Ellen de Haas

Topical 5-Aminolaevulinic Acid Mediated Photodynamic Therapy of Superficial Basal Cell Carcinoma Using Two Light Fractions with a Two-hour Interval: Long-term Follow-up

Photodynamic therapy (PDT) of superficial basal cell carcinoma using topical 5-aminolaevulinic acid (ALA) and 75-100 J/cm(2) light dose yields unsatisfactory longterm results. In several animal models, illumination with two light fractions approximately 2 h apart was considerably more effective than single illumination, suggesting the need for a pilot clinical study. Fifteen patients with a total of 86 primary superficial basal cell carcinomas, received topical ALA and were illuminated 4 and 6 h later, both with 45 J/cm(2) laser light (633 +/- 1 nm). Fluorescence spectra were measured before and immediately after each illumination. At a mean follow-up of 59 months (range 44-82), 67 lesions could be evaluated, 56 of which showed a complete response (84%). Cosmesis was good/excellent in 88% of the complete response group and fair in 12%. There was no correlation between protoporphyrin fluorescence and response, but a significant correlation between the percentage of fluorescence left after photobleaching by the first illumination and the amount of protoporphyrin re-synthesized 2 h later. In conclusion, the long-term complete remission rate of fractionated ALA-mediated PDT of superficial basal cell carcinoma as reported here is significantly better than after PDT with single illumination previously reported by others, but equal to studies using single illumination with a much higher light fluence. Further improvement may be possible by reducing the fluence of the first fraction, with constant total fluence

Light fractionation significantly improves the response of superficial basal cell carcinoma to aminolaevulinic acid photodynamic therapy: five-year follow-up of a randomized, prospective trial.

Photodynamic therapy (PDT) using topical porphyrin-precursors is a promising treatment for superficial basal cell carcinoma (sBCC), but it needs further optimization. The aim of this study was to compare 5-year lesion (complete) response rates of sBCC treated with topical aminolaevulinic acid (ALA)-PDT using a single illumination vs. ALA-PDT using a 2-fold illumination scheme. A prospective, randomized study was performed, in which 91 patients with 299 lesions were treated with a 2-fold illumination scheme with 2 light fractions of 20 and 80 J/cm2 delivered 4 and 6 h after a single application of 20% ALA, and 106 patients with 274 lesions were treated with a single illumination of 75 J/cm2 4 h after a single application of 20% ALA. All lesions were treated at a fluence rate of 50 mW/cm2. An interim time to event analysis of complete response (CR) rates at 12 months showed encouraging results, and therefore lesions were followed for 5 years post-therapy. A third group of 50 patients with 172 lesions treated with 2-fold illumination were included after the initial period and analysed separately. The CR rate was significantly greater following the 2-fold illumination than the single illumination (p = 0.0002, log-rank test). Five years after therapy the CR rate after 2-fold illumination was 88%, whereas the CR rate after single illumination was 75%. The CR rate in the third group of lesions, treated with 2-fold illumination was 97% and 88% at 12 months and 5 years after therapy, respectively. Long-term follow-up indicates superior efficacy in sBCC of ALA-PDT with 2-fold illumination compared with ALA-PDT with single illumination.

