Does primary neoadjuvant systemic therapy eradicate minimal residual disease? Analysis of disseminated and circulating tumor cells before and after therapy

Abstract

Background: Patients with breast cancer (BC) undergoing neoadjuvant chemotherapy (NACT) may experience metastatic relapse despite achieving a pathologic complete response. We analyzed patients with BC before and after NACT for disseminated tumor cells (DTCs) in the bone marrow(BM); comprehensively characterized circulating tumor cells (CTCs), including stem cell-like CTCs (slCTCs), in blood to prove the effectiveness of treatment on these cells; and correlated these findings with response to therapy, progression-free survival (PFS), and overall survival (OS).

Methods: CTCs (n = 135) and slCTCs (n = 91) before and after NACT were analyzed using the AdnaTest BreastCancer, AdnaTest TumorStemCell, and epithelial-mesenchymal transition (QIAGEN Hannover GmbH Germany). The expression of estrogen receptor, progesterone receptor, and the resistance marker excision repair cross-complementing rodent repair deficiency, complementation group 1 (ERCC1), nuclease were studied in separate single-plex reverse transcription polymerase chain reaction experiments. DTCs were evaluated in 142 patients before and 165 patients after NACT using the pan-cytokeratin antibody A45-B/B3 for immunocytochemistry.

Results: The positivity rates for DTCs, CTCs, and slCTCs were 27 %, 24 %, and 51 % before and 20 %, 8 %, and 20 % after NACT, respectively. Interestingly, 72 % of CTCs present after therapy were positive for ERCC1, and CTCs before (p = 0.005) and after NACT (p = 0.05) were significantly associated with the presence of slCTCs. Whereas no significant associations with clinical parameters were found for CTCs and slCTCs, DTCs were significantly associated with nodal status (p = 0.03) and histology (0.046) before NACT and with the immunohistochemical subtype (p = 0.02) after NACT. Univariable Cox regression analysis revealed that age (p = 0.0065), tumor size before NACT (p = 0.0473), nodal status after NACT (p = 0.0137), and response to NACT (p = 0.0136) were significantly correlated with PFS, whereas age (p = 0.0162) and nodal status after NACT (p = 0.0243) were significantly associated with OS. No significant correlations were found for DTCs or any CTCs before and after therapy with regard to PFS and OS.

Conclusions: Although CTCs were eradicated more effectively than DTCs, CTCs detected after treatment seemed to be associated with tumor cells showing tumor stem cell characteristics as well as with resistant tumor cell populations that might indicate a worse outcome in the future. Thus, these patients might benefit from additional second-line treatment protocols including bisphosphonates for the eradication of DTCs.

Fig. 1

Comparison of expression profiles of circulating tumor cells (CTCs) before and after neoadjuvant chemotherapy (NACT). The expression rates of the different transcripts analyzed on CTCs are shown (a) before and (b) after NACT. c The presence of stem cell–like circulating tumor cell (slCTCs) is shown, including aldehyde dehydrogenase 1 (ALDH1)-positive cells and CTCs in epithelial–mesenchymal transition (EMT) before and after NACT. The identification of EMT markers was considered positive if at least one marker (phosphoinositide 3-kinase, AKT2, Twist-related protein 1) was detected in the sample. EpCAM epithelial cell adhesion molecule, ERCC1 excision repair cross-complementing rodent repair deficiency, complementation group 1, HER2 human epidermal growth factor receptor 2, MUC1 mucin 1, cell surface associated

Fig. 2

Prognostic capability of DTCs, CTCs, and slCTCs before and after NACT with regard to PFS. Kaplan-Meier curves were drawn to compare PFS with regard to CTCs/slCTCs in blood and DTCs in BM before and after NACT. No significant associations were found with regard to PFS for every cell type tested (for DTCs, HR 1.065, 95 % CI 0.411–2.763, p = 0.9190 before therapy; HR 1.053, 95 % CI 0.393–2.821, p = 0.8969 after therapy; for CTCs, HR 1.298, 95 % CI 0.466–3.620, p = 0.6179 before NACT; HR 0.607, 95 % CI 0.080–4.582, p = 0.6285 after NACT; and for slCTCs, HR 1.346, 95 % CI 0.450–4.032, p = 0.5950 before therapy; HR 0.254, 95 % CI 0.033–1.942, p = 0.1865 after therapy). In the univariable Cox regression model estimated survival curves, blue line = DTC/CTC/slCTC-negative patients and red line = DTC/CTC/slCTC-positive patients. BM bone marrow, CI confidence interval, CTC circulating tumor cell, DTC disseminated tumor cell, HR hazard ratio, NACT neoadjuvant chemotherapy, PFS progression-free survival, slCTC stem cell–like circulating tumor cell

Fig. 3

Prognostic capability of DTCs, CTCs, and slCTCs before and after NACT with regard to OS. Estimated survival curves adjusted for cell type status were drawn to compare OS with regard to CTCs/slCTCs in blood and DTCs in BM before and after NACT. No significant associations were found with regard to OS for every cell type tested [for DTCs, HR 1.404, 95 % CI 0.422–4.664, p = 0.5800 before therapy; HR 0.628, 95 % CI 0.142–2.786, p = 0.5406 after therapy; for CTCs, HR 1.795, 95 % CI 0.538–5.987, p = 0.3414 before NACT; HR 1.083, 95 % CI 0.137–8.550, p = 0.9400 after NACT; for slCTCs, HR 0.830, 95 % CI 0.186–3.710, p = 0.8068 before and after therapy; and not applicable (no events for slCTC-positive patients)]. BM bone marrow, CI confidence interval, CTC circulating tumor cell, DTC disseminated tumor cell, HR hazard ratio, NACT neoadjuvant chemotherapy, OS overall survival, slCTC stem cell–like circulating tumor cell