Development of a functional assay for homologous recombination status in primary cultures of epithelial ovarian tumor and correlation with sensitivity to poly(ADP-ribose) polymerase inhibitors.
OBJECTIVE
Poly(ADP-ribose) polymerase (PARP) inhibitors selectively target homologous recombination (HR)-defective cells and show good clinical activity in hereditary breast and ovarian cancer associated with BRCA1 or BRCA2 mutations. A high proportion (up to 50%) of sporadic epithelial ovarian cancers (EOC) could be deficient in HR due to genetic or epigenetic inactivation of BRCA1/BRCA2 or other HR genes. Therefore, there is a potential for extending the use of PARP inhibitors to these patients if HR status can be identified. We developed a functional assay of HR status in primary cultures of EOCs based on Rad51 focus formation that correlates well with sensitivity to the potent PARP inhibitor AG014699.


METHODS
Primary cultures were derived from ascitic fluid from patients with EOCs. HR status was investigated by gammaH2AX and Rad51 focus formation by immunofluorescence. Cytotoxicity to PARP inhibitors was tested by sulforhodamine B and survival assay.


RESULTS
Twenty-five cultures were evaluated for HR status and cytotoxicity to PARP inhibitor. Following exposure to AG014699, there was an increase in Rad51 foci (HR competent) in 9 of 24 (36%) but no increase (HR deficient) in 16 of 24 (64%) cultures. Cytotoxicity was observed in 15 of 16 (93%) HR-deficient samples but not in 9 of 9 HR-competent samples following 24-hour exposure to 10 mumol/L AG014699.


CONCLUSIONS
HR status can be determined in primary cancer samples by Rad51 focus formation, and this correlates with in vitro response to PARP inhibition. Use of this assay as a biomarker now needs testing in the setting of a clinical trial.
Mukhopadhyay
Asima
A
Northern Gynaecological Oncology Centre, Queen Elizabeth Hospital, Gateshead, UK.
Elattar
Ahmed
A
Cerbinskaite
Aiste
A
Wilkinson
Sarah J
SJ
Drew
Yvette
Y
Kyle
Suzanne
S
Los
Gerrit
G
Hostomsky
Zdenek
Z
Edmondson
Richard J
RJ
Curtin
Nicola J
NJ
D019313:BRCA1 Protein / Q000235:genetics; D024682:BRCA2 Protein / Q000235:genetics; D001681:Biological Assay; D014408:Biomarkers, Tumor / Q000378:metabolism; D002470:Cell Survival; D019008:Drug Resistance, Neoplasm; D005260:Female; D006657:Histones / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D010051:Ovarian Neoplasms / Q000235:genetics* / Q000378:metabolism; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism; D051135:Rad51 Recombinase / Q000378:metabolism; D011995:Recombination, Genetic; D012235:Rhodamines / Q000378:metabolism; D014407:Tumor Cells, Cultured
D019313:BRCA1 Protein; D024682:BRCA2 Protein; D014408:Biomarkers, Tumor; C468783:H2AX protein, human; D006657:Histones; D007211:Indoles; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D012235:Rhodamines; C022027:lissamine rhodamine B; C531549:rucaparib; D011065:Poly(ADP-ribose) Polymerases; D051135:Rad51 Recombinase