Gene expression profiling and histopathological characterization of triple-negative/basal-like breast carcinomas

Introduction  

Breast cancer is a heterogeneous group of tumors, and can be subdivided on the basis of histopathological features, genetic alterations and gene-expression profiles. One well-defined subtype of breast cancer is characterized by a lack of HER2 gene amplification and estrogen and progesterone receptor expression ('triple-negative tumors'). We examined the histopathological and gene-expression profile of triple-negative tumors to define subgroups with specific characteristics, including risk of developing distant metastases.     

Glycan gene expression signatures in normal and malignant breast tissue; possible role in diagnosis and progression

Glycosylation is the stepwise procedure of covalent attachment of oligosaccharide chains to proteins or lipids, and alterations in this process have been associated with malignant transformation. Simultaneous analysis of the expression of all glycan-related genes clearly gives the advantage of enabling a comprehensive view of the genetic background of the glycobiological changes in cancer cells. Studies focusing on the expression of the whole glycome have now become possible, which prompted us to review the present knowledge on glycosylation in relation to breast cancer diagnosis and progression, in the light of available expression data from tumors and breast tissue of healthy individuals. We used various data resources to select a set of 419 functionally relevant genes involved in synthesis, degradation and binding of N-linked and O-linked glycans, Lewis antigens, glycosaminoglycans (chondroitin, heparin and keratan sulfate in addition to hyaluronan) and glycosphingolipids. Such glycans are involved in a number of processes relevant to carcinogenesis, including regulation of growth factors/growth factor receptors, cell–cell adhesion and motility as well as immune system modulation. Expression analysis of these glycan-related genes revealed that mRNA levels for many of them differ significantly between normal and malignant breast tissue. An associative analysis of these genes in the context of current knowledge of their function in protein glycosylation and connection(s) to cancer indicated that synthesis, degradation and adhesion mediated by glycans may be altered drastically in mammary carcinomas. Although further analysis is needed to assess how changes in mRNA levels of glycan genes influence a cell's glycome and the precise role that such altered glycan structures play in the pathogenesis of the disease, lessons drawn from this study may help in determining directions for future research in the rapidly-developing field of glycobiology.     

P-cadherin expression and basal-like subtype in breast cancers

Breast cancer is considered as one of the multifactorial diseases. The aim of the current study is to investigate the association between P-cadherin and molecular subtypes of breast cancer, especially the basal-like subtype. Two hundred and thirteen breast–invasive ductal carcinomas were involved in this study. The expressions of P-cadherin were detected via immunohistochemistry. The 213 cases were divided into luminal A, luminal B, HER2 overexpression subtype, and normal breast-like and basal-like subtypes according to the standard of molecular breast cancer subtypes. In addition, the expressions of CK5/6 and CK14 were detected to distinguish between the normal breast-like and the basal-like subtypes. P-cadherin expression was found in 91 cases of 213 breast–invasive ductal carcinomas, with a positive rate of 42.7 %. P-cadherin correlated negatively with estrogen receptor (ER) (p = 0.001) and progesterone receptor (p = 0.001), whereas it positively correlated with histologic grade (p = 0.003), NPI (p = 0.005), p53 (p = 0.038), and Ki67 (p = 0.022). P-cadherin expression showed a strong correlation with recurrence and distant metastasis (p = 0.009), and invasion of the vascular and soft tissues (p = 0.004). Moreover, P-cadherin expression existed in the basal-like and non-basal-like subtypes. During prognosis, P-cadherin expression was associated with decreased disease-free survival in patients (p = 0.009) and overall survival (OS) (p = 0.005). In addition, multivariate analysis showed that tumor grade (p = 0.021), ER (p = 0.015), clinical stage (p = 0.001), and P-cadherin (p = 0.033) were significant predictors of OS. The current data suggest that P-cadherin may be used to distinguish the basal-like subtype and to predict the outcome in view of the relationship with DFS and OS. Furthermore, P-cadherin expression may be useful in making treatment decisions.     

Nuclear CSPP1 expression defined subtypes of basal-like breast cancer

Background:

The multi-exon CSPP1 gene, encoding for centrosome and microtubule-associated proteins involved in ciliogenesis and cell division, is a candidate oncogene in luminal breast cancer but expression of CSPP1 proteins remained unexplored.

