Karijn P.M. Suijkerbuijk

The TWIST1 oncogene is a direct target of hypoxia-inducible factor-2α

Hypoxia-inducible factors (HIFs) are highly conserved transcription factors that play a crucial role in oxygen homeostasis. Intratumoral hypoxia and genetic alterations lead to HIF activity, which is a hallmark of solid cancer and is associated with poor clinical outcome. HIF activity is regulated by an evolutionary conserved mechanism involving oxygen-dependent HIFα protein degradation. To identify novel components of the HIF pathway, we performed a genome-wide RNA interference screen in Caenorhabditis elegans, to suppress HIF-dependent phenotypes, like egg-laying defects and hypoxia survival. In addition to hif-1 (HIFα) and aha-1 (HIFβ), we identified hlh-8, gska-3 and spe-8. The hlh-8 gene is homologous to the human oncogene TWIST1. We show that TWIST1 expression in human cancer cells is enhanced by hypoxia in a HIF-2α-dependent manner. Furthermore, intronic hypoxia response elements of TWIST1 are regulated by HIF-2α, but not HIF-1α. These results identify TWIST1 as a direct target gene of HIF-2α, which may provide insight into the acquired metastatic capacity of hypoxic tumors.

Methylation of the TWIST1 Promoter, TWIST1 mRNA Levels, and Immunohistochemical Expression of TWIST1 in Breast Cancer

TWIST1, an antiapoptotic and prometastatic transcription factor, is overexpressed in many epithelial cancers including breast. Only little is known regarding the regulation of TWIST1 in these cancers. Recently, an increase in the TWIST1 promoter methylation has been shown in breast cancers. To correlate the percentage of TWIST1 promoter methylation to the protein levels, we analyzed simultaneously the methylation status as well as the mRNA and the percentage of cells expressing TWIST1 in normal breast tissue and 76 invasive breast cancers. We found that TWIST1 promoter methylation is significantly more prevalent in malignant compared with healthy breast tissue. Furthermore, the percentage of cells expressing TWIST1 was greater in breast malignancy compared with matched healthy tissue from the same patients. There was no correlation, however, between TWIST1 promoter methylation and TWIST1 protein or RNA expression. This indicates that although TWIST1 CpG methylation is useful as a biomarker in breast cancer diagnosis, there is no direct correlation with TWIST1 expression. (Cancer Epidemiol Biomarkers Prev 2008;17(12):3325–30)

Methylation is less abundant in BRCA1-associated compared with sporadic breast cancer

BACKGROUND Promoter methylation is a common epigenetic mechanism to silence tumor suppressor genes during breast cancer development. We investigated whether BRCA1-associated breast tumors show cancer-predictive methylation patterns similar to those found in sporadic tumors. PATIENTS AND METHODS Quantitative multiplex methylation-specific PCR of 11 genes involved in breast carcinogenesis (RARB, RASSF1, TWIST1, CCND2, ESR1, SCGB3A1, BRCA1, BRCA2, CDKN2A, APC, CDH1) was carried out on 32 BRCA1-associated and 46 sporadic breast carcinomas and on normal breast tissue from seven BRCA1 mutation carriers and 13 non-carriers. RESULTS The extent of cumulative methylation increased with age (P < 0.001). The median cumulative methylation index (CMI) of all studied genes was significantly higher in tumors (89) than in normal tissue (13, P < 0.001). The median CMI was significantly lower in BRCA1-associated (59) than in sporadic breast tumors (122, P = 0.001), in estrogen receptor (ER)-negative tumors (73) than in ER-positive tumors (122, P = 0.005) and in lymph node-negative (77) compared with lymph node-positive tumors (137, P = 0.007). In subgroup analysis, the effect of a BRCA1 germline mutation on methylation proved to be independent of ER status, lymph node status and age. CONCLUSIONS These data indicate that BRCA1-associated breast cancers show less promoter methylation compared with sporadic breast carcinomas indicating a difference in disease etiology.

Improving early breast cancer detection: focus on methylation

The need for additional breast cancer screening tools is indisputably high, as one may conclude from the high rate of interval malignancies in women undergoing regular screening. DNA promoter methylation frequently occurs during breast carcinogenesis and is an early event in this process. Moreover, a field defect for methylation has been described and methylation values can reliably be assessed in limited amounts of DNA. Simultaneous detection of methylation of a panel of genes in breast fluids and/or blood derivatives could be both sufficiently specific and sensitive to be of additive value to current imaging-based screening methods. This review describes the recent developments in methylation detection in breast fluids, serum and plasma that paved the way for large prospective studies. These studies will provide us with the definite answer as to what will be the additive value of defining the methylation status of specific genes to current imaging-based screening methods.

