Presence of circulating tumor cells (CTCs) is a validated prognostic marker in metastatic breast cancer. Additional prognostic information may be obtained by morphologic characterization of CTCs. We explored whether apoptotic CTCs, CTC clusters and leukocytes attached to CTCs are associated with breast cancer subtype and prognosis at base-line (BL) and in follow-up (FU) blood samples in patients with metastatic breast cancer scheduled for first-line systemic treatment. Patients with a first metastatic breast cancer event were enrolled in a prospective observational study prior to therapy initiation and the CellSearch system (Janssen Diagnostics) was used for CTC enumeration and characterization. We enrolled patients (N = 52) with ≥5 CTC/7.5 ml blood at BL (median 45, range 5–668) and followed them with blood sampling for 6 months during therapy. CTCs were evaluated for apoptotic changes, CTC clusters (≥3 nuclei), and leukocytes associated with CTC (WBC-CTC, ≥1 CTC + ≥1 leukocytes) at all time-points by visual examination of the galleries generated by the CellTracks Analyzer. At BL, patients with triple-negative and HER2-positive breast cancer had blood CTC clusters present more frequently than patients with hormone receptor-positive cancer (P = 0.010). No morphologic characteristics were associated with prognosis at BL, whereas patients with apoptotic CTCs or clusters in FU samples had worse prognosis compared to patients without these characteristics with respect to progression-free (PFS) and overall survival (OS) (log-rank test: P = 0.0012 or lower). Patients with apoptotic or clustered CTCs at any time-point had impaired prognosis in multivariable analyses adjusting for number of CTCs and other prognostic factors (apoptosis: HROS = 25, P < 0.001; cluster: HROS = 7.0, P = 0.006). The presence of WBC-CTCs was significantly associated with an inferior prognosis in terms of OS at 6 months in multivariable analysis. Patients with a continuous presence of apoptotic or clustered CTCs in FU samples after systemic therapy initiation had worse prognosis than patients without these CTC characteristics. In patients with ≥5 CTC/7.5 ml blood at BL, morphologic characterization of persistent CTCs could be an important prognostic marker during treatment, in addition to CTC enumeration alone. Clinical Trials (NCT01322893), registration date 21 March 2011

Background: PAM50 breast cancer intrinsic subtyping adds prognostic information in early breast cancer; however, the role in metastatic disease is unclear. We aimed to identify PAM50 subtypes in primary tumors (PTs) and metastases to outline subtype changes and their prognostic role. Methods: RNA was isolated from PTs, lymph node metastases (LNMs), and distant metastases (DMs) in metastatic breast cancer patients (n = 140) included in a prospective study (NCT01322893). Gene expression analyses were performed using the Breast Cancer 360 (BC360) assay from Nano-String. The subtype shifts were evaluated using McNemar and symmetry tests, and clinical outcomes were evaluated with log-rank tests and Cox regression. Results: The PAM50 subtype changed in 25/59 of paired samples between PTs and LNMs (P_symmetry = 0.002), in 31/61 between PTs and DMs (P_symmetry < 0.001), and in 16/38 between LNMs and DMs (P_symmetry = 0.004). Shifts toward subtypes with worse outcomes were the most common. Patients with shifts from the luminal PT to non-luminal DM subtypes had worse progression-free survival compared to patients with a stable subtype (hazard ratio (HR): 2.3; 95% confidence interval (CI): 1.14–4.68, p = 0.02). Conclusion: Strong evidence of PAM50 subtype shifts toward unfavorable subtypes were seen between PTs and metastatic samples. For patients with a shift in subtype from luminal PT to non-luminal DM, a worse prognosis was noted.

Mammographic tumour appearance may provide prognostic useful information. For example, spiculation indicates invasiveness, but also better survival compared to tumours with other appearances. We aimed to study the relationship between mammographic tumour appearance and established clinicopathological factors, including surrogate molecular breast cancer subtypes, in the large Malmö Diet and Cancer Study. A total of 1116 women with invasive breast cancer, diagnosed between 1991 and 2014, were included. Mammographic tumour appearance in relation to status for oestrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2, histological grade, Ki67 and molecular subtype was analysed using various regression models. All models were adjusted for relevant confounders, including breast density, which can affect mammographic appearance. The results consistently showed that spiculated tumours are indicative of favourable characteristics, as they are more likely to be ER and PR positive, and more often exhibit lower histological grade and lower Ki67 expression. Furthermore, spiculated tumours tend to be of luminal A-like subtype, which is associated with a good prognosis. The establishment of associations between mammographic tumour appearance and clinico­pathological factors may aid in characterizing breast cancer at an earlier stage. This could contribute to more individualized breast cancer treatment in the future.

