Limits...
Development of a multiparameter flow cytometric assay as a potential biomarker for homologous recombination deficiency in women with high-grade serous ovarian cancer.

Lee JM, Gordon N, Trepel JB, Lee MJ, Yu M, Kohn EC - J Transl Med (2015)

Bottom Line: Patients who did not respond to PARPi therapy had a significantly higher pre-treatment level of γH2AX (p = 0.01), and a higher ratio of γH2AX/MRE11 (11.0 [3.5-13.2] v. 3.3 [2.8-9.9], p < 0.03) compared with responders.We successfully developed and applied a multiparameter flow cytometry assay to measure γH2AX and MRE11 in PBMCs.Prospective studies will be required to validate this surrogate biomarker assay as a potential predictive biomarker of PARPi-based therapy.

View Article: PubMed Central - PubMed

Affiliation: Women's Malignancies Branch, Center for Cancer Research, National Cancer Institute, 10 Center Dr. MSC1906, Building 10, Room 12N/226, Bethesda, MD, 20892-1906, USA. leej6@mail.nih.gov.

ABSTRACT

Objectives: PARP inhibitors (PARPi) are a novel class of drugs with activity in patients with acquired or germline homologous recombination (HR) deficiency-associated high-grade serous ovarian cancer (HGSOC). We hypothesized that measuring γH2AX as an indicator of DNA double-strand breaks (DSB), and MRE11 or RAD51 as an indicator of DSB repair, would reflect HR status and predict response to PARPi-based therapy. Our aim was to develop and use high-throughput multiparametric flow cytometry to quantify γH2AX with MRE11 or RAD51 in PBMCs as a readily available surrogate.

Methods: Healthy donor PBMCs were used for assay development and optimization. We validated induction of γH2AX, MRE11 and RAD51 by staining with fluorophore-conjugated antibodies. The multiparameter flow cytometric method was applied to PBMC samples from recurrent HGSOC patients who were treated with PARPi, olaparib and carboplatin.

Results: Stimulation was necessary for quantification of a DNA damage response to olaparib/carboplatin in healthy donor PBMCs. The flow cytometric protocol could not distinguish between cytoplasmic and nuclear RAD51, erroneously indicating activation in response to injury. Thus, MRE11 was selected as the marker of DSB repair. PBMCs from 15 recurrent HGSOC patients were then examined. Patients who did not respond to PARPi therapy had a significantly higher pre-treatment level of γH2AX (p = 0.01), and a higher ratio of γH2AX/MRE11 (11.0 [3.5-13.2] v. 3.3 [2.8-9.9], p < 0.03) compared with responders.

Conclusions: We successfully developed and applied a multiparameter flow cytometry assay to measure γH2AX and MRE11 in PBMCs. Prospective studies will be required to validate this surrogate biomarker assay as a potential predictive biomarker of PARPi-based therapy.

No MeSH data available.


Related in: MedlinePlus

Demonstration of target proteins by IF confocal microscopy. Cells were collected prior to stimulation with PMA/I (−4 h), after stimulation but prior to treatment with O/C (0 h) and every 12 h afterwards for 48 h. a γH2AX; b MRE11; c RAD51.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4508767&req=5

Fig2: Demonstration of target proteins by IF confocal microscopy. Cells were collected prior to stimulation with PMA/I (−4 h), after stimulation but prior to treatment with O/C (0 h) and every 12 h afterwards for 48 h. a γH2AX; b MRE11; c RAD51.

Mentions: Immunofluorescence was used to both confirm the temporal and qualitative induction of the signals for γH2AX, RAD51, and MRE11, and also to evaluate the subcellular localization of the IF signal for evaluation of off-target non-nuclear staining that could bias the flow cytometric interpretation. Figure 2 shows representative panels of single stain IF confocal images obtained over the same treatment time course as replicate multiparameter flow cytometric studies. γH2AX and MRE11 (Figure 2a, b) stained nuclear foci, and focus formation increased progressively over injury time. RAD51 foci were both nuclear and cytoplasmic (Figure 2c, arrowheads). Few nuclear RAD51 foci were visible at 12 and 24 h, with discreet perinuclear cytoplasmic foci seen at 24 h, peaking at 36 h. By 48 h, all RAD51 was nuclear. The flow cytometric protocol used cannot distinguish between cytoplasmic and nuclear RAD51 and interpretation of RAD51 results would erroneously include cytoplasmic RAD51 staining as activation in response to injury in a clinical setting where there may not be control for exposure time that this experiment suggests to be necessary. Thus, MRE11 was selected as the marker of DSB repair for analysis of patient PBMCs by multiparameter flow cytometry.Figure 2


Development of a multiparameter flow cytometric assay as a potential biomarker for homologous recombination deficiency in women with high-grade serous ovarian cancer.

