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Prognostic importance of cell-free DNA in chemotherapy resistant ovarian cancer treated with bevacizumab
European Journal of Cancer (2014) 50, 2611– 2618
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Original Research
Prognostic importance of cell-free DNA in chemotherapy resistant ovarian cancer treated with bevacizumab Karina Dahl Steffensen a,d,⇑, Christine Vestergaard Madsen a,d, Rikke Fredslund Andersen b, Marianne Waldstrøm c, Parvin Adimi a, Anders Jakobsen a,d a
Department of Oncology, Vejle Hospital, Kabbeltoft 25, DK-7100 Vejle, Denmark Department of Clinical Biochemistry, Vejle Hospital, Kabbeltoft 25, DK-7100 Vejle, Denmark c Department of Pathology, Vejle Hospital, Kabbeltoft 25, DK-7100 Vejle, Denmark d Institute of Regional Health Research, University of Southern Denmark, Winsløwparken 19,3, DK-5000 Odense C, Denmark b
Received 9 April 2014; received in revised form 25 June 2014; accepted 30 June 2014 Available online 30 July 2014
KEYWORDS cfDNA Bevacizumab Ovarian cancer Prognosis Multiresistant
Abstract Aim: Treatment of multiresistant epithelial ovarian cancer (EOC) is palliative and patients who have become resistant after multiple lines of chemotherapy often have an unmet need for further and less toxic treatment. Anti-angiogenic therapy has attracted considerable attention in the treatment of EOC in combination with chemotherapy. However, only a minor subgroup will benefit from the treatment and there is an obvious need for new markers to select such patients. The purpose of this study was to investigate the effect of single-agent bevacizumab in multiresistant EOC and the importance of circulating cell-free DNA (cfDNA) in predicting treatment response. Methods: One hundred and forty-four patients with multi-resistant EOC were treated with single-agent bevacizumab 10 mg/kg every three weeks. Baseline plasma samples were analysed for levels of cfDNA by real-time polymerase chain reaction (PCR). Results: Eighteen percent responded to treatment according to CA125 and 5.6% had partial response by Response Evaluation Criteria in Solid Tumours (RECIST). Stable disease was seen in 53.5% and 48.6% of the patients by CA125 and RECIST, respectively. Median progression free survival (PFS) and overall survival (OS) were 4.2 and 6.7 months, respectively.
⇑ Corresponding author at: Department of Oncology, Vejle Hospital, Kabbeltoft 25, DK-7100 Vejle, Denmark. Tel.: +45 79406038/+45 21770727; fax: +45 79406907. E-mail addresses: Karina.Dahl.Steff[email protected] (K.D. Steffensen), [email protected] (C.V. Madsen), Rikke. [email protected] (R.F. Andersen), [email protected] (M. Waldstrøm), [email protected] (P. Adimi), Anders. [email protected] (A. Jakobsen).
http://dx.doi.org/10.1016/j.ejca.2014.06.022 0959-8049/Ó 2014 Elsevier Ltd. All rights reserved.
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Cell-free DNA was highly correlated to PFS (p = 0.0004) and OS (p = 0.005) in both univariate and multivariate analyses (PFS, hazard ratio (HR) = 1.98, p = 0.002; OS, HR = 1.66, p = 0.02), as patients with high cfDNA had a poor outcome. Conclusions: Single-agent bevacizumab treatment in multiresistant EOC appears to be a valuable treatment option with acceptable side-effects. Cell-free DNA showed independent prognostic importance in patients treated with bevacizumab and could be applied as an adjunct for treatment selection. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Internationally, there is broad consensus on first-line cytotoxic treatment of epithelial ovarian cancer and the vast majority of the patients are treated with platinum and taxane combination chemotherapy. Unfortunately, most patients with advanced ovarian cancer will relapse within a few years after diagnosis and since metastatic ovarian cancer after platinum containing first-line chemotherapy is a disease with a very poor prognosis, these patients are often treated with several lines of palliative chemotherapy. Platinum sensitive patients with late relapse are typically retreated with either single agent platinum or platinum containing combination chemotherapy. Patients with early relapse and platinum resistant disease are often treated with single agent pegylated liposomal doxorubicin or topotecan with relatively low response rates [1]. There are a number of other chemotherapeutic agents to offer to patients with recurrent ovarian cancer and common characteristics are poor response rates and short progression-free survival (PFS). The average ovarian cancer patient without major comorbidity will therefore often receive multiple treatment regimens in the course of therapy. Patients who have received multiple lines of chemotherapy and therefore have become multiresistant frequently have an unmet need for further treatment. The treatment of multiresistant ovarian cancer patients is palliative and quality of life is crucial in this setting. Treatment without haematological toxicity and neurological side-effects is thus preferred. A number of biological treatment regimens, including anti-angiogenic therapy, have the advantage of rare haematological toxicity and usually a completely different and less toxic sideeffect profile compared to conventional chemotherapy. Anti-angiogenic therapy has recently attracted considerable attention in the treatment of ovarian cancer with significant results in both the first line setting [2,3] and in the treatment of recurrence [4,5]. Bevacizumab treatment has shown improvement of PFS in first as well as in second line treatment but it seems that increased PFS (both hazard ratio (HR) and PFS duration) is more striking in treatment of relapse [6]. Bevacizumab has been approved in Europe for frontline treatment of epithelial ovarian, fallopian tube and peritoneal cancer when administered in addition to
carboplatin and paclitaxel for up to six treatment cycles followed by single agent bevacizumab until disease progression, unacceptable toxicity or for a maximum of 15 months. Based on the OCEANS study [5], bevacizumab has also been approved for treatment of recurrent platinumsensitive disease in combination with carboplatin and gemcitabine for six cycles and up to 10 cycles followed by single agent bevacizumab until disease progression. There is currently no approval for bevacizumab use in platinum-resistant recurrence although positive data from the AURELIA study [4] presented at ASCO 2012 showed prolonged PFS using bevacizumab in combination with different cytostatics (HR = 0.48). We therefore initiated the present study to offer palliative treatment with single agent bevacizumab as part of a translational biomarker protocol with the objective of identifying biomarkers for estimation of tumour burden and treatment outcome. Circulating cell-free DNA (cfDNA) is an emerging new biomarker. In cancer patients both wild-type (normal) and tumour derived DNA is released into the circulation. The main source of cfDNA is not yet fully clarified but thought to be related to necrosis and apoptosis of cancer cells or alternatively, secreted by different cells [7–9]. Cell-free DNA is probably a prognostic variable as indicated in several studies [10], but contradictory results have also been published and the area needs further research. The issue has only been sparsely investigated in ovarian cancer and to our knowledge no other studies have explored the value of cfDNA in patients treated with bevacizumab or any other antiangiogenic treatment. In addition to the clinical results the purpose of this study was to investigate whether baseline cfDNA in heavily pretreated platinum-resistant patients is correlated with prognosis and with treatment response to single agent bevacizumab. 2. Materials and methods 2.1. Patient population One hundred and forty-four patients with multiresistant ovarian cancer were treated with single agent
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bevacizumab 10 mg/kg every three weeks. The treatment continued until progression, intolerable side-effects or patient wish for discontinuation of therapy. They were all treated at the Department of Oncology, Vejle Hospital during the period from 18th July 2007 to 15th February 2013. At the time of inclusion a CT scan was performed to identify and measure target lesions according to the Response Evaluation Criteria in Solid Tumours (RECIST) criteria and to rule out tumour invasion into the bowel wall or major vessels, which were exclusion criteria due to concerns as to bowel perforation or bleeding. CT scans were also performed after every three cycles for response evaluation, and CA125 was measured at each cycle. Most patients were evaluable by both the RECIST and GCIG CA125 criteria. The clinical study was an integrated part of a biomarker protocol. The protocol was approved by the Regional Scientific Ethics Committee for Southern Denmark (S-20070070). Written informed consent was obtained from all patients and the Helsinki II Declaration was observed. Blood samples for translational analyses were collected at baseline and prior to each treatment cycle. Only the baseline samples were analysed for the present study. After half an hour the blood samples were centrifuged at 2000g for 10 min at room temperature and EDTA whole blood, EDTA plasma and serum were subsequently stored at 80 °C until use. 2.2. Methods 2.2.1. Cell-free DNA Cell-free DNA was purified from 1 ml of plasma using the QIAsymphony DSP Virus/Pathogen Kit on a QiaSymphony robot (Qiagen, Hilden, Germany). DNA was eluted in 110 ll elution buffer. The level of cfDNA was assessed by real-time polymerase chain reaction (PCR). The cyclophilin A gene was used as target as previously described [11] and Ct values were used for calculation of the number of DNA alleles per mL plasma. All DNA samples were tested for DNA from lymphocytes by a multiplex qPCR assay targeting the unique immunoglobulin DNA rearrangements in B-cells [12]. This was done to determine if pre-analytical factors resulting in contamination of the plasma with blood cell DNA had occurred. Blood cell lysis can occur during blood draw or because of prolonged storage prior to plasma isolation, and blood cells can accidentally be included in the plasma fraction by pipetting into the buffy coat. Samples with contaminating DNA from lymphocytes were excluded from further analysis. A pool of whole blood DNA from healthy subjects was used as positive control and water as negative control.
