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


METHODS
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.
Wang
Lihua H
LH
Laboratory of Human Toxicology and Pharmacology, Science Applications International Corporation, Frederick, MD, USA.
Pfister
Thomas D
TD
Parchment
Ralph E
RE
Kummar
Shivaani
S
Rubinstein
Larry
L
Evrard
Yvonne A
YA
Gutierrez
Martin E
ME
Murgo
Anthony J
AJ
Tomaszewski
Joseph E
JE
Doroshow
James H
JH
Kinders
Robert J
RJ
D000970:Antineoplastic Agents / Q000494:pharmacology / Q000627:therapeutic use*; D014408:Biomarkers, Tumor / Q000097:blood*; D045744:Cell Line, Tumor; D004249:DNA Damage; D016903:Drug Monitoring / Q000379:methods*; D006657:Histones / Q000097:blood*; D006801:Humans; D009369:Neoplasms / Q000097:blood* / Q000188:drug therapy; D009360:Neoplastic Cells, Circulating / Q000737:chemistry*; D015588:Observer Variation
D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; C468783:H2AX protein, human; D006657:Histones