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Clinical Responses to Focused Ultrasound Applied to Women WithVulval Intraepithelial Neoplasia

ulval intraepithelial neoplasia (VIN) is a premalignant skindisorder that often causes severe and long-lasting pruritus,pain, and psychosexual dysfunction. It has a spectrum of

clinical and histopathologic manifestations and is divided into 2 sub-types: the usual type, which is caused by a persistent human papil-lomavirus (HPV) infection; and the differentiated type, which isassociated with lichen sclerosus.1 The types differ in etiology, mor-phologic characteristics, biological characteristics, clinical features,and malignant potential.2 Although the classification of 3 subtypes(VIN 1–3) by the World Health Organization is still widely used,3VIN nowadays only applies to histologically “high-grade” squa-mous lesions (VIN 2 and 3); VIN 1 is considered nonexistent.1

Ying Jia, MD, Jin Wu, MS, Man Xu, MD, Liangdan Tang, MD, Chengzhi Li, MD, Ming Luo, MS, Meng Lou, MS

Received October 30, 2013, from the Departmentof Obstetrics and Gynecology, First AffiliatedHospital of Chongqing Medical University,Chongqing, China (Y.J., J.W., L.T., M.Lu., M.Lo.);and Department of Pathology, College of BasicMedicine (M.X.), and Biomedical EngineeringCollege (C.L.), Chongqing Medical University,Chongqing, China. Revision requested December2, 2013. Revised manuscript accepted for publica-tion February 25, 2014.

We thank Wenjun Zong, MD, PhD(Department of Obstetrics and Gynecology,Wyckoff Heights Medical Center, Brooklyn, NY),for assistance with manuscript preparation, theDepartment of Pathology of Chongqing MedicalUniversity and the staff in the outpatient clinics ofthe Department of Obstetrics and Gynecology ofFirst Affiliated Hospital of Chongqing MedicalUniversity for assistance, the Public Health Bureauof Chongqing City for financial support, and, mostimportantly, our patients for their participation inthis study.

Address correspondence to Liangdan Tang,MD, Department of Obstetrics and Gynecology,First Affiliated Hospital of Chongqing MedicalUniversity, 400016 Chongqing, China.

E-mail: [email protected]

AbbreviationsHPV, human papillomavirus; VIN, vulvalintraepithelial neoplasia

V

©2014 by the American Institute of Ultrasound in Medicine | J Ultrasound Med 2014; 33:1903–1908 | 0278-4297 | www.aium.org

ORIGINAL RESEARCH

Objectives—Focused ultrasound waves penetrate superficial tissues and are aimedtoward the target tissues at specific depths to exert their biological effects. Focused ultra-sound has been applied for a number of clinical indications, including vulval dystro-phies and low-grade vulval disease. This study aimed to assess the efficacy and safety offocused ultrasound treatment of high-grade vulval intraepithelial neoplasia (VIN).

Methods—Eighteen women with high-grade VIN were recruited and treated withfocused ultrasound. During each posttreatment follow-up, the safety of, side effects of,and clinical responses to focused ultrasound were evaluated by a standardized protocol,including symptoms, clinical appearance, and histologic findings.

Results—All patients completed the designed follow-ups. In most cases, superficial mildto moderate swelling and blisters were seen in the focused ultrasound–treated skin butnot in adjacent normal skin. Of the 18 patients, 16 showed complete histologic regres-sion and resolution of symptoms 6 months after treatment. Of the other 2 patients, 1showed complete regression after a second focused ultrasound treatment. The otherpatient did not respond to the focused ultrasound treatment and underwent a partial vul-vectomy 6 months after treatment. None of the patients developed invasive carcinomaof the vulva during the follow-up period. One patient had local pruritus that was notalleviated by anti-inflammatory medication and local care.

Conclusions—The complete responses observed in women with high-grade VINtreated by focused ultrasound, together with the preservation of adjacent normal tissue,suggest that focused ultrasound may be considered for treatment of high-grade VIN.

