The ZNF217 Biomarker Predicts Low- and High-Risk Oncotype DX® Recurrence Score in ER-Positive Invasive Breast Cancers

We assessed mRNA and protein expression levels of the ZN217 oncogene in 17 clinical FFPE ER-positive invasive breast cancer specimens with known (low or high) Oncotype DX^® Recurrence Scores. This study shows that mRNA or nuclear protein levels of the ZNF217 significantly correlate with Oncotype DX^® Recurrence Score.

Report

Breast cancer (BC) is the most frequent cancer among women. Expression of Estrogen Receptor α (ERα) is found in 60–80% of BC patients, and allows an accurate prediction of response to endocrine therapy (ET). However, between 10 and 50% of ER⁺ BC treated patients will later relapse. Thus, a more precise method for stratifying patients based on their prognosis and for predicting their response to therapy remains needed.

The Oncotype DX^® (ODX) genomic assay tests for the expression of 21 genes and calculates a Recurrence Score (RS), which predicts the risk of distant disease recurrence in ER⁺ BC. A high RS value indicates a poor prognosis and a higher probability of distant recurrence at 10 years in patients treated with adjuvant ET (Paik et al., 2004). We have shown that high expression levels of the ZNF217 oncogenic transcription factor are associated with poor prognosis, recurrent distant metastases and can predict response to ET in ER⁺ BC (Vendrell et al., 2012; Nguyen et al., 2014). This novel snapshot report investigates the correlation between ZNF217 expression levels (protein or mRNA) and ODX RS.

After approval by the Institutional Review Board, the pathology database of the Montefiore Medical Center (NY, USA) was searched to identify ER⁺ BC cases with: (i) low-risk (<18) or high-risk (>31) ODX RS; (ii) sufficient tissue for both ZNF217 immunohistochemistry (IHC) (Nguyen et al., 2014) and ZNF217 RTQ-PCR (Loudig et al., 2007; Kotorashvili et al., 2012; Vendrell et al., 2012) investigations. Seventeen FFPE clinical specimens were selected (Figure 1A). After ZNF217 IHC analysis, the percentage of positive staining of tumor nuclei was estimated (range: 0–80%). ZNF217 mRNA levels ranged from 0.5 to 22.5 (arbitrary units) and the mean value was used as a cutoff. Figure 1 illustrates that: (i) all the clinical specimens with low-risk ODX RS displayed low ZNF217 mRNA levels (<4) or low percentage of IHC stained nuclei (<5%); (ii) ZNF217 nuclear staining and ZNF217 mRNA levels were significantly associated with ODX RS; (iii) combining both IHC analysis and ZNF217 mRNA levels allowed the stratification of the samples with a better accuracy, with 100 and 80%, respectively, of low-risk ODX RS and high-risk ODX RS correctly classified and significantly association with ZNF217 expression levels (P = 0.002). Strikingly, two high ODX RS specimens displaying the highest ZNF217 mRNA levels (9.2 and 22.5) also displayed the highest ZNF217 IHC staining (70–80%) and pejorative clinical record (T2 with invaded nodes and recurrent breast cancer, respectively).

FIGURE 1

Figure 1. (A) ZNF217 predicts low- and high-risk Oncotype DX^® Recurrence Score. (B) Illustrative examples of ZNF217 IHC staining in two representative ER⁺ invasive breast carcinoma tumor samples with high-risk Oncotype DX^® Recurrence Score with positive nuclear staining (specimen A) or low-risk Oncotype DX^® Recurrence Score without nuclear staining (specimen B). (C) RTQ-PCR assessment of ZNF217 mRNA levels (arbitrary units) in specimen A and specimen B.

Altogether, while these exploratory results were obtained in a small cohort, our preliminary data indicate a correlation between ZNF217 expression levels and ODX RS. This is in agreement with previous observations that both ZNF217 expression levels and the ODX RS are prognostic and predictive of ET response in ER⁺ BC. Supporting recent observations indicate that ZNF217 expression levels also predict neoadjuvant ET response in these patients (Vendrell et al., 2018). However, it is necessary to extend our study to a larger cohort including low-, high- but also intermediate- ODX RS specimens to investigate whether assessing ZNF217 levels (alone or in combination with ODX) could provide additional information to the current well-established ODX genomic assay.

Ethics Statement

The protocol was approved by the Institutional Review Board, Montefiore Medical Center and Albert Einstein College of Medicine (NY, USA).

Author Contributions

PC and SF conceived the study. SF supervised and analyzed the IHC experiments performed by JA. OL supervised and analyzed the RTQ-PCR experiments performed by CL. PC and SF co-analyzed the data. PC wrote the manuscript.

Conflict of Interest Statement

SF served in an expert advisory panel for Genomic Health.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

The French-American Fulbright Program and the French Ministry of Higher Education, Research and Innovation supported PC. This work was supported by the French Ligue contre le cancer (committees 71 and 42). We thank Dr. B. Manship for critical reading of the manuscript.

Abbreviations

BC, Breast Cancer; ERα, Estrogen receptor α; ER+, Estrogen receptor α-positive; ET, endocrine therapy; ODX, Oncotype DX^®; RS, Recurrence Score; FFPE, Formalin-Fixed, Paraffin-Embedded; IHC, immunohistochemistry; RTQ-PCR, real-time quantitative polymerase chain reaction.

References

Kotorashvili, A., Ramnauth, A., Liu, C., Lin, J., Ye, K., Kim, R., et al. (2012). Effective DNA/RNA co-extraction for analysis of microRNAs, mRNAs, and genomic DNA from formalin-fixed paraffin-embedded specimens. PLoS ONE 7:e34683. doi: 10.1371/journal.pone.0034683

PubMed Abstract | CrossRef Full Text | Google Scholar

Loudig, O., Milova, E., Brandwein-Gensler, M., Massimi, A., Belbin, T. J., Childs, G., et al. (2007). Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT). Nucleic Acids Res. 35:e94. doi: 10.1093/nar/gkm510

PubMed Abstract | CrossRef Full Text | Google Scholar

Nguyen, N. T., Vendrell, J. A., Poulard, C., Gyorffy, B., Goddard-Leon, S., Bieche, I., et al. (2014). A functional interplay between ZNF217 and estrogen receptor alpha exists in luminal breast cancers. Mol. Oncol. 8, 1441–1457. doi: 10.1016/j.molonc.2014.05.013

PubMed Abstract | CrossRef Full Text | Google Scholar

Paik, S., Shak, S., Tang, G., Kim, C., Baker, J., Cronin, M., et al. (2004). A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. N. Engl. J. Med. 351, 2817–2826. doi: 10.1056/NEJMoa041588

PubMed Abstract | CrossRef Full Text | Google Scholar

Vendrell, J. A., Solassol, J., Gyorffy, B., Vilquin, P., Jarlier, M., Donini, C. F., et al. (2018). Evaluating ZNF217 mRNA expression levels as a predictor of response to endocrine therapy in ER+ breast cancer. Front. Pharmacol. 9:1581. doi: 10.3389/fphar.2018.01581

PubMed Abstract | CrossRef Full Text | Google Scholar

Vendrell, J. A., Thollet, A., Nguyen, N. T., Ghayad, S. E., Vinot, S., Bieche, I., et al. (2012). ZNF217 is a marker of poor prognosis in breast cancer that drives epithelial-mesenchymal transition and invasion. Cancer Res. 72, 3593–3606. doi: 10.1158/0008-5472.CAN-11-3095

PubMed Abstract | CrossRef Full Text | Google Scholar