A PET imaging agent for evaluating PARP-1 expression in ovarian cancer

Abstract

Background: Poly(ADP-ribose) polymerase (PARP) inhibitors are effective in a broad population of patients with ovarian cancer; however, resistance caused by low enzyme expression of the drug target PARP-1 remains to be clinically evaluated in this context. We hypothesize that PARP-1 expression is variable in ovarian cancer and can be quantified in primary and metastatic disease using a novel PET imaging agent.

Methods: We used a translational approach to describe the significance of PET imaging of PARP-1 in ovarian cancer. First, we produced PARP1-KO ovarian cancer cell lines using CRISPR/Cas9 gene editing to test the loss of PARP-1 as a resistance mechanism to all clinically used PARP inhibitors. Next, we performed preclinical microPET imaging studies using ovarian cancer patient-derived xenografts in mouse models. Finally, in a phase I PET imaging clinical trial we explored PET imaging as a regional marker of PARP-1 expression in primary and metastatic disease through correlative tissue histology.

Results: We found that deletion of PARP1 causes resistance to all PARP inhibitors in vitro, and microPET imaging provides proof of concept as an approach to quantify PARP-1 in vivo. Clinically, we observed a spectrum of standard uptake values (SUVs) ranging from 2-12 for PARP-1 in tumors. In addition, we found a positive correlation between PET SUVs and fluorescent immunohistochemistry for PARP-1 (r2 = 0.60).

Conclusion: This work confirms the translational potential of a PARP-1 PET imaging agent and supports future clinical trials to test PARP-1 expression as a method to stratify patients for PARP inhibitor therapy.

Trial registration: Clinicaltrials.gov NCT02637934.

Funding: Research reported in this publication was supported by the Department of Defense OC160269, a Basser Center team science grant, NIH National Cancer Institute R01CA174904, a Department of Energy training grant DE-SC0012476, Abramson Cancer Center Radiation Oncology pilot grants, the Marsha Rivkin Foundation, Kaleidoscope of Hope Foundation, and Paul Calabresi K12 Career Development Award 5K12CA076931.

Keywords: Diagnostic imaging; Molecular biology; Oncology; Pharmacology; Therapeutics.

Figure 1. The characterization of PARP1-KO ovarian cancer cell lines and in vitro evaluation of PARP inhibitor efficacy.

(A) Immunofluorescence showed PARP-1 was absent in more than 90% of single cells in PARP1-KO polyclonal populations (ANOVA, ****P < 0.0001) and was reduced in BRCA1-restored cells compared with parent control (ANOVA, ****P < 0.0001). (B) Polyclonal populations of PARP1-KO cell lines had reduced PARP-1 by Western blot compared with parent control. (C) [¹²⁵I]KX1 radioligand binding assays showed a significant reduction in radiotracer binding in PARP1-KO and UWB1.289 BRCA1-restored cell lines compared with parent control (ANOVA, P < 0.0001). (D) Immunofluorescence of olaparib-treated UWB1.289 PARP1-KO and UWB1.289 BRCA1-restored cells showed no increase in γH2AX compared with DMSO controls. Olaparib-treated OVCAR8 PARP1-KO G1 and G3 cells showed a 1.3 times increase (ANOVA, **P < 0.01 and ***P < 0.001, respectively) in γH2AX from DMSO controls. This was in contrast to olaparib-treated UWB1.289 and OVCAR8 cells that showed a 2.6 times (ANOVA, ****P < 0.0001) and 2.2 times (ANOVA, ****P < 0.0001) increase in γH2AX from DMSO controls. (E) Cell viability assays showed that PARP1-KO cells were equally resistant to olaparib compared with BRCA1-restored cells and all clinical PARP inhibitors required PARP-1 for maximum efficacy. Loss of PARP1 caused the greatest change in efficacy for niraparib and talazoparib. Cisplatin sensitivity was used as a positive control and remained unchanged after loss of PARP1. All in vitro experiments were completed 3 independent times. Cell lines shown in A–D, from left to right, are: UWB1.289, UWB1.289 BRCA1 restored, UWB1.289 PARP1-KO G1, UWB1.289 PARP1-KO G2, UWB1.289 PARP1-KO G3, OVCAR8, OVCAR8 PARP1-KO G1, OVCAR8 PARP1-KO G2, and OVCAR8 PARP1-KO G3. –, BRCA1 mutant. +, BRCA1 restored.

