Distribution and Clustering of Cutaneous T-Cell Lymphoma (CTCL) Cases in Canada During 1992 to 2010.

BACKGROUND: Clustering of patients with cutaneous T-cell lymphoma (CTCL) was reported in several jurisdictions around the world. This rare cancer is known to affect spouses and in some cases multiple members of the same family. These combined results suggest the existence of external disease triggers/promoters. We recently conducted the first comprehensive analysis of CTCL incidence and mortality in Canada, which revealed case clustering in several regions. OBJECTIVES: To extend our previous analysis on CTCL incidence across Canada and to provide all the collected data on CTCL patient incidence in Canada during the period of 1992 to 2010. METHODS: Clinical parameters for patients with CTCL in Canada were analyzed using 2 independent population-based cancer registries: Canadian Cancer Registry and Le Registre Québécois du Cancer. The CTCL incidence rates were examined on different geographical levels, including provinces/territories, cities, and forward sortation areas. RESULTS: Our findings further corroborate our earlier observations of higher CTCL incidence in Newfoundland and Labrador, maritime provinces (Nova Scotia and New Brunswick), and prairie provinces (Manitoba and Saskatchewan). Also, most cities with high CTCL incidence were located in these provinces. Extensive mapping of high-incidence postal codes supports case clustering in a number of communities that are located in the proximity of industrial centres and seaports. CONCLUSIONS: Detailed analysis of CTCL incidence in Canada is critical to fully understand the burden of this disease in our country, to begin the search for a possible external trigger for this lymphoma, and to reform how health care resources are distributed throughout the country to better serve Canadian patients with CTCL.

Comprehensive analysis of cutaneous T-cell lymphoma (CTCL) incidence and mortality in Canada reveals changing trends and geographic clustering for this malignancy.

BACKGROUND: Previous reports of geographic clustering of cutaneous T-cell lymphoma (CTCL) in Texas, Pittsburgh, and Sweden as well as the occurrence of CTCL in married couples and family members raise a possibility of the existence of an external and potentially preventable trigger(s) for this rare skin cancer. METHODS: The authors studied CTCL incidence and mortality in Canada using 3 distinct population-based cancer databases. Data on patients' sex, age at the time of diagnosis, subtype of CTCL malignancy, reporting province, city, and postal code were analyzed. CTCL cases were mapped across Canada using geographic information systems software. RESULTS: In total, 6685 patients with CTCL were identified in Canada during 1992 through 2010 (CTCL incidence rate, 11.32 cases per million individuals per year), of which 58% were males. The mean age at diagnosis was 59.4 ± 21.5 years. Geographic analysis of patients revealed increased CTCL incidence on the provincial and city levels in several eastern provinces and in Manitoba. An analysis according to postal codes (Forward Sortation Area [FSA]) identified select communities in which several high-incidence FSAs were contiguous or adjacent. Several of these FSAs were located in industrial regions of Canadian cities. Conversely, 3 of 8 low-incidence FSAs were clustered in Ottawa, Ontario, which has very little industrial presence. An analysis of CTCL mortality in Canada corroborated the current incidence findings. CONCLUSIONS: The current results provide a comprehensive analysis of CTCL burden in Canada and highlight several important areas of geographic case clustering. These findings argue that industrial exposures may play an important role in promoting CTCL pathogenesis. Cancer 2017;123:3550-67. © 2017 American Cancer Society.

AAV-mediated Gene Therapy Halts Retinal Degeneration in PDE6ß-deficient Dogs.

We previously reported that subretinal injection of AAV2/5 RK.cpde6ß allowed long-term preservation of photoreceptor function and vision in the rod-cone dysplasia type 1 (rcd1) dog, a large animal model of naturally occurring PDE6ß deficiency. The present study builds on these earlier findings to provide a detailed assessment of the long-term effects of gene therapy on the spatiotemporal pattern of retinal degeneration in rcd1 dogs treated at 20 days of age. We analyzed the density distribution of the retinal layers and of particular photoreceptor cells in 3.5-year-old treated and untreated rcd1 dogs. Whereas no rods were observed outside the bleb or in untreated eyes, gene transfer halted rod degeneration in all vector-exposed regions. Moreover, while gene therapy resulted in the preservation of cones, glial cells and both the inner nuclear and ganglion cell layers, no cells remained in vector-unexposed retinas, except in the visual streak. Finally, the retinal structure of treated 3.5-year-old rcd1 dogs was identical to that of unaffected 4-month-old rcd1 dogs, indicating near complete preservation. Our findings indicate that gene therapy arrests the degenerative process even if intervention is initiated after the onset of photoreceptor degeneration, and point to significant potential of this therapeutic approach in future clinical trials.

