 is a mitochondrial chaperone protein, found in the cytoplasm, endoplasmic reticulum and cytoplasmic vesicles. It functions in many cellular processes such as mitochondrial biogenesis, intracellular trafficking, cell proliferation, signaling, immortalization and tumorigenesis. Thus, inhibition of mortalin is a promising avenue for cancer therapy. Previous studies in our lab have suggested that mortalin contributes to breast cancer development and progression. We showed that tumor extracellular vesicle secretion was decreased by knockdown of mortalin expression using HIV-1 Nef SMR peptides. Specifically, these peptides can block extracellular vesicle secretion and mediate cell cycle arrest in MDA-MB-231 and MCF-7 breast cancer cells. Aims: This study aims to investigate further the function and mechanism of interaction of PEG-SMR-CLU and SMR-CPP peptides with the chaperone protein mortalin and to explore the effect of SMR-derived peptides and mortalin expression on extracellular vesicle release and complement dependent cell toxicity in human breast cancer and leukemia cell lines. Results: Our results demonstrated additional effects reversing the tumorigenicity of these cells. First, the modified SMRwt peptides reduced the expression of the mesenchymal marker vimentin (VIM). Second, exposure to the SMRwt peptide inhibited mortalin and complement C9 expression in MDA-MB-231, MCF-7 breast cancer cells and K562 leukemia cells as measured by the Western blot analysis. Third, the SMRwt peptides blocked the cancer cells’ ability to release extracellular vesicles, which we observed blocked extracellular vesicle-mediated release of complement, re-establishing complements mediated cell death in those peptide-treated cells. Methods: We developed a series of peptides derived from the Secretion Modification Region (SMR) of HIV-1 Nef protein, modified by the addition of either a cell-penetrating peptide (CPP), a positively charged arginine-rich peptide derived from HIV-1 regulatory protein Tat, or a Clusterin-binding peptide (CLU), a molecular chaperone involved in protein secretion. Both CPP and CLU peptide sequences were added at the C-terminus of the Nef SMR peptide. The CLU-containing peptides were also modified with polyethylene glycol (PEG) to enhance solubility. After treatment of cells with the peptides, we used the MTT cell viability and complement-mediated cytotoxicity assays to confirm the inhibitory role of modified SMRwt peptides on the proliferation of MDA-MB-231 and MCF-7 breast cancer cells and K562 leukemia cells. Flow cytometry was used to determine complement mediated cell apoptosis and death. Western blot analysis was used to track SMR peptides impact on expression of mortalin, vimentin and complement C9 and to measure the expression of extracellular vesicle proteins. NanoSight analysis and acetylcholinesterase (AChE) assay were used for measuring extracellular vesicles particle size and concentration and acetylcholinesterase. Conclusions: Mortalin promotes cell proliferation, metastasis, angiogenesis, downregulate apoptotic signaling. Thus, mortalin is a potential therapeutic target for cancer immunotherapy. The novel SMRwt peptides antagonize the functions of mortalin, blocking tumor extracellular vesicle release and extracellular vesicle-mediated release of complement. This leads to decreases in breast cancer cell metastasis and allows standard treatment of these late stage tumor cells, thus having important clinical implications for late stage breast cancer chemotherapy. These findings support further investigation into the therapeutic value of the SMR peptide in cancer metastasis.

Background: Phosphatase and tensin homologue deleted from chromosome 10 (PTEN) negatively regulates the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway. Triple negative breast cancers (TNBC) are often PTEN-deficient, making mTOR a compelling target. We evaluated the efficacy of catalytic mTOR inhibitor TAK228 alone and in combination with eribulin in TNBC. Results: Five of eight triple negative breast cell lines were sensitive to TAK228, independent of PIK3CA/PTEN status. Western blotting demonstrated inhibition of mTORC1/2 signaling as demonstrated by decreased phospho-AKT, phospho-S6 and phospho-4EBP1. In vitro, TAK228 was synergistic with eribulin in all eight TNBC cell lines. The combination of TAK228 and eribulin did not enhance apoptosis but increased G2/M growth arrest. In vivo, TAK228 led to modest growth inhibition in TNBC patient-derived xenografts (PDXs) with no tumor regression observed. In two TNBC PDXs with PTEN loss, one with intrinsic eribulin sensitivity, another eribulin resistance, TAK228 in combination with eribulin did not enhance in vivo efficacy. In a third PTEN-negative TNBC model, eribulin alone achieved disease stabilization, but the combination of TAK228 and eribulin led to significantly smaller tumor volumes compared to eribulin alone (p < 0.001). Methods: We tested in vitro efficacy of TAK228 in a panel of TNBC cell lines with cell proliferation assays. In vivo antitumor efficacy of TAK228 was evaluated alone and in combination with eribulin. Conclusion: TAK228 enhances the antitumor efficacy of eribulin in TNBC models in vitro, and enhanced in vivo activity in selected models. Further study is needed to determine the potential of this combination, and optimal patient selection strategies.

