Primary Breast Cancer Biopsies Research Articles

Tissue factor in cancer progression and angiogenesis

Constitutive expression of tissue factor (TF) by cancer cells triggers local and systemic activation of the coagulation cascade and is a major cause of cancer-associated thrombosis. Primary breast cancer biopsies show a marked upregulation of TF and protease activated receptor (PAR) 2, as well as increased TF cytoplasmic domain phosphorylation that is correlated with cancer relapse. TF signaling involving PAR2 and integrins has multiple effects on angiogenesis and tumor progression. The non-coagulant, alternatively spliced form of TF retains an integrin-binding site and, upon deposition into the tumor stroma, stimulates angiogenesis by ligating endothelial integrins α vβ 3 and α 6β 1. On tumor cells, full-length TF is constitutively associated with laminin-binding β 1 integrins that support TF-VIIa-PAR2 signaling leading to upregulation of pro-angiogenic and immune modulatory cytokines and growth factors. Deficiency of PAR2, but not of the thrombin receptor PAR1, delays spontaneous breast cancer development and the angiogenic switch in mice. In addition, human xenograft breast cancer growth and angiogenesis is suppressed by selective antibody inhibition of TF-VIIa-PAR2 signaling, but not by blocking TF initiated coagulation. Thus, interruption of TF signaling represents a potential anti-angiogenic strategy that does not carry an increased risk of bleeding associated with prolonged inhibition of the TF coagulation pathway.

Antiestrogens Induce Transforming Growth Factor β–Mediated Immunosuppression in Breast Cancer

Antiestrogens are universally used to treat estrogen receptor--positive breast cancer, but relapses occur commonly due to the development of drug resistance. The ability of antiestrogen to induce transforming growth factor beta (TGFbeta) in breast cancer cells may be relevant to the emergence of resistance, not only at the level of cell autonomous effects of TGFbeta on cancer progression but also at the level of its effects on the host immune system. To evaluate the potential role of tumor-derived, antiestrogen-induced TGFbeta as an immune suppressor, we established in vitro mixed lymphocyte tumor reactions (MLTR) using MCF-7 cells and peripheral blood mononuclear cells (PBMC), as well as tumor tissue and autologous tumor infiltrating lymphocytes (TIL) obtained from primary breast cancer biopsies. In allogeneic MLTR, antiestrogen-treated MCF-7 cells caused downregulation of the effector molecules granzyme B, perforin, and Fas ligand in CD8(+) T cells, and suppressed the generation of cytotoxic effector cells in a TGFbeta-dependent manner. Furthermore, we documented induction of regulatory T cells in CD4(+) T cells, based on Foxp3 expression and T-cell activation in cocultures. In autologous MLTR, antiestrogen treatment gave rise to enhanced Foxp3 expression of TIL/PBMC and decreased the number of apoptotic tumor cells. These effects were reversed by addition of a TGFbeta neutralizing antibody. Our findings offer evidence that antiestrogen induces immunosuppression in the tumor microenvironment, through a TGFbeta-dependent mechanism that may contribute to the development of antiestrogen resistance in breast cancer.

Netrin-1: a promising therapeutic target in metastatic breast cancer. Expression analysis in localized and metastatic breast tumors and preclinical validation.

Abstract Abstract #3075 Netrin-1, an axon navigation cue was proposed to play a crucial role during colorectal tumorigenesis by regulating apoptosis. The netrin-1 receptors DCC and UNC5H belong to the family of dependence receptors that share the ability to induce apoptosis in the absence of their ligand. Cells expressing one of these dependence receptors are thus dependent on ligand availability to survive. Such a trait confers on these receptors a tumor suppressor activity by preventing uncontrolled cell proliferation.
 We have previously shown in breast cancer that gaining autocrine expression of the ligand netrin-1 would represent for cancer cells a selective advantage. Human and murine netrin-1-expressing mammary metastatic cell lines undergo apoptosis when netrin-1 expression is experimentally decreased or when decoy soluble receptor ectodomains are used. Such treatments prevent metastasis formation in a syngenic mouse model of lung colonization by a mammary cancer cell line (Fitamant et al., PNAS 2008, vol 105).
 In order to assess the clinical importance of netrin-1 expression in human breast cancer, we quantified by Q-RT-PCR the mRNA levels of netrin-1 in primary breast cancer biopsies and studied their correlation to pathological and clinical data. We used frozen tumor samples from 120 breast cancer patients treated in our institution between 1993 and 2000. At diagnosis, median age was 50, median tumor size was 40 mm. 11% of the patients had SBR1 tumor, 46% had SBR2 tumor and 43% had SBR3 tumor. Estrogen receptor was positive for 62% of the patients. 17% of the patients were pN0, M0; 71% were pN+, M0 and 12% already had distant metastasis (M+) at time of diagnosis. Although there is no correlation between netrin-1 expression and tumor size, histological SBR grade or hormone receptor status, there is a strong correlation between high netrin-1 expression and the presence of nodal involvement (p=0,007) or distant metastasis at the time of diagnosis (p<0,0001). We hypothesize that high netrin-1 expression allows an early and rapid metastatic dissemination during tumor development. We are currently exploring netrin-1 expression levels at different metastatic sites and are completing survival analysis of our cohort of patients.
 In parallel, preclinical mice models of xenografts either of human breast cancer cell lines or fresh human breast tumors were used to evaluate netrin-1 targeting strategy. The preliminary results show that disrupting netrin-1 activity appears to be more efficient than a conventional Adryamicin-Cyclophosphamide treatment in decreasing tumor sizes and suppressing lung metastasis.
 These results support netrin-1 as a marker of metastatic dissemination and as a promising therapeutic target in breast cancer progression. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3075.