Multiple Xanthogranulomas in an Adult: Known Entity, New Association.

BACKGROUND: Multiple xanthogranulomas (XGs) in adults are rare, although an increasing number of case reports are being published. The most frequent association is hematologic malignancies, but the majority of cases remain idiopathic, with occasional spontaneous resolution. OBJECTIVE: The aims of this report are to describe a case of eruptive XG in a woman with a solid neoplasia who was receiving imatinib and to review the literature. METHODS AND RESULTS: This 33-year-old woman had a gastrointestinal stromal tumor. After undergoing surgical removal and being on imatinib for 1 year, the patient developed multiple slightly erythematous papules with an orange hue on the axillary region, trunk, abdomen, and thighs. A biopsy confirmed the diagnosis of XG. CONCLUSION: This is to the investigators' knowledge the first case of eruptive XG in the setting of a solid neoplasia. The possibility of drug-induced XG lesions due to imatinib cannot be excluded. This presentation could be added to the list of associations of adult XG.

Successful gene therapy in the RPGRIP1-deficient dog: a large model of cone-rod dystrophy.

For the development of new therapies, proof-of-concept studies in large animal models that share clinical features with their human counterparts represent a pivotal step. For inherited retinal dystrophies primarily involving photoreceptor cells, the efficacy of gene therapy has been demonstrated in canine models of stationary cone dystrophies and progressive rod-cone dystrophies but not in large models of progressive cone-rod dystrophies, another important cause of blindness. To address the last issue, we evaluated gene therapy in the retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1)-deficient dog, a model exhibiting a severe cone-rod dystrophy similar to that seen in humans. Subretinal injection of AAV5 (n = 5) or AAV8 (n = 2) encoding the canine Rpgrip1 improved photoreceptor survival in transduced areas of treated retinas. Cone function was significantly and stably rescued in all treated eyes (18-72% of those recorded in normal eyes) up to 24 months postinjection. Rod function was also preserved (22-29% of baseline function) in four of the five treated dogs up to 24 months postinjection. No detectable rod function remained in untreated contralateral eyes. More importantly, treatment preserved bright- and dim-light vision. Efficacy of gene therapy in this large animal model of cone-rod dystrophy provides great promise for human treatment.

Mannose-coupled AAV2: A second-generation AAV vector for increased retinal gene therapy efficiency.

Inherited retinal diseases are a leading and untreatable cause of blindness and are therefore candidate diseases for gene therapy. Recombinant vectors derived from adeno-associated virus (rAAV) are currently the most promising vehicles for in vivo therapeutic gene delivery to the retina. However, there is a need for novel AAV-based vectors with greater efficacy for ophthalmic applications, as underscored by recent reports of dose-related inflammatory responses in clinical trials of rAAV-based ocular gene therapies. Improved therapeutic efficacy of vectors would allow for decreases in the dose delivered, with consequent reductions in inflammatory reactions. Here, we describe the development of new rAAV vectors using bioconjugation chemistry to modify the rAAV capsid, thereby improving the therapeutic index. Covalent coupling of a mannose ligand, via the formation of a thiourea bond, to the amino groups of the rAAV capsid significantly increases vector transduction efficiency of both rat and nonhuman primate retinas. These optimized rAAV vectors have important implications for the treatment of a wide range of retinal diseases.

Restoration of vision in the pde6ß-deficient dog, a large animal model of rod-cone dystrophy.

