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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

Optimization of PBMC flow cytometry method. a Unstimulated PBMCs. Cells were seeded and incubated overnight without PMA/I stimulation before treatment with olaparib/carboplatin (O/C) for 48 h. b–d Optimization of stimulation conditions. Condition 1: PMA/I × 4 h followed by PBS washout and subsequent O/C × 48 h (P/I × 4 h > O/C × 48 h); condition 2: PMA/I × 4 h then O/C × 24 h followed by washout and subsequent O/C × 24 h (P/I × 4 h–O/C × 24 h > O/C × 24 h); condition 3: PMA/I × 4 h followed by O/C × 48 h with no washout (P/I × 4 h–O/C × 48 h). Cells were collected after 24, 36 and 48 h of treatment before undergoing the single-stain flow cytometry protocol. b γH2AX; c MRE11; d RAD51. e, f Demonstration of optimized dual-stain. Cells were plated and treated according to condition 1 before undergoing the dual-stain process for MRE11 and γH2AX (e), or for RAD51 and γH2AX (f).
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Fig1: Optimization of PBMC flow cytometry method. a Unstimulated PBMCs. Cells were seeded and incubated overnight without PMA/I stimulation before treatment with olaparib/carboplatin (O/C) for 48 h. b–d Optimization of stimulation conditions. Condition 1: PMA/I × 4 h followed by PBS washout and subsequent O/C × 48 h (P/I × 4 h > O/C × 48 h); condition 2: PMA/I × 4 h then O/C × 24 h followed by washout and subsequent O/C × 24 h (P/I × 4 h–O/C × 24 h > O/C × 24 h); condition 3: PMA/I × 4 h followed by O/C × 48 h with no washout (P/I × 4 h–O/C × 48 h). Cells were collected after 24, 36 and 48 h of treatment before undergoing the single-stain flow cytometry protocol. b γH2AX; c MRE11; d RAD51. e, f Demonstration of optimized dual-stain. Cells were plated and treated according to condition 1 before undergoing the dual-stain process for MRE11 and γH2AX (e), or for RAD51 and γH2AX (f).

Mentions: The use of healthy volunteer PBMCs as a surrogate resource for biomarker development and application required demonstration that they could acquire DNA damage. We thus began by modeling the treatment effect of olaparib/carboplatin (O/C) on the expression of γH2AX, MRE11, and RAD51 in healthy donor PBMCs. Figure 1a shows no induction of γH2AX, MRE11, or RAD51 expression after exposure to O/C at physiologically attainable concentrations for 48 h. Expression of the three proteins was induced in response to O/C only when PBMCs were stimulated with PMA/I (Figure 1b–d). Three conditions of stimulation were examined. In the first, PBMCs were stimulated with PMA/I, washed and then exposed to O/C for 48 h; the second introduced O/C at 4 h for 24 h, then cells were washed and re-exposed to O/C for an additional 24 h; the third introduced O/C for 48 h after the initial 4 h stimulation with no washouts. All three conditions induced statistically significant changes in γH2AX, MRE11 and RAD51 expression after 24 h (all p < 0.001). Notably, stimulation for 4 h followed by washout and subsequent O/C treatment for up to 48 h (condition 1) yielded a greater expression of γH2AX, MRE11, and RAD51 after 48 h treatment compared to the other two conditions. Flow cytometry secondary antibody and fluorophore negative controls showed no significant signal (data not shown).Figure 1


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)

Optimization of PBMC flow cytometry method. a Unstimulated PBMCs. Cells were seeded and incubated overnight without PMA/I stimulation before treatment with olaparib/carboplatin (O/C) for 48 h. b–d Optimization of stimulation conditions. Condition 1: PMA/I × 4 h followed by PBS washout and subsequent O/C × 48 h (P/I × 4 h > O/C × 48 h); condition 2: PMA/I × 4 h then O/C × 24 h followed by washout and subsequent O/C × 24 h (P/I × 4 h–O/C × 24 h > O/C × 24 h); condition 3: PMA/I × 4 h followed by O/C × 48 h with no washout (P/I × 4 h–O/C × 48 h). Cells were collected after 24, 36 and 48 h of treatment before undergoing the single-stain flow cytometry protocol. b γH2AX; c MRE11; d RAD51. e, f Demonstration of optimized dual-stain. Cells were plated and treated according to condition 1 before undergoing the dual-stain process for MRE11 and γH2AX (e), or for RAD51 and γH2AX (f).
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getmorefigures.php?uid=PMC4508767&req=5

Fig1: Optimization of PBMC flow cytometry method. a Unstimulated PBMCs. Cells were seeded and incubated overnight without PMA/I stimulation before treatment with olaparib/carboplatin (O/C) for 48 h. b–d Optimization of stimulation conditions. Condition 1: PMA/I × 4 h followed by PBS washout and subsequent O/C × 48 h (P/I × 4 h > O/C × 48 h); condition 2: PMA/I × 4 h then O/C × 24 h followed by washout and subsequent O/C × 24 h (P/I × 4 h–O/C × 24 h > O/C × 24 h); condition 3: PMA/I × 4 h followed by O/C × 48 h with no washout (P/I × 4 h–O/C × 48 h). Cells were collected after 24, 36 and 48 h of treatment before undergoing the single-stain flow cytometry protocol. b γH2AX; c MRE11; d RAD51. e, f Demonstration of optimized dual-stain. Cells were plated and treated according to condition 1 before undergoing the dual-stain process for MRE11 and γH2AX (e), or for RAD51 and γH2AX (f).
Mentions: The use of healthy volunteer PBMCs as a surrogate resource for biomarker development and application required demonstration that they could acquire DNA damage. We thus began by modeling the treatment effect of olaparib/carboplatin (O/C) on the expression of γH2AX, MRE11, and RAD51 in healthy donor PBMCs. Figure 1a shows no induction of γH2AX, MRE11, or RAD51 expression after exposure to O/C at physiologically attainable concentrations for 48 h. Expression of the three proteins was induced in response to O/C only when PBMCs were stimulated with PMA/I (Figure 1b–d). Three conditions of stimulation were examined. In the first, PBMCs were stimulated with PMA/I, washed and then exposed to O/C for 48 h; the second introduced O/C at 4 h for 24 h, then cells were washed and re-exposed to O/C for an additional 24 h; the third introduced O/C for 48 h after the initial 4 h stimulation with no washouts. All three conditions induced statistically significant changes in γH2AX, MRE11 and RAD51 expression after 24 h (all p < 0.001). Notably, stimulation for 4 h followed by washout and subsequent O/C treatment for up to 48 h (condition 1) yielded a greater expression of γH2AX, MRE11, and RAD51 after 48 h treatment compared to the other two conditions. Flow cytometry secondary antibody and fluorophore negative controls showed no significant signal (data not shown).Figure 1

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