Feasibility of focused ultrasound therapy for recurrent cervicitis with high-risk human papillomavirus infection

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1 Ultrasound Obstet Gynecol 2009; 34: Published online in Wiley InterScience ( DOI: /uog.7357 Feasibility of focused ultrasound therapy for recurrent cervicitis with high-risk human papillomavirus infection C.-Z. LI*, Z.-B. WANG*, X. YANG*, Y. TANG, D. WANG, Y. HUANG and X.-F. FAN* *Institute of Ultrasonic Engineering in Medicine, Chongqing Medical University and Department of Obstetrics & Gynecology, Chongqing Tumor Hospital, Chongqing, China KEYWORDS: cervicitis; cervix; focused ultrasound; human papillomaviruses; infection; therapy ABSTRACT INTRODUCTION Objective To investigate the feasibility of focused ultrasound therapy for recurrent cervicitis with high-risk human papillomavirus (HR-HPV) infection. Methods This was a prospective clinical study, in which 20 patients with HR-HPV-positive recurrent cervicitis were enrolled. Focused ultrasound therapy was performed by one gynecologist. All patients were followed up for 6 months after ultrasound therapy. Telephone interviews, colposcopic examinations and Hybrid Capture II tests were performed to assess the safety and effectiveness of focused ultrasound therapy for HR-HPV-positive cervicitis. Results Ultrasound therapy was tolerated well, and no severe complications were observed in any patient. Vaginal discharge was found intermittently in 95% of patients. Without any intervention this disappeared 1 month after ultrasound therapy. Patients symptoms were relieved significantly by ultrasound therapy, including 88.9% patients who had abundant leukorrhea, 80% who had pelvic pain and 87.5% who had postcoital bleeding. No colposcopic evidence of cervicitis remained postoperatively in 75% of patients, and cytological examination showed that the lesions had disappeared in 80% of patients. Follow-up HPV testing revealed that 75% of patients presented negative HR-HPV infection following Conclusions Focused ultrasound therapy is feasible and effective in the treatment of patients with HR-HPVpositive cervicitis. It may provide a useful non-invasive treatment for recurrent cervicitis with HR-HPV infection. Copyright 2009 ISUOG. Published by John Wiley & Sons, Ltd. Human papillomavirus (HPV) infection is very common among women worldwide. In the United States, it is estimated to be the most common sexually transmitted infection 1. The overall prevalence of HPV is 26.8% among US females aged 14 to 59 years, with the highest prevalence being among those aged 20 to 24 years 2. Similar rates of occurrence have been found in China, with an overall prevalence of 29.1% among women and the highest prevalence being among those aged 18 to 20 years 3. There are more than 120 genotypes of recognized HPVs. Epidemiological studies have shown that genital HPV infection is the main cause of cervical cancer 4,5, which is the second most common cancer among women worldwide 6. According to their epidemiological association with cervical cancer, genital HPVs are generally categorized into low-risk and high-risk types. Infections with high-risk types, such as HPV types 16 and 18, can cause cervical, anal and other genital cancers 7,8. Although HPV virus itself cannot be treated, management approaches for clinically relevant HPV infection are necessary if the infection causes abnormal cell changes such as lesions and warts in anogenital areas. These approaches are commonly used in patients with HPV lesions, focusing on the symptomatic relief of the infection 9. Currently, both surgical and non-surgical therapies including cryosurgery, laser ablation and electrocautery are standard treatment strategies for the cervical lesions. The goal of these local therapies is to remove all the abnormal cells, thus removing all or most of the HPV-infected cells. Therapy is usually performed in a way that is tailored to the individual, based on the extent of the disease and the needs of each patient 10. As a non-invasive tool, focused ultrasound therapy is a new and promising approach in clinical practice Correspondence to: Dr C.-Z. Li, Box 153, Institute of Ultrasonic Engineering in Medicine, Chongqing Medical University, 1 Medical College Road, Yuzhong District, Chongqing , People s Republic of China ( licz@haifu.com.cn) Accepted: 22 July 2009 Copyright 2009 ISUOG. Published by John Wiley & Sons, Ltd. ORIGINAL PAPER

