Anti-PD-1 immunotherapy with adjuvant ablative fractional laser displays increased tumour clearance of squamous cell carcinoma, a murine study.

PD-1 checkpoint inhibitors are used as systemic immunotherapy for locally advanced and metastatic cutaneous squamous cell carcinoma (SCC); however, improved treatment efficacy is urgently needed. In this study, we aimed to investigate the effect of combining systemic anti-PD-1 treatment with adjuvant ablative fractional laser (AFL) in a spontaneous SCC mouse model. Tumours induced by ultraviolet radiation in the strain C3.Cg-Hrhr /TifBomTac were divided into four groups: anti-PD-1-antibody+AFL (n = 33), AFL alone (n = 22) anti-PD-1-antibody alone (n = 31) and untreated controls (n = 46). AFL was given at Day 0 (100 mJ/mb, 5% density), while anti-PD-1-antibody (ip, 200 µg) at Days 0, 2, 4, 6 and 8. Response to treatment was evaluated by tumour growth, survival time and by dividing response to treatment into complete responders (clinically cleared tumours), partial responders (reduced tumour growth rate compared to untreated controls) and non-responders (no decrease in tumour growth rate compared to untreated controls). The strongest tumour response was observed following the combination of systemic anti-PD-1 treatment combined with laser exposure, resulting in the highest percentage of complete responders (24%) compared with untreated controls (0%, p < 0.01), AFL monotherapy (13%, p > 0.05) and anti-PD-1-antibody monotherapy (3%, p > 0.05). Moreover, all three treatment interventions demonstrated significantly reduced tumour growth rates compared with untreated controls (p < 0.01), and the mice had significantly longer survival times (p < 0.01). In conclusion, the combination treatment revealed an improved treatment effect that significantly enhanced the complete tumour clearance not observed with the monotherapies, indicating a possible additive effect of anti-PD-1 with adjuvant AFL in treatment of SCC.

Ablative fractional laser treatment reduces hedgehog pathway gene expression in murine basal cell carcinomas.

This study aimed to investigate the impact of ablative fractional laser (AFL) on hedgehog pathway gene expression in murine microscopic basal cell carcinomas (BCCs) and compare these results to the effect of topical treatment with vismodegib, an FDA-approved hedgehog inhibitor. In 25 mice, 1 cm2 skin test sites (n = 44) containing microscopic BCCs were exposed to one of three interventions: a single CO2 AFL treatment (1 pulse, 40 mJ/microbeam, wavelength 10.6 µm, 5% density, pulse rate 250 Hz, n = 12), eight topical vismodegib treatments (3.8 mg/mL, n = 8), or combination of AFL and vismodegib treatments (n = 9). Untreated controls were included for comparison (n = 15). After 4 days, skin samples were analyzed for hedgehog gene expression (Gli1, Gli2, and Ptch1) by qPCR and vismodegib concentrations by liquid chromatography mass spectrometry (data analyzed with two-tailed t-tests and linear regression). A single treatment with AFL monotherapy significantly reduced hedgehog gene expression compared to untreated controls (Gli1 72.4% reduction, p = 0.003; Gli2 55.2%, p = 0.010; Ptch1 70.9%, p < 0.001). Vismodegib treatment also reduced hedgehog gene expression (Gli1 91.6%; Gli2 83.3%; Ptch1 83.0%), significantly surpassing AFL monotherapy for two out of three genes (Gli1, p = 0.017; Gli2, p = 0.007; Ptch1, p = 0.15). AFL and vismodegib combination mirrored the effects of vismodegib monotherapy (Gli1, p = 0.424; Gli2, p = 0.289; Ptch1, p = 0.593), possibly due to comparable cutaneous vismodegib concentrations (mean ± SD, vismodegib monotherapy 850 ± 475 µmol/L; combination 1036 ± 824 µmol/L; p = 0.573). In conclusion, a single AFL treatment significantly reduced hedgehog gene expression in murine BCCs mimicking the effects of eight topical applications of vismodegib. Further studies are needed to assess whether AFL can be utilized for BCC treatment, either as monotherapy or in combination with other drugs.