Phosphorylation of Estrogen Receptor-α at Ser167 Is Indicative of Longer Disease-Free and Overall Survival in Breast Cancer Patients

Ser(167) was first identified as a major phosphorylation site of the estrogen receptor -alpha (ER) positive in the MCF7 breast cancer cell line. Subsequent studies have shown that Ser(167) phosphorylation is important in the regulation of ER activity and have identified p90RSK and AKT as protein kinases that phosphorylate Ser(167). The purpose of this study was to determine the importance of Ser(167) phosphorylation in breast cancer progression. Immunohistochemical staining of primary breast cancer biopsies (n = 290) was carried out using antibodies specific for ER phosphorylated at Ser(167) and for phosphorylated p44/p42 mitogen-activated protein kinase (MAPK), phosphorylated p90RSK, and phosphorylated AKT. In ER-positive breast cancer patients, Ser(167) phosphorylation was associated with low tumor grade (P = 0.011), lymph node negativity (P = 0.034), and relapse-free (P = 0.006) and overall (P = 0.023) survival. Further, Ser(167) phosphorylation was strongly associated with phosphorylated p90RSK (P < 0.001), previously shown to phosphorylate Ser(167) in vitro, as well as being associated with phosphorylated MAPK (P < 0.0005). The activities of both kinases also seemed to be indicative of better prognosis. There was, however, no association between HER2 positivity and Ser(167) phosphorylation nor were the activities of MAPK or p90RSK associated with HER2 status, suggesting that other cell surface receptors may be important in regulating these activities in breast cancer. These findings show that phosphorylation at Ser(167) of ER predicts for likelihood of response of ER-positive breast cancer patients to endocrine therapies.

Hypermethylation of theBreast Cancer–Associated Gene 1Promoter Does Not Predict Cytologic Atypia or Correlate with Surrogate End Points of Breast Cancer Risk

Mutation of the breast cancer-associated gene 1 (BRCA1) plays an important role in familial breast cancer. Although hypermethylation of the BRCA1 promoter has been observed in sporadic breast cancer, its exact role in breast cancer initiation and association with breast cancer risk is unknown. The frequency of BRCA1 promoter hypermethylation was tested in (a) 14 primary breast cancer biopsies and (b) the initial random periareolar fine-needle aspiration (RPFNA) cytologic samples obtained from 61 asymptomatic women who were at increased risk for breast cancer. BRCA1 promoter hypermethylation was assessed from nucleotide -150 to nucleotide +32 relative to the transcription start site. RPFNA specimens were stratified for cytologic atypia using the Masood cytology index. BRCA1 promoter hypermethylation was observed at similar frequency in nonproliferative (normal; Masood <or=10: 18%, 2 of 11), hyperplastic (Masood 11-13: 15%, 6 of 41), and atypical cytology (Masood 14-17: 22%, 4 of 18; P = 0.79). BRCA1 promoter hypermethylation was not associated with (a) family history of breast or ovarian cancer or (b) calculated Gail or BRCAPRO risk score. BRCA1 promoter hypermethylation was associated with (a) age (P = 0.028) and (b) the combined frequency of promoter hypermethylation of the retinoic acid receptor-beta2 (RARB) gene, estrogen receptor-alpha (ESR1) gene, and p16 (INK4A) gene (P = 0.003). These observations show that BRCA1 promoter hypermethylation (a) is not associated with breast cancer risk as measured by mathematical risk models and (b) does not predict mammary atypia in RPFNA cytologic samples obtained from high-risk women.