Methylation profiles of hereditary and sporadic ovarian cancer.

Bol G M, Suijkerbuijk K P M, Bart J, Vooijs M, van der Wall E & van Diest P J
(2010) Histopathology 57, 363–370
Methylation profiles of hereditary and sporadic ovarian cancer

Physical Activity in Relation to Mammographic Density in the Dutch Prospect-European Prospective Investigation into Cancer and Nutrition Cohort

Background: Evidence accumulates that physical inactivity is one of the few modifiable risk factors for breast cancer. The mechanism through which physical inactivity affects breast cancer risk is not clear. The study aim was to investigate the association between physical activity and breast density because mammographic density is strongly associated with breast cancer risk. Methods: We did a cross-sectional study in 620 women, of ages 49 to 68 years and participants of the Dutch Prospect-European Prospective Investigation into Cancer and Nutrition cohort. A self-administered questionnaire was used to obtain information on duration and intensity of physical activity (recreational, household, and occupational) during the year preceding study recruitment. A total activity index (inactive, moderately inactive, moderately active, and active) was estimated by combining all activity types. Percent and absolute breast density were determined on screening mammograms using a computer-aided method. Multivariate linear regression was used to examine the association between physical activity and breast density. Results: Mean percent density was 35.3% [95% confidence interval (95% CI), 31.8-38.8] for the inactive category compared with 36.1% (95% CI, 33.0-39.2) for the active category. Mean absolute density values for the inactive and active category were 45.8 cm2 (95% CI, 40.9-50.7) and 42.6 cm2 (95% CI, 38.3-47.0), respectively. Subgroup analysis for postmenopausal women showed similar results, as did separate analyses for recreational and household activity. Conclusions: The result does not support a relation between current physical activity and mammographic density in postmenopausal women. (Cancer Epidemiol Biomarkers Prev 2006;15(3):456–60)

Comparison of different promoter methylation assays in breast cancer.

Background: Promoter hypermethylation has emerged as a promising cancer biomarker. Currently, a large variety of quantitative and non-quantitative techniques is used to measure methylation in clinical specimens. Here we directly compared three commonly used methylation assays and assessed the influence of tissue fixation, target sequence location and the amount of DNA on their performance. Methods: We used Methylation-Specific PCR (MSP), Quantitative Multiplex MSP (QM-MSP) and Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA) to compare methylation of CCND2, SCGB3A1, RARB and RASSF1 on DNA from 40 breast carcinomas. Results: A comparison between MSP and QM-MSP on the same samples showed a high discrepancy: 20% of tumors that showed no methylation in MSP gave >10% methylation in QM-MSP. In contrast, QM-MSP correlated strongly with MS-MLPA when targeting the same sequence in DNA from paraffin embedded as well as fresh frozen tissue. This correlation declined when target sequences were non-overlapping. In titration experiments, MSP and MS-MLPA performed robust with 10 ng of DNA, while QM-MSP was at least ten-fold more sensitive. Conclusion: Despite the difference in molecular basis, QM-MSP and MS-MLPA showed moderate to strong correlations. In contrast, there was a poor concordance between either of these techniques and non-quantitative MSP. For biological samples with scarce DNA, QM-MSP is the method of choice.

Molecular analysis of nipple fluid for breast cancer screening.

Lack of sensitivity and specificity of image-based breast cancer screening has urged the exploration of alternate screening modalities. Nipple fluid, which contains breast epithelial cells, is produced in small amounts in the breast ducts of nonlactating women and can be collected by noninvasive vacuum aspiration. After administration of nasal oxytocin, nipple aspiration yields sufficient material for molecular analysis in the large majority of women. Whereas nipple fluid cytology appears to have only a moderate correlation with breast cancer development, methylation holds promise as a more appropriate biomarker, since methylation aberrations occur as an early and frequent event during carcinogenesis. Using quantitative multiplex methylation-specific PCR, methylation can be detected in minute amounts of DNA extracted from nipple aspirates, precluding the need for more invasive intraductal approaches such as ductal lavage and random periareolar fine needle aspiration. The application of genomic and proteomic diagnostics to nipple aspirates therefore provides unprecedented opportunities for early breast cancer diagnosis amendable to population-based screening.