Impact of skin hydration on patterns of microthermal injury produced by fractional CO2 laser.

OBJECTIVES: The impact of skin hydration on patterns of thermal injury produced by ablative fractional lasers (AFLs) is insufficiently examined under standardized conditions. Using skin with three different hydration levels, this study assessed the effect of hydration status on microchannel dimensions generated by a fractional CO2 laser. METHODS: A hydration model (hyperhydrated-, dehydrated- and control) was established in ex vivo porcine skin, validated by changes in surface conductance and sample mass. After, samples underwent AFL exposure using a CO2 laser (10,600 nm) at two examined pulse energies (10 and 30 mJ/mb, fixed 10% density, six repetitions per group). Histological assessment of distinct microchannels (n = 60) determined three standardized endpoints in H&E sections: (1) depth of microthermal treatment zones (MTZs), (2) depth of microscopic ablation zones (MAZs), and (3) coagulation zone (CZ) thickness. As a supplemental in vivo assessment, the same laser settings were applied to hyperhydrated- (7-h occlusion) and normohydrated forearm skin (no pretreatment) of a human volunteer. Blinded measurement of MAZ depth (n = 30) was performed using noninvasive optical coherence tomography (OCT). RESULTS: Modest differences in microchannel dimensions were shown between hyperhydrated, dehydrated and control skin at both high and low pulse energy. Compared to controls, hyperhydration led to median reductions in MTZ and MAZ depth ranging from 5% to 8% (control vs. hyperhydrated at 30 mJ/mb; 848 vs. 797 µm (p < 0.003) (MAZ); 928 vs. 856 µm (p < 0.003) (MTZ)), while 14%-16% reductions were shown in dehydrated skin (control vs. dehydrated at 30 mJ/mb; MAZ: 848 vs. 727 µm (p < 0.003); MTZ: 928 vs. 782 µm (p < 0.003)). The impact of skin hydration on CZ thickness was in contrast limited. Corresponding with ex vivo findings, hyperhydration was similarly associated with lower ablative depth in vivo skin. Thus, median MAZ depth in hydrated skin was 10% and 14% lower than in control areas at 10 and 30 mJ/mb pulse energy, respectively (10 mJ: 210 vs. 180 µm (p < 0.001); 30 mJ: 335 vs. 300 µm (p < 0.001)). CONCLUSION: Skin hydration status can exert a minimal impact on patterns of microthermal injury produced by fractional CO2 lasers, although the clinical implication in the context of laser therapy requires further study.

Topical Delivery of Hedgehog Inhibitors: Current Status and Perspectives.

Systemic treatment with hedgehog inhibitors (HHis) is available to treat basal cell carcinomas but their utility is limited by adverse effects. Topical delivery methods may reduce adverse effects, but successful topical treatment depends on sufficient skin uptake, biological response, and time in tumor tissue. The aim of this review was to evaluate the current status of topical HHi delivery for BCCs and discuss barriers for translating systemic HHis into topical treatments. A literature search identified 16 preclinical studies and 7 clinical trials on the topical delivery of 12 HHis that have been clinically tested on BCCs. Preclinical studies on drug uptake demonstrated that novel formulations, and delivery- and pre-treatment techniques enhanced topical HHi delivery. Murine studies showed that the topical delivery of sonidegib, itraconazole, vitamin D3 and CUR-61414 led to biological responses and tumor remission. In clinical trials, only topical patidegib and sonidegib led to at least a partial response in 26/86 BCCs and 30/34 patients, respectively. However, histological clearance was not observed in the samples analyzed. In conclusion, the incomplete clinical response could be due to poor HHi uptake, biodistribution or biological response over time. Novel topical delivery techniques may improve HHi delivery, but additional research on cutaneous pharmacokinetics and biological response is needed.

Optical Imaging Visualizes a Homogeneous and Horizontal Band-Like Biodistribution of Large- and Small-Size Hydrophilic Compounds Delivered by Ablative Fractional Laser.