Clinical Staging and Prognostic Factors in Folliculotropic Mycosis Fungoides

IMPORTANCE Large case series suggest that patients with folliculotropic mycosis fungoides (FMF) have a worse prognosis than patients with classic mycosis fungoides (MF). However, recent studies described a subgroup of patients with FMF with a more favorable prognosis. Distinction between indolent and aggressive FMF may have important therapeutic consequences but is hampered by the inability of the current tumor-node-metastasis-blood (TNMB) staging system to classify patients with FMF in a clinically meaningful way. OBJECTIVE To differentiate between indolent and aggressive FMF using clinicopathological criteria and to define prognostic factors in patients with FMF. DESIGN, SETTING, AND PARTICIPANTS In this prospective cohort study, we followed 203 patients with FMF, included in the Dutch Cutaneous Lymphoma Registry between October 1985 and May 2014 at a tertiary referral center hosting the Dutch Cutaneous Lymphoma Registry. Overall, 220 patients with FMF had been registered, but 17 patients with incomplete follow-up data or a history of classic MF were excluded. MAIN OUTCOMES AND MEASURES Main outcomes included clinical and histological characteristics, disease progression, and survival. Prognostic factors were investigated using Cox proportional hazard regression analysis. Distinction between early plaque-stage FMF and advanced plaque-stage FMF was made by a blinded review of skin biopsy specimens from patients presenting with plaques. RESULTS In a cohort of 147 men and 56 women (median [range] age, 59 [15-93] years), patients with histologically early plaque-stage FMF had a very similar overall survival (OS) rate to patients with only patches and/or follicular papules (10-year OS, 71% vs 80%), while the survival rate of patients with histologically advanced plaque-stage FMF was almost identical to that of patients presenting with tumors (10-year OS, 25% vs 27%). Subsequently, 3 clinical subgroups with significantly different survival data were distinguished: early skin-limited FMF (group A; n = 84; 5-year and 10-year OS, 92% and 72%); advanced skin-limited FMF (group B; n = 102; 5-year and 10-year OS, 55% and 28%); and FMF presenting with extracutaneous disease (group C; n = 17; 5-year and 10-year OS, 23% and 2%). Age at diagnosis, large cell transformation and secondary bacterial infection were independent risk factors for disease progression and/or poor survival. CONCLUSIONS AND RELEVANCE The results of this study provide useful criteria to differentiate between indolent and aggressive FMF and confirm the existence of a subgroup of FMF with a favorable prognosis.

Light fractionation does not enhance the efficacy of methyl 5-aminolevulinate mediated photodynamic therapy in normal mouse skin

Previous work demonstrated that fractionated illumination using two fractions separated by a dark interval of 2 h, significantly enhanced the clinical efficacy of photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA). Considering the increasing clinical use of methyl 5-aminolevulinate (MAL) and the expected gain in efficacy by light fractionation we have investigated the response to MAL-PDT using a single and a two-fold illumination scheme and compared that with ALA-PDT. Our results show that fractionated illumination does not enhance the efficacy of PDT using MAL as it does using ALA despite the comparable fluorescence intensities at the end of the first light fraction and at the start of the second light fraction. Only the initial rate of photobleaching was slightly greater during ALA-PDT although the difference was small. Previously we hypothesized that cells surviving the first fraction are more susceptible to the second fraction. Since this is not true for MAL-PDT our data suggest that the distribution of MAL and ALA in tissues, and therefore the site of PDT induced damage, is an important parameter in the mechanism underlying the 2-fold illumination scheme.

Fractionated illumination at low fluence rate photodynamic therapy in mice.

Photodynamic therapy (PDT) for actinic field cancerization is effective but painful. Pain mechanisms remain unclear but fluence rate has been shown to be a critical factor. Lower fluence rates also utilize available oxygen more efficiently. We investigated PDT effect in normal SKH1‐HR mice using low and high fluence rate aminolevulinic acid (ALA) PDT and a fractionated illumination scheme. Six groups of six mice with different light treatment parameters were studied. Visual skin damage was assessed up to 7 days post‐PDT. Fluorescence and reflectance spectroscopy during illuminations provided us with real‐time information about protoporphyrin IX (PpIX) photobleaching. A novel dosing approach was introduced in that we used a photobleaching percentage instead of a preset fluence. Data show similar total and maximum damage scores in high and low fluence rate groups. Photobleaching of PpIX in the low fluence rate groups shows a trend toward more efficient photobleaching. Results indicate that low fluence rate PDT is as effective as and more efficient than high fluence rate PDT in normal mouse skin. Low fluence rate PDT light protocols need to be explored in human studies in search for an effective and well‐tolerated treatment for actinic field cancerization.