Defects in the ß subunit of rod cGMP phosphodiesterase 6 (PDE6ß) are associated with autosomal recessive retinitis pigmentosa (RP), a childhood blinding disease with early retinal degeneration and vision loss. To date, there is no treatment for this pathology. The aim of this preclinical study was to test recombinant adeno-associated virus (AAV)-mediated gene addition therapy in the rod-cone dysplasia type 1 (rcd1) dog, a large animal model of naturally occurring PDE6ß deficiency that strongly resembles the human pathology. A total of eight rcd1 dogs were injected subretinally with AAV2/5RK.cpde6ß (n = 4) or AAV2/8RK.cpde6ß (n = 4). In vivo and post-mortem morphological analysis showed a significant preservation of the retinal structure in transduced areas of both AAV2/5RK.cpde6ß- and AAV2/8RK.cpde6ß-treated retinas. Moreover, substantial rod-derived electroretinography (ERG) signals were recorded as soon as 1 month postinjection (35% of normal eyes) and remained stable for at least 18 months (the duration of the study) in treated eyes. Rod-responses were undetectable in untreated contralateral eyes. Most importantly, dim-light vision was restored in all treated rcd1 dogs. These results demonstrate for the first time that gene therapy effectively restores long-term retinal function and vision in a large animal model of autosomal recessive rod-cone dystrophy, and provide great promise for human treatment.

Regulation of retinal function but nonrescue of vision in RPE65-deficient dogs treated with doxycycline-regulatable AAV vectors.

In previous studies, we demonstrated that recombinant adeno-associated virus (rAAV)-mediated gene transfer of the doxycycline (Dox)-regulatable system allows for the regulation of erythropoietin (EPO) expression in the retina of nonhuman primates after intravenous or oral administration of Dox. In addition, it was shown that administrating different amounts of Dox resulted in a dose-response dynamic of transgene expression. Adeno-associated viral gene therapy has raised hope for the treatment of patients with Leber congenital amaurosis, caused by mutations in the retinal pigment epithelium (RPE)-specific gene RPE65. The preliminary results of three clinical trials suggest some improvement in visual function. However, further improvements might be necessary to optimize vision recovery and this means developing vectors able to generate transgene expression at physiological levels. The purpose of this study was to investigate the ability of the Dox-regulatable system to regulate retinal function in RPE65(-/-) Briard dogs. rAAV vectors expressing RPE65 under the control of either the TetOff and TetOn Dox-regulated promoters or the cytomegalovirus (CMV) constitutive promoter were generated and administered subretinally to seven RPE65-deficient dogs. We demonstrate that the induction and deinduction of retinal function, as assessed by electroretinography (ERG), can be achieved using a Dox-regulatable system, but do not lead to any recovery of vision.

A case of durvalumab-induced lichenoid eruption evolving to bullous eruption after phototherapy: A case report.

Immune checkpoint inhibitor therapy nowadays became a treatment for a wide range of cancers, and may be responsible for various dermatologic adverse effects, including bullous eruptions. In our report, we present a case of late-onset immunotherapy-induced eruption in a 62-year-old woman treated with anti-programmed cell death-L1 agent durvalumab for metastatic squamous cell carcinoma. Diagnosed as lichenoid dermatitis upon initial presentation, this eruption evolved into necrotic bullous dermatitis after several weeks of phototherapy, with histology and direct immunofluorescence study favoring lichen planus pemphigoides. Thus, this case may be regarded as durvalumab-induced lichenoid dermatitis with phototherapy-triggered progression to necrotic lichen planus pemphigoides-like eruption. The patient's eruption responded to oral prednisone and immunotherapy interruption. Interestingly, durvalumab reintroduction in this patient led to recurrent lichenoid dermatitis without bullous component. This case of immunotherapy skin toxicity is rather distinctive by its clinical and histopathologic features, with phototherapy as an additional triggering factor.

Detection of intact rAAV particles up to 6 years after successful gene transfer in the retina of dogs and primates.

Gene transfer to the retina using recombinant adeno-associated viral (rAAV) vectors has proven to be an effective option for the treatment of retinal degenerative diseases in several animal models and has recently advanced into clinical trials in humans. To date, intracellular trafficking of AAV vectors and subsequent capsid degradation has been studied only in vitro, but the fate of AAV particles in transduced cells following subretinal injection has yet to be elucidated. Using electron microscopy and western blot, we analyzed retinas of one primate and four dogs that had been subretinally injected with AAV2/4, -2/5, or -2/2 serotypes and that displayed efficient gene transfer over several years. We show that intact AAV particles are still present in retinal cells, for up to 6 years after successful gene transfer in these large animals. The persistence of intact vector particles in the target organ, several years postadministration, is totally unexpected and, therefore, represents a new and unanticipated safety issue to consider at a time when gene therapy clinical trials raise new immunological concerns.