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2.3. Statistics Statistical analysis was performed with the Number Cruncher Statistical System (NCSS), version 2007, (Kaysville, UT, United States of America (USA)). Categorical variables were compared by means of the v2-test and the Mann–Whitney U test was used for comparing levels of cfDNA between the different patient characteristics. For association of cfDNA with PFS and overall survival (OS), cfDNA was dichotomised as low or high according to levels below or above the median. Progression free survival was calculated from the first day of bevacizumab treatment until progression or death from any cause. The date of progression was both reported based on RECIST alone and as a combination of RECIST and CA125 or clinical progression (whichever came first). Overall survival was calculated as the interval from bevacizumab initiation until death from any cause. Univariate overall survival analysis was performed using the Kaplan–Meier method and log-rank statistics were used for comparison of survival plots. Multivariate overall survival analysis was determined by the Cox regression model. The parameters entered in the Cox analysis were cfDNA (below and above the median), FIGO stage, performance status (PS), number of previous treatment regimens as categorical variables and age at diagnosis as a continuous variable. FIGO stage was divided into stage I/II versus stage III versus stage IV, performance status into PS 0 versus PS1 versus PS2+3 and previous treatment regimens into 62 versus >2 to avoid both entering groups with very low numbers and to avoid too many parameters entered into the Cox model according to the number of events if not merged into fewer variables. Grade and histology were not entered into the Cox model since these parameters were not significant in the univariate analysis. A value of p < 0.05 was considered statistically significant. 3. Results 3.1. Patient characteristics Table 1 shows the major patient characteristics. On average, the bevacizumab therapy was the fifth treatment regimen as all 144 patients were multiresistant to chemotherapy and had received a median of four prior (range 1–9) chemotherapy containing regimens. All patients were carboplatin resistant at the time of inclusion. The average age at the time of bevacizumab initiation was 62 years (range 27–79) and most patients were still in a good general condition (PS 0–1 = 85%). The median time span between initial diagnosis of ovarian cancer and study inclusion was 3.6 years, and between
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Table 1 Patient characteristics. Patient characteristics FIGO stage I II III IV
N = 144
%
13 12 91 28
9.0 8.3 63.2 19.4
123 9 12
85.4 6.3 8.3
13 44 59
11.2 37.9 50.9
Number of prior regimens 1 2 3 4 5 6 7 8 9
1 16 17 43 37 16 10 3 1
0.7 11.1 11.8 29.9 25.7 11.1 6.9 2.1 0.7
Performance status 0 1 2 3
63 61 17 3
43.8 42.4 11.8 2.1
Histology Serous Endometrioid Other Clear cell (1), mixed (3), carcinosarcoma (2), transition cellular (1), adenocarcinoma not further classified (2), borderline tumour with invasive implants (3) Tumour grade 1 2 3 Not graded: 28 (biopsy only, clear cell or carcinosarcoma)
study inclusion and last chemotherapy treatment was 1.8 months. 3.2. Treatment effect The median number of bevacizumab cycles was four (range 1–85, mean 7). At the time of analysis, seven patients were still receiving bevacizumab. Thirty-nine patients (27%) received at least nine cycles of treatment (>6 months), and 18 patients (9.7%) received bevacizumab for more than one year without significant toxicity (18 cycles). Three patients continued bevacizumab beyond progression verified by CA125 or RECIST for 11, 13.5 and 24.3 months, respectively, and one patient is currently still in treatment 24 months after progression without clinical deterioration. These four patients had continued major clinical effect, improved general condition or cessation of ascites production. One patient still on treatment has received 85 bevacizumab cycles and still has partial response according to RECIST. This patient had a ‘bevacizumab holiday’ for a period of 6 months after cycle 72 due to elective
surgery for mitral valve insufficiency. During this pause CA125 increased 10-fold, but decreased by 80% after resuming bevacizumab treatment. The CA125 response rate (RR) was 18% (26 out of 144 patients) according to the GCIG CA125 criteria. Seventy-seven (53.5%) patients had stable CA125 levels during treatment as their best response, 17 patients (11.8%) had CA125 progression and 24 patients did not have measurable disease by CA125. Partial response (PR) evaluated by RECIST was seen in 5.6% of the patients (eight out of 144 intention to treat patients) and 48.6% had stable disease (SD) as best response. Thirty-six patients (25%) had progressive disease and 30 patients (20.8%) were not evaluable by RECIST but included in the above response reporting. The same applied to the CA125 response analysis. For all included patients, the median PFS was 3.4 months (95% confidence interval (CI) 2.7–4.0) when assessed by either CA125, RECIST or clinical deterioration, whichever came first and 4.2 months (95% CI 3.5– 4.6) using the RECIST criteria only. The median OS was 6.7 months (95% CI 5.9–7.5) (Fig. 1). 3.3. Side-effects As expected, the most common side-effects were hypertension and proteinuria. Hypertension grade 2 (140–159/90–99 mmHg) was seen in 28 (19.4%) of the patients and 10 (6.9%) had grade 3 (160/100 mmHg). No patients stopped treatment due to uncontrollable hypertension. Proteinuria was measured by a urine dipstick test prior to each treatment and in 33 patients (22.9%) the dipstick showed 2+ and in one patient 3+. None of the patients presented >1 g protein/24 h during the following urine collection. The serious side-effects were; gastrointestinal perforation 4.2% (N = 6), fistula 3.5% (N = 5), venous thromboembolism 3.5% (N = 5), pulmonary embolism 2.1% (N = 3), cerebral thrombosis 0.7% (N = 1) and one patient (0.7%) experienced an episode of a transient ischaemic attack. There were no treatment-related deaths. 3.4. cfDNA and correlation to response, PFS and OS Baseline levels of cfDNA were available in 110 out of the 144 patients. Nine patients did not have a baseline plasma sample and 25 plasma samples showed contamination with DNA from leucocytes and were therefore excluded from further analysis. The median cfDNA level was 5150 alleles per mL plasma, range 1000–85,000. There was no correlation between levels of baseline CA125 and levels of baseline cfDNA (Spearman rank correlation coefficient, r = 0.14). Cell-free DNA levels were not correlated with FIGO stage, histology, tumour grade or number of previous
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Fig. 1. Kaplan–Meier estimates of progression free survival (PFS) and overall survival (OS). For PFS two survival plots are shown; left side based on Response Evaluation Criteria in Solid Tumours (RECIST) alone and right side based on CA125, RECIST or clinical deterioration, whichever came first.
