Breast Cancer Research - Latest articles

A robust classifier of high predictive value to identify good prognosis patients in ER negative breast cancer.

Andrew E Teschendorff and Carlos Caldas — 2008-08-28

IntroductionPatients with primary operable estrogen receptor negative (ER-) breast cancer account for approximately 30% of all cases and generally have worse prognosis than ER+ patients. Nevertheless, a significant proportion of ER- cases have favourable outcome and could potentially benefit from a less aggressive course of therapy. Identification of such good prognosis patients however remains difficult and at present only possible through histopathological factors. Methods: Building on a previously identified 7-gene prognostic IR-module for ER negative breast cancer, we develop a novel statistical tool based on Mixture Discriminant Analysis in order to build a classifier that can accurately identify ER- patients with good prognosis. Results: We report the construction of a 7-gene expression classifier that accurately predicts, across a training cohort of 183 ER- tumors and six independent test cohorts (a total of 469 ER- tumors), ER- patients of good prognosis (in test sets, average predictive value = 94% (range 85%-100%), average hazard ratio = 0.15 (0.07-0.36) P<0.000001) independently of lymph node status and treatment. Conclusions: This 7-gene classifier could be used in a PCR-based clinical assay to identify good prognosis ER- patients, who may therefore benefit from less aggressive treatment regimens.

Variable size CAD prompts and mammography film reader decisions

2008-08-25

IntroductionThe purpose of this study was to investigate the effect of CAD prompts on reader behaviour in a large sample of breast screening mammograms by analysing the relationship of the presence and size of prompts to the recall decision. Methods: Local research ethics committee approval was obtained; informed consent was not required. Mammograms were from women attending routine mammography at two breast screening centres in 1996. Films, previously double read, were re-read by a different reader using CAD. The study material included 315 cancer cases comprising all screen detected cancer cases, all subsequent interval cancers and 861 normal cases randomly selected from 10,267 cases.. Ground truth data were used to assess the efficacy of CAD prompting. Associations between prompt attributes and tumour features or reader recall decisions were assessed by Chi squared tests. Results: There was a highly significant relationship between prompting and a decision to recall for cancer cases and for a random sample of normal cases (p<0.001). 64% of all cases contained at least one CAD prompt. In cancer cases, larger prompts were more likely to be recalled (p=0.02) for masses but there was no such association for calcifications (p=0.9). In a random sample of 861 normal cases, larger prompts were more likely to be recalled (p=0.02) for both mass and calcification prompts. Significant associations were observed with prompting and breast density (p=0.009) for cancer cases but not for normal cases (p=0.05). Conclusions: For both normal and cancer cases, prompted mammograms were more likely to be recalled and prompt size was also associated with a recall decision.

Relaxin reduces xenograft tumor growth of human MDA-MB-231 breast cancer cells

Yvonne Y Radestock, Cuong C Hoang-Vu and Sabine S Hombach-Klonisch — 2008-08-21

IntroductionRelaxin is increased in human breast cancer and was shown to promote cancer cell migration in carcinoma cells of the breast, prostate and thyroid. In estrogen receptor alpha-negative MDA-MB-231 human breast cancer cells, relaxin was shown to down-regulate the metastasis-promoting protein S100A4 (metastasin), a highly significant prognostic factor for poor survival in breast cancer patients. The cellular mechanisms of relaxin exposure in breast cancer cells are not fully understood. The aim of this study was to investigate short-term and long-term effects of relaxin on cancer cell motility and S100A4 expression and to determine the long-term effects of relaxin on in vivo tumor growth in an estrogen-independent context. Methods: We have established stable transfectants of highly invasive estrogen receptor alpha-negative MDA-MB-231 human breast cancer cells with constitutive expression of bioactive H2-relaxin (MDA/RLN2). RLN2 secretion was determined by ELISA. Relaxin receptor RXFP1 (Relaxin-family-peptide) was detected by RT-PCR and its activation was assessed by induction of cyclic-AMP. Stable MDA/RLN2 clones and RLN2 treated MDA-MB-231 cells were subjected to motility and in vitro-invasion assays. Proliferation was assessed in bromodeoxyuridine (BrdU) and MTT assays. S100A4 expression was determined by RT-PCR and Western Blot. Specific small interfering RNA was employed to down-regulate relaxin receptor and S100A4. MDA/EGFP vector control and two MDA/RLN2 clones were injected subcutaneously in nude mice to determine tumor growth and cancer cell invasiveness in vivo. Xenograft tumor tissues were assessed by histology and immunohistochemistry and frozed tissues were used for the detection of S100A4 and RLN2. Results: Short-term exposure to relaxin for 24 hours increased cell motility in a relaxin receptor-dependent manner. This increase in cell motility was mediated by S100A4. Long-term exposure to relaxin secreted from stable transfectants reduced cell motility and in vitro invasiveness. Relaxin decreased cell proliferation and down-regulated cellular S100A4 levels in MDA-MB-231 and T47D breast cancer cells. Stable MDA/RLN2 transfectants produced smaller xenograft tumors containing reduced S100A4 protein levels in vivo. Conclusions: Our results indicate that long-term exposure to relaxin confers growth inhibitory and anti-invasive properties in estrogen-independent tumors in vivo which may in part be mediated through a down-regulation of S100A4.

