BMC - Breast Cancer Research

IntroductionA number of breast cancer risk prediction models have been developed to provide insight into a woman's individual breast cancer risk. Although circulating levels of estradiol in postmenopausal women predict subsequent breast cancer risk, whether the addition of estradiol levels adds significantly to a model's predictive power has not previously been evaluated. Methods: Using linear regression, the authors developed an imputed estradiol score using measured estradiol levels (the outcome) and both case status and risk factor data (e.g., body mass index) from a nested case-control study conducted within a large prospective cohort study and used multiple imputation methods to develop an overall risk model including both risk factor data from the main cohort and estradiol levels from the nested case-control study. Results: The authors evaluated the addition of imputed estradiol level to the previously published Rosner and Colditz log incidence model for breast cancer risk prediction within the larger Nurses' Health Study cohort. Follow-up was from 1980-2000; during this time 1559 invasive estrogen receptor positive breast cancer cases were confirmed. The addition of imputed estradiol levels significantly improved risk prediction; the age-specific concordance statistic increased from 0.635+/-0.007 to 0.645+/-0.007 (p

Expression profiling and biomarker(s) discovery aim to provide means for tumour diagnosis, classification, therapy response and prognosis. The identification of novel markers could potentially lead to the building of robust early detection strategies and personalized, effective breast cancer therapies that would improve patient outcome. Recent evidence supports the hypothesis that genomic expression profiling using microarray analysis is a reliable method for breast cancer classification and prognostication. However, genes clearly do not act by themselves, or indeed they do not have catalytic or signalling capabilities. Hence, genetic biomarker information alone cannot perfectly predict cancer and its response to treatment. Genes clearly exert their effect after transcription through translation into active proteins. Consequently, postgenomic projects correlating protein expression profiles with tumour classification have led to some established biomarkers. In this regard, these biomarkers associate with disease prediction and can be associated with treatment response. Recently, Brozokova and colleagues demonstrated that surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF MS) profiling of breast cancer tissue proteomes can potentially expand the biomarker repertoire and our knowledge of breast cancer behaviour.

IntroductionThe use of cultured cell lines as model systems for normal tissue is limited by the molecular alterations accompanying the immortalisation process, including changes in the mRNA and miRNA repertoire. Therefore, identification of cell lines with normal-like expression profiles is of paramount importance in studies of normal gene regulation. Methods: The mRNA and miRNA expression profiles of several breast cell lines of cancerous or normal origin were measured using printed slide arrays, Luminex bead arrays and real-time RT-PCR. Results: We demonstrate that the mRNA expression profiles of two breast cell lines are similar to that of normal breast tissue: HB4a, immortalised normal breast epithelium, and PMC42, a breast cancer cell line that retains progenitor pluripotency allowing in culture differentiation to both secretory and myoepithelial fates. In contrast, only PMC42 exhibits a normal-like miRNA expression profile. We identified a group of miRNAs that are highly expressed in normal breast tissue and PMC42 but are lost in all other cancerous and normal-origin breast cell lines, and observed a similar loss in immortalised lymphoblastoid cell lines compared to healthy uncultured B cells. Moreover, like tumour suppressor genes, these miRNAs are lost in a variety of tumours. We show that the mechanism leading to the loss of these miRNAs in breast cancer cell lines has genomic, transcriptional and post-transcriptional components. Conclusion: We propose that despite its neoplastic origin, PMC42 is an excellent molecular model for normal breast epithelium, providing a unique tool to study breast differentiation and the function of key miRNAs which are typically lost in cancer.

