Clinical and immunologic results of local hyperthermia at 44 °C for extensive genital warts in patients with diabetes mellitus

Abstract

Background: Genital warts are more extensive and difficult to treat in patients with diabetes mellitus due to defective immune responses.

Purpose: Our aim was to confirm the suitability of local hyperthermia for the treatment of genital warts in patients with diabetes mellitus and to investigate the immune cells in lesional areas at different time intervals after treatment.

Methods: We treated three diabetic patients with extensive genital warts by local hyperthermia at 44 °C for 30 min a day for 3 consecutive days plus 2 additional days 1 week later, then once a week till there showed signs of clinical clearance. Immunohistochemical profile was described on serial biopsies from a patient with confluent plaques.

Results: The warty lesions in the patients resolved in 6, 4 and 9 weeks, respectively. Immunohistochemical staining in the regressing warts revealed abundant infiltrating CD4+ T and CD8+ T lymphocytes (P < 0.01), as well as macrophages and CD1a+ dendritic cells.

Conclusions: This preliminary study suggested that local hyperthermia was a safe and effective single modality in the treatment of genital warts in diabetic patients and could induce a rapid immune response in lesional skin.

• Genital warts
• hyperthermia
• diabetes mellitus
• immunological

Introduction

Genital warts are a common infection of the ano-genital epithelium caused by human papillomavirus (HPV). Patients with diabetes mellitus (DM), once they acquire a genital HPV infection, tend to suffer from more extensive warts, require more treatments, and have more recurrences compared with patients without DM [1], possibly due to defective immune responses [2]. Ablation/destruction of infected tissue, antivirals and immune response modifiers are the therapeutic methods used most often, with reported clearance rates varying between 30–90%, and recurrence rates between 5% and 40% [3].

Hyperthermia alone, or in combination with other treatment, has been utilised in the treatment of some types of neoplasms, fungal and HPV infections [4–6]. Local hyperthermia has been used in treating different clinical types of HPV infection. The cure rates vary greatly from 41% to 93.5%, attributable to different hyperthermia levels (from 40 °C to 150 °C), protocols (successive or intermittent hyperthermia), and responsiveness of specific conditions [7]. We recently reported that intermittent mild local hyperthermia at 44 °C for 30 min cleared 53–57% of plantar warts, significantly superior to the sham treatment group (clearance rate of 11.54%) [8].

The present study aims to explore the feasibility of local hyperthermia at 44 °C to treat extensive genital warts in patients with DM. In addition, immunohistochemical evaluation was performed sequentially on wart tissue sections from distant sites away from the local hyperthermia targeted site.

Patients and methods

Three patients (two male, one female; mean age 22 years) with histologically confirmed genital warts were enrolled. Lesions were located on the foreskin/glans/urethral meatus/labias or perianal regions. Multiple lesions (>10) were documented in the three patients. Serological tests for HIV and syphilis were all negative. HPV type 6 and 11 were detected from lesional scrapings by flow-through hybridisation and gene chip (HybriMax), respectively [9]. None of the three patients had received any previous treatment. This trial was approved by the Ethics Committee of China Medical University (no. 22, 2009). All patients signed informed consent for the study.

Two had history of type II DM and one was type I DM for several years. Two had received regular insulin treatment since the establishment of the diagnosis. One had been taking antidiabetic drugs. The serum glucose level was controlled well in one patient. However, the serum glucose level was not controlled well in the other two patients; their fasting blood glucose levels ranged between 9.0–12.0 mmol/L and 13.4.0–21.41 mmol/L, postprandial glucose between 14.5–20.0 mmol/L and 17.9–33.88 mmol/L, urine sugar between ++ and +++, urinary ketone between + and +++.

In this study, we employed a patented hyperthermia device (China Medical University, Shenyang) with an energy source from a tungsten-halogen lamp (>90% wavelength between 760–2300 nm, peak wavelength 1200 nm). Heat emitted by the device concentrated locally on a target/treatment area of 0.5 × 0.5 cm without direct contact. The device was designed to sustain the heated skin surface at temperatures as desired by an infrared temperature monitor and a feedback circuit. We chose a confluent plaque as target site in each patient. The target lesion was subjected to local hyperthermia at 44 °C, once a day for 3 consecutive days, each treatment lasted 30 min. A week later, the patient received two more consecutive treatments. Then the patient received treatment once a week, until there was complete disappearance of the lesions. The patients scored the severity of pain by a 0–10 ascending visual analogue scale (0, no pain; 10, excruciating pain) during the treatments.

Biopsy specimens were obtained before and at 2, 4 and 6 weeks after local hyperthermia in untreated lesions distant from the target lesions in one patient. Skin specimens were fixed in buffered 10% formalin, embedded in paraffin, and sectioned. Monoclonal antibodies anti-CD4 (1:20), anti-CD8 (1:20), anti-CD1a (1:20) and anti-CD68 (1:20) were employed [10]. An eyepiece graticule was used in conjunction with a high-power (×40) objective lens, and a minimum of 10 fields was assessed per case.

Results

The three patients experienced apparent regression of their warts in 3, 2 and 4 weeks, respectively. Complete clearance of lesions was in 6, 4 and 9 weeks (Figure 1), respectively. All three patients tolerated the treatments well, albeit the patients experiencing moderate tolerable pain. The patients scored the severity of pain at 4–5, 2–2.5 and 3.5–5.5 by a 0–10 ascending visual analogue scale, respectively. No other significant side effects were observed. There was no sign of recurrence during a 6-month follow-up.