Activated Akt1 accelerates MMTV-c-ErbB2 mammary tumourigenesis in mice without activation of ErbB3

2008-08-13

IntroductionErbB2, a member of the epidermal growth factor receptor (EGFR) family, is overexpressed in 20-30% of human breast cancer cases and forms oncogenic signaling complexes when dimerized to ErbB3 or other EGFR family members. Methods: We have crossed the MMTV-myr-Akt1 transgenic mice (which express constitutively active Akt1 in the mammary gland) with MMTV-c-ErbB2 transgenic mice to evaluate the role of Akt1 activation in ErbB2-induced mammary carcinoma utilizing immunoblot analysis, magnetic resonance spectroscopy, and histological analyses. Results: Bitransgenic MMTV-c-ErbB2, MMTV-myr-Akt1 mice develop mammary tumors twice as quickly as the MMTV-c-ErbB2 mice. The bitransgenic tumors were less organized, had more mitotic figures and fewer apoptotic cells. However, many bitransgenic tumors displayed areas of extensive necrosis as compared to the tumors from MMTV-c-ErbB2 mice. The two tumor types demonstrate dramatically different expression and activation of EGFR family members as well as different metabolic profiles. c-ErbB2 tumors demonstrate overexpression of EGFR, ErbB2, ErbB3 and ErbB4 and activation/phosphorylation of both ErbB2 and ErbB3, underscoring the importance of the entire EGFR family in ErbB2-induced tumorigenesis. Tumors from bitransgenic mice demonstrate overexpression of the myr-Akt1 and ErbB2 transgenes, however there was dramatically less overexpression and phosphorylation of ErbB3, the phosphorylation of ErbB2 was diminished, the level of EGFR protein was decreased and ErbB4 protein was undetectable. There was also an observable attenuation in a subset of tyrosine-phosphorylated secondary signaling molecules and Src in the bitransgenic tumors as compared to the c-ErbB2 tumors, but Erk was activated/phosphorylated in both tumor types. Finally, the bitransgenic tumors were metabolically more active as indicated by increased glucose transporter (GLUT1) expression, elevated lactate production and decreased intracellular glucose (suggesting increased glycolysis). Conclusions: Expression of activated Akt1 in MMTV-c-ErbB2 mice accelerates tumorigenesis with a reduced requirement for signaling through the EGFR family as well as a subset of downstream signaling molecules with a metabolic shift in the tumors from bitransgenic mice. The reduction in signaling downstream of ErbB2 when Akt is activated suggest a possible mechanism by which tumor cells can become resistant to ErbB2-targeted therapies, necessitating therapies that target oncogenic signaling events downstream of ErbB2.

Multifunctional roles of insulin-like growth factor binding protein 5 in breast cancer

2008-08-11

The insulin-like growth factor axis, which has been shown to protect cells from apoptosis, plays an essential role in normal cell physiology and in cancer development. The family of insulin-like growth factor binding proteins (IGFBPs) has been shown to have a diverse spectrum of functions in cell growth, death, motility, and tissue remodeling. Among the six IGFBP family members, IGFBP-5 has recently been shown to play an important role in the biology of breast cancer, especially in breast cancer metastasis; however, the exact mechanisms of action remain obscure and sometimes paradoxical. An in-depth understanding of IGFBP-5 would shed light on its potential role as a target for breast cancer therapeutics.

