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Purpose: Early evaluation of tumor heterogeneity related to metastasis and outcomes is a major challenge in the management of advanced breast cancer (BCa) in the clinic. In this study, we introduced the value of baseline circulating tumor cells (CTC) and ctDNA for early differentiation of clinical stages, tumor heterogeneity, and prognosis in clinic., Experimental Design: A total of 292 patients with BCa were enrolled in this study, including 254 Stage IV and 38 Stage III patients, and examined the baseline levels of CTCs, CTC-clusters, and plasma ctDNA before initiating therapies. Outcomes including progression-free survival (PFS) and overall survival were evaluated using proportional hazards regression analysis., Results: The baseline CTCs, including HER2+ CTCs, in Stage IV patients were approximately 9.5 times higher than those detected in Stage III patients. Baseline CTC counts with a cutoff of 5 were significantly associated with the prognosis. Within each stage, patients with <5 CTCs had significantly longer PFS. Stage III patients with no CTCs exhibited the longest survival compared with patients with ≥1 CTC. CTC-clusters were only found in Stage IV patients, among whom 15 Stage IV patients with ≥5 CTC-clusters had the worst PFS compared with the 239 Stage IV patients with <5 CTC-clusters. Similar outcomes were observed in 28 out of 254 Stage IV patients who had at least one CTC-cluster detected, as these patients had shorter PFS compared with CTC-cluster negative group. The major differences in ctDNA mutations between patients with Stage III and Stage IV BCa were in PIK3CA and ESR1, which were associated with specific organ metastasis and worse outcomes., Conclusions: Assessing the baseline levels of CTCs, CTC-clusters, and mutational ctDNA profile could reliably aid in differentiation of clinical stage and early prediction of metastasis and outcomes in advanced BCa., (©2024 American Association for Cancer Research.)

N6-methyladenosine (m6A) methylation modification affects the tumorigenesis and metastasis of breast cancer (BC). This study investigated the association between m6A regulator-mediated methylation modification patterns and characterization of the tumour microenvironment in BC, as well as their prognostic importance. Public gene expression data and clinical annotations were collected from The Cancer Genome Atlas (TCGA) database, the Gene Expression Omnibus website and the METABRIC program. We analysed the genetic expression, gene-gene interactions, gene mutations and copy number variations using R software. The data were screened for risk genes using the Cox risk regression model, and we developed an algorithm for risk score and its predictive value. Compared to adjacent normal tissue, we identified 16 differentially expressed m6A regulators in BC, including six writers and 10 readers. Under unsupervised clustering, two distinguished modification patterns were identified, cluster C1 and C2. Compared to m6A cluster C2, cluster C1 was found to be more involved in immune-related pathways, with a relatively higher immune score and stromal score (P < 0.05). Patients were divided into two groups based on their risk scores for survival analysis. The patients in the high-risk score group had significantly worse overall survival than patients in the low-risk score group, (P < 0.0001). The TCGA database validation revealed the same prognostic tendency. In summary, our study showed distinct m6A regulator modification patterns contribute to the immunological heterogeneity and diversity of BC. The development of m6A gene signatures and the m6A score aid in the prognostic prediction of patients with BC., (© 2024 John Wiley & Sons Australia, Ltd.)

The gut microbiota has been demonstrated to be correlated with the clinical phenotypes of diseases, including cancers. However, there are few studies on clinical subtyping based on the gut microbiota, especially in breast cancer (BC) patients. Here, using machine learning methods, we analysed the gut microbiota of BC, colorectal cancer (CRC), and gastric cancer (GC) patients to identify their shared metabolic pathways and the importance of these pathways in cancer development. Based on the gut microbiota-related metabolic pathways, human gene expression profile and patient prognosis, we established a novel BC subtyping system and identified a subtype called "challenging BC". Tumours with this subtype have more genetic mutations and a more complex immune environment than those of other subtypes. A score index was proposed for in-depth analysis and showed a significant negative correlation with patient prognosis. Notably, activation of the TPK1-FOXP3-mediated Hedgehog signalling pathway and TPK1-ITGAE-mediated mTOR signalling pathway was linked to poor prognosis in "challenging BC" patients with high scores, as validated in a patient-derived xenograft (PDX) model. Furthermore, our subtyping system and score index are effective predictors of the response to current neoadjuvant therapy regimens, with the score index significantly negatively correlated with both treatment efficacy and the number of immune cells. Therefore, our findings provide valuable insights into predicting molecular characteristics and treatment responses in "challenging BC" patients., (© 2024. The Author(s).)

Intratumoral hypoxia is a microenvironmental feature that promotes breast cancer progression and is associated with cancer mortality. Plexin B3 (PLXNB3) is highly expressed in estrogen receptor-negative breast cancer, but the underlying mechanisms and consequences have not been thoroughly investigated. Here, we report that PLXNB3 expression is increased in response to hypoxia and that PLXNB3 is a direct target gene of hypoxia-inducible factor 1 (HIF-1) in human breast cancer cells. PLXNB3 expression is correlated with HIF-1α immunohistochemistry, breast cancer grade and stage, and patient mortality. Mechanistically, PLXNB3 is required for hypoxia-induced MET/SRC/focal adhesion kinase (FAK) and MET/SRC/STAT3/NANOG signaling as well as hypoxia-induced breast cancer cell migration, invasion, and cancer stem cell specification. PLXNB3 knockdown impairs tumor formation and lung metastasis in orthotopic breast cancer mouse models., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Hypoxia is a key characteristic of the breast cancer microenvironment that promotes expression of the transcriptional activator hypoxia-inducible factor 1 (HIF-1) and is associated with poor patient outcome. HIF-1 increases the expression or activity of stem cell pluripotency factors, which control breast cancer stem cell (BCSC) specification and are required for cancer metastasis. Here, we identify nuclear prelamin A recognition factor ( NARF ) as a hypoxia-inducible, HIF-1 target gene in human breast cancer cells. NARF functions as an essential coactivator by recruiting the histone demethylase KDM6A to OCT4 bound to genes encoding the pluripotency factors NANOG, KLF4, and SOX2, leading to demethylation of histone H3 trimethylated at lysine-27 (H3K27me3), thereby increasing the expression of NANOG, KLF4, and SOX2, which, together with OCT4, mediate BCSC specification. Knockdown of NARF significantly decreased the BCSC population in vitro and markedly impaired tumor initiation capacity and lung metastasis in orthotopic mouse models.

O-linked β-N-acetyl glucosamine (O-GlcNAc) is attached to proteins under glucose-replete conditions; this posttranslational modification results in molecular and physiological changes that affect cell fate. Here we show that posttranslational modification of serine/arginine-rich protein kinase 2 (SRPK2) by O-GlcNAc regulates de novo lipogenesis by regulating pre-mRNA splicing. We found that O-GlcNAc transferase O-GlcNAcylated SRPK2 at a nuclear localization signal (NLS), which triggers binding of SRPK2 to importin α. Consequently, O-GlcNAcylated SRPK2 was imported into the nucleus, where it phosphorylated serine/arginine-rich proteins and promoted splicing of lipogenic pre-mRNAs. We determined that protein nuclear import by O-GlcNAcylation-dependent binding of cargo protein to importin α might be a general mechanism in cells. This work reveals a role of O-GlcNAc in posttranscriptional regulation of de novo lipogenesis, and our findings indicate that importin α is a "reader" of an O-GlcNAcylated NLS., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: The background parenchymal enhancement at breast magnetic resonance imaging use to predict breast cancer attracts many searchers to draw a possible relationship. However, the results of their relationships were conflicting. This meta-analysis was performed to assess breast cancer frequency associations with background parenchymal enhancement., Methods: A systematic literature search up to January 2020 was performed to detect studies recording associations between breast cancer frequency and background parenchymal enhancement. We found thirteen studies including 13,788 women at the start with 4046 breast cancer. We calculated the odds ratio (OR) and the 95% confidence intervals (CIs) between breast cancer frequency and background parenchymal enhancement by the dichotomous technique with a random or fixed-effect model., Results: Women with minimal or mild background parenchymal enhancement at breast magnetic resonance imaging did not have any risk of breast cancer compared to control women (OR, 1.20; 95% CI 0.54-2.67). However, high background parenchymal enhancement at breast magnetic resonance imaging (OR, 2.66; 95% CI 1.36-5.19) and moderate (OR, 2.51; 95% CI 1.49-4.21) was associated with a significantly higher rate of breast cancer frequency compared to control women., Conclusions: Our meta-analysis showed that the women with high and moderate background parenchymal enhancement at breast magnetic resonance imaging have higher risks, up to 2.66 fold, of breast cancer. We suggest that women with high or moderate background parenchymal enhancement at breast magnetic resonance imaging to be scheduled for more frequent follow-up and screening for breast cancer to avoid any complications.