Key Words—efficacy; focused ultrasound; local treatment; safety; vulval intraepithe-lial neoplasia

doi:10.7863/ultra.33.11.1903

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The augmented incidence of VIN parallels the increase inthe trend of HPV infection.4 Despite the spontaneousregression observed in some young women,5 the usual typeof VIN is thought to be a premalignant condition; thus,treatment is usually recommended for all women with VIN.6Current practice supports the early treatment of VINlesions to prevent later development of invasive vulvalcancer and to allow less invasive treatment for preserva-tion of normal vulval anatomy and function.

Surgery has been considered the best treatment forhigh-grade VIN.7 However, surgical treatment often leads tosexual dysfunction and anatomic distortions. Furthermore,the usual type of VIN is often multifocal and multicentricand more commonly diagnosed in younger womenbecause of the spread of HPV infection. In addition, sur-gery does not clear the persistent infection, which is theleading cause of frequent recurrences after treatment.8,9

Topical application of 5% imiquimod has been reportedto successfully treat VIN10 and may be suitable for wide-spread disease. Cidofovir often causes intense localinflammatory reactions, and patients tend to reject it.11

Other medical treatments for VIN include interferon α,1% 5-fluorouracil, and retinoids, with variable outcomes.12

Therefore, an ideal treatment of VIN should have highcure rates and the ability to cover a wide area without caus-ing substantial tissue damage or deformity.

As a recently developed noninvasive technique, focusedultrasound waves penetrate the superficial tissues and focuson the target tissues at specific depths to exert biologicaleffects without a substantial impact on the adjacent tissues,which can facilitate recovery of diseased tissues.13 Focusedultrasound waves result in a temperature rise in less than 1second, mechanical alteration, and cavitation, which togetherdenature diseased tissues, promote coagulative necrosis,and allow the tissues to be replaced by surroundingnormal healthy tissues. Focused ultrasound therapy is sub-grouped into high- and low-intensity focused ultrasound.The parameters of high-intensity focused ultrasound ther-apy include a working frequency of 0.8 to 1 MHz, ultra-sound power output of 160 to 350 W, and a treatmentdepth of greater than 10 mm. It has been reported to bemainly used to treat deep-located benign and malignanttumors, such as uterine myoma, liver cancer, pancreaticcancer, and osteosarcoma. On the other hand, the param-eters of low-intensity focused ultrasound therapy include aworking frequency of 8 to 12 MHz, ultrasound power out-put of 50 W or lower, and a treatment depth of 2 to 4 mm.It has been used for treatment of vulval lesions and cer-vicitis; as such, in our study, we chose to investigatelow-intensity focused ultrasound. At certain spectra, low-

intensity focused ultrasound can also specifically damagecells of the diseased tissues while maintaining the integrityof normal anatomic structures to permit tissue repair andrestore normal anatomy and function.14,15 Wang et al16

showed that focused ultrasound was remarkably effectivein treating vulval condyloma acuminatum. Studies haveshown that focused ultrasound can successfully treat vulvaldystrophies with efficacy of 96% and minimal side effects.17

Moreover, focused ultrasound treatments have been foundto be particularly useful for epidermal diseases, since theselesions do not involve the deep structures of the skin.Recently, we published a report using a mouse model, inwhich focused ultrasound had successful efficacy (100%)in treating high-grade VIN lesions.18 On the basis of theintriguing animal data, in this pilot study, we aimed toinvestigate whether focused ultrasound is safe and effec-tive for women with high-grade VIN.

Materials and Methods

The study was approved by the Ethics Committee ofChongqing Medical University. Written informed consentwas obtained from all patients. The low-intensity focusedultrasound equipment (CZF version) was manufactured byChongqing Haifu Technology Co, Ltd (Chongqing, China).

