Gene expression in extratumoral microenvironment predicts clinical outcome in breast cancer patients

Department of Epidemiology, University of North Carolina at Chapel Hill, Campus Box 7435, Chapel Hill, NC 27599, USA. .
Breast cancer research: BCR (Impact Factor: 5.49). 03/2012; 14(2):R51. DOI: 10.1186/bcr3152
Source: PubMed

ABSTRACT A gene expression signature indicative of activated wound responses is common to more than 90% of non-neoplastic tissues adjacent to breast cancer, but these tissues also exhibit substantial heterogeneity. We hypothesized that gene expression subtypes of breast cancer microenvironment can be defined and that these microenvironment subtypes have clinical relevance.
Gene expression was evaluated in 72 patient-derived breast tissue samples adjacent to invasive breast cancer or ductal carcinoma in situ. Unsupervised clustering identified two distinct gene expression subgroups that differed in expression of genes involved in activation of fibrosis, cellular movement, cell adhesion and cell-cell contact. We evaluated the prognostic relevance of extratumoral subtype (comparing the Active group, defined by high expression of fibrosis and cellular movement genes, to the Inactive group, defined by high expression of claudins and other cellular adhesion and cell-cell contact genes) using clinical data. To establish the biological characteristics of these subtypes, gene expression profiles were compared against published and novel tumor and tumor stroma-derived signatures (Twist-related protein 1 (TWIST1) overexpression, transforming growth factor beta (TGF-β)-induced fibroblast activation, breast fibrosis, claudin-low tumor subtype and estrogen response). Histological and immunohistochemical analyses of tissues representing each microenvironment subtype were performed to evaluate protein expression and compositional differences between microenvironment subtypes.
Extratumoral Active versus Inactive subtypes were not significantly associated with overall survival among all patients (hazard ratio (HR) = 1.4, 95% CI 0.6 to 2.8, P = 0.337), but there was a strong association with overall survival among estrogen receptor (ER) positive patients (HR = 2.5, 95% CI 0.9 to 6.7, P = 0.062) and hormone-treated patients (HR = 2.6, 95% CI 1.0 to 7.0, P = 0.045). The Active subtype of breast microenvironment is correlated with TWIST-overexpression signatures and shares features of claudin-low breast cancers. The Active subtype was also associated with expression of TGF-β induced fibroblast activation signatures, but there was no significant association between Active/Inactive microenvironment and desmoid type fibrosis or estrogen response gene expression signatures. Consistent with the RNA expression profiles, Active cancer-adjacent tissues exhibited higher density of TWIST nuclear staining, predominantly in epithelium, and no evidence of increased fibrosis.
These results document the presence of two distinct subtypes of microenvironment, with Active versus Inactive cancer-adjacent extratumoral microenvironment influencing the aggressiveness and outcome of ER-positive human breast cancers.

