HDAC inhibition prevents transgene expression downregulation and loss-of-function in T-cell-receptor-transduced T cells.

T cells that are gene-modified with tumor-specific T cell receptors are a promising treatment for metastatic melanoma patients. In a clinical trial, we treated seven metastatic melanoma patients with autologous T cells transduced to express a tyrosinase-reactive T cell receptor (TCR) (TIL 1383I) and a truncated CD34 molecule as a selection marker. We followed transgene expression in the TCR-transduced T cells after infusion and observed that both lentiviral- and retroviral-transduced T cells lost transgene expression over time, so that by 4 weeks post-transfer, few T cells expressed either lentiviral or retroviral transgenes. Transgene expression was reactivated by stimulation with anti-CD3/anti-CD28 beads and cytokines. TCR-transduced T cell lentiviral and retroviral transgene expression was also downregulated in vitro when T cells were cultured without cytokines. Transduced T cells cultured with interleukin (IL)-15 maintained transgene expression. Culturing gene-modified T cells in the presence of histone deacetylase (HDAC) inhibitors maintained transgene expression and functional TCR-transduced T cell responses to tumor. These results implicate epigenetic processes in the loss of transgene expression in lentiviral- and retroviral-transduced T cells.

Regulatory T-cells in alopecia areata.

BACKGROUND: Alopecia areata (AA) is believed to have an autoimmune mechanism in which the hair follicles are targeted by CD4+ and CD8+ lymphocytes. Studies investigating the autoimmune mechanism of other cutaneous diseases, including vitiligo, showed that Treg is a component of cutaneous immune privilege. Our study uses immunohistochemical staining in formalin-fixed, paraffin-embedded tissue to examine the percentage of CD4+ FoxP3+ , CD25+ FoxP3+ , and CD8+ FoxP3+ Treg in AA in human specimens. METHODS: Immunohistochemical double staining for CD4+ FoxP3+ , CD25+ FoxP3+ , and CD8+ FoxP3+ was performed on 12 AA cases and 12 other autoimmune and non-autoimmune cutaneous diseases. The frequency of CD4+ FoxP3+ , CD25+ FoxP3+ , and CD8+ FoxP3+ Treg was counted and expressed as a percentage of total CD4+ , CD25+ , and CD8+ lymphocytes, respectively, in order to account for intersample inflammatory response variability. RESULTS: There was a significant reduction in the mean frequency of CD4+ FoxP3+ and CD25+ FoxP3+ in AA when compared to other autoimmune and non-autoimmune cutaneous diseases. CONCLUSION: Treg is significantly lower in AA when compared to other cutaneous diseases. Additionally, this immunohistochemical-staining protocol may be useful to evaluate Treg in formalin-fixed, paraffin-embedded specimens for other cutaneous diseases. Studies examining Treg in AA and other cutaneous diseases may have implications for future interventions.

Cutaneous Cytomegalovirus Infection in an Immunocompetent Patient: Innocent Bystander or Culprit?

We present a rare case of cutaneous cytomegalovirus (CMV) infection in a nonimmunocompromised patient. A 74-year-old woman with a history of diabetes presented with an ulcer on the right lateral tibia that occurred at the site of a nerve core biopsy. Subsequent biopsy of the ulcer edge showed granulation tissue with neutrophilic inflammation. The patient underwent extensive antibiotic treatment for possible infection with weekly wound care. However, the ulceration persisted and enlarged. A repeat biopsy 1 year later showed superficial and deep mixed inflammation with an associated vasculitis. On close examination, endothelial and eccrine ducts cells showed characteristic CMV viral cytopathic changes with positivity on CMV immunohistochemical stain. Although the patient was started on valganciclovir, the ulceration did not resolve with treatment and slightly enlarged. Treatment modalities included dapsone, prednisone, weekly wound care, wound vacuum, and eventually a skin graft of the ulcer site. This case highlights the presence of CMV infection in a cutaneous ulceration in a relatively immunocompetent patient, and the lack of response to treatment raises the question whether CMV was causative, partially contributory, or simply an innocent bystander.

Correction to: Clinical and immunologic evaluation of three metastatic melanoma patients treated with autologous melanoma-reactive TCR-transduced T cells.

