Germline mutations in the BRCA1 and BRCA2 genes are responsible for approximately 10% of epithelial ovarian cancers.1,2 These genes are involved in repair of double-strand DNA breaks through homologous recombination.3 Loss of BRCA1 or 2 function contributes to genomic instability and tumorigenesis as evidenced by the increased rates of breast and ovarian cancers in patients who inherit germ-line mutations.3 Survival of women with BRCA1/2-associated ovarian cancers is superior to that of sporadic cases after treatment with platinum-based chemotherapy.4 This is attributable to the enhanced cytotoxic efficacy of platin drugs in the context of a diminished capacity to repair chemotherapy-induced DNA damage.
An alternative DNA damage repair mechanism is through poly(adenosine diphosphate [ADP]-ribose) polymerase–mediated repair of single strand DNA breaks. Poly(ADP-ribose) polymerase inhibitors have been shown to potentiate chemotherapy-induced DNA damage in the absence of BRCA1/2.5,6 Unrepaired DNA damage caused by poly(ADP-ribose) polymerase inhibition would normally be repaired through BRCA-mediated homologous recombination; patients with BRCA mutations lose this repair mechanism, making them more sensitive to poly(ADP-ribose) polymerase inhibition.7 Poly(ADP-ribose) polymerase inhibitors cause chromosomal instability, cell cycle arrest, and apoptosis when administered to BRCA-deficient tumor cell lines.5,6
Recently reported clinical trials in BRCA1/2 carriers with breast or ovarian cancer have demonstrated efficacy of poly(ADP-ribose) polymerase inhibitors both as a single agent and in combination with cytotoxic chemotherapy (O'Shaughnessy J, Osborne C, Pippen J, Yoffe M, Patt D, Monaghan G, et al. Efficacy of BSI-201, a poly (ADP-ribose) polymerase-1 inhibitor, in combination with gemcitabine/carboplatin (G/C) in patients with metastatic triple-negative breast cancer (TNBC): results of a randomized phase II trial [abstract 3]. J Clin Oncol 2009;27 suppl:18s and Audeh MW, Penson RT, Friedlander M, Powell B, Bell-McGuinn KM, Scott C, et al. Phase II trial of the oral PARP inhibitor olaparib (AZD2281) in BRCA-deficient advanced ovarian cancer [abstract 5500]. J Clin Oncol 2009;27 suppl:15s). Although these agents seem to be active primarily against cancers of mutation carriers, little is known regarding the variation in poly(ADP-ribose) polymerase expression in hereditary and sporadic ovarian cancers. The aim of this study was to estimate the range of poly(ADP-ribose) polymerase expression in serous ovarian cancers and to determine whether expression is associated with response to therapy and outcome. If poly(ADP-ribose) polymerase expression is heterogeneous between cancers, it is possible that assessment of the level of expression could help to guide the use of this therapy in addition to BRCA1/2 mutation status.
MATERIALS AND METHODS
Primary serous epithelial ovarian cancer tissue specimens were obtained at the time of primary surgery from 186 women treated at Duke University Medical Center between January 1995 and December 2003. The study was approved by the Duke University institutional review board. Specimens were acquired through the institutional review board–approved tissue acquisition protocol and were frozen at −70°C in the Duke Gynecology Oncology frozen tissue bank. Frozen tumor specimens were formalin fixed and paraffin embedded and were histologically evaluated by a gynecologic pathologist to confirm correct pathologic diagnosis. All of the patients in this study received platinum-based chemotherapy regimens, and all were followed until death or for more than 5 years if still living.
Immunohistochemistry for poly(ADP-ribose) polymerase was performed using formalin-fixed, paraffin-embedded tissues. Sections 4–5 micrometers thick were placed in 0.01 M citrate buffer, pH 6.0, for a 15-minute heat-induced antigen retrieval in the Decloaking Chamber (Biocare Medical, Concord, CA). Mouse monoclonal antibodies to poly(ADP-ribose) polymerase (clone A6.4.12; NeoMarkers, Fremont, CA) were used at a dilution of 1:200 and incubated overnight at 4°C, followed by antibody detection using the two-step Universal 4plusHPR horseradish peroxidase kit (Biocare Medical). Slides were developed using chromogen diaminobenzidine and then counterstained with methyl green. Mouse monoclonal immunoglobulin G antibodies were used as a control for each tumor specimen. The level of poly(ADP-ribose) polymerase immunostaining was evaluated on a scale of 0–12 based on the product of intensity (0=absent, 1=light, 2=moderate, 3=heavy) and the percentage of tumor cells stained (0=0%, 1=less than 10%, 2=10–50%, 3=51–80%, 4=more than 80%) as assessed by one reviewer, who was blinded to the clinicopathologic features. A score of 8 or above was considered high poly(ADP-ribose) polymerase staining, indicating at least moderate to heavy staining of 50% or more tumor cells.