// Sierra A. Colavito 1 , James T. Platt 2 , Matthew A. Held 3 , Zongzhi Liu 2 , Ryan Sokup 1 and David F. Stern 4 , 5 1 Department of Biology, University of Wisconsin-La Crosse, La Crosse, WI, USA 2 Department of Internal Medicine and Yale Cancer Center, Yale School of Medicine, New Haven, CT, USA 3 Department of Molecular, Cellular and Biomedical Sciences, University of New Hampshire, Durham, NH, USA 4 Department of Pathology, Yale School of Medicine, New Haven, CT, USA 5 Yale Cancer Center, New Haven, CT, USA Correspondence to: Sierra A. Colavito, email: scolavito@uwlax.edu Keywords: epithelial-mesenchymal transition; triple-negative breast cancer; targeted drug combination screen; claudin-low breast cancer; CHK1 Received: June 25, 2019 Accepted: July 05, 2019 Published: August 06, 2019 ABSTRACT Mesenchymal stem-like (MSL) breast cancers are enriched for cells with tumor reconstituting and mesenchymal characteristics. These cancers are often triple-negative and have a poor prognosis. Few effective targeted treatment options exist for patients with these cancers, and even when targeted therapies exist, resistance often arises and tumors recur, due in part to drug-tolerant persisting tumor cells with self-renewal capability. Effective treatment strategies will combine agents that target the bulk-tumor and reconstituting cells. In order to identify such a combination therapy, we conducted an inhibitor screen using 40 targeted agents at three different doses in all pairwise combinations. Checkpoint Kinase 1 (CHK1) inhibitors were identified as potent inhibitors of MSL breast cancers. When combined with a pro-apoptotic agent/B Cell Lymphoma 2 (BCL2) inhibitor, the effectiveness of the combination regimen was super-additive compared to either treatment alone and was selective for MSL cancers. Treatment of MSL breast cancer cells results in DNA damage, cell-cycle defects characterized by a prolonged S-phase, increased apoptosis and decreased colony forming abilities compared to untreated cells. These data suggest that a combination of a CHK1 and BCL2 inhibitor could be an effective treatment for patients with MSL breast cancer. Several other effective drug combinations were also identified.

There is consensus on the routine use of postmastectomy radiotherapy (PMRT) in patients with four or more positive axillary lymph nodes. However, the benefits of PMRT in patients with T1-2 and 1-3 involved lymph nodes still remain controversial. Data from the Surveillance, Epidemiology, and End Results Program (SEER) of the United States between 2010 and 2012 were used to analyze the outcomes of 675 triple-negative breast cancer (TNBC) patients with T1-2 and 1-3 lymph nodes involved. Those patients were subdivided into radiotherapy (RT) (312) and no-RT groups (363). After a median follow-up time of 37 months, Kaplan-Meier analysis showed that PMRT significantly improved overall survival (OS) but not breast cancer-specific survival (BCSS) in the total cohort of 675 patients (P=0.033 and P=0.063). And it was demonstrated that PMRT were independently associated with increased OS according to univariate and multivariate analyses. However, no significant differences in BCSS or OS were observed between the groups stratified by the number of positive lymph nodes. In conclusion, PMRT significantly improved OS for TNBC patients with T1-2 and 1-3 lymph nodes involved. Additional prospective studies are needed to provide a stronger evidence base for choosing patients for PMRT.

