BackgroundTriple-negative breast cancers (TNBC) neither express hormone receptors, nor overexpress HER2. They are associated with poor prognosis, as defined by low five-year survival and high recurrence rates after adjuvant therapy. Overall, TNBC share striking similarities with basal-like breast cancers (BBC), so a number of studies considered them being the same. The purpose of this review is to summarise the latest findings on TNBC concerning its relation and delineation to BBC, discuss the developmental pathways involved and address clinical implications for this complex type of breast cancer.MethodsThe recent literature from PubMed and Medline databases was reviewed.ResultsNot all TNBC are of the intrinsic BBC subtype (nonbasal (NB)-TNBC), nor are all BBC triple-negative (non-triple-negative (NTN)-BBC). There is increasing evidence that a triple-negative, basal-like breast cancer (TNBBC) subtype develops mainly through a BRCA1-related pathway. Somatic mutations that contribute to NTN-BBC and NB-TNBC development are possibly not related to this pathway, but may occur randomly due to increased genomic instability in these tumours. Several therapeutic options exist for TNBBC, which exhibited promising results in recent clinical trials. Cytotoxic therapies, e.g. combined treatment with anthracyclines or taxanes, achieved good tumour regression rates in the neo-adjuvant setting, but also showed considerable recurrence during the first 5 years after therapy. Targeted therapy options involve PARP1 and EGFR inhibition, although both approaches still need further investigation.ConclusionsTNBC and BBC are not the same disease entity. The TNBBC subtype shows the largest homogeneity in terms of tumour development, prognosis and clinical intervention options.

NDRG4 is a candidate tumor suppressor gene in colorectal cancer whose expression is frequently inactivated by promoter methylation. NDRG4 promoter methylation is a potential biomarker for the noninvasive detection of colorectal cancer in stool samples.

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Current genome-wide methods to detect DNA-methylation in healthy and diseased tissue require high-quality DNA from fresh-frozen (FF) samples. However, well-annotated clinical samples are mostly available as formalin-fixed, paraffinembedded (FFPE) tissues containing poor-quality DNA. To overcome this limitation, we here aimed to evaluate a DNA restoration protocol for usage with the genome-wide Infinium HumanMethylation450 BeadChip assay (HM-450K). Sixty-six DNA samples from normal colon (n = 9) and breast cancer (n = 11) were interrogated separately using HM-450K. Analyses included matched FF/FFPE samples and technical duplicates. FFPE DNA was processed with (FFPEr) or without a DNA restoration protocol (Illumina). Differentially methylated genes were finally validated in 24 additional FFPE tissues using nested methylation-specific PCR (MSP). In summary, β-values correlation between FFPEr duplicates was high (ρ = 0.9927 (s.d. ± 0.0015)). Matched FF/FFPEr correlation was also high (ρ = 0.9590 (s.d. ± 0.0184)) compared with matched FF/FFPE (ρ = 0.8051 (s.d. ± 0.1028). Probe detection rate in FFPEr samples (98.37%, s.d. ± 0.66) was comparable to FF samples (99.98%, s.d. ± 0.019) and substantially lower in FFPE samples (82.31%, s.d. ± 18.65). Assay robustness was not decreased by sample archival age up to 10 years. We could also demonstrate no decrease in assay robustness when using 100 ng of DNA input only. Four out of the five selected differentially methylated genes could be validated by MSP. The gene failing validation by PCR showed high variation of CpG β-values in primer-binding sites. In conclusion, by using the FFPE DNA restoration protocol, HM-450K assays provide robust, accurate and reproducible results with FFPE tissue-derived DNA, which are comparable to those obtained with FF tissue. Most importantly, differentially methylated genes can be validated using more sensitive techniques, such as nested MSP, altogether providing an epigenomics platform for molecular pathological epidemiology research on archived samples with limited tissue amount. Epigenomic changes are recognised as important factors in tumour initiation, growth and progression. Global and local DNA-methylation patterns are frequently altered in cancer cells, resulting in genomic instability and diminished or elevated expression of tumour-suppressor genes or oncogenes, respectively, driving malignant transformation, growth promotion and metastasis (reviewed in Jones and Baylin 1 and Petronis 2 ). In the clinical setting, cancer-specific DNA-methylation changes are increasingly evaluated as biomarkers for early detection, staging or patient prognosis.In addition, a role for DNA-methylation changes in mediating either cancer drug resistance or drug sensitivity has been hypothesised and verified. [3][4][5][6] Therefore, the in-depth study of the cancer DNA-methylome holds promise to provide important clues as to which genes and biological networks are affected at tumour initiation and progression. Furthermore, it will provide clues as to which g...

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Recently, DNA methylation has been suggested as a potential mechanism involved in the transcriptional regulation of SHH gene expression in cancer. However, detailed analyses on the underlying transcriptional mechanisms of SHH expression have not been presented so far and were therefore the focus of this study. We found that the genomic region of SHH contains two different transcriptional start sites and four CpG islands spread from the 5' promoter region to the 3' end of the SHH gene. Based on this CpG island topology we analyzed the influence of DNA methylation within the promoter region as well as in exon 2 and exon 3 on SHH mRNA expression in a large set (n = 14) of benign and malignant human cell lines, and further elucidated the functionality of the two identified SHH transcription initiation sites. Methylation-specific PCR (MSP) clearly showed that SHH is expressed independently of DNA methylation within exon 2 and exon 3 of its genomic region, while methylation of the promoter region is able to abrogate SHH expression. Most interesting, we found activation of the upstream SHH promoter in several breast cancer cell lines when the downstream SHH promoter is methylated. These observations lead us to propose a transcriptional model for the SHH gene, in which combined mechanisms of DNA methylation and alternative promoter usage coordinate the transcriptional activity of this important developmental gene.