Quantification of Free Plasma DNA Before and After Chemotherapy in Patients With Advanced Epithelial Ovarian Cancer : Diagnostic Molecular Pathology

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00019606-200803000-00006ArticleDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2008 by Lippincott Williams & Wilkins.17March 2008 p 34-38Quantification of Free Plasma DNA Before and After Chemotherapy in Patients With Advanced Epithelial Ovarian CancerOriginal ArticlesCapizzi, Elisa MSc*; Gabusi, Elena PhD*; Grigioni, Antonia D'Errico MD*; De Iaco, Pierandrea MD†; Rosati, Marta MD‡; Zamagni, Claudio MD‡; Fiorentino, Michelangelo MD, PhD**Molecular Pathology Laboratory, “Addarii” Institute of Oncology†Department of Obstetrics and Gynecology‡Medical Oncology Unit, S. Orsola-Malpighi Teaching Hospital, University of Bologna, Bologna, ItalyWe state that there are no financial conflicts of interest for the present paper.Reprint: Michelangelo Fiorentino, MD, PhD, Istituto Oncologico “Addarii”, Viale Ercolani 4/2, 40138, Bologna, Italy (e-mail: [email protected]).AbstractObjectivesA nonrandomized trial was planned to investigate the role of free plasma DNA (FPDNA) in patients with epithelial ovarian cancer before and after chemotherapy. Twenty-two patients with advanced stage ovarian cancer not suitable for debulking were treated with a neoadjuvant platinum/taxanes chemotherapy. Patients with clinical complete or partial response underwent radical hystero-oophorectomy, omentectomy, and lymphadenectomy and were followed up every 3 to 6 months.MethodsBlood samples were obtained from each patient before chemotherapy, before each cycle, before and after surgery. FPDNA was quantified by real-time quantitative polymerase chain reaction using the Quantifiler Human Quantification Kit and expressed in ng/mL. Fifty female healthy blood donor volunteers were used as controls.ResultsMedian FPDNA quantities discriminated between patients before chemotherapy (29.6±22.7 ng/mL) and controls (6.4±4.0 ng/mL) using a 14.5 ng/mL cutoff with 77% sensitivity and 96% specificity (P<0.001). Mean FPDNA concentrations significantly decreased after chemotherapy (17.9±14.5 ng/mL, P=0.001). A peak of FPDNA levels (66.2±45.2 ng/mL) was observed in association with surgery (P<0.001). Median follow-up and median progression-free survival time were 13.4±5.1 and 11.7±5.6 months, respectively. Eight patients with FPDNA values above the cutoff after chemotherapy showed disease progression or died, whereas 7 patients with FPDNA below the cutoff were free from disease. Patients with FPDNA levels above and below the cutoff showed significantly separated progression-free survival curves (P=0.007, log-rank test).ConclusionsFPDNA quantification significantly discriminates between cancer patients and controls and correlates with response to chemotherapy. Although performed in a limited series, we demonstrated a correlation between FPDNA values and clinical behavior of ovarian cancer patients.Ovarian cancer is the leading cause of death for gynecologic malignancies in Italy.1 Owing to the absence of reliable population screening protocols, the majority of ovarian cancer patients are diagnosed at an advanced stage, with an expected 5-year survival rarely exceeding 30%.2 Standard care of advanced ovarian cancer is based on a combination of surgery and chemotherapy with regimens including platinum compounds and taxanes.3 Ovarian cancer diagnosis and monitoring of recurrence and response to therapy is generally assessed by computed tomography (CT) scan, transvaginal ultrasound, and serum CA-125.4 The combined use of these techniques is generally associated with high specificity while the sensitivity might be improved especially in case of early stage tumors, and for the detection of minimal residual neoplastic disease after therapy.5 Therefore, novel serum markers and molecular staging techniques are required to improve the sensitivity of detecting recurrent disease and to facilitate earlier detection of ovarian cancer.The detection of free plasma DNA (FPDNA) in the blood has been proposed as a noninvasive test for