treatment regimens. Higher levels of cfDNA were found in patients with PS 2 (median cfDNA, 9600 alleles per mL plasma) compared to patients with PS 0 (median cfDNA 4700 alleles per mL plasma), p = 0.006 and PS 1 (median cfDNA 5250 alleles per mL plasma), p = 0.02. It appears from Table 2 that cfDNA levels were correlated with CA125 response. Higher CA125 response rates were seen in patients with cfDNA below the median than in patients with high cfDNA levels. On the
other hand, there was no correlation between cfDNA and RECIST response. Fig. 2 illustrates that cfDNA was highly correlated to both PFS and OS, as patients with high cfDNA had a poor outcome with respect to PFS (p = 0.0004) as well as OS (p = 0.005). Patients with low cfDNA had a median PFS (CA125, RECIST or clinical) of 4.2 months (95% CI 3.0–4.8) compared to 2.9 months (95% CI 2.1–3.5) in patients with high levels. The same was true for the OS with a
Table 2 Correlation of baseline cell-free DNA (cfDNA) with Response Evaluation Criteria in Solid Tumours (RECIST) (A) and CA125 response (B). P = 0.55, v2
Partial response
Stable disease
Progression
Total
4 (8.5%) 2 (5.0%)
30 (63.8%) 23 (57.5%)
13 (27.7%) 15 (37.5%)
47 (100%) 40 (100%) 87b
CA125 response
CA125 stabilisation
Ca125 progression
Total
14 (28.6%) 3 (6.8%)
32 (65.3%) 33 (75.0%)
3 (6.1%) 8 (18.2%)
49 (100%) 44 (100%) 93c
A Low cfDNAa High cfDNA P = 0.010, v2 B Low cfDNAa High cfDNA a b c
Low: below median of 5150 alleles per mL plasma. 30 patients were not evaluable by CT and an additional 27 patients did not have a baseline plasma sample. 24 patients were not evaluable by CA125 and an additional 27 patients did not have a baseline plasma sample.
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Fig. 2. Kaplan–Meier survival plots for progression free survival (PFS) and overall survival (OS) according to cell-free DNA (cfDNA) below or above the median plasma level.
median OS of 8.1 months (95% CI 6.5–11.3) in patients with low cfDNA compared to 5.0 months (95% CI 3.9– 6.5) in high cfDNA patients. In addition to cfDNA, performance status and number of prior regimens were correlated with PFS and OS in the univariate analysis. FIGO was only correlated with OS and not PFS (data not shown). In particular, there was a significant difference in outcome between patients who received less and patients who received more than two prior treatment regimens. Patients with 6two prior regimens had a significantly worse prognosis (p = 0.0015 for PFS and p = 0.0004 for OS), possibly due to the fact that these patients became platinum resistance early in the course of treatment. When controlling for the effect of one or more variables in the multivariate analysis both the number of previous treatment regimens and poor performance status remained prognostic of outcome. Moreover, a high level of baseline cfDNA remained an independent prognostic factor of poor PFS and OS (HR = 1.98, p = 0.002) and (HR = 1.66, p = 0.02), respectively (Table 3). 4. Discussion Single-agent bevacizumab for multiresistant ovarian cancer patients appears to be a valuable treatment option with tolerable side-effects in line with other studies. The GOG170D study included 62 patients, who had received one or two previous regimens, to be treated with single-agent bevacizumab 15 mg/kg. A response rate of 21% was seen and 25 patients (40.3%) were progression free for at least six months. The median PFS and OS were 4.7 and 17 months, respectively [13].
Cannistra et al. [14] reported a phase 2 study of singleagent bevacizumab in platinum-resistant patients with up to three previous regimens, in which a response rate of 15.9% was seen. The median PFS and OS were 4.4 months and 10.7 months, respectively. In comparison our PFS was 4.2 months evaluated by the RECIST criteria. When evaluating PFS based on worst-case scenario, specified as the date with the first sign of progression according to CA125, RECIST or clinical progression, whichever came first, the PFS was 3.4 months. Overall survival in our study was somewhat shorter than in the two phase II studies, but a direct comparison is not possible since the patients presented here were heavily pretreated and also included patients with performance status 2. The GOG170D study only included patients who had received up to two prior lines of chemotherapy and 42% were still platinum-sensitive. In the study by Cannistra et al. the maximum number of prior regimens was three. It should be noted that also in our study, approximately 50% of the treated patients achieved stable disease as was the case in the abovementioned phase II studies. In patients who have progressed on multiple lines of chemotherapy, stable disease in a palliative setting is often clinically meaningful, especially when treated for a relatively long period as in our study with 27% of all included patients receiving at least nine cycles of bevacizumab (>6 months) and 18 patients (9.7%) receiving the treatment for more than one year without significant toxicity (18 cycles). This is considerably longer than the periods in which many patients have experienced stable disease during treatment with conventional chemotherapy. Anti-angiogenic therapy has shown several positive results in the treatment of ovarian cancer and there are now four positive randomised phase III trials
K.D. Steffensen et al. / European Journal of Cancer 50 (2014) 2611–2618 Table 3 Multivariate analysis. Progression free survival (PFS)
Hazard ratio (HR)
95% confidence interval (CI) HR
p
Age
1.01
0.99–1.03
0.46
FIGO stage I–II III IV
1.00 1.30 1.81
0.73–2.31 0.88–3.74
0.37 0.11
Number of prior regimens 62 1.00 >2 0.51
0.28–0.92
0.02
Performance status 0 1 2+3
1.00 0.95 1.94
0.61–1.48 1.06–3.52
0.88 0.03
Cell-free DNA (cfDNA) Low 1.00 High 1.98
1.29–3.04
0.002
Overall survival (OS)
HR
95% CI HR
p
Age
1.00
0.98–1.02
0.84
FIGO stage I–II III IV
1.00 1.32 2.10
0.74–2.35 1.02–4.35
0.34 0.04
Number of prior regimens 62 1.00 >2 0.43
0.24–0.79
0.006
Performance status 0 1 2+3
1.00 1.30 5.05
0.83–2.03 2.67–9.55
0.25 <10
cfDNA Low High
1.00 1.66
1.09–2.52
0.02
4
combining bevacizumab with chemotherapy in the treatment of both frontline (GOG 218 [3] and ICON7 [2]) and recurrent platinum-resistant (AURELIA [4]) or platinum-sensitive (OCEANS [5]) epithelial ovarian cancer. Furthermore, three randomised phase III results adding anti-angiogenic tyrosine kinase inhibitors to chemotherapy have recently been presented at meetings. They include results from two frontline studies; one study of maintenance pazopanib (AGO-OVAR16 [15]), which increased PFS by 5.6 months (HR 0.77) and a second study with frontline nintedanib (AGO-OVAR12/ LUME-Ovar1 [16]) in combination with carboplatin/ paclitaxel plus maintenance nintedanib showing an increased PFS of 0.7 months (HR 0.84). Furthermore, the ICON6 study [17] demonstrated positive results with an increase in PFS and OS of 2.0 months (HR 0.68) and 2.7 months (HR 0.70), respectively, adding cediranib (concurrent and maintenance) to platinum-based chemotherapy in recurrent, platinum-sensitive patients.
