p53-autoantibody may be more sensitive than CA-125 in monitoring microscopic and macroscopic residual disease after primary therapy for epithelial ovarian cancer.

Purpose To evaluate the use of p53-autoantibodies (p53-aab) for monitoring minimal disease after standard therapy of advanced epithelial ovarian cancer (EOC).

Retrospective analysis of p53-aab in preoperative and long-term follow-up serum samples from 10 patients selected for representing three relevant EOC subgroups: platinum-sensitive disease after macroscopic complete debulking (n = 4) and platinum-sensitive (n = 3) or platinum-resistant disease (n = 3), both after suboptimal debulking with residual tumor of <1 cm diameter. p53-aab levels were quantified by a sandwich ELISA in two independent experiments. CA-125 values of all samples and clinical information were retrieved from medical records.

Patients with early relapse (median PFS 7 months, n = 8) had high p53-aab levels throughout follow-up while CA-125 values had dropped below the cut-off after primary surgery during or after chemotherapy in these cases. Patients with seroconversion to p53-aab negativity experienced prolonged PFS (n = 2; #1: 50 months, #2: no evidence of disease for 36 months until last follow-up). Continued p53-aab positivity was not related to the resection status or platinum sensitivity.

p53-autoantibodies may be a highly sensitive marker for minimal residual tumor mass after surgery and/or chemotherapy rather than standard CA-125, possibly due to the different nature of these markers. CA-125 released by cancer cells is related to tumor mass, whereas p53-aab levels can indicate the presence of few tumor cells due to amplification by the immune system. Seroconversion of p53-aab could be associated with long-term survival.

Epithelial ovarian cancer, p53- autoantibody, Biomarker

Highly sensitive serologic markers based on, for example, autoimmunity could be indicative of minimal residual disease and may be helpful to decide on the duration of maintenance or consolidation therapy in epithelial ovarian cancer (EOC). The best evaluated biomarker available CA-125 cannot reliably detect small tumor masses <8 cm3 (Hori and Gambhir 2011). Thus, CA-125 is not suitable as an indicator for the presence of microscopic minimal residual disease following radical surgery and/or chemotherapy. A valid indicator of tumor load at the end of therapy is needed for optimizing treatment, follow-up decisions and cost-effectiveness in particular using targeted therapies for consolidation or maintenance. The effectiveness of, for example, bevacizumab containing consolidation therapy in cohorts with postoperative minimal and with macroscopic residual disease has recently been shown by phase III trials (Burger et al. 2011; Perren et al. 2011). To better understand and monitor the effect of such extended therapy, highly sensitive biomarkers are needed. Autoantibodies against p53 (p53-aab) are associated with the overexpression or accumulation of the mutated protein and are highly specific for certain cancer types such as EOC, particularly EOC type II (Soussi 2000; Vogl et al. 2000; Bauerschlag et al. 2010; Lu et al. 2011). Yet, the low prevalence excludes its use as a screening marker for early diagnosis. However, one published study described the applicability of p53-aab as biomarker to monitor EOC patients during treatment but did not report individual changes of p53-aab levels during follow-up (Gadducci et al. 1998). Studies on other tumor entities have revealed associations of p53-aab level changes and therapy outcome (Takeda et al. 2001; Gumus et al. 2004). The majority of colorectal cancer patients converted from p53-aab positivity to serum levels below cut-off after complete resection (28/30 cases) (Takeda et al. 2001). In patients with invasive bladder cancer, seroconversion of p53-aab positivity was significantly related to an improved outcome in comparison with both, initially p53-aab negative cases and patients with positive serum levels after therapy (p < 0.05) (Gumus et al. 2004). To investigate the potential usefulness of p53-aab as a biomarker for therapy monitoring in EOC, we retrospectively analyzed follow-up samples from decidedly representative patients after optimal tumor debulking who tested positive for p53-aab as well as for the standard marker CA125 in preoperative sera.

Clinical samples We retrospectively analyzed serum samples from 10 patients collected during follow-up, positive for p53-aab as well as CA125 in preoperative sera and representative for three clinically important subgroups. Patients were selected as either being after complete or being after incomplete tumor resection and with different sensitivity status regarding platinum-based chemotherapy: Group #1 platinum-sensitive disease after macroscopic complete debulking (n = 4), group #2 platinum-sensitive (n = 3) and group #3 platinum-resistant disease (n = 3), each after suboptimal debulking with residual tumor of <1 cm diameter. p53-aab levels were quantified using a sandwich ELISA in two independent experiments. CA-125 values of all samples and clinical information were retrieved from medical records of our hospital. For control, serum samples from 12 patients negative for p53-aab at primary diagnosis were included in the ELISA testing. The Ethics Committee of the Jena University Hospital approved the use of these serum samples stored without personal data. Serum samples were obtained before surgery of primary and during regular follow-up visits. Samples were stored at −30 °C. p53-autoantibody ELISA A p53-aab ELISA (Steinbeis-Transferzentrum Angewandte Biologische Chemie, Mannheim, Germany) was used to quantify the level of p53-aab. This ELISA has previously been shown to be superior in comparison with other available systems (Rohayem et al. 1999). In addition to the clinical samples, a calibrator dilution series and both negative and positive control samples were included. Serum samples were diluted 1:100 and analyzed in two independent experiments. Samples with mean p53-aab levels >120 U/ml were judged as positive.

