First-in-human trial of nanoelectroablation therapy for basal cell carcinoma: Proof of method

Abstract

This nanoelectroablation therapy effectively treats subdermal murine allograft tumors, autochthonous basal cell carcinoma (BCC) tumors in Ptch1+/−K14-Cre-ER p53 fl/fl mice, and UV-induced melanomas in C57/BL6 HGF/SF mice. Here we describe the first human trial of this modality. We treated 10 BCCs on three subjects with 100–1000 electric pulses 100 ns in duration, 30 kV/cm in amplitude, applied at 2 pulses per second. Seven of the 10 treated lesions were completely free of basaloid cells when biopsied and two partially regressed. Two of the 7 exhibited seborrheic keratosis in the absence of basaloid cells. One of the 10 treated lesions recurred by week 10 and histologically had the appearance of a squamous cell carcinoma. No scars were visible at the healed sites of any of the successfully ablated lesions. One hundred pulses were sufficient for complete ablation of BCCs with a single, one minute nanoelectroablation treatment.

Background

Very short electric pulses in the nanosecond domain have been found to penetrate into cells and tissues and permeabilize both organelle membranes and the plasma membrane if the electric field strength is at least 10 kV/cm (1). One cellular response to the non-thermal application of hundreds of 100 ns pulses at 30 kV/cm is the initiation of apoptosis (2) and this nanoelectroablation therapy is very effective in eliminating murine skin tumors without scarring (3–7). Direct measurement of mouse skin temperature during the application of 100 pulses indicated a temperature increase of only a few degrees (3;5) Therefore this is a non-hyperthermic therapy.

Questions Addressed

The goals of this study were to test a new modality for the treatment of BCCs for the first-in-human trial, and to demonstrate safety and efficacy in treating human tumors leading to the application of this therapy to internal organ malignancies.

Experimental Design

This first Safety Clinical Trial of the NanoBlate System as a non-significant risk device was approved by the Children’s Hospital Oakland Research Institute’s IRB and was registered on Clinicaltrials.gov (NCT01463709). Three subjects were recruited to participate and provided written informed consent. About 14 weeks after treatment of 10 BCCs, the treated skin regions were removed and processed for histological examination by a dermatopathologist to provide an objective evaluation of the treated region. Pre-treatment biopsies were not conducted but post-treatment biopsies confirmed that the partially ablated lesions were BCCs by the presence of basaloid cells. Additional details regarding the methodology can be found in the supplementary material.

Results

We treated ten basal cell carcinomas (BCC) on three Caucasian patients with 100 to 1000 electric pulses, each 30 kV/cm in amplitude and 100 ns long, at a rate of 2 pulses per second (pps) (See supplemental material for electrode and pulse shape images). We found that a single treatment of 100 pulses per treatment zone was sufficient to cause most of these lesions to disappear during the ensuing several weeks. Edema was present following treatment and the penetration points of the electrode needles were also evident (fig. 1). A crust appeared over the treatment region within 2 days and began to fall off in 2 weeks. The skin was initially slightly pink in color and after a few weeks appeared normal such that some treated lesions could not be located several weeks after treatment without reference to a pre-treatment map. However, in one patient with skin that was slightly darker than that of the other two patients, there was a hyperpigmentation at the two treatment sites (fig. 2A,B). Histological analysis confirmed that this discoloration was not indicative of persisting tumor. Rather it is most likely due to inflammation-induced stimulation of melanin production by melanocytes that is subsequently taken up by macrophages that remain long after inflammation has ended. About 14 weeks after treatment we removed skin from the treated region and collected serial thin sections every 100 µm through the entire region. These H&E-stained sections were subjected to independent histological analysis by a dermatopathologist. Seven of the 10 lesions were completely free of basaloid cells and 2 were partially ablated. Both of these two had not been treated over their entire surface. One of these was lesion C in fig. 1. That lesion was slightly larger than our electrode; hence the single treatment of 380 pulses did not cover the entire lesion. The second partial ablation is shown in fig. 2C. This lesion was 2 cm long by 1 cm wide and, while we treated it with 100 pulses in 4 different regions, this treatment did not cover the entire lesion.

Figure 1. Five BCCs treated on a 55 year old woman with basal cell nevus syndrome.

