Circulating tumor cells in newly diagnosed inflammatory breast cancer

This retrospective study was conducted under Institutional Review Board (IRB)-approved protocol DR10-0227 by using the Clinic Station, the MD Anderson Cancer Center (MDACC) electronic medical record database. Patients were identified by using two databases; database of newly diagnosed IBC patients treated in MD Anderson Cancer Center between 1989 and 2011, as described in the previous study [3], and the MDACC IBC database with available data from 2007 to 2012 (Figure 1). A population of consecutive stage III IBC and metastatic IBC patients with CTCs measurement before starting neoadjuvant or first-line treatment, was eligible. Only treatment-naïve patients with newly diagnosed disease, starting treatment with neoadjuvant or first-line chemotherapy, were included in this study. Patients underwent systemic therapy, as appropriate for their malignancies, irrespective of CTCs. Patients with concurrent malignancy other than nonmelanoma skin cancer in the previous 5 years were excluded.

All patients underwent pretreatment diagnostic biopsy. The diagnosis of IBC was based on clinical signs such as diffuse erythema, peau d'orange, tenderness, induration, and warmth [26,27]. The presence of dermal lymphatic emboli in the diagnostic pathology report was not mandatory for the pathological diagnosis of IBC. Clinical stage at diagnosis of primary disease was coded according to the criteria set forth in the sixth edition of the American Joint Committee on Cancer’s AJCC Cancer Staging Manual [28].

In all patients, data regarding age, menopausal status, tumor histologic subtype, hormone-receptor status, HER2 amplification status, type and number of sites of metastases, delivery of systemic therapy, and outcome (progression, survival, pathological complete remission) were recorded and compared with the presence and number of CTCs. Because statins might have an antitumor effect in IBC, we also recorded statin use before the diagnosis of IBC [25]. Lipophilic statins (simvastatin, fluvastatin, and lovastatin) were classified as L-statins, and weakly lipophilic-to-hydrophilic statins (atorvastatin, pravastatin, and rosuvastatin) were classified as H-statins, as described previously [25].

The retrospective study was approved by the Institutional Review Board of the University of Texas, MD Anderson Cancer Center, and a waiver of consent form was granted.

The CellSearch system (Veridex Corporation, Warren, NJ, USA) was used to detect and enumerate CTCs in 7.5 ml of whole peripheral blood. Samples were subjected to enrichment of epithelial cells with anti-EpCAM-coated ferrous particles. CTCs were defined as nucleated cells (DAPI+) expressing cytoplasmic cytokeratins 8, 18, or 19 and lacking surface expression of the common leukocyte antigen (CD45) [18]. Specimens were stored at room temperature and processed for detection of CTCs by using CellSearch within 1 day of phlebotomy.

Patient characteristics were tabulated. Baseline CTC count was defined as the earliest CTC measurement obtained before the start of a new line of therapy. We dichotomized baseline CTC counts in two different ways: as <1 or ≥1 and as <5 or ≥5. The cut-off at 1 CTC was chosen because it has been investigated in other settings, such as primary breast cancer, including locally advanced breast cancer [29,30]. The cut-off at 5 CTCs was established as prognostic for PFS and OS in patients with metastatic breast cancer in a previous study [18].

In an exploratory analysis, we correlated baseline CTC counts with PFS and OS. Median follow-up period was calculated as a median observation time among all patients and among those still alive at the time of their last follow-up. PFS was calculated from the date of baseline CTC enumeration to the date of progression or death or the date of last adequate follow-up. OS was calculated from the date of baseline CTC enumeration to the date of death or last follow-up. PFS and OS were estimated by using the Kaplan-Meier product-limit method and compared between groups by using the log-rank test. Univariate analyses were performed with either χ² or Fisher Exact test, as appropriate. A multivariate Cox proportional hazards model for PFS and OS was used to assess differences in outcome on the basis of baseline CTC counts, hormone-receptor status (positive for either or negative for both), HER-2 status (overexpressed or negative), stage (stage III versus metastatic IBC), site of metastasis (visceral versus non-visceral), and number of metastatic sites. Visceral metastases were defined here as lung, liver, adrenal gland, brain, kidney, pancreas, and/or peritoneal involvement with or without ascites and/or pleural effusions. Nonvisceral metastases were defined as involvement of any of the following sites without visceral metastases: breast, lymph nodes, chest wall, bone, skin, and/or abdomen. Step-wise regression techniques were used to build multivariate models by using a significance level of 0.10 to remain in the model. Wilcoxon matched-pairs signed-ranks test was used to compare baseline CTC counts with CTC counts at the time of progression, and Mann–Whitney U test was used to compare CTC counts between patients with stage III IBC and metastatic IBC. All statistical tests were two-sided, and P values <0.05 were considered statistically significant.