The authors would like to make the following corrections to the published article.

Clinical and immunologic evaluation of three metastatic melanoma patients treated with autologous melanoma-reactive TCR-transduced T cells.

Malignant melanoma incidence has been increasing for over 30 years, and despite promising new therapies, metastatic disease remains difficult to treat. We describe preliminary results from a Phase I clinical trial (NCT01586403) of adoptive cell therapy in which three patients received autologous CD4+ and CD8+ T cells transduced with a lentivirus carrying a tyrosinase-specific TCR and a marker protein, truncated CD34 (CD34t). This unusual MHC Class I-restricted TCR produces functional responses in both CD4+ and CD8+ T cells. Parameters monitored on transduced T cells included activation (CD25, CD69), inhibitory (PD-1, TIM-3, CTLA-4), costimulatory (OX40), and memory (CCR7) markers. For the clinical trial, T cells were activated, transduced, selected for CD34t+ cells, then re-activated, and expanded in IL-2 and IL-15. After lymphodepleting chemotherapy, patients were given transduced T cells and IL-2, and were followed for clinical and biological responses. Transduced T cells were detected in the circulation of three treated patients for the duration of observation (42, 523, and 255 days). Patient 1 tolerated the infusion well but died from progressive disease after 6 weeks. Patient 2 had a partial response by RECIST criteria then progressed. After progressing, Patient 2 was given high-dose IL-2 and subsequently achieved complete remission, coinciding with the development of vitiligo. Patient 3 had a mixed response that did not meet RECIST criteria for a clinical response and developed vitiligo. In two of these three patients, adoptive transfer of tyrosinase-reactive TCR-transduced T cells into metastatic melanoma patients had clinical and/or biological activity without serious adverse events.

The microenvironment in primary cutaneous melanoma with associated spontaneous tumor regression: evaluation for T-regulatory cells and the presence of an immunosuppressive microenvironment.

Spontaneous tumor regression, regression in the absence of therapeutic intervention, can be identified histologically in over 25% of primary cutaneous melanomas at initial diagnosis. A unique subset of T lymphocytes found in areas of regression can be histologically distinguished from tumor-infiltrating T lymphocytes (TIL) found in areas of tumor progression. We call this unique subset of T lymphocytes regression-associated T lymphocytes (RATs). The aim of this study is to determine the phenotype of lymphocytes and the density of specific cell types linked to immunosuppression in areas of tumor progression compared with areas of tumor regression. These specific cell types include T-regulatory cells (Tregs) and S100A9 cells. A total of 14 primary cutaneous melanomas with areas of progression and regression were used. Immunohistochemistry staining was used to identify CD4 cells, CD8 cells, Tregs, and S100A9 cells. Two independent observers manually counted three high-powered ×40 fields. There was no predominance of CD4 or CD8 T lymphocytes in either RATs or TIL. We identified a lower density of Tregs in RATs compared with TIL when using the FOXP3/CD4 Treg marker (P=0.04) and a marginal difference when using our second, confirmatory Treg marker, FOXP3/CD25 (P=0.11). We observed a lower density of S100A9 cells in RATs compared with TIL (P=0.002). There was an observable difference in the tumor microenvironments of RATs and TIL, with RATs having a significantly lower density of Tregs and S100A9 cells. We deduce that the absence of immunosuppression in areas of regression allows for a more robust immune response and thus effective eradication of tumor cells.

Changing Dermatopathology Protocols for Processing Small Skin Biopsies: Decreases in Slide Quantity Does Not Affect Patient Care and Allows for Optimal Efficiency.