Clinicopathologic features were analyzed using the Fisher exact test to compare categorical variables. Kaplan-Meier analysis was performed to evaluate the relationship between poly(ADP-ribose) polymerase and overall survival. Two-tailed P values were used, and statistical significance was set at P=.05. Survival analysis was performed using the log-rank (Mantel-Cox) test. Analyses were performed with GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA).
The clinicopathologic characteristics of the 186 patients are reported in Table 1. The median age of the patient population was 61 years. Patients had predominantly advanced-stage (stage III/IV) and high-grade (grade 2/3) disease. Of the patients with stage III/IV cancer, 43% were optimally cytoreduced (less than 1 cm maximal diameter residual tumors) at primary surgery. Of the 174 patients with advanced-stage disease, 145 (83%) had clinical response data; 70% had a complete clinical response with their initial treatment. All patients have at least 5 years of survival data, and 153 patients (71%) have died. The median survival for the entire group was 42.3 months. Because BRCA1/2 mutation status was not known in these patients, family history was used as a surrogate for BRCA1/2 mutation status. There were 45 patients (24%) who had at least one first-degree relative with breast or ovarian cancer.
Representative pictures of poly(ADP-ribose) polymerase immunostaining are shown in Figure 1, and the relationship between poly(ADP-ribose) polymerase expression and clinical features is detailed in Table 2. High poly(ADP-ribose) polymerase expression was present in 54% of the cases; there was no relationship with stage, grade, debulking status, or family history of breast or ovarian cancer. There was also no difference in the rate of complete clinical response to primary chemotherapy between cases with low poly(ADP-ribose) polymerase expression (70%) compared with those with high poly(ADP-ribose) polymerase expression (71%).
Although not statistically significant, there was shortened progression-free survival in cases with high poly(ADP-ribose) polymerase expression (9 compared with 16 months, P=.16; Fig. 2). Kaplan-Meier survival analysis demonstrated a relationship between high poly(ADP-ribose) polymerase staining and poor survival. Patients with high poly(ADP-ribose) polymerase expression scores over 8 (moderate or strong staining of 50% or more) had a median survival of 36 months, compared with 43 months for cancers with low poly(ADP-ribose) polymerase expression (P=.04; Fig. 2). This relationship also was observed when higher cutoff points for high poly(ADP-ribose) polymerase expression were examined. For example, survival of patients with poly(ADP-ribose) polymerase expression scores of 12 (strong staining of 80% or more of tumor cells) was 35 months, compared with 43 months in patients with lower scores (P=.02). Poor survival with high poly(ADP-ribose) polymerase expression was also seen after excluding patients with a family history of breast or ovarian cancer (P=.07), although this did not reach statistical significance.
Poly(ADP-ribose) polymerase inhibitors have recently been shown to be active as single-agent therapy and in combination with platin-based chemotherapy in BRCA1/2-deficient cancers (O'Shaughnessy et al. J Clin Oncol 2009;27 suppl:18s and Audeh et al. J Clin Oncol 2009;27 suppl:15s). Poly(ADP-ribose) polymerase normally functions as an enzyme in base excision repair to correct DNA single-strand breaks, whereas BRCA1 and BRCA2 are key contributors to DNA double-strand break repair by homologous recombination (O'Shaughnessy et al. J Clin Oncol 2009;27 suppl:18s).5–8 With poly(ADP-ribose) polymerase inhibition, single-strand DNA breaks persist, resulting in stalled replication forks and double-strand DNA breaks. Intact BRCA1 and BRCA2 pathways allow these lesions to be repaired through homologous recombination, but in the absence of this backup mechanism, BRCA-deficient cells undergo cell cycle arrest and apoptosis as a result of poly(ADP-ribose) polymerase inhibition. Loss of platinum/poly(ADP-ribose) polymerase sensitivity in some BRCA1/2-deficient cancers has been shown to be attributable to back mutations in which functional BRCA1/2 protein is restored.9,10
At least one small study has evaluated clinicopathologic features and outcome related to poly(ADP-ribose) polymerase expression in ovarian cancer. Among 50 serous ovarian cancers, of which only 18 were advanced stage (stage III), Brustmann11 reported that strong poly(ADP-ribose) polymerase expression was associated with a trend toward advanced-stage, high-grade tumors and worse survival. The aim of our present study was to determine whether there was a relationship between poly(ADP-ribose) polymerase expression and response to chemotherapy and survival in a group of serous ovarian cancers with unknown BRCA1/2 status. We found that 54% of 186 serous ovarian cancers had high poly(ADP-ribose) polymerase expression. Although high poly(ADP-ribose) polymerase expression was not predictive of a worse response to primary chemotherapy, high poly(ADP-ribose) polymerase expression was associated with a 7-month decrease in overall survival.