Figure 2. In vivo [¹⁸F]FTT microPET imaging and ex vivo autoradiography of 2 xenografts derived from patients with ovarian cancer.

(A) Tumor-bearing mice underwent microPET imaging with [¹⁸F]FTT before (top images) and after (bottom images) olaparib treatment. White arrows point to patient-derived xenograft tumors. (B) Ex vivo autoradiographs of tumor and muscle from untreated (–) versus olaparib-treated (+) mice. (C) Significant differences were observed in the tumor-to-muscle ratios calculated before and after olaparib treatment from microPET images (4.2 ± 0.32 vs. 2.5 ± 0.11, parametric paired t test, **P < 0.0025, n = 4). (D) Ex vivo autoradiographs of untreated (–) versus olaparib-treated (+) mice shown in B also showed a statistically significant difference between groups (5.14 ± 0.13 vs. 2.41 ± 0.18, n = 2, 10 sections/tumor, parametric unpaired t test, ****P < 0.0001).

Figure 3. Diagram overview and flow chart of the pilot clinical trial of [¹⁸F]FTT PET/CT imaging in ovarian cancer.

Ten patients who were enrolled and underwent PET imaging were excluded from this subanalysis due to lack of clinical tissue samples available for correlative studies.

Figure 4. Immunohistochemistry and autoradiography analysis on clinical tissue.

Distance bars represent 275 μM. (A) HE, PARP-1, and [¹²⁵I]KX-1 autoradiograph on adjacent tissue sections showed colocalization between [¹²⁵I]KX1 and PARP-1 c-IHC. (B) In vitro autoradiography showed a difference in PARP-1 expression that was also confirmed by PARP-1 f-IHC. Max intensity by autoradiograph was 0.28 vs. 0.20 μCi/mg and fluorescent intensity of PARP-1 from whole-section f-IHC was 9.6 vs. 6.7 RFU.

Figure 5. Clinical [¹⁸F]FTT and [¹⁸F]FDG PET/CT images of a patient with ovarian cancer with vaginal cuff lesion.

Minimal radiotracer in the urinary bladder with [¹⁸F]FTT PET allowed for clear visualization of the lesion (green arrow) with no interference, despite some bowel uptake (yellow arrow on [¹⁸F]FTT image). Note excreted radiotracer in the bladder on [¹⁸F]FDG PET (yellow arrow on [¹⁸F]FDG PET).

Figure 6. Patients 2 and 11 underwent [¹⁸F]FTT PET/CT imaging within 2 weeks of completing 4 cycles of carboplatin and paclitaxel.

Omental metastases in patient 2 showed higher uptake of [¹⁸F]FTT than [¹⁸F]FDG with maximum SUVs of (A) 7.8 vs. 3.4 and (B) 5.1 vs. 2.0. Patient 2 was platinum resistant and relapsed within 4 months of therapy. Patient 11 showed low uptake on both [¹⁸F]FTT and [¹⁸F]FDG with maximum SUVs of (C) 2.4 vs. 3.7 and (D) 2.3 vs. 2.9. Patient 11 received 2 additional cycles of chemotherapy and was platinum sensitive. Yellow arrows indicate sites of disease.

Figure 7. [¹⁸F]FTT PET imaging of PARP-1 in ovarian cancer patients and tissue correlates.

(A) The spectrum of PARP-1 expression as determined by [¹⁸F]FTT PET/CT imaging with maximum SUVs ranging from approximately 2 to 12. Yellow arrows indicate sites of disease. (B) We found a positive correlation between PARP-1 immunofluorescence versus [¹⁸F]FTT PET or [¹²⁵I]KX1 autoradiography (linear regression, r² = 0.60, 0.79). No associations were observed between PARP-1 immunofluorescence, [¹⁸F]FTT imaging, or [¹²⁵I]KX1 autoradiography and [¹⁸F]FDG.