The functional loss of the retinoblastoma tumour suppressor is a common event in basal-like and luminal B breast carcinomas

Jason I Herschkowitz, Xiaping He, Cheng Fan, Charles M Perou, Jason I Herschkowitz, Xiaping He, Cheng Fan, Charles M Perou

Abstract

Introduction: Breast cancers can be classified using whole genome expression into distinct subtypes that show differences in prognosis. One of these groups, the basal-like subtype, is poorly differentiated, highly metastatic, genomically unstable, and contains specific genetic alterations such as the loss of tumour protein 53 (TP53). The loss of the retinoblastoma tumour suppressor encoded by the RB1 locus is a well-characterised occurrence in many tumour types; however, its role in breast cancer is less clear with many reports demonstrating a loss of heterozygosity that does not correlate with a loss of RB1 protein expression.

Methods: We used gene expression analysis for tumour subtyping and polymorphic markers located at the RB1 locus to assess the frequency of loss of heterozygosity in 88 primary human breast carcinomas and their normal tissue genomic DNA samples.

Results: RB1 loss of heterozygosity was observed at an overall frequency of 39%, with a high frequency in basal-like (72%) and luminal B (62%) tumours. These tumours also concurrently showed low expression of RB1 mRNA. p16INK4a was highly expressed in basal-like tumours, presumably due to a previously reported feedback loop caused by RB1 loss. An RB1 loss of heterozygosity signature was developed and shown to be highly prognostic, and was potentially a predictive marker of response to neoadjuvant chemotherapy.

Conclusions: These results suggest that the functional loss of RB1 is common in basal-like tumours, which may play a key role in dictating their aggressive biology and unique therapeutic responses.

Figures

Figure 1
Figure 1
The expression of retinoblastoma pathway members varies across breast cancer intrinsic subtypes. Two-hundred and thirty-two human samples are ordered by subtype according to the five-class single sample predictor from Hu and colleagues [9]. Samples are coloured according to their subtype: red = basal-like, dark blue = luminal A, light blue = luminal B, pink = human epidermal growth factor receptor 2 (HER2)-enriched and green = normal breast-like. (a) proliferation gene cluster. (b) Retinoblastoma (RB)-pathway genes which are present on the array and passed data quality filtering criteria of showing a signal intensity of more than 30 units in both channels.
Figure 2
Figure 2
The expression of RB1, p16INK4a and cyclin D1 varies across the breast cancer intrinsic subtypes. Box plot comparisons of (a) Retinoblastoma (RB) 1, (b) proliferation signature, (c) p16INK4a and (d) cyclin D1 mRNA expression relative to the five intrinsic subtypes as defined by the five-class centroid predictor.
Figure 3
Figure 3
Retinoblastoma (RB) 1 loss of heterozygosity (LOH) is associated with high proliferation. (a) An example of LOH detected in a breast tumour sample at RB1 intron 20. The arrows point to the missing allele with A7-N being DNA from normal tissue and A7-T is tumour DNA from the same patient. (b) Two-way unsupervised hierarchical clustering of breast tumour samples with informative RB1 LOH status (LOH + = blue, LOH - = yellow) using the proliferation gene cluster. (c) Box plot comparison of the average proliferation cluster expression to RB1 LOH status.
Figure 4
Figure 4
High p16INK4a mRNA and protein levels are associated with retinoblastoma (RB) 1 loss of heterozygosity (LOH). (a) RB1 staining of normal breast tissue, (b) RB1 LOH+ basal-like tumour lacking RB1 staining, and (c) the same RB1 LOH+ basal-like tumour showing staining for p16INK4a both nuclear and cytoplasmic.(d) Box plot comparison showing high p16INK4a mRNA expression in RB1 LOH + breast tumours.
Figure 5
Figure 5
Venn diagram showing the overlap between the retinoblastoma (RB) 1 loss of heterozygosity (LOH), proliferation, and RB1-loss gene lists.
Figure 6
Figure 6
Retinoblastoma (RB) 1 loss of heterozygosity (LOH) gene list is highly predictive of breast cancer patient outcome. Kaplan-Meier survival curves showing overall survival (OS) or relapse-free survival (RFS) by dividing the patients in thirds (a, c) or in halves (b, d) using the rank order average expression values of the RB-LOH signature using the NKI295 breast cancer data set.

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