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. 2010 Feb 1;16(3):1073-84.
doi: 10.1158/1078-0432.CCR-09-2799. Epub 2010 Jan 26.

Monitoring drug-induced gammaH2AX as a pharmacodynamic biomarker in individual circulating tumor cells

Lihua H Wang  1 , Thomas D Pfister, Ralph E Parchment, Shivaani Kummar, Larry Rubinstein, Yvonne A Evrard, Martin E Gutierrez, Anthony J Murgo, Joseph E Tomaszewski, James H Doroshow, Robert J Kinders
Affiliations

Monitoring drug-induced gammaH2AX as a pharmacodynamic biomarker in individual circulating tumor cells

Lihua H Wang et al. Clin Cancer Res. .

Abstract

Purpose: Circulating tumor cells (CTC) in peripheral blood of patients potentially represent a fraction of solid tumor cells available for more frequent pharmacodynamic assessment of drug action than is possible using tumor biopsy. However, currently available CTC assays are limited to cell membrane antigens. Here, we describe an assay that directly examines changes in levels of the nuclear DNA damage marker gammaH2AX in individual CTCs of patients treated with chemotherapeutic agents.

Experimental design: An Alexa Fluor 488-conjugated monoclonal gammaH2AX antibody and epithelial cancer cell lines treated with topotecan and spiked into whole blood were used to measure DNA damage-dependent nuclear gammaH2AX signals in individual CTCs. Time-course changes in both CTC number and gammaH2AX levels in CTCs were also evaluated in blood samples from patients undergoing treatment.

Results: The percentage of gammaH2AX-positive CTCs increased in a concentration-dependent manner in cells treated with therapeutically relevant concentrations of topotecan ex vivo. In samples from five patients, percent gammaH2AX-positive cells increased post-treatment from a mean of 2% at baseline (range, 0-6%) to a mean of 38% (range, 22-64%) after a single day of drug administration; this increase was irrespective of increases or decreases in the total CTC count.

Conclusions: These data show promise for monitoring dynamic changes in nuclear biomarkers in CTCs (in addition to CTC count) for rapidly assessing drug activity in clinical trials of molecularly targeted anticancer therapeutics as well as for translational research.

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Figures

Fig. 1
Fig. 1
Induction of γH2AX can be detected in topotecan-treated tumor cell lines and whole blood samples spiked with treated tumor cell lines using the γH2AX-AF488 antibody. A, FACS analysis of untreated (left) or 1 μM topotecan-treated (right) MCF7, HT-29, and SKOV-3 tumor cells. FACS traces of γH2AX-AF488 (blue) and isotype control (black). Histogram presented for quantified FACS data from a representative experiment; data from three independent experiments were collected. B, Photomicrographs of control and 1 μM topotecan-treated MCF7 cells stained with γH2AX-AF488 (green) and DAPI (blue). 200x. C, MCF7 or PC-3 cells were treated with 1 μM topotecan for 2 h, spiked into whole blood from healthy donors, and stained using antibodies against CK-PE, CD45-APC, γH2AX-AF488, and DAPI. Check boxes adjacent to images: a checked box next to the composite image (CK-PE/DAPI) indicates a CTC; next to the CD45-APC image indicates the presence of a contaminating leukocyte; and next to an image for γH2AX-AF488 staining indicates a γH2AX-positive cell. Four representative fields of cells isolated using the CellTracks AutoPrep and detected with the CellTracks Analyzer II are presented for each cell line (i–iv).
Fig. 2
Fig. 2
γH2AX-AF488 staining was nuclear-specific, DNA damage specific, and increased in a dose-dependent manner in cells isolated from whole blood from healthy donors spiked with topotecan-treated tumor cell lines. A–C, Representative images of three different cell fields captured using the CellTracks Analyzer II from cells stained with HER2-FITC, γH2AX-AF488, or no antibody (control) using the online staining protocol. SKOV-3 cells were treated with 1 μM topotecan for 2 h and stained with anti-HER2-FITC (A); 1 μM topotecan-treated MCF7 cells were stained with γH2AX-AF488 (B). Control MCF7 cells were treated with 0.1–100 μM topotecan for 2 h and processed without antibody; 100 μM data shown (C). Check boxes adjacent to images: CTCs have a check next to the composite CK-PE/DAPI image; contaminating leukocytes would have a check next to the CD45-APC images. Checks next to images of antibody-specific staining of HER2-FITC or γH2AX-AF488 indicate positive staining. D, MCF7 or PC-3 cells were treated with 0.01–10 μM topotecan for 2 h, spiked into whole blood from healthy donors, and processed in the CellSearch system with anti-γH2AX-AF488 (top). Graph of data from one representative experiment. PC-3 cells were treated with 0.5–4 mM temozolomide or 0.01–10 μM rapamycin for 2 h, spiked into whole blood from healthy donors, and then processed in the CellSearch system. The isolated cells were stained with anti-γH2AX-AF488 (bottom).
Fig. 3
Fig. 3
Real-time detection of chemotherapy-induced time-course changes in γH2AX: correlation between total number of CTCs (A) and percent γH2AX-positive CTCs (B). Data presented for five patients on three different chemotherapeutic regimens (Table 3). For patient #4, ten representative pre-treatment (C) and ten post-treatment (D) CTC images captured with the CellTracks Analyzer II are presented (i – x). Patient blood samples enriched for CTCs were stained using antibodies for CK-PE, CD45-APC, DAPI, and γH2AX-AF488. Check boxes adjacent to images: a checked box next to the composite image (CK-PE/DAPI) indicates a CTC; next to the CD45-APC images would indicate the presence of a contaminating leukocyte; and next to an image for γH2AX-AF488 staining indicates a γH2AX-positive cell.
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