The online version of this article (https://doi.org/10.1186/s13058-018-1049-0) contains supplementary material, which is available to authorized users.
Elin Borgen, Maria C. Rypdal and Maria Soledad Sosa contributed equally as first authors, and Julio A. Aguirre-Ghiso and Bjørn Naume contributed equally as last authors to this work.
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.
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.
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).
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.
Additional file 1: Table S1. Descriptive data from all tested patients. (XLSX 15 kb)13058_2018_1049_MOESM1_ESM.xlsx
Additional file 2: Figure S1. AE1AE3/NR2F1 DIF staining on normal MNCs spiked with breast cancer cell line cells. The first row shows normal MNCs (AE1AE2-negative) and one breast cancer cell line cell (MCF7; AE1AE3-positive). The MNCs contain 0–3 small/weak NR2F1 signals per nucleus and the cancer cell two similarly small signals. These cells are all defined as being NR2F1low cells in the present study. Occasionally, normal BM cells harbored up to 5 small signals (not shown in the figure). The second row shows MNCs with a cluster of four breast cancer cell line cells (SKBR3), of which the lower left cell does not contain any NR2F1 signals and is therefore defined as NR2F1low. The third cell from the left contains 7–8 small signals, with a tendency to signal clustering, and satisfies the criteria for an NR2F1high cell. In cell numbers 2 and 4 from the left, 4–5 small signals are seen. Although the signals of these cells tend to melt together in clusters/larger signals they still represent expressions below the cut-off for NR2F1high classification, but are approaching the cut-off level. The cells in the second row of this figure therefore illustrate the a priori defined cut-off between NR2F1-positive and -negative cells. (NR2F1 signals in MNCs in the second row are out of focus and therefore not visible on the images). For illustration of the NR2F1 classification of DTCs within the study, see Fig. 2a and Additional file 7: Figure S2. (PDF 684 kb)
Additional file 3: Figure S2. Illustration of the classification system for NR2F1 expression of DTC prospectively chosen for the present study. NR2F1low DTC (A–C). (A) Cluster of three DTCs identified by AE1AE3 in red fluorescence and a morphology compatible with tumor cells. Two of the DTCs have no NR2F1 signals and one has one small NR2F1 signal. Surrounding BM MNCs have 0–1 NR2F1 signals of a similar size. (B, C) One DTC with 2–3 small NR2F1 signals. Adjacent normal BM MNCs with 0–1 small NR2F1 signals. NR2F1high DTC (D, E): (D) Cluster of two DTCs with coarse, partly confluent NR2F1 signals of varying sizes (signals in BM MNCs not visualized because of not being in focus). (E) Cluster of 5 DTCs, three of them defined as NR2F1high because of > 5 NR2F1 signals, partly of large signal size. The remaining two DTCs, with no NR2F1 signals, are assigned NR2F1low, as well as the adjacent normal BM MNCs with 0–1 small NR2F1 signals. (PDF 337 kb)13058_2018_1049_MOESM3_ESM.pdf
Additional file 4: Table S2. Overview of patient material and DTC results. (DOCX 34 kb)13058_2018_1049_MOESM4_ESM.docx
Additional file 5: Table S3. Characteristics of the DTC-positive cases by double immunofluorescence (DIF). (DOCX 33 kb)13058_2018_1049_MOESM5_ESM.docx
Additional file 6: Figure S3. Serial BM samples: number of DTCs detected in the original DTC analysis (APAAP-ICC technique). (PPTX 128 kb)13058_2018_1049_MOESM6_ESM.pptx
Additional file 7: Table S4. Overview of received treatment between the two BM aspiration time points for the patients presented in Fig. 3. (DOCX 33 kb)
Additional file 8: Table S5. Additional results from the serial DTC analyses on samples presented in Fig. 3b and c (in the same order). (DOCX 39 kb)
Additional file 9: Figure S4. (A) Survival analyses (time to systemic relapse/breast cancer death) in relation to NR2F1 profile of DTCs for patients being nonmetastatic at the time point of last DIF DTC-positive BMA and having no subsequent BM analyzed; patients harboring ≥ 500 DTC excluded. (B) Survival analyses (time to systemic relapse/breast cancer death) in relation to NR2F1 profile of DTCs for patients being nonmetastatic at time point of last DIF DTC-positive BMA and having no subsequent BM analyzed; NeoTax study patients excluded. (PPTX 114 kb)13058_2018_1049_MOESM9_ESM.pptx
Additional file 10: Figure S5. Survival analyses according to NR2F1 and Ki67 DTC profiles of patients being nonmetastatic at the time of last DIF DTC-positive BMA, having no subsequent BM analyzed, and no chemotherapy after last BMA. (PPTX 120 kb)13058_2018_1049_MOESM10_ESM.pptx
Additional file 11: Figure S6. (A) Survival analyses (time to systemic relapse/breast cancer death) in relation to NR2F1 profile at last DIF DTC-positive BMA, restricted to those with Ki67 DTC analysis available (only patients being nonmetastatic at last DIF DTC-positive BMA included). (B) As A, but analysis restricted to patients having no subsequent BMA analyzed. (PPTX 68 kb)13058_2018_1049_MOESM11_ESM.pptx
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- NR2F1 stratifies dormant disseminated tumor cells in breast cancer patients
Maria C. Rypdal
Maria Soledad Sosa
Per E. Lønning
Julio A. Aguirre-Ghiso
- BioMed Central
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