Next-generation Sequencing

A Powerful Tool for the Discovery of Molecular Markers in Breast Ductal Carcinoma In Situ

Hitchintan Kaur; Shihong Mao; Seema Shah; David H Gorski; Stephen A Krawetz; Bonnie F Sloane; Raymond R Mattingly

Disclosures

Expert Rev Mol Diagn. 2013;13(2):151-165. 

In This Article

Molecular Markers of DCIS

Personalized approaches that combine molecular profiling and histological observations are being implemented to improve risk stratification and therapy for patients with DCIS. Using a combination of molecular and morphological features along with genomic and immunohistochemical data, most preinvasive and invasive lesions can be stratified into a low-grade molecular pathway or a high-grade molecular pathway. The low-grade molecular subtype is characterized by loss of 16q, gain of 1q and expression of estrogen receptors (ERs) and progesterone receptors (PRs). The lesions of this subtype are referred to as 'luminal'. Furthermore, depending on the absence or presence of Her-2, they are classified as luminal A or luminal B, respectively. The high-grade variety has gain of 11q13, loss of 13q, amplification of 17q12 and infrequent expression of ER and PR. Lesions with these molecular and gene expression signatures are referred to as 'basal' or 'Her-2' depending on the absence or presence of Her-2 expression, respectively.[67–69] Meta-analysis of microarray gene expression profiles of several triple-negative breast cancer (TNBC) cases has revealed further intrinsic differences in gene signatures and ontologies. On the basis of gene expression profiles, Chen et al. have developed a subtyping tool that can classify TNBC into six molecular subtypes: namely two basal-like (BL1 and BL2) subtypes, an immunomodulatory subtype, a mesenchymal subtype, a mesenchymal stem-like subtype and a luminal androgen receptor subtype.[70] The TNBC subtypes exhibit differential sensitivities to therapeutic agents and hence may explain differences in clinical behaviors of TNBC.[70] Analysis of publicly available gene expression datasets of DCIS cases using similar subtyping tools could provide an insight into the distinct genetic subtypes of DCIS that would require specific targeted agents to block progression to invasive cancer.

Some of the important molecular markers that have previously been identified in patients with DCIS are described in further detail below.

Her-2

Her-2 expression is primarily associated with high-grade comedo-type DCIS, in the absence or presence of concomitant IDC, and hence is regarded as an independent prognostic factor.[71] Since Her-2 immunoreactivity has been reported to be significantly higher in DCIS cases than in IDC, Allred et al. proposed that Her-2/neu amplification plays a more important role in initiation than in progression of IDC.[72,73] Her-2 status is regarded as an important prognostic and predictive marker, with its overexpression predicting local recurrence.[74]

Estrogen Receptor

ER expression is inversely related to the grade of DCIS lesions,[75] and treating DCIS that expresses ER with tamoxifen significantly reduces the risk of subsequent breast cancer by 40–50%.[76]

Progesterone Receptor

PR expression also has an inverse relationship with nuclear grade and its presence is associated with expression of ER and lack of comedo-necrosis in DCIS.[77,78]

Cyclin D1

Amplification of cyclin D1 has been observed in 10–18% cases of DCIS.[79,80] There have, however, been conflicting reports on the correlation between expression of cyclin D1 and ER.[81,82] There is not a correlation between cyclin D1 expression and risk of local recurrence.[83]

Bcl-2

Bcl-2 has been shown to be an independent prognostic marker in early stages of breast carcinoma. It is present in the continuum of breast lesions from ADH to well-differentiated DCIS,[84,85] and its expression gradually decreases as lesions become more aggressive.[86]

p53

Inactivating mutations of p53 have been observed in a large percentage (40%) of high-grade DCIS. Low-grade DCIS does not exhibit any alterations and the frequency of these mutations is very low (5%) in the intermediate-grade lesions.[87,88]

Ki67

Well-differentiated DCIS lesions generally have lower expression of Ki67, whereas poorly differentiated lesions have higher levels. Quantitative assessment by automated image analysis of multiple DCIS tumor samples showed that invasion is associated with a significant increase in Ki67 expression and decreases in ER, PR and Her-2 expression.[89]

c-met & VEGF

Increased levels of c-met are associated with VEGFA and FGFR-1, all of which contribute to angiogenic processes.[90] High expression of VEGF correlates with biological aggressiveness of DCIS lesions.[91,92] Increased frequency of FGFR1 amplification is associated with progression of DCIS to IDC and poor prognosis.[93]

Myc

Although increased myc expression is associated with poor prognosis in IDC, its role in preinvasive lesions is not clear. There have been conflicting reports regarding amplification of the c-myc gene in premalignant lesions of the breast. No c-myc amplification was observed in DCIS in two independent studies,[80,94] whereas another group reported c-myc amplification in DCIS lesions adjacent to invasive lesions.[95] Altintas et al. reported that high expression of c-myc in DCIS did not predict local recurrence.[96]

Combinations

A direct positive relationship has been observed for the expression of ER, PR and Bcl-2.[97] Ringberg et al. suggested that a molecular signature with lack of ER and PR, Her-2 overexpression, accumulation of p53 and high Ki67 expression is a strong predictor of local recurrence in DCIS.[98] In a retrospective study of DCIS cases, DCIS lesions that were positive for p16, COX-2 and Ki67 expression are significantly associated with risk of subsequent invasive cancer. In contrast, DCIS lesions that either lacked ER but were positive for ERBB2 and Ki67, or that lacked COX2, yet were positive for p16 and Ki67, were associated with recurrence of DCIS.[99] There are additional studies that associate p16 expression with progression.[100,101]

Other Markers

The results are conflicting on the status of other molecular markers such as TGFβ,[102,103] p27 and p21, and it is currently hard to interpret the roles of these molecules in DCIS progression.[81] Efforts are ongoing to decipher the molecular events associated with progression of DCIS. Lu et al. implicated 14-3-3-sigma in conjunction with Her-2 in the progression to IDC.[104] Qi et al. analyzed miRNA expression patterns in preinvasive and invasive lesions of the breast. They found a consistent increase at each successive stage in the expression of miR-21 along with its targets (PTEN, PCCD4 and TMI).[105] Other examples of noncoding RNAs (ncRNAs) are given below.

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