Circulating Cancer-Associated Macrophage-Like Cells Differentiate Malignant Breast Cancer and Benign Breast Conditions

Abstract

Background: Blood-based testing can be used as a noninvasive method to recover and analyze circulating tumor-derived cells for clinical use. Circulating cancer-associated macrophage-like cells (CAML) are specialized myeloid cells found in peripheral blood and associated with the presence of solid malignancies. We measured CAMLs prospectively in peripheral blood to ascertain their prevalence, specificity, and sensitivity in relation to breast disease status at clinical presentation.

Methods: We report on two related but separate studies: 1) CellSieve microfilters were used to isolate CAMLs from blood samples of patients with known malignant disease (n = 41). Prevalence and specificity was compared against healthy volunteers (n = 16). 2) A follow-up double-blind pilot study was conducted on women (n = 41) undergoing core-needle biopsy to diagnose suspicious breast masses.

Results: CAMLs were found in 93% of known malignant patients (n = 38/41), averaging 19.4 cells per sample, but none in the healthy controls. In subjects undergoing core biopsy for initial diagnosis, CAMLs were found in 88% of subjects with invasive carcinoma (n = 15/17) and 26% with benign breast conditions (n = 5/19).

Conclusion: These preliminary pilot studies suggest that the presence of CAMLs may differentiate patients with malignant disease, benign breast conditions, and healthy individuals.

Impact: We supply evidence that this previously unidentified circulating stromal cell may have utility as a screening tool to detect breast cancer in various malignancies, irrespective of disease stage. Cancer Epidemiol Biomarkers Prev; 25(7); 1037-42. ©2016 AACR.

Figure 1. Overview of sensitivity and specificity determination based on our two studies, either Study A: populations of women with diagnosed breast cancer or Study B: populations of women with positive Clinical Breast Exam (CBE)/mammography but before diagnosis

(A) Patients with invasive breast malignancy were used to determine sensitivity of an assay based on CAML presence. Healthy controls were used to determine the specificity. (B) The assay was then tested using patients with positive breast mammography, or positive CBEs, for CAML presence by standard blood draw at the time core biopsy was taken. Standard pathological assessment was used to determine invasive malignant disease, benign condition, or non-invasive disease/Stage 0. Patient information was then unblinded and sensitivity/specificity of CAMLs in relation to pathological assessment was compared.

Figure 2. Appearance of CAMLs isolated on filters from cancer patients

Single ~100 µm CAML from a breast cancer patient with a polymorphonuclear DAPI stained structure (blue), intense CD14 (red) staining, some cytokeratin staining (green) and weak CD45 (purple). Box size=100 µm.

Figure 3. Study A: Presence, sensitivity and specificity of CAMLs in patients with known invasive breast cancer versus healthy control volunteers

(A) CAMLs were found in 38 of 41 patients with known invasive breast cancer, but in none of the 16 healthy controls. (B) The number of CAMLs ranged 0–105 per 7.5 mL blood sample in patients with cancer. (C) ROC curve shows a clear stratification between a healthy control population from a malignant population, AUC=0.96. Sensitivity: 93% (CI95% 80–99%) Specificity: 100% (CI95% 79–100%) PPV: 100% (CI95% 91–100%) NPV: 84% (CI95% 60–97%).

Figure 4. Study B: Presence, sensitivity and specificity of CAMLs in patients with positive mammograms/CBEs compared to standard pathological assessment

(A) CAMLs were found in all patients with non-invasive disease/Stage 0, in 88% of patients with invasive disease, and 26% of patients with benign conditions. (B) CAMLs were found in a range of 0–8 cells per 7.5 mL sample. (C) ROC curves comparing invasive breast cancer (n=17) to benign conditions (n=19) showed an AUC=0.78. Sensitivity: 88% (CI95% 64–99%) Specificity: 74% (CI95% 49–91%) PPV: 75% (CI95% 51–91%) NPV: 88% (CI95% 62–99%).