Peritumoral Skin Research Articles

Melanoma-associated repair-like Schwann cells suppress anti-tumor T-cells via 12/15-LOX/COX2-associated eicosanoid production

ABSTRACT Peripheral glia, specifically the Schwann cells (SCs), have been implicated in the formation of the tumor microenvironment (TME) and in cancer progression. However, in vivo and ex vivo analyses of how cancers reprogram SC functions in different organs of tumor-bearing mice are lacking. We generated Plp1-CreERT/tdTomato mice which harbor fluorescently labeled myelinated and non-myelin forming SCs. We show that this model enables the isolation of the SCs with high purity from the skin and multiple other organs. We used this model to study phenotypic and functional reprogramming of the SCs in the skin adjacent to melanoma tumors. Transcriptomic analyses of the peritumoral skin SCs versus skin SCs from tumor-free mice revealed that the former existed in a repair-like state typically activated during nerve and tissue injury. Peritumoral skin SCs also downregulated pro-inflammatory genes and pathways related to protective anti-tumor responses. In vivo and ex vivo functional assays confirmed immunosuppressive activities of the peritumoral skin SCs. Specifically, melanoma-reprogrammed SCs upregulated 12/15-lipoxygenase (12/15-LOX) and cyclooxygenase (COX)-2, and increased production of anti-inflammatory polyunsaturated fatty acid (PUFA) metabolites prostaglandin E2 (PGE2) and lipoxins A4/B4. Inhibition of 12/15-LOX or COX2 in SCs, or EP4 receptor on lymphocytes reversed SC-dependent suppression of anti-tumor T-cell activation. Therefore, SCs within the skin adjacent to melanoma tumors demonstrate functional switching to repair-like immunosuppressive cells with dysregulated lipid oxidation. Our study suggests the involvement of the melanoma-associated repair-like peritumoral SCs in the modulation of locoregional and systemic anti-tumor immune responses.

Micro-nevi in peri-tumoral skin of cutaneous melanoma and basal cell carcinoma

Background: Harboring many melanocytic nevi is a risk factor for melanoma. The prevalence of micro-nevi, melanocytic nevi that can only be detected microscopically, has been studied scarcely and the significance is unknown. Objectives: To systematically analyze the presence of micro-nevi in cutaneous excisions from cutaneous melanoma and basal cell carcinoma to achieve an insight in the density of micro-nevi per mm peri-tumoral skin. Also to speculate in the relevance and significance of micro-nevi for evolving melanoma at the same anatomical site. Methods: We re-examined histologically peri-tumoral skin of primary and re-excised melanomas and primary excisions of basal cell carcinomas for comparison. The size of a micro-nevus was defined to a diameter < 2 mm, of either a junctional, compound or intradermal nevus. Results: 104 melanoma and 208 basal cell carcinoma cases were microscopically re-examined. In the melanoma group 6 micro-nevi were found. The sum of the peri-tumoral skin was 1768 mm, giving a density of 1 nevus per 295 mm. In the basal cell carcinoma group 5 micro-nevi were found. The sum of the peri-tumoral skin was 1370 mm, giving a density of 1 nevus per 274 mm. No difference in the density of micro-nevi in the two groups of skin cancer was found. Conclusions: The significance of these micro-nevi remains unclear. The aim with this study is to draw attention to the existence of micro-nevi and further studies are needed to determine the significance of the presence of micro-nevi.