2 Focused ultrasound therapy for cervicitis with HPV infection 591 While ultrasound beams propagate through human tissue, they can be brought to a tight focus at a distance from their source. If the concentrated energy is sufficient, this leads to the thermal destruction of targeted tissue at depth, without damage to surrounding or overlying tissues 14. Our previous clinical study has shown that focused ultrasound therapy is feasible and effective in the treatment of patients with vulvar dystrophy 15 and symptomatic cervical ectopy 16. In this study, we hypothesized that focused ultrasound therapy could treat chronic cervicitis caused by HPV infection that was uncontrollable by conventional therapy. Thus, the goal of this study was to investigate the safety, effectiveness and feasibility of focused ultrasound therapy as a noninvasive tool for the management of recurrent cervicitis with high-risk HPV (HR-HPV) infection. PATIENTS AND METHODS Clinical study design This was a prospective clinical study of a series of patients with the aim of investigating the safety and feasibility of focused ultrasound in the treatment of cervicitis with HR-HPV infection. It was approved by ethics committees of both Chongqing Medical University and Chongqing Tumor Hospital, and all patients gave written informed consent before being admitted into the clinical study. Patients From May to October 2007, a total of 20 women with HR-HPV-positive cervicitis were enrolled into the study. The characteristics of enrolled patients are shown in Table 1. Patients who met the inclusion criteria were recruited and evaluated by one of three senior gynecologists. Inclusion criteria were: recurrence of cervicitis after at least two attempts at conventional therapy, positive HR- HPV test, histologically confirmed cervical intraepithelial neoplasia (CIN) Grade 1 or chronic inflammation, clinical symptoms such as pelvic pain, abundant leukorrhea and contact bleeding, negative pregnancy test, general Table 1 Characteristics of the enrolled patients with HR-HPV positive cervicitis Characteristic n or mean ± SD Number of patients 20 Age (years) ± 6.61 Cytology Inflammation 9 Atypical squamous cells of undetermined 8 significance Low-grade squamous intraepithelial lesion 2 High-grade squamous intraepithelial lesion 1 Pathology (biopsy) Inflammation 12 Cervical intraepithelial neoplasia 1 6 good health, laboratory values for hematology and biochemistry within normal ranges, and no treatment for cervicitis and HPV in the last 3 months. The patients had previously received at least two courses of antibiotics including azithromycin and doxycycline for chlamydial and other non-gonococcal infection, but the symptoms related to cervicitis had persisted or recurred with no evidence of gonococcal infection. Excluded were women who had high-grade CIN (Grade 2 or 3) or cervical cancer diagnosed by histopathological examination, women who were pregnant or breastfeeding, and those who had other gynecological disorders including cervical dysplasia at the time of enrollment. Colposcopic examination of the cervix was performed before ultrasound therapy. A red cervical ectropion with an inflamed appearance was observed in all patients. Along with the red appearance, underlying blood vessels were seen clearly, and sustained endocervical bleeding was induced easily by gentle passage of a cotton swab. Lesions in the transformation zone of the cervix were assessed by applying 5% acetic acid and iodine solution. During the colposcopy procedure cytological examination of the abnormal cervix was performed in 20 patients, and a punch biopsy was taken from the key points of the abnormal areas in 18 patients. The Hybrid Capture II (HC-2) test (Digene Corporation, Gaithersburg, MD, USA) was used to detect HR-HPV DNA in all patients before and 6 months after ultrasound therapy. A sample of cells from the ectocervix and endocervix was obtained with a cervical cytobrush during colposcopy. The cytobrush was then immersed in transport medium, and kept at 20 C until analysis. Focused ultrasound therapy The Seapostar ultrasound therapy device (Chongqing Haifu (HIFU) Technology Co. Ltd, Chongqing, China) was used to treat all patients in the study. It comprised a power generator, therapeutic transducer, central console and circulating degassed water system. The ultrasound energy was produced by a 10-mm-diameter focused ultrasound transducer with a focal length of 4 mm, operating at frequencies of 8 12 MHz. The acoustic power was 3 5 W. Ultrasound therapy was performed without anesthesia on an outpatient basis by one gynecologist. All patients underwent the therapeutic procedure 3 7 days after their menstrual period. A sterile speculum was placed in the vagina so the cervix could be seen clearly, and cervical mucus and discharge were removed using cotton balls. The vaginal wall and cervical surface were then sterilized with iodophor, and the size of the cervical lesion was confirmed using an iodine test. The hand-held ultrasound transducer, which was completely enveloped in a disposable metal cover, was in direct contact with the cervix using a coupling medium. A spiral scanning track, directed by the operator, was used to treat the cervical lesion and included a 2-mm ring of normal tissue surrounding the lesion. The therapy procedure time ranged from 5 to 10 min.