Photodynamic therapy with methylaminolevulinate 80 mg/g without occlusion improves acne vulgaris.

BACKGROUND: Photodynamic therapy (PDT) with methylaminolevulinate (MAL) under occlusion is effective for the treatment of acne vulgaris but is associated with significant phototoxic side effects. OBJECTIVE: To evaluate the safety and efficacy of topical MAL with or without occlusion followed by red light exposure in patients with facial acne vulgaris. PATIENTS/METHODS: Forty-four patients with facial acne vulgaris were randomized to receive four MAL applications (80 mg/g) at two-week intervals with occlusion on either the right or left side followed 90 minutes later by either 25 or 37 J/cm2 of red light. RESULTS: At 18 weeks after the first MAL-PDT treatment, the percentage of inflammatory lesions was reduced by a median of 31.7, 59.4, 58.1 and 55.8 percent for patients randomized to 25 J/cm2 without occlusion, 25 J/cm2 with occlusion, 37 J/cm2 without occlusion and 37 J/cm2 with occlusion respectively. MAL-PDT was, in general, well tolerated and only two patients discontinued their participation due to adverse events. CONCLUSION: PDT with MAL at 80 mg/g without occlusion reduces the number of inflammatory lesions in patients with facial acne vulgaris.

The RPGRIP1-deficient dog, a promising canine model for gene therapy.

PURPOSE: To evaluate the RPGRIP1-deficient miniature longhaired dachshund (MLHD) dog as a potential candidate for gene therapy. METHODS: Six RPGRIP1-deficient MLHD dogs from our dog colony have been observed for two years using a variety of noninvasive procedures. These included bilateral full-field electroretinograms (ERG) to evaluate retinal function, fundus photographs to evaluate retinal vascularization, and optical coherence tomographs (OCT) to evaluate retinal thickness. We also performed histological examination of hematoxylin- and eosin-stained retinal sections as well as sections labeled in situ by the terminal dUTP nick end labeling (TUNEL) method. RESULTS: ERG findings showed that as early as 2 months of age, cone function was lost while rod function was preserved. However, by 9 months of age, both cone and rod functions could not be detected. Functional visual assessment based on the ability to avoid obstacles showed that vision was retained up to the age of 11 months. Both OCT and histopathology studies revealed a progressive thinning of the outer nuclear layer (ONL) over the first 2 years of age. TUNEL labeling identified apoptotic photoreceptor cell death as the cause of this thinning of the ONL. CONCLUSIONS: A treatment strategy should consist in initiating gene therapy as early as possible after birth to prevent or delay the loss of rod function. In the MLHD, successful subretinal delivery of a therapeutic vector is feasible at 2 months of age and may prevent or delay the loss of rod function.

Subretinal delivery of recombinant AAV serotype 8 vector in dogs results in gene transfer to neurons in the brain.

Recombinant adeno-associated virus (rAAV) vectors are among the most efficient gene delivery vehicles for gene transfer to the retina. This study evaluates the behavior of the rAAV8 serotype vector with regard to intraocular delivery in rats and dogs. Subretinal delivery of an AAV2/8.gfp vector results in efficient gene transfer in the retinal pigment epithelium (RPE), the photoreceptors and, surprisingly, in the cells of the inner nuclear layer as well as in ganglion cells. Most importantly, in dogs, gene transfer also occurred distal to the injection site in neurons of the lateral geniculate nucleus of the brain. Because green fluorescent protein (GFP) was detected along the visual pathway within the brain, we analyzed total DNA extracted from various brain slices using PCR. Vector sequences were detected in many parts of the brain, but chiefly in the contralateral hemisphere.

Gene expression profiling and immune cell-type deconvolution highlight robust disease progression and survival markers in multiple cohorts of CTCL patients.