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Therefore, one of the most debated issues today is not whether to treat ovarian cancer patients with bevacizumab or other anti-angiogenic agents, but when. Another unresolved issue is whether treatment results will change with the increasing amount of patients having received anti-angiogenic therapy as part of frontline or relapse treatment. The question of response to continued or re-initiated anti-angiogenic therapy remains unsettled, but two ongoing studies, AGO-OVAR 2.21 (NCT01837251) and MITO-16/MaNGO OV-2 (NCT01802749) both examine bevacizumab beyond progression after first-line carboplatin/paclitaxel plus bevacizumab and hopefully, some answers will appear from these studies. To our knowledge this is the first study illustrating the prognostic value of cfDNA in patients treated with bevacizumab. We found that both PFS and OS were significantly shorter in patients with high levels of cfDNA in the blood (p = 0.004 and p = 0.0005, respectively), and this was also the case in the multivariate analysis (p = 0.02 and p = 0.0002). The literature on the clinical value of cfDNA in ovarian cancer is very scarce. There are a few studies [18–21] that illustrate the diagnostic value of cfDNA investigating the hypothesis that cfDNA is higher in the blood of patients with ovarian cancer than in patients with benign ovarian tumours and healthy controls. However, the results are divergent. The studies by Kamat et al. [18] and Zachariah et al. [19] showed higher levels of cfDNA in cancer patients, while the study by No et al. [21] could not demonstrate a statistical difference in the preoperative serum levels of cfDNA between patients with ovarian cancer and patients with benign tumours. The latter study, however, was able to demonstrate an independent prognostic factor for disease-free survival (p = 0.01) and overall survival (p = 0.02) in patients with lower gene expression levels of the RAS oncogene family (RAB25) in cfDNA. Of prognostic studies, Dobrzycka et al. (N = 55 samples analysed for cfDNA) showed significantly worse survival (p = 0.022) in cfDNA positive patients compared to cfDNA negative patients, although no independent multivariate analysis was presented [22]. Another study by Kamat et al. [23] reported that preoperative cell-free DNA was an independent predictor of disease-specific survival in both a training (p = 0.02) and a validation set (p = 0.01). Furthermore, preoperative plasma total cell-free DNA levels were significantly elevated in patients with epithelial ovarian cancer compared to benign ovarian tumours and healthy controls. Our study supports these data, as we found a significant and independent association between elevated cfDNA and outcome in multiresistant ovarian cancer patients treated with single-agent bevacizumab as a ‘last-line’ treatment attempt. The detection of cfDNA therefore provides prognostic value in this setting.
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Treatment of multiresistant ovarian cancer remains a considerable challenge. In many situations a patient may be better off with best supportive care and no active anti-cancer treatment, which is justified only if there is a reasonable chance of benefit. The results presented here suggest that cfDNA may serve as an adjunct in the selection of this treatment option. Conflict of interest statement None declared. Acknowledgements We would like to thank our colleagues in oncology departments all over Denmark for referring their patients to experimental treatment with bevacizumab at our cancer center and the patients for their participation and contribution of blood samples. We are also grateful to Karin Larsen for her careful proofreading. The study was supported in part by the Department of Clinical Oncology, Vejle Hospital and The Cancer Foundation. References [1] Gordon AN, Fleagle JT, Guthrie D, Parkin DE, Gore ME, Lacave AJ. Recurrent epithelial ovarian carcinoma: a randomized phase III study of pegylated liposomal doxorubicin versus topotecan. J Clin Oncol 2001;19(14):3312–22. [2] Perren TJ, Swart AM, Pfisterer J, Ledermann JA, PujadeLauraine E, Kristensen G, et al. A phase 3 trial of bevacizumab in ovarian cancer. N Engl J Med 2011;365(26):2484–96. [3] Burger RA, Brady MF, Bookman MA, Fleming GF, Monk BJ, Huang H, et al. Incorporation of bevacizumab in the primary treatment of ovarian cancer. N Engl J Med 2011;365(26):2473–83. [4] Pujade-Lauraine E, Hilpert F, Weber B, Reuss A, Poveda A, Kristensen G, et al. AURELIA: a randomized phase III trial evaluating bevacizumab (BEV) plus chemotherapy (CT) for platinum (PT)-resistant recurrent ovarian cancer (OC). J Clin Oncol 2012;30(18 Suppl., Gynecol Oncol Section) [abstr. LBA5002; Ref Type: Abstract]. [5] Aghajanian C, Blank SV, Goff BA, Judson PL, Teneriello MG, Husain A, et al. OCEANS: a randomized, double-blind, placebocontrolled phase III trial of chemotherapy with or without bevacizumab in patients with platinum-sensitive recurrent epithelial ovarian, primary peritoneal, or fallopian tube cancer. J Clin Oncol 2012;30(17):2039–45. [6] Monk BJ, Pujade-Lauraine E, Burger RA. Integrating bevacizumab into the management of epithelial ovarian cancer: the controversy of front-line versus recurrent disease. Ann Oncol 2013;24(Suppl. 10):x53–8. [7] Gonzalez-Masia JA, Garcia-Olmo D, Garcia-Olmo DC. Circulating nucleic acids in plasma and serum (CNAPS): applications in oncology. Onco Targets Ther 2013;6:819–32. [8] Stroun M, Maurice P, Vasioukhin V, Lyautey J, Lederrey C, Lefort F, et al. The origin and mechanism of circulating DNA. Ann N Y Acad Sci 2000;906:161–8.