3 592 Li et al. The cervical lesion became edematous immediately after ultrasound treatment, and the cervix presented concentric contraction with modest introversion of the external cervical aperture. Follow-up During follow-up, any new symptoms or changes in previous symptoms, such as pelvic pain, leukorrhea, menstrual pattern and postcoital bleeding, were carefully recorded for each patient. Individual telephone interviews were conducted with all patients 7 days after A follow-up visit was carried out in the outpatient clinic monthly during the 6-month follow-up period, and colposcopic examination was performed to observe any signs of local complication resulting from the ultrasound therapy and any postoperative changes in the cervical lesion. The site, character and size of the lesions, the number of cervical quadrants involved and the patterns of vasculature were recorded for each patient. The recovery of the cervical lesion and the size of the residual lesion were also measured. Each patient underwent an HPV test 6 months after Patients were advised that sexual intercourse and vaginal douches should be avoided during the 1 st month after In order to avoid HPV reinfection, we also advised that during sexual intercourse a condom should be used during the 2 nd and 3 rd months after The sexual partner of each patient did not undergo assessment or treatment for HPV infection. Statistical analysis All observed data are reported as mean ± SD. The statistical significance of any observed difference in percentage data was analyzed using McNemar s test. Statistical significance was defined as a P < RESULTS Safety evaluation Ultrasound therapy was tolerated well in all patients. During the therapeutic procedure there was no pain but discomfort was observed in five patients. Four patients had mild colporrhagia 1 week after ultrasound therapy. This lasted for 3 5 days, and the volume was less than one third that of a normal menstrual period. Vaginal discharge began immediately after treatment and was observed intermittently for 1 3 weeks postoperatively in 19 of 20 patients. This usually consisted of a clear and watery fluid and may have resulted from thermal reaction of the treated cervix. The number of menstrual napkins required to absorb this discharge at its peak averaged from 3 to 5 per day in 12 patients (the other seven did not require napkins). No intervention was required to bring about its resolution. No severe complications requiring treatment were observed in the patients during the follow-up period. Symptom relief Abundant leukorrhea was recorded in 18 patients, pelvic pain in 10, and postcoital bleeding in eight before After ultrasound therapy, there was a marked decrease of vaginal discharge in 16 patients, pain relief in eight, and decreased postcoital bleeding in seven (Table 2). Macroscopic and cytological changes in cervical lesion In each patient colposcopic examination was performed monthly during the follow-up period to observe the healing process of the cervical ectropion. A typical example of cervicitis is seen in Figure 1, showing gross differences in a cervical lesion before and 3 months after Following ultrasound therapy, no colposcopic evidence of cervicitis was observed during 6 months of follow-up in 15 of 20 patients, including 11 patients with cervicitis and four with CIN Grade 1. Cytological examination showed that the lesions disappeared 1 2 months after ultrasound therapy in 16 of 20 patients, including seven patients with inflammation, six patients with atypical squamous cells of undetermined significance, two patients with low-grade squamous intraepithelial lesion and one case with high-grade squamous intraepithelial lesion. Four women had persisting abnormal appearance on cytology, but with no evidence of disease progression during the follow-up period (Table 2). HPV test Before treatment all patients were found to be HR-HPVpositive. A follow-up HC-2 test performed 6 months after ultrasound therapy showed that 15 of 20 patients were negative for HR-HPV infection, with a decrease to 1pg/mL HPV-DNA after Table 2 Changes in symptoms and cytology before and after ultrasound therapy in patients with HR-HPV positive cervicitis Symptom/ cytology Before treatment (n) After treatment (n) Cure rate (%) Symptom 36 5* 86 Abundant leukorrhea 18 2* 89 Pelvic pain 10 2* 80 Postcoital bleeding 8 1* 88 Abnormal cytology 20 4* 80 Inflammation 9 2* 78 Atypical squamous cells 8 2* 75 of undetermined significance Low-grade squamous intraepithelial lesion High-grade squamous intraepithelial lesion *P < 0.01 compared with symptom or cytological result before