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Available from: Patricia Casbas-Hernandez, Jul 16, 2014
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    • "In the current study, expression of PIR in tumor tissues was virtually similar to the non-cancerous tissues. Previous studies reported that some gene expression patterns in the invasive tissues are comparable to their non-invasive breast tissues, suggesting that these signatures may predict progression of early premalignant lesions in non-cancerous tissues [28,34,35]. The PIR functions as a transcriptional regulator whose expression is deregulated in several cancer types. "
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    ABSTRACT: Background Previously, we performed analysis of gene expression in 46 axillary lymph node negative tumors and identified molecular gene signatures that resulted in different clinical outcomes. The aim of this study was to determine the correlation of γ-glutamyl hydrolase (GGH), fatty acid amide hydrolase (FAAH), Pirin (PIR) and TAF5-like RNA polymerase II, p300/CBP-associated factor (PCAF)-associated factor, 65 kDa (TAF5L), selected from identified gene signatures, with clinical outcomes as well as classical clinicopathological characteristics in primary invasive breast cancer patients. Methods The protein levels of GGH, FAAH, PIR and TAF5L were assessed by immunohistochemistry (IHC) on a panel of 80 primary invasive breast tumors. Quantitative real-time PCR (qRT-PCR) and western blot analysis were performed to verify the expression levels of the candidate biomarkers. Patient disease-specific survival (DSS) and recurrence-free survival (RFS) were evaluated using the Kaplan-Meier method. The prognostic biomarkers were identified by univariate analysis with a log-rank test and by multivariate analysis with Cox proportional hazards regression models. Results The GGH and FAAH protein levels were significantly up-regulated in invasive breast cancer tumors compared with adjacent non-cancerous tissues. Furthermore, the protein levels of GGH and FAAH were significantly correlated in tumor tissues. Tumoral GGH protein expression was significantly correlated with shorter DSS and RFS. Furthermore, the protein expression of GGH was positively correlated with undifferentiated tumors (BRE grade III) and ER/PR expressing tumors. Multivariate regression analysis showed that only GGH protein expression independently predicts DSS. No such correlations were found for FAAH, PIR and TAF5L protein expression. However, elevated protein levels of FAAH were positively associated with high number of lymph node involvement and upregulated levels of PIR were positively related with lymph node metastasis. The TAF5L was pronouncedly down-regulated in primary invasive breast cancer tissues compared to matched adjacent non-cancerous tissues. Conclusion These data show for the first time that cytoplasmic GGH might play a relevant role in the development and progression of invasive breast cancer, warranting further investigations. Our findings suggest that GGH serve as a potential biomarker of unfavorable clinical outcomes over short-term follow-up in breast cancer. The GGH may be a very attractive targeted therapy for selected patients.
    BMC Cancer 02/2013; 13(1):47. DOI:10.1186/1471-2407-13-47 · 3.36 Impact Factor
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    • "Later genetic studies subsequently showed that shortened telomeric DNA and four to five times more prevalent LOH loci characterized the field cancerization of normal-appearing tissues within 1 cm of microscopically defined tumor margins [12]. More recently, gene expression profiling of the peritumor breast microenvironment by different groups has revealed the presence of two distinct RNA expression subtypes [15, 16]: one “normal” microenvironment, independent of distance from tumor, and another “active” microenvironment, its presence dependent on distance from the tumor edge and independent of tumor phenotype yet associated with poorer patient prognosis, and its signature consistent with extracellular remodeling, wound healing, fibrosis, and epithelial-to-mesenchymal transition (EMT) [16]. Whether or not this epigenetic EMT-like phenotype within histologically normal cancer-adjacent mammary tissue also reflects any underlying genetic field cancerization effect remains unclear, as neither of those two gene expression studies evaluated genomic aberrations in the microdissected peritumor tissue, and the investigators concluded that this EMT-like phenotype was a host reaction unrelated to any specific breast cancer phenotype [15, 16]. "
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    ABSTRACT: Field cancerization effects as well as isolated tumor cell foci extending well beyond the invasive tumor margin have been described previously to account for local recurrence rates following breast conserving surgery despite adequate surgical margins and breast radiotherapy. To look for evidence of possible tumor cell contamination or field cancerization by genetic effects, a pilot study (Study 1: 12 sample pairs) followed by a verification study (Study 2: 20 sample pairs) were performed on DNA extracted from HER2-positive breast tumors and matching normal adjacent mammary tissue samples excised 1–3 cm beyond the invasive tumor margin. High-resolution molecular inversion probe (MIP) arrays were used to compare genomic copy number variations, including increased HER2 gene copies, between the paired samples; as well, a detailed histologic and immunohistochemical (IHC) re-evaluation of all Study 2 samples was performed blinded to the genomic results to characterize the adjacent normal tissue composition bracketing the DNA-extracted samples. Overall, 14/32 (44 %) sample pairs from both studies produced genome-wide evidence of genetic aberrations including HER2 copy number gains within the adjacent normal tissue samples. The observed single-parental origin of monoallelic HER2 amplicon haplotypes shared by informative tumor–normal pairs, as well as commonly gained loci elsewhere on 17q, suggested the presence of contaminating tumor cells in the genomically aberrant normal samples. Histologic and IHC analyses identified occult 25–200 μm tumor cell clusters overexpressing HER2 scattered in more than half, but not all, of the genomically aberrant normal samples re-evaluated, but in none of the genomically normal samples. These genomic and microscopic findings support the conclusion that tumor cell contamination rather than genetic field cancerization represents the likeliest cause of local clinical recurrence rates following breast conserving surgery, and mandate caution in assuming the genomic normalcy of histologically benign appearing peritumor breast tissue. Electronic supplementary material The online version of this article (doi:10.1007/s10549-012-2290-3) contains supplementary material, which is available to authorized users.