[9] Schwarzenbach H, Hoon DS, Pantel K. Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer 2011;11(6):426–37. [10] Marzese DM, Hirose H, Hoon DS. Diagnostic and prognostic value of circulating tumor-related DNA in cancer patients. Expert Rev Mol Diagn 2013;13(8):827–44. [11] Spindler KL, Pallisgaard N, Vogelius I, Jakobsen A. Quantitative cell-free DNA, KRAS, and BRAF mutations in plasma from patients with metastatic colorectal cancer during treatment with cetuximab and irinotecan. Clin Cancer Res 2012;18(4):1177–85. [12] Pallisgaard N, Andersen RF, Spindler KL, Brandslund I, Jakobsen A. Controls for quantification of total cell free DNA qPCR analyses in plasma. Eur J Cancer 2013;49(Suppl. 4):S36 [abstr. MC13-0088; Ref Type: Abstract]. [13] Burger RA, Sill MW, Monk BJ, Greer BE, Sorosky JI. Phase II trial of bevacizumab in persistent or recurrent epithelial ovarian cancer or primary peritoneal cancer: a Gynecologic Oncology Group Study. J Clin Oncol 2007;25(33):5165–71. [14] Cannistra SA, Matulonis UA, Penson RT, Hambleton J, Dupont J, Mackey H, et al. Phase II study of bevacizumab in patients with platinum-resistant ovarian cancer or peritoneal serous cancer. J Clin Oncol 2007;25(33):5180–6. [15] Du Bois A, Floquet A, Kim JW, Rau J, del Campo JM, Friedlander M, et al. Randomized, double-blind, phase III trial of pazopanib versus placebo in women who have not progressed after first-line chemotherapy for advanced epithelial ovarian, fallopian tube, or primary peritoneal cancer (AEOC): Results of an international Intergroup trial (AGO-OVAR16). Clin Oncol 2013;31(Suppl.) [abstr. LBA5503; Ref Type: Abstract]. [16] Du Bois A, Kristensen G, Ray-Coquard I, Reuss A, Pignata S, Colombo N, et al. AGO-OVAR 12: A randomized placebocontrolled GCIG/ENGOT intergroup phase III trial of standard frontline chemotherapy +/ nintedanib for advanced ovarian cancer. Int J Gynecol Cancer 2013;23(8 Suppl. 1) [Ref Type: Abstract]. [17] Ledermann J, Perren TJ, Raja FA, Embleton A, Rustin GJ, Jayson G, et al. Randomised double-blind phase III trial of cediranib (AZD 2171) in relapsed platinum sensitive ovarian cancer: Results of the ICON6 trial. European Cancer Congress 2013 Late Breaking Abstract #10. 2013. Ref Type: Abstract. [18] Kamat AA, Sood AK, Dang D, Gershenson DM, Simpson JL, Bischoff FZ. Quantification of total plasma cell-free DNA in ovarian cancer using real-time PCR. Ann N Y Acad Sci 2006;1075:230–4. [19] Zachariah RR, Schmid S, Buerki N, Radpour R, Holzgreve W, Zhong X. Levels of circulating cell-free nuclear and mitochondrial DNA in benign and malignant ovarian tumors. Obstet Gynecol 2008;112(4):843–50. [20] Liggett TE, Melnikov A, Yi Q, Replogle C, Hu W, Rotmensch J, et al. Distinctive DNA methylation patterns of cell-free plasma DNA in women with malignant ovarian tumors. Gynecol Oncol 2011;120(1):113–20. [21] No JH, Kim K, Park KH, Kim YB. Cell-free DNA level as a prognostic biomarker for epithelial ovarian cancer. Anticancer Res 2012;32(8):3467–71. [22] Dobrzycka B, Terlikowski SJ, Kinalski M, Kowalczuk O, Niklinska W, Chyczewski L. Circulating free DNA and p53 antibodies in plasma of patients with ovarian epithelial cancers. Ann Oncol 2011;22(5):1133–40. [23] Kamat AA, Baldwin M, Urbauer D, Dang D, Han LY, Godwin A, et al. Plasma cell-free DNA in ovarian cancer: an independent prognostic biomarker. Cancer 2010;116(8):1918–25.
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Original Research
Prognostic importance of cell-free DNA in chemotherapy resistant ovarian cancer treated with bevacizumab Karina Dahl Steffensen a,d,⇑, Christine Vestergaard Madsen a,d, Rikke Fredslund Andersen b, Marianne Waldstrøm c, Parvin Adimi a, Anders Jakobsen a,d a
Department of Oncology, Vejle Hospital, Kabbeltoft 25, DK-7100 Vejle, Denmark Department of Clinical Biochemistry, Vejle Hospital, Kabbeltoft 25, DK-7100 Vejle, Denmark c Department of Pathology, Vejle Hospital, Kabbeltoft 25, DK-7100 Vejle, Denmark d Institute of Regional Health Research, University of Southern Denmark, Winsløwparken 19,3, DK-5000 Odense C, Denmark b
Received 9 April 2014; received in revised form 25 June 2014; accepted 30 June 2014 Available online 30 July 2014
KEYWORDS cfDNA Bevacizumab Ovarian cancer Prognosis Multiresistant
Abstract Aim: Treatment of multiresistant epithelial ovarian cancer (EOC) is palliative and patients who have become resistant after multiple lines of chemotherapy often have an unmet need for further and less toxic treatment. Anti-angiogenic therapy has attracted considerable attention in the treatment of EOC in combination with chemotherapy. However, only a minor subgroup will benefit from the treatment and there is an obvious need for new markers to select such patients. The purpose of this study was to investigate the effect of single-agent bevacizumab in multiresistant EOC and the importance of circulating cell-free DNA (cfDNA) in predicting treatment response. Methods: One hundred and forty-four patients with multi-resistant EOC were treated with single-agent bevacizumab 10 mg/kg every three weeks. Baseline plasma samples were analysed for levels of cfDNA by real-time polymerase chain reaction (PCR). Results: Eighteen percent responded to treatment according to CA125 and 5.6% had partial response by Response Evaluation Criteria in Solid Tumours (RECIST). Stable disease was seen in 53.5% and 48.6% of the patients by CA125 and RECIST, respectively. Median progression free survival (PFS) and overall survival (OS) were 4.2 and 6.7 months, respectively.
⇑ Corresponding author at: Department of Oncology, Vejle Hospital, Kabbeltoft 25, DK-7100 Vejle, Denmark. Tel.: +45 79406038/+45 21770727; fax: +45 79406907. E-mail addresses: Karina.Dahl.Steff[email protected] (K.D. Steffensen), [email protected] (C.V. Madsen), Rikke. [email protected] (R.F. Andersen), [email protected] (M. Waldstrøm), [email protected] (P. Adimi), Anders. [email protected] (A. Jakobsen).
http://dx.doi.org/10.1016/j.ejca.2014.06.022 0959-8049/Ó 2014 Elsevier Ltd. All rights reserved.
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Cell-free DNA was highly correlated to PFS (p = 0.0004) and OS (p = 0.005) in both univariate and multivariate analyses (PFS, hazard ratio (HR) = 1.98, p = 0.002; OS, HR = 1.66, p = 0.02), as patients with high cfDNA had a poor outcome. Conclusions: Single-agent bevacizumab treatment in multiresistant EOC appears to be a valuable treatment option with acceptable side-effects. Cell-free DNA showed independent prognostic importance in patients treated with bevacizumab and could be applied as an adjunct for treatment selection. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Internationally, there is broad consensus on first-line cytotoxic treatment of epithelial ovarian cancer and the vast majority of the patients are treated with platinum and taxane combination chemotherapy. Unfortunately, most patients with advanced ovarian cancer will relapse within a few years after diagnosis and since metastatic ovarian cancer after platinum containing first-line chemotherapy is a disease with a very poor prognosis, these patients are often treated with several lines of palliative chemotherapy. Platinum sensitive patients with late relapse are typically retreated with either single agent platinum or platinum containing combination chemotherapy. Patients with early relapse and platinum resistant disease are often treated with single agent pegylated liposomal doxorubicin or topotecan with relatively low response rates [1]. There are a number of other chemotherapeutic agents to offer to patients with recurrent ovarian cancer and common characteristics are poor response rates and short progression-free survival (PFS). The average ovarian cancer patient without major comorbidity will therefore often receive multiple treatment regimens in the course of therapy. Patients who have received multiple lines of chemotherapy and therefore have become multiresistant frequently have an unmet need for further treatment. The treatment of multiresistant ovarian cancer patients is palliative and quality of life is crucial in this setting. Treatment without haematological toxicity and neurological side-effects is thus preferred. A number of biological treatment regimens, including anti-angiogenic therapy, have the advantage of rare haematological toxicity and usually a completely different and less toxic sideeffect profile compared to conventional chemotherapy. Anti-angiogenic therapy has recently attracted considerable attention in the treatment of ovarian cancer with significant results in both the first line setting [2,3] and in the treatment of recurrence [4,5]. Bevacizumab treatment has shown improvement of PFS in first as well as in second line treatment but it seems that increased PFS (both hazard ratio (HR) and PFS duration) is more striking in treatment of relapse [6]. Bevacizumab has been approved in Europe for frontline treatment of epithelial ovarian, fallopian tube and peritoneal cancer when administered in addition to