CTCL follows different courses depending on the clinical stage at the time of diagnosis. Patients with early stage Mycosis Fungoides (MF) variant of CTCL may experience an indolent course over decades, whereas patients with advanced MF and Sézary Syndrome (SS) disease at diagnosis, often succumb within 5 years. Even within early stage CTCL/MF, a minority of patients will progress to more advanced stages. We recently generated RNA sequencing data on 284 CTCL-relevant genes for 157 patients and identified differentially expressed genes across stages I-IV. In this study, we aim to validate robust molecular markers linked to disease progression and survival. We performed multiple hypothesis testing-corrected analysis of variance (ANOVA) on the expression of individual genes across all CTCL samples and early stage (≤IIA) CTCL/MF patients. We used in silico immune cell-type deconvolution from gene expression data to estimate immune cell populations. Based on the analysis of all CTCL samples, we identified TOX, FYB, and CD52 as predictors of disease progression and poor survival. Among early stage (≤IIA) CTCL/MF patients, these 3 genes, along with CCR4, were valuable to predict disease progression. We validated these 4 genes in 3 independent, external Sézary Syndrome patient cohorts with RNA-Sequencing data. In silico immune cell-type deconvolution revealed that neutrophil infiltration in early stage MF conveyed a higher risk for disease progression. Also, NK cell infiltration in late stage MF/SS correlated with improved survival. TOX, FYB, CCR4 and CD52 are robust disease progression and decreased survival biomarkers in CTCL.

TruSeq-Based Gene Expression Analysis of Formalin-Fixed Paraffin-Embedded (FFPE) Cutaneous T-Cell Lymphoma Samples: Subgroup Analysis Results and Elucidation of Biases from FFPE Sample Processing on the TruSeq Platform.

Cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of malignancies with courses ranging from indolent to potentially lethal. We recently studied in a 157 patient cohort gene expression profiles generated by the TruSeq targeted RNA gene expression sequencing. We observed that the sequencing library quality and depth from formalin-fixed paraffin-embedded (FFPE) skin samples were significantly lower when biopsies were obtained prior to 2009. We also observed that the fresh CTCL samples clustered together, even though they included stage I-IV disease. In this study, we compared TruSeq gene expression patterns in older (≤2008) vs. more recent (≥2009) FFPE samples to determine whether these clustering analyses and earlier described differentially expressed gene findings are robust when analyzed based on the year of biopsy. We also explored biases found in FFPE samples when subjected to the TruSeq analysis of gene expression. Our results showed that ≤2008 and ≥2009 samples clustered equally well to the full data set and, importantly, both analyses produced nearly identical trends and findings. Specifically, both analyses enriched nearly identical DEGs when comparing benign vs. (1) stage I-IV and (2) stage IV (alone) CTCL samples. Results obtained using either ≤2008 or ≥2009 samples were strongly correlated. Furthermore, by using subgroup analyses, we were able to identify additional novel differentially expressed genes (DEGs), which did not reach statistical significance in the prior full data set analysis. Those included CTCL-upregulated BCL11A, SELL, IRF1, SMAD1, CASP1, BIRC5, and MAX and CTCL-downregulated MDM4, SERPINB3, and THBS4 genes. With respect to sample biases, no matter if we performed subgroup analyses or full data set analysis, fresh samples tightly clustered together. While principal component analysis revealed that fresh samples were spatially closer together, indicating some preprocessing batch effect, they remained in the proximity to other normal/benign and FFPE CTCL samples and were not clustering as outliers by themselves. Notably, this did not affect the determination of DEGs when analyzing ≥2009 samples (fresh and FFPE biopsies) vs. ≥2009 FFPE samples alone.

Gene expression analysis in Cutaneous T-Cell Lymphomas (CTCL) highlights disease heterogeneity and potential diagnostic and prognostic indicators.

