CD38 Expression by Circulating and Skin-Infiltrating Lymphocytes from Sezary Syndrome Patients: A Flow Cytometry and Immunohistochemistry Study.

BACKGROUND: Reports on the expression of CD38 in Sézary syndrome (SS), erythrodermic primary cutaneous T cell lymphoma with leukemic involvement, are limited. The aim of the present study is the analysis of the expression of CD38 by skin-infiltrating mononuclear cells and circulating T lymphocytes in a cohort of SS patients. METHODS: SS patients diagnosed since 1985 in our clinic were retrospectively analyzed for CD38 expression in biopsy and blood samples by immunohistochemistry and flow cytometry, respectively. RESULTS: SS patients show a predominant CD38-negative phenotype on both skin and blood. A subgroup of patients was found expressing CD38 (12 cases) in either the skin (>25% cell infiltrate) or blood (CD4+CD38+ >50%), among whom 4 in the blood, 7 in the skin, and 1 in both blood and skin. CONCLUSION: The implications of these observations may be twofold: the relevance in basic science is related to a potential role in immune defense regulation, whilst in perspective CD38 may become a target for antibody therapy, considering the availability of different anti-CD38 monoclonal antibodies.

HERV-E expression in peripheral mononuclear cells of patients with psoriasis.

BACKGROUND: Psoriasis is a common inflammatory skin disease characterized by uncontrolled proliferation of keratinocytes and recruitment of T lymphocytes into the skin. Possible triggers for psoriasis have been attributed to drugs or pathogens such as bacteria and possibly virus. Human endogenous retroviruses (HERVs) might play a role in triggering these antiviral immune responses, since the role of HERVs in the pathogenesis of autoimmune diseases has generated considerable interest. Some studies have also reported an association of HERV-E and psoriasis. None of them investigate the HERV-E expression in peripheral blood of psoriasis. All these considerations have prompted us to perform a survey for HERV-E expression in PBMC from psoriatic patients. METHODS: Peripheral blood mononuclear cells from 69 psoriatic patients were analyzed. Total RNA was extracted and amplified with reverse transcription polymerase chain reaction. Results were compared with those obtained in a cohort of 20 healthy donors. RESULTS: HERV E was expressed in all samples analyzed but the level of expression was much lower in the psoriasis that in HC P<0.0001. CONCLUSIONS: The reasons for the unexpected, low levels of HERV expression in psoriatic patients are unclear and might be in part a consequence of antiviral defense mechanisms.

Phenotypical Markers, Molecular Mutations, and Immune Microenvironment as Targets for New Treatments in Patients with Mycosis Fungoides and/or Sézary Syndrome.

Primary cutaneous lymphomas encompass a wide spectrum of rare lymphoproliferative disorders originating in the skin, among which, mycosis fungoides (MF) is the most common subtype. The treatment of this disease is based on skin-directed therapies eventually in association with biologic response modifiers in the early phases, whereas in patients with the advanced stages, several therapeutic strategies can be used including mono and/or polychemotherapy and bone marrow transplantation. In recent years, the identification of specific markers (phenotypical, immunological, and molecular) has led to the development of several studies (including two randomized phase III trials). The results of these studies are modifying our therapeutic strategy toward a personalized treatment approach in which the clinical characteristics of the patients and tumor-node-metastasis-blood stage are considered together with the expression of specific markers (i.e., a CD30-positive expression for the use of brentuximab vedotin). This review will provide a comprehensive scenario of the main phenotypical, molecular, and immunological markers related to MF pathogenesis and disease evolution, which could represent the target for the development of innovative effective treatments in this disease.

Isolation of extracellular vesicles improves the detection of mutant DNA from plasma of metastatic melanoma patients.

Detection of BRAFV600E within cell free tumor DNA (ctDNA) is emerging as a promising means to improve patients' stratification or enable BRAF inhibitor (BRAFi) therapeutic monitoring in a minimally invasive manner. Here, we investigated whether extracellular vesicle-(EV)-associated-DNA (EV-DNA) has value as an alternative source of circulating BRAFV600E. To do so, we identified a clinical practice-compatible protocol for the isolation of EV-DNA and assessed BRAF gene status on plasma samples from metastatic melanoma patients at the beginning and during BRAFi therapy. This protocol uses a peptide with high affinity for EVs and it has been found to recover more mutant DNA from plasma than standard ultracentrifugation. Molecular analyses revealed that mutant DNA is largely unprotected from nuclease digestion, interacting with the outer side of the EV membrane or directly with the peptide. When used on clinical samples, we found that the protocol improves the detection of BRAFV600E gene copies in comparison to the reference protocol for ctDNA isolation. Taken together, these findings indicate that EVs are a promising source of mutant DNA and should be considered for the development of next-generation liquid biopsy approaches.

Is HERV-K and HERV-W expression regulated by mir-155 in Sézary Syndrome?

BACKGROUND: According to the latest update, 2578 unique mature miRNAs are currently annotated in the human genome and participate in the regulation of multiple events, such as cellular proliferation or apoptosis. A previous study analyzing global miRNA expression patterns in GH cells (high HERV-K versus low) showed that two miRNAs (miR-663 and miR-638) are differentially regulated and exhibit expression parallel to that of HERV-K. The aim of this study was to evaluate HERV-K and -W pol gene and mir-155 expression in SS patients and possible relationship between them. METHODS: The comparison between SS patients and healthy donor showed a significant difference in terms of mir-155 expression P=0.0003 as previously reported by our groups. RESULTS: We demonstrated that HERV-K and -W pol gene expression was significantly higher in SS patients vs. healthy donor as previously reported by our groups. Our correlation data suggest that miR-155 are not directly involved in regulating the HERVs. CONCLUSIONS: Furthermore, further studies including other cohorts of pathology with mir-155 and HERVs involvement such as inflammatory diseases are needed to investigate the role of mir-155 in the cross-activations of HERVs.