Phosphorylation of tyrosine 1248-ERBB2 measured by chemiluminescence-linked immunoassay is an independent predictor of poor prognosis in primary breast cancer patients

ERBB2 (HER2/Neu) gene amplification and overexpression is associated with increased risk of metastases and shorter survival in breast cancer. Tyrosine 1248 is a major phosphorylation site of ERBB2 and reflects the activation status of the receptor. The aim of this study was to investigate the relationships between quantitative levels of pY1248-ERBB2 (p-ERBB2) and the expression of epidermal growth factor receptor (EGFR)-family members, and whether p-ERBB2 could provide additional prognostic value compared with established prognostic markers. For this purpose we developed a highly sensitive chemiluminescence-linked immunoassay (CLISA) and detected p-ERBB2 levels in 70 primary breast cancer biopsies. Phosphorylated ERBB2 correlated with EGFR and ERBB2, and inversely with oestrogen receptor (ER), progesterone receptor (PgR) and ERBB4 expression levels. Additionally, p-ERBB2 was associated with poor clinical outcome in univariate and multivariate Cox regression analysis. Further studies are needed to evaluate the predictive value of p-ERBB2.

Does a truncated form of the transcription factor Ets1 exist in breast cancer cells?

Does a truncated form of the transcription factor Ets1 exist in breast cancer cells?

Expression and prognostic value of lactoferrin mRNA isoforms in human breast cancer

We investigated the expression levels of human lactoferrin (Lf), a steroid hormone-inducible gene product the expression of which is often altered during oncogenesis, and of Delta-lactoferrin (DeltaLf), its alternative isoform, which has been shown to be absent from tumor cell lines in commonly used human breast epithelial cell lines, using semiquantitative RT-PCR. Both mRNAs were detected but with levels of expression lower than those found in normal breast epithelial cells. This downregulation was much more visible for DeltaLf since its expression was either significantly diminished (BT-20, MCF-7 cell lines) or practically absent (MDA-MB-231, T-47D, HBL 100 cell lines). In order to determine whether Lf gene products are useful prognosic tools, we further analyzed their expression levels in 99 primary breast cancer biopsies. DeltaLf transcripts were found in all of the samples, whereas Lf transcripts were found in 88% of them. Lf and DeltaLf expression levels were positively correlated (p = 0.003). Lf expression was related to tumor type with a higher recovery in lobular-type tumors (p = 0.04). DeltaLf expression was related to the histoprognostic grading (p = 0.02). In univariate analyses, DeltaLf and Lf expressions were prognosis parameters, high concentrations being associated with a longer overall survival.

Genes that co-cluster with estrogen receptor alpha in microarray analysis of breast biopsies.

The estrogen receptor plays a critical role in the pathogenesis and clinical behavior of breast cancer. To better understand the molecular basis of estrogen-dependent forms of this disease we studied gene expression profiles from 53 primary breast cancer biopsies. Gene expression data for more than 7000 genes were generated from each tumor sample with oligo microarrays. A standard correlation-clustering algorithm identified 18 genes that co-clustered with estrogen receptor alpha. Eleven of these genes had previously been associated with estrogen regulation or breast tumorigenesis including trefoil factor 1 and estrogen regulated LIV-1. Additional study of these 18 genes may further delineate the role of estrogen receptor in breast cancer, generate new predictive biomarkers for response to endocrine therapies and identify novel therapeutic targets.

Algorithms for quantitation of protein expression variation in normal versus tumor tissue as a prognostic factor in cancer: Met oncogene expression, and breast cancer as a model

Immunohistochemistry and immunofluorescence (IF) assays frequently rely on subjective observer evaluation for grading. The aim of our study was to develop an objective quantitative index based on confocal laser scanning microscopy (CLSM) and image analysis of an IF assay to determine alteration in protein expression levels in normal versus tumor tissue. The relative levels of Met expression, a prognostic factor in breast cancer, were used as a model for evaluating image analysis algorithms. Primary human breast cancer biopsies were collected. Sections containing tumor and adjacent uninvolved normal regions were immunostained for Met and digital images were acquired by CLSM. Subsequently, the digital data were manipulated using several different algorithms to calculate prognostic indexes. The results were correlated with the clinical outcome to determine the prognostic value of these indexes. Different algorithms were used to obtain quantitative indexes to evaluate the relative levels of Met expression. We report a statistical correlation between patient prognosis and relative Met level in normal versus tumor tissue as determined by three distinct algorithms using Kaplan-Meier analysis (log-rank): calculations based on intensity levels differences DV (P = 0.002), DIntensity (P = 0.014), and entropy divergence (Dentropy; P = 0.0023). Using adjacent normal tissue as an internal reference, a quantitative index of tumor Met level divergence can be objectively determined to have a prognostic value. Moreover, this methodology can be used for other proteins in a variety of different diseases.