Eighteen women with VIN confirmed by pathologicanalysis who wished to avoid surgical treatment were enrolledin this study. They had diagnosis and treatment at either theFirst or Second Affiliated Hospital of Chongqing MedicalUniversity from May 2009 through July 2012. The diagnosiswas made according to the histologic criteria of the revisedInternational Society for the Study of Vulvovaginal Disease1;accordingly, VIN 1 lesions were excluded. Other exclusioncriteria were invasive disease or an inability to exclude invasivedisease, pregnancy, current participation in another clinicaltrial, and treatment of VIN within the preceding 3 months.Age, menopausal status, lesion size, and disease focus andduration were not specific eligibility criteria.

Vulvoscopy with application of acetic acid was per-formed before biopsy and focused ultrasound treatment;the lesion area was recorded on a standardized diagram;and a digital image of the lesion was taken. Before treat-ment, the patients were anesthetized locally with a lido-caine injection. The focused ultrasound treatment wasdelivered at power of 4.0 to 4.5 W and a frequency of9.7 MHz and was extended to 4 mm beneath the epidermisand 5 mm outside the lesion margin, which was deter-mined by application of acetic acid. The entire treatmentarea underwent linear scanning until the treated skin sur-face temperature reached 40°C to 43°C, which was simulta-

Jia et al—Focused Ultrasound Applied to Women With Vulval Intraepithelial Neoplasia

J Ultrasound Med 2014; 33:1903–19081904


neously measured with a TES-1310 thermometer (TES,Taiwan). At this point, the treated skin and mucusappeared congested and edematous.18 The duration ofultrasound treatment was 20 to 30 minutes. Immediatelyafter treatment, each patient was advised to void, and thevulval area was sterilized with povidone-iodine. A sterilepetroleum gauze was applied to the treatment area, and icepacks were used intermittently during the day with a 5-minute-on, 5-minute-off cycle for 12 hours.16 Cold injuryfrom the ice was prevented by real-time observation of skincolor. The ice pack was removed when the skin becamepale. Patients then received hip baths of 1:5000 potassiumpermanganate twice daily. Sterile oil gauze and warm moistcompresses of 50% magnesium sulfate were applied to thetreatment area twice daily until the local edema disap-peared. Symptomatic treatments were administered for 3to 5 days.

All 18 patients completed the scheduled follow-ups ata 4-week interval for the first 3 months, with biopsy per-formed at the third visit. The follow-ups included patients’symptoms and local visualization of the treated area undera vulvoscope (with the application of acetic acid). Patientswere then followed at the sixth month and regularly at 6-month intervals thereafter. During follow-up, biopsy wasperformed when there was any positive result.

Results

The 18 women recruited had a mean age of 48.83 (range,31–71) years. The VIN was diagnosed with pathologic con-firmation before focused ultrasound treatment. There were14 patients with VIN 2 and 4 with VIN 3. Of the 14 patientswith VIN 2, 10 had the usual type of VIN 2, and 4 had the dif-ferentiated of type VIN 2. Of the 4 patients with VIN 3 , 2had usual VIN 3, and 2 had differentiated VIN 3 (Table 1).

Skin congestion and edema peaked at 24 hours aftertreatment and gradually improved after 3 days. Five patientspresented with blisters, and all showed superficial second-degree burns. One week after treatment, congestion andedema of the vulva disappeared in 11 patients, whereas theother 7 patients had mild to moderate edema that lasted foranother week. None of the patients developed any infec-tion, skin rupture, or coagulation necrosis in the treated areaor any injuries to the untreated area. Moderate pain waspresent for the first several days but usually disappeared bythe end of the first week. The pruritus disappeared withindays of treatment. Four patients developed recurrent vulvalpruritus 1 week later, but pruritus markedly improved withlocal medication in 3 patients and was persistent in theother (Table 2).