A. 5 × 10 mm lesion treated with 100 p, 30 kV/cm over two regions to cover the entire lesion. Images indicate lesion appearance before and after nsPEF treatment as well as 2, 10 and 14 wk later. A histological section of each lesion collected at 14 wk is shown on the bottom. Lesion “A” was undetectable by 10 wk and histological analysis at 14 wk indicated a dermal scar characterized by horizontally oriented collagen bundles and an increased number of vertically oriented vessels. Carcinoma was not identified within the biopsied portion of this lesion. B. 5 mm-wide lesion treated with 100p, 30 kV/cm. Lesion was nearly gone by 10 wk and undetectable at 14 wk. Carcinoma was not identified within the biopsied portion of this lesion. C. 6 mm wide lesion treated with 500 pulses, 30 kV/cm. Histological analysis of biopsy indicated invasive squamous cell carcinoma, keratoacanthoma type, narrowly excised on plane of section examined. The cup-shared lesion invaginates into the dermis and is filled with keratin. Squamous cells show minimal atypia and cells at base of lesion have a glassy-appearing cytoplasm. D. 8 mm-long lesion treated with 380 pulses, 30 kV/cm. The electrode did not cover the entire lesion. Histology conducted at 14 wk shows skin with multiple buds and irregular proliferations of basaloid cells attached to the undersurface of the epidermis. The peripheral layer of basaloid cells shows nuclear palisading and there is focal retraction artifact between the tumor islands and surrounding stroma. Necrotic keratinocytes and mitotic figures are present among the tumor cells. E. 5 mm-wide lesion treated with 1000 pulses, 30 kV/cm. This lesion took longer to fade away. Histology conducted at 14 wk indicated a dermal scar, characterized by horizontally oriented collagen bundles and an increased number of vertically oriented vessels. Carcinoma is not identified within the biopsied portion of this lesion.

Figure 2. Five BCCs treated on two subjects.

A–B: 46 year old woman. Lesion A was treated with 100 pulses, 30 kV/cm over two regions to cover the entire lesion with the 5 mm × 7 mm electrode coverage. Lesion was gone by 7 wk and histological analysis of the treated region at 11 wk indicated dermal scar, characterized by horizontally oriented collagen bundles and an increased number of vertically oriented vessels. Dermal melanophages are present within the superficial dermis. There is minimal lymphohistiocytic infiltrate present. Carcinoma is not identified within the biopsied portion of this lesion. Lesion B was treated with 500 pulses, 30 kV/cm. The treated region was removed for biopsy at 11 weeks. Sections show a dermal scar, characterized by horizontally oriented collagen bundles and an increased number of vertically oriented vessels. There is focal calcification and keloidal collagen present within the dermis. A lymphohistiocytic infiltrate is present surrounding the dermal fibrosis. Carcinoma is not identified within the biopsied portion of this lesion. C–E: Three BCCs treated on the scalp of a 60 year old male. Lesion C was treated in four regions with 100 pulses, 30 kV/cm. At 15 wk the entire treated region was removed for histology. Sections show skin with buds and irregular proliferations of basaloid cells attached to the undersurface of the epidermis. The peripheral layer of basaloid cells shows nuclear palisading and there is focal retraction artifact between the tumor islands and surrounding stroma. Necrotic keratinocytes and mitotic figures are present among the tumor cells. Lesion D was treated with 1000 pulses, 30 kV/cm. Entire treated region was removed at 15 wk and histological analysis indicated skin with seborrheic keratosis, including orthokeratotic hyperkeratosis, acanthosis, horn pseudocysts, and an epidermal proliferation composed of small, bland, cuboidal keratinocytes. This proliferation lacks significant cytologic atypia or mitotic activity. Dermal fibrosis is present. Lesion E was treated in two regions with 100 pulses, 30 kV/cm. The entire treated region was removed at 15 wk and histological analysis indicated seborrheic keratosis, including orthokeratotic hyperkeratosis, acanthosis, horn pseudocysts, and an epidermal proliferation composed of small, bland, cuboidal keratinocytes. This proliferation lacks significant cytologic atypia or mitotic activity. Dermal fibrosis is present.

Lesion C in figure 1 appeared to be completely covered by our electrode and was not visible on week 2. However, by week 10 a recurrent tumor was visible which histologically had the appearance of a squamous cell carcinoma (SCC)

Conclusions

This first-in-human safety trial of nanoelectroablation indicated that this new therapy is safe and may offer a fast and scarless alternative to the current standard of care for small BCCs. The main advantages of this therapy over surgical excision or electro-desiccation and curettage are the reduced pain, the short treatment time and the absence of scarring. The efficacy of this treatment modality was good. Assuming that lesion C (fig. 1) was actually a SCC that recurred, seven of the nine BCCs treated (78%) exhibited no basaloid cells when biopsied at 14 weeks after treatment. Three of these lesions disappeared completely, two exhibited hyperpigmentation and two exhibited seborrheic keratosis.

We still need to conduct a real validation of this method with sufficient clinical data. Thereafter we can compare it with other methods with relevant information for clinicians.”

This therapy could also be used to ablate other lesion types. BioElectroMed has had success ablating angiomas, moles and lentigines. Another group has used 20 ns pulses to successfully ablate several other tumor types in mice and one BCC on a human subject (8;9)

Abbreviations

nsPEF

nanosecond pulsed electric fields

pps

pulses per second