Hormone therapy and anti-ErbB2 therapies are prescribed according to the hormone receptor [estrogen receptor α (ERα)/progesterone receptor] and ErbB2 status of the initial tumor, but it appears that circulating tumor cells (CTCs) and, consequently, the metastatic cells may have a different receptor status. As an attempt to meet the crucial need for identification of the subpopulation of patients that will benefit from more individualized therapies, rapidly evolving therapies should allow a profiling of the tumors and/or of the CTCs. We established a triple fluorescence staining using eight cell lines to visualize the CTCs (cytokeratin detection) and then to define their individual ERα and ErbB2 status. Afterward, we used this method for blood samples from 26 metastatic breast cancer patients. We identified major differences of ERα levels between the cell lines and even within one cell line. For the metastatic patients, we detected and characterized CTCs in 38.5% of the patients with a total of 92 CTCs. We could demonstrate that at least 69.6% of the CTCs exhibit an ERα and/or ErbB2 status different from the status of the primary tumor and that the CTCs from only 30% of the patients had no change of receptor status. Strikingly, heterogeneities of the status, aggregation, and size clearly appear within the CTCs. The data we generated outline the importance of a profiling not only of tumors but also of CTCs to establish individualized treatments. CTCs may then appear as new prognosis and treatment marker for both metastatic and adjuvant breast cancers.

BACKGROUND: We evaluated both estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) status on disseminated tumor cells (DTCs) in the bone marrow of 54 patients with early breast cancer and compared these with the corresponding primary tumor (PT). MATERIALS AND METHODS: Bone marrow aspirates were obtained at the time of first surgery, and ER and HER2 status on DTCs was assessed simultaneously by immunocytochemistry using a triple fluorescence staining method. RESULTS: The median number of DTCs was 13 (range 1-95). The concordance rate between ER status on DTC and PT was 74%. Patients with an ER-positive PT were significantly more likely to have at least one ER-positive DTC (34 out of 42) than patients with an ER-negative PT (6 out of 12; P = .031). Thirty-nine (93%) of the 42 patients with ER-positive PT had at least one ER-negative DTC. The concordance rate between HER2 status on DTC and PT was 52%. The probability of having at least one HER2-positive DTC was not related to the HER2 status of the PT (P = 0.56). Twenty-two (46%) of the 48 patients with a HER2-negative PT had at least one HER2-positive DTC. All the six patients with a HER2-positive PT had at least one HER2-negative DTC. CONCLUSION: Taken together, our study confirms that ER and/or HER2 status may differ between DTC and PT. This discordance could be important for patients lacking ER or HER2 expression on the PT but showing ER-positive or HER2-positive DTC because they might benefit from an endocrine and/or HER2-targeted therapy.

