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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

De novo production of MRE11 in response to DNA damage. a Cycloheximide (CHM) abolishes MRE11 response to O/C injury. b CHM does not inhibit the expression of γH2AX. 24 and 48 h time points compared to 0 time point, and comparison of O/C treatment only (O/C) to CHM followed by O/C (CHM > O/C) for MRE11 or γH2AX at the same time point.
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Fig3: De novo production of MRE11 in response to DNA damage. a Cycloheximide (CHM) abolishes MRE11 response to O/C injury. b CHM does not inhibit the expression of γH2AX. 24 and 48 h time points compared to 0 time point, and comparison of O/C treatment only (O/C) to CHM followed by O/C (CHM > O/C) for MRE11 or γH2AX at the same time point.

Mentions: MRE11 is the repair protein recruited in response to DNA injury. The delay in expression of MRE11 detected by flow cytometry seen in Figure 1e raised the possibility of the need for de novo production of MRE11 upon DNA DSB. Cycloheximide (100 μM) was used to block de novo protein synthesis. This concentration inhibits lymphocyte protein synthesis up to 92% with a minimal induction of apoptosis [23]. Figure 3a shows no induction of MRE11 in cells pre-treated with PMA/I in the presence of cycloheximide for 4 h prior to treatment with O/C. Cycloheximide did not have a significant effect on the expression of γH2AX over the same time interval, consistent with the fact that γH2AX arises as the result of post-translational modification and should therefore be unaffected by the inhibition of protein synthesis (Figure 3b). These results indicate that induction of MRE11 protein is a result of exposure to DNA damaging agents and is measurable in stimulated PBMCs.Figure 3


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)

De novo production of MRE11 in response to DNA damage. a Cycloheximide (CHM) abolishes MRE11 response to O/C injury. b CHM does not inhibit the expression of γH2AX. 24 and 48 h time points compared to 0 time point, and comparison of O/C treatment only (O/C) to CHM followed by O/C (CHM > O/C) for MRE11 or γH2AX at the same time point.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: De novo production of MRE11 in response to DNA damage. a Cycloheximide (CHM) abolishes MRE11 response to O/C injury. b CHM does not inhibit the expression of γH2AX. 24 and 48 h time points compared to 0 time point, and comparison of O/C treatment only (O/C) to CHM followed by O/C (CHM > O/C) for MRE11 or γH2AX at the same time point.
Mentions: MRE11 is the repair protein recruited in response to DNA injury. The delay in expression of MRE11 detected by flow cytometry seen in Figure 1e raised the possibility of the need for de novo production of MRE11 upon DNA DSB. Cycloheximide (100 μM) was used to block de novo protein synthesis. This concentration inhibits lymphocyte protein synthesis up to 92% with a minimal induction of apoptosis [23]. Figure 3a shows no induction of MRE11 in cells pre-treated with PMA/I in the presence of cycloheximide for 4 h prior to treatment with O/C. Cycloheximide did not have a significant effect on the expression of γH2AX over the same time interval, consistent with the fact that γH2AX arises as the result of post-translational modification and should therefore be unaffected by the inhibition of protein synthesis (Figure 3b). These results indicate that induction of MRE11 protein is a result of exposure to DNA damaging agents and is measurable in stimulated PBMCs.Figure 3

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