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Warawdekar et al. J Cancer Metastasis Treat 2017;3:23-33 Journal of
DOI: 10.20517/2394-4722.2016.66
Cancer Metastasis and Treatment

www.jcmtjournal.com
Topic: Circulating Tumor Cells: Diagnostics and Clinical Applications Open Access

A versatile method for enumeration and

characterization of circulating tumor cells

from patients with breast cancer

Ujjwala M. Warawdekar , Vani Parmar , Aruna Prabhu , Abhay Kulkarni 1,3* , Meenal Chaudhari 1,3* ,
2,3
1,3
2,3
Rajendra A. Badwe 2,3
1 CRI Lab Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Navi Mumbai 410208, India.
2 Department of Surgery, Breast Unit, Tata Memorial Hospital, Tata Memorial Centre, Parel, Mumbai 400012, India.
3 Homi Bhabha National Institute, Anushakti Nagar, Mumbai 400085, India.
* The authors contributed equally to this paper.
Correspondence to: Dr. Ujjwala M. Warawdekar, CRI Lab Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata
Memorial Centre, Kharghar, Navi Mumbai 410208, India. E-mail: uwarawdekar@actrec.gov.in

How to cite this article: Warawdekar UM, Parmar V, Prabhu A, Kulkarni A, Chaudhari M, Badwe RA. A versatile methodology for the enumeration
and characterization of circulating tumor cells from patients with breast cancer. J Cancer Metastasis Treat 2017;3:23-33.
ABSTRACT

Article history: Aim: To establish a standardized protocol for the isolation and enumeration of circulating
Received: 16-11-2016 tumor cells (CTCs) from peripheral blood of patients with metastatic breast cancer.
Accepted: 12-01-2017 Methods: The protocol used tumor cells spiked in a lymphoid cell line with detection by flow
Published: 23-02-2017 cytometry and quantitative reverse transcription polymerase chain reaction (QRT-PCR). Cells
of the human mammary cancer subtypes were spiked into Jurkat cells, which served as the
Key words: 5
Circulating tumor cells, lymphocyte designate in numbers from 10 to 500 per 10 Jurkat cells. This mixed population
was probed for CD45, EpCAM, and pancytokeratin acquired from flow cytometry and
cytokeratin-19, characterized by microscopy. QRT-PCR was done for CK-19, MUC-1, EpCAM, and GAPDH.
flow cytometry, Validation was attained with blood samples from 22 patients with metastatic breast cancer
circulating tumor cell enumeration, and 20 healthy individuals. Results: Flow cytometry could detect 1 breast cancer cell
quantitative reverse transcription per 100,000 Jurkat cells, with similar detection levels in the breast cancer subtypes.
polymerase chain reaction, Samples from patients with breast cancer showed a range of CTCs from 1-85 per 10 mL of
EpCAM
blood. Quantitation of expression for EpCAM, CK-19, Muc-1, and Her2neu confirmed the
presence of CTCs in 76% of samples. Conclusion: Density gradient and immunomagnetic
enrichment accomplished isolation of CTCs and quantitation was achieved using flow
cytometry. Combined QRT-PCR and imaging further validated these findings, rendering
a robust methodology.
INTRODUCTION and their association with tumor progression and
metastatic development. [5-7] Reports have also shown
Circulating tumor cells (CTCs) in peripheral blood has that a change in CTCs number predicts response
emerged as an important surrogate marker for prognosis to therapy and can evaluate residual disease. [8-11]
of cancer. [1-4] Various studies have demonstrated the Hence, to establish CTC number and molecular
presence of CTCs in peripheral blood of patients characteristics, a necessary requirement is a feasible
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