early tumor detection and monitoring of tumor recurrence and response to therapy.6–11 Despite the occurrence of false positives, particularly in subjects with autoimmune or inflammatory diseases or recent surgical procedures, significantly increased FPDNA levels have been observed in patients with epithelial cancers, including ovarian cancer, compared with healthy subjects.12–14 In particular, levels of circulating cell-free DNA were found to correlate with tumor burden and to decline after therapy in an orthotopic ovarian cancer mouse model.15 Furthermore, specific p53 mutations were detected in the free circulating DNA of approximately one third of women with epithelial ovarian cancer.16 The association between FPDNA quantity and tumor recurrence and/or response to therapy is the key point for the application of this test in the clinical oncology routine of ovarian cancer patients.We, therefore, analyzed the variations of FPDNA quantity in a prospective, nonrandomized study on 22 patients with advanced (stage III-IV) epithelial ovarian cancer treated with carboplatin or with a platinum/taxane-based regimen as neoadjuvant (primary) chemotherapy before surgery.PATIENT POPULATION AND STUDY DESIGNWe designed a prospective nonrandomized clinical and bio-pathologic study on 22 consecutive patients who received clinical diagnosis of advanced epithelial ovarian cancer (FIGO stage IIIC or IV) between September 2004 and June 2006. Eligibility criteria for patient enrollment were (1) histologic diagnosis of serous epithelial ovarian cancer; (2) clinical tumor stage IIIC or IV according to FIGO classification not suitable for optimal surgical debulking; (3) no treatment with chemotherapy or surgery (except for diagnostic laparoscopy) before first blood collection; (4) availability of complete clinical data, CT scan, and CA-125 dosage for each patient.The mean age of the patients was 64±8 years (range 46 to 75). At the time of enrollment, 11 (50%) patients were in clinical tumor stage IIIC and 11 (50%) in stage IV. Diagnostic laparoscopy before the enrollment was performed in 21 of the 22 patients (95%). At histologic examination, 1 case (5%) had tumor differentiation grade 1, 1 (5%) grade 2, and 19 (90%) grade 3.Twenty patients were treated with 6 cycles of carboplatin (area under the curve 4 or 5) and paclitaxel (135 to 175 mg/mq), whereas 2 elderly patients were treated with carboplatin area under the curve 5 as single agent. Cycles were repeated for every 3 weeks. Clinical response to therapy was assessed by serum CA-125 dosage after every each cycle while CT scan, fluorodeoxyglucose-positron emission tomography scan, pelvic ultrasound, and pelvic magnetic resonance imaging were performed at baseline and after the third and the sixth cycle. All patients with a clinical complete or partial response (according to WHO criteria) underwent laparotomic hysterectomy, bilateral salpingo-oophorectomy, omentectomy and lymph-node dissection.Once enrolled, 4 mL of blood samples were drawn from all patients according to the following schedule: (i) before any treatment (time zero); (ii) before each chemotherapy cycle; (iii) 1 week before surgery; (iv) 24 hours, 10 days, and 1 month after surgery (total samples for each patient n=10).Four mL blood samples of 50 healthy females were retrieved from the blood bank of our hospital and used as negative controls for the technique. Control samples were all from white females and were matched for age (mean age 42 y, range 35 to 62), collected, extracted, and processed alongside and alike those of the patients.FPDNA QUANTIFICATIONFresh blood samples for FPDNA quantification were collected into a Vacutainer EDTA tube and processed within 1 hour from collection. Whole blood from patients and controls was centrifuged twice at 2500g for 10 minutes at 4°C to separate plasma from the cellular fraction. DNA was purified from 1 mL of plasma (QIAamp Blood Mini Kit, Qiagen, Hilden, Germany) and eluted in 50 μL. Quantification of FPDNA was accomplished by Real-time quantitative polymerase chain reaction (qPCR) amplification of the human telomerase reverse