Biological Effects of Low Dose Radiation (LDR) on Peri-Tumoral and Tumoral Areas with Squamous Cell Skin Cancer

<h3>Purpose/Objective(s)</h3> Low dose radiation (LDR) may have paradoxically beneficial effects via an adaptive response. We aimed to assess the adaptive response of healthy and malignant skin tissue to low doses of radiation and explore the molecular details behind the induced adaptive response. <h3>Materials/Methods</h3> We conducted a prospective, randomized pilot study (N=8) to ascertain the biological effects of integrating 0.1 Gy of LDR into standard radiotherapy sessions (2.0 Gy fractions) over the first two weeks of radiotherapy for patients 18-80 years of age (both sexes) diagnosed with squamous cell carcinoma of skin (lesions of 1 – 10 cm). Radiation treatment was done with a linear accelerator. The patients received definitive radiation for the tumor as determined by the treating physician using 2.0 Gy fractions. In addition, 0.1 Gy of LDR was integrated into the standard radiotherapy course on day 0 and day 6 over the first two weeks of treatment. Radiation delivery to healthy tissue was with a 1.5 cm margin around the tumor using a mask over the tumor. We obtained partial thickness skin biopsy samples of the tumor and peri-tumoral normal tissue before treatment from four patients (for control). An additional four patients (actively treated) were biopsied before and 24 hours after 0.1 Gy of LDR (day 0, 1 and day 6, 7). RNA was obtained from skin biopsies and processed for genetic expression (138K genes). <h3>Results</h3> The change in gene expression in healthy peri-tumoral tissues due to LDR exposure was relatively low (only 325 genes: 170 upregulated, 155 downregulated). The <i>senescence and autophagy in cancer</i> and <i>genotoxicity pathway</i> genes showed the most significant downregulation in peritumoral healthy tissue comparing days 0 and 1. The biological pathways in peritumoral healthy tissue comparing days 6 and 7 showed predominantly downregulation of genes with most pronounced downregulation seen in <i>electron transport chain in mitochondria</i> and genes involved in <i>non-alcoholic fatty liver disease</i>. In contrast to normal peritumoral tissue, the difference in gene expression in tumor tissue due to LDR exposure was significantly more pronounced (5,651 genes: 2,896 upregulated, 2,755 downregulated) The <i>allograft rejection</i> genes and <i>nuclear receptor meta-pathway</i> were the most upregulated in the tumoral tissues comparing days 0 and 1. The nuclear receptor meta-pathway were the most upregulated in the tumoral tissues on days 6 and 7. The <i>Allograft rejection pathway in skin cancer</i> was among those upregulated. <h3>Conclusion</h3> LDR had a modest effect on miRNA expression when applied to healthy peritumoral skin. Downregulated pathways predominate, and DNA repair mechanisms are stimulated by the first dose in healthy tissue. In contrast, a more pronounced effect on gene expression (as measured by miRNA profiles), including upregulation of several key relevant pathways, was induced by LDR to malignant tissue.

Autofluorescence identifies highly phagocytic tissue-resident macrophages in mouse and human skin and cutaneous squamous cell carcinoma.

Macrophages from human and mouse skin share phenotypic and functional features, but remain to be characterized in pathological skin conditions. Skin-resident macrophages are known to derive from embryonic precursors or from adult hematopoiesis. In this report, we investigated the origins, phenotypes and functions of macrophage subsets in mouse and human skin and in cutaneous squamous cell carcinoma (cSCC) using the spectral flow cytometry technology that enables cell autofluorescence to be considered as a full-fledged parameter. Autofluorescence identifies macrophage subsets expressing the CD206 mannose receptor in human peri-tumoral skin and cSCC. In mouse, all AF+ macrophages express the CD206 marker, a subset of which also displaying the TIM-4 marker. While TIM-4-CD206+ AF+ macrophages can differentiate from bone-marrow monocytes and infiltrate skin and tumor, TIM-4 identifies exclusively a skin-resident AF+ macrophage subset that can derive from prenatal hematopoiesis which is absent in tumor core. In mouse and human, AF+ macrophages from perilesional skin and cSCC are highly phagocytic cells contrary to their AF- counterpart, thus identifying autofluorescence as a bona fide marker for phagocytosis. Our data bring to light autofluorescence as a functional marker characterizing subsets of phagocytic macrophages in skin and cSCC. Autofluorescence can thus be considered as an attractive marker of function of macrophage subsets in pathological context.