Methods:

CSPP1 gene and protein expression was examined in normal mammary tissue, human breast cancer cell lines, and primary breast cancer biopsies from two patient cohorts. Cell type and epitope-dependent subcellular-specific CSPP1 staining pattern in normal mammary gland epithelium and cancer biopsies were correlated to molecular and clinical parameters.

Results:

A novel, nuclear localised CSPP1 isoform was exclusively detected in luminal epithelial cells, whereas cytoplasmic CSPP-L was generally expressed in normal mammary epithelium. Luminal cell-related nuclear CSPP1 expression was preserved in type-matched cell lines and carcinomas, and correlated to gene copy number and mRNA expression. In contrast, basal-like carcinomas displayed generally lower CSPP1 mRNA expression. Yet, a subgroup of basal-like breast carcinomas depicted nuclear CSPP1 expression, displayed luminal traits, and differed from nuclear CSPP1 devoid counterparts in expression of eight genes. Eight-gene signature defined groups of basal-like tumours from an independent cohort showed significant differences in survival.

Conclusions:

Differential expression of a nuclear CSPP1 isoform identified biologically and clinically distinct subgroups of basal-like breast carcinoma.     

Gene signatures of breast cancer progression and metastasis

Breast cancer is a heterogeneous disease. Patient outcome varies significantly, depending on prognostic features of patients and their tumors, including patient age, menopausal status, tumor size and histology, nodal status, and so on. Response to treatment also depends on a series of predictive factors, such as hormone receptor and HER2 status. Current treatment guidelines use these features to determine treatment. However, these guidelines are imperfect, and do not always predict response to treatment or survival. Evolving technologies are permitting increasingly large amounts of molecular data to be obtained from tumors, which may enable more personalized treatment decisions to be made. The challenge is to learn what information leads to improved prognostic accuracy and treatment outcome for individual patients.     

Systematic characterisation of GABRP expression in sporadic breast cancer and normal breast tissue

The GABRP gene has been previously identified by in silico analysis of four million ESTs as a candidate gene differentially expressed in breast cancer. GABRP is located on chromosome 5q34 and it encodes the pi-subunit of the gamma-aminobutyric acid (GABA) receptor, a transmembrane protein expressed in the brain and several nonneuronal tissues. Using cDNA dot blot hybridisation (cancer profiling array), quantitative RT-PCR and non-radioisotopic in situ hybridisation (ISH), we have analysed GABRP expression in breast cancer and normal breast tissues as well as in nontumorigenic and tumorigenic breast cell lines. Analysis of the cancer profiling array revealed a more than 2-fold downregulation of GABRP (p < 0.001) in 76% of primary breast carcinomas (n = 50) compared to corresponding normal tissues. Quantitative RT-PCR in a panel of 23 normal human tissues showed that the GABRP expression level was most abundant in the normal breast tissues compared to other human tissues. GABRP downregulation in breast cancer was confirmed by quantitative RT-PCR in cryopreserved breast tumour and normal breast tissue specimens (n = 22), in archival formalin-fixed, paraffin-embedded tissue specimens (n = 32), as well as in breast cancer cell lines (n = 8). Furthermore, a significant downregulation of GABRP was noted in large (pT3-pT4) (p = 0.044) primary breast tumours. Non-radioisotopic ISH showed strong GABRP expression in normal epithelial and benign papilloma breast cells, but no signal could be detected in invasive ductal carcinoma. Altogether, these data suggest that GABRP is progressively down-regulated with tumour-progression, and that it may be useful as a prognostic marker in breast cancer.     