transcriptase gene (hTERT) using the Quantifiler Human DNA Quantification Kit (Applied Biosystems, Foster City, CA) on a ABI PRISM 7000 instrument (Applied Biosystems). For each reaction, 2 μL of the eluted DNA were used in a final volume of 25 μL and analyzed in duplicate. FPDNA concentrations were calculated by interpolation with the standard amplification curve. qPCR conditions were 50°C for 2 minutes, 95°C for 10 minutes, 95°C for 15 seconds, 60°C for 60 seconds for 50 cycles according to the instructions of the manufacturer. Results were expressed as DNA ng/mL of plasma.STATISTICAL ANALYSISData are reported as means±SD, ranges, and frequencies. The Mann-Whitney test and the Wilcoxon test correlation were applied. Progression-free survival (PFS) rate was calculated using the Kaplan-Meier method and the log-rank test was used. Data analysis was performed using SPSS for Windows (Version 13.0). A 2-tailed P value less than 0.05 was used to define statistical significance.ETHICSThe study (a subprotocol of the Arianna 02 Project) was approved by the Ethical Committee of the S. Orsola-Malpighi Hospital. Informed consent was obtained from all patients by signature of the specific form provided by the Ethical Committee. The study protocol conformed to the ethical guidelines of the “World Medical Association Declaration of Helsinki-Ethical Principles for Medical Research Involving Human Subjects” adopted by the 18th WMA General Assembly, Helsinki, Finland, June 1964, as revised in Tokyo 2004.RESULTSClinical OutcomeAll the 22 patients accomplished the 6 planned chemotherapy cycles. Fourteen of the 22 (64%) patients showed clinical total or partial response after therapy and underwent surgery. Six (27%) patients had a stable disease (tumor unchanged or reduction <50%) and 4 of them were also submitted to debulking surgery after neoadjuvant chemotherapy. The 2 patients (9%) who progressed on chemotherapy were not eligible for debulking surgery and started a second-line treatment. Median follow-up was 13.4±5.1 months (range 4 to 27) and median PFS was 11.7±5.6 months (range 4 to 27). At the time of the last available follow-up, 3 patients were dead for disease progression (2 treated with surgery and 1 during the second line of chemotherapy), 9 were alive with disease progression, and 10 were alive and free of disease progression (Table 1).JOURNAL/dimp/04.03/00019606-200803000-00006/table1-6/v/2021-02-17T195944Z/r/image-tiff FPDNA Variations According to Clinical Pathological ParametersFPDNA in Cancer Patients and ControlsMean FPDNA levels of the ovarian cancer patients before chemotherapy (time zero) were compared with controls. The best cutoff value to discriminate between patients and healthy controls was calculated as the mean FPDNA value of the controls plus 2 standard deviations and was fixed at 14.5 ng/mL. Seventeen (77%) of the 22 patients showed FPDNA values above the cutoff, whereas 48 (96%) of the 50 controls showed FPDNA values below the cutoff. The mean levels of FPDNA were significantly higher in ovarian cancer patients before any therapy (29.6±22.7 ng/mL, range 8.5 to 92.9) compared with healthy controls (6.4±4.0 ng/mL, range 0.7 to 16.5) (P<0.001, Mann-Whitney test) (see box-plot in Fig. 1). Seven patients in stage IIIC and 10 in stage IV had FPDNA baseline values above the cutoff.JOURNAL/dimp/04.03/00019606-200803000-00006/figure1-6/v/2021-02-17T195944Z/r/image-jpeg Box-plots of FPDNA concentrations in healthy controls, ovarian cancer patients before therapy (time zero) and ovarian cancer patients after 6 cycles of chemotherapy (before surgery).FPDNA Values Before and After ChemotherapyWe evaluated the variations of FPDNA at time zero and after the sixth cycle of chemotherapy. At time zero, 17 of the 22 (77%) patients had FPDNA values above the cutoff, whereas after chemotherapy 11 (50%) patients had FPDNA levels above the cutoff and 11 below the cutoff. Mean FPDNA quantities after chemotherapy (17.9±14.5 ng/mL, range 2.7 to 57.2) were significantly decreased compared with the time zero values (P=0.001, Wilcoxon test) (box-plot in Fig. 1). Similar results were also