461 Telomerase Reverse Transcriptase Mutations, Enzyme Activity and Telomere Length in Cutaneous Basal Cell Carcinoma

We aimed to:1) Analyse telomerase reverse transcriptase (TERT) mutations in cutaneous basal cell carcinoma (CBC) tumours and describe its relationship with intra-tumour enzyme activity and telomere length (TL). 2) Compare TERT activity between tumour and clinically healthy peritumoral skin. 3) Compare TL between CBC and peripheral blood. A cohort of 45 patients with 50 CBC were included. All tumours were located in the neck, face or scalp. Usual clinicopathological variables were recorded for each patient and tumour. TERT mutations (by direct sequencying) and TERT enzyme activity (by qPCR) were studied in all 50 CBC and its peritumoral clinically healthy skin. TL were measured in 44 tumours and in peripheral blood leukocytes in 30 patients. TERT mutations existed in 32 of 43 (69,76%) of the validly sequenced tumours (sequencyng failed in 7 CBC), with 8 of 43 (18,6%) carrying two mutations, mostly showing the UV signature (C→T and CC→TT). TERT activity was significantly more active in tumoral CBC tissue than in peritumoral clinically healthy skin samples (p=0,012). Telomeres were significantly longer in tumour samples than in peripheral blood (p=0.002). TERT mutations are frequent in CBC. TERT is activated in most CBC, mainly due to activating mutations in TERT although alternate pathways may be involved since it is also active in CBC without TERT mutations. Telomere length is longer in tumoral tissue than in peripheral blood in patients with CBC.

Beta Human Papillomavirus Infection Is Prevalent in Elephantiasis and Exhibits a Productive Phenotype: A Case-Control Study.

Elephantiasis is considered a cutaneous region of immune deficiency with cobblestone-like surface caused by a wart-like eruption. Verrucosis is a diffuse human papillomavirus (HPV) infection linked to immunodeficiency disorders. The objective of this study was to examine the prevalence of HPV infection in lymphedema and its pathogenic role in elephantiasis. A retrospective case-control study was performed examining lymphedematous skin and controls of peritumoral normal skin. HPV infection was evaluated at the DNA, protein, and histopathologic levels by polymerase chain reaction, immunohistochemistry, and light microscopy, respectively. Overall, 540 HPV DNAs were detected in 120 of 122 cutaneous samples (median 4 HPV DNAs per sample, range 0-9). Compared with controls, no differences existed in type or number of HPVs identified. Instead, a diverse spectrum of HPV-related histopathologies were evident, likely reflecting the multiplicity of HPV genotypes detected. Most notably, increasing histopathologic lymphedema stage significantly correlated with markers of productive HPV infection such as altered keratohyaline granules and HPV L1 capsid expression. Limitations of this study are the absence of normal skin controls not associated with neoplasia or subclinical lymphedema, and lack of assessment of HPV copy number per keratinocyte infected. In conclusion, productive HPV infection, not HPV type or numbers detected, distinguished lymphedematous skin from controls. These findings support the theory that lymphedema creates a region of depressed immunity that permits productive HPV infection, manifested clinically by diffuse papillomatosis, characteristic of elephantiasis.

Peripheral Mohs micrographic surgery for the treatment of extramammary Paget's disease

Objective To evaluate effectiveness of peripheral Mohs micrographic surgery for the treatment of extramammary Paget's disease (EMPD). Methods A total of 28 patients with EMPD were treated with peripheral Mohs micrographic surgery. The depth and extent of tumor infiltration were evaluated before the surgery. One day before the surgery, 20% aminolevulinic acid hydrochloride was topically applied to determine and label surgical margins under a Wood's lamp. After fluorescence-based localization, peritumoral skin tissues were resected and underwent frozen-section examination according to the protocol for Mohs micrographic surgery. Meanwhile, the tumor was resected. After surgery, patients were followed up every 3 - 6 months to detect local recurrence and metastasis. Results Of the 28 patients, 25 were male and 3 were female. Six patients each underwent 3 sessions of frozen-section examination, and 12 patients each received 2 sessions, with an average of 1.86 sessions for each patient. During the follow-up for 5-72 months, local recurrence occurred in 3 cases, and 1 patient died of tumor metastasis and uremia after 2 years of follow-up. Conclusion Peripheral Mohs micrographic surgery is a time-saving and effective treatment for EMPD. Key words: Mohs surgery; Paget disease, extramammary; Treatment outcome; Recurrence