The skin barrier generally limits the topical delivery of hydrophilic molecules. Ablative fractional laser (AFL) facilitates cutaneous drug uptake of smaller hydrophilic compounds in several studies. In this imaging-based study, we aim to investigate the cutaneous biodistribution of two different-sized hydrophilic compounds delivered by an ablative fractional CO2 laser at minimally invasive settings. Intact or CO2 AFL-pretreated (2.5 mJ/mb and 5% density) ex vivo porcine skin was topically applied with a large or small hydrophilic compound (fluorescence labeled antibody nivolumab (150,000 g/mol, n = 4) or ATTO 647N (746 g/mol, n = 3)). Samples were incubated for 20 h in a Franz cell setup, whereafter optical coherence tomography (OCT) was used to assess laser channel depth, and ex vivo confocal microscopy (EVCM) was used to assess epidermal thickness and cutaneous biodistribution of nivolumab and ATTO 647N. With an EVCM-assessed median epidermal thickness of 70.3 µm and OCT-assessed ablation depth of 31.9 µm, minimally invasive settings enabled shallow penetration into the mid-epidermis. The AFL-assisted uptake of the antibody nivolumab and the smaller compound ATTO 647N showed a similar homogenous and horizontal band-like biodistribution pattern that reached mid-dermis. No uptake of nivolumab or ATTO 647N was observed in intact skin. In conclusion, AFL-induced mid-epidermal laser channels facilitates the cutaneous delivery of two hydrophilic compounds that are distributed in a similar homogeneous and horizontal band-like pattern, irrespective of their molecular size.

Anti-PD-1 Therapy with Adjuvant Ablative Fractional Laser Improves Anti-Tumor Response in Basal Cell Carcinomas.

The efficacy of anti-programmedcelldeath1therapy (aPD-1), which was recently approved for basal cell carcinoma (BCC) treatment, can be enhanced by adjuvant ablative fractional laser (AFL) in syngeneic murine tumor models. In this explorative study, we aimed to assess locally applied AFL as an adjuvant to systemic aPD-1 treatment in a clinically relevant autochthonous BCC model. BCC tumors (n = 72) were induced in Ptch1+/-K14-CreER2p53fl/fl-mice (n = 34), and the mice subsequently received aPD-1 alone, AFL alone, aPD-1+AFL, or no treatment. The outcome measures included mouse survival time, tumor clearance, tumor growth rates, and tumor immune infiltration. Both aPD-1 and AFL alone significantly increased survival time relative to untreated controls (31 d and 34.5 d, respectively vs. 14 d, p = 0.0348-0.0392). Complementing aPD-1 with AFL further promoted survival (60 d, p = 0.0198 vs. aPD-1) and improved tumor clearance and growth rates. The BCCs were poorly immune infiltrated, but aPD-1 with adjuvant AFL and AFL alone induced substantial immune cell infiltration in the tumors. Similar to AFL alone, combined aPD-1 and AFL increased neutrophil counts (4-fold, p = 0.0242), the proportion of MHCII-positive neutrophils (p = 0.0121), and concordantly, CD4+ and CD8+ T-cell infiltration (p = 0.0061-0.0242). These descriptive results suggest that the anti-tumor response that is generated by aPD-1 with adjuvant AFL is potentially promoted by increased neutrophil and T-cell engraftment in tumors. In conclusion, local AFL shows substantial promise as an adjuvant to systemic aPD-1 therapy in a clinically relevant preclinical BCC model.

Laser Immunotherapy: A Potential Treatment Modality for Keratinocyte Carcinoma.

The role of the immune system in cancer growth is well recognized and the development of immunotherapy represents a breakthrough in cancer treatment. Recently, the use of systemic immunotherapy was extended to keratinocyte carcinoma (KC), specifically locally advanced and metastasizing basal and squamous cell carcinoma. However, since most KC lesions are non-aggressive, systemic treatment with associated side effects is rarely justified. Conversely, topical immunotherapy with imiquimod remains restricted to premalignant and superficial lesions. Use of laser in the treatment of KC has evolved from physical tumor destruction and laser-assisted drug delivery to laser-mediated immune modulation. Evidence indicates that laser monotherapy can lead to immune cell infiltration, tumor reduction and resistance to tumor re-inoculation. Combining laser with immunotherapeutic agents, termed laser immunotherapy (LIT), may further potentiate immune activation and tumor response. Studies on LIT show not only direct anti-tumor effects but systemic adaptive immunity, illustrated by the prevention of tumor recurrence and regression in distant untreated tumors. These findings imply a therapeutic potential for both local and metastatic disease. This work provides rationales for immune-based treatment of KC and presents the current status of KC immunotherapy. Aiming to expand the field of KC immunotherapy, the review discusses the literature on immune activation following laser monotherapy and LIT.

