Identify γH2AX and pKAP1 in CTC populations for blood-based investigation of DNA damage response

With the increasing investigation of DNA damage response (DDR)/PARP inhibitors, alone or in combination with chemotherapy or immunotherapy, there is a need for blood-based, robust, and repeatable DDR assays.

ANGLE has developed immunofluorescence assays to detect two DNA damage markers – phosphorylated histone variant H2AX (γH2AX) and phosphorylated KRAB-associated protein 1 (pKAP1) – in circulating tumor cells (CTCs) enriched using ANGLE’s Parsortix® technology.

For use in the research setting as an endpoint in clinical studies, these assays make longitudinal, repeatable monitoring of treatment response possible. The process involves shipping patient blood samples to ANGLE’s ISO 15189-accredited laboratory* in the US where the CTC enrichment and immunofluorescent detection are carried out, and a results report provided.

Assay performance

The assays have been fully evaluated and verified in cancer cell models and ested for feasibility in cancer patient samples. They demonstrate high analytical sensitivity and specificity, with positive nuclear staining in mesenchymal CTCs.

Mean performance metrics using etoposide-treated and untreated cancer cell lines (MCF7 – γH2AX; H226 – pKAP1) spiked into peripheral blood and recovered using the Parsortix® technology.

Sample staining

Patient sample showing γH2AX-positive nuclear staining (diffuse and foci) in a mesenchymal CTC (blue = nucleus, purple = mesenchymal markers, white = blood cell markers, orange = γH2AX)

Patient sample showing pKAP1-positive nuclear staining in a cluster of mesenchymal CTCs (blue = nucleus, purple = mesenchymal markers, white = blood cell markers, orange = pKAP1)

Assay performance

The assays exhibit a dose response with increasing doses of DNA-damaging agent and a positive linear relationship is seen between cancer cells spiked and cells harvested.

Dose response

Dose response of γH2AX or pKAP1 positivity in cancer cells (MCF7 – γH2AX; H226 – pKAP1) following treatment with increasing doses of etoposide.

Linearity analysis

Increasing numbers of γH2AX-expressing MCF7 cells or pKAP1-expressing H226 cells were spiked into whole blood, recovered using Parsortix® technology, harvested onto slides and stained using an epithelial/mesenchymal antibody panel and γH2AX or pKAP1.

Clinical findings

The markers and assays are built on a foundation of robust evidence from clinical studies:

  • An increase in γH2AX-positive CTCs was seen after a single dose and there were dynamic changes in γH2AX positivity over time with different chemotherapy regimens.1
  • In a study of radium-223 therapy, an increase in γH2AX-positive CTCs was associated with a lower risk of death.2
  • In patients with breast cancer, γH2AX positivity increased after treatment with veliparib, doxorubicin and cyclophosphamide in tumor tissue although there was only sufficient tissue in 6 out of 10 patients.3  In contrast, CTCs were isolated from 27 patients and treatment increased γH2AX expression at day 7 and 14.

These studies highlight the utility of CTCs and DDR marker analysis for minimally invasive and rapid assessment of treatment response over time.


For Research Use Only. Not For Use in Diagnostic Procedures.

*The ANGLE US Lab ISO15189 accreditation scope is specific for the CTC Pap Stain Assay
1. Wang LH, Pfister TD, Parchment RE, et al. Monitoring drug-induced gammaH2AX as a pharmacodynamic biomarker in individual circulating tumor cells. Clin Cancer Res. 2010;16(3):1073-84. doi: 10.1158/1078-0432.CCR-09-2799;

2. Chatzkel J, Mocha J, Smith J, et al. Circulating tumor cells and γH2AX as biomarkers for responsiveness to radium-223 in advanced prostate cancer patients. Future Sci OA. 2019;6(1):FSO437. doi: 10.2144/fsoa-2019-0092;

3. Tan AR, Chan N, Kiesel BF, et al. A phase I study of veliparib with cyclophosphamide and veliparib combined with doxorubicin and cyclophosphamide in advanced malignancies. Cancer Chemother Pharmacol. 2022;89(1):49-58. doi: 10.1007/s00280-021-04350-x