Novel CARD11 Mutations in Human Cutaneous Squamous Cell Carcinoma Lead to Aberrant NF-κB Regulation

NF-κB signaling plays a crucial role in regulating proliferation and differentiation in the epidermis. Alterations in the NF-κB pathway can lead to skin pathologies with a significant burden to human health such as psoriasis and cutaneous squamous cell carcinoma (cSCC). Caspase recruitment domain (CARD)-containing scaffold proteins are key regulators of NF-κB signaling by providing a link between membrane receptors and NF-κB transcriptional subunits. Mutations in the CARD family member, CARD14, have been identified in patients with the inflammatory skin diseases psoriasis and pityriasis rubra pilaris. Here, we describe that the gene coding for another CARD scaffold protein, CARD11, is mutated in more than 38% of 111 cSCCs, and show that novel variants outside of the coiled-coil domain lead to constitutively activated NF-κB signaling. CARD11 protein expression was detectable in normal skin and increased in all cSCCs tested. CARD11 mRNA levels were comparable with CARD14 in normal skin and CARD11 mRNA was increased in cSCC. In addition, we identified CARD11 mutations in peritumoral and sun-exposed skin, suggesting that CARD11-mediated alterations in NF-κB signaling may be an early event in the development of cSCC.

In vivo skin fluorescence imaging in young Caucasian adults with early malignant melanomas.

BackgroundHuman cutaneous malignant melanoma (CMM) is an aggressive cancer showing a dramatic worldwide increase in incidence over the past few decades. The most prominent relative epidemiological increase has been disclosed in young women. The aim of the study was to assess the effects of chronic sun exposures in order to rate the extend of melanocytic stimulations in the vicinity of CMM.MethodsThe study was designed to evaluate the melanin distribution and density using ultraviolet light illumination. The present study was performed on surgical excision specimens of thin CMM lesion removed from the upper limbs of 55 Caucasian adults (37 women and 18 men). Two control groups comprised 23 men and 21 women of similar ages who had medium-size congenital melanocytic nevi, also present on the upper limbs. The peritumoral skin was scrutinized using a Visioscan® VC98 device, revealing the faint mosaic melanoderma (FMM) pattern that grossly indicates early signs of chronic photodamage in epidermal melanin units.ResultsThe median extent of relative FMM was significantly higher in the CMM male group. By contrast, the CMM female group showed a reverse bimodal distribution in FMM size. Only 12/37 (32.5%) of the CMM female group had an increased FMM size, whereas 25/37 (67.5%) of females with CMM had a global FMM extent in the normal range, relative to the controls.ConclusionThin CMM supervening in young women appear unrelated to repeat photoexposure. Other mechanisms are possibly involved.

Expression of proteins involved in epigenetic regulation in human cutaneous melanoma and peritumoral skin.