4 Focused ultrasound therapy for cervicitis with HPV infection 593 Figure 1 Macroscopic appearance of the cervix before (a) and 3 months after (b) focused ultrasound therapy. (a) Viable cervical lesion: a red area of cervical erosion (arrows) is visible in the transformation zone of the cervix, with an inflamed appearance and cervical ectropion; (b) 3 months after ultrasound therapy the red cervical erosion has disappeared and the cervix presents normal appearance and elasticity. DISCUSSION Cervical cancer is the second most common cancer among women worldwide, with an estimated new cases and deaths in the year Inthe United States, there has been a 70% decrease in cervical cancer-related mortality over the past five decades due to well-developed screening programs 17. A decline is also evident in some developing countries such as China, where the estimated age-standardized incidence rate in 2002 was 6.8, compared with 17.8 in Itisclear that the major etiological agents are high-risk subtypes of HPV such as type 16 and 18, and that the biology of HPV cervical cancer is characterized by a long precancerous phase with quantifiable risk factors for HPV persistence and for progression of HPV-related CIN to cervical cancer 4,18,19. Management approaches for clinically relevant HPV infection of the cervix include local therapies targeting the HPV lesions and systemic therapies such as immunotherapy and antiviral treatment for HPV infection. Surgical removal and minimally invasive ablative techniques, such as laser, cryoablation and electrocautery, are the standard therapeutic strategies that focus on treating the symptoms of the cervical infection 10. They are performed individually in clinical practice, based on the extent of disease and the needs of the patient. The novel approach of focused ultrasound is a non-invasive therapy for targeted tissues, and our preliminary results showed that it was safe and effective in the treatment of subcutaneously diseased tissues in patients with vulvar dystrophy 15,withnodamage to the overlying skin. Compared with laser ablation, focused ultrasound therapy had the equivalent therapeutic efficacy in patients with symptomatic cervicitis, but postoperative bleeding and reactive discharge were much less in the patients who underwent ultrasound therapy 16.Due to its non-invasive nature, without loss of normal tissue during the therapeutic procedure, the diseased cervix was anatomically well-preserved, with normal elasticity, and the physiological function of the cervix was not influenced after ultrasound therapy. However, as the diseased tissues are completely destroyed by ultrasound energy, it is very important to screen the patients thoroughly using colposcopy and biopsy before ultrasound therapy, to make sure that the treated lesion is benign. In this study conventional antibiotic therapy had failed to control the cervicitis symptoms, such as abundant leukorrhea, pelvic pain and postcoital bleeding, in all patients before enrollment. We found that after ultrasound therapy the symptoms were relieved significantly, including in 89% (16/18) of patients with abundant leukorrhea, 80% (8/10) of those with pelvic pain and 88% (7/8) of those with postcoital bleeding. Colposcopic examination showed no evidence of cervicitis in 75% (15/20) patients, and cytological follow-up found that the HPV lesions had disappeared in 80% (16/20) patients after ultrasound therapy. The significant improvement of previous symptoms in the patients suggest that the ultrasound therapy could have cured the cervical HPV lesions. However, due to safety considerations, such as the possibility of ultrasound-induced abnormality of the cervical canal post-treatment, middle-aged patients who already had children were selected for the study. Our further research aims to use focused ultrasound for the treatment of young patients, aged years, in whom the highest prevalence of HPV infection is observed. We also found that HR-HPV infection of the cervix was negative 6 months after focused ultrasound therapy in 75% (15/20) patients who had HR-HPVpositive cervicitis previously. This result suggests that ultrasound therapy could locally clear HR-HPV infection by removing all abnormally HPV-infected cervical cells. However, the infection persisted in the remaining 25% (5/20) patients. This may have been due to a multifocal infection, with one or more foci still being in some kind of incubation period, or being at an earlier