Increasing evidence supports that long noncoding RNAs (lncRNAs) act as master regulators involved in tumorigenesis and development at the N6-methyladenine (m 6 A) epigenetic modification level. However, the underlying regulatory mechanism in breast cancer (BRCA) remains elusive. Here, we unveil that LINC00942 (LNC942) exerts its functions as an oncogene in promoting METTL14-mediated m 6 A methylation and regulating the expression and stability of its target genes CXCR4 and CYP1B1 in BRCA initiation and progression. Specifically, LNC942 and METTL14 were significantly upregulated accompanied with the upregulation of m 6 A levels in BRCA cells and our included BRCA cohorts (n = 150). Functionally, LNC942 elicits potent oncogenic effects on promoting cell proliferation and colony formation and inhibiting cell apoptosis, subsequently elevating METTL14-mediated m 6 A methylation levels and its associated mRNA stability and protein expression of CXCR4 and CYP1B1 in BRCA cells. Mechanistically, LNC942 directly recruits METTL14 protein by harboring the specific recognize sequence (+176-+265), thereby stabilized the expression of downstream targets of LNC942 including CXCR4 and CYP1B1 through posttranscriptional m 6 A methylation modification in vitro and in vivo. Therefore, our results uncover a novel LNC942-METTL14-CXCR4/CYP1B1 signaling axis, which provides new targets and crosstalk m 6 A epigenetic modification mechanism for BRCA prevention and treatment.

Background: To evaluate the feasibility and safety of ultrasound-guided totally implantable venous access port (TIVAP) implantation via the right innominate vein in patients with breast cancer., Methods: Sixty-seven breast cancer patients underwent ultrasound-guided implantation of TIVAPs via the right innominate vein for administration of chemotherapy. Clinical data including technical success, success rate for the first attempt, periprocedural, and postoperative complications were recorded and retrospectively studied., Results: All patients underwent successful surgery. The success rate of the first attempt was 95.52% (64/67). The operation time was 28 to 45 min, with an average of 36 ± 6 min. Periprocedural complications included artery punctures in 1 (1.50%, 1/67) patient. Prior to this study, the mean TIVAP time was 257 ± 3 days (range 41 to 705 days). The rate of postoperative complications was 4.48% (3/67), including catheter-related infections in 1 case and fibrin sheath formation in 2 cases. Up to the present study, three people had unplanned port withdrawal due to complications, and the TIVAPs for 25 patients were still in normal use., Conclusions: The success rate of ultrasound-guided TIVAPs via the right innominate vein is high with low complications, thus safe and feasible. This technique can provide a new option for chemotherapy of breast cancer patients.

Cancer stem cells (CSCs) are considered to be tumor‑initiating cells, responsible for tumor invasive growth and dissemination to distant organ sites. Typically, radiation treatment and chemotherapy should target CSCs. However, current research investigating CSCs is impeded by the difficulty of isolating pure CSCs and maintaining them in vitro. In the present study, the synergistic inhibition of glycogen synthase kinase 3 and mitogen‑activated protein kinase kinase using small molecules, CHIR99021 and PD184352, efficiently generated CSCs from immortalized human mammary epithelial cells (HMLEs) and resulted in the acquisition of mesenchymal traits and the expression of epithelial‑mesenchymal transition markers. The cell proliferation, invasion and migration of HMLE cells were significantly promoted by CHIR99021 and PD184352 (P<0.05). Furthermore, the cell cycle was shifted from the G0/G1 phase to the G2/M phase, and the apoptotic rate was suppressed in HMLE cells following treatment with CHIR99021 and PD184352. Compared with control group, the stimulated cells exhibited an increased ability to form mammospheres and regenerate a tumor. In addition to these properties, the induced cells also exhibited notable chemotherapy resistance. In vivo, the treatment of cells with CHIR99021 and PD184352 promoted the growth of HMLE‑engrafted tumor types. These results provide a practical strategy for the generation of CSCs using small molecules in vitro, which provides a cell resource that may be used for drug screening. Additionally, the present results additionally highlighted the synergistic functions of Wnt and mitogen‑activated protein kinase kinase signaling pathways in tumorigenesis.

Estrogen receptor α (ERα) is a crucial target for ERα positive breast cancer treatment. Previous drug discovery efforts were focused on developing inhibitors that targeted the canonical ligand binding pockets of the ligand binding domain (LBD) of ERα. However, significant percentage of patients developed cancer relapse with drug-resistance. ERα peptidomimetic modulators have been considered as promising treatments for drug resistant breast cancers as they are targeting ERα-coactivator interacting interface instead of the ligand binding pocket of ERα. Herein, we reviewed the recent development of ERα peptidomimetics antagonists., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

This research aims to address the problem of discriminating benign cysts from malignant masses in breast ultrasound (BUS) images based on Convolutional Neural Networks (CNNs). The biopsy-proven benchmarking dataset was built from 1422 patient cases containing a total of 2058 breast ultrasound masses, comprising 1370 benign and 688 malignant lesions. Three transferred models, InceptionV3, ResNet50, and Xception, a CNN model with three convolutional layers (CNN3), and traditional machine learning-based model with hand-crafted features were developed for differentiating benign and malignant tumors from BUS data. Cross-validation results have demonstrated that the transfer learning method outperformed the traditional machine learning model and the CNN3 model, where the transferred InceptionV3 achieved the best performance with an accuracy of 85.13% and an AUC of 0.91. Moreover, classification models based on deep features extracted from the transferred models were also built, where the model with combined features extracted from all three transferred models achieved the best performance with an accuracy of 89.44% and an AUC of 0.93 on an independent test set.

Due to the low contrast and ambiguous boundaries of the tumors in breast ultrasound (BUS) images, it is still a challenging task to automatically segment the breast tumors from the ultrasound. In this paper, we proposed a novel computational framework that can detect and segment breast lesions fully automatic in the whole ultrasound images. This framework includes several key components: pre-processing, contour initialization, and tumor segmentation. In the pre-processing step, we applied non-local low-rank (NLLR) filter to reduce the speckle noise. In contour initialization step, we cascaded a two-step Otsu-based adaptive thresholding (OBAT) algorithm with morphologic operations to effectively locate the tumor regions and initialize the tumor contours. Finally, given the initial tumor contours, the improved Chan-Vese model based on the ratio of exponentially weighted averages (CV-ROEWA) method was utilized. This pipeline was tested on a set of 61 breast ultrasound (BUS) images with diagnosed tumors. The experimental results in clinical ultrasound images prove the high accuracy and robustness of the proposed framework, indicating its potential applications in clinical practice. Graphical abstract ᅟ.