Epigenetic processes play a critical role in melanoma development. However, little is known about proteins responsible for epigenetic transformations in melanoma cells. The processes in the peritumoral skin within the excision margin are almost unstudied. We studied the changes in expression of 112 proteins involved in epigenetic regulation of gene expression in the human cutaneous melanoma and its peritumoral zone using "The Proteomic Antibody Microarrays" (GRAA2, Sigma-Aldrich). Dimethylated histone H3 at lysines 4 and 9 as well as proteins involved in the regulation of transcription (histone deacetylases HDAC-1 and HDAC-11, DNA methyl-binding protein Kaiso), cell cycle control (protein kinases Aurora-В and PKR, chromosome protein CENP-E , and phosphorylated and acetylated histone H3), DNA repair (phosphorylated histone H2AX), and nuclear protein import (importins α3 and α5/7) were over-expressed in the melanoma tissue as compared with normal skin. At the same time, HDAC-10 and proliferating cell nuclear antigen PCNA were downregulated. In the peritumoral skin, at the excision margin (1-2 cm from the melanoma edge), we observed similar changes in expression of these proteins and, additionally, over-expression of arginine methyltransferases PRMT5 and NAD-dependent histone deacetylase SIR2. Histone methyltransferase G9a and metastasis-associated protein 2 were downregulated. Therefore, epigenetic regulation that requires histone modifications and expression of some regulatory proteins is of importance for melanoma development and propagation. The observed changes in the peritumoral skin may indicate the epigenetic pre-tuning in this zone possibly involved in malignant transformation. These results can be potentially useful for melanoma diagnostics and targeted therapy.

Langerhans Cells from Human Cutaneous Squamous Cell Carcinoma Induce Strong Type 1 Immunity

Langerhans cells (LCs) are dendritic cells (DCs) localized to the epidermis. They should be the first antigen-presenting cells to encounter squamous cell carcinoma (SCC). The aim of this study was to investigate the ability of LCs isolated from human SCC to induce T-cell proliferation and polarization. We investigated the ability of LCs from SCC and peritumoral skin to induce T-cell proliferation and polarization. We also studied the effect of SCC supernatant on the ability of LCs from normal skin, in vitro-generated LCs, and DCs to activate and polarize T cells. LCs from SCC were stronger inducers of allogeneic CD4(+) and CD8(+) T-cell proliferation and IFN-γ production than LCs from peritumoral skin. We found that tumor supernatants (TSNs) were rich in immunosuppressive cytokines; despite this, allogeneic CD4(+) and CD8(+) T-cell proliferation and IFN-γ induction by LCs were augmented by TSN. Moreover, TSN facilitated IFN-γ induction by in vitro-generated LCs, but suppressed the ability of in vitro-generated DCs to expand allogeneic CD4(+) and CD8(+) T cells. We have demonstrated that LCs from SCC can induce type 1 immunity. TSN induces IFN-γ induction by in vitro-generated LCs. This contrasts greatly with prior studies showing that DCs from SCC cannot stimulate T cells. These data indicate that LCs may be superior to DCs for SCC immunotherapy and may provide a new rationale for harnessing LCs for the treatment of cancer patients.

Lymphangiectases Are Common Underlying Warts and in Normal Peritumoral Skin: Histologic Evidence of Decreased Immune Surveillance

Lymphangiectases are a histologic sign of lymphostasis, which is associated with decreased immune cell trafficking and cell-mediated immunity. To determine if latent lymphedema is apparent underlying warts and in skin affected by cutaneous neoplasia. The number and maximal dilation of lymphangiectases were measured in the upper half of the dermis of 51 consecutive biopsies of warts, 230 consecutive normal skin samples from primary skin tumor excisions, and 14 normal skin samples from breast reduction (11) and panniculectomy (3) specimens. All warts had one or more underlying lymphangiectases compared with 79% of peritumor normal skin samples and 50% of cosmetic specimens. The mean number of lymphangiectases and mean maximal dilation were significantly greater in warts than in peritumor skin, which was significantly greater than cosmetic skin samples (3.6 vs. 1.3 vs. 0.12 lymphangiectases per square millimeter and 54 vs. 23 vs. 1 μm, respectively; P = 0.0001). Warts exhibited mild fibrosis significantly more frequently than peritumor skin (57% vs. 5%; P = 0.0001). For peritumor (normal) skin, age, solar elastosis, and adjacent malignancy correlated with greater dilation of lymphatics. Solar elastosis also correlated with increased number of lymphangiectases. Minor trauma and solar elastosis from chronic ultraviolet radiation exposure are likely the etiologic factors in the development of lymphostasis. By decreasing immune surveillance, latent lymphedema ostensibly facilitates human papillomavirus infection and carcinogenesis.