Epidemiology of basal-like breast cancer

Risk factors for the newly identified “intrinsic” breast cancer subtypes (luminal A, luminal B, basal-like and human epidermal growth factor receptor 2-positive/estrogen receptor-negative) were determined in the Carolina Breast Cancer Study, a population-based, case–control study of African-American and white women. Immunohistochemical markers were used to subtype 1,424 cases of invasive and in situ breast cancer, and case subtypes were compared to 2,022 controls. Luminal A, the most common subtype, exhibited risk factors typically reported for breast cancer in previous studies, including inverse associations for increased parity and younger age at first full-term pregnancy. Basal-like cases exhibited several associations that were opposite to those observed for luminal A, including increased risk for parity and younger age at first term full-term pregnancy. Longer duration breastfeeding, increasing number of children breastfed, and increasing number of months breastfeeding per child were each associated with reduced risk of basal-like breast cancer, but not luminal A. Women with multiple live births who did not breastfeed and women who used medications to suppress lactation were at increased risk of basal-like, but not luminal A, breast cancer. Elevated waist-hip ratio was associated with increased risk of luminal A in postmenopausal women, and increased risk of basal-like breast cancer in pre- and postmenopausal women. The prevalence of basal-like breast cancer was highest among premenopausal African-American women, who also showed the highest prevalence of basal-like risk factors. Among younger African-American women, we estimate that up to 68% of basal-like breast cancer could be prevented by promoting breastfeeding and reducing abdominal adiposity. An erratum to this article can be found at     

Gene expression profiling of paired ovarian tumors obtained prior to and following adjuvant chemotherapy: molecular signatures of chemoresistant tumors

Chemotherapy (CT) resistance in ovarian cancer is related to multiple factors, and assessment of these factors is necessary for the development of new drugs and therapeutic regimens. In an effort to identify such determinants, we evaluated the expression of approximately 21,000 genes using DNA microarray screening in paired tumor samples taken prior to and after CT treatment from 6 patients with predominantly advanced stage, high-grade epithelial ovarian cancer. A subset of differentially expressed genes was selected from all microarray data by initial filtering on confidence at p=0.05, followed by filtering on expression level (>or=2-fold). Using these selection criteria, we found 121 genes to be commonly up-regulated and 54 genes to be down-regulated in the post-CT tumors, compared to primary tumors. Up-regulated genes in post-CT tumors included substantial number of genes with previously known implication in mechanisms of chemoresistance (TOP2A, ETV4, ABCF2, PRDX2, COX2, COX7B, MUC1, MT3, MT2A), and tumorigenesis (SCGB2A2, S100A9, YWHAE, SFN, ATP6AP1, MGC5528, ASS, TACC3, ARHGAP4, SRA1; MGC35136, PSAP, SPTAN1, LGALS3BP, TUBA4, AMY2B, PPIA, COX1, GRB2, CTSL). Down-regulated genes in post-CT samples mostly included genes implicated in chemosensitivity (GRP, TRA1, ADPRTL1, TRF4-2), cell proliferation and cell cycle control (NGFRAP1, TPD52L1, TAX1BP1) and tumor suppression and apoptosis (SMOC2, TIMP3, AXIN1, CASP4, P53SCV). Additionally, gene clustering analysis revealed the existence of two distinct expression signatures of chemoresistant tumors, which was further confirmed by assessment of some genetic (p53 gene mutation status) and clinical parameters (CT regimens). Our data suggest that intrinsic and acquired chemoresistant phenotypes of post-CT tumors may be attributed to the combined action of different factors implicated in mechanisms of chemoresistance, tumor invasion/progression and control of cell proliferation. This type of molecular profiling could have important clinical implications in resolving chemoresistance and the development of novel treatment strategies designed to prevent its emergence.     

Analysis of integrin beta4 expression in human breast cancer: association with basal-like tumors and prognostic significance.

PURPOSE: The beta4 integrin has been implicated in functions associated with the genesis and progression of carcinomas based on data obtained from cell lines and mouse models. Data on its expression and relevance to human carcinomas, however, are relatively scant. The aim of this study was to assess its expression and prognostic significance in human breast carcinomas. EXPERIMENTAL DESIGN: We integrated data on beta4 expression from multiple gene profiling studies of breast tumors of known clinical outcome with immunohistochemical analysis of 105 breast carcinomas, and we identified genes whose expression correlates with that of beta4. RESULTS: The expression of both beta4 mRNA and protein is not homogeneous in breast cancer and it associates most significantly with the "basal-like" subtype of breast tumors (P = 0.008). No association between beta4 and HER2 expression was evident from either gene profiling or immunohistochemical analysis. To gain insight into the relevance of beta4 expression to human breast carcinomas, we generated a 65-gene "beta4 signature" based on integration of four published gene profiling studies that included the top 0.1% of genes that correlated with beta4, either positively or negatively. This beta4 signature predicted decreased time to tumor recurrence and survival of patients when applied to four data sets including two independent ones. CONCLUSIONS: These observations indicate that beta4 expression in human breast cancer is restricted and associated with basal-like cancers, and they support the hypothesis that beta4 may function in concert with a discrete set of proteins to facilitate the aggressive behavior of a subset of tumors.     