Objectives: To compare the diagnostic efficacies of B-mode ultrasound (US), strain elastography (SE), contrast-enhanced ultrasound (CEUS) and the combination of these modalities for breast lesions <1 cm in size., Methods: Between January 2013 and October 2015, 203 inpatients with 209 sub-centimetre breast lesions categorised as BI-RADS-US (Breast Imaging Reporting and Data System for Ultrasound) 3-5 were included. US, SE and CEUS were performed to evaluate each lesion. The diagnostic performances of different ultrasonic modalities were compared. The diagnostic efficacies of BI-RADS-US and our re-rating systems were also compared. The pathology findings were used as the reference standard., Results: The specificities of US, SE and CEUS for tumour differentiation were 17.4 %, 56.2 % and 86.0 %, respectively (P < 0.05); and the sensitivities were 100 %, 93.2 % and 93.2 % for US, SE and CEUS, respectively (P < 0.05). The area under the curve (AUC) of the receiver operating characteristic (ROC) curve was 0.867 for original BI-RADS-US, 0.882 for BI-RADS-US combined with only SE, 0.953 for BI-RADS-US combined with only CEUS and 0.924 for BI-RADS-US combined with both SE and CEUS. The best combination was BI-RADS-US combined with only CEUS., Conclusions: Evaluating sub-centimetre breast lesions with SE and CEUS could increase the diagnostic specificity while retaining high sensitivity compared with B-mode ultrasound., Key Points: • Evaluating breast lesions with SE and CEUS could increase the diagnostic specificity • SE and CEUS offer alternatives to biopsy and possibly allow shorter-interval follow-ups • BI-RADS-US combined with CEUS exhibited the best diagnostic performance.

Background: Pregnancy-associated breast cancers (PABCs), especially those diagnosed after childbirth, are often aggressive, with a poor prognosis. Factors influencing PABC are largely unknown. Micro(mi)RNAs are present in many human body fluids and shown to influence cancer development and/or growth., Subjects and Methods: In six nursing mothers, we determined if breast cancer-associated miRNAs were (1) detectable in human breast milk and (2) if detectable, their relative expression in milk fractions compared to matched serum. We evaluated by quantitative PCR the expression of 11 cancer-associated miRNAs (10a-5p, 16, 21, 100, 140, 145, 155, 181, 199, 205, 212) in breast milk cells, fat and supernatant (skim milk), and matched serum., Results: miRNA expression was detectable in all samples. For 10/11 miRNAs, mean relative expression compared to control (ΔCt) values was lowest in milk cells, the exception being miR205. Relative concentration was highest in the skim fraction of milk for all miRNAs. Expression was higher in skim milk than matched serum for 7/11 miRNAs and in serum for 4/11 miRNAs. miR205 expression was higher in all milk fractions than in matched serum., Conclusion: In conclusion, the expression of breast cancer-associated miRNAs is detectable in human breast milk and serum samples. The concentration is highest in skim milk, but is also detectable in milk fat and milk cells.

Background/aim: We reported that vitamin D3 increased transforming growth factor (TGF)β2 and decreased prostaglandin (PG)E 2 in the breast of normal-risk women, suggesting a protective effect. We determined if the findings held for higher risk women., Patients and Methods: Seventy-eight women received daily for one month/menstrual cycle: placebo, 400 international units (IU) vitamin D3, 2,000 IU vitamin D3 or 2,000 IU vitamin D3/400 mg celecoxib. Nipple aspirate fluid (NAF) and/or serum were analyzed for PGE 2 , TGFβ1,-2, vitaminD binding protein (DBP) 25(OH)D; and plasma for celecoxib., Results: 25(OH)D increased (p<0.001) in women receiving 2,000 IU vitamin D3. Two thousand IU vitamin D3 lowered NAF PGE 2 in normal-risk women (p=0.029), whereas 2,000 IU vitamin D3/celecoxib lowered NAF PGE 2 in high-risk women (p=0.063). Serum TGFβ1 was influenced by treatment (p=0.011). NAF TGFβ2 increase correlated with increase in 25(OH)D. DBP serum levels were higher than matched NAF, regardless of race, and did not appreciably change with treatment., Conclusion: Vitamin D3 influenced TGFβ1 and -β2 expression. PGE 2 response to vitamin D3 treatment was influenced by a participant's breast cancer risk. The implications of these observations regarding breast cancer risk should be further evaluated., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Objective: To evaluate the diagnostic performance of superb microvascular imaging (SMI) in breast lesions, comparing with contrast-enhanced ultrasonographic microvascular imaging (MVI)., Methods: From April to November 2015, 132 patients (with 132 breast lesions) were enrolled in the retrospective study. All lesions were evaluated with colour Doppler flow imaging (CDFI), colour SMI (cSMI), monochrome SMI (mSMI) and contrast-enhanced ultrasonographic MVI. Receiver-operating characteristic curve analysis was performed to compare the diagnostic performance of SMI and MVI for discrimination between benign and malignant breast lesions., Results: Histological analysis showed 58 malignant and 74 benign lesions. mSMI was more sensitive in detecting blood flow signals in breast lesions than CDFI (p < 0.001) and cSMI (p < 0.001). Differences of vessels inside breast lesions and morphologic features of vessels between benign and malignant lesions were statistically significant on mSMI (p < 0.001). Using root hair-like and crab claw-like patterns as the criteria for malignant lesions, the sensitivity, specificity and accuracy for differentiation based on the microvascular architecture patterns were 77.6, 90.5 and 84.8% for mSMI and 89.6, 87.8 and 88.6% for MVI. Areas under curve of mSMI and MVI were not significantly different (p = 0.129)., Conclusion: mSMI can increase blood flow detection and depict the microvascular architecture of breast lesions. The diagnostic performance of mSMI was not significantly different from MVI. SMI has potential in the differential diagnosis of breast lesions., Advances in Knowledge: mSMI is a non-invasive technique for vascularity evaluation of breast tumours and it is beneficial for breast tumour differentiation.

Breast cancer is a heterogeneous disease associated with diverse clinical, biological and molecular features, presenting huge challenges for prognosis and treatment. Here we found that perilipin-1 (PLIN1) mRNA expression is significantly downregulated in human breast cancer. Kaplan-Meier analysis indicated that patients presenting with reduced PLIN1 expression exhibited poorer overall metastatic relapse-free survival (p = 0.03). Further Cox proportional hazard models analysis revealed that the reduced expression of PLIN1 is an independent predictor of overall survival in estrogen receptor positive (p < 0.0001, HR = 0.87, 95% CI = 0.81-0.92, N = 3,600) and luminal A-subtype (p = 0.02, HR = 0.88, 95% CI = 0.78-0.98, N = 1,469) breast cancer patients. We also demonstrated that the exogenous expression of PLIN1 in human breast cancer MCF-7 and MDA-MB-231 cells significantly inhibits cell proliferation, migration, invasion and in vivo tumorigenesis in mice. Together, these data provide novel insights into a prognostic significance of PLIN1 in human breast cancer and reveal a potentially new gene therapy target for breast cancer.

Chemoresistance in breast cancer has been of great interest in past studies. However, the development of rational therapeutic strategies targeting chemoresistant cells is still a challenge in clinical oncology. By integrating data from global differences of gene expression and phospho-receptor tyrosine kinases between sensitive parental cells (MCF-7) and doxorubicin-resistant cells (MCF-7/ADR), we identified Axl as a potential target for chemoresistance and metastasis in multidrug resistant breast cancer cells. We analyzed Axl expression in 57 breast cancer cell lines and detected a dramatic increase in its expression level in mesenchymal breast cancer cell lines. Axl silencing suppressed invasive and metastatic potentials of chemoresistant breast cancer cells as well as increased elimination of cancer cells when combined with doxorubicin. Furthermore, in preclinical assays, an Axl inhibitor R428 showed increased cell death upon doxorubicin treatment. Additionally, using phospho-kinase array based proteomic analysis, we identified that Akt/GSK-3β/β-catenin cascade was responsible for Axl-induced cell invasion. Nuclear translocation of β-catenin then induced transcriptional upregulation of ZEB1, which in turn regulated DNA damage repair and doxorubicin-resistance in breast cancer cells. Most importantly, Axl was correlated with its downstream targets in tumor samples and was associated with poor prognosis in breast cancer patients. These results demonstrate that Gas6/Axl axis confers aggressiveness in breast cancer and may represent a therapeutic target for chemoresistance and metastasis.