Lymphangiogenesis Linked to VEGF-C from Tumor-Associated Macrophages: Accomplices to Metastasis by Cutaneous Squamous Cell Carcinoma?

During wound healing, dermal macrophages secrete lymphangiogenic vascular endothelial growth factor (VEGF)-C, and lymphatic vessels transport cytokines and cells to draining lymph nodes. In this issue, Moussai et al. show that macrophages in peritumoral nonlesional skin near squamous cell carcinoma secrete prolymphangiogenic VEGF-C. Their study suggests how tumor-associated macrophages and neolymphatic vessels may coordinate metastasis starting early in cutaneous squamous cell carcinoma.

O.595 SCC of external ear – to what extent should we investigate?

NF-κB signaling plays a crucial role in regulating proliferation and differentiation in the epidermis. Alterations in the NF-κB pathway can lead to skin pathologies with a significant burden to human health such as psoriasis and cutaneous squamous cell carcinoma (cSCC). Caspase recruitment domain (CARD)-containing scaffold proteins are key regulators of NF-κB signaling by providing a link between membrane receptors and NF-κB transcriptional subunits. Mutations in the CARD family member, CARD14, have been identified in patients with the inflammatory skin diseases psoriasis and pityriasis rubra pilaris. Here, we describe that the gene coding for another CARD scaffold protein, CARD11, is mutated in more than 38% of 111 cSCCs, and show that novel variants outside of the coiled-coil domain lead to constitutively activated NF-κB signaling. CARD11 protein expression was detectable in normal skin and increased in all cSCCs tested. CARD11 mRNA levels were comparable with CARD14 in normal skin and CARD11 mRNA was increased in cSCC. In addition, we identified CARD11 mutations in peritumoral and sun-exposed skin, suggesting that CARD11-mediated alterations in NF-κB signaling may be an early event in the development of cSCC.

Muscarinic receptors autoantibodies purified from mammary adenocarcinoma-bearing mice sera stimulate tumor progression

The ability of tumor cells to stimulate adaptive immunity, particularly by inducing anti-tumor antibodies (Abs), has been extensively reviewed. LM3 is a tumorigenic cell line derived from a murine mammary metastatic adenocarcinoma that spontaneously overexpressed mAchR. Here we investigate the ability of Abs purified from the sera of LM3 tumor-bearing mice, directed against muscarinic acetylcholine receptors (mAchR) to modulate tumor cells' proliferation and angiogenesis. We observed that IgG from early tumor bearers (ETB), 14-day LM3 tumor, and from late tumor bearers (LTB), 28-day LM3 tumor, displaced tritiated quinuclidinyl benzilate binding to LM3 tumor cells, confirming Abs interaction with cholinoceptors, while IgG from normal mice did not modify the antagonist binding to mAchR at any concentration tested. In addition, Abs from ETB and LTB immunoblotted a protein of 70 kDa on murine tumor cells and on heart homogenates that was also recognized by a specific anti-M 2 receptor monoclonal antibody. We also observed that IgG purified from ETB-stimulated LM3 cells' proliferation in a more effective manner than the muscarinic agonist carbachol (CARB) did. IgG from LTB-potentiated LM3 cells induced angiogenesis by increasing the number of blood vessels and VEGF-A production in peritumoral skin “via” mAchR, in an agonist similar manner. All effects were blocked by preincubating cells with the non-selective antagonist atropine. In conclusion, autoAbs purified from LM3 tumor-bearing mice sera exert different pro-tumor actions depending on the stage of tumor development: in ETB, they stimulate tumor cells' proliferation, while in LTB they potentiate tumor neovascularization.