In dermatopathology, no standard protocol exists for processing small biopsy specimens. In our original protocol, 2 routine initial slides per biopsy were prepared. For 1003 biopsies, we noted how often the second slide helped in diagnosis or eliminated the need for additional deeper sections. After obtaining these data, we switched to processing only 1 initial slide (new protocol) and again evaluated 1003 biopsies. During the original protocol, the second slide never helped to make a diagnosis that was not apparent on the first slide. When deeper sections were ordered (10.4% of cases), they helped in the diagnosis 34.6% of the time. In the new protocol, deeper sections were ordered in 15.9% of cases and helped in the diagnosis 32.7% of the time when ordered. Comparing rates of deeper sections ordered showed no significant difference for benign, inflammatory/reactive, and premalignant/malignant groups (P > 0.1). However, there was a significant increase in deeper sections ordered for melanocytic lesions from 16.9% to 32.3% (P < 0.05). Also, a significantly greater percentage of punch biopsies (31.5% and 42.0% in the respective protocols) required deeper sections than shave biopsies (7.4% and 12.6% in the respective protocols). Switching protocols, the estimated annual cost savings is $2890. The majority of cases at our institution are properly diagnosed using only 1 slide. From our study findings, we conclude that 1 slide preparation for small biopsies is the best practice for our institution and one that does not affect diagnostic accuracy, reduces costs, and helps in effective time management.

Multiple Primary Merkel Cell Carcinomas Presenting as Pruritic, Painful Lower Leg Tumors.

Merkel cell carcinoma (MCC) is a rare and highly aggressive neuroendocrine tumor of the skin which almost exclusively presents as a solitary tumor. It is most often seen on sun-exposed regions, historically almost exclusively on the head and neck, with only rare case reports on the extremities. Although recent studies have shown increased incidence with up to 20% on the extremities, here we present one of these rare emerging presentations, with the addition of a unique treatment option. Our patient is an 80-year-old male with a 3-month history of multiple raised, rapidly enlarging tumors on the right ankle. Two separate biopsies were performed and demonstrated sheets and clusters of small blue cells filling the dermis with scant cytoplasm, dusty chromatin, and nuclear molding. Subsequent immunohistochemical stains confirmed the diagnosis of multiple primary MCC. Despite the characteristic immunohistochemical profile of primary MCC, the possibility of a metastatic neuroendocrine carcinoma from an alternate primary site was entertained, given his unusual clinical presentation. A complete clinical workup including CT scans of the chest, abdomen, and pelvis showed no evidence of disease elsewhere. Instead of amputation, the patient opted for nonsurgical treatment with radiation therapy alone, resulting in a rapid and complete response. This case represents an unusual presentation of primary MCC and demonstrates further evidence that radiation as monotherapy is an effective local treatment option for inoperable MCC.

Actinic Keratosis, Transected: What Lies Beneath?

Evaluation of superficial transected shave biopsies of squamous neoplasms often presents a diagnostic dilemma for the dermatopathologist because of the lack of complete visualization of the base of the epidermis. Fear of "missing" an invasive carcinoma must be balanced with avoidance of overdiagnosis of precancerous actinic keratosis (AK), especially on cosmetically sensitive areas such as the face. If a concern exists that a more invasive component may be present, a diagnosis of AK transected at the base (AKT) will often be rendered to alert the dermatologist of this concern. Because of lack of objective data regarding the malignant transformation of this diagnosis, the method of treatment is often based on the clinical appearance of the residual lesion or the lesion is rebiopsied to establish a more definitive diagnosis, costing patient and physician time and increasing health care costs. This study aims to provide objective data regarding (1) how often dermatologists are resampling these lesions and (2) how accurately an AKT diagnosis identifies patient risk for a more aggressive lesion, to establish whether there is a benefit in providing more tissue for the initial biopsy. We performed a retrospective study examining 274 biopsies with a diagnosis of AKT. We found only 27% had follow-up rebiopsy or excision. Of these 73 cases, 63% showed residual AK and 20% showed a more serious lesion, which warrants more aggressive treatment. Because the health care culture slowly shifts to metric-driven medicine and value-based payment, providing objective data for the progression of diagnosis, such as AKT, will be important (1) to aid the clinician in taking adequate tissue samples for diagnosis to make adequately informed management decisions, (2) to reduce the conversion of AK to squamous cell carcinoma, the resultant depth of squamous cell invasion, and the patient's risk of metastases to improve the patient's long-term outcomes, and (3) to decrease the overall cost of the patient's health care by improving the patient's long-term outcomes.

Targeting Fyn in Ras-transformed cells induces F-actin to promote adherens junction-mediated cell-cell adhesion.