Background: Women diagnosed with pregnancy-associated breast cancer postpartum have a worse prognosis, stage for stage, than other women with breast cancer. The time of breast involution is tumor promotional. The extracellular matrix protein tenascin-C is upregulated during involution in animal models and promotes breast cancer progression. It interacts with transforming growth factor (TGF)β, which also is involved in breast involution and breast cancer progression. Little is known about the expression of tenascin-C during human breast involution, nor its relationship to TGFβ. The purpose of this study was to investigate the expression of tenascin-C throughout lactation, as well as its relationship to TGFβ1 and TGFβ2., Material and Methodology: Three milk samples from 25 lactating women (transitional, whole, and wean) were collected, separated into components (cells, fat, and skim), and the skim fraction analyzed for total protein, tenascin-C, TGFβ1, and TGFβ2. Tenascin-C, TGFβ1, and TGFβ2 were detectable in all milk samples., Results: Highest tenascin-C levels on average were found in whole milk, whereas highest mean TGFβ1 and TGFβ2 levels were in wean milk. Wean samples on average had higher levels of both TGFβ1 (26%) and TGFβ2 (>500%) than matched transitional milk samples. Tenascin-C levels in wean milk were associated with nursing length (p = 0.048). Combining all three milk collection time points, tenascin-C exhibited a weak inverse correlation with TGFβ1 and TGFβ2 (p < 0.1). The inverse correlation at the wean time point was stronger for TGFβ2 than -1 (-0.37 versus -0.25). Tenascin-C, a protein known to promote breast cancer progression, is expressed throughout lactation., Conclusion: The inverse correlation with TGFβ2 in wean milk suggests a possible interaction during breast involution, which should be further investigated.

Objective: Although positive association between abortion and breast cancer was frequently reported from case-control studies, results from prospective studies were still unclear. This study aimed to evaluate this association based on prospective studies., Methods: PubMed, ISI Web of Knowledge and Embase were systematically searched for prospective studies on the association between abortion and breast cancer up to April 2014, supplemented by manual searches on the references. Two reviewers independently conducted the literature search, study selection, data extraction, and quality assessment of included studies. Random effects models were used to estimate the combined relative risks (RRs) and corresponding 95 % confidence intervals (95 % CIs)., Results: Fifteen prospective studies [14 focused on induced abortion (IA), and 12 focused on spontaneous abortion (SA)] were included in the final analysis. The combined RRs (95 % CIs) of breast cancer risk were 1.00 (0.94-1.05) [1.00 (0.92-1.08) for cumulative-incidence data and 1.00 (0.94-1.05) for incidence-rate data] for IA, and 1.02 (0.95-1.09) [1.06 (0.96-1.16) for cumulative-incidence data and 1.01 (0.92-1.09) for incidence-rate data] for SA, respectively. Non-significant associations of breast cancer with IA and SA were also found among nulliparous women, women with abortion before or after the first full-term pregnancy, women with one or ≥ 2 abortions, and women with first abortion after 30 years old., Conclusion: The current prospective evidences are not sufficient to support the positive association between abortion (including IA and SA) and breast cancer risk.

Cancer immunotherapy has shown great promise as a new standard cancer therapeutic modality. However, the response rates are limited for current approach that depends on enhancing spontaneous antitumor immune responses. Therefore, increasing tumor immunogenicity by expressing appropriate cytokines should further improve the current immunotherapy. IL-33 is a member of the IL-1 family of cytokines and is released by necrotic epithelial cells or activated innate immune cells and is thus considered a "danger" signal. The role of IL-33 in promoting type 2 immune responses and tissue inflammation has been well established. However, whether IL-33 drives antitumor immune responses is controversial. Our previous work established that IL-33 promoted the function of CD8(+) T cells. In this study, we showed that the expression of IL-33 in two types of cancer cells potently inhibited tumor growth and metastasis. Mechanistically, IL-33 increased numbers and IFN-γ production by CD8(+) T and NK cells in tumor tissues, thereby inducing a tumor microenvironment favoring tumor eradication. Importantly, IL-33 greatly increased tumor Ag-specific CD8(+) T cells. Furthermore, both NK and CD8(+) T cells were required for the antitumor effect of IL-33. Moreover, depletion of regulatory T cells worked synergistically with IL-33 expression for tumor elimination. Our studies established "alarmin" IL-33 as a promising new cytokine for tumor immunotherapy through promoting cancer-eradicating type 1 immune responses., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Background: Pregnancy-associated breast cancer (PABC) is aggressive and difficult to diagnose. High intake of most types of dietary fat is thought to increase breast cancer risk; however, results in humans supporting this premise remain equivocal. Fatty acid (FA) concentrations in the body comprise both dietary intake and endogenous FA production. Most assessments of FA levels have been performed on blood, with little information on the effect of FA levels in breast milk on PABC risk., Objective: This study aimed to determine if FA concentrations in the milk from women diagnosed with breast cancer while nursing were different in the cancer-containing breast and opposite breast., Methods: We quantified 16 long-chain FA and soluble FA synthase (sFAS) enzyme levels from 4 women diagnosed with PABC, comparing results from the cancer-containing breast to those from the normal breast., Results: Fatty acid concentrations consistently exceeded and trended higher (P < .10) in each cancer-containing breast for 20:4n-6 (arachidonic acid [AA]), 20:5n-3 (eicosapentaenoic acid [EPA]), and 22:5n-6 (docosapentaenoic acid [DPA]). Soluble FA synthase levels were similar in the cancer-containing and normal breasts., Conclusion: Breast milk concentrations of AA, EPA, and DPA increased in the cancer-containing breast of women with PABC. This increase was not associated with higher sFAS levels., (© The Author(s) 2014.)

We reported that resveratrol decreased DNA methyltransferase (DNMT) 1 and 3b expression in vitro and demethylates tumor suppressor RASSF-1a in women at increased breast cancer risk. We investigated the effects of resveratrol on DNMT and miRNA expression in normal and tumor mammary tissue in a rodent model of estrogen dependent mammary carcinoma. Eighty-nine female ACI rats received estradiol plus: low dose (lo) resveratrol, high dose (hi) resveratrol, 5-aza-2-deoxycytidine (Aza), a known inhibitor of DNMTs, or control (no additional treatment). After 21 wk of treatment, animals were sacrificed and mammary glands harvested. Matched tumor/normal tissues were available from 36 rats. DMNT3b (but not DNMT1) differed in tumor vs. normal tissue after lo (P = .04) and hi (P = .007) resveratrol and Aza treatment. With hi resveratrol, DNMT3b decreased in tumor but increased normal tissue. Hi resveratrol increased miR21, -129, -204, and -489 >twofold in tumor and decreased the same miRs in normal tissue 10-50% compared to control. There was an inverse association between DNMT3b and miR129, -204, and -489 in normal and/or tumor tissue. Treatment with resveratrol differentially influences tumor vs. normal tissue DNMT3b and miRNA expression. This mechanism of action of resveratrol to influence mammary carcinogenesis warrants further investigation.

A main obstacle to overcome during the treatment of tumors is drug resistance to chemotherapy; emerging studies indicate that a key factor contributing to this problem is the acidic tumor microenvironment. Here, we found that LASS2 expression was significantly lower in drug-resistant Michigan Cancer Foundation-7/adriamycin (MCF-7/ADR) human breast cancer cells than the drug-sensitive MCF-7 cells, and low expression of LASS2 was associated with poor prognosis in patients with breast cancer. Our results showed that the overexpression of LASS2 in MCF-7/ADR cells increased the chemosensitivity to multiple chemotherapeutic agents, including doxorubicin (Dox), whereas LASS2 knockdown in MCF-7 cells decreased the chemosensitivity. Cell-cycle analysis revealed a corresponding increase in apoptosis in the LASS2-overexpressing cells following Dox exposure, showing that the overexpression of LASS2 increased the susceptibility to Dox cytotoxicity. This effect was mediated by a significant increase in pHe (extracellular pH) and lysosomal pH, and more Dox entered the cells and stayed in the nuclei of cells. In nude mice, the combination of LASS2 overexpression and Dox significantly inhibited the growth of xenografts. Our findings suggest that LASS2 is involved in chemotherapeutic outcomes and low LASS2 expression may predict chemoresistance.

