Thomas J. Flotte

Damage to hair follicles by normal-mode ruby laser pulses

BACKGROUND Although many temporary treatments exist for hirsutism and hypertrichosis, a practical and permanent hair removal treatment is needed. OBJECTIVE Our purpose was to study the use of normal-mode ruby laser pulses (694 nm, 270 microseconds, 6 mm beam diameter) for hair follicle destruction by selective photothermolysis. METHODS Histologically assessed damage in ex vivo black-haired dog skin after the use of different laser fluences was used to design a human study; 13 volunteers with brown or black hair were exposed to normal-mode ruby laser pulses at fluences of 30 to 60 J/cm2, delivered to both shaved and wax-epilated skin sites. An optical delivery device designed to maximize light delivery to the reticular dermis was used. Hair regrowth was assessed at 1, 3, and 6 months after exposure by counting terminal hairs. RESULTS Fluence-dependent selective thermal injury to follicles was observed histologically. There was a significant delay in hair growth in all subjects at all laser-treated sites compared with the unexposed shaven and epilated control sites. At 6 months, there was significant hair loss only in the areas shaved before treatment at the highest fluence. At 6 months, four subjects had less than 50% regrowth, two of whom showed no change between 3 and 6 months. Transient pigmentary changes were observed; there was no scarring. CONCLUSION Selective photothermolysis of hair follicles with the normal-mode ruby laser produces a growth delay consistent with induction of prolonged telogen with apparently permanent hair removal in some cases.

Comparative Oncogenomics Identifies NEDD9 as a Melanoma Metastasis Gene

Genomes of human cancer cells are characterized by numerous chromosomal aberrations of uncertain pathogenetic significance. Here, in an inducible mouse model of melanoma, we characterized metastatic variants with an acquired focal chromosomal amplification that corresponds to a much larger amplification in human metastatic melanomas. Further analyses identified Nedd9, an adaptor protein related to p130CAS, as the only gene within the minimal common region that exhibited amplification-associated overexpression. A series of functional, biochemical, and clinical studies established NEDD9 as a bona fide melanoma metastasis gene. NEDD9 enhanced invasion in vitro and metastasis in vivo of both normal and transformed melanocytes, functionally interacted with focal adhesion kinase and modulated focal contact formation, and exhibited frequent robust overexpression in human metastatic melanoma relative to primary melanoma. Thus, comparative oncogenomics has enabled the identification and facilitated the validation of a highly relevant cancer gene governing metastatic potential in human melanoma.

Vascular Targeting in Photodynamic Occlusion of Subretinal Vessels

PURPOSE To evaluate the potential of photodynamic therapy (PDT) using benzoporphyrin derivative (BPD) for occlusion of subretinal neovascular membranes, the authors studied efficiency and collateral damage of PDT-induced photothrombosis in the rabbit choriocapillary layer. METHOD Benzoporphyrin derivative, a new photosensitizer, currently in clinical trials for tumor therapy, was used. Low-density lipoprotein served as a carrier to enhance selective targeting of vascular endothelial cells. RESULTS Complete choriocapillary occlusion was achieved at a BPD dose of 2 mg/kg and a radiant exposure as low as 10 J/cm2. When PDT was performed 3 hours after BPD application, damage to the neural retina was minimal. Only inner photoreceptor segments showed mitochondrial swelling probably secondary to choroidal ischemia. Bruchs membrane remained intact. Retinal pigment epithelium was invariably damaged as seen with other photosensitizers. CONCLUSION Compared with photocoagulation BPD-PDT allows endothelial-bound intraluminal photothrombosis, sparing important structures such as neural retina and Bruchs membrane. It may thus provide a more selective treatment of juxtafoveal and subfoveal neovascular membranes.

Detection of microscopic melanoma metastases in sentinel lymph nodes

Sentinel lymph node biopsy following radioisotope labeling is a recently developed, minimally invasive surgical staging procedure used in the management of primary cutaneous malignant melanoma. If histologic analysis reveals melanoma metastasis in the sentinel lymph node, completion lymphadenectomy is performed and adjuvant therapy considered. The routine pathologic assessment of the sentinel lymph node consists of bisecting the lymph node along its long axis and histologic examination of one hematoxylin and eosin–stained section of each cut surface.