Enrichment with anti-cytokeratin alone or combined with anti-EpCAM antibodies significantly increases the sensitivity for circulating tumor cell detection in metastatic breast cancer patients

2008-08-07

IntroductionCirculating tumor cells (CTCs) can be detected in most cancer patients and they may offer to meet an existing medical need to monitor cancer patients during a course of treatment and for helping to determine recurrent disease. CTCs are rare in cancer patient blood and enrichment is necessary for sensitive CTCs detection. Most CTC enrichment technologies are based on binding to anti-EpCAM antibodies even though CTC identification criteria are cytokeratin positive (CK+), CD45 negative (CD45-) and DAPI (nuclear stain) positive (DAPI+). However, some tumor cells express low or no EpCAM. Here we present a highly sensitive and reproducible enrichment method that is based on binding to anti-CK alone or the combination of anti-CK and anti-EpCAM antibodies. Methods: Blood samples from 49 metastatic breast cancer patients were processed with the CellSearchTM system and in parallel by our CTC assay method. We used anti-CK alone or in combination with anti-EpCAM antibodies for CTC enrichment. Brightfield and fluorescence labeled anti-CK, anti-CD45 and DAPI (nuclear stain) images were used for CTC identification. The Ariol(R) system was used for automated cell image capture and analysis of CTCs on glass slides. Results: Our method has the capability to enrich three types of CTCs including CK+ & EpCAM+, CK+ & EpCAM-/low and CK-/low & EpCAM+ cells. In the blind method comparison, our anti-CK antibody enrichment method showed a significantly higher CTC positive rate (49% vs. 29%) and a larger dynamic CTC detected range (1 to 571 vs. 1 to 270) than that of the CellSearchTM system in the total 49 breast cancer patients. 15 to 111% more CTCs were detected by our method than with the CellSearchTM method in the patients with higher CTC counts (>20 CTCs per 7.5ml of blood). The three fluorescent and brightfield images capability of the Ariol(R) system reduced false positive CTC events according to the established CTC criteria. Conclusions: Our data indicate that the tumor specific intracellular CK marker can be used for efficient CTC enrichment. Enrichment with anti-CK alone or combined with anti-EpCAM antibodies significantly enhances assay sensitivity. The three fluorescent and brightfield superior images with the Ariol(R) system reduce false positive CTC events.

Prolactin receptor antagonism reduces the clonogenic capacity of breast cancer cells and potentiates doxorubicin and paclitaxel cytotoxicity

2008-08-05

IntroductionExogenous prolactin is mitogenic and antiapoptotic in breast cancer cells and over-expression of autocrine prolactin cDNA in breast cancer cell lines has been shown to stimulate their growth and protect against chemotherapy induced apoptosis. We examined the effects of the 'pure' prolactin receptor (PRLR) antagonist Delta1-9-G129R-hPrl (Delta1-9) on breast cancer cell cell number and clonogenicity, alone and in combination with chemotherapy. Methods: The effects of doxorubicin, paclitaxel and Delta1-9 on the growth of breast cancer cell lines (MCF-7, T47D, MDA-MB-453, MDA-MB-468 and SK-BR-3) in monolayer culture were assessed by sulphorhodamine B (SRB) assay. Effects on clonogenicity were assessed by soft agar assay for the cell lines and the mammosphere assay for disaggregated primary ductal carcinoma in situ (DCIS) samples. Dual fluorescence immunocytochemistry was used to identify subpopulations of cells expressing the PRLR and autocrine prolactin. Results: Delta1-9 as a single agent had no effect on cell number in monolayer culture, but potentiated the cytotoxic effects of doxorubicin and paclitaxel. Accordingly doxorubicin induced expression of prolactin mRNA and protein in all 5 breast cancer cell lines tested. Delta1-9 alone inhibited the clonogenicity in soft agar of cell lines by ~90% and of mammosphere forming efficiency of six disaggregated primary DCIS samples by a median of 56% (range 32 to 88%). Subpopulations of cells could be identified in the cell lines based on PRLR and prolactin expression. Conclusions: Autocrine prolactin appears to act as an inducible survival factor in a clonogenic sub-population of breast cancer cells. Thus the rational combination of cytotoxics and Delta1-9 may improve outcomes in breast cancer therapy by targeting this cell population.

Comparison of molecular phenotypes of ductal carcinoma in situ and invasive breast cancer