Breast cancer that develops during or shortly after pregnancy is frequently more aggressive than cancer diagnosed at other times in a woman's life. To better understand the patterns of cancer-related protein expression in the breasts of lactating women, we determined the differences in total and individual protein expression in milk based on (a) three time points during lactation (early, mid, and late), (b) length of lactation, and (c) parity. Breastmilk was collected from 72 healthy lactating women within 10 days of starting lactation (transitional [T]), 2 months after lactation started, and during breast weaning (W). Sixteen proteins whose expression is altered in breast cancer (11 kallikreins [KLKs], basic fibroblast growth factor [bFGF], YKL-40, neutrophil gelatinase-associated lipocalin, and transforming growth factor [TGF] β1 and β2) were evaluated. The concentration of total milk protein decreased over time (p<0.01 at 2 months and W compared with T). After we controlled for total protein, KLK6 and TGFβ2 significantly increased, and bFGF decreased from T to W. Neither length of nursing nor parity significantly influenced individual protein expression at the W time point. On the other hand, length of nursing did influence the difference in KLK6, -7, and -8 expression between the W and T time points. Total milk protein concentration is lower in the mid and late phases of nursing. Biomarker differences between T and W milk samples in KLK6, TGFβ2, and bFGF are consistent with a protective effect of nursing.

Background: Breast cancer risk increases during pregnancy and remains elevated for a number of years thereafter. Cancer-associated proteins that are secreted into breast milk may provide a means to detect cancer in the lactating breast or to assess future breast cancer risk., Objective: To determine whether proteins linked to breast cancer would be differentially expressed in matched (both breasts from each participant) human milk samples collected from women with unilateral breast cancer., Methods: Five cancer-associated proteins (basic fibroblast growth factor [bFGF], YKL-40, neutrophil gelatinase-associated lipocalin, and transforming growth factor β1 and β2) were analyzed in milk provided by 5 lactating women, 4 of whom were known to have cancer in 1 breast (and the opposite breast clinically disease free) at the time of milk collection and 1 who developed breast cancer 2 years after milk collection., Results: Expression was significantly higher for TGFβ2 (P = .03) and bFGF (P =.03) in the breasts with cancer., Conclusion: These proteins may play a role in assessing a woman's risk of pregnancy-associated breast cancer. Because of variable protein concentration among patients and the limited sample size, the results are considered preliminary.

Trans-resveratrol, present in high concentration in the skin of red grapes and red wine, has a dose-dependent antiproliferative effect in vitro, prevents the formation of mammary tumors, and has been touted as a chemopreventive agent. Based upon in vitro studies demonstrating that trans-resveratrol downregulates the expression of 1) DNA methyltransferases and 2) the cancer promoting prostaglandin (PG)E(2), we determined if trans-resveratrol had a dose-related effect on DNA methylation and prostaglandin expression in humans. Thirty-nine adult women at increased breast cancer risk were randomized in double-blind fashion to placebo, 5 or 50 mg trans-resveratrol twice daily for 12 wk. Methylation assessment of 4 cancer-related genes (p16, RASSF-1α, APC, CCND2) was performed on mammary ductoscopy specimens. The predominant resveratrol species in serum was the glucuronide metabolite. Total trans-resveratrol and glucuronide metabolite serum levels increased after consuming both trans-resveratrol doses (P < .001 for both). RASSF-1α methylation decreased with increasing levels of serum trans-resveratrol (P = .047). The change in RASSF-1α methylation was directly related to the change in PGE(2) (P = .045). This work provides novel insights into the effects of trans-resveratrol on the breast of women at increased breast cancer risk, including a decrease in methylation of the tumor suppressor gene RASSF-1α. Because of the limited sample size, our findings should be validated in a larger study.

Background: First full term pregnancy (FFTP) completed at a young age has been linked to low long term breast cancer risk, whereas late FFTP pregnancy age confers high long term risk, compared to nulliparity. Our hypothesis was that proteins linked to breast cancer would be differentially expressed in human milk collected at three time points during lactation based on age at FFTP., Methods: We analyzed breast milk from 72 lactating women. Samples were collected within 10 days of the onset of lactation (baseline-BL), two months after lactation started and during breast weaning (W). We measured 16 proteins (11 kallikreins (KLKs), basic fibroblast growth factor, YKL-40, neutrophil gelatinase-associated lipocalin and transforming growth factor (TGF) β-1 and -2) associated with breast cancer, most known to be secreted into milk., Results: During lactation there was a significant change in the expression of 14 proteins in women < 26 years old and 9 proteins in women > = 26 at FFTP. The most significant (p < .001) changes from BL to W in women divided by FFTP age (< 26 vs. > = 26) were in KLK3,6, 8, and TGFβ2 in women < 26; and KLK6, 8, and TGFβ2 in women > = 26. There was a significant increase (p = .022) in KLK8 expression from BL to W depending on FFTP age. Examination of DNA methylation in the promoter region of KLK6 revealed high levels of methylation that did not explain the observed changes in protein levels. On the other hand, KLK6 and TGFβ1 expression were significantly associated (r2 = .43, p = .0050)., Conclusions: The expression profile of milk proteins linked to breast cancer is influenced by age at FFTP. These proteins may play a role in future cancer risk.

Background: Herein we present the results of two related investigations. The first study determined if concentrations in breast nipple discharge (ND) of two proteins (urinary plasminogen activator, uPA and its inhibitor, PAI-1) predicted the presence of breast atypia and cancer in pre- and/or postmenopausal women requiring surgery because of a suspicious breast lesion. The second study assessed if these proteins increased the predictive ability of a carbohydrate (Thomsen Friedenreich, TF) which we previously demonstrated predicted the presence of disease in postmenopausal women requiring surgery., Methods: In the first study we prospectively enrolled 79 participants from whom we collected ND, measured uPA and PAI-1 and correlated expression with pathologic findings. In the second study we analyzed 35 (uPA and PAI-1 in 24, uPA in an additional 11) ND samples collected from different participants requiring breast surgery, all of whom also had TF results., Results: uPA expression was higher in pre- and PAI-1 in postmenopausal women with 1) cancer (DCIS or invasive) vs. either no cancer (atypia or benign pathology, p = .018 and .025, respectively), or benign pathology (p = .017 and .033, respectively); and 2) abnormal (atypia or cancer) versus benign pathology (p = .018 and .052, respectively). High uPA and PAI-1 concentrations and age were independent predictors of disease in premenopausal women, with an area under the curve (AUC) of 83-87% when comparing diseased vs. benign pathology. uPA, TF, and age correctly classified 35 pre- and postmenopausal women as having disease or not 84-91% of the time, whereas combining uPA+PAI-1+TF correctly classified 24 women 97-100% of the time., Conclusions: uPA and PAI-1 concentrations in ND were higher in women with atypia and cancer compared to women with benign disease. Combining uPA, PAI-1 and TF in the assessment of women requiring diagnostic breast surgery maximized disease prediction. The assessment of these markers may prove useful in early breast cancer detection.

Background and Aim: We investigated the prognostic importance of dickkopf-1(DKK1) and beta-catenin expression in triple negative breast cancers., Methods: The expression of DKK1 and beta-catenin was evaluated in breast cell lines using RT-PCR and western blot. Immunohistochemistry was used to characterize the expression pattern of DKK1 and beta-catenin in 85 triple negative breast cancers and prognostic significance was assessed by Kaplan-Meier analysis and Cox proportional hazards regression modeling., Results: The expression of DKK1 was confirmed in hormone-resistant breast cell lines MDA-MB-231, MDA-MB-231-HM and MDA-MB-435. Expression of DKK1 in triple negative breast cancers correlated with cytoplasmic/nuclear beta-catenin (p = 0.000). Elevated expression of DKK1 and cytoplasmic/nuclear beta-catenin in triple negative cancers indicate poor outcome of patients. DKK1 was also a prognostic factor for patients with earlier stage or no lymph node metastasis., Conclusion: DKK1 together with beta-catenin might be important prognostic factors in triple negative breast carcinoma. DKK1 might be a valuable biomarker in predicting the prognosis of patients with earlier stage or no lymph node metastasis. It is possible that through further understanding of the role of Wnt/beta-catenin pathway activation, beta-catenin would be a potential therapeutic target for the triple negative breast cancer.