EpCAM expression is an indicator of recurrence in basal-like breast cancer

Advances in the understanding of the molecular basis of breast cancer have necessitated a definition of more sensitive and specific indicators of prognosis that are central to the underlying cancer biology and that reflect the complicated and heterogeneous nature of the disease. This study investigates the expression of epithelial cell adhesion molecule (EpCAM) in breast cancer particularly basal-like phenotype group which remains unclear. EpCAM expression was assessed using immunohistochemistry in a large well-characterised series of breast carcinomas prepared as tissue microarrays. Relationships between EpCAM expression with molecular subtypes, clinicopathological variables and patients' outcome were examined. EpCAM expression was associated with higher tumour grade (P < 0.001), larger tumour size (P < 0.001) and basal phenotype (P = 0.03). Importantly, within the basal-like tumours those positive for EpCAM showed a significantly shorter DFI (LR = 7.97, P = 0.005) and MFS (LR 4.01, P = 0.045). EpCAM may play a role in breast cancer progression and its expression is associated with poor patient outcome in basal-like breast cancer, independent of other prognostic factors.     

Gene methylation profiles of normal mucosa, and benign and malignant colorectal tumors identify early onset markers

Background

CD30, a 120 kDa surface phosphorylated protein is a member of tumour necrosis/nerve growth factor receptor (TNF/NGFR) family and constitutively expressed by Hodgkin and Reed-Sternberg (HRS) cells of Hodgkin lymphoma (HL) and the neoplastic cells of Anaplastic Large Cell Lymphoma (ALCL). A disease-specific protein marker is yet to be identified in Hodgkin lymphoma cells. In order to define HL-specific biomarkers, novel murine monoclonal antibodies were developed in our laboratory.

Results

Murine monoclonal antibodies (mabs) were raised against the B3 sub clone of HL-derived cell line KM-H2. Two of these mabs (clone R23.1 mab and clone R24.1 mab) are IgG₁ class antibodies that recognize a 21 kDa protein present at the cell membrane and in the cytoplasm in HL-derived cell lines. Clone R24.1 mab recognizes a formalin-resistant epitope and labels HRS cells in tissue samples from patients with HL of the classical type, ALCL, and subsets of T and B cell aggressive Non-Hodgkin Lymphomas (NHL). The antigen recognized by the clone R23.1 mab and clone R24.1 mab does not share epitopes with CD30 cluster regions A, B, or C, and, unlike CD30, is not expressed by phytohemagglutinin (PHA) activated T cells.

Conclusion

The 21 kDa protein detected by clone R23.1 and clone R24.1 mabs is a novel membrane-associated protein that may be a potential marker for the diagnosis and targeted therapy of HL and aggressive T and B cell NHL.     

Calpain system protein expression in basal-like and triple-negative invasive breast cancer

BackgroundBasal-like and triple-negative breast tumours encompass an important clinical subgroup and biomarkers that can prognostically stratify these patients are required.Materials and methodsWe investigated two breast cancer tissue microarrays for the expression of calpain-1, calpain-2 and calpastatin using immunohistochemistry. The first microarray was comprised of invasive tumours from 1371 unselected patients, and the verification microarray was comprised of invasive tumours from 387 oestrogen receptor (ER)-negative patients.ResultsThe calpain system contains a number of proteases and an endogenous inhibitor, calpastatin. Calpain activity is implicated in important cellular processes including cytoskeletal remodelling, apoptosis and survival. Our results show that the expression of calpastatin and calpain-1 are significantly associated with various clinicopathological criteria including tumour grade and ER expression. High expression of calpain-2 in basal-like or triple-negative disease was associated with adverse breast cancer-specific survival (P = 0.003 and <0.001, respectively) and was verified in an independent cohort of patients. Interestingly, those patients with basal-like or triple-negative disease with a low level of calpain-2 expression had similar breast cancer-specific survival to non-basal- or receptor- (oestrogen, progesterone or human epidermal growth factor receptor 2 (HER2)) positive disease.ConclusionsExpression of the large catalytic subunit of m-calpain (calpain-2) is significantly associated with clinical outcome of patients with triple-negative and basal-like disease.