Expression profiling to predict outcome in breast cancer: the influence of sample selection

Gene expression profiling of tumors using DNA microarrays is a promising method for predicting prognosis and treatment response in cancer patients. It was recently reported that expression profiles of sporadic breast cancers could be used to predict disease recurrence better than currently available clinical and histopathological prognostic factors. Having observed an overlap in those data between the genes that predict outcome and those that predict estrogen receptor-α status, we examined their predictive power in an independent data set. We conclude that it may be important to define prognostic expression profiles separately for estrogen receptor-α-positive and estrogen receptor-α-negative tumors

Basal-like phenotype is not associated with patient survival in estrogen-receptor-negative breast cancers

INTRODUCTION: Basal-phenotype or basal-like breast cancers are characterized by basal epithelium cytokeratin (CK5/14/17) expression, negative estrogen receptor (ER) status and distinct gene expression signature. We studied the clinical and biological features of the basal-phenotype tumors determined by immunohistochemistry (IHC) and cDNA microarrays especially within the ER-negative subgroup. METHODS: IHC was used to evaluate the CK5/14 status of 445 stage II breast cancers. The gene expression signature of the CK5/14 immunopositive tumors was investigated within a subset (100) of the breast tumors (including 50 ER-negative tumors) with a cDNA microarray. Survival for basal-phenotype tumors as determined by CK5/14 IHC and gene expression signature was assessed. RESULTS: From the 375 analyzable tumor specimens, 48 (13%) were immunohistochemically positive for CK5/14. We found adverse distant disease-free survival for the CK5/14-positive tumors during the first years (3 years hazard ratio (HR) 2.23, 95% confidence interval (CI) 1.17 to 4.24, p = 0.01; 5 years HR 1.80, 95% CI 1.02 to 3.15, p = 0.04) but the significance was lost at the end of the follow-up period (10 years HR 1.43, 95% CI 0.84 to 2.43, p = 0.19). Gene expression profiles of immunohistochemically determined CK5/14-positive tumors within the ER-negative tumor group implicated 1,713 differently expressed genes (p < 0.05). Hierarchical clustering analysis with the top 500 of these genes formed one basal-like and a non-basal-like cluster also within the ER-negative tumor entity. A highly concordant classification could be constructed with a published gene set (Sorlie's intrinsic gene set, concordance 90%). Both gene sets identified a basal-like cluster that included most of the CK5/14-positive tumors, but also immunohistochemically CK5/14-negative tumors. Within the ER-negative tumor entity there was no survival difference between the non-basal and basal-like tumors as identified by immunohistochemical or gene-expression-based classification. CONCLUSION: Basal cytokeratin-positive tumors have a biologically distinct gene expression signature from other ER-negative tumors. Even if basal cytokeratin expression predicts early relapse among non-selected tumors, the clinical outcome of basal tumors is similar to non-basal ER-negative tumors. Immunohistochemically basal cytokeratin-positive tumors almost always belong to the basal-like gene expression profile, but this cluster also includes few basal cytokeratin-negative tumors

3-Phosphoinositide–Dependent Kinase 1 Potentiates Upstream Lesions on the Phosphatidylinositol 3-Kinase Pathway in Breast Carcinoma

Lesions of ERBB2, PTEN, and PIK3CA activate the phosphati- dylinositol 3-kinase (PI3K) pathway during cancer development by increasing levels of phosphatidylinositol-3,4,5-triphosphate (PIP3). 3-Phosphoinositide-dependent kinase 1 (PDK1) is the first node of the PI3K signal output and is required for activation of AKT. PIP3 recruits PDK1 and AKT to the cell membrane through interactions with their pleckstrin homology domains, allowing PDK1 to activate AKT by phosphorylating it at residue threonine-308. We show that total PDK1 protein and mRNA were overexpressed in a majority of human breast cancers and that 21% of tumors had five or more copies of the gene encoding PDK1, PDPK1. We found that increased PDPK1 copy number was associated with upstream pathway lesions (ERBB2 amplification, PTEN loss, or PIK3CA mutation), as well as patient survival. Examination of an independent set of breast cancers and tumor cell lines derived from multiple forms of human cancers also found increased PDK1 protein levels associated with such upstream pathway lesions. In human mammary cells, PDK1 enhanced the ability of upstream lesions to signal to AKT, stimulate cell growth and migration, and rendered cells more resistant to PDK1 and PI3K inhibition. After orthotopic transplantation, PDK1 overexpression was not oncogenic but dramatically enhanced the ability of ERBB2 to form tumors. Our studies argue that PDK1 overexpression and increased PDPK1 copy number are common occurrences in cancer that potentiate the oncogenic effect of upstream lesions on the PI3K pathway. Therefore, we conclude that alteration of PDK1 is a critical component of oncogenic PI3K signaling in breast cancer