Fyn, a member of the Src family kinases (SFK), is an oncogene in murine epidermis and is associated with cell-cell adhesion turnover and induction of cell migration. Additionally, Fyn upregulation has been reported in multiple tumor types, including cutaneous squamous cell carcinoma (cSCC). Introduction of active H-Ras(G12V) into the HaCaT human keratinocyte cell line resulted in upregulation of Fyn mRNA (200-fold) and protein, while expression of other SFKs remained unaltered. Transduction of active Ras or Fyn was sufficient to induce an epithelial-to-mesenchymal transition in HaCaT cells. Inhibition of Fyn activity, using siRNA or the clinical SFK inhibitor Dasatinib, increased cell-cell adhesion and rapidly (5-60 min) increased levels of cortical F-actin. Fyn inhibition with siRNA or Dasatinib also induced F-actin in MDA-MB-231 breast cancer cells, which have elevated Fyn. F-actin co-localized with adherens junction proteins, and Dasatinib-induced cell-cell adhesion could be blocked by Cytochalasin D, indicating that F-actin polymerization was a key initiator of cell-cell adhesion through the adherens junction. Conversely, inhibiting cell-cell adhesion with low Ca(2+) media did not block Dasatinib-induced F-actin polymerization. Inhibition of the Rho effector kinase ROCK blocked Dasatinib-induced F-actin and cell-cell adhesion, implicating relief of Rho GTPase inhibition as a mechanism of Dasatinib-induced cell-cell adhesion. Finally, topical Dasatinib treatment significantly reduced total tumor burden in the SKH1 mouse model of UV-induced skin carcinogenesis. Together these results identify the promotion of actin-based cell-cell adhesion as a newly described mechanism of action for Dasatinib and suggest that Fyn inhibition may be an effective therapeutic approach in treating cSCC.

The effectiveness of annotated (vs. non-annotated) digital pathology slides as a teaching tool during dermatology and pathology residencies.

BACKGROUND: With today's technology, paraffin-embedded, hematoxylin & eosin-stained pathology slides can be scanned to generate high quality virtual slides. Using proprietary software, digital images can also be annotated with arrows, circles and boxes to highlight certain diagnostic features. Previous studies assessing digital microscopy as a teaching tool did not involve the annotation of digital images. The objective of this study was to compare the effectiveness of annotated digital pathology slides versus non-annotated digital pathology slides as a teaching tool during dermatology and pathology residencies. METHODS: A study group composed of 31 dermatology and pathology residents was asked to complete an online pre-quiz consisting of 20 multiple choice style questions, each associated with a static digital pathology image. After completion, participants were given access to an online tutorial composed of digitally annotated pathology slides and subsequently asked to complete a post-quiz. A control group of 12 residents completed a non-annotated version of the tutorial. RESULTS: Nearly all participants in the study group improved their quiz score, with an average improvement of 17%, versus only 3% (P = 0.005) in the control group. CONCLUSIONS: These results support the notion that annotated digital pathology slides are superior to non-annotated slides for the purpose of resident education.

Resolution of recalcitrant pyogenic granuloma with laser, corticosteroid, and timolol therapy.

A pyogenic granuloma (PG) is a rapidly growing benign vascular tumor that can be found on the skin or subcutaneous tissue. While some pyogenic granulomas may resolve spontaneously, most have a tendency to bleed easily and require treatment. Current therapeutic modalities include topical imiquimod, cryotherapy, electrodessication, curettage, excision, laser therapy, sclerotherapy, and microembolization. We report a recalcitrant case of chronic pyogenic granuloma occurring on the scalp of a healthy young male which was unresponsive to conventional surgical and non-surgical modalities. Ultimately, aggressive laser therapy, intralesional triamcinolone acetonide injections, and topical timolol application led to complete resolution and healing.

Mobile teledermatopathology: using a tablet PC as a novel and cost-efficient method to remotely diagnose dermatopathology cases.