Enumeration of circulating tumor cells (CTCs) obtained from minimally invasive blood samples has been well established as a valuable monitoring tool in metastatic and early breast cancer, as well as in several other cancer types. The gold standard technology for detecting CTCs in blood against a backdrop of millions of leukocytes is the FDA-approved CellSearch system (Janssen Diagnostics), which relies on EpCAM-based immunomagnetic separation. Secondary characterization of these cells could enable treatment selection based on specific targets in these cells, as well as providing a real time window into the metastatic process and offering unique insights into tumor heterogeneity. The objective of this study was to develop a method for downstream characterization of CTCs following isolation with the CellSearch system. An in vitro CTC model system focusing on clinically useful treatment predictive biomarkers in breast cancer, specifically the estrogen receptor α (ERα) and the human epidermal growth factor receptor 2 (HER2), was established using healthy donor blood spiked with breast cancer cell lines MCF7 (ERα+/HER2−) and SKBr3 (ERα−/HER2+). Following CTC isolation by CellSearch, the captured CTCs were further enriched and fixed on a microscope slide using the in-house-developed CTC-DropMount technique. The recovery rate of CTCs after CellSearch Profile analysis and CTC-DropMount was 87%. A selective and consistent triple-immunostaining protocol was optimized. Cells positive for DAPI, cytokeratin (CK) 8, 18 and 19, but negative for the leukocyte-specific marker CD45, were classified as CTCs and subsequently analyzed for ERα and HER2 expression. The method was verified in breast cancer patient samples, thus demonstrating its clinical relevance. Our results show that it is possible to ascertain the status of important predictive biomarkers expressed in breast cancer CTCs using the newly developed CTC-DropMount technique. Downstream characterization of multiple biomarkers using a standard fluorescence microscope demonstrates that important clinical and biological information may be obtained from a single patient blood sample following either CellSearch epithelial or profile analyses. Clinical Trials NCT01322893

The rapid development of new therapies in metastatic breast cancer (MBC), entails a need for improved prognostic and monitoring tools. Thymidine kinase 1 (TK1) is involved in DNA synthesis and its activity correlates to outcome in cancer patients. The aim of this study was to evaluate serum TK1 activity (sTK1) levels in MBC patients as a tool for prognostication and treatment monitoring. 142 women with MBC scheduled for 1^st line systemic treatment were included in a prospective observational study. sTK1 was measured at baseline (BL) and at 1, 3 and 6 months and correlations to progression-free and overall survival (PFS, OS) evaluated. High sTK1 levels (above median) correlated to worse PFS and OS at BL, also after adjusting for other prognostic factors. sTK1 levels were significantly associated with PFS and OS measured from follow-up time points during therapy. Changes from 3 to 6 months during therapy significantly correlated to PFS and OS, whereas early changes did not. We could demonstrate sTK1 level as an independent prognostic factor in patients with newly diagnosed MBC. Changes in sTK1 levels from 3 to 6 months correlated to PFS and OS. Future studies of sTK1 are warranted to further define its clinical utility.

Background: Metastatic breast cancer (MBC) is a challenging disease, and despite new therapies, prognosis is still poor for a majority of patients. There is a clinical need for improved prognostication where immuno-oncology markers can provide important information. The aim of this study was to evaluate serum immuno-oncology markers in MBC patients and their respective relevance for prediction of survival. Patients and methods: We investigated a broad panel of 92 immuno-oncology proteins in serum from 136 MBC patients included in a prospective observational study (NCT01322893) with long-term follow-up. Serum samples were collected before start of systemic therapy and analyzed using multiplex proximity extension assay (Olink Target 96 Immuno-Oncology panel). Multiple machine learning techniques were used to identify serum markers with highest importance for prediction of overall and progression-free survival (OS and PFS), and associations to survival were further evaluated using Cox regression analyses. False discovery rate was then used to adjust for multiple comparisons. Results: Using random forest and random survival forest analyses, we identified the top nine and ten variables of highest predictive importance for OS and PFS, respectively. Cox regression analyses revealed significant associations (P < 0.005) of higher serum levels of IL-8, IL-10 and CAIX with worse OS in multivariable analyses, adjusted for established clinical prognostic factors including circulating tumor cells (CTCs). Similarly, high serum levels of IL-8, IL-10, ADA and CASP8 significantly associated with worse PFS. Interestingly, high serum levels of FasL significantly associated with improved OS and PFS. In addition, CSF-1, IL-6, MUC16, TFNSFR4 and CD244 showed suggestive evidence (P < 0.05) for an association to survival in multivariable analyses. After correction for multiple comparisons, IL-8 still showed strong evidence for correlation to survival. Conclusion: To conclude, we found six serum immuno-oncology markers that were significantly associated with OS and/or PFS in MBC patients, independently of other established prognostic factors including CTCs. Furthermore, an additional five serum immuno-oncology markers provided suggestive evidence for an independent association to survival. These findings highlight the relevance of immuno-oncology serum markers in MBC patients and support their usefulness for improved prognostication. Trial registration Clinical Trials (NCT01322893), registered March 25, 2011.