Topical Photodynamic Therapy Using Different Porphyrin Precursors Leads to Differences in Vascular Photosensitization and Vascular Damage in Normal Mouse Skin

Different distributions of hexyl aminolevulinate (HAL), aminolevulinic acid (ALA) and methyl aminolevulinate (MAL) in the superficial vasculature are not well studied but they are hypothesized to play an important role in topical photodynamic therapy (PDT). The colocalization of fluorescent CD31 and protoporphyrin IX (PpIX) was calculated using confocal microscopy of mouse skin sections to investigate the vascular distribution after topical application. Vascular damage leads to disruption of the normal endothelial adherens junction complex, of which CD144 is an integral component. Therefore, normal CD31 combined with loss of normal fluorescent CD144 staining was visually scored to assess vascular damage. Both the vascular PpIX concentration and the vascular damage were highest for HAL, then ALA and then MAL. Vascular damage in MAL was not different from normal contralateral control skin. This pattern is consistent with literature data on vasoconstriction after PDT, and with the hypothesis that the vasculature plays a role in light fractionation that increases efficacy for HAL and ALA‐PDT but not for MAL. These findings indicate that endothelial cells of superficial blood vessels synthesize biologically relevant PpIX concentrations, leading to vascular damage. Such vascular effects are expected to influence the oxygenation of tissue after PDT which can be important for treatment efficacy.

Microscopic Distribution of Protoporphyrin (PpIX) Fluorescence in Superficial Basal Cell Carcinoma During Light-fractionated Aminolaevulinic Acid Photodynamic Therapy

Light fractionation in aminolaevulinic acid photodynamic therapy (PDT) of superficial basal cell carcinoma has been shown to enhance the therapeutic efficacy significantly. We have shown previously that this increase in efficacy is not simply due to an increase in the amount of protoporphyrin utilized during therapy. The present study investigated the spatial distribution of protoporphyrin in 32 superficial basal cell carcinomas undergoing light-fractionated PDT. Superficial fluorescence imaging performed during therapy was compared with the microscopic analysis of protoporphyrin fluorescence in biopsies acquired immediately before the second of two light fractions. The microscopic distribution of fluorescence was also compared with tumour sections immunohistochemically stained for Ki-67. Large variations in superficial and microscopic protoporphyrin fluorescence were found in both control and treated lesions. Despite these variations there was a reasonable correlation between the two techniques (R2=0.86). The mean fluorescence intensity in control biopsies was greater than in illuminated lesions before the second light fraction, but there was no significant difference in the variation within and between regions of interest in each of these sets of lesions. There was no clear trend in depth of protoporphyrin reappearance during the dark interval between light fractions. The general distribution of cells stained positive for Ki-67 followed the protoporphyrin fluorescence that was associated with islands of basal cell carcinoma. In conclusion, this study confirms that the mean relative re-synthesis of protoporphyrin after PDT is consistent with that found previously in pre-clinical models. There are large variations in fluorescence within superficial basal cell carcinoma that include regions of tumour cells that do not synthesize protoporphyrin.

Monitoring blood volume and saturation using superficial fibre optic reflectance spectroscopy during PDT of actinic keratosis

Optically monitoring the vascular physiology during photodynamic therapy (PDT) may help understand patient-specific treatment outcome. However, diffuse optical techniques have failed to observe changes herein, probably by optically sampling too deep. Therefore, we investigated using differential path-length spectroscopy (DPS) to obtain superficial measurements of vascular physiology in actinic keratosis (AK) skin. The AK-specific DPS interrogation depth was chosen up to 400 microns in depth, based on the thickness of AK histology samples. During light fractionated aminolevulinic acid-PDT, reflectance spectra were analyzed to yield quantitative estimates of blood volume and saturation. Blood volume showed significant lesion-specific changes during PDT without a general trend for all lesions and saturation remained high during PDT. This study shows that DPS allows optically monitoring the superficial blood volume and saturation during skin PDT. The patient-specific variability supports the need for dosimetric measurements. In DPS, the lesion-specific optimal interrogation depth can be varied based on lesion thickness.

