Ultrasonics 54 (2014) 451–454

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A study of ovarian cancer biomarker amplification using ultrasound for early stage detection q
Danhong Peng a,b, Tian Xu a, Timothy J. Mason c, Wei Wu a,⇑

Key Laboratory of Environment Medicine and Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing 210009, PR China Department of Obstetrics and Gynecology, Zhongda Hospital, Southeast University, Nanjing 210009, PR China c Sonochemistry Centre, Faculty of Health and Life Science, Coventry University, Coventry CV1 5FB, UK

a r t i c l e

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a b s t r a c t
The application of serum biomarker to ovarian tumors for early stage detection and clinical diagnosis is a rapidly expanding research area. The problem with conventional markers is that they are often released too late or at too low a level to be detected in time to trigger effective treatment. Ultrasound has been used to influence bio-effects in living cells, but there is only one reported case of the use of ultrasound to enhance the release of a biomarker (Carcinoembryonic antigen CEA). In this study we report the use of ultrasound to enhance the release of a combination of ovarian cancer biomarkers (CA125 and CA199) to help in the diagnosis of ovarian cancer at an early stage. The results indicated that after 5 min sonication at a frequency of 1 MHz and intensity of 0.3 W cmÀ2, the CA125 and CA19-9 levels were increased by 2.02 and 4.21-fold respectively. These findings suggest that ultrasonic treatment can be used to enhance the release of serum biomarkers from ovarian tumors. Ó 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Article history: Received 29 July 2012 Received in revised form 30 May 2013 Accepted 30 May 2013 Available online 20 June 2013 Keywords: Ovarian cancer cell line Biomarker Amplification Ultrasound

1. Introduction Ovarian cancer is one of the leading causes of cancer deaths among women but if the disease is treated at an early stage there is an excellent prognosis for survival following treatment. However some 70% of patients are diagnosed at an advanced stage and in this case there is a poor survival rate of only 10–30% therefore it is important to detect ovarian cancer at early stage [1,2]. Current diagnosis methods for ovarian cancer are the CA-125 blood test and transvaginal ultrasound [3]. But neither of these tests are sufficiently sensitive or specific [4]. Recently, biomarkers such as CA125 and CA19-9 have been used to help in the early screening and diagnosis of ovarian cancer i.e. at a stage when it is the most treatable and before it has had a chance to grow and spread [5–8]. CA125 is the most widely used tumor marker in ovarian cancer but its sensitivity and specificity are not ideal because the levels of this marker are raised to approximately 80% of all epithelial ovarian cancers (EOC) and in only 50% of stage I EOC. The sensitivity and specificity rates of another biomarker CA19-9 are both lower than those of CA125. From this it can be argued that the results from individual existing markers are not specific

q This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike License, which permits noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ⇑ Corresponding author. Tel.: +86 13073420218. E-mail address: 101005800@seu.edu.cn (W. Wu).

enough because they can be affected by other malignancies, benign conditions such as diverticulitis, liver cirrhosis and also by physiological conditions including menstruation and pregnancy. In addition the concentration of a biomarker signal in the blood is often very low and so is difficult to detect. In order to be used in screening for early stage cancer the tumor marker must be sufficiently detectable to provide a positive result. Research [9–16] suggests that the use of a combination of biomarkers improves sensitivity for the detection of ovarian cancer. Ultrasound has been shown to be effective in a range of cancer therapies including the direct killing of cancer cells, enhanced drug delivery and for improving membrane permeability for the uptake of drugs. In 2009, researchers [17] reported the use of ultrasound to increase the release of biomarker Carcinoembryonic antigen (CEA). In this study, ultrasound at 1 MHz with an intensity of 0.3 W cmÀ2 was applied in cell culture experiments using the human colon cancer cell line LS174T. The biomarker CEA concentration was measured before and after exposure of the tumor cells to ultrasound. After 30 min treatment there was a 4-fold increase in the CEA levels when compared with untreated control samples. Mouse tumor xenograft models were also tested using 1 MHz ultrasound at an intensity of 2 W cmÀ2 applied directly to the site of the tumor for 6 min. The CEA level was increased by more than 10-fold, which may have been due to the somewhat higher intensity used. The ultrasonic transducer applied in this work was a commercial device (Sonitron 2000, Belgium), which can provide a well collimated beam penetrating deep into the tissue. This work indicated the possibilities that the concentration of tumor