Evaluation of Hypocrellin B in a human bladder tumor model in experimental photodynamic therapy: Biodistribution, light dose and drug-light interval effects

Hypocrellin B (HB), a monomeric perylenequinone pigment, is a promising second-generation photosensitizer for photodynamic therapy. We have evaluated the efficacy of HB mediated PDT by experimenting with various drug-light intervals, based on the biodistribution analysis in human bladder tumor (MGH cell line) models. Tumor growth rates were assessed at 10-day post treatment followed by morphometric analysis. Biodistribution of HB was evaluated using spectrofluorophotometry analysis (Ex: 480 nm, Em: 620-630 nm). The level of HB peaked at 6 h postinjection in tumor, peritumoral skin and normal muscle followed by a decline over the next 42 h. Concurrently, the ratio of drug in tumor versus skin was relatively low at all times in comparison to tumor to muscle ratio. In serum, concentration of HB peaked at 1 h. Almost 88% of its original uptake level was cleared at 48 h. The level of PDT response revealed a strong dependence on the drug-light intervals (DLI) and light dose. For both high and low fluence/fluence rate, comparable tumor response was observed at 1 h DLI; treated tumors exhibited significant tumor regression compared to 6 and 24 h DLI. The absence of tumor response was observed at 24 h DLI even at high light dose (100 J/cm(2); 100 mW/cm(2)). Tumor response detected at low light dose (12 J/cm(2); 12 mW/cm(2)) at short DLI suggests that the tumor vasculature is a more sensitive target compared to the cellular compartment of the tumor, correlating significantly with the bioavailability of the drug in serum. Therefore, HB mediated PDT effect is characteristics of a predominantly vascular mediated effect. This study confirms that for short drug-light intervals, PDT seems to target tumor vasculature, which contributes to tumor destruction.

Reduced Expression of Insulin-Like Growth Factor-Binding Protein-3 (IGFBP-3) in Squamous Cell Carcinoma Complicating Recessive Dystrophic Epidermolysis Bullosa

Squamous cell carcinoma (SCC) is a common complication in individuals with recessive dystrophic epidermolysis bullosa (RDEB). For the severe Hallopeau-Siemens subtype, the mortality rate from SCC is over 55% by the age of 40 y. Currently, little is known about the molecular pathology or cell biology of SCC in RDEB. In this study, we compared gene expression in RDEB SCC (n=3) and non-EB SCC (n=3) with corresponding RDEB and non-EB peri-tumoral skin, with microarray analysis using DermArray membranes as well as semi-quantitative and real-time RT-PCR. Both tumor sets showed downregulation of epidermal differentiation markers (e.g., profilaggrin, keratins 1 and 10) as well as certain pro-apoptotic genes (e.g., death-associated kinase-3 or ZIP kinase). Likewise, in both groups there was upregulation of matrix metalloproteinase 1 and laminin 5 in the tumors. But we found that the expression of insulin-like growth factor-binding protein-3 (IGFBP-3) was lower (mean of 5.8-fold) in RDEB SCC compared with non-EB SCC. These data were verified by immunohistochemistry. IGFBP-3 has an important role in cancer cell apoptosis mediated via the nuclear retinoid X receptor alpha (RXRalpha). Reduced expression of IGFBP-3 in RDEB SCC may provide a partial explanation for the aggressive behavior and poor prognosis of these tumors in this genodermatosis.

P53 Patches are Not Increased in Patients with Multiple Nonmelanoma Skin Cancers

p53 Patches are Not Increased in Patients with Multiple Nonmelanoma Skin Cancers

Pharmacokinetics, Tissue Distribution and Photodynamic Therapy Efficacy of Liposomal‐Delivered Hypocrellin A, a Potential Photosensitizer for Tumor Therapy