Cutaneous T-Cell Lymphomas (CTCL) are rare, but potentially devastating malignancies, whose pathogenesis remains poorly elucidated. Unfortunately, currently it is not possible to predict based on the available criteria in which patients the cancer will progress and which patients will experience an indolent disease course. Furthermore, at early stages this malignancy often masquerades as psoriasis, chronic eczema or other benign inflammatory dermatoses. As a result, it takes on average 6 y to diagnose this lymphoma since its initial presentation. In this study, we performed transcription expression profiling using TruSeq targeted RNA gene expression on 181 fresh and formalin-fixed and paraffin-embedded (FFPE) skin samples from CTCL patients and patients affected by benign inflammatory dermatoses that often mimic CTCL clinically and on histology (e.g., psoriasis, chronic eczema, etc.) We also analyzed multiple longitudinal biopsies that were obtained from the same patients over time. Our results underscore significant molecular heterogeneity with respect to gene expression between different patients and even within the same patients over time. Our study also confirmed TOX, FYB, LEF1, CCR4, ITK, EED, POU2AF, IL26, STAT5, BLK, GTSF1 and PSORS1C2 genes as being differentially expressed between CTCL and benign skin biopsies. In addition, we found that differential expression for a subset of these markers (e.g., TOX, FYB, GTSF1 and CCR4) may be useful in prognosticating this disease. This research, combined with other molecular analyses, prepares the foundation for the development of personalized molecular approach toward diagnosis and management of CTCL.

Diversity of cutaneous human papillomavirus types in individuals with and without skin lesion.

BACKGROUND: Human papillomavirus (HPV) is ubiquitous on the skin of normal and immunosuppressed hosts. OBJECTIVE: We describe the diversity of HPV types in skin specimens using PCR-sequencing directly and after cloning with FAP59/64 or HVP2/B5 primers. STUDY DESIGN: Cross-sectional analysis of skin swabs. RESULTS: Seventy-five (92.6%) of 81 subjects provided samples that could be analysed with PCR (34 healthy controls <50 years old, 13 healthy controls > or =50 years old, 12 with actinic keratosis (AK), 8 with squamous cell carcinoma (SCC) and 8 renal transplant recipients). HPV DNA was detected more frequently with FAP59/64 (68/75, 91%) than with HVP2/B5 (9/75, 12%) (p<0.001). Agreement of typing results using FAP59/64 primers with both sequencing strategies was fair (mean kappa 0.56+/-0.19, 95% CI: 0.46-0.65). HPV species 1 and 2 of the beta-papillomavirus genus were associated with the presence of AK (OR=24.8, 95% CI: 2.3-262.6). A greater number of HPV types per sample was found in individuals with AK or SCC (p=0.046) or AK alone (p=0.02), than in healthy participants. CONCLUSION: HPV infection on the skin is best evaluated with a combination of primers and sequencing strategies. Novel putative types were frequently detected in SCC. Skin lesions have a greater number of HPV types than normal skin.

Gene therapeutic prospects in early onset of severe retinal dystrophy: restoration of vision in RPE65 Briard dogs using an AAV serotype 4 vector that specifically targets the retinal pigmented epithelium.

Previous studies have tested gene replacement therapy in RPE65 deficient dogs using recombinant adeno-associated virus 2/2 (rAAV2/2), -2/1 or -2/5 mediated delivery of the RPE65 gene. They all documented restoration of dark- and light-adapted ERG responses and improved psychophysical outcomes. Use of a specific RPE65 promoter and a rAAV vector that targets transgene expression specifically to the retinal pigmented epithelium (RPE) may, however, provide a safer setting for the long-term therapeutic expression of RPE65. Subretinal injection of rAAV2 pseudotyped with serotype 4 (rAAV2/4) specifically targets the RPE. The purpose of our study was to evaluate a rAAV2/4 vector carrying a human RPE65cDNA driven by a human RPE65 promoter, for the ability to restore vision in RPE65-/- purebred Briard dogs. Recombinant rAAV2/4 and rAAV2/2 vectors containing similar human RPE65 promoter and cDNA cassettes were generated and administered subretinally in 8 affected dogs, ages 8 to 30 months (n = 6 with rAAV2/4, n = 2 with rAAV2/2). Although fluorescein angiography and OCT examinations displayed retinal abnormalities in treated retinas, electrophysiological analysis demonstrated that restoration of rod and cone photoreceptor function started as soon as 15 days post-injection, reaching maximal function at 3 months post-injection, and remaining stable thereafter in all animals treated at 8 to 11 months of age. As assessed by the ability of these animals to avoid obstacles in both dim and normal light, functional vision was restored in the treated eye, while the untreated contralateral eye served as an internal control. The dog treated at a later age (30 months) did not recover retinal function or vision, suggesting that there might be a therapeutic window for the successful treatment of RPE65 -/- dogs by gene replacement therapy.