Biopsies at the 3-month visit after focused ultrasoundtreatment showed that 10 of the 18 patients had normalvulval skin histologic results, and 8 had positive results: 4VIN 1, 3 VIN 2, and 1 VIN 3. At 6 months, biopsies of the8 patients with VIN revealed that 4 patients had normalvulval skin, and the other 4 had positive results: 2 with VIN1 and 2 with the same conditions as before focused ultra-sound treatment. Of the latter 2 patients, the patient withdifferentiated VIN 3 chose to undergo partial vulvectomy,whereas the other with usual VIN 2 chose a second focusedultrasound treatment, and vulval biopsy results turned outto be normal after 6 months (Table 3). The clinical andpathologic features of VIN before and after focused ultra-sound therapy are shown in Figures 1 and 2. All 18 womencompleted variable periods of follow-up, which rangedfrom 10 months to 4 years, and none of the patients devel-oped invasive carcinoma of the vulva. According to VINhistologic staging and clinical scoring standards, 14 patientswere cured; the responses in 2 patients were remarkable;and the responses in the other 2 patients were ineffective.

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Table 1. Patient Demographics and VIN Types (n = 18)

Characteristic Value

Age, y 48.83 (31–71)Disease duration, y 6 (1–20)Prior diseases, n

Condyloma 3Immunosuppressive disease 1Vulval dystrophy 6

Pathologic type, nVIN 2

Usual VIN 2 10Differentiated VIN 2 4

VIN 3Usual VIN 3 2Differentiated VIN 3 2

Data are presented as mean (range) where applicable.

Table 2. Adverse Reactions After Focused Ultrasound Treatment

Time After Focused Ultrasound

Appearance Immediately 1 wk 1 mo

Symptoms, nVulval pain 18 0 0Vulval pruritus 0 4 1

Signs, nVulval swelling

Mild to moderate 16 5 0Severe 2 2a 0

Skin congestion 18 0 0Blisters 5 0 0

aThe 2 patients with severe swelling had moderate edema that waspersistent for 1 week after treatment.


Discussion

Vulval intraepithelial neoplasia is a precancerous disorderof the vulva that is closely related to HPV infection anddysplasia of the keratinocytes. Approximately 35% to 50%of VIN disorders are associated with high-risk strains ofHPV. For example, HPV 6, 11, and 16 are related to thepathogenesis that causes vulval disorders.19 Clinical presen-tations of VIN vary and lack specificity. Patients with VINgenerally are asymptomatic, and the most common symp-tom is pruritus in more than 60% of patients. All of thepatients in our study presented with pruritus. The diagnosis,treatment, and pathologic classification of VIN have alwaysbeen a challenge to gynecologists. For patients whose diag-nosis is in doubt, colposcopy and biopsy guided by aceticacid are frequently performed to ensure proper treatment.

Focused ultrasound can effectively damage the dermisof vulvar lesions, promote repair and regeneration of capil-laries and small blood vessels in the dermis, improve nutri-tion of nerve ends, and maintain the integrity of local tissuesand organs. Ultramicroscopic morphologic studies con-firmed that ultrasound treatment could substantially affectvascular endothelial cells and basal cells in the dermis.


J Ultrasound Med 2014; 33:1903–19081906

Figure 1. Clinical and pathologic features of patient 1, who underwent a large lesion biopsy and follow-up for more than 10 months. A, Clinicalappearance before biopsy. B, Clinical appearance at the focused ultrasound treatment. C, Moderate vulval swelling seen right after the focused ultra-sound treatment. D, Follow-up at the 3-month visit. E, Confirmation of VIN 3 (hematoxylin-eosin, original magnification ×400). F, Three months aftertreatment, the structures of the squamous epithelium had recovered normally (hematoxylin-eosin, original magnification ×100).