All patients had increased CA-125 values (>35 U/ml) at primary diagnosis and were optimally resected either with minimal residual tumor mass (<1 cm, n = 6) or without macroscopic disease (n = 4). Four patients without macroscopic disease at the end of primary surgery and platinum sensitivity were defined as one representative group. Platinum-resistant as well as platinum-sensitive disease as judged by the progression-free time interval after chemotherapy (6 months) was included within the group of patients with postoperative minimal macroscopic residual disease (each phenotype n = 3). In this study on patients’ representative of the three relevant subgroups, p53-aab levels stayed detectable in patients experiencing early clinical relapse but declined below cut-off in patients with prolonged PFS. A relation of p53-aab positivity to the resection status or platinum sensitivity did not become evident. The 8 patients with early relapse exhibited high p53-aab levels during follow-up (median PFS 7 months) while CA-125 values had dropped below the cut-off after primary surgery and chemotherapy in these cases. One patient showed negative CA-125 measurements even during tumor progression as detected by imaging studies and clinical presentation (Fig. 1a). Patients with minimal macroscopic residual tumor and platinum resistance (n = 3) had the shortest progression-free survival (PFS; median 3.5 months). Slightly higher but similar PFS for platinum-sensitive patients with minimal residual tumor mass (n = 2, median PFS 6.9 months) and without macroscopic disease (n = 3, median PFS 8.9 months) was observed if the patients had elevated p53-aab levels after therapy. The only patients with extended PFS were the two patients with p53-aab seronegativity after the end of therapy during follow-up. These two patients had p53-aab and CA-125 serum levels below the cut-off values after therapy. Patient #9 (platinum-sensitive, <1 cm residual tumor) did not show an increase in serum markers during follow-up and showed progression-free survival for 36 months until the last follow-up data. Patient #10 (platinum-sensitive, no macroscopic residual tumor) experienced a prolonged progression-free survival and serum markers were below cut-off values over a longer time period following primary treatment (up to 38 months). Both markers tested positive 50 months post-surgery when the relapse was clinically diagnosed (Fig. 1b). Control patients, negative for p53-aab at primary diagnosis (n = 12), did not show p53-aab at any time irrespective of the progression status except for one patient. She showed positive p53-aab levels at relapse despite negative results at primary diagnosis and a follow-up visit (109 and 86 months before relapse, respectively).

Based on these data, it could be expected that the p53-aab test is more sensitive for the detection of small residual tumor mass after surgery and chemotherapy than CA-125. The potential release of CA-125 by non-malignant tissue, that is, the endometrium, endometriosis or benign ovarian cysts prevents the use of lower cut-off values within the range of the detection limit (1 U/ml) of available assays (Mongia et al. 2006) and thus the possible identification of minimal residual disease. Available published data did not reveal basic differences in (1) half-life times for CA-125 and autoantibodies (ca. 7 days) or (2) the time period to normalization of serum levels after surgery (one to several months) (Mastropaolo et al. 1986; Zalcman et al. 1998; Gumus et al. 2004; Anderson and LaBaer 2005; Riedinger et al. 2006; Tan et al. 2009). These data are confirmed by our analyses of two patients showing a seroconversion after <7 months (earliest available serum samples). Thus, we would suggest that steadily elevated p53-aab levels are not caused by long-lived plasma cells (“memory”) but by a sustained immune response of effector B cells to a sufficient amount of residual tumor cells. A constant stimulation of the immune system is necessary to maintain a high level of p53-aab (Soussi 2000). Levels of autoantibodies produced by memory cells even in patients without residual tumor cells may be below the cut-off value (120 U/ml). In this pilot cohort of patients negative for p53-aab at primary diagnosis, 1 of 12 patients (8 %) developed p53-aab during follow-up and was scored as p53-aab positive at relapse. This frequency of seroconversion is equal to data presented by Gadducci et al. (2/25 patients, 8 %) but lower than the general frequency of p53-aab in EOC patients (20–40 %) (Gadducci et al. 1998; Soussi 2000; Vogl et al. 2000). Thus, most but not all cases of p53-autoimmune reaction in EOC patients could be related to inherent properties. Whether seroconversion of initially p53-aab negative patients is related to tumor cell (i.e. p53 mutation) or immune system properties is not quite understood. These promising results warrant a multicentric prospective observational study on p53-aab as a biomarker for therapy monitoring. Such a study should gather a broad database to answer questions about changes of p53-aab levels at different time points: immediately after surgery, during chemotherapy and during maintenance or consolidation therapy. First results from such setting show variable changes of p53-aab levels after surgery and during chemotherapy although analyses for a correlation to patients’ outcome have to be postponed until follow-up data are available. In view of the present drawbacks of classical biomarkers that are released by the tumor cell itself (Cramer et al. 2011; Hori and Gambhir 2011), the possible use of autoantibody panels as early detection markers should be evaluated for EOC. The presence of autoantibodies directed against p53 and other tumor antigens has been shown in prediagnostic sera from breast cancer and colorectal cancer patients (Lu et al. 2012; Pedersen et al. 2012). In conclusion, this pilot study supports the hypothesis that p53-aab could be a highly sensitive marker for the detection of small residual tumor mass after surgery and chemotherapy rather than CA-125 due to the different nature of these markers. CA-125 released by cancer cells is related to the tumor mass, whereas p53-aab levels can indicate the presence of few tumor cells due to amplification by the immune system. Seroconversion of p53-aab seems to be associated with long-term survival, whereas elevated levels of p53-aab after primary therapy could not only help identify patients gaining benefit from the novel maintenance or consolidation therapies employing, for example, antibodies targeting tumor antigens such as bevacizumab but also could help to decide on the optimal timing to end therapy. Conflict of interest MF is employed by Steinbeis-Transferzentrum commercializing the p53-AAB ELISA. The Steinbeis-Transferzentrum had no influence on data acquisition, analysis or interpretation and manuscript drafting. All other authors have no conflict of interest to disclose.

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