0041-624X/$ - see front matter Ó 2013 The Authors. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ultras.2013.05.014

Ltd. Statistical analysis Statistical analyses were done with SPSS17. USA) for the measurement of frequency. Ultrasonic equipment The parameter settings of the HM-1 ultrasonic equipment (Jiangsu Hanmei Science and Technology Co. An ultrasound power meter (Model UPM-DF-1E. the media was removed and the cells were rinsed three times with 2–3 mL fresh media. Ltd. 2. USA) at 37 °C and under 5% CO2 concentration. 2). / Ultrasonics 54 (2014) 451–454 biomarkers in blood could be increased leading to a more sensitive and specific diagnosis. continuous-wave ultrasound (8 W cmÀ2) for 30 s treatment has been suggested to induce transient pores in the cell membranes of some surviving cells [18]. Tektronix Co. A significance level of 0. Ltd. The diameter of the ultrasonic probe was 21 mm.0. Cell death detection Cell death was determined immediately after sonication.5. The release of CA125 was also seen to increase slightly (P > 0. As an extension of this it would be of interest to determine whether ultrasonic treatment can enhance the release of two other ovarian tumor biomarkers CA125 and CA19-9 to a concentration enabling early stage screening and a consequent improvement in patient survival rate. Results and discussion 3. Time release of CA125 from ovarian cancer cells in culture in the presence and absence of ultrasound. USA).. 2. Ohmic Co.6. The average concentration of CA125 after sonication was 6. Cells Human ovarian carcinoma cell lines. Fig.02. the cells were harvested and cell death was determined by adding 10% Trypan blue to the cell suspension.01) continued to increase slowly over the remaining 30 min sonication. The sensitivity of this method was 0. 2.26 U/mL. USA). we decided to set the sonication time in all further experiments for 5 min. This was the driving force for our work which was to use this effect to provide a safe but effective ultrasonic treatment for the enhanced release of ovarian tumor biomarkers without causing permanent damage to the human cells involved.2. Cells grew in incubator (SERIES 8000 WJ. Materials and methods 2. Hyclone Laboratories Inc. China) was used between the bottom of the flasks and the ultrasonic probe.15 U/mL).05) without sonication but this was significantly lower compared with sonicated samples (Fig. SKOV3 were cultured in McCOY’s 5A medium (12400024.3 W cmÀ2 and 50% duty cycle. One of the bio-effects of ultrasound is to induce a temporary increase in the permeability of the cell membrane.1. Gibco. Thermo Scientific. All data are presented as the mean ± standard deviation (SD). with the probe placed flush against the bottom of the flasks (Fig. The media was removed. 1. Immediately after treatment 0. 1 MHz. 2. which was a 3fold increase in CA125 levels compared with control samples (2. Ultrasonic treatment of cells Cells were seeded at 5 Â 106 in culture flasks (Gibco. Similar reports of ultrasonic effects on CEA were published by D’Souza et al. Only dead cells with disrupted cell membranes are stained by the dye. Switzerland). and grown overnight in complete media forming a 100% confluent monolayer of cells. [17]. 3. Taizhou. Time release of CA125 from ovarian cancer cells in culture treated with ultrasound To study the time release of biomarkers.1... 2.6 U/mL. A significant increase of CA125 was observed from a treatment time of 5 min and the biomarker concentration (P < 0.. Thus. ultrasound coupling gel (Shengyou.3. cells were sonicated using the ultrasonic probe system described above.. Schematic diagram of experimental set-up. China) were: frequency of 1 MHz.05). Ultrasonic treatment was applied from the bottom of the flask for 5 min. Life Technologies. To achieve good sonication. Peng et al.5 mL samples of suspension were removed for analysis. In our study we found that the major increase occurred after 5 min treatment but after this the continued increase of CA125 concentration was slow (P > 0. intensity of 0. 1). Analysis of variance (Univariate ANOVA) and t test were adopted. 5 mL cells were then added into culture flasks immediately before sonication.. 2. Fig.452 D. USA) supplemented with 10% fetal calf serum (SV30087. . Control samples were run in parallel without sonication. Taizhou. The percentage of dead cells was counted using an inverted microscope (1X51.05 was used. OLYMPUS.. CA125/CA19-9 quantification CA125 and CA19-9 concentration in media were determined using a Roche automatic electrochemiluminescence immunoassay analyzer (COBAS6000. 2. USA) was used for intensity measurement and an oscilloscope (model TDS1000B. Japan) within 3 min after staining. On the following day.4.