Photodynamic and Nail Penetration Enhancing Effects of Novel Multifunctional Photosensitizers Designed for The Treatment of Onychomycosis.

Novel multifunctional photosensitizers (MFPSs), 5,10,15‐tris(4‐N‐methylpyridinium)‐20‐(4‐phenylthio)‐[21H,23H]‐porphine trichloride (PORTH) and 5,10,15‐tris(4‐N‐methylpyridinium)‐20‐(4‐(butyramido‐methylcysteinyl)‐hydroxyphenyl)‐[21H,23H]‐porphine trichloride (PORTHE), derived from 5,10,15‐Tris(4‐methylpyridinium)‐20‐phenyl‐[21H,23H]‐porphine trichloride (Sylsens B) and designed for treatment of onychomycosis were characterized and their functionality evaluated. MFPSs should function as nail penetration enhancer and as photosensitizer for photodynamic treatment (PDT) of onychomycosis. Spectrophotometry was used to characterize MFPSs with and without 532 nm continuous‐wave 5 mW cm−2 laser light (± argon/mannitol/NaN3). Nail penetration enhancement was screened (pH 5, pH 8) using water uptake in nails and fluorescence microscopy. PDT efficacy was tested (pH 5, ± argon/mannitol/NaN3) in vitro with Trichophyton mentagrophytus microconida (532 nm, 5 mW cm−2). A light‐dependent absorbance decrease and fluorescence increase were found, PORTH being less photostable. Argon and mannitol increased PORTH and PORTHE photostability; NaN3 had no effect. PDT (0.6 J cm−2, 2 μm) showed 4.6 log kill for PORTH, 4.4 for Sylsens B and 3.2 for PORTHE (4.1 for 10 μm). Argon increased PORTHE, but decreased PORTH PDT efficacy; NaN3 increased PDT effect of both MFPSs whereas mannitol increased PDT effect of PORTHE only. Similar penetration enhancement effects were observed for PORTH (pH 5 and 8) and PORTHE (pH 8). PORTHE is more photostable, effective under low oxygen conditions and thus realistic candidate for onychomycosis PDT.

Light Fractionation Significantly Increases the Efficacy of Photodynamic Therapy Using BF-200 ALA in Normal Mouse Skin.

Background Light fractionation significantly increases the efficacy of 5-aminolevulinic acid (ALA) based photodynamic therapy (PDT) using the nano-emulsion based gel formulation BF-200. PDT using BF-200 ALA has recently been clinically approved and is under investigation in several phase III trials for the treatment of actinic keratosis. This study is the first to compare BF-200 ALA with ALA in preclinical models. Results In hairless mouse skin there is no difference in the temporal and spatial distribution of protoporphyrin IX determined by superficial imaging and fluorescence microscopy in frozen sections. In the skin-fold chamber model, BF-200 ALA leads to more PpIX fluorescence at depth in the skin compared to ALA suggesting an enhanced penetration of BF-200 ALA. Light fractionated PDT after BF-200 ALA application results in significantly more visual skin damage following PDT compared to a single illumination. Both ALA formulations show the same visual skin damage, rate of photobleaching and change in vascular volume immediately after PDT. Fluorescence immunohistochemical imaging shows loss of VE-cadherin in the vasculature at day 1 post PDT which is greater after BF-200 ALA compared to ALA and more profound after light fractionation compared to a single illumination. Discussion The present study illustrates the clinical potential of light fractionated PDT using BF-200 ALA for enhancing PDT efficacy in (pre-) malignant skin conditions such as basal cell carcinoma and vulval intraepithelial neoplasia and its application in other lesion such as cervical intraepithelial neoplasia and oral squamous cell carcinoma where current approaches have limited efficacy.