The assessment of DNA had demonstrated altered methylation in malignant compared to benign breast tissue. The purpose of our study was to (i) confirm the predictive ability of methylation assessment in breast tissue, and (ii) use the genes found to be cancer predictive in tissue to evaluate the diagnostic potential of hypermethylation assessment in nipple aspirate fluid (NAF) and mammary ductoscopic (MD) samples. Quantitative methylation specific (qMS)-PCR was conducted on three specimen sets: 44 malignant (CA) and 34 normal (NL) tissue specimens, 18 matched CA, adjacent normal (ANL) tissue and NAF specimens, and 119 MD specimens. Training and validation tissue sets were analyzed to determine the optimal group of cancer predictive genes for NAF and MD analysis. NAF and MD cytologic review were also performed. Methylation of CCND-2, p16, RAR-beta and RASSF-1a was significantly more prevalent in tumor than in normal tissue specimens. Receiver operating characteristic curve analysis demonstrated an area under the curve of 0.96. For the 18 matched CA, ANL and NAF specimens, the four predictive genes identified in cancer tissue contained increased methylation in CA vs. ANL tissue; NAF samples had higher methylation than ANL specimens. Methylation frequency was higher in MD specimens from breasts with cancer than benign samples for p16 and RASSF-1a. In summary, i) routine quantitative DNA methylation assessment in NAF and MD samples is possible, and ii) genes hypermethylated in malignant breast tissue are also altered in matched NAF and in MD samples, and may be useful to assist in early breast cancer detection.

One of the mechanisms of multiple drug resistance (MDR) is inappropriate sequestration of basic chemotherapeutic agents in acidic endo-lysosomes of cells. The protonation, sequestration, and secretion (PSS) model indicates that drug distribution can be affected by intracellular pH such as lysosomal pH. The vacuolar-H(+)-ATPase (V-ATPase) plays an important role in regulation of intracellular pH by pumping protons into acidic endosomes via an ATP-driven process. In this study, ATP6L, the 16kDa subunit of V-ATPase, was knocked-down by anti-ATP6L small interfering RNA (siRNA) to study the effect on chemosensitivity in the human drug-resistant breast cancer cells MCF-7/ADR. Introduction of anti-ATP6L small interfering RNA duplex into drug-resistant cancer cells significantly inhibited the expression of ATP6L mRNA and protein, as detected by qRT-PCR and Western blot. Inhibition of ATP6L expression by siRNA in MCF-7/ADR sensitized the cells to the cytotoxicity of basic chemotherapeutic agents like doxorobicin, 5-fluorourocil and vincristine. This effect was mediated by a significant increase in lysosomal pH and retention of anticancer drugs into nuclei of cells. These results support the role of tumor acidity in resistance to chemotherapy and provide a rationale for the use of tumor pH modifier agents as coadjuvants in novel anticancer therapies.

Background: While increased urokinase-type plasminogen activator (uPA) expression in breast cancer tissue is directly associated with poor prognosis, recent evidence suggests that uPA overexpression may suppress tumor growth and prolong survival. Celecoxib has been shown to have antiangiogenic and antiproliferative properties. We sought to determine if uPA, PA inhibitor (PAI)-1 and prostaglandin (PG)E2 expression in nipple aspirate fluid (NAF) and uPA and PGE2 expression in plasma were altered by celecoxib dose and concentration in women at increased breast cancer risk., Methods: NAF and plasma samples were collected in women at increased breast cancer risk before and 2 weeks after taking celecoxib 200 or 400 mg twice daily (bid). uPA, PAI-1 and PGE2 were measured before and after intervention., Results: Celecoxib concentrations trended higher in women taking 400 mg (median 1025.0 ng/mL) compared to 200 mg bid (median 227.3 ng/mL), and in post- (534.6 ng/mL) compared to premenopausal (227.3 ng/mL) women. In postmenopausal women treated with the higher (400 mg bid) celecoxib dose, uPA concentrations increased, while PAI-1 and PGE2 decreased. In women taking the higher dose, both PAI-1 (r = -.97, p = .0048) and PGE2 (r = -.69, p = .019) in NAF and uPA in plasma (r = .45, p = .023) were correlated with celecoxib concentrations., Conclusion: Celecoxib concentrations after treatment correlate inversely with the change in PAI-1 and PGE2 in the breast and directly with the change in uPA in the circulation. uPA upregulation, in concert with PAI-1 and PGE2 downregulation, may have a cancer preventive effect.

Circulating mitochondrial DNA (mtDNA) affected by mutations have been detected in melanoma, prostate cancer, and digestive neoplasms involving the pancreas, liver, and the colon. We sought to detect such mutations in women with breast cancer to assess if the method could be used to aid in the diagnosis of breast cancer. Blood was collected and mtDNA extracted; 27 samples included 14 patients who had breast cancer and 13 healthy controls. White blood cells and serum were separated. The mitochondrial D-loop region was amplified using PCR followed by automated DNA sequencing. The collected data was analyzed with computer software to detect both polymorphisms and mutations. mtDNA sequencing was successful in 93% of the samples (n=23). No mutations were found in any of the study groups. Polymorphisms were detected in all specimens, three of which had not been previously reported. The method used did not detect mtDNA mutations in the blood of women with breast cancer, but was extremely sensitive in polymorphism detection.

Arachidonic acid, a bioactive molecule metabolized into prostaglandins and leukotrienes, contributes to cellular proliferation and tumor progression. Group IIa secretory phospholipase A2 (sPLA2-IIa) can facilitate arachidonate release from cellular phospholipids, suggesting its involvement in tumor evolution. Analysis of breast nipple aspirate fluid (NAF), a noninvasive research tool, allows the identification of biomarkers of breast cancer. We sought to determine whether sPLA2-IIa expression might be related to breast cancer development or progression. sPLA2-IIa expression was evaluated in NAF samples from 110 women (57 women with and 53 without breast cancer) using ELISA and western blotting; ultrastructural immunolocalization was performed in epithelial cells floating in NAF. Immunocytochemistry revealed that sPLA2-IIa is a constitutive intracellular protein suggesting that breast ductal cells synthesize and secrete the 14 kDa protein in NAF. Among all 110 subjects, sPLA2-IIa expression was significantly increased both in NAF and within ductal epithelial cells from cancer containing breasts. While in healthy women menopausal status did not influence sPLA2-IIa expression (P = 0.457), among patients with breast cancer there was a significant down-regulation in postmenopausal subjects (P < 0.0001). Moreover, sPLA2-IIa concentration in NAF from breast cancer patients was positively correlated with tumor stage (r (2) = 0.979, P = 0.0012), suggesting an active secretion/accumulation of the enzyme in NAF based on tumor burden. sPLA2-IIa activity may serve a dual role in breast carcinogenesis, beneficial in its release of arachidonate and detrimental in the metabolic conversion of arachadonic acid into prostaglandins and leukotrienes.