Background: Metastatic triple negative breast cancer (mTNBC) is a heterogeneous disease with poor prognosis. Molecular evolution of TNBC through chemotherapy selection pressure is well recognized but poorly understood. PI3K/AKT/mTOR is one of the most commonly identified oncogenic-driver pathways in breast cancer. The current study is designed to understand the genomic and transcriptomic changes, focusing on the PI3K/AKT/mTOR pathway alterations in paired primary and metastatic TNBCs. Results: Genomic analysis of 7 paired specimens identified 67 known mutations including those from the following signaling pathways: cell cycle, p53, PI3K/AKT/mTOR, RAS/MAPK, and RTK/GF. Principle coordinate analysis (PCoA) identified 4 distinctive molecular groups based on the gene expression patterns of PI3K/AKT/mTOR pathway. Key differentially-expressed genes included AKT3, GSK3B, GNA11, PI3KR1, and GNAQ. Importantly, AKT-targeted therapy showed efficacy in a patient-derived xenograft (PDX) model of TNBC in vivo. Conclusion: Genomic discordance of paired primary and metastatic TNBCs was identified, with significant increase in tumor proliferation pathways seen in metastases. Among the differentially expressed genes, AKT3 can potentially serve as a target for novel combination therapy for treatment of metastatic TNBC. Methods: Paired specimens from 10 patients with TNBCs were identified through an IRB-approved protocol (2002–2015). FoundationOneTM sequencing was performed for genomic profiling, and Affymetrix Human Genechip 2.0st was used for mRNA expression profiling. The similarity among samples was calculated based on Pearson correlation coefficients, which were used to construct hierarchical clustering and heat maps.

Patient-derived xenografts (PDX) are generated in immune deficient mice and demonstrate histologic and molecular features similar to their corresponding human tumors. However, murine tumors (non-human) spontaneously occur in these models. 120 consecutive patients with high-risk primary breast cancer enrolled in the prospective neoadjuvant BEAUTY study had tumor tissue obtained at the time of diagnosis. These tumor cells, including initial tissue and subsequent generations, were injected into either NSG (n = 365) or NOD-SCID (n = 396) female mice. Mice with initial tumor growth sufficient for transfer to the 2nd generation underwent histologic review by pathologists, including Ki67 staining. After passaging the tumors for up to 4 generations, at least one primary mouse tumor was detected from 24 of the 54 PDX-lines, for a frequency of 3.2% (24 mice out of 761 mice), including murine lymphomas (n = 13), mammary tumors (n = 7), osteosarcomas (n = 2), and hemangiosarcomas (n = 2). While true PDX showed scattered strong staining with Ki67, murine tumors were Ki67 negative. No significant differences (p = 0.062) were observed comparing development of murine tumors in NOD-SCID (n = 8) vs NSG mice (n = 16). While PDX are a useful tool in cancer research, there is a potential for spontaneous murine tumors to arise, which could alter results of studies utilizing PDX. Morphologic review by a pathologist, potentially along with Ki67 staining, is necessary to ensure that tumor growth represents the desired PDX prior to use in downstream studies. This study is the first prospective study evaluating the frequency, type, and time frame for development of non-human tumors.

Background: Breast cancer is the most frequent and one of the most fatal malignancies among women. Within the concept of personalized medicine, molecular characterization of tumors is usually performed by analyzing somatic mutations, RNA gene expression signatures or the proteome by mass-spectrometry. Alternatively, the immunological fingerprint of the patients can be analyzed by protein microarrays, which is able to provide another layer of molecular pathological information without invasive intervention. Results: We have investigated the immune signature of breast cancer patients and compared them with healthy controls, using protein microarray-based IgG profiling. The identified differentially reactive antigens (n=517) were further evaluated by means of various pathway analysis tools. Our results indicate that the immune signature of breast cancer patients shows a clear distinction from healthy individuals characterized by differentially reactive antigens involved in known disease relevant signaling pathways, such as VEGF, AKT/PI3K/mTOR or c-KIT, which is in close agreement with the findings from RNA-based expression profiles. Conclusion: Differential antigenic properties between breast cancer patients and healthy individual classes can be defined by serum-IgG profiling on protein microarrays. These immunome profiles provide an additional layer of molecular pathological information, which has the potential to refine and complete the systems biological map of neoplastic disease.