Estrogen and progesterone receptor mrna levels in primary breast cancer: Association with patient survival and other clinical and tumor features

The relative expression of estrogen receptor (ER) and progesterone receptor (PR) mRNA transcripts was measured in 71 primary breast-cancer biopsies. ER and PR binding activity were estimated in parallel by the dextran-coated-charcoal method. There was a close correlation between the amount of ER mRNA and estradiol binding activity. Tumors from post-menopausal patients contained higher levels of ER mRNA than those from pre-menopausal patients. Northern-blot analysis indicated the presence of a major band of 6.3 kb in all ER mRNA-positive tumors. Some tumors showed, in addition, 3.7- and 2.4-kb transcripts. PR binding activity and overall PR mRNA levels correlated moderately. PR mRNA and ER mRNA were associated. Four PR mRNA species with estimated sizes of 11.4, 4.5, 3.7 and 2.5 kb were detected in 14% of the PR mRNA-positive tumors. The 3.7-kb transcript was detected to varying degrees in all PR mRNA-positive biopsies, accompanied in some tumors by the 2.5-kb species. ER and PR mRNA levels > or = 50 pg/5 micrograms total RNA correlated with prolonged survival of the patients. In addition, high ER mRNA levels were associated with absence or minimal necrosis and vascular invasion together with absence or minimal level of tumor lymphocytic infiltration, but not with age, clinical stage, tumor size or overexpression of c-myc or c-erbB-2 mRNA. PR mRNA was not statistically associated with any of the above clinicopathological features. A bivariate analysis showed that both ER and PR mRNA levels were able to predict overall survival independently of the lymph-node status.

Immunoradiometric detection of pS2 and total cathepsin D in primary breast cancer biopsies: their correlation with steroid receptors.

Commercially available immunoradiometric assays were used for pS2 and total cathepsin D determination in the cytosol fraction obtained from 266 primary breast cancers. We show that pS2 and cathepsin D values were significantly associated (Spearman's rank correlation: P < 0.0001) in tumours from lymph node-positive patients (N+), while such association did not reach significance in tumours taken from patients with negative lymph nodes (N-). Moreover, cathepsin D concentrations in pS2-rich tumours (pS2 above the median value, 5 ng mg-1 protein) were significantly higher (Mann-Whitney-Wilcoxon's rank-sum test: P = 0.00001) than those obtained in the samples expressing less than 5 ng of pS2 per mg of protein. pS2 was also correlated to both the oestrogen receptor (ER) (Spearman's rank correlation: P < 0.0001) and the progesterone receptor (PR) (Spearman's rank correlation: P = 0.022). No significant differences in the expression of pS2 and cathepsin D taken from N+ and N- patients were found. Furthermore, no significant differences in pS2 and cathepsin D expression were obtained by stratifying tumours on the basis of their size (T). pS2 and cathepsin D values obtained in ER-positive/PR-positive tumours did not significantly differ from the values obtained in ER-positive/PR-negative and in ER-negative/PR-positive tumours. We conclude that pS2 could have a role in cathepsin D expression, and that it can be used in the assessment of a functioning oestrogen response machinery in those tumours that express only ER.

Relationships between tamoxifen binding proteins in primary breast cancer biopsies

Using high-resolution isoelectric focusing gel electrophoresis (IEF), two tamoxifen binding sites (TBS) with isoelectric point (pI) values of 4.5 and 4.3 were identified, with different affinities for tamoxifen. The form at pI 4.3 (HTBS) displayed high affinity for the ligand (kD ≈ 5 nM), while the protein at pI 4.5 (LTBS) had lower affinity (kD ≈ 50 nM). LTBS was found in the microsomal fraction and HTBS in the cytosol. Of a total of 319 tumours studied, 257 were oestrogen receptor (ER) positive and 106 HTBS positive. In this combined group, thus able to bind tamoxifen either through the presence of ER or HTBS (or both), ER and PR were both negatively correlated with HTBS ( P < 0.0001). The oestrogen-induced protein pS2 was assayed in 92 of the 319 tumours, and was also negatively ( P < 0.0001) correlated with HTBS. The levels of HTBS were similar between infiltrating ductal carcinomas without special features (NOS) and non-NOS forms. However, HTBS concentrations were significantly higher in poorly differentiated grade 3 carcinomas than grade 2 ( P < 0.05) and grade 1 ( P < 0.01) forms. Conversely, ER concentration was lower in grade 3 than grade 1 forms ( P < 0.05). Both the relationship between high affinity TBS and ER and the high concentration of HTBS in ER-poor grade 3 carcinomas may have a bearing on the known variability of tumour response to endocrine therapy and prognosis.