carboplatin and paclitaxel for up to six treatment cycles followed by single agent bevacizumab until disease progression, unacceptable toxicity or for a maximum of 15 months. Based on the OCEANS study [5], bevacizumab has also been approved for treatment of recurrent platinumsensitive disease in combination with carboplatin and gemcitabine for six cycles and up to 10 cycles followed by single agent bevacizumab until disease progression. There is currently no approval for bevacizumab use in platinum-resistant recurrence although positive data from the AURELIA study [4] presented at ASCO 2012 showed prolonged PFS using bevacizumab in combination with different cytostatics (HR = 0.48). We therefore initiated the present study to offer palliative treatment with single agent bevacizumab as part of a translational biomarker protocol with the objective of identifying biomarkers for estimation of tumour burden and treatment outcome. Circulating cell-free DNA (cfDNA) is an emerging new biomarker. In cancer patients both wild-type (normal) and tumour derived DNA is released into the circulation. The main source of cfDNA is not yet fully clarified but thought to be related to necrosis and apoptosis of cancer cells or alternatively, secreted by different cells [7–9]. Cell-free DNA is probably a prognostic variable as indicated in several studies [10], but contradictory results have also been published and the area needs further research. The issue has only been sparsely investigated in ovarian cancer and to our knowledge no other studies have explored the value of cfDNA in patients treated with bevacizumab or any other antiangiogenic treatment. In addition to the clinical results the purpose of this study was to investigate whether baseline cfDNA in heavily pretreated platinum-resistant patients is correlated with prognosis and with treatment response to single agent bevacizumab. 2. Materials and methods 2.1. Patient population One hundred and forty-four patients with multiresistant ovarian cancer were treated with single agent
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bevacizumab 10 mg/kg every three weeks. The treatment continued until progression, intolerable side-effects or patient wish for discontinuation of therapy. They were all treated at the Department of Oncology, Vejle Hospital during the period from 18th July 2007 to 15th February 2013. At the time of inclusion a CT scan was performed to identify and measure target lesions according to the Response Evaluation Criteria in Solid Tumours (RECIST) criteria and to rule out tumour invasion into the bowel wall or major vessels, which were exclusion criteria due to concerns as to bowel perforation or bleeding. CT scans were also performed after every three cycles for response evaluation, and CA125 was measured at each cycle. Most patients were evaluable by both the RECIST and GCIG CA125 criteria. The clinical study was an integrated part of a biomarker protocol. The protocol was approved by the Regional Scientific Ethics Committee for Southern Denmark (S-20070070). Written informed consent was obtained from all patients and the Helsinki II Declaration was observed. Blood samples for translational analyses were collected at baseline and prior to each treatment cycle. Only the baseline samples were analysed for the present study. After half an hour the blood samples were centrifuged at 2000g for 10 min at room temperature and EDTA whole blood, EDTA plasma and serum were subsequently stored at 80 °C until use. 2.2. Methods 2.2.1. Cell-free DNA Cell-free DNA was purified from 1 ml of plasma using the QIAsymphony DSP Virus/Pathogen Kit on a QiaSymphony robot (Qiagen, Hilden, Germany). DNA was eluted in 110 ll elution buffer. The level of cfDNA was assessed by real-time polymerase chain reaction (PCR). The cyclophilin A gene was used as target as previously described [11] and Ct values were used for calculation of the number of DNA alleles per mL plasma. All DNA samples were tested for DNA from lymphocytes by a multiplex qPCR assay targeting the unique immunoglobulin DNA rearrangements in B-cells [12]. This was done to determine if pre-analytical factors resulting in contamination of the plasma with blood cell DNA had occurred. Blood cell lysis can occur during blood draw or because of prolonged storage prior to plasma isolation, and blood cells can accidentally be included in the plasma fraction by pipetting into the buffy coat. Samples with contaminating DNA from lymphocytes were excluded from further analysis. A pool of whole blood DNA from healthy subjects was used as positive control and water as negative control.
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2.3. Statistics Statistical analysis was performed with the Number Cruncher Statistical System (NCSS), version 2007, (Kaysville, UT, United States of America (USA)). Categorical variables were compared by means of the v2-test and the Mann–Whitney U test was used for comparing levels of cfDNA between the different patient characteristics. For association of cfDNA with PFS and overall survival (OS), cfDNA was dichotomised as low or high according to levels below or above the median. Progression free survival was calculated from the first day of bevacizumab treatment until progression or death from any cause. The date of progression was both reported based on RECIST alone and as a combination of RECIST and CA125 or clinical progression (whichever came first). Overall survival was calculated as the interval from bevacizumab initiation until death from any cause. Univariate overall survival analysis was performed using the Kaplan–Meier method and log-rank statistics were used for comparison of survival plots. Multivariate overall survival analysis was determined by the Cox regression model. The parameters entered in the Cox analysis were cfDNA (below and above the median), FIGO stage, performance status (PS), number of previous treatment regimens as categorical variables and age at diagnosis as a continuous variable. FIGO stage was divided into stage I/II versus stage III versus stage IV, performance status into PS 0 versus PS1 versus PS2+3 and previous treatment regimens into 62 versus >2 to avoid both entering groups with very low numbers and to avoid too many parameters entered into the Cox model according to the number of events if not merged into fewer variables. Grade and histology were not entered into the Cox model since these parameters were not significant in the univariate analysis. A value of p < 0.05 was considered statistically significant. 3. Results 3.1. Patient characteristics Table 1 shows the major patient characteristics. On average, the bevacizumab therapy was the fifth treatment regimen as all 144 patients were multiresistant to chemotherapy and had received a median of four prior (range 1–9) chemotherapy containing regimens. All patients were carboplatin resistant at the time of inclusion. The average age at the time of bevacizumab initiation was 62 years (range 27–79) and most patients were still in a good general condition (PS 0–1 = 85%). The median time span between initial diagnosis of ovarian cancer and study inclusion was 3.6 years, and between