5 594 Li et al. and undetectable stage of development at the time of ultrasound therapy. There were some weaknesses in this clinical study. The follow-up period was 6 months, which is relatively short for patients with HPV-infected cervicitis because of the potential risk of local recurrence. We have recently undertaken a Phase III clinical trial to investigate the long-term effects of ultrasound therapy on HPV-infected cervicitis, including local recurrence of the HPV-infected lesions. All enrolled patients will be followed up for 2 years after ultrasound therapy. Our next step will be to perform multiple randomized clinical trials to compare focused ultrasound therapy with other local approaches, such as laser, for the treatment of HR- HPV-positive cervicitis in terms of long-term efficacy. The second weakness of the study was that there were no numerical gradations of the treatment responses, which were based on changes in patients symptoms and histological diagnosis before and after ultrasound Quantitive assessment will be established in our ongoing randomized clinical trial for evaluation of local responses after In summary, we have reported for the first time that focused ultrasound therapy could be safe, effective and feasible in the treatment of patients with HPVinfected cervicitis. It could provide a non-invasive approach for treating recurrent cervicitis with HR-HPV infection. ACKNOWLEDGMENTS This work was supported by the Foundation of Ministry of Education of China (grant No. IRT0454) and the National Natural Science Foundation of China (grant No ). REFERENCES 1. Weinstock H, Berman S, Cates W. Sexually transmitted infections in American youth: incidence and prevalence estimates, Perspect Sex Reprod Health 2004; 36: Dunne EF, Unger ER, Sternberg M, McQuillan G, Swan DC, Patel SS, Markowitz LE. Prevalence of HPV infection among females in the United States. JAMA 2007; 297: Zhang WY, Xue YZ, Chen M, Han L, Luo M. Prevalence of high-risk human papillomavirus infection in different cervical lesion among organized health-examination women in Shanghai, China. Chin Med J 2008; 121: Schiffman M, Castle PE, Jeronimo J, Rodriguez AC, Wacholder S. Human papillomavirus and cervical cancer. Lancet 2007; 370: Bosch FX, Manos MM, Munoz N, Sherman M, Jansen AM, Peto J, Schiffman MH, Moreno V, Kurman R, Shah KV, International Biological Study on Cervical Cancer (IBSCC) Study Group. Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. J Natl Cancer Inst 1995; 87: Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, CA Cancer J Clin 2005; 55: Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, Snijders PJ, Meijer CJ, International Agency for Research on Cancer Multicenter Cervical Cancer Study Group. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003; 348: Bosch FX, Castellsague X, de Sanjose S. HPV and cervical cancer: screening or vaccination? BrJCancer2008; 98: Snoeck R. Papillomavirus and Antivir Res 2006; 71: Spitzer M. Screening and management of women and girls with human papillomavirus infection. Gynecol Oncol 2007; 107: S14 S Kennedy JE. High-intensity focused ultrasound in the treatment of solid tumours. Nat Rev Cancer 2005; 5: Chaussy C, Thuroff S, Rebillard X, Gelet A. Technology insight: High-intensity focused ultrasound for urologic cancers. NatClinPractUrol2005; 2: Wu F. Extracorporeal high intensity focused ultrasound in the treatment of patients with solid malignancy. Minim Invasive Ther Allied Technol 2006; 15: ter Haar G. Therapeutic applications of ultrasound. Prog Biophys Mol Biol 2007; 93: Li C, Bian D, Chen W, Zhao C, Yin N, Wang Z. Focused ultrasound therapy of vulvar dystrophies: a feasibility study. Obstet Gynecol 2004; 104: Chen J, Zhou D, Liu Y, Peng J, Li C, Chen W, Wang Z. A comparison between ultrasound therapy and laser therapy for symptomatic cervical ectopy. Ultrasound Med Biol 2008; 34: Solomon D, Breen N, McNeel T. Cervical cancer screening rates in the United States and the potential impact of implementation of screening guidelines. CA Cancer J Clin 2007; 57: Woodman CB, Collins SI, Young LS. The natural history of cervical HPV infection: unresolved issues. Nat Rev Cancer 2007; 7: de Sanjose S, Diaz M, Castellsague X, Clifford G, Bruni L, Munoz N, Bosch FX. Worldwide prevalence and genotype distribution of cervical human papillomavirus DNA in women with normal cytology: a meta-analysis. Lancet Infect Dis 2007; 7: