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. 2018 Oct 16;20(1):120.
doi: 10.1186/s13058-018-1049-0.

NR2F1 stratifies dormant disseminated tumor cells in breast cancer patients

Elin Borgen  1 , Maria C Rypdal  1 , Maria Soledad Sosa  2   3 , Anne Renolen  1 , Ellen Schlichting  4 , Per E Lønning  5   6 , Marit Synnestvedt  7 , Julio A Aguirre-Ghiso  8 , Bjørn Naume  9   10
Affiliations

NR2F1 stratifies dormant disseminated tumor cells in breast cancer patients

Elin Borgen et al. Breast Cancer Res. .

Abstract

Background: The presence of disseminated tumor cells (DTCs) in bone marrow (BM) is an independent prognostic factor in early breast cancer but does not uniformly predict outcome. Tumor cells can persist in a quiescent state over time, but clinical studies of markers predicting the awakening potential of DTCs are lacking. Recently, experiments have shown that NR2F1 (COUP-TF1) plays a key role in dormancy signaling.

Methods: We analyzed the NR2F1 expression in DTCs by double immunofluorescence (DIF) staining of extra cytospins prepared from 114 BM samples from 86 selected DTC-positive breast cancer patients. Samples collected at two or more time points were available for 24 patients. Fifteen samples were also analyzed for the proliferation marker Ki67.

Results: Of the patients with detectable DTCs by DIF, 27% had ≥ 50% NR2F1high DTCs, chosen a priori as the cut-off for "dormant profile" classification. All patients with systemic relapse within 12 months after BM aspiration carried ≤ 1% NR2F1high DTCs, including patients who transitioned from having NR2F1high-expressing DTCs in previous BM samples. Of the patients with serial samples, half of those with no relapse at follow-up had ≥ 50% NR2F1high DTCs in the last BM aspiration analyzed. Among the 18 relapse-free patients at the time of the last DTC-positive BM aspiration with no subsequent BM analysis performed, distant disease-free intervals were favorable for patients carrying ≥ 50% NR2F1high DTCs compared with those with predominantly NR2F1low DTCs (p = 0.007, log-rank). No survival difference was observed by classification according to Ki67-expressing DTCs (p = 0.520).

Conclusions: Our study translates findings from basic biological analysis of DTC dormancy to the clinical situation and supports further clinical studies of NR2F1 as a marker of dormancy.

Keywords: Bone marrow; Breast cancer; DTC; Disseminated tumor cells; Dormancy; Micrometastasis; NR2F1; Occult disease.

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Conflict of interest statement

Ethics approval and consent to participate

NeoTax Study: Norwegian Regional Ethics Committee approval number 273/96–82.96 REK Helseregion III. Oslo1 Study: Norwegian Regional Ethics Committee approval number S-97103. SATT Study: Norwegian Regional Ethics Committee approval number S-03032. Informed consent to participate was given by all patients.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Clinical studies overview. Overview of the clinical studies, number of patients, and number of samples analyzed by DIF in the present study. Bone marrow aspiration (BMA) time points are indicated, as well as therapy administered. *One patient had a BMA performed at an unknown time point; however not harboring any disseminated tumor cells (DTC) by DIF. ER estrogen receptor
Fig. 2
Fig. 2
Images of disseminated tumor cells (DTCs) stained by double immunofluorescence (AE1AE3/NR2F1 and AE1AE3/Ki67) and correlation between Ki67 and NR2F1 expression. a DTCs from the BM of study patients analyzed by DIF. The strong and irregular cytoplasmic cytokeratin staining (AE1AE3 antibody, in red fluorescence) identifies these cells as DTCs among the normal BM MNCs (AE1AE3-negative). The two upper rows show NR2F1high DTCs with the presence of nuclear NR2F1 signal clusters (in green fluorescence; first row) or one large size NR2F1 signal (i.e., larger than the size range observed in normal BM MNCs; second row). Third row shows two DTCs classified as NR2F1low containing only two (lower cell) or three (upper cell) small NR2F1 signals, i.e., expression not exceeding what may be observed in normal BM MNCs. The bottom row shows two DTCs positive for Ki67 (in green). b Comparison of the expression on DTC of NR2F1 versus the proliferation marker Ki67. Results from DIF analysis of Ki67/AE1AE3 versus NR2F1/AE1AE3, respectively, on 15 of the BM samples presented in Table 1, where additional cytospins were available for both analyses
Fig. 3
Fig. 3
Disseminated tumor cell (DTC) status by DIF and NR2F1 expression in patients with bone marrow (BM) samples available at two time points. Results of AE1AE3/NR2F1 DIF analysis performed on 24 patients classified as DTC-positive in the original DTC analysis, and with available BM samples from two aspiration time points. The number of DIF cytokeratin-positive DTCs (a), the proportion of NR2F1high DTCs in patients with DIF DTC-positive status at both BM aspiration (BMA) time points (b), and the proportion of NR2F1high DTCs in patients with DIF DTC-positive status in the second but not the first BMA (c) are presented. The right sections of b and c show time to relapse or last observation and additional clinical information for the patients presented in b and c. Chemo chemotherapy, N/A not applicable, neg negative, pos positive, Pt patient
Fig. 4
Fig. 4
Survival analyses in relation to DTC dormancy profile and Ki67 status. Survival analyses (time to systemic relapse/breast cancer death) in relation to NR2F1 (a,c) and Ki67 profile (b,d) of DTCs (at last DIF DTC-positive bone marrow (BM) aspiration). a,b Patients being nonmetastatic at last DIF DTC-positive BMA. c,d Patients being  nonmetastatic at last DIF DTC-positive BMA with no subsequent BM analysis performed. Cum cumulative
See this image and copyright information in PMC

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