Enhanced and Sustained Cutaneous Delivery of Vismodegib by Ablative Fractional Laser and Microemulsion Formulation.

Oral administration of vismodegib for basal cell carcinoma treatment is limited by significant class-specific systemic side effects. We investigated the approach of combining ablative fractional laser-assisted drug delivery with an extended-release microemulsion formulation of vismodegib to provide efficient cutaneous delivery in vivo. The developed formulation consisted of an oil-in-water microemulsion stabilized by Tween-80. Pig skin was exposed to ablative fractional laser followed by topical application of vismodegib microemulsion for 4 hours. At 4 hours, 2 days, 5 days, and 9 days, we evaluated vismodegib biodistribution in superficial, mid, and deep dermis and plasma (n = 189 measurements) and assessed local skin reactions. Sustained topical delivery of vismodegib was detected in all depths of ablative fractional laser-exposed skin over the course of the study, with peak concentrations found at 5 days and 9 days. The highest vismodegib concentrations reached 1,409.7 µmol/liter in superficial dermis and 62.3 µmol/liter in deep dermis, exceeding steady-state plasma concentrations previously reported for oral administration of vismodegib (5.5-56.0 µmol/liter). Ablative fractional laser increased vismodegib uptake up to 16.6-fold compared with intact skin. Only mild local skin responses to vismodegib were observed, and no vismodegib was detected in plasma. We report sustained topical delivery of vismodegib in vivo at high concentrations with favorable skin tolerability, suggesting a future safer vismodegib treatment.

Vehicle type affects filling of fractional laser-ablated channels imaged by optical coherence tomography.

Ablative fractional laser (AFXL) is an emerging method that enhances topical drug delivery. Penetrating the skin in microscopic, vertical channels, termed microscopic treatment zones (MTZs), the fractional technique circumvents the skin barrier and allows increased uptake of topically applied drugs. This study aims to elucidate the impact of vehicle type on the filling of MTZs from application of liquid, gel, and cream vehicles. Ex vivo pig skin was exposed to 10,600 nm fractional CO2 laser at 5% density, 120 µm beam diameter, and fluences of 40 and 80 mJ/microbeam (mJ/mb). Six repetitions were performed for each of six interventions (2 fluences and 3 vehicle types, n = 36). MTZ dimensions and filling by vehicle type were evaluated by optical coherence tomography, using blue tissue dye as a contrast-enhancing agent. Outcome measure was degree of MTZ filling assessed as percentages of empty, partially filled, and completely filled MTZs (108-127 MTZs/intervention analyzed) and evaluated statistically using Kruskal-Wallis and Dunn's tests. MTZs reached mid-dermal levels of 225 µm (40 mJ/mb) and 375 µm (80 mJ/mb) penetration depths (p < 0.0001). Filling of MTZs depended on type of applied vehicle. At 80 mJ/mb, liquid (67% completely filled, p < 0.01) and gel (60%, p < 0.05) formulations filled MTZs significantly better than cream formulation (31%). At 40 mJ/mb, liquid and gel formulations filled 90% (p < 0.05) and 77% (p > 0.05) of MTZs completely versus 55% for cream formulation. Thus, filling was overall greater for more superficial MTZs. In conclusion, vehicle type affects filling of MTZs, which may be of importance for AFXL-assisted drug delivery.

Expression patterns of nicotinamide phosphoribosyltransferase and nicotinic acid phosphoribosyltransferase in human malignant lymphomas.

