Accurate Screening for Early-Stage Breast Cancer by Detection and Profiling of Circulating Tumor Cells

Timothy Crook, Robert Leonard, Kefah Mokbel, Alastair Thompson, Michael Michell, Raymond Page, Ashok Vaid, Ravi Mehrotra, Anantbhushan Ranade, Sewanti Limaye, Darshana Patil, Dadasaheb Akolkar, Vineet Datta, Pradip Fulmali, Sachin Apurwa, Stefan Schuster, Ajay Srinivasan, Rajan Datar, Timothy Crook, Robert Leonard, Kefah Mokbel, Alastair Thompson, Michael Michell, Raymond Page, Ashok Vaid, Ravi Mehrotra, Anantbhushan Ranade, Sewanti Limaye, Darshana Patil, Dadasaheb Akolkar, Vineet Datta, Pradip Fulmali, Sachin Apurwa, Stefan Schuster, Ajay Srinivasan, Rajan Datar

Abstract

Background: The early detection of breast cancer (BrC) is associated with improved survival. We describe a blood-based breast cancer detection test based on functional enrichment of breast-adenocarcinoma-associated circulating tumor cells (BrAD-CTCs) and their identification via multiplexed fluorescence immunocytochemistry (ICC) profiling for GCDFP15, GATA3, EpCAM, PanCK, and CD45 status.

Methods: The ability of the test to differentiate BrC cases (N = 548) from healthy women (N = 9632) was evaluated in a case-control clinical study. The ability of the test to differentiate BrC cases from those with benign breast conditions was evaluated in a prospective clinical study of women (N = 141) suspected of BrC.

Results: The test accurately detects BrAD-CTCs in breast cancers, irrespective of age, ethnicity, disease stage, grade, or hormone receptor status. Analytical validation established the high accuracy and reliability of the test under intended use conditions. The test detects and differentiates BrC cases from healthy women with 100% specificity and 92.07% overall sensitivity in a case-control study. In a prospective clinical study, the test shows 93.1% specificity and 94.64% overall sensitivity in differentiating breast cancer cases (N = 112) from benign breast conditions (N = 29).

Conclusion: The findings reported in this manuscript support the clinical potential of this test for blood-based BrC detection.

Keywords: breast cancer; circulating tumor cells; immunocytochemistry; screening.

Conflict of interest statement

Timothy Crook, Robert Leonard, Alastair Thompson, Michael Michell, Ashok Vaid, Ravi Mehrotra, Anantbhushan Ranade, and Sewanti Limaye have no competing interests. Kefah Mokbel and Raymond Page are fractional stock holders of the Study Sponsor. Darshana Patil, Dadasaheb Akolkar, Vineet Datta, Pradip Fulmali, Sachin Apurwa, Stefan Schuster, and Ajay Srinivasan are employees of the Study Sponsor. Rajan Datar is the founder of the Study Sponsor.

Figures

Figure 1
Figure 1
Schema of test. Functional enrichment of circulating tumor cells (CTCs) is achieved using a cell culture medium that is cytotoxic towards all non-malignant cells, and permits survival of tumor-derived malignant cells. Peripheral blood mononuclear cells (PBMC) isolated from whole blood are treated with the medium for 120 h, after which the surviving cells and cell clusters are harvested and evaluated by multiplexed immunocytochemistry (ICC) profiling, to determine presence of breast-adenocarcinoma-associated CTCs (BrAD-CTCs), which are identified as CD45-negative cells that express GATA3, GCDFP15, and EpCAM in combination with PanCK.
Figure 2
Figure 2
Decision matrix for classifying samples. The detection threshold for breast-adenocarcinoma-associated CTCs (BrAD-CTCs) is ≥ 15 PanCK cells/5 mL, which is constituted by the detection of ≥ 5 GATA3+, PanCK+, and CD45-cells, along with ≥ 5 GCDFP15+, PanCK+, and CD45- cells, as well as ≥ 5 EpCAM+, PanCK+, and CD45-cells in the respective aliquots. Depending on the number of each type of marker positive cells, samples are marked as positive, equivocal or negative. The decision matrix bestows priority to GATA3 and GCDFP15 over EpCAM while classifying samples to ensure specificity for BrAD over other epithelial malignancies where EpCAM+ cells may be detected but breast-specific markers would be absent. Thus, while the test can detect EpCAM+, PanCK+, and CD45-cells, which may be present in various epithelial malignancies, it specifically reports only BrAD-CTCs.

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