Physical characteristics and biological effects of laser-induced stress waves

Laser-induced stress waves can be generated by one of the following mechanisms: optical breakdown, ablation, or rapid heating of an absorbing medium. These three modes of laser interaction with matter allow the investigation of cellular and tissue responses to stress waves with different characteristics and under different conditions. The effects of stress waves on cells and tissues can be quite disparate. Stress waves can fracture tissue, kill cells, decrease cell viability and increase the permeability of the plasma membrane. They can induce deleterious effects during medical procedures of high power, short pulse lasers or, alternatively, may facilitate new therapeutic modalities, such as drug delivery and gene therapy. This review covers the generation of laser-induced stress waves and their effects on cell cultures and tissue.

Merkel cell neoplasms. Histology, electron microscopy, biology, and histogenesis.

Seven primary malignant neoplasms of the skin composed of cells with features of Merkel cells are described. Each of them developed in older individuals, resembled either a large cell lymphoma or an undifferentiated carcinoma histologically, and contained APUD cell-type dense-core granules and intercellular junctions when studied by electron microscopy. Six neoplasms were situated in the dermis, but the seventh seemed to arise from the epidermis and, in addition, had a squamous component. Three neoplasms recurred following local resection and four metastasized to regional lymph nodes. A review of published reports of similar cases supports the impression that these neoplasms have a predilection for the skin of the face. The clinical behavior of these neoplasms, the likely relationship of the cells which constitute them to Merkel cells, and the implications of their differentiation for the embryological origin of Merkel cells arc discussed.

Lack of selectivity of protoporphyrin IX fluorescence for basal cell carcinoma after topical application of 5-aminolevulinic acid: implications for photodynamic treatment

Clinical trials of topical ALA in photodynamic therapy (PDT) of basal cell carcinoma (BCC) show significant recurrence rates. Exogenous 5-aminolevulinic acid (ALA) is converted by intracellular enzymes to photoactive protoporphyrin IX (PpIX) in human tissues. PpIX generates cytotoxic singlet oxygen when irradiated with visible light in the 400–640 nm range. To evaluate variability and heterogeneity in PpIX production by tumors in such trials, and to assess the usefulness of PpIX for marking skin tumors, we measured PpIX fluorescence distribution in BCC after topical application of 20% ALA cream. ALA cream was applied under occlusion for periods ranging from 3 to 18 h (average 6.9 h, SD 4 h) to 16 BCCs. ALA conversion to PpIX in the BCCs was assessed by in vivo photography, ex vivo video fluorescence imaging, and fluorescence microscopy. External macroscopic PpIX fluorescence, as assessed by in vivo and ex vivo imaging, correlated with the clinical presence of BCC. Examination by a digital imaging fluorescence microscope revealed inter- and intratumor fluorescence variability and heterogeneity. PpIX fluorescence corresponding to full tomor thickness was found in six superficial and four nodular tumors, and partial-thickness fluorescence was observed in five nodular tumors, but no PpIX fluorescence was observed in some areas of superficial, nodular and infiltrating tumors. In a significant number of nodular and infiltrating BCCs, topical ALA appeared to provide little or no PpIX in deep tumor lobules. In addition, no selectivity for tumor tissue versus normal epidermis was seen. The grossly brighter external PpIX fluorescence over tumors may be due, therefore, to enhanced penetration through tumor-reactive stratum corneum and to the tumor thickness. The absence of reproducible fluorescence marking of nodular and infiltrating BCC suggests that topical ALA, at least under the present delivery protocols, may not be a reliable regimen for photodynamic treatment of these BCCs.

Photodynamic Therapy of Experimental Choroidal Melanoma Using Lipoprotein-delivered Benzoporphyrin

BACKGROUND Benzoporphyrin derivative monoacid (BPD) is a new photosensitizer currently undergoing clinical trial for cutaneous malignancies. Compared with the clinically most frequently used sensitizer, Photofrin, BPD may offer higher tumor phototoxicity, better tissue penetration, and absence of significant skin sensitization. Low-density lipoprotein (LDL) carriers heighten efficiency and selectivity of BPD because neovascular and tumor cells express an increased number of LDL receptors. Hence, in addition to the vaso-occlusive effects similar to most other photosensitizers, LDL-BPD also has been shown to cause direct tumor cell damage. METHODS Benzoporphyrin derivative monoacid was complexed with human LDL and used in photodynamic treatment of choroidal melanomas experimentally induced in eight albino rabbits. Five rabbits served as controls. Three hours after intravenous injection of 2 mg/kg body weight of LDL-BPD, eight tumors were irradiated at 692 nm and 100 J/cm2 via an argon-pumped dye laser coupled into a slit lamp. RESULTS Angiography and histologic findings showed immediate photothrombosis after disintegration of endothelial membranes. After complete necrosis of tumor cells within 24 hours, a small fibrotic scar slowly developed. No tumor regrowth was noted up to 6 weeks when animals were killed. CONCLUSION These data suggest that photodynamic treatment with LDL-BPD may be a promising modality for multiple clinical applications, including tumors and neovascularizations II.