The purpose of the study was to determine in human malignant lymphomas the expression patterns of nicotinamide phosphoribosyltransferase (NAMPT) and nicotinic acid phosphoribosyltransferase (NAPRT), the primary, rate-limiting enzymes in the synthesis of NAD+. NAMPT is a potential biomarker for sensitivity to NAMPT inhibitors and NAPRT is a biomarker for the use of nicotinic acid as a chemoprotectant in treatment with NAMPT inhibitors. The NAMPT inhibitor, APO866, is currently in clinical phase II trials in lymphomas. The expression of NAMPT and NAPRT was investigated in 53 samples of malignant lymphomas (diffuse large B-cell lymphoma, follicular B-cell lymphoma, Hodgkin's lymphoma and peripheral T-cell lymphoma). The expression of NAMPT was generally high in the more aggressive malignant lymphomas, with >80% strong expression, whereas the expression in the more indolent follicular lymphoma (FL) was significantly lower (>75% moderate or low expression, p = 0.0002). NAMPT was very highly expressed in Hodgkin Reed-Sternberg cells in Hodgkin's lymphoma. NAPRT expression was more varied (p > 0.0001) with 30-50% low expression except for Hodgkin's lymphoma where 85% displayed low expression (p = 0.0024). In conclusion, FL are a promising target for NAMPT inhibitors whereas substantial subsets of malignant lymphomas especially in Hodgkin lymphoma may be suitable for a combination treatment with nicotinic acid and NAMPT inhibitors.

A preclinical study on the rescue of normal tissue by nicotinic acid in high-dose treatment with APO866, a specific nicotinamide phosphoribosyltransferase inhibitor.

Inhibitor of nicotinamide phosphoribosyltransferase APO866 is a promising cancer drug currently in phase II clinical trials in oncology. Here, we present a strategy for increasing the therapeutic potential of APO866 through the rescue of normal tissues by coadministration of nicotinic acid (Vitamin B(3)). We examined the toxicity profile of APO866 in B6D2F1 mice and the effect of oral administration of nicotinic acid on tissue toxicity. Nicotinic acid (50 mg/kg) protects mice from death and severe toxicity from an APO866 dose (60 mg/kg) four times the monotherapy maximum tolerated dose (15 mg/kg). In a panel of six cancer cell lines, we find that three (including ML-2 cells) are protected by nicotinic acid in vitro, whereas the cytotoxicity of APO866 remains unaffected in the remaining three (including A2780 cells). A selective biomarker for the protection by nicotinic acid was subsequently identified by quantitative RT-PCR. The expression of nicotinic acid phosphoribosyltransferase is low in the cell lines not rescued from APO866 by nicotinic acid compared with protected cell lines. The findings in cell lines translated into xenograft models in which the combination of 50 mg/kg nicotinic acid and 50 mg/kg APO866 in mouse xenografts of A2780 cells increased life span by >3-fold compared with standard treatment of 15 mg/kg, and the effect of APO866 was clearly decreased when using the same treatment paradigm in ML-2 xenografts. In conclusion, the combination of high doses of APO866 with rescue by nicotinic acid may significantly increase the therapeutic potential in a subset of cancers with low expression of nicotinic acid phosphoribosyltransferase.

Anticancer agent CHS-828 inhibits cellular synthesis of NAD.

Malignant cells display increased demands for energy production and DNA repair. Nicotinamide adenine dinucleotide (NAD) is required for both processes and is also continuously degraded by cellular enzymes. Nicotinamide phosphoribosyltransferase (Nampt) is a crucial factor in the resynthesis of NAD, and thus in cancer cell survival. Here, we establish the cytotoxic mechanism of action of the small molecule inhibitor CHS-828 to result from impaired synthesis of NAD. Initially, we detected cross-resistance in cells between CHS-828 and a known inhibitor of Nampt, FK866, a compound of a structurally different class. We then showed that nicotinamide protects against CHS-828-mediated cytotoxicity. Finally, we observed that treatment with CHS-828 depletes cellular NAD levels in sensitive cancer cells. In conclusion, these results strongly suggest that, like FK866, CHS-828 kills cancer cells by depleting NAD.