: Dermatopathology has relatively few studies regarding teledermatopathology and none have addressed the use of new technologies, such as the tablet PC. We hypothesized that the combination of our existing dynamic nonrobotic system with a tablet PC could provide a novel and cost-efficient method to remotely diagnose dermatopathology cases. 93 cases diagnosed by conventional light microscopy at least 5 months earlier by the participating dermatopathologist were retrieved by an electronic pathology database search. A high-resolution video camera (Nikon DS-L2, version 4.4) mounted on a microscope was used to transmit digital video of a slide to an Apple iPAD2 (Apple Inc, Cupertino, CA) at the pathologist's remote location via live streaming at an interval time of 500 ms and a resolution of 1280/960 pixels. Concordance to the original diagnosis and the seconds elapsed to reaching the diagnosis were recorded. 24.7% (23/93) of cases were melanocytic, 70.9% (66/93) were nonmelanocytic, and 4.4% (4/93) were inflammatory. About 92.5% (86/93) of cases were diagnosed on immediate viewing (<5 seconds), with the average time to diagnosis at 40.2 seconds (range: 10-218 seconds). Of the cases diagnosed immediately, 98.8% (85/86) of the telediagnoses were concordant with the original. Telepathology performed via a tablet PC may serve as a reliable and rapid technique for the diagnosis of routine cases with some diagnostic caveats in mind. Our study established a novel and cost-efficient solution for those institutions that may not have the capital to purchase either a dynamic robotic system or a virtual slide system.

Unusual clinical presentation of cutaneous angiosarcoma masquerading as eczema: a case report and review of the literature.

An unusual case of cutaneous angiosarcoma clinically mimicking eczema is described. A 98-year-old Caucasian male presented with a 6-month history of a flesh-colored, subcutaneous nodule on his left forehead with contralateral facial erythema and scaling that had been previously diagnosed as eczema. Despite treatments with topical steroids and moisturizers, the condition did not resolve. At our clinic, excisional biopsy of the forehead lesion and scouting biopsies from the contralateral cheek were performed which revealed cutaneous angiosarcoma. The described case illustrates that dermatitis-like features should be considered as a rare clinical manifestation of cutaneous angiosarcoma. It also demonstrates that these lesions may respond well to radiotherapy as a single modality.

The new AJCC guidelines in practice: utility of the MITF immunohistochemical stain in the evaluation of single-cell metastasis in melanoma sentinel lymph nodes.

The American Joint Commission on Cancer 2010 guideline changes recommend all patients with single-cell melanoma metastasis identified by immunhistochemical staining of sentinel lymph nodes to be classified as N1 stage. In this study we evaluated the utility of adding microphthalmia transcription factor (MITF) to our current sentinel node evaluation protocol. Twenty benign lymph nodes from nonmelanoma patients were stained with MITF, MART-1/Melan-A, and HMB-45. Hematoxylin and eosin-stained and immunohistochemically stained slides were assessed for the frequency of positive staining, pattern and location of staining, and for the presence of pigment within the node. Fifteen of 20 (75%) lymph nodes evaluated showed >1 cell staining with MITF in variable locations and patterns throughout the lymph node. MART-1/Melan-A-positive cells were identified in 4 of 20 cases (20.0%), mostly in the sinusoidal location (3/4). Fifty percent (10/20) of the lymph nodes contained cells that stained positive for HMB-45. Nine of 10 of the HMB-45-positive cells were in a sinusoidal location corresponding to easily discernible pigmented histiocytes. We conclude that MITF stains nonmelanocytic cells in benign lymph nodes 75% of the time. MITF has a much higher rate of positive staining in benign lymph nodes compared with MART-1/Melan-A, which had positive staining only 20% of the time. MITF showed 2 different staining patterns, type A and type B. Both patterns could be difficult to distinguish from true metastasis, and thus adding MITF to sentinel node evaluation panels is not helpful and may be histologically vexing.

Eosinophilic cytoplasmic inclusion bodies in vesicular multinucleated melanocytes: a clue to the diagnosis of benign melanocytic lesions.

Different types of multinucleated melanocytes have been described in benign and malignant melanocytic lesions. Here we describe a relatively common, though underappreciated, type of multinucleated melanocyte characterized by abundant vesicular and fibrillary-appearing cytoplasm containing one or multiple eosinophilic inclusion bodies. In our experience, these vesicular multinucleated melanocytes with inclusion bodies are invariably seen in nevi of long duration. The presence of these cells can be a reassuring histological finding when evaluating a melanocytic lesion.