Hypocrellin A, from Hypocrella bambusae, is a novel photosensitizer of high singlet oxygen quantum yield for photodynamic therapy (PDT). Tissue distributions were studied in tumor-bearing mice as a function of time following administration. The tumor model was S-180 sarcoma transplanted into one hind leg of male Kunming mice; hypocrellin A (HA) was delivered to the mice by intravenous injection of 5 mg/kg of body weight as a suspension either as a unilamellar liposome or in dimethyl sulfoxide (DMSO)-solubilized saline. The HA was isolated from several tissues and organs, as well as tumors and peritumoral muscles and skin. Quantitation was performed by a high-performance liquid chromatographic technique with detection that utilizes the native fluorescence of HA. Independent of the delivery system, the dye was retained in tumors at higher concentrations than in normal tissues, except for kidney, liver, lung and spleen. The dye retention in tumors was high and was vehicle dependent. For the liposomal system, the maximal accumulation in tumor and maximal ratios of dye in tumor versus peritumoral muscle and skin occurred 12 h postinjection; for the DMSO saline system, the maximal ratio occurred earlier, 6 h postadministration. Liposomal delivery improved the selective accumulation of the dye in tumor with higher maximal levels in tumor and higher ratios of tumor-to-muscle and tumor-to-skin. Levels of dye were very low or not detectable in the brain. The PDT efficacy of HA in the liposome and DMSO saline systems was determined by evaluating the tumor volume regression percent. The PDT efficacy of HA in liposomes was highest when light treatment was performed at 12 h postinjection, consistent with the highest retention of HA in tumors. Similarly, the maximal PDT efficacy in DMSO saline was attained at 6 h postinjection, the highest HA retention point in tumor. Moreover, the peak PDT efficacy of HA in liposomes was much higher than that of HA in DMSO saline and even hematoporphyrin monomethylether.

Leishmaniasis and Kaposi's Sarcoma in an HIV-Infected Patient

To the Editor: The presence of Kaposi sarcoma and leishmaniasis in the same cutaneous lesion, which has been observed in six cases (1-6), often reveals infection of peritumoral skin (1,3,4) (i.e., within the noninvolved areas immediately surrounding the abnormal portion of the skin biopsy). Subsequent to our previous report (4), we have seen two additional instances of leishmaniasis in perineoplastic dermis of Kaposi's sarcoma. A similar situation was described in the letter of Abajo et al. In contrast, the presence of Leishmania in clinically uninvolved skin (i.e., within a biopsy specimen taken distant from lesional skin) of human immunodeficiency virus (HIV)-infected patient with visceral leishmaniasis (VL) has not been looked for. As we mentioned in our article (4), only one patient had Leishmania in such "normal" skin documented with the technique of split-skin smear. However, with this procedure, a sharp distinction cannot be made between simple peripheral blood infestation of the dermis versus actual cutaneous involvement. Since our previous report (4), we have seen one additional case of "silent" leishmaniasis in two normal skin biopsy specimens of a HIV-infected patient with a prolonged fever, who had refused bone marrow aspiration. All cases demonstrating Leishmania in "normal" skin showed an extravascular pattern of infestation. This finding demonstrates, in HIV-infected patients with VL, a widespread involvement of skin by Leishmania, which differs from a simple peripheral blood dissemination. In comparison to bone marrow aspirate, the diagnostic value of peripheral blood smears for discovery of VL in HIV-infected subjects remain low (7). As with both marrow aspiration, normal results on skin biopsy could be useful in confirming the diagnosis of VL. On the other hand, in HIV-infected patients with Mediterranean VL the skin could act as a reservoir of infection and allow human-to-human transmission. Abajo et al. mention a new example of presumed "silent" leishmaniasis (E. F. Dandén, P. Penas, L. Rios, J. Fraga, J. Alvar, A. Garcia-Diez, unpublished observations). Although their report is not yet published, on the basis of their letter, two questions still remain to be answered. First, are their cases best categorized as cutaneous leishmaniasis or VL with a cutaneous involvement? The other unknown is whether or not the biopsy specimens demonstrating the "silent" leishmaniasis were obtained adjacent to or distant from the clinically apparent cutaneous lesion. C. Perrin, M.D. P. Del Giudice, M.D. B. Taillan, M.D. Y. Lefichoux, M.D. JF. Michiels, M.D. Departments of Pathology, Infectious Disease, Internal Medicine, and Parasitology Laboratory; University of Nice; Nice, France