2008-08-05

IntroductionAt least four major categories of invasive breast cancer that are associated with different clinical outcomes have been identified by gene expression profiling: luminal A, luminal B, human epidermal growth factor receptor 2 (HER2) and basal-like. However, the prevalence of these phenotypes among cases of ductal carcinoma in situ (DCIS) has not been previously evaluated in detail. The purpose of this study was to compare the prevalence of these distinct molecular subtypes among cases of DCIS and invasive breast cancer. Methods: We constructed tissue microarrays (TMAs) from breast cancers that developed in 2,897 women enrolled in the Nurses' Health Study (1976-1996). TMA slides were immunostained for estrogen receptor (ER), progesterone receptor (PR), HER2, cytokeratin 5/6 (CK5/6), and epidermal growth factor receptor (EGFR). Using these immunostain results, cases were grouped into molecularly defined subtypes. Results: The prevalence of the distinct molecular phenotypes differed significantly between DCIS (n=272) and invasive breast cancers (n=2,249). The luminal A phenotype was significantly more frequent among invasive cancers (73.4%) than among DCIS lesions (62.5%) (p= 0.0002). In contrast, luminal B and HER2 molecular phenotypes were both more frequent among DCIS (13.2% and 13.6%, respectively) as compared to invasive tumors (5.2% and 5.7%, respectively) (p <0.0001). The basal-like phenotype was more frequent among the invasive cancers (10.9%) than DCIS (7.7%), although this difference was not statistically significant (p=0.15). High-grade DCIS and invasive tumors were more likely to be HER2 type and basal-like than low- or intermediate-grade lesions. Among invasive tumors, basal-like and HER2 type tumors were more likely to be >2 cm in size, high-grade, and have nodal involvement compared with luminal A tumors. Conclusions: The major molecular phenotypes previously identified among invasive breast cancers were also identified among cases of DCIS. However, the prevalence of the luminal A, luminal B, and HER2 phenotypes differed significantly between DCIS and invasive breast cancers.

Breast cancer susceptibility loci and mammographic density

2008-08-05

IntroductionRecently, the Breast Cancer Association Consortium (BCAC) conducted a multi-stage genome-wide association study and identified 11 single nucleotide polymorphisms (SNPs) associated with breast cancer risk. Given the high degree of heritability of mammographic density and its strong association with breast cancer, it was hypothesised that breast cancer susceptibility loci may also be associated with breast density and provide insight into the biology of breast density and how it influences breast cancer risk. Methods: We conducted an analysis in the Nurses' Health Study (n = 1121) to assess the relation between 11 breast cancer susceptibility loci and mammographic density. At the time of their mammogram, 217 women were premenopausal and 904 women were postmenopausal. We used generalised linear models adjusted for covariates to determine the mean percentage of breast density according to genotype. Results: Overall, no association between the 11 breast cancer susceptibility loci and mammographic density was seen. Among the premenopausal women, three SNPs (rs12443621 [TNRc9/LOC643714], rs3817198 [lymphocyte-specific protein-1] and rs4666451) were marginally associated with mammographic density (p < 0.10). All three of these SNPs showed an association that was consistent with the direction in which these alleles influence breast cancer risk. The difference in mean percentage mammographic density comparing homozygous wildtypes to homozygous variants ranged from 6.3 to 8.0%. None of the 11 breast cancer loci were associated with postmenopausal breast density. Conclusion: Overall, breast cancer susceptibility loci identified through a genome-wide association study do not appear to be associated with breast cancer risk.

Meta-analysis of gene expression profiles in breast cancer: toward a unified understanding of breast cancer subtyping and prognosis signatures

2008-07-28

IntroductionBreast cancer subtyping and prognosis have been studied extensively by gene expression profiling, resulting in disparate signatures with little overlap in their constituent genes. Although a previous study demonstrated a prognostic concordance among gene expression signatures, it was limited to only one dataset and did not fully elucidate how the different genes were related to one another nor did it examine the contribution of well-known biological processes of breast cancer tumorigenesis to their prognostic performance.MethodTo address the above issues and to further validate these initial findings, we performed the largest meta-analysis of publicly available breast cancer gene expression and clinical data, which are comprised of 2,833 breast tumors. Gene coexpression modules of three key biological processes in breast cancer (namely, proliferation, estrogen receptor [ER], and HER2 signaling) were used to dissect the role of constituent genes of nine prognostic signatures. Results: Using a meta-analytical approach, we consolidated the signatures associated with ER signaling, ERBB2 amplification, and proliferation. Previously published expression-based nomenclature of breast cancer 'intrinsic' subtypes can be mapped to the three modules, namely, the ER-/HER2- (basal-like), the HER2+ (HER2-like), and the low- and high-proliferation ER+/HER2- subtypes (luminal A and B). We showed that all nine prognostic signatures exhibited a similar prognostic performance in the entire dataset. Their prognostic abilities are due mostly to the detection of proliferation activity. Although ER- status (basal-like) and ERBB2+ expression status correspond to bad outcome, they seem to act through elevated expression of proliferation genes and thus contain only indirect information about prognosis. Clinical variables measuring the extent of tumor progression, such as tumor size and nodal status, still add independent prognostic information to proliferation genes. Conclusion: This meta-analysis unifies various results of previous gene expression studies in breast cancer. It reveals connections between traditional prognostic factors, expression-based subtyping, and prognostic signatures, highlighting the important role of proliferation in breast cancer prognosis.