Mammographic density has been strongly associated with increased risk of breast cancer. Furthermore, density is inversely correlated with the accuracy of mammography and, therefore, a measurement of density conveys information about the difficulty of detecting cancer in a mammogram. Initial methods for assessing mammographic density were entirely subjective and qualitative; however, in the past few years methods have been developed to provide more objective and quantitative density measurements. Research is now underway to create and validate techniques for volumetric measurement of density. It is also possible to measure breast density with other imaging modalities, such as ultrasound and MRI, which do not require the use of ionizing radiation and may, therefore, be more suitable for use in young women or where it is desirable to perform measurements more frequently. In this article, the techniques for measurement of density are reviewed and some consideration is given to their strengths and limitations.

IntroductionHuman breast tumors are heterogeneous and consist of phenotypically diverse cells. Breast cancer cells with a CD44+/CD24- phenotype have been suggested to have tumor-initiating properties with stem cell-like and invasive features, although it is unclear whether their presence within a tumor has clinical implications. There is also a large heterogeneity between tumors, illustrated by reproducible stratification into various subtypes based on gene expression profiles or histopathological features. We have explored the prevalence of cells with different CD44/CD24 phenotypes within breast cancer subtypes. Methods: Double-staining immunohistochemistry was used to quantify CD44 and CD24 expression in 240 human breast tumors for which information on other tumor markers and clinical characteristics was available. Gene expression data was also accessible for a cohort of the material. Results: A considerable heterogeneity in CD44 and CD24 expression was seen both between and within tumors. A complete lack of both proteins was evident in 35% of the tumors, while 13% contained cells of more than one of the CD44+/CD24-, CD44-/CD24+ and CD44+/CD24+ phenotypes. CD44+/CD24- cells were detected in 31% of the tumors, ranging in proportion from only a few to close to 100% of tumor cells. The CD44+/CD24- phenotype was most common in the basal-like subgroup characterized as negative for the estrogen and progesterone receptors as well as for HER2 and positive for cytokeratin 5/14 and/or EGFR, and particularly common in BRCA1 hereditary tumors of which 94% contained CD44+/CD24- cells. The CD44+/CD24- phenotype was surprisingly scarce in HER2+ tumors, which had a predominantly CD24+ status. A CD44+/CD24- gene expression signature was generated, which included CD44 and alpha-6 integrin (CD49f) among the top-ranked overexpressed genes. Conclusions: We demonstrate an association between basal-like and particularly BRCA1 hereditary breast cancer and presence of CD44+/CD24- cells. However, not all basal-like and very few HER2+ tumors contain CD44+/CD24- cells, which emphasizes that a putative tumorigenic ability may not be confined to cells of this phenotype and that other breast cancer stem cell markers remain to be identified.

IntroductionThe identification of potential breast cancer stem cells is of importance as the characteristics of stem cells suggest that they are resistant to conventional forms of therapy. Several techniques have been proposed to isolate or enrich for tumorigenic breast cancer stem cells, including (a) culture of cells in non-adherent non-differentiating conditions to form mammospheres and (b) sorting of the cells by their surface phenotype (expression of CD24 and CD44). Methods: We have cultured metastatic cells found in pleural effusions from breast cancer patients in non-adherent conditions without serum to form mammospheres. Dissociated cells from these mammospheres were used to determine the tumorigenicity of these cultures. Expression of CD24 and CD44 on uncultured cells and mammospheres derived from the pleural effusions was documented. Results: We found that the majority (20/27) of the pleural effusions tested contained cells capable of forming mammospheres of varying sizes that could be passaged. After dissociation and plating with serum onto adherent dishes, the cells can differentiate, as determined by the increased expression of cytokeratins and MUC1. Analysis of surface expression of CD24 and CD44 on uncultured cells from 21 of the samples showed that the cells from some samples separated into two populations, but some did not. The proportion of cells that could be considered CD44+/CD24low/- was highly variable and did not appear to correlate with the ability to form the larger mammospheres. Of eight pleural effusion mammospheres tested in severe combined immunodeficiency disease (SCID) mice, four were found to induce tumours when only 5,000 or fewer cells were injected, whereas the same number of uncultured cells did not form tumours. The ability to induce tumours appeared to correlate with the ability to produce the larger mammospheres. Uncultured cells from a highly tumorigenic sample (PE14) were uniformly negative for surface expression of both CD24 and CD44. Conclusion: This paper shows, for the first time, that mammosphere culture of pleural effusions enriches for cells capable of inducing tumours in SCID mice. The data suggest that mammosphere culture of these metastatic cells could provide a highly appropriate model for studying the sensitivity of the tumorigenic 'stem' cells to therapeutic agents and for further characterisation of the tumour-inducing subpopulation of breast cancer cells.

