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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

Injury and repair surrogates trend with outcome in PBMCs from women treated with O/C. PBMCs obtained from 15 women pretreated with O/C and viably frozen were available and analyzed according to our demonstrated SOP. The graphed values in y axis are raw MFI levels as they represent data from patient samples taken at only one time point (before O/C treatment). a γH2AX; b MRE11. Median MFI levels of γH2AX (c) and MRE11 (d) were lower in patients with objective measurable responses to O/C. A pretreatment ratio of γH2AX/MRE11 was significantly higher in patients with no measurable response to O/C (e). Pre-treatment γH2AX (f), MRE11 (g) and a ratio of γH2AX/MRE11 (h) by dual-label flow cytometry are lower in gBRCAm patients compared with BRCAwt patients.
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Fig4: Injury and repair surrogates trend with outcome in PBMCs from women treated with O/C. PBMCs obtained from 15 women pretreated with O/C and viably frozen were available and analyzed according to our demonstrated SOP. The graphed values in y axis are raw MFI levels as they represent data from patient samples taken at only one time point (before O/C treatment). a γH2AX; b MRE11. Median MFI levels of γH2AX (c) and MRE11 (d) were lower in patients with objective measurable responses to O/C. A pretreatment ratio of γH2AX/MRE11 was significantly higher in patients with no measurable response to O/C (e). Pre-treatment γH2AX (f), MRE11 (g) and a ratio of γH2AX/MRE11 (h) by dual-label flow cytometry are lower in gBRCAm patients compared with BRCAwt patients.

Mentions: We applied our multiparameter flow cytometric γH2AX/MRE11 assay to archival PBMC samples from 15 patients with recurrent HGSOC who received the PARPi, olaparib and carboplatin [22]. Tables 1 and 2 summarize the characteristics and clinical responses of this subset of patients. The global distribution of γH2AX and MRE11 expression across all the patients is shown in Figure 4a, b. In this small group, patients whose best response was stable disease or progression expressed higher pre-treatment levels of both γH2AX and MRE11 compared to patients who had complete or partial responses (p = 0.01 and p = 0.11; Figure 4c, d). We proposed that the ratio of the values of injury to repair, γH2AX/MRE11, may provide a better view of the DNA repair status in the patient PBMCs. The median value of the γH2AX/MRE11 ratio of non-responders was significantly higher compared to responders, indicating more injury than repair (11.0 [range 3.5–13.2] v. 3.3 [range 2.8–9.9], p = 0.026; Figure 4e). Figures 4f–h show the distribution of γH2AX and MRE11 expression and ratio of γH2AX/MRE11 in patients as a function of gBRCAm status. Five of the 15 patients were gBRCAm carriers, and showed significantly lower pre-treatment levels of both γH2AX and MRE11 compared to non-mutation carriers (all p < 0.017; Figure 4f, g). The median value of the γH2AX/MRE11 ratio of BRCAwt was significantly higher compared to gBRCAm carriers (10.5 [range 3.5–13.2] v. 2.9 [range 2.8–8.4], p < 0.01; Figure 4h).Table 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)

Injury and repair surrogates trend with outcome in PBMCs from women treated with O/C. PBMCs obtained from 15 women pretreated with O/C and viably frozen were available and analyzed according to our demonstrated SOP. The graphed values in y axis are raw MFI levels as they represent data from patient samples taken at only one time point (before O/C treatment). a γH2AX; b MRE11. Median MFI levels of γH2AX (c) and MRE11 (d) were lower in patients with objective measurable responses to O/C. A pretreatment ratio of γH2AX/MRE11 was significantly higher in patients with no measurable response to O/C (e). Pre-treatment γH2AX (f), MRE11 (g) and a ratio of γH2AX/MRE11 (h) by dual-label flow cytometry are lower in gBRCAm patients compared with BRCAwt patients.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4508767&req=5

Fig4: Injury and repair surrogates trend with outcome in PBMCs from women treated with O/C. PBMCs obtained from 15 women pretreated with O/C and viably frozen were available and analyzed according to our demonstrated SOP. The graphed values in y axis are raw MFI levels as they represent data from patient samples taken at only one time point (before O/C treatment). a γH2AX; b MRE11. Median MFI levels of γH2AX (c) and MRE11 (d) were lower in patients with objective measurable responses to O/C. A pretreatment ratio of γH2AX/MRE11 was significantly higher in patients with no measurable response to O/C (e). Pre-treatment γH2AX (f), MRE11 (g) and a ratio of γH2AX/MRE11 (h) by dual-label flow cytometry are lower in gBRCAm patients compared with BRCAwt patients.
Mentions: We applied our multiparameter flow cytometric γH2AX/MRE11 assay to archival PBMC samples from 15 patients with recurrent HGSOC who received the PARPi, olaparib and carboplatin [22]. Tables 1 and 2 summarize the characteristics and clinical responses of this subset of patients. The global distribution of γH2AX and MRE11 expression across all the patients is shown in Figure 4a, b. In this small group, patients whose best response was stable disease or progression expressed higher pre-treatment levels of both γH2AX and MRE11 compared to patients who had complete or partial responses (p = 0.01 and p = 0.11; Figure 4c, d). We proposed that the ratio of the values of injury to repair, γH2AX/MRE11, may provide a better view of the DNA repair status in the patient PBMCs. The median value of the γH2AX/MRE11 ratio of non-responders was significantly higher compared to responders, indicating more injury than repair (11.0 [range 3.5–13.2] v. 3.3 [range 2.8–9.9], p = 0.026; Figure 4e). Figures 4f–h show the distribution of γH2AX and MRE11 expression and ratio of γH2AX/MRE11 in patients as a function of gBRCAm status. Five of the 15 patients were gBRCAm carriers, and showed significantly lower pre-treatment levels of both γH2AX and MRE11 compared to non-mutation carriers (all p < 0.017; Figure 4f, g). The median value of the γH2AX/MRE11 ratio of BRCAwt was significantly higher compared to gBRCAm carriers (10.5 [range 3.5–13.2] v. 2.9 [range 2.8–8.4], p < 0.01; Figure 4h).Table 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