Expression of activated oncogenes in the murine mammary gland: transgenic models for human breast cancer.

Breast cancer is the leading cause of death among non-smoking women and thus has been the focus of intensive research. It has been generally accepted that the deregulation of oncogenes or their regulators play a pivotal role in progression of this prevalent disease. For example, amplification and overexpression of a number of oncogenes has been observed in a proportion of primary breast cancer biopsies. More recently, there has also been reports of inactivation tumor suppressor genes in human breast cancer. While there is compelling evidence for a role of these genes in breast cancer tumor progression due to limitations inherent in these studies it is difficult to establish a direct causal association between expression of a certain oncogene and tumor progression. For this reason many groups have employed the transgenic mouse as a model system to directly study effects of oncogene expression in the murine mammary gland. This review will attempt to highlight some of the important lessons and potential applications that have emerged from the study of oncogene expression in the mammary epithelium of transgenic mice. The utility of the transgenic system to assess the transforming potential of oncogenes, to investigate the multi-step nature of malignant progression, and to be used as models for therapeutic intervention will be discussed.

Cellular effects of tamoxifen in primary breast cancer.

We have investigated the effect of tamoxifen on the biological characteristics of the primary tumour in 33 patients with breast cancer. All patients had pre-treatment biopsy of the primary breast cancer and subsequent trucut biopsy of the primary tumour after 1-4 months on tamoxifen therapy. The staining patterns in the primary tumour of each of the tumour antigens 115D8, DF3, NCRC-11, and carcinoembryonic antigen (CEA) were unaffected by tamoxifen therapy: there was 16%-31% change before vs. during tamoxifen therapy, but this did not reach significance for any of the four antigens. Comparison of the pattern of staining between the four antigens showed 115D8, DF3, and NCRC-11 to be similar to each other both before and during tamoxifen therapy. All three antigens were significantly different from the staining pattern shown by CEA before and during therapy. Tumour antigen expression pre-treatment did not correlate with therapeutic response for any of the four antigens. However, NCRC-11 staining during tamoxifen therapy did correlate with response (p = 0.004). There was no significant difference in oestrogen receptor status before and during tamoxifen therapy, both of which correlated with response. DNA ploidy of the tumour before tamoxifen did not correlate with response to therapy, but there was a weak correlation between response and DNA ploidy measured during tamoxifen therapy. In only a minority of tumours (up to 30%) did tamoxifen exert an effect on the biological characteristics studied. Changes in these biological markers were mainly in tumours which responded to tamoxifen, and the changes seen were a tendency to greater expression of differentiation antigens.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of the menstrual cycle on the concentrations of estrogen and progesterone receptors in primary breast cancer biopsies.

There is controversy in the literature regarding the effects of endogenous hormones on estrogen receptors (ER) and progesterone receptors (PR) in young women with breast cancer. We studied 117 young women with primary breast cancer and assessed their breast biopsies for ER and PR. The women had a record of their last menstrual period prior to breast biopsy. The menstrual cycle was divided into four phases--early proliferative (days 1-7), late proliferative (days 8-15), early secretory (days 16-22), and late secretory (days 23-30). There were lower levels of both ER and PR in biopsies excised during the early secretory phase than in other phases of the cycle; early proliferative phase receptor positive medians of ER = 77 fmol/mg protein and PR = 467 fmol/mg protein fell to ER = 28 fmol/mg and PR = 128 fmol/mg protein in the early secretory phase.

Enzyme activity, acidic nuclear proteins, and prognosis in human breast cancer.

The activities of the enzymes lactate dehydrogenase, 6-phosphogluconate dehydrogenase, and phosphohexose isomerase in primary human breast cancer biopsies are shown to be related to the time between mastectomy and recurrence of the cancer. These enzymes have higher activity in malignant breast tissues generally than in non-malignant breast tissues. In tumours from patients with long free periods these differences are not apparent.Evidence is presented which suggests that two different types of breast cancer can be distinguished according to the relative amounts of phosphohexose isomerase and acidic nuclear proteins. It is suggested that this difference may be related to hormone responsiveness.