Introductionc-Jun activation domain-binding protein-1 (Jab1) is a multifunctional signaling protein that previously has been shown to be a master regulator of a poor prognostic gene signature in invasive breast cancer and to mediate the action of S100A7. Since epidermal growth factor receptor (EGFR), like S100A7, is often expressed in estrogen receptor-alpha-negative (ER?-) breast cancer, we set out to investigate the role of Jab1 in mediating EGFR signaling, another facet of the ER?- phenotype. Methods: MDA-MB-231 and MDA-MB-468 ER?-/EGFR+ cell lines were assessed for localization of Jab1 and levels of downstream genes by immunofluorescence and nuclear protein extract assay following treatment with epidermal growth factor (EGF) and extracellular signal-regulated kinase (ERK) pathway inhibitor. A cohort of 424 human breast tumors was also assessed by immunohistochemistry. Results: EGF treatment of cell lines resulted in increased Jab1 nuclear expression. This effect was inhibited by the ERK pathway inhibitor, PD98059. EGF treatment was also associated with colocalization of pERK (phosphorylated ERK) and Jab1 as well as regulation of the Jab1 downstream target gene, p27. When Jab1 activity was knocked down, p27 levels were restored to pre-EGF treatment level. Analysis of EGFR and Jab1 expression in a cohort of invasive breast tumors by tissue microarray and immunohistochemistry confirmed a relationship between EGFR and increased nuclear Jab1 within the ER?- subset (n = 154, P = 0.019). The same association was also confirmed for S100A7 and Jab1 (P = 0.036), and high Jab1 nuclear expression was most frequent in tumors that were positive for both EGFR and S100A7 (P = 0.004). Conclusion: Jab1 is a target of EGFR signaling in ER?- cell lines and breast tumors and therefore may be a common central factor and potential therapeutic target for important cell signaling pathways in ER?- breast cancer.

A woman typically presents for genetic counselling because she has a strong family history and is interested in knowing the probability she will develop disease in the future; that is, her absolute risk. Relative risk for a given factor refers to risk compared with either population average risk (sense a), or risk when not having the factor, with all other factors held constant (sense b). Not understanding that these are three distinct concepts can result in failure to correctly appreciate the consequences of studies on clinical genetic testing. Several studies found that the frequencies of mutations in ATM, BRIP1, PALB2 and CHEK2 were many times greater for cases with a strong family history than for controls. To account for the selected case sampling (ascertainment), a statistical model that assumes that the effect of any measured variant multiplies the effect of unmeasured variants was applied. This multiplicative polygenic model in effect estimated the relative risk in the sense b, not sense a, and found it was in the range of 1.7 to 2.4. The authors concluded that the variants are "low penetrance". They failed to note that their model fits predicted that, for some women, absolute risk may be as high as for BRCA2 mutation carriers. This is because the relative risk multiplies polygenic risk, and the latter is predicted by family history. Therefore, mutation testing of these genes for women with a strong family history, especially if it is of early onset, may be as clinically relevant as it is for BRCA1 and BRCA2.