Background: Epidemiologic studies suggest that long term low dose celecoxib use significantly lowers breast cancer risk. We previously demonstrated that 400 mg celecoxib taken twice daily for 2 weeks lowered circulating plasma and breast nipple aspirate fluid (NAF) prostaglandin (PG)E2 concentrations in post- but not premenopausal high risk women. We hypothesized that circulating concentrations of celecoxib influenced PGE2 response, and that plasma levels of the drug are influenced by menopausal status. To address these hypotheses, the aims of the study were to determine: 1) if circulating plasma concentrations of celecoxib correlated with the change in plasma or NAF PGE2 concentrations from baseline to end of treatment, and 2) whether menopausal status influenced circulating levels of celecoxib., Methods: Matched NAF and plasma were collected from 46 high risk women who were administered celecoxib twice daily for two weeks, 20 subjects receiving 200 mg and 26 subjects 400 mg of the agent. NAF and plasma samples were collected before and 2 weeks after taking celecoxib., Results: In women taking 400 mg bid celecoxib, plasma concentrations of the agent correlated inversely with the change in NAF PGE2 levels from pre- to posttreatment. Nonsignificant trends toward higher celecoxib levels were observed in post- compared to premenopausal women. There was a significant decrease in NAF but not plasma PGE2 concentrations in postmenopausal women who took 400 mg celecoxib (p = 0.03)., Conclusion: In high risk women taking 400 mg celecoxib twice daily, plasma concentrations of celecoxib correlated with downregulation of PGE2 production by breast tissue. Strategies synergistic with celecoxib to downregulate PGE2 are of interest, in order to minimize the celecoxib dose required to have an effect.

Serum levels of YKL-40 have been associated with inflammatory diseases and breast cancer. Our purpose was to determine if YKL-40 in breast tissue, nipple aspirate fluid (NAF) and serum is (i) concentrated in NAF compared to matched serum, (ii) increased in the NAF, serum or tissue of women with biopsy proven precancer or cancer compared to healthy women and (iii) influenced by menopausal status. 118 women (61 healthy subjects, 10 with precancer and 47 with breast cancer) aged 17-95 years provided NAF with or without serum samples for analysis. Matched tissue was analyzed from a subset of subjects who underwent breast biopsy. All NAF and serum samples had detectable levels of YKL-40. Median YKL-40 levels for the entire cohort were 683 fold higher in NAF than serum. Premenopausal subjects had higher NAF and lower serum levels of YKL-40 than postmenopausal subjects. YKL-40 levels in NAF but not serum were higher in women with precancer (atypical hyperplasia and lobular carcinoma in situ) than in either healthy subjects (p = 0.025) or subjects with breast cancer (p = 0.015). In women with precancer, YKL-40 distribution in tissue correlated with YKL serum level (p = 0.043). YKL-40 is concentrated in NAF, with the highest concentrations in premenopausal women. NAF levels of YKL-40 are significantly higher in women with precancers than healthy subjects, suggesting that measuring YKL-40 in NAF may improve the identification of women at increased breast cancer risk.

Breast cancer (BC), a worldwide disease with increasing incidence, develops from ductal/lobular epithelium. Nipple aspirate fluid (NAF), secreted from the breast ducts and lobules, can be analyzed to assess breast metabolic activity. Whether lipid peroxidation in the mammary gland promotes or prevents tumorigenesis is unclear. Malondialdehyde (MDA) and the 8-epimer of Prostaglandin F(2alpha) (8-iso-PGF(2alpha)), two lipid peroxidation markers, were studied in milk (n = 10), NAF (n = 140) and plasma (n = 35) samples. MDA was detected in all plasma, in 80% of milk samples and in 95% of NAF samples. MDA levels in NAF and plasma were significantly higher than in milk (p = 0.016 and p = 0.029, respectively). We found no significant difference between levels of MDA in NAF samples from BC patients compared to healthy controls. 8-iso-PGF(2alpha) was detectable in all samples. 8-iso-PGF(2alpha) median levels in NAF were significantly higher than in both milk and plasma (p < 0.0001). The highest 8-iso-PGF(2alpha) levels were found in NAF from healthy women, significantly higher than in women with BC (p < 0.0001). No significant differences were found in both markers after the age-adjustment. High levels of lipid peroxidation products in NAF suggest their in situ production in the nonlactating breast. Active lipid peroxidation may have a physiologic role in the normal mammary gland. Lower levels of 8-iso-PGF(2alpha) in NAF from BC patients suggest altered production of arachidonic acid metabolites during breast carcinogenesis., ((c) 2007 Wiley-Liss, Inc.)

Background: Celecoxib inhibits PGE2 production in cancerous tissue. We previously reported that PGE2 levels in nipple aspirate fluid (NAF) and plasma were not decreased in women at increased breast cancer risk who received celecoxib 200 mg twice daily (bid). The endpoints of the current study were to determine if a short course of celecoxib 400 mg bid would decrease PGE2 levels in women 1) at increased breast cancer risk, and 2) with established breast cancer., Methods: NAF and plasma samples were collected before, 2 weeks after taking celecoxib 400 mg bid, and two weeks after washout from 26 women who were at increased breast cancer risk. From 13 women with newly diagnosed breast cancer, NAF from the incident breast and plasma were collected before and on average 2 weeks after taking celecoxib. Additionally, in nine of the 13 women with breast cancer, NAF was collected from the contralateral breast., Results: No consistent change in NAF or plasma PGE2 levels was noted in high risk premenopausal women. NAF PGE2 levels decreased after celecoxib administration in postmenopausal high risk women (p = 0.02), and in both the NAF (p = 0.02) and plasma (p = 0.03) of women with breast cancer., Conclusion: Celecoxib 400 mg bid taken on average for 2 weeks significantly decreased NAF, but not plasma, PGE2 levels in postmenopausal high risk women, and decreased both NAF and plasma PGE2 levels in women with newly diagnosed breast cancer. PGE2 levels may predict celecoxib breast cancer prevention and treatment efficacy. Our observations are preliminary, and larger studies to confirm and extend these findings are warranted.

Human estrogen receptor-positive breast cancer cells typically display elevated levels of Myc protein due to overexpression of MYC mRNA, and elevated insulin-like growth factor 1 receptor (IGF1R) due to overexpression of IGF1R mRNA. We hypothesized that scintigraphic detection of MYC peptide nucleic acid (PNA) probes with an IGF1 peptide loop on the C-terminus, and a [99mTc]chelator peptide on the N-terminus, could measure levels of MYC mRNA noninvasively in human IGF1R-overexpressing MCF7 breast cancer xenografts in nude mice. We prepared the chelator-MYC PNA-IGF1 peptide, as well as a 4-nt mismatch PNA control, by solid-phase synthesis. We imaged MCF7 xenografts scintigraphically and measured the distribution of [99mTc]probes by scintillation counting of dissected tissues. MCF7 xenografts in nude mice were visualized at 4 and 24 h after tail vein administration of the [99mTc]PNA probe specific for MYC mRNA, but not with the mismatch control. The [99mTc]probes distributed normally to the kidneys, livers, tumors, and other tissues. Molecular imaging of oncogene mRNAs in solid tumors with radiolabel-PNA-peptide chimeras might provide additional genetic characterization of preinvasive and invasive breast cancers.

Unlike nuclear (n)DNA, of which there is one paired copy per cell, there are many copies of mitochondrial (mt)DNA per cell, making PCR amplification of mtDNA easier in samples of limited cellularity. The aims of this study were to (i) determine the mutation patterns of breast cancers through a comprehensive screen of mtDNA mutations, and (ii) assess if mutations in the cancers are also detectable in breast nipple aspirate fluid (NAF), a physiologic fluid which contains shed ductal epithelial cells. Fifteen breast cancers, matched benign tissues and NAF were collected. Nine overlapping primer sets were used to sequence the entire mitochondrial genome from tissue samples. For NAF samples, we focused on the 19 nucleotide positions (np) where mutations were found in a 3701 bp region (np 15331 to 2463), which includes the displacement (D)-loop, a mtDNA mutation hot spot. Fourteen of the fifteen (93%) cancer samples had > or =1 somatic mtDNA mutation for a total of 45 at 35 np (9 np reported previously, 26 new). Nine of fifteen tumors had > or =2 mutations. The D-loop contained 17 of 45 (38%) and non-D-loop (coding) regions contained 28 (62%) mutations. Of the 28 mutations in the coding loci, 11 led to an amino acid change. The frequency of mtDNA mutations was higher in the D-loop region (1.5 versus 0.18% of loci). 155 polymorphisms were identified (98 reported previously, 57 new). Sixteen of forty-five (36%) mutations were located at polymorphism sites. Four of nineteen mtDNA mutations in 10 cancers located between np 15331 and 2463 were found in matched NAF (two of eleven mutations in the D-loop and two of eight in non-D-loop regions). No mutations were found in five matched NAF samples from women whose cancers lacked a mutation in the same region. In conclusion, mtDNA mutations in breast cancer occur both within and outside of the D-loop, though the mutation rate in the D-loop is over 7-fold higher than in coding areas. We identified 26 new mutation loci (25 in regions sequenced by others, one in an area not). The high frequency of mtDNA mutations at polymorphic loci requires further investigation. Specific mtDNA mutations can be detected in a subset of NAF samples from women with breast cancer.