Table 3. Pathologic Findings

Before Focused Ultrasound, After Focused Ultrasound, n

Histologic Type 3 mo 6 mo

VIN 2Usual VIN 2 (10)

Normal vulva 7Usual VIN 1 3 3 normal vulvaUsual VIN 2 0

Differentiated VIN 2 (4)Normal vulva 2Differentiated VIN 1 1 1Differentiated VIN 2 1 1 differentiated VIN 1

VIN 3Usual VIN 3 (2)

Normal vulva 1Usual VIN 1 0Usual VIN 2 1 1a

Usual VIN 3 0Differentiated VIN 3 (2)

Normal vulva 0Differentiated VIN 1 0Differentiated VIN 2 1 1 differentiated VIN 1Differentiated VIN 3 1 1b

aPatient with usual VIN 2 at 6-month biopsy had a second focusedultrasound treatment and a normal vulva after another 6 months.bPatient with differentiated VIN 3 at 6-month biopsy chose partialvulvectomy.


Skin lesions showed various degrees of recovery. Epidermalcells were arranged in an orderly pattern, and both desmo-somes and melanin granules in the cytoplasm increased to acertain degree. Melanocytes can often be found on the basallayer. Similar effects were observed in treating vulvar condy-loma acuminatum and other diseases of the epidermis withfocused ultrasound.16 To the best of our knowledge, this arti-cle is the first report of treatment of high-grade VIN byfocused ultrasound. Of the 18 patients with VIN treated by focused ultrasound, 16 (89%) showed complete clinicaland histologic clearance 6 months after the treatment. Noneof the 18 patients with multifocal VIN treated by focusedultrasound developed invasive cancer in this study. Ineffec-tive treatment is mainly attributed to involvement of the hairfollicles, which can contain VIN and extend into the subcu-taneous fat 3 mm or deeper. Consequently, it is possible thatlarge VIN lesions involving hair-bearing areas may be pref-erentially treated by other modalities.6 In this data set, a lackof concurrent HPV detection was a defect, but VIN remis-sion and no signs of reoccurrence might indicate a likelihoodof viral regression. Nevertheless, we are in the process of

studying the HPV status of the biopsies and will include thedata in a future report with an expanded sample size andlonger follow-up.

In our study, local side effects were limited to skinswelling and blisters of the affected mucosa, with no effectseen in the surrounding normal tissue, and these effectswere often minor and well tolerated. Skin congestion andpain were reported by all patients but usually lasted a fewdays after treatment. It is worth noting that the patientshad to stay home with a certain degree of discomfort for 2to 3 weeks, which may be a limiting factor for applicationof focused ultrasound. Future studies, therefore, shouldinclude an analgesic algorithm as part of the protocol.Focused ultrasound therapy can be easily applied to lesionswith a wide area and repeated as needed. Because of itseffectiveness and minimal side effects, as demonstrated inthis investigation, we believe that focused ultrasound is use-ful for treating VIN lesions in the external genital regions,with additional benefits of maximum preservation ofanatomy and function. However, we recommend repeti-tive treatment until the lesion regression completes.

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Figure 2. Clinical and pathologic features of patient 2 over more than 40 months of follow-up. A, Clinical appearance before the biopsy. B, Clinicalappearance at the focused ultrasound treatment. C, Severe vulval swelling and blistering right after focused ultrasound treatment. D, Follow-up at 6to 7 months after focused ultrasound treatment. E, The biopsy result was focal VIN 2 (hematoxylin-eosin, original magnification ×400). F, Six monthsafter treatment, the structures of the squamous epithelium had recovered (hematoxylin-eosin, original magnification ×100).


Finally, despite the appealing results reported here, closefollow-up visits are strongly encouraged, as no definitiveconclusion can be drawn at this time with regard to long-term effects and recurrence rates after focused ultrasoundtreatment of VIN.

In conclusion, high-grade VIN represents a difficultmanagement problem. The complete clinical responses inwomen with long-standing VIN treated by focused ultra-sound, together with preservation of vulval anatomy andfunction, suggest that focused ultrasound treatment mayhave a place in the therapeutic strategies for VIN. Furtherstudies are warranted to investigate its validity, safety, andworking mechanisms.

