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To better understand the biology of hormone receptor-positive and-negative breast cancer and to identify methylated gene markers of disease progression, we carried out a genome-wide methylation array analysis on 103 primary invasive breast cancers and 21 normal breast samples, using the Illumina Infinium HumanMethylation27 array that queried 27,578 CpG loci. Estrogen and/or progesterone receptor-positive tumors displayed more hypermethylated loci than estrogen receptor (ER)-negative tumors. However, the hypermethylated loci in ER-negative tumors were clustered closer to the transcriptional start site compared with ER-positive tumors. An ER-classifier set of CpG loci was identified, which independently partitioned primary tumors into ER subtypes. A total of 40 (32 novel and 8 previously known) CpG loci showed differential methylation specific to either ER-positive or ER-negative tumors. Each of the 40 ER subtype-specific loci was validated in silico, using an independent, publicly available methylome dataset from the Cancer Genome Atlas. In addition, we identified 100 methylated CpG loci that were significantly associated with disease progression; the majority of these loci were informative particularly in ER-negative breast cancer. Overall, the set was highly enriched in homeobox containing genes. This pilot study shows the robustness of the breast cancer methylome and illustrates its potential to stratify and reveal biological differences between ER subtypes of breast cancer. Furthermore, it defines candidate ER-specific markers and identifies potential markers predictive of outcome within ER subgroups.

Cyclin D2 is a member of the D-type cyclins, implicated in cell cycle regulation, differentiation, and malignant transformation. It was noted previously that cyclin D2 is not expressed in the majority of breast cancer cell lines, whereas abundant expression was detected in finite life span human mammary epithelial cells. By reverse transcription-PCR and Western blot analysis, we extended this finding to primary breast carcinomas and show that the majority of these tumors lack expression of cyclin D2 mRNA (18 of 24) and protein (10 of 13). In contrast, both luminal and myoepithelial subpopulations of normal breast tissues expressed cyclin D2. Hypermethylation of the CpG island in the promoter was detected by methylation-specific PCR in nearly half of the breast cancers (49 of 106) and was associated with silencing of cyclin D2 gene expression. Promoter hypermethylation was also detected in ductal carcinoma in situ, suggesting that loss of cyclin D2 expression is an early event in tumorigenesis. Our results suggest that loss of cyclin D2 expression is associated with the evolution of breast cancer.

Telomerase is a reverse transcriptase that maintains chromosome ends, compensating for the progressive loss of DNA that occurs during replication. High telomerase enzyme activity is an unfavorable prognostic feature for several types of cancers. We investigated whether telomerase level predicts outcome for patients with the pediatric renal malignancy Wilms' tumor. In a case-cohort study of 78 patients with favorable histology Wilms' tumor, we compared tumor telomerase levels in patients with and without eventual recurrence. Three measures of telomerase were used: (a) telomerase enzyme activity; (b) expression of hTR, the RNA component of telomerase; and (c) mRNA expression of hTERT, the gene that encodes the catalytic component of the enzyme. Of the evaluable samples, 81% had detectable telomerase activity, 97% had detectable hTERT transcript, and 100% had detectable hTR. Weak correlations were observed between telomerase activity and hTR level (r = 0.34, P = 0.02) and between telomerase activity and hTERT mRNA level (r = 0.32, P = 0.04). Of the variables assessed, only hTERT mRNA expression correlated with outcome. The median hTERT mRNA level in tumors with recurrence was higher than that in tumors without recurrence (1.42 versus 0.97 units, P = 0.023, Wilcoxon). Univariate analysis of hTERT mRNA level as a continuous variable suggested that each unit increase in hTERT mRNA level increased the risk of recurrence (RR) by a factor of 1.66 [95% confidence interval (CI), 1.2-2.3; P < 0.005]. Compared with tumors with hTERT mRNA levels of 0-1 units, tumors with hTERT mRNA levels of 1-2 units had a RR of 2.72 (95% CI, 0.91-8.13; P = 0.074), and tumors with hTERT mRNA levels >2 units had a RR of 6.40 (95% CI, 1.49-27.67, P = 0.013). Multivariate analysis of hTERT mRNA level as a predictor of recurrence, adjusted for tumor stage and age at diagnosis, revealed a RR of 1.48 (95% CI, 0.9-2.6; P = 0.16). Measurement of hTERT mRNA level may, therefore, enable clinicians to identify a population of patients at high risk for recurrence and to adjust their therapy accordingly. A larger study will be necessary to determine whether hTERT expression is an independent prognostic indicator. Further biological investigation is warranted to discern whether the link between high hTERT expression and unfavorable prognosis is causative or correlative.

The 1-methyl-1-nitrosourea-induced rat mammary tumor model system is well studied, reproducible, and widely used. We have investigated whether these tumors possess higher telomerase activity than normal mammary tissue. Using the telomeric repeat amplification protocol assay, we found significantly higher telomerase activity in 36 mammary carcinomas than in 72 mammary glands of virgin rats. The level of telomerase activity in virgin rats was unaffected by strain, age, stage of the estrous cycle, or ovariectomy. However, mammary glands obtained from pregnant rats exhibited telomerase activity comparable to that found in the tumors, possibly reflecting the high epithelial content of these tissues. Indeed, isolated epithelial cells from virgin and pregnant mammary glands and from carcinomas had similar telomerase activities. Thus, telomerase activity is constitutive in the rat mammary epithelium and is not a unique characteristic of malignant transformation in this tissue. These results underscore the importance of attributing biochemical properties to specific cell types in a tissue, a situation not paralleled in the interpretation of data from in vitro models.

The inability to distinguish microinvasive follicular thyroid cancer from benign follicular tumors preoperatively presents an important surgical dilemma. We examined 44 follicular tumors and found telomerase activity in all 11 follicular carcinomas and in 8 of 33 benign follicular tumors. It was undetectable in 22 normal thyroid tissues adjacent to the tumors. Telomerase activity may thus provide a diagnostic marker distinguishing benign from malignant follicular thyroid tumors. The ability to identify invasive follicular thyroid tumors could avert over 14,000 thyroidectomies annually in the United States, thereby significantly decreasing morbidity and health care costs.