Integrated molecular pathway analysis informs a synergistic combination therapy targeting PTEN/PI3K and EGFR pathways for basal-like breast cancer

The basal-like breast cancer (BLBC) subtype is characterized by positive staining for basal mammary epithelial cytokeratin markers, lack of hormone receptor and HER2 expression, and poor prognosis with currently no approved molecularly-targeted therapies. The oncogenic signaling pathways driving basal-like tumorigenesis are not fully elucidated. Methods One hundred sixteen unselected breast tumors were subjected to integrated analysis of phosphoinositide 3-kinase (PI3K) pathway related molecular aberrations by immunohistochemistry, mutation analysis, and gene expression profiling. Incidence and relationships between molecular biomarkers were characterized. Findings for select biomarkers were validated in an independent series. Synergistic cell killing in vitro and in vivo tumor therapy was investigated in breast cancer cell lines and mouse xenograft models, respectively. Results Sixty-four % of cases had an oncogenic alteration to PIK3CA, PTEN, or INPP4B; when including upstream kinases HER2 and EGFR, 75 % of cases had one or more aberration including 97 % of estrogen receptor (ER)-negative tumors. PTEN-loss was significantly associated to stathmin and EGFR overexpression, positivity for the BLBC markers cytokeratin 5/14, and the BLBC molecular subtype by gene expression profiling, informing a potential therapeutic combination targeting these pathways in BLBC. Combination treatment of BLBC cell lines with the EGFR-inhibitor gefitinib plus the PI3K pathway inhibitor LY294002 was synergistic, and correspondingly, in an in vivo BLBC xenograft mouse model, gefitinib plus PI3K-inhibitor PWT-458 was more effective than either monotherapy and caused tumor regression. Conclusions Our study emphasizes the importance of PI3K/PTEN pathway activity in ER-negative and basal-like breast cancer and supports the future clinical evaluation of combining EGFR and PI3K pathway inhibitors for the treatment of BLBC. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2609-2) contains supplementary material, which is available to authorized users.BioMed Central open acces

Recurrent gross mutations of the PTEN tumor suppressor gene in breast cancers with deficient DSB repair

Basal-like breast cancer (BBC) is a subtype of breast cancer with poor prognosis. Inherited mutations of BRCA1, a cancer susceptibility gene involved in double-strand DNA break (DSB) repair, lead to breast cancers that are nearly always of the BBC subtype; however, the precise molecular lesions and oncogenic consequences of BRCA1 dysfunction are poorly understood. Here we show that heterozygous inactivation of the tumor suppressor gene Pten leads to the formation of basal-like mammary tumors in mice, and that loss of PTEN expression is significantly associated with the BBC subtype in human sporadic and BRCA1-associated hereditary breast cancers. In addition, we identify frequent gross PTEN mutations, involving intragenic chromosome breaks, inversions, deletions and micro copy number aberrations, specifically in BRCA1-deficient tumors. These data provide an example of a specific and recurrent oncogenic consequence of BRCA1-dependent dysfunction in DNA repair and provide insight into the pathogenesis of BBC with therapeutic implications. These findings also argue that obtaining an accurate census of genes mutated in cancer will require a systematic examination for gross gene rearrangements, particularly in tumors with deficient DSB repair

Is the small heat shock protein αB-crystallin an oncogene?

In the last 5 years, global gene expression profiling has allowed for the subclassification of the heterogeneous disease of breast cancer into new subgroups with prognostic significance. However, for most subgroups, the nature of the contributions of individual genes to the clinical phenotypes remains largely unknown. In this issue of the JCI, Moyano and colleagues further examine the oncogenic potential of the small heat shock protein α-basic–crystallin, commonly expressed in tumors of the basal-like breast cancer subtype associated with poor prognosis, and show that it is an oncogenic protein in the breast

Vascular endothelial growth factor is a strong predictor of early distant recurrences in a prospective study of premenopausal women with lymph-node negative breast cancer

We investigate the prognostic significance of the pro-angiogenic cytokine vascular endothelial growth factor (VEGF), urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1), and S-phase fraction (SPF) for distant disease free survival (DDFS) in 219 premenopausal patients with node-negative breast cancer (NNBC). In univariate analysis significantly shorter DDFS was observed for patients with high VEGF (p = 0.006), high uPA (p = 0.001). and high SPF (p < 0.001). The prognostic significance of VEGF varied over time being very, strong for early relapses (0-2.25 years follow-up) (HR = 7.9: p = 0.006) while no difference was seen in the subsequent follow-up period (HR = 1.3: p = 0.62). In a series of bivariate analyses VEGF provided prognostic information during the whole observation period (0-72 months) in addition to age, tumor size, oestrogen receptor (ER), progesterone receptor (PgR), and uPA. Also this effect was more pronounced during the first follow-up period suggesting VEGF as a marker of early recurrences. (C) 2008 Elsevier Ltd. All rights reserved