Thus the combined use of CA125 and CA19-9 offers the possibility of improvements in sensitivity of ovarian cancer detection without significant loss in specificity. the CA125 concentration was 4. After sonication. 4. .g.19 ± 0. Although it is not used as widely as CA125 in ovarian cancer CA19-9 has been studied in the gynecological field since the 1980s and shown to have prognostic significance for tumor cells.2. 0. 2).2 s on Xenopuslaevis oocytes revealed that 110 nm pores were generated in the membrane [20]. / Ultrasonics 54 (2014) 451–454 453 Fig.38 ± 0. 3.D. Comparisons on time release of CA125 and CA19-9 after sonication CA125 and CA19-9 levels observed after sonication were higher than these without sonication (P < 0. The initial concentration of CA125 and CA19-9 were 2.075 MHz (intensity not reported) for 0.25 U/mL and 0.3 W cmÀ2 intensity and 50% duty cycle) over a period of 5 min increased the concentration of both of these biomarkers. Direct observation of the effects of sonication at 1. SF and AFP. The use of tumor biomarkers can improve the accuracy of diagnosis and management of patients with ovarian cancer. Based on this observation we deduce that this type of effect can also enhance the movement of biomarkers out of the cell. Compared with CA125 alone (64%).41 ± 0. Flow chart of experimental design. confirming that ultrasound can amplify tumor biomarker release (see Fig. Although the mechanism of the formation of transient pores in biological membranes induced by ultrasonic cavitation is not fully understood.01 U/mL. CA19-9 is another tumor antigen. Our results also suggest the possibility that ultrasonic treatment could also increase the levels of other markers e.61 ± 0. It is not easy to diagnose at early stage although the cure/survival rate would be much higher if treatment could be applied at this stage. respectively.09 U/mL and CA19-9 concentration was 2. Unfortunately at an early stage the biomarker levels are usually too low to be detected. 3. Ovarian cancer is the most frequent cause of death from gynecological cancer and the fourth most common cancer-related killer for female patients. it is believed that they are associated with the generation of shockwaves and microjects produced on bubble Fig. a combination of CA19-9 and CA125 presents an increase in positivity rate to 100% [13]. CA72-4. The most widely used and extensively researched tumor marker in ovarian cancer is CA125 but it cannot be used alone for cancer detection because only 50–60% of patients with stage I epithelial ovarian cancer will have levels high enough to register positive. These transient pores in cell membranes provided channels through which macromolecules could pass. The ultrasonic technique has been shown to produce pores in the cell membrane without immediate killing effects and so can cause a temporary increase in permeability [19].01 in both cases).10 U/mL (Fig. 3). Peng et al. The results indicated that ultrasonic treatment (1 MHz frequency. Comparisons on time release of CA125 and CA19-9 in the presence and absence of ultrasound. HE4.

Pauly. Methods in Molecular Biology 472 (2009) 413–437. For this reason it is important to maintain the ultrasonic parameters at levels sufficient to achieve amplification of biomarker release but to minimize cell death.H. Further investigations in this work are outlined in the flow chart (Fig. Liu. Food and drug administration center for devices and radiological health: information for manufacturers seeking market clearance of diagnostic ultrasound systems. Hollema. H.W. B. Nyborg. [2] F. There have been no previous reports about the use of ultrasound in connection with CA125 and CA19-9 expression and transfer process.results from a series of reproductive age women undergoing laparoscopic surgery for benign gynaecological conditions. In addition.43). S.J. Retrospective analysis of 67 consecutive cases of pure ovarian immature teratoma. A.M.A. A. I. Hirai. Tang. E.A. <http:// www. ten Hoor. D’Souza.L. [13] A.A. CA19-9 and interleukin-6 to detect the presence of endometriosis. 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