Background: Differences in ER- and HER2-expression on metastases compared to the primary tumor (PT) are a known phenomenon and may have clinical implications in respect of targeted systemic treatment approaches. The aim of this study was to evaluate both ER- and HER2-status on disseminated tumor cells (DTCs) in the bone marrow (BM) of patients (pts) with early breast cancer (EBC) and to compare these with the corresponding PT. Methods: BM aspirates were obtained at the time of first surgery. After Ficoll enrichment for mononuclear cells two cytospins with 106 BM cells were evaluated for ER-, HER2- and cytokeratin (CK) -expressions simultaneously by immunocytochemistry using a triple fluorescence staining method with antibodies directed against human ER (secondly labeled with Cy3, red), HER2 (Coumarin-AMCA, blue) and CK (DyLight488, green). The manual analysis was conducted using a computerized fluorescence microscope (Axioskop, Zeiss, Germany). Criteria for CK- and HER2-positivity were the ring-like appearance of the respective membrane stainings and for ER-expression a nuclear staining. Only pts with the detection of CK positive cells (DTC+) and known ER- and HER2-status of the PT (n = 54) were selected for this analysis. Results: The median number of DTCs was 13 (range 1-95; total number of DTCs detected: 1082). 40 (74%) of the pts had at least one ER-positive (pos) DTC, 24 (44%) at least one HER2-pos DTC, 14 (26%) at least one ER-pos/HER2-pos DTC, and 50 (93%) at least one ER-negative/HER2-negative (neg) DTC, while 10 (19%) pts had only ER-neg/HER2-neg DTCs. The concordance rate between ER-status on DTCs and PT was 74%. Pts with an ER-pos PT were significantly more likely to have at least one ER-pos DTC (34 out of 42) than pts with an ER-neg PT (6 out of 12; Chi-square test, χ2 = 4.66, p = 0.031). 39 (93%) of the 42 pts with ER-pos PT had at least 1 ER-neg DTC. The concordance rate between HER2-status on DTCs and PT was 52%. The probability of having at least one HER2-pos DTC was not related to the HER2-status of the PT (Chi-square test, χ2 = 0.34, p = 0.56). 22 (46%) of the 48 pts with a HER2-neg PT had at least one HER2-pos DTC. All of the 6 pts with a HER2-pos PT had at least one HER2-neg DTC. 7 out of 10 pts with a triple-neg PT had at least one DTC pos for ER, HER2 or both. Further the heterogeneity of the ER- and HER2-expression on DTCs compared to the PT for different DTC counts was evaluated. We detected all possible combinations of ER- and HER2-experssion on DTCs regardless of the respective status of the PT. Conclusions: Our study confirms that the ER- and/or HER2-status on DTCs may differ compared to the PT. This discordance could be especially important for pts with a triple-neg PT and ER-pos or HER2-pos DTCs, since they might respond favorably to an endocrine or HER2-targeted therapy. On the other hand, the presence of ER-neg or HER2-neg DTCs in pts with ER-pos or HER2-pos PT might explain some of the failures of adjuvant endocrine or HER2 targeted therapy. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-06.

e22059 Background: The goal of this study was to evaluate both ER- and HER2-status on DTCs in the bone marrow of patients with early BC and to compare these with the corresponding PT. In case of discordant expression, DTCs might help in properly selecting targeted therapy according to their phenotype. Methods: Bone marrow (BM) samples were obtained from early BC patients at the time of first breast surgery (years 2006 to 2009). Two cytospins with 106 BM cells were prepared after Ficoll enrichment for mononuclear cells. We were able to determine the presence of DTCs (cytokeratin (CK)+cells) in a total of 58 patients. Simultaneously ER- and HER2-expressions were analyzed by immunocytochemistry using a triple fluorescence staining method. Briefly, antibodies (AB) directed against human ER (secondly labeled with Cy3, red), HER2 (DyLight488, blue) and CK (Coumarin-AMCA, green) were used. The manual analysis was conducted using a computerized fluorescence microscope (Axioskop, Zeiss, Germany). Criteria for CK-positivity and HER2-overexpression were the ring-like appearance of the respective membrane-staining and for ER-expression a nuclear staining. Data on ER- and HER2-status of the PT were available for all 58 and 54 of the patients respectively. Results: The median number of DTCs detected was 12 (range 1-95; total number of DTCs 1132). 41 (71%) of the patients had at least one ER+ DTC, 25 (43%) at least one HER2+ DTC, 15 (26%) at least one ER+/HER2+ DTC and 13 (22%) had DTCs neither expressing ER nor HER2. The concordance rate between ER-status on DTCs and PT was 74%. 6 (43%) patients with ER- PT had ER+ DTCs. The concordance rate between HER2-status on DTCs and PT was 52% and 22 (46%) patients with HER2- PT had HER2+DTCs. Seven out of 10 patients with a triple-negative PT had at least one DTC positive for ER, HER2, or both. Conclusions: Our study confirms that the ER- and/or HER2-status on DTCs might differ compared to the PT. This discordance, which we demonstrated at the protein level, could be especially important for patients with a triple-negative PT and ER+ or HER2+ DTCs, since they might respond favorably to an endocrine or HER2-targeted therapy.