References

1. Sideri M, Jones RW, Wilkinson EJ, et al. Squamous vulvar intraepithelialneoplasia: 2004 modified terminology, ISSVD Vulvar OncologySubcommittee. J Reprod Med 2005; 50:807–810.

2. Scurry J, Wilkinson EJ. Review of terminology of precursors of vulvar squa-mous cell carcinoma. J Low Genit Tract Dis 2006; 10:161–169.

3. Wilkinson EJ, Teixeira MR. Tumors of the vulva. In: Tavassoli FA, DevileeP (eds). Pathology and Genetics of Tumours of the Breast and Female GenitalOrgans: World Health Organization Classification of Tumours. Lyon, France:IARC Press; 2003:316.

4. Judson PL, Habermann EB, Baxter NN, Durham SB, Virnig BA. Trendsin the incidence of invasive and in situ vulvar carcinoma. Obstet Gynecol2006; 107:1018–1022.

5. Jones RW, Rowan DM. Spontaneous regression of vulvar intraepithelialneoplasia 2-3. Obstet Gynecol 2000; 96:470–472.

6. Committee on Gynecologic Practice of the American College of Obstetricians and Gynecologists and the American Society for Colposcopy and Cervical Pathology. Management of vulvar intraepithe-lial neoplasia. J Low Genit Tract Dis 2012; 16:1–3.

7. Frega A, Sesti F, Sopracordevole F, et al. Imiquimod 5% cream versus coldknife excision for treatment of VIN 2/3: a five-year follow-up. Eur Rev MedPharmacol Sci 2013; 17:936–940.

8. van Seters M, van Beurden M, de Craen AJ. Is the assumed natural historyof vulvar intraepithelial neoplasia III based on enough evidence? A sys-tematic review of 3322 published patients. Gynecol Oncol 2005; 97:645–651.

9. Jones RW, Rowan DM, Stewart AW. Vulvar intraepithelial neoplasia:aspects of the natural history and outcome in 405 women. Obstet Gynecol2005; 106:1319–1326.

10. van Seters M, van Beurden M, ten Kate FJ, et al. Treatment of vulvarintraepithelial neoplasia with topical imiquimod. N Engl J Med 2008;358:1465–1473.

11. Tomson N, Sterling J, Miller R, Wilson C, Baldwin P. Vulval and perianalintraepithelial neoplasia: response to chemoradiotherapy in a womanwith idiopathic CD4 lymphocytopenia. Clin Exp Dermatol 2012; 37:865–868.

12. Todd RW, Luesley DM. Medical management of vulvar intraepithelialneoplasia. J Low Genit Tract Dis 2005; 9:206–212.

13. ter Haar G, Sinnett D, Rivens I. High intensity focused ultrasound: a sur-gical technique for the treatment of discrete liver tumours. Phys Med Biol1989; 34:1743–1750.

14. Doan N, Reher P, Meghji S, et al. In vitro effects of therapeutic ultrasoundon cell proliferation, protein synthesis, and cytokine production by humanfibroblasts, osteoblasts, and monocytes. J Oral Maxillofac Surg 1999;57:409–419.

15. Wang ZB. Application of high intensity focused ultrasound in the treat-ment of gynecology. Chin J Obstet Gynecol 2003; 38:510–513

16. Wang F, Li CZ, Wu YJ. Clinical and pathological analysis of focused ultra-sound in the treatment of vulval condyloma acuminatum. J ChongqingMed University 2005; 5:56–57.

17. Li C, Bian D, Chen W, Zhao C, Yin N, Wang Z. Focused ultrasound ther-apy of vulvar dystrophies: a feasibility study. Obstet Gynecol 2004;104:915–921.

18. Jia Y, Wu J, Chen L, et al. Focused ultrasound therapy for vulvar intraep-ithelial neoplasia in a mice model. Am J Med Sci 2013; 346:303–307.

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