IntroductionThe "MINO" mouse model of ductal carcinoma in situ (DCIS) consists of six lines with distinct morphologic phenotypes and behavior each meeting experimentally defined criteria for "pre-cancer". Specifically, these lines grow orthotopically in cleared mammary fat pads and consistently progress to an invasive phenotype capable of ectopic growth. Transition to carcinoma has a consistent latency for each line and three of the lines also show pulmonary metastatic potential. Methods: Gland cleared orthotopic transplanted precancer MINO tissues were analyzed by BAC and oligo array CGH, microsatellite PCR, and telomerase repeat amplification assay. MINO cells were dissociated and cultured in three dimensional (3D) culture and transplanted in syngeneic gland cleared mammary fat pads. Results: Comparative genomic hybridization shows that the pre-cancer and invasive tumors are genetically stable with low level changes including whole chromosome gains in some lines. No changes are associated with progression, although spontaneous focal amplifications and deletions were detected occasionally. Microsatellite analysis shows a low frequency of alterations which are predominantly permanent within a MINO line. Telomerase activity is increased in both the MINO and the derived tumors when compared to normal mouse mammary gland. Dissociation of the precancer lesion cells and three dimensional "spheroid" culture of single cells reveals a bipotential for myoepithelial and luminal differentiation and the formation of unique 3D "MINOspheres." These "MINOspheres" show features that are intermediate between spheroids that are derived from normal and carcinoma cells. Transplantation of a single cell derived MINOsphere recapitulates the outgrowth of the precancer morphology and progression to carcinoma. Conclusion: These data establish a pre-cancer stem cell capable of self renewal and multilineage differentiation as the origin of invasive cancer. In the context of this model, these cells have programmed potential for latency and metastasis that does not appear to require sequential genetic hits for transformation.

IntroductionEpidermal growth factor receptor (EGFR) is involved in regulating cell growth in breast carcinomas. Its activated form, phosphorylated EGFR (pEGFR), is correlated with poor prognosis in lung cancer, but it has not yet been fully investigated in breast cancer. The aim of this study was to investigate the expressions of EGFR and pEGFR and their correlation with overall and disease-free survival, clinicopathological parameters and biological markers of invasion and angiogenesis (phosphorylated Akt [pAkt], urokinase plasminogen activator receptor [uPAR], matrix metalloproteinase [MMP]-14, vascular endothelial growth factor receptor [VEGFR]-1/Flt-1). Methods: A three-step immunohistochemical method was applied to paraffin-embedded sections from 154 patients with invasive breast carcinoma in order to detect expressions of the proteins EGFR, pEGFR, oestrogen receptor, progesterone receptor, c-erbB-2, pAkt, VEGFR-1/Flt-1, MMP-14 and uPAR. The results were evaluated statistically using the ?2 test. Overall and disease-free survival distribution curves were assessed using the Kaplan-Meier test and log-rank statistics, followed by Cox proportional hazards regression model. Results: EGFR and pEGFR proteins were immunodetected in the membrane of the malignant cells (11.3% and 35.7%, respectively). EGFR expression was positively correlated with nuclear grade (P = 0.001) and negatively correlated with the hormonal receptor oestrogen receptor (P = 0.005). pEGFR was positively related to the Akt pathway (P = 0.008) and appeared to participate in invasion and metastasis (uPAR, P = 0.049; MMP-14, P = 0.025; VEGFR-1/Flt-1, P = 0.016). Univariate analysis showed that the EGFR/pEGFR phenotype was associated with poor overall survival (P = 0.019), a finding further supported by multivariate analysis (P = 0.013). Conclusion: These data provide evidence that pEGFR expression is related to angiogenesis (via VEGFR-1/Flt-1, MMP-14 and pAkt pathways) and invasiveness (via uPAR, MMP-14 and pAkt pathways) and that the EGFR/pEGFR phenotype is associated with poor patient survival in invasive breast cancer.

