Assessment of FANCD2 nuclear foci formation in paraffin-embedded tumors: a potential patient-enrichment strategy for treatment with DNA interstrand crosslinking agents.
A major mechanism of DNA repair related to homologous recombination is the Fanconi anemia (FA) pathway. FA genes collaborate with BRCA genes to form foci of DNA repair on chromatin after DNA damage or during the S phase of the cell cycle. Our goal was to develop a method capable of evaluating the functional status of the pathway in patients' tumor tissue, which could also be practically incorporated into large-scale screening. To develop this method, we first used Western immunoblot to detect FANCD2 protein monoubiquitination in fresh tumor specimens of patients with ovarian cancer undergoing surgery and stained formalin-fixed paraffin-embedded tumor tissue simultaneously with 4',6-diamidino-2-phenylindole, FANCD2, and Ki67 antibodies, eventually extending this method to other solid tumors. This triple stain permitted evaluation of the presence, or lack thereof, of FANCD2 subnuclear repair foci in proliferating cells by immunofluorescence microscopy. Overall, we evaluated 156 formalin-fixed paraffin-embedded tumor samples using the FA triple-staining immunofluorescence method. The ratios of FANCD2 foci-negative tumors in ovarian, lung, and breast tumor samples were 21%, 20%, and 29.4%, respectively. Our studies have led to the development of a suitable method for screening, capable of identifying tumors with somatic functional defects in the FA pathway. The use of paraffin-embedded tissues renders the reported method suitable for large-scale screening to select patients for treatment with DNA interstrand crosslinking agents, poly ADP-ribose polymerase inhibitors, or their combination.
Comprehensive Cancer Center, Division of Medical Oncology, The Ohio State University College of Medicine and Public Health, Columbus, Ohio, USA.
Wenrui
W
Duan
Li
L
Gao
Weiqiang
W
Zhao
Marino
M
Leon
Wolfgang
W
Sadee
Amy
A
Webb
Kimberly
K
Resnick
Xin
X
Wu
Bhuvaneswari
B
Ramaswamy
David E
DE
Cohn
Charles
C
Shapiro
Paul R
PR
Andreassen
Gregory A
GA
Otterson
Miguel A
MA
Villalona-Calero
D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000627:therapeutic use; D004260:DNA Repair / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005199:Fanconi Anemia / Q000235:genetics / Q000378:metabolism; D052236:Fanconi Anemia Complementation Group D2 Protein / Q000378:metabolism*; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008175:Lung Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D009369:Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D010051:Ovarian Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D016612:Paraffin Embedding; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D057170:Translational Research, Biomedical; D054875:Ubiquitination
D003432:Cross-Linking Reagents; D004791:Enzyme Inhibitors; C499905:FANCD2 protein, human; D052236:Fanconi Anemia Complementation Group D2 Protein; D000067856:Poly(ADP-ribose) Polymerase Inhibitors