    Breast Cancer Research and Treatment 10/2012; 136(3). DOI:10.1007/s10549-012-2290-3 · 3.94 Impact Factor
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    • "Furthermore, our work also shows that the stroma is a significant source of TWIST transcript expression. Indeed, the current data reinforce recent observations made by Roman-Perez and colleagues after evaluating gene expression patterns in 72 breast tissue samples [36]. They identified two distinct subtypes, Active and Inactive, in the cancer-adjacent extra-tumoral microenvironment of breast tissues and demonstrated that TWIST1 was highly expressed along with other stromal associated genes, such as VIM, ADAMTS2, COL4A2, COL4A1, ITGA7 and ITGB1, in the Active subtype. "
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    ABSTRACT: Introduction The TWIST homolog 1 (TWIST1) is a transcription factor that induces epithelial to mesenchymal transition (EMT), a key process in metastasis. The purpose of this study was to investigate whether TWIST1 expression predicts disease progression in a large breast cancer cohort with long-term clinical follow-up, and to reveal the biology related to TWIST1 mediated disease progression. Methods TWIST1 mRNA expression level was analyzed by quantitative real-time reverse polymerase chain reaction (RT-PCR) in 1,427 primary breast cancers. In uni- and multivariate analysis using Cox regression, TWIST1 mRNA expression level was associated with metastasis-free survival (MFS), disease-free survival (DFS) and overall survival (OS). Separate analyses in lymph node-negative patients (LNN, n = 778) who did not receive adjuvant systemic therapy, before and after stratification into estrogen receptor (ER)-positive (n = 552) and ER-negative (n = 226) disease, were also performed. The association of TWIST1 mRNA with survival endpoints was assessed using Kaplan-Meier analysis. Using gene expression arrays, genes showing a significant Spearman rank correlation with TWIST1 were used to identify overrepresented Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG)-annotated biological pathways. Results Increased mRNA expression level of TWIST1 analyzed as a continuous variable in both uni- and multivariate analysis was associated with shorter MFS in all patients (hazard ratio (HR): 1.17, 95% confidence interval, (95% CI):1.09 to 1.26; and HR: 1.17, 95% CI: 1.08 to 1.26; respectively), in LNN patients (HR: 1.22, 95% CI: 1.09 to 1.36; and HR: 1.21, 95% CI: 1.07 to 1.36; respectively) and in the ER-positive subgroup of LNN patients (HR: 1.34, 95% CI: 1.17 to 1.53; and HR: 1.32, 95% CI: 1.14 to 1.53; respectively). Similarly, high TWIST1 expression was associated with shorter DFS and OS in all patients and in the LNN/ER-positive subgroup. In contrast, no association of TWIST1 mRNA expression with MFS, DFS or OS was observed in ER-negative patients. Genes highly correlated with TWIST1 were significantly enriched for cell adhesion and ECM-related signaling pathways. Furthermore, TWIST1 mRNA was highly expressed in tumor stroma and positively related to tumor stromal content (P <0.001). Conclusions TWIST1 mRNA expression is an independent prognostic factor for poor prognosis in LNN/ER-positive breast cancer. The biological associations suggest an involvement of the tumor microenvironment in TWIST1's adverse role in breast cancer.
    Breast cancer research: BCR 09/2012; 14(5):R123. DOI:10.1186/bcr3317 · 5.49 Impact Factor
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