Progesterone receptor status is a marker for hormone responsiveness and disease prognosis in breast cancer. Progesterone receptor negative tumours have generally been shown to have a poorer prognosis than progesterone receptor positive tumours. The observed loss of progesterone receptor could be through a range of mechanisms, including the generation of alternatively spliced progesterone receptor variants that are not detectable by current screening methods. Many progesterone receptor mRNA variants have been described with deletions of various whole, multiple or partial exons that encode differing protein functional domains. These variants may alter the progestin responsiveness of a tissue and contribute to the abnormal growth associated with breast cancer. Absence of specific functional domains from these spliced variants may also make them undetectable or indistinguishable from full length progesterone receptor by conventional antibodies. A comprehensive investigation into the expression profile and activity of progesterone receptor spliced variants in breast cancer is required to advance our understanding of tumour hormone receptor status. This, in turn, may aid the development of new biomarkers of disease prognosis and improve adjuvant treatment decisions.

IntroductionMicroarray-based gene expression profiling represents a major breakthrough for understanding the molecular complexity of breast cancer. cDNA expression profiles cannot detect changes in activities that arise from post-translational modifications, however, and therefore do not provide a complete picture of all biologically important changes that occur in tumors. Additional opportunities to identify and/or validate molecular signatures of breast carcinomas are provided by proteomic approaches. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) offers high-throughput protein profiling, leading to extraction of protein array data, calling for effective and appropriate use of bioinformatics and statistical tools. Methods: Whole tissue lysates of 105 breast carcinomas were analyzed on IMAC 30 ProteinChip Arrays (Bio-Rad, Hercules, CA, USA) using the ProteinChip Reader Model PBS IIc (Bio-Rad) and Ciphergen ProteinChip software (Bio-Rad, Hercules, CA, USA). Cluster analysis of protein spectra was performed to identify protein patterns potentially related to established clinicopathological variables and/or tumor markers. Results: Unsupervised hierarchical clustering of 130 peaks detected in spectra from breast cancer tissue lysates provided six clusters of peaks and five groups of patients differing significantly in tumor type, nuclear grade, presence of hormonal receptors, mucin 1 and cytokeratin 5/6 or cytokeratin 14. These tumor groups resembled closely luminal types A and B, basal and HER2-like carcinomas. Conclusion: Our results show similar clustering of tumors to those provided by cDNA expression profiles of breast carcinomas. This fact testifies the validity of the SELDI-TOF MS proteomic approach in such a type of study. As SELDI-TOF MS provides different information from cDNA expression profiles, the results suggest the technique's potential to supplement and expand our knowledge of breast cancer, to identify novel biomarkers and to produce clinically useful classifications of breast carcinomas.

The letter from Drs. Lindeman, Visvader, Smalley and Eaves conveys their concern regarding the future of the prospective isolation and characterization of individual cells that may be characterized as mammary stem cells upon in vivo transplantation. As they have pointed out, the difficulties encountered in identifying specific mammary epithelial subtypes by different levels of fluorescence (particularly for membrane components that decorate most if not all mammary epithelial cells) leads to differential reporting and "resultant confusion" and "underscores the need for improved standardization".

Not applicable.

IntroductionSomatic alterations have been shown to correlate with breast cancer prognosis and survival, but less is known about the effects of common inherited genetic variation. Of particular interest are genes involved in cell cycle pathways, which regulate cell division. Methods: We examined associations between common germline genetic variation in 13 genes involved in cell cycle control (CCND1, CCND2, CCND3, CCNE1, CDK2 [p33], CDK4, CDK6, CDKN1A [p21, Cip1], CDKN1B [p27, Kip1], CDKN2A [p16], CDKN2B [p15], CDKN2C [p18], and CDKN2D [p19]) and survival among women diagnosed with invasive breast cancer participating in the SEARCH (Studies of Epidemiology and Risk factors in Cancer Heredity) breast cancer study. DNA from up to 4,470 women was genotyped for 85 polymorphisms that tag the known common polymorphisms (minor allele frequency > 0.05) in the genes. The genotypes of each polymorphism were tested for association with survival using Cox regression analysis. Results: The rare allele of the tagging single nucleotide polymorphism (SNP) rs2479717 is associated with an increased risk of death (hazard ratio = 1.26 per rare allele carried, 95% confidence interval: 1.12 to 1.42; P = 0.0001), which was not attenuated after adjusting for tumour stage, grade, and treatment. This SNP is part of a large linkage disequilibrium block, which contains CCND3, BYSL, TRFP, USP49, C6ofr49, FRS3, and PGC. We evaluated the association of survival and somatic expression of these genes in breast tumours using expression microarray data from seven published datasets. Elevated expression of the C6orf49 transcript was associated with breast cancer survival, adding biological interest to the finding. Conclusion: It is possible that CCND3 rs2479717, or another variant it tags, is associated with prognosis after a diagnosis of breast cancer. Further study is required to validate this finding.

IntroductionEstrogen receptor (ER)-positive breast cancers are considered prognostically more favorable than ER-negative tumors, whereas human epidermal growth factor receptor (HER)2/neu-positive breast cancers are associated with worse prognosis. The objective of the present study was to determine whether ER-positive and ER-negative status relates to epigenetic changes in breast cancer-related genes. To evaluate epigenetic differences in tumor-related genes relating to ER and HER2/neu status of primary tumors, we examined the promoter methylation status of the promoter region CpG islands of eight major breast tumor-related genes (RASSF1A, CCND2, GSPT1, TWIST, APC, NES1, RAR?2, and CDH1). Methods: Paired ER-positive (n = 65) and ER-negative (n = 65) primary breast tumors (n = 130) matched for prognostic factors were assessed. DNA was extracted from paraffin-embedded tumor tissue after microdissection, and methylation-specific PCR and capillary-array electrophoresis analysis were performed. Results: In early stages of tumor progression (T1 and N0), RASSF1A and CCND2 were significantly (P

IntroductionNeoadjuvant chemotherapy has become the standard of care for the diverse population of women diagnosed with locally advanced breast cancer. Serum biomarker levels are increasingly being investigated for their ability to predict therapy response and aid in the development of individualized treatment regimens. Multianalyte profiles may offer greater predictive power for neoadjuvant treatment response than the individual biomarkers currently in use. Materials and Methods: Serum samples were collected from 44 patients enrolled in a phase I-II, open-label study of liposomal doxorubicin and paclitaxel in combination with whole breast hyperthermia for the neoadjuvant treatment of locally advanced breast cancer (stage IIB or III). Samples were collected prior to each of four rounds of treatment and prior to definitive surgery. Samples were assayed by Luminex for 55 serum biomarkers including: cancer antigens, growth/angiogenic factors, apoptosis-related molecules, metastasis-related molecules, adhesion molecules, adipokines, cytokines, chemokines, hormones, and other proteins. Results: Biomarker levels were compared retrospectively with clinical and pathologic treatment response. Univariate analysis of the data identified several groups of biomarkers that differed significantly among treatment outcome groups early in the course of neoadjuvant chemotherapy. Multivariate statistical analysis revealed multi-biomarker panels that could differentiate between treatment response groups with high sensitivity and specificity. Conclusions: We demonstrate here that serum biomarker profiles may offer predictive power concerning treatment response and outcome in the neoadjuvant setting. The continued development of these findings will be of considerable clinical utility in the design of treatment regimens for individual breast cancer patients. Trial Registration: #NCT00346229.