Studies have primarily evaluated the role of poly(ADP-ribose) polymerase inhibitors in BRCA1/2-deficient cancers, whereas it is less well known whether these agents have efficacy in other cancers. In addition to inactivation by germ-line mutation, loss of BRCA1/2 function may occur via other mechanisms. First, somatic mutations in BRCA1 have been described in some sporadic ovarian cancers.12 Hennessy et al recently reported BRCA1/2 gene sequencing from 235 ovarian cancers and found mutations in 20% of the cancers, a higher than expected prevalence based on the germline rate (Hennessy B, Timms K, Carey MS, Gutin A, Broaddus R, Gonzalez-Angulo A, et al. Somatic BRCA status in ovarian tumors [abstract 5528]. J Clin Oncol 2009;27 suppl:15s). Twenty-five percent of these mutations were novel and had never been found as germline mutations. In addition, epigenetic silencing of BRCA1 occurs in approximately 15% of sporadic breast and ovarian cancers and has been associated with worse outcome in patients with ovarian cancer.13,14 Likewise, patterns of gene expression have been described using microarrays that characterize “BRCA-like” cancers that lack mutations.15 These studies suggest that there may be patients without a germline BRCA1/2 mutation who might benefit from the addition of a poly(ADP-ribose) polymerase inhibitor because of functional BRCA1/2 inactivation. In addition, it is possible that cancers with intact BRCA1/2 but high poly(ADP-ribose) polymerase expression might also benefit from poly(ADP-ribose) polymerase inhibitor therapy. One limitation of the present study is that we did not know the BRCA1/2 mutation status of the patients. In view of this, we used a family history of breast or ovarian cancer as a surrogate for BRCA1/2 mutation status. After excluding these patients, there was still a trend toward poor survival. This suggests that high poly(ADP-ribose) polymerase expression may be a poor prognostic factor in sporadic ovarian cancers.
Currently, there are at least seven phase clinical trials under way evaluating the use of poly(ADP-ribose) polymerase inhibitors in cancer treatment, four of which specifically include patients with BRCA1/2 germline mutations (O'Shaughnessy et al. J Clin Oncol 2009;27 suppl:18s and Audeh et al. J Clin Oncol 2009;27 suppl:15s).8 A recent abstract was published reporting the interim results of a phase 2 trial evaluating the poly(ADP-ribose) polymerase inhibitor olaparib (AZD2281) in patients with BRCA-deficient ovarian cancer; this treatment was well tolerated and highly active in advanced, chemotherapy-refractory cancers (Audeh et al. J Clin Oncol 2009;27 suppl:15s). Fong et al16 recently reported results from a phase 1 trial of a poly(ADP-ribose) polymerase inhibitor that demonstrated antitumor activity in BRCA1/2 mutation carriers with minimal adverse effects, whereas there was no response in cancers in patients who did not have BRCA mutations.
The present study demonstrates that there is significant heterogeneity of poly(ADP-ribose) polymerase expression among advanced serous ovarian cancers. This heterogeneity was noted in the entire group, including the subset with a strong family history of breast or ovarian cancers, which was used as a surrogate for germline BRCA1/2 mutation. Poly(ADP-ribose) polymerase inhibitors are clearly emerging as a promising treatment for ovarian cancers in patients with germline BRCA1/2 mutations. However, not all BRCA1/2-associated cancers respond to poly(ADP-ribose) polymerase inhibitors, and conversely, it is possible that there is a subset of sporadic cases that might respond. There are several mechanisms by which sporadic ovarian cancers might have functional BRCA1/2 inactivation, and whether these or other sporadic ovarian cancers would benefit from the use of poly(ADP-ribose) polymerase inhibitors has not been determined. In view of this, it is possible that evaluation of the level of poly(ADP-ribose) polymerase expression, along with BRCA1/2 status, may prove useful in guiding the use of poly(ADP-ribose) polymerase inhibitors. This clinical paradigm has become the standard of care with respect to treatment with agents that target the estrogen, progesterone, and androgen receptors as well as human epidermal growth factor receptor 2/neu. Further studies are needed to determine the relationship between poly(ADP-ribose) polymerase expression, BRCA1/2 mutational and functional status, and the efficacy of poly(ADP-ribose) polymerase inhibitors.
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