Verteporfin Photodynamic Therapy Retreatment of Normal Retina and Choroid in the Cynomolgus Monkey

OBJECTIVE This study evaluated the effect of repeated photodynamic therapy (PDT) applications on normal primate retina and choroid using an intravenous infusion of liposomal benzoporphyrin derivative (verteporfin). DESIGN This was an experimental study in a primate model. ANIMALS/CONTROLS: Six cynomolgus monkeys were used as experimental subjects and one monkey was used as a control subject. INTERVENTION Three consecutive PDT treatments at 2-week intervals were applied over the center of the fovea or the optic nerve of each eye. Verteporfin was delivered by intravenous infusion at a dose of 6 mg/m2, 12 mg/m2, or 18 mg/m2. Laser irradiation was then applied using a diode laser (689 nm) with light doses and spot sizes kept constant. MAIN OUTCOME MEASURES Findings were documented by fundus photography, fluorescein angiography, and light and electron microscopy. RESULTS A cumulative dose response was seen angiographically and histologically with more severe damage to the retina and choroid noted at higher dye doses. Photodynamic therapy applied to the macula using the 6-mg/m2 verteporfin dose showed recovery of choriocapillaris, with mild retinal pigment epithelium and outer photoreceptor damage at 6 weeks. At this dose, the optic nerve showed few focal sites of axon atrophy and capillary loss. Treatments over the macula using the 12-mg/m2 and 18-mg/m2 doses led to chronic absence of choriocapillaris and photoreceptors at 6 weeks. One of two optic nerves became atrophic after PDT applications using dye doses of 12 mg/m2, and both optic nerves became atrophic in the 18-mg/m2 dye dose group. CONCLUSION Limited damage to the retina, choroid, and optic nerve was present in primates treated with multiple PDT sessions using 6 mg/m2 verteporfin with light doses and the timing of irradiation kept constant. However, PDT using higher dye doses of 12 mg/m2 and 18 mg/m2 led to significant chronic damage to the normal retina, choroid, and optic nerve.

Photodynamic therapy of arteries. A novel approach for treatment of experimental intimal hyperplasia.

BACKGROUND Photodynamic therapy (PDT) uses light activation of otherwise nontoxic dyes for the production of reactive oxygen species that cause cell injury and death. METHODS AND RESULTS The inhibition of intimal hyperplasia (IH) by PDT was studied in the balloon injury model of the rat carotid artery. Chloroaluminum-sulfonated phthalocyanine (CASPc) was the drug chosen for PDT because it does not produce skin photosensitivity and has a high absorption peak of light at 675 nm, a wavelength with good tissue penetration. A pilot study indicated that CASPc administration with laser radiant exposure of 100 J/cm2 resulted in a homogeneous, circumferential effect on the whole artery. Male Sprague-Dawley rats received the balloon catheter injury to the left common carotid artery (day 0) and were equally divided into two groups. Nine rats received either CASPc (5 mg/kg i.v., n = 6) or saline (n = 3) at day 2, before IH was present, and nine rats received CASPc or saline in the same manner on day 7, when IH was already present. Twenty minutes after drug injection, the distal left common carotid artery was irradiated under saline with 675-nm laser light at 100 mW/cm2 for 10(3) seconds (100 J/cm2). At this low laser irradiance, there are no thermal effects, but photoactivation of CASPc occurs. The rats were killed at day 14 after balloon injury when IH reaches a maximum. The arteries were harvested after perfusion-fixation for light microscopy, histological and computerized morphometric evaluation, and transmission electron microscopy (TEM) analysis. The cross-sectional areas of the neointima were measured in the PDT-treated arteries and in the laser-only control arteries. There was a significant mean +/- SD decrease of IH in the PDT-irradiated segments of the arteries (0.06 +/- 0.05 mm2) versus the laser-only control ones (0.17 +/- 0.07 mm2) (t test, p less than 0.001), with no statistical difference between the day 2 and day 7 treated rats. Lack of IH was correlated in 90% of cases with histological absence of medial smooth muscle cells or inflammatory cells, but no other structural injury was identified. TEM analysis showed early evidence of PDT-mediated cytotoxic effects at 4 hours and the absence of collagen or elastic tissue structural alterations. CONCLUSIONS These data demonstrated that PDT can effectively inhibit the IH response when it is used before or during induction of cellular proliferation in this acute model. Although the long-term implications of PDT in arteries need to be defined, this technique may offer a new method for understanding and treating IH.