obtained comparing the samples at time zero with those after the first cycle of chemotherapy (mean values 18.1±12.9 ng/mL, range 2.7 to 58.7, P=0.003 at Wilcoxon test). The 4 patients not suitable for debulking surgery after neoadjuvant chemotherapy also had FPDNA levels above the cutoff during all the 6 cycles. FPDNA variations according to chemotherapy are summarized in Table 1.FPDNA and Disease ProgressionFPDNA values obtained after the sixth cycle of chemotherapy (before surgery) were compared with disease progression. PFS was calculated from the day of the first chemotherapy cycle to the time of disease progression or to the most recent follow-up. A general trend of correlation was found between high levels of FPDNA after the sixth cycle of chemotherapy and ovarian cancer progression. In particular, 8 of the 11 (73%) patients with FPDNA values above the cutoff after chemotherapy showed disease progression or died, whereas 7 of the 11 (64%) patients with FPDNA levels below the cutoff after chemotherapy were free from disease at the time of the last available follow-up. Patients with FPDNA levels above and below the cutoff after chemotherapy showed significantly separated Kaplan-Meier PFS curves (P=0.007, log-rank test) (Fig. 2).JOURNAL/dimp/04.03/00019606-200803000-00006/figure2-6/v/2021-02-17T195944Z/r/image-tiff PFS Kaplan-Meier curves of ovarian cancer patients according to FPDNA levels above and below the cutoff (14.5 ng/mL) after the sixth cycle of chemotherapy (before surgery). PFS time was calculated from the day of the first chemotherapy cycle (time zero).Association Between FPDNA Levels and SurgeryA strong increase of FPDNA levels was observed after surgery in the 18 patients who underwent bilateral hystero-oophorectomy. In particular, mean FPDNA values 24 hours after surgery (66.2±45.2 ng/mL, range 17.6 to 198.0) were significantly higher compared with the blood samples drawn 1 week before surgery (P<0.001, Wilcoxon test). FPDNA quantities were still high 10 days after surgery (mean values 54.9±45.0, range 11.4 to 204.1, P<0.001), whereas a decrease was observed after 1 month (mean values 23.3±31.3, range 4.4 to 140.4) (Table 1).CA-125 Before and After ChemotherapyThe cutoff level of serum CA-125 was stated at 35 U/mL. At the time of diagnosis (time zero), all the 22 patients had CA-125 values ≥35 U/mL (mean 1086.5±1353.8, range 47 to 6000). At the end of the sixth cycle of chemotherapy, 15 (68%) of the 22 patients showed serum CA-125 decrease below the cutoff. CA-125 values after the sixth cycle of chemotherapy (before surgery) were compared with disease progression. Four of the 12 patients with tumor progression had CA-125 values ≥35 U/mL, whereas 7 of 10 patients free of disease progression had CA-125 ≤35 U/mL (Table 1).DISCUSSIONOvarian cancer is one of the most deadly tumors of the female, because more than 70% of the patients are diagnosed in an advanced stage. Prognosis of ovarian cancer depends on the extent of tumor burden at the time of diagnosis and the clinical response to platinum/taxane chemotherapy. Early diagnosis of ovarian cancer and its recurrences is currently based on the combination of CT scan, transvaginal ultrasound, and serum CA-125. However, the assessment of CA-125 in the serum seems to be highly specific but not as much sensitive, being negative in approximately 50% of early ovarian cancer cases at the time of diagnosis.17 Novel molecular markers are therefore required to improve early diagnosis and monitoring of ovarian cancer.Here, we report on the value of cell-FPDNA for the diagnosis and monitoring of response to therapy in advanced ovarian cancer patients. Our data show that the mean levels of FPDNA were significantly higher in ovarian cancer patients than in controls, with a sensitivity of 77% and a specificity of 96%. Although this finding needs to be confirmed in a larger series, it is in keeping with other reports and thus strengthens the promising diagnostic value of FPDNA testing in ovarian cancer.14–19A major limitation to FPDNA testing in human tumors is the detection of circulating cell-free DNA in nontumor conditions