DNA from Human Polyomaviruses, MWPyV, HPyV6, HPyV7, HPyV9 and HPyV12 in Cutaneous T-cell Lymphomas.

BACKGROUND/AIM: The etiopathogenesis of mycosis fungoides and Sézary syndrome remains obscure. Different viruses have been proposed to have a role in the etiopathogenesis of cutaneous T-cell lymphomas (CTCL). In the present study, the presence of five recently discovered human polyomaviruses 6 (HPyV6), human polyomaviruses 7 (HPyV7), human polyomaviruses 9 (HPyV9), human polyomaviruses 12 (HPyV12), and Malawi polyomavirus (MWPyV), have been analyzed in 55 CTCL in order to confirm the skin tropism and the possible pathological association of these new polyomaviruses. MATERIALS AND METHODS: Human polyomaviruses DNA were amplified from skin lesions were recovered from a total of 55 patients (32 males and 23 females, average age 63±15 years) affected by CTCL. RESULTS: When assayed for the presence of 5 different HPyVs, (HPyV6, HPyV7, HPyV9, MWPyV, and HPyV12) HPyV9, HPyV10 and HPyV12 DNA sequences were not found in any skin specimens. HPyV6 and 7 DNA was detected in 1/55 (1.8%) of skin specimens. CONCLUSION: The low-level presence of HPyV6 and HPyV7 DNA, and lack of detection of polyomaviruses HPyV9, MWPyV and HPyV12 in our series do not support a significant role of these HPyVs subtypes in the etiopathogenesis of skin cancers.

BRAF and MEK Inhibitors Increase PD-1-Positive Melanoma Cells Leading to a Potential Lymphocyte-Independent Synergism with Anti-PD-1 Antibody.

Purpose: BRAF and MEK inhibitors (BRAF/MEKi) favor melanoma-infiltrating lymphocytes, providing the rationale for current combinatorial trials with anti-PD-1 antibody. A portion of melanoma cells may express PD-1, and anti-PD-1 antibody could have a direct antitumor effect. Here, we explore whether BRAF/MEKi modulate rates of PD-1+ melanoma cells, supporting an additional-lymphocyte-independent-basis for their therapeutic combination with anti-PD-1 antibody.Experimental Design: With data mining and flow cytometry, we assessed PD-1, PD-L1/2 expression on melanoma cell lines (CCLE, N = 61; validation cell lines, N = 7) and melanoma tumors (TCGA, N = 214). We explored in vitro how BRAF/MEKi affect rates of PD-1+, PD-L1/2+ melanoma cells, and characterized the proliferative and putative stemness features of PD-1+ melanoma cells. We tested the functional lymphocyte-independent effect of anti-PD-1 antibody alone and in combination with BRAF/MEKi in vitro and in an in vivo immunodeficient murine model.Results: PD-1 is consistently expressed on a small subset of melanoma cells, but PD-1+ cells increase to relevant rates during BRAF/MEKi treatment [7.3% (5.6-14.2) vs. 1.5% (0.7-3.2), P = 0.0156; N = 7], together with PD-L2+ melanoma cells [8.5% (0.0-63.0) vs. 1.5% (0.2-43.3), P = 0.0312; N = 7]. PD-1+ cells proliferate less than PD-1- cells (avg. 65% less; t = 7 days) and are preferentially endowed with stemness features. In vivo, the direct anti-melanoma activity of PD-1 blockage as monotherapy was negligible, but its association with BRAF/MEKi significantly delayed the development of drug resistance and tumor relapse.Conclusions: BRAF/MEKi increase the rates of PD-1+ melanoma cells that may sustain tumor relapse, providing a lymphocyte-independent rationale to explore combinatory strategies with anti-PD-1 antibody. Clin Cancer Res; 24(14); 3377-85. ©2018 AACR.

Erythema multiforme-like lesions in primary cutaneous aggressive cytotoxic epidermotropic CD8+ T-cell lymphoma: A diagnostic and therapeutic challenge.

Primary cutaneous aggressive cytotoxic epidermotropic CD8+ T-cell lymphoma is an extremely rare, rapidly progressing, cutaneous lymphoma, with frequent systemic involvement and poor prognosis, that still represents a diagnostic and therapeutic challenge, especially in the early stage. Herein, we report a case of an elderly woman with a fulminant course, who at onset presented with clinical and pathological features mimicking erythema multiforme (EM) and treated with cyclosporine that led to rapid deterioration with fatal outcome 6 months after disease onset. Histopathology showed a lichenoid, epidermotropic and nodular, angiocentric, dermal and subcutaneous infiltrate of sF1, CD8+, CD45RA+ small to medium-sized atypical lymphoid cells, which strongly expressed cytotoxic markers. Monoclonal T-cell-γ receptor was clonally rearranged and array-CGH showed numerous chromosomal imbalances. This case evidences the clinical, pathological and therapeutic challenges involved in this tumor. The first biopsy showed an interface dermatitis-like pattern, revealing the deceptive features that early cutaneous infiltrates of this aggressive lymphoma may have. A high suspicion for aggressive CTCL and a low threshold for repeat biopsies should be maintained when faced with rapidly progressing and