Receptor Conversion in Distant Breast Cancer Metastases: A Systematic Review and Meta-analysis

Background In metastatic breast cancer, hormone and/or human epidermal growth factor receptor 2 (HER2)-targeted therapy decision-making is still largely based on tissue characteristics of the primary tumor. However, a change of estrogen receptor alpha (ERα), progesterone receptor (PR), and HER2 status in distant metastases has frequently been reported. The actual incidence of this phenomenon has been debated. Methods We performed a meta-analysis including 39 studies assessing receptor conversion from primary breast tumors to paired distant breast cancer metastases. We noted the direction of change (positive to negative or vice versa) and performed subgroup analyses for different thresholds for positivity, the type of test used to assess HER2 receptor status, and metastasis location-specific differences (two-sided tests). Results Overall, the incidence of receptor conversion varied largely between studies. For ERα, PR, and HER2, we found that random effects pooled positive to negative conversion percentages of 22.5% (95% confidence interval [CI] = 16.4% to 30.0%), 49.4% (95% CI = 40.5% to 58.2%), and 21.3% (95% CI = 14.3% to 30.5%), respectively. Negative to positive conversion percentages were 21.5% (95% CI = 18.1% to 25.5%), 15.9% (95% CI = 11.3% to 22.0%), and 9.5% (95% CI = 7.4% to 12.1%). Furthermore, ERα discordance was statistically significantly higher in the central nervous system and bone compared with liver metastases (20.8%, 95% CI = 15.0% to 28.0%, and 29.3%, 95% CI = 13.0% to 53.5%, vs 14.3%, 95% CI = 11.3% to 18.1, P = .008 and P < .001, respectively), and PR discordance was higher in bone (42.7%, 95% CI = 35.1% to 50.6%, P < .001) and liver metastases (47.0%, 95% CI = 41.0% to 53.0%, P < .001) compared with central nervous system metastases (23.3%, 95% CI = 16.0% to 32.6%). Conclusions Receptor conversion for ERα, PR, and HER2 occurs frequently in the course of disease progression in breast cancer. Large prospective studies assessing the impact of receptor conversion on treatment efficacy and survival are needed. Meanwhile, reassessing receptor status in metastases is strongly encouraged.

Low levels of BNIP3 promoter hypermethylation in invasive breast cancer.

Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) is a pro-apoptotic member of the Bcl-2 family induced under hypoxia [3,5,6]. Low or absent expression has been described in several human tumors, resulting in poor prognosis [1]. We previously reported that BNIP3 expression is lost in a significant portion of invasive breast cancers, which was correlated with poor prognostic features such as positive lymph node status and high proliferation [9]. Since DNA promoter hypermethylation (“methylation”) is a common mechanism to silence gene expression, contributing to tumor-progression and invasion, we further studied the relation between BNIP3 methylation and gene expression by RNA in situ hybridization (ISH) in invasive breast cancer. In 40 cases of invasive breast cancer, BNIP3 methylation was studied by Methylation Specific Multiplex Ligation-dependent Probe Amplification [2,4,8, 11] with 2 BNIP3 probes (157 and 291) located in the CpG island harboring the BNIP3 promoter. The 157 probe is located 50 nucleotides before exon 1, while probe 291 is located in exon 1. Both probes target sequences located in the BNIP3 promoter that is located within a 1700-bp CpG island, which spans from −1162 to +538 bp of the transcription start site and contains the first exon of the BNIP3 gene. This promoter area has been reported to be methylated in pancreatic cancer [12] and in gastric and colorectal cancer [10]. Data on BNIP3 mRNA in situ hybridization were derived from a previous study [9]. BNIP3 methylation levels in 8 normal tissue samples was on average 0.08. BNIP3 methylation was overall low (between 0.3 and 15% for probe 157 and between 0 and 31% for probe 291), while other genes included in this assay (CCND2, RASSF1, GSTP1, HIN1) showed methylation percentages up to 100% (means 24–51%) in the same tumors. Cases that were negative (N = 4) in BNIP3 RNA ISH showed higher BNIP3 Fig. 1. Relation between BNIP3 promoter hypermethylation (probe 157) and BNIP3 RNA expression by in situ hybridization (4 cases were negative and 36 positive).