like autoimmune diseases, trauma, or surgery.18 We observed a strong increase of FPDNA levels in association with surgery in our series. Doubtless, the spike of FPDNA 24 hours after hystero-oophorectomy was observed in all the patients who underwent surgery with a progressive decrease in the blood samples taken 10 days and 1 month after the operation. Studies on many human cancers have successfully demonstrated the presence of tumor-specific mutations or epigenetic modifications in the FPDNA to better identify its neoplastic derivation. Unfortunately, there are very few reports of FPDNA characterization in ovarian cancer and no specific molecular markers for this tumor type have been identified so far.16–19 Nevertheless, we think that the surgery-related FPDNA increase detected in our series was sufficiently clear-cut and reproducible in all cases to warrant the reliability of the test.Our prospective study was designed to assess the variations of FPDNA according to therapy. We found a significant decrease in the mean levels of FPDNA in the blood samples taken either after the first and the sixth cycle of chemotherapy compared with the time zero values. Conversely, 4 nonresponder patients also showed FPDNA levels above the cutoff for the entire length of the neoadjuvant chemotherapy. These data confirm the neoplastic origin of the cell-free circulating DNA in these women. Comparison between serum CA-125 and FPDNA at time zero showed that the former is quite specific being ≥35 U/mL in all patients. By contrast, among the 12 patients who developed disease progression at the time of last available follow-up, 8 (67%) had FPDNA levels above the cutoff, whereas only 4 (33%) showed serum CA-125 ≥35 U/mL in the samples taken after the sixth cycle of chemotherapy (before surgery). We thus demonstrated that the FPDNA test is a candidate potential indicator of response to chemotherapy in advanced ovarian cancer.Although our series of patients is too limited to provide conclusive survival data, we found an interesting correlation between high FPDNA levels and a shorter PFS time. The Kaplan-Meier survival curves were significantly separated in 2 groups with a median follow-up of more than 13 months (Fig. 2). However, the present results require confirmation from prolonged follow-up data.The introduction of cell-free DNA testing in the routine clinical oncology practice of ovarian cancer awaits further confirmation in other series. In the meantime, we propose PCR quantification of FPDNA as a promising, noninvasive, and cost-effective test for the diagnosis and the monitoring of response to therapy in advanced ovarian cancer.REFERENCES1. Zambon P, La Rosa F. Gynecological cancers: cervix, corpus uteri, ovary. Epidemiol Prev. 2004;28(2 suppl):68–74.[Context Link][Medline Link]2. Berkenblit A, Cannistra SA. Advances in the management of epithelial ovarian cancer. J Reprod Med. 2005;50:426–438.[Context Link][Medline Link]3. Herzog TJ, Pothuri B. Ovarian cancer: a focus on management of recurrent disease. Nat Clin Pract Oncol. 2006;3:604–611.[Context Link]4. Bast RC Jr, Badgwell D, Lu Z, et al. New tumor markers: CA125 and beyond. Int J Gynecol Cancer. 2005;15(suppl 3):274–281.[Context Link][Full Text][CrossRef][Medline Link]5. Jacobs I, Stabile I, Bridges J, et al. Multimodal approach to screening for ovarian cancer. Lancet. 1988;6:268–271.[Context Link][CrossRef][Medline Link]6. Sidransky D. Circulating DNA. 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PFS Kaplan-Meier curves of ovarian cancer patients according to FPDNA levels above and below the cutoff (14.5 ng/mL) after the sixth cycle of chemotherapy (before surgery). PFS time was calculated from the day of the first chemotherapy cycle (time zero).Quantification of Free Plasma DNA Before and After Chemotherapy in Patients With Advanced Epithelial Ovarian CancerCapizzi Elisa MSc; Gabusi, Elena PhD; Grigioni, Antonia D'Errico MD; De Iaco, Pierandrea MD; Rosati, Marta MD; Zamagni, Claudio MD; Fiorentino, Michelangelo MD, PhDOriginal ArticlesOriginal Articles117p 34-38

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