Lee JM, Gordon N, Trepel JB, Lee MJ, Yu M, Kohn EC - J Transl Med (2015)

Demonstration of target proteins by IF confocal microscopy. Cells were collected prior to stimulation with PMA/I (−4 h), after stimulation but prior to treatment with O/C (0 h) and every 12 h afterwards for 48 h. a γH2AX; b MRE11; c RAD51.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4508767&req=5

Fig2: Demonstration of target proteins by IF confocal microscopy. Cells were collected prior to stimulation with PMA/I (−4 h), after stimulation but prior to treatment with O/C (0 h) and every 12 h afterwards for 48 h. a γH2AX; b MRE11; c RAD51.
Mentions: Immunofluorescence was used to both confirm the temporal and qualitative induction of the signals for γH2AX, RAD51, and MRE11, and also to evaluate the subcellular localization of the IF signal for evaluation of off-target non-nuclear staining that could bias the flow cytometric interpretation. Figure 2 shows representative panels of single stain IF confocal images obtained over the same treatment time course as replicate multiparameter flow cytometric studies. γH2AX and MRE11 (Figure 2a, b) stained nuclear foci, and focus formation increased progressively over injury time. RAD51 foci were both nuclear and cytoplasmic (Figure 2c, arrowheads). Few nuclear RAD51 foci were visible at 12 and 24 h, with discreet perinuclear cytoplasmic foci seen at 24 h, peaking at 36 h. By 48 h, all RAD51 was nuclear. The flow cytometric protocol used cannot distinguish between cytoplasmic and nuclear RAD51 and interpretation of RAD51 results would erroneously include cytoplasmic RAD51 staining as activation in response to injury in a clinical setting where there may not be control for exposure time that this experiment suggests to be necessary. Thus, MRE11 was selected as the marker of DSB repair for analysis of patient PBMCs by multiparameter flow cytometry.Figure 2

Bottom Line: Patients who did not respond to PARPi therapy had a significantly higher pre-treatment level of γH2AX (p = 0.01), and a higher ratio of γH2AX/MRE11 (11.0 [3.5-13.2] v. 3.3 [2.8-9.9], p < 0.03) compared with responders.We successfully developed and applied a multiparameter flow cytometry assay to measure γH2AX and MRE11 in PBMCs.Prospective studies will be required to validate this surrogate biomarker assay as a potential predictive biomarker of PARPi-based therapy.

View Article: PubMed Central - PubMed

Affiliation: Women's Malignancies Branch, Center for Cancer Research, National Cancer Institute, 10 Center Dr. MSC1906, Building 10, Room 12N/226, Bethesda, MD, 20892-1906, USA. leej6@mail.nih.gov.

ABSTRACT

Objectives: PARP inhibitors (PARPi) are a novel class of drugs with activity in patients with acquired or germline homologous recombination (HR) deficiency-associated high-grade serous ovarian cancer (HGSOC). We hypothesized that measuring γH2AX as an indicator of DNA double-strand breaks (DSB), and MRE11 or RAD51 as an indicator of DSB repair, would reflect HR status and predict response to PARPi-based therapy. Our aim was to develop and use high-throughput multiparametric flow cytometry to quantify γH2AX with MRE11 or RAD51 in PBMCs as a readily available surrogate.

Methods: Healthy donor PBMCs were used for assay development and optimization. We validated induction of γH2AX, MRE11 and RAD51 by staining with fluorophore-conjugated antibodies. The multiparameter flow cytometric method was applied to PBMC samples from recurrent HGSOC patients who were treated with PARPi, olaparib and carboplatin.

Results: Stimulation was necessary for quantification of a DNA damage response to olaparib/carboplatin in healthy donor PBMCs. The flow cytometric protocol could not distinguish between cytoplasmic and nuclear RAD51, erroneously indicating activation in response to injury. Thus, MRE11 was selected as the marker of DSB repair. PBMCs from 15 recurrent HGSOC patients were then examined. Patients who did not respond to PARPi therapy had a significantly higher pre-treatment level of γH2AX (p = 0.01), and a higher ratio of γH2AX/MRE11 (11.0 [3.5-13.2] v. 3.3 [2.8-9.9], p < 0.03) compared with responders.

Conclusions: We successfully developed and applied a multiparameter flow cytometry assay to measure γH2AX and MRE11 in PBMCs. Prospective studies will be required to validate this surrogate biomarker assay as a potential predictive biomarker of PARPi-based therapy.

No MeSH data available.


Related in: MedlinePlus