BackgroundPatients 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).MethodsCTCs (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.ResultsThe 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.ConclusionsAlthough 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0679-3) contains supplementary material, which is available to authorized users.

Disseminated tumor cells (DTCs) in the bone marrow (BM) and circulating tumor cells (CTCs) in bloodof breast cancer patients (pts) are known to correlate with worse outcome. Here we demonstrate a different prognostic value of DTCs and CTCs and explain these findings by early clodronate intake. CTCs (n = 376 pts) were determined using the AdnaTest BreastCancer (Qiagen Hannover GmbH, Germany) and DTCs (n = 525 pts) were analyzed by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. Clodronate intake was recommended in case of DTC-positivity. CTCs were detected in 22% and DTCs in 40% of the pts, respectively. DTCs were significantly associated with nodal status (p = 0. Despite major improvements in diagnosis and treatment, about 30% of primary breast cancer patients show a relapse of the disease years after first diagnosis which is explained by micrometastatic spread to the bone marrow (BM) by disseminated tumor cells (DTCs) being present in up to 40% of the patients 1 . After the publication of three large cohorts of primary breast cancer patients, the presence and persistence of DTCs during recurrencefree follow-up has been widely accepted as an independent prognostic marker with regard to increased risk for progression free survival (PFS) and overall survival (OS) 2-4 . However, although DTCs have even been used as a monitoring tool for treatment in primary breast cancer BM aspiration is very invasive and less accepted by patients due to pain and discomfort 5 . In this regard, increasing evidence suggested that circulating tumor cells (CTCs) in blood could be a useful biomarker to estimate risk for recurrence in breast cancer. The prognostic significance of these cells has already been demonstrated by different groups 6-8 and very recently, the independent prognostic relevance of CTCs, both before and after adjuvant chemotherapy, has been demonstrated in an impressive cohort of 2026 primary breast cancer patients participating in a large prospective clinical trial 9 . Phenotyping of both cell types in primary breast cancer has demonstrated that a proportion of DTCs and CTCs are present in a non-proliferative state and have stem-cell like characteristics, which may explain resistance to conventional chemotherapeutic drugs [10][11][12][13] . Furthermore, a discordant receptor status between the primary tumor and these cells with regard to the human epidermal growth factor receptor 2 (HER2) and the estrogen receptor (ER) has been demonstrated which might lead to uneffective trastuzumab and/or antihormonal

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