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Table 1 Patient characteristics. Patient characteristics FIGO stage I II III IV
N = 144
%
13 12 91 28
9.0 8.3 63.2 19.4
123 9 12
85.4 6.3 8.3
13 44 59
11.2 37.9 50.9
Number of prior regimens 1 2 3 4 5 6 7 8 9
1 16 17 43 37 16 10 3 1
0.7 11.1 11.8 29.9 25.7 11.1 6.9 2.1 0.7
Performance status 0 1 2 3
63 61 17 3
43.8 42.4 11.8 2.1
Histology Serous Endometrioid Other Clear cell (1), mixed (3), carcinosarcoma (2), transition cellular (1), adenocarcinoma not further classified (2), borderline tumour with invasive implants (3) Tumour grade 1 2 3 Not graded: 28 (biopsy only, clear cell or carcinosarcoma)
study inclusion and last chemotherapy treatment was 1.8 months. 3.2. Treatment effect The median number of bevacizumab cycles was four (range 1–85, mean 7). At the time of analysis, seven patients were still receiving bevacizumab. Thirty-nine patients (27%) received at least nine cycles of treatment (>6 months), and 18 patients (9.7%) received bevacizumab for more than one year without significant toxicity (18 cycles). Three patients continued bevacizumab beyond progression verified by CA125 or RECIST for 11, 13.5 and 24.3 months, respectively, and one patient is currently still in treatment 24 months after progression without clinical deterioration. These four patients had continued major clinical effect, improved general condition or cessation of ascites production. One patient still on treatment has received 85 bevacizumab cycles and still has partial response according to RECIST. This patient had a ‘bevacizumab holiday’ for a period of 6 months after cycle 72 due to elective
surgery for mitral valve insufficiency. During this pause CA125 increased 10-fold, but decreased by 80% after resuming bevacizumab treatment. The CA125 response rate (RR) was 18% (26 out of 144 patients) according to the GCIG CA125 criteria. Seventy-seven (53.5%) patients had stable CA125 levels during treatment as their best response, 17 patients (11.8%) had CA125 progression and 24 patients did not have measurable disease by CA125. Partial response (PR) evaluated by RECIST was seen in 5.6% of the patients (eight out of 144 intention to treat patients) and 48.6% had stable disease (SD) as best response. Thirty-six patients (25%) had progressive disease and 30 patients (20.8%) were not evaluable by RECIST but included in the above response reporting. The same applied to the CA125 response analysis. For all included patients, the median PFS was 3.4 months (95% confidence interval (CI) 2.7–4.0) when assessed by either CA125, RECIST or clinical deterioration, whichever came first and 4.2 months (95% CI 3.5– 4.6) using the RECIST criteria only. The median OS was 6.7 months (95% CI 5.9–7.5) (Fig. 1). 3.3. Side-effects As expected, the most common side-effects were hypertension and proteinuria. Hypertension grade 2 (140–159/90–99 mmHg) was seen in 28 (19.4%) of the patients and 10 (6.9%) had grade 3 (160/100 mmHg). No patients stopped treatment due to uncontrollable hypertension. Proteinuria was measured by a urine dipstick test prior to each treatment and in 33 patients (22.9%) the dipstick showed 2+ and in one patient 3+. None of the patients presented >1 g protein/24 h during the following urine collection. The serious side-effects were; gastrointestinal perforation 4.2% (N = 6), fistula 3.5% (N = 5), venous thromboembolism 3.5% (N = 5), pulmonary embolism 2.1% (N = 3), cerebral thrombosis 0.7% (N = 1) and one patient (0.7%) experienced an episode of a transient ischaemic attack. There were no treatment-related deaths. 3.4. cfDNA and correlation to response, PFS and OS Baseline levels of cfDNA were available in 110 out of the 144 patients. Nine patients did not have a baseline plasma sample and 25 plasma samples showed contamination with DNA from leucocytes and were therefore excluded from further analysis. The median cfDNA level was 5150 alleles per mL plasma, range 1000–85,000. There was no correlation between levels of baseline CA125 and levels of baseline cfDNA (Spearman rank correlation coefficient, r = 0.14). Cell-free DNA levels were not correlated with FIGO stage, histology, tumour grade or number of previous
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Fig. 1. Kaplan–Meier estimates of progression free survival (PFS) and overall survival (OS). For PFS two survival plots are shown; left side based on Response Evaluation Criteria in Solid Tumours (RECIST) alone and right side based on CA125, RECIST or clinical deterioration, whichever came first.
treatment regimens. Higher levels of cfDNA were found in patients with PS 2 (median cfDNA, 9600 alleles per mL plasma) compared to patients with PS 0 (median cfDNA 4700 alleles per mL plasma), p = 0.006 and PS 1 (median cfDNA 5250 alleles per mL plasma), p = 0.02. It appears from Table 2 that cfDNA levels were correlated with CA125 response. Higher CA125 response rates were seen in patients with cfDNA below the median than in patients with high cfDNA levels. On the
other hand, there was no correlation between cfDNA and RECIST response. Fig. 2 illustrates that cfDNA was highly correlated to both PFS and OS, as patients with high cfDNA had a poor outcome with respect to PFS (p = 0.0004) as well as OS (p = 0.005). Patients with low cfDNA had a median PFS (CA125, RECIST or clinical) of 4.2 months (95% CI 3.0–4.8) compared to 2.9 months (95% CI 2.1–3.5) in patients with high levels. The same was true for the OS with a
Table 2 Correlation of baseline cell-free DNA (cfDNA) with Response Evaluation Criteria in Solid Tumours (RECIST) (A) and CA125 response (B). P = 0.55, v2
Partial response
Stable disease
Progression
Total
4 (8.5%) 2 (5.0%)
30 (63.8%) 23 (57.5%)
13 (27.7%) 15 (37.5%)
47 (100%) 40 (100%) 87b
CA125 response
CA125 stabilisation
Ca125 progression
Total
14 (28.6%) 3 (6.8%)
32 (65.3%) 33 (75.0%)
3 (6.1%) 8 (18.2%)
49 (100%) 44 (100%) 93c
A Low cfDNAa High cfDNA P = 0.010, v2 B Low cfDNAa High cfDNA a b c
Low: below median of 5150 alleles per mL plasma. 30 patients were not evaluable by CT and an additional 27 patients did not have a baseline plasma sample. 24 patients were not evaluable by CA125 and an additional 27 patients did not have a baseline plasma sample.
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Fig. 2. Kaplan–Meier survival plots for progression free survival (PFS) and overall survival (OS) according to cell-free DNA (cfDNA) below or above the median plasma level.
median OS of 8.1 months (95% CI 6.5–11.3) in patients with low cfDNA compared to 5.0 months (95% CI 3.9– 6.5) in high cfDNA patients. In addition to cfDNA, performance status and number of prior regimens were correlated with PFS and OS in the univariate analysis. FIGO was only correlated with OS and not PFS (data not shown). In particular, there was a significant difference in outcome between patients who received less and patients who received more than two prior treatment regimens. Patients with 6two prior regimens had a significantly worse prognosis (p = 0.0015 for PFS and p = 0.0004 for OS), possibly due to the fact that these patients became platinum resistance early in the course of treatment. When controlling for the effect of one or more variables in the multivariate analysis both the number of previous treatment regimens and poor performance status remained prognostic of outcome. Moreover, a high level of baseline cfDNA remained an independent prognostic factor of poor PFS and OS (HR = 1.98, p = 0.002) and (HR = 1.66, p = 0.02), respectively (Table 3). 4. Discussion Single-agent bevacizumab for multiresistant ovarian cancer patients appears to be a valuable treatment option with tolerable side-effects in line with other studies. The GOG170D study included 62 patients, who had received one or two previous regimens, to be treated with single-agent bevacizumab 15 mg/kg. A response rate of 21% was seen and 25 patients (40.3%) were progression free for at least six months. The median PFS and OS were 4.7 and 17 months, respectively [13].
