Immunophenotyping invasive breast cancer: paving the road for molecular imaging

Abstract

BackgroundMammographic population screening in The Netherlands has increased the number of breast cancer patients with small and non-palpable breast tumors. Nevertheless, mammography is not ultimately sensitive and specific for distinct subtypes. Molecular imaging with targeted tracers might increase specificity and sensitivity of detection. Because development of new tracers is labor-intensive and costly, we searched for the smallest panel of tumor membrane markers that would allow detection of the wide spectrum of invasive breast cancers.MethodsTissue microarrays containing 483 invasive breast cancers were stained by immunohistochemistry for a selected set of membrane proteins known to be expressed in breast cancer.ResultsThe combination of highly tumor-specific markers glucose transporter 1 (GLUT1), epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1-R), human epidermal growth factor receptor 2 (HER2), hepatocyte growth factor receptor (MET), and carbonic anhydrase 9 (CAIX) 'detected' 45.5% of tumors, especially basal/triple negative and HER2-driven ductal cancers. Addition of markers with a 2-fold tumor-to-normal ratio increased the detection rate to 98%. Including only markers with >3 fold tumor-to-normal ratio (CD44v6) resulted in an 80% detection rate. The detection rate of the panel containing both tumor-specific and less tumor-specific markers was not dependent on age, tumor grade, tumor size, or lymph node status.ConclusionsIn search of the minimal panel of targeted probes needed for the highest possible detection rate, we showed that 80% of all breast cancers express at least one of a panel of membrane markers (CD44v6, GLUT1, EGFR, HER2, and IGF1-R) that may therefore be suitable for molecular imaging strategies. This study thereby serves as a starting point for further development of a set of antibody-based optical tracers with a high breast cancer detection rate.

Differential Expression of Growth Factor Receptors and Membrane-Bound Tumor Markers for Imaging in Male and Female Breast Cancer

“…However, we observed FGFR2 expression in 3 cases (9.4%), IGF1-R expression in 1 case (3.1%), and a clear membranous staining of CAXII in 2 cases (6.3%). Gynecomastia was positive for CD44v6 (predominant staining of the myoepithelium) in all cases like normal female breast epithelium, which is probably not influencing the sensitivity of detection as we previously stated [24]. No difference in expression between the subtypes of gynecomastia was observed.…”

“…From the previous results it became clear that all growth factor receptors are too infrequently expressed in male breast cancer to serve as individual targets for molecular imaging strategies, meaning that a panel of growth factor receptors supplemented with membrane-bound tumor markers is required to obtain a sufficient detection rate like in female breast cancer [24]. Our previously proposed panel consisting of CD44v6, EGFR, HER2, IGF1-R, and GLUT1 for imaging of female breast cancer was significantly less sensitive for imaging of male breast cancer (female breast cancer 79.3% vs. male breast cancer 69.1%, p = 0.025; Figure 2A).…”

“…All scoring was done by JFV, RK and PJvD who were blinded to patient characteristics and results of other stainings. Expression data for the female breast cancers and for the hypoxia proteins in male breast cancer were derived from our previous studies [24], [31].…”

“…Further, near-infrared fluorescently labeled antibodies can be used for image-guided surgery, thereby enhancing radical resection of breast cancer and lymph node metastases [21]–[23]. We recently described that in addition to growth factor receptors, hypoxia upregulated proteins (carbonic anhydase IX (CAIX) and XII (CAXII), and GLUT1) and CD44 variants might be useful for molecular imaging of female breast cancer [24]. Because fluorescently labeled antibodies and antibody-fragments are not easily internalized, ERα and PR are not considered for optical imaging strategies.…”

Production and Characterization of a Peptide-based Monoclonal Antibody Against CD44 Variant 6

“…(4,(26)(27)(28)(29) AntiCD44v6 antibodies have also been found to have diagnostic applications in some malignancies such as head and neck squamous cell carcinoma (HNSCC) (30)(31)(32) and in noninvasive imaging of breast cancer. (33,34) Because of its high expression in HNSCC, CD44v6 has been targeted with a monoclonal antibody (U36) for diagnosis of this cancer in humans. (31,32) The humanized anti-CD44v6 monoclonal antibody has also been conjugated with the near-infrared fluorescent dye and was used for molecular imaging of ductal carcinoma in mice.…”