Unlabelled: Detection of a new or recurrent breast cancer lesion relies on physical examination and imaging studies, primarily mammography, followed by histopathologic evaluation of biopsy tissue for morphologic confirmation. Approximately 66%-85% of detected lesions are not malignant. Therefore, biopsies are unnecessary for at least two thirds of patients. Human estrogen receptor-positive breast cancer cells typically display an elevated level of cyclin D1 protein because of the overexpression of CCND1 messenger RNA (mRNA) and an elevated level of insulin-like growth factor 1 (IGF1) receptor (IGF1R) because of the overexpression of IGF1R mRNA. We hypothesized that scintigraphic detection of CCND1 peptide nucleic acid (PNA) hybridization probes with a (99m)Tc-chelating peptide on the N terminus and an IGF1 peptide loop on the C terminus could detect CCND1 mRNA in human MCF7 breast cancer xenografts in nude mice from outside the body., Methods: We prepared the CCND1 probes as well as mismatched controls by solid-phase synthesis. We used fluorescence microscopy to detect the cellular uptake of fluoresceinyl probes and quantitative reverse transcription-polymerase chain reaction to detect the hybridization of probes to mRNA. We imaged (99m)Tc-probes in MCF7 xenografts scintigraphically and measured distribution by scintillation counting of dissected tissues., Results: IGF1R-overexpressing MCF7 cells internalized the fluorescein-chelator-CCND1 PNA-IGF1 peptide but not the mismatched control peptide. The chelator-CCND1 PNA-IGF1 peptide but not the control peptide lowered the level of cyclin D1 protein in IGF1R-overexpressing MCF7 xenografts in nude mice after intratumoral injection. IGF1R-overexpressing MCF7 xenografts in nude mice were visualized at 4, 12, and 24 h after tail vein administration of the (99m)Tc-CCND1 antisense probe but not the control probe. (99m)Tc-chimeras were distributed normally in the kidneys, liver, tumors, and other tissues., Conclusion: Cancer gene activity can be detected from outside the body by probing with radionuclide-chelator-PNA-peptide chimeras.

Deletion mutations in mitochondrial (mt) DNA (mtDNA) as well as microsatellite instability (MSI) and loss of heterozygosity (LOH) in nuclear DNA (nDNA) exist in human cancer. We determined if: (1) large-scale mtDNA deletion mutations were present in cancerous and not in normal breast tissue, and (2) combining mt- and nDNA findings would provide complementary information to identify breast cancer. Thirty-nine matched breast cancer/histologically normal and 23 "true" normal tissue samples from women without breast cancer were microdissected and DNA extracted. 4977, approximately 3938, approximately 4388 and approximately 4576bp deletions were observed, with the 4576bp deletion being present in 0% of true normal, 13% of histologically normal specimens from a cancerous breast and 77% of breast cancers. The other three deletions were not specific to a breast containing cancer. LOH was found in 66.7% and MSI in 38.5% of samples. 38/39 (97.4%) tumors had at least one nDNA or 4576bp mtDNA alteration, suggesting that mt- and nDNA analysis provides complementary information in breast cancer detection. The 4576bp deletion appears to indicate cancer in the breast. The higher mtDNA copy number in cancer coupled with a mtDNA deletion mutation which appears specific for breasts which contain cancer may prove to be a good target to screen for cancer in the breast, including specimens of low and/or mixed cellularity.

Background: Tumor expression of urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and uPA receptor (uPAR) are breast cancer prognostic factors. Less is known about their usefulness in breast cancer diagnosis. Nipple aspirate fluid (NAF) is secreted into the breast duct and collected noninvasively, making it potentially useful both in breast cancer diagnosis and prognosis. We determined the association of uPA, PAI-1, and uPAR levels in NAF with breast cancer (1) detection and (2) advanced disease., Methods: A total of 88 NAF specimens were collected from women with or without breast cancer, and uPA, PAI-1, and uPAR expression were measured by enzyme-linked immunosorbent assay., Results: uPA and uPAR were independent predictors of cancer presence; uPAR was also an independent predictor of advanced disease stage. Higher PAI-1 expression in breast cancer that was found with univariate analysis was not observed after logistic regression was applied., Conclusions: NAF evaluation of uPA, uPAR, and, perhaps, PAI-1 (significant only in univariate analysis) may provide useful breast cancer diagnostic and prognostic information.

Purpose: Loss of heterozygosity (LOH) and microsatellite instability (MSI) have been identified in a variety of human cancers. The purpose of this prospective study was to determine whether (a) DNA can be isolated from nipple aspirate fluid (NAF) and PCR amplified to large fragments, (b) LOH and MSI are detectable in NAF, and (c) LOH and MSI in tissue and NAF increase with disease progression from precursor lesions to cancer., Experimental Design: Forty-six matched samples from breast lesions, normal breast, and NAF were microdissected, and DNA was extracted. Eleven microsatellite markers from seven chromosomes that have a high frequency of LOH/MSI in breast cancer were designed and respectively amplified., Results: LOH and/or MSI were identified in 22 of 46 (48%) breast lesions, including LOH in 8 of 36 (22%) proliferative/papilloma (P/Pap) and 7 of 10 (70%) cancer specimens, whereas MSI was found in 14 of 36 (39%) P/Pap and 6 of 10 (60%) cancer specimens. LOH/MSI loci in which alterations were detected in the 22 tissue specimens were PCR amplified using matched NAF DNA. LOH/MSI was detected in NAF from both P/Pap (5 of 15; 33%) and breast cancer (3 of 7; 43%) samples., Conclusions: Our findings suggest that (a) DNA from NAF, a physiological fluid collected noninvasively, can be PCR amplified and used to screen for LOH and MSI alterations that are known to be linked to breast cancer, suggesting that this methodology might prove useful for breast cancer screening, and (b) similar to findings in breast tissue, LOH and MSI alterations increase in frequency with disease progression in NAF, which suggests that NAF is a surrogate for breast tissue which has important prognostic implications.

Imaging oncogene mRNA in tumours would provide a powerful tool for the early detection of occult malignant lesions. The goal was to prepare a chimera consisting of a dodecamer antisense peptide nucleic acid (PNA) specific for c-MYC oncogene overexpressed in human breast cancer cells and a chelating moiety that facilitates quantitative radiolabelling with 99mTc and evaluate it for hybridization and tissue distribution in laboratory animals. The pentapeptide chelator-PNA dodecamer specific for c-MYC mRNA was extended from a solid support by 9-fluorenylmethyloxycarbonyl (Fmoc) coupling. Similarly, a chelator-PNA chimera with four central mismatches was also prepared which served as a control. The chimeras were purified, characterized and evaluated for hybridization to c-MYC mRNA by fluorescent, real-time polymerase chain reaction (RT-PCR). The chimeras were labelled with 99mTc and their tissue distribution was examined in athymic nude mice bearing experimental human breast tumours. 99mTc radiolabelling was quantitative and presented a single peak in reversed phase liquid chromatography. Fluorescent real-time polymerase chain reactions using primer and fluorescent probe sets previously calculated for c-MYC mRNA demonstrated inhibition of reverse transcription by the c-MYC specific chimera as compared to that of the control. Tissue distribution studies of antisense and mismatch chimeras at 4 h and 24 h after administration displayed modest accumulation in the liver, and appreciable levels in tumours. These observations suggest that 99mTc-peptide-PNA probes might be useful for imaging gene expression in tumours, and the approach is worthy of further investigation.