Cannistra et al. [14] reported a phase 2 study of singleagent bevacizumab in platinum-resistant patients with up to three previous regimens, in which a response rate of 15.9% was seen. The median PFS and OS were 4.4 months and 10.7 months, respectively. In comparison our PFS was 4.2 months evaluated by the RECIST criteria. When evaluating PFS based on worst-case scenario, specified as the date with the first sign of progression according to CA125, RECIST or clinical progression, whichever came first, the PFS was 3.4 months. Overall survival in our study was somewhat shorter than in the two phase II studies, but a direct comparison is not possible since the patients presented here were heavily pretreated and also included patients with performance status 2. The GOG170D study only included patients who had received up to two prior lines of chemotherapy and 42% were still platinum-sensitive. In the study by Cannistra et al. the maximum number of prior regimens was three. It should be noted that also in our study, approximately 50% of the treated patients achieved stable disease as was the case in the abovementioned phase II studies. In patients who have progressed on multiple lines of chemotherapy, stable disease in a palliative setting is often clinically meaningful, especially when treated for a relatively long period as in our study with 27% of all included patients receiving at least nine cycles of bevacizumab (>6 months) and 18 patients (9.7%) receiving the treatment for more than one year without significant toxicity (18 cycles). This is considerably longer than the periods in which many patients have experienced stable disease during treatment with conventional chemotherapy. Anti-angiogenic therapy has shown several positive results in the treatment of ovarian cancer and there are now four positive randomised phase III trials
K.D. Steffensen et al. / European Journal of Cancer 50 (2014) 2611–2618 Table 3 Multivariate analysis. Progression free survival (PFS)
Hazard ratio (HR)
95% confidence interval (CI) HR
p
Age
1.01
0.99–1.03
0.46
FIGO stage I–II III IV
1.00 1.30 1.81
0.73–2.31 0.88–3.74
0.37 0.11
Number of prior regimens 62 1.00 >2 0.51
0.28–0.92
0.02
Performance status 0 1 2+3
1.00 0.95 1.94
0.61–1.48 1.06–3.52
0.88 0.03
Cell-free DNA (cfDNA) Low 1.00 High 1.98
1.29–3.04
0.002
Overall survival (OS)
HR
95% CI HR
p
Age
1.00
0.98–1.02
0.84
FIGO stage I–II III IV
1.00 1.32 2.10
0.74–2.35 1.02–4.35
0.34 0.04
Number of prior regimens 62 1.00 >2 0.43
0.24–0.79
0.006
Performance status 0 1 2+3
1.00 1.30 5.05
0.83–2.03 2.67–9.55
0.25 <10
cfDNA Low High
1.00 1.66
1.09–2.52
0.02
4
combining bevacizumab with chemotherapy in the treatment of both frontline (GOG 218 [3] and ICON7 [2]) and recurrent platinum-resistant (AURELIA [4]) or platinum-sensitive (OCEANS [5]) epithelial ovarian cancer. Furthermore, three randomised phase III results adding anti-angiogenic tyrosine kinase inhibitors to chemotherapy have recently been presented at meetings. They include results from two frontline studies; one study of maintenance pazopanib (AGO-OVAR16 [15]), which increased PFS by 5.6 months (HR 0.77) and a second study with frontline nintedanib (AGO-OVAR12/ LUME-Ovar1 [16]) in combination with carboplatin/ paclitaxel plus maintenance nintedanib showing an increased PFS of 0.7 months (HR 0.84). Furthermore, the ICON6 study [17] demonstrated positive results with an increase in PFS and OS of 2.0 months (HR 0.68) and 2.7 months (HR 0.70), respectively, adding cediranib (concurrent and maintenance) to platinum-based chemotherapy in recurrent, platinum-sensitive patients.
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Therefore, one of the most debated issues today is not whether to treat ovarian cancer patients with bevacizumab or other anti-angiogenic agents, but when. Another unresolved issue is whether treatment results will change with the increasing amount of patients having received anti-angiogenic therapy as part of frontline or relapse treatment. The question of response to continued or re-initiated anti-angiogenic therapy remains unsettled, but two ongoing studies, AGO-OVAR 2.21 (NCT01837251) and MITO-16/MaNGO OV-2 (NCT01802749) both examine bevacizumab beyond progression after first-line carboplatin/paclitaxel plus bevacizumab and hopefully, some answers will appear from these studies. To our knowledge this is the first study illustrating the prognostic value of cfDNA in patients treated with bevacizumab. We found that both PFS and OS were significantly shorter in patients with high levels of cfDNA in the blood (p = 0.004 and p = 0.0005, respectively), and this was also the case in the multivariate analysis (p = 0.02 and p = 0.0002). The literature on the clinical value of cfDNA in ovarian cancer is very scarce. There are a few studies [18–21] that illustrate the diagnostic value of cfDNA investigating the hypothesis that cfDNA is higher in the blood of patients with ovarian cancer than in patients with benign ovarian tumours and healthy controls. However, the results are divergent. The studies by Kamat et al. [18] and Zachariah et al. [19] showed higher levels of cfDNA in cancer patients, while the study by No et al. [21] could not demonstrate a statistical difference in the preoperative serum levels of cfDNA between patients with ovarian cancer and patients with benign tumours. The latter study, however, was able to demonstrate an independent prognostic factor for disease-free survival (p = 0.01) and overall survival (p = 0.02) in patients with lower gene expression levels of the RAS oncogene family (RAB25) in cfDNA. Of prognostic studies, Dobrzycka et al. (N = 55 samples analysed for cfDNA) showed significantly worse survival (p = 0.022) in cfDNA positive patients compared to cfDNA negative patients, although no independent multivariate analysis was presented [22]. Another study by Kamat et al. [23] reported that preoperative cell-free DNA was an independent predictor of disease-specific survival in both a training (p = 0.02) and a validation set (p = 0.01). Furthermore, preoperative plasma total cell-free DNA levels were significantly elevated in patients with epithelial ovarian cancer compared to benign ovarian tumours and healthy controls. Our study supports these data, as we found a significant and independent association between elevated cfDNA and outcome in multiresistant ovarian cancer patients treated with single-agent bevacizumab as a ‘last-line’ treatment attempt. The detection of cfDNA therefore provides prognostic value in this setting.
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Treatment of multiresistant ovarian cancer remains a considerable challenge. In many situations a patient may be better off with best supportive care and no active anti-cancer treatment, which is justified only if there is a reasonable chance of benefit. The results presented here suggest that cfDNA may serve as an adjunct in the selection of this treatment option. Conflict of interest statement None declared. Acknowledgements We would like to thank our colleagues in oncology departments all over Denmark for referring their patients to experimental treatment with bevacizumab at our cancer center and the patients for their participation and contribution of blood samples. We are also grateful to Karin Larsen for her careful proofreading. The study was supported in part by the Department of Clinical Oncology, Vejle Hospital and The Cancer Foundation. References [1] Gordon AN, Fleagle JT, Guthrie D, Parkin DE, Gore ME, Lacave AJ. Recurrent epithelial ovarian carcinoma: a randomized phase III study of pegylated liposomal doxorubicin versus topotecan. J Clin Oncol 2001;19(14):3312–22. [2] Perren TJ, Swart AM, Pfisterer J, Ledermann JA, PujadeLauraine E, Kristensen G, et al. A phase 3 trial of bevacizumab in ovarian cancer. N Engl J Med 2011;365(26):2484–96. [3] Burger RA, Brady MF, Bookman MA, Fleming GF, Monk BJ, Huang H, et al. Incorporation of bevacizumab in the primary treatment of ovarian cancer. N Engl J Med 2011;365(26):2473–83. [4] Pujade-Lauraine E, Hilpert F, Weber B, Reuss A, Poveda A, Kristensen G, et al. AURELIA: a randomized phase III trial evaluating bevacizumab (BEV) plus chemotherapy (CT) for platinum (PT)-resistant recurrent ovarian cancer (OC). J Clin Oncol 2012;30(18 Suppl., Gynecol Oncol Section) [abstr. LBA5002; Ref Type: Abstract]. [5] Aghajanian C, Blank SV, Goff BA, Judson PL, Teneriello MG, Husain A, et al. OCEANS: a randomized, double-blind, placebocontrolled phase III trial of chemotherapy with or without bevacizumab in patients with platinum-sensitive recurrent epithelial ovarian, primary peritoneal, or fallopian tube cancer. J Clin Oncol 2012;30(17):2039–45. [6] Monk BJ, Pujade-Lauraine E, Burger RA. Integrating bevacizumab into the management of epithelial ovarian cancer: the controversy of front-line versus recurrent disease. Ann Oncol 2013;24(Suppl. 10):x53–8. [7] Gonzalez-Masia JA, Garcia-Olmo D, Garcia-Olmo DC. Circulating nucleic acids in plasma and serum (CNAPS): applications in oncology. Onco Targets Ther 2013;6:819–32. [8] Stroun M, Maurice P, Vasioukhin V, Lyautey J, Lederrey C, Lefort F, et al. The origin and mechanism of circulating DNA. Ann N Y Acad Sci 2000;906:161–8.
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