IntroductionTumor invasion and metastasis remain a major cause of mortality in breast cancer patients. High concentrations of nitric oxide (NO) suppress tumor invasion and metastasis in vivo. NO prodrugs generate large amounts of NO upon metabolism by appropriate intracellular enzymes, and therefore could have potential in the prevention and therapy of metastatic breast cancer. Methods: This study was designed to determine the effects of the NO-releasing prodrug JS-K on breast cancer invasion and the mechanisms involved. MDA-MB-231, MDA-MB-231/F10, and MCF-7/COX-2 were the three breast cancer cell lines tested. NO levels were determined spectrophotometrically using a NO assay kit. Invasion was determined using Matrigel invasion assays. The expression of matrix metalloproteinases (MMPs) and tissue inhibitor of MMPs (TIMPs) were determined using an MMP array kit and enzyme-linked immunosorbent assays. The activity and expression of extracellular signal-regulated kinase (ERK)1/2, p38, and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs) were determined using Western blot. Results: Under conditions by which JS-K was not cytotoxic, JS-K significantly decreased (p

IntroductionTreatment with estrogen and progesterone (E+P) mimics the protective effect of parity on mammary tumors in rodents and depends upon the activity of p53. The following experiments tested whether exogenous E+P primes p53 to be more responsive to DNA damage and whether these pathways confer resistance to mammary tumors in a mouse model of Li-Fraumeni syndrome. Methods: Mice that differ in p53 status (Trp53+/+, Trp53+/-, Trp53-/-) were treated with E+P for 14 days and then were tested for p53-dependent responses to ionizing radiation. Responses were also examined in parous and age-matched virgins. The effects of hormonal exposures on tumor incidence were examined in BALB/c-Trp53+/- mammary tissues. Results: Nuclear accumulation of p53 and apoptotic responses were increased similarly in the mammary epithelium from E+P-treated and parous mice compared with placebo and age-matched virgins. This effect was sustained for at least 7 weeks after E+P treatment and did not depend on the continued presence of ovarian hormones. Hormone stimulation also enhanced apoptotic responses to ionizing radiation in BALB/c-Trp53+/- mice but these responses were intermediate compared with Trp53+/+ and Trp-/- tissues, indicating haploinsufficiency. The appearance of spontaneous mammary tumors was delayed by parity in BALB/c-Trp53+/- mice. The majority of tumors lacked estrogen receptor (ER), but ER+ tumors were observed in both nulliparous and parous mice. However, apoptotic responses to ionizing radiation and tumor incidence did not differ among outgrowths of epithelial transplants from E+P-treated donors and nulliparous donors. Conclusion: Therefore, E+P and parity confer a sustained increase in p53-mediated apoptosis within the mammary epithelium and suppress mammary tumorigenesis, but this effect was not retained in epithelial outgrowths.

IntroductionLevels of insulin-like growth factor (IGF)-I and its main binding protein (IGFBP-3) have been associated with breast cancer risk among premenopausal women. However, associations of IGFBP-3 levels with breast cancer risk have been inconsistent, possibly due to the different predominant forms of circulating IGFBP-3 (intact versus fragmented) that were measured in these studies. Here, we examine the association of breast cancer risk factors with intact and total IGFBP-3 levels. Methods: This cross-sectional study includes 737 premenopausal women recruited at screening mammography. Plasma intact and total IGFBP-3 and IGF-I levels were measured by enzyme-linked immunosorbent assay methods. Percent and absolute breast density were estimated using a computer-assisted method. The associations were evaluated using generalized linear models and Pearson (r) or Spearman (rs) partial correlation coefficients. Results: Means ± standard deviations of intact and total IGFBP-3 levels (ng/mL) were 1,044 ± 234 and 4,806 ± 910, respectively. Intact and total IGFBP-3 levels were correlated with age and smoking. Levels of intact IGFBP-3 were negatively correlated with waist-to-hip ratio (WHR) (r = -0.128; P = 0.0005), parity (rs = -0.078; P = 0.04), and alcohol intake (r = -0.137; P = 0.0002) and positively correlated with energy intake (r = 0.075; P = 0.04). In contrast, total IGFBP-3 levels were positively correlated with WHR (r = 0.115; P = 0.002), parity (rs = 0.089; P = 0.02), body mass index (BMI) (r = 0.115; P = 0.002), physical activity (r = 0.118; P = 0.002), and IGF-I levels (r = 0.588; P