As shown in Table I and Figure 2, there were significant increases of CD4+ and CD8+ T cell infiltration at weeks 2 and 4 after initiation of hyperthermia. Epidermal CD1a+ and dermal CD68+ cells were also increased at week 4. At week 6, cellular infiltration recovered to baseline levels.

Figure 1. Extensive genital warts in patients with type I DM. Warty lesions before treatment (A), four weeks after hyperthermia (B), complete clearance of lesions nine weeks after hyperthermia (C).

Figure 2. Immunohistochemical staining for CD4, CD8, CD1a and CD68 cells in untreated lesions distant from the target lesions (10×).Thumbnails were taken from the same picture (40×).

Table I.  Counts of infiltrating immune cells in lesions before and 2, 4, 6 weeks after local hyperthermia in one patient. CD1a positive cells were distributed in both the epidermis and dermis, whereas CD4+, CD8+ and CD68+ cells were mostly distributed in the upper dermis close to the epidermis. The numbers of positive staining cells were counted under an eyepiece graticule with a high-power (×40) objective lens. Counts were done in at least 10 representative fields and cell numbers were expressed as mean ± SD.

	Pretreatment		Post-treatment (weeks)
	0	2	4	6
CD4 (dermis)	120.70 ± 28.23	193.40 ± 36.75**	214.80 ± 28.98**	135.60 ± 18.47
CD8 (dermis)	49.00 ± 21.71	101.70 ± 53.27**	100.40 ± 32.03**	69.60 ± 22.05
CD1a (dermis)	12.80 ± 10.01	16.90 ± 10.08	26.60 ± 15.99	27.00 ± 14.06*
CD1a (epidermis)	41.40 ± 15.00	27.20 ± 5.88	54.60 ± 16.08	76.00 ± 17.07**
CD68 (dermis)	33.50 ± 8.61	43.50 ± 10.89	60.20 ± 22.60**	31.60 ± 6.45

Dunnett's t test from the statistical package of one-way ANOVA was used to compare the number differences of infiltrating cells between the pretreatment (0 week) and those at 2 weeks/4 weeks/6 weeks, respectively. A p value of <0.05 was regarded as statistically significant (**P < 0.01; *P < 0.05).

Conclusion

There is a perception that the skin and mucosa of diabetes patients are colonised (and can be infected) by pathogenic microorganisms such as bacteria, viruses and fungi [11]. Selection of treatment for patients with genital warts complicated with DM is cautious, due to its possible subsequent infection, delayed wound healing, or recurrence. Mild local hyperthermia at a temperature of 44 °C achieved clearance of the warts in this case series. In our previous trial on immunocompetent patients with warts we designed a two-session treatment, i.e. the target lesion was subjected to local hyperthermia at 44 °C for 30 min, once a day for 3 consecutive days. In an interval of 1 week or 2 weeks the patient received two more consecutive treatments [8]. In the present trial we (as well as the patients) noted the warts continued to grow in size and number during the first two sessions of treatment. We then continued to apply local hyperthermia once a week until there were signs of clearance.

The mechanisms by which local hyperthermia successfully treats warts are not clear. In the present and previous reports we noted that patients with multiple warts experienced almost simultaneous clearance of targeted warts as well as the remaining untargeted ones, suggesting the establishment of a specific immune response against HPV infected tissues. Local hyperthermia could promote migrational maturation of Langerhans cells, rendering a higher possibility of antigen presentation in an ex vivo culture condition [12]. Hyperthermia may stimulate cytotoxic and apoptotic effects, which may help eliminate HPV-infected tissue [13], [14] and induce production of endogenous interferon in condyloma acuminatea [15].

By immunohistochemical staining on sequential biopsy specimens we noted that the regressing warts contained significantly more CD4+ and CD8+ T lymphocytes (P < 0.01 compared to baseline), lasting at least 2 weeks during the observation period. In addition, there was increased infiltration of CD68 positive (macrophages) and epidermal CD1a positive cells (most probably Langerhans cells) at a later stage. A similar phenomenon was also observed in photodynamic therapy for HPV infected skin, for which the elicitation of a specific host response to the infection was speculated [10].

There are some limitations in the present study. First, it is not a controlled study. Secondly, this pilot study conducted on a small series. A randomised controlled study on a larger series will be necessary to consolidate this pilot trial. In conclusion, this preliminary study suggested that local hyperthermia was a safe and effective single modality in the treatment of genital warts in patients with DM and could induce a rapid activation of specific immunity in lesional skin, while more detailed mechanisms of action remain to be investigated.

Acknowledgement

We thank Robert A. Schwartz of New Jersey Medical School for his critical reading of the manuscript.

Declaration of interest: This work was supported by the National Natural Science Foundation of China (30972659), Innovative Research Teams in Universities, Liaoning Bureau of Education (LT2012012) and Public Welfare Research Fund for Healthcare, Ministry of Health (201202013). The sponsors had no role in the design and conduct of the study; in the collection, analysis, and interpretation of data, or in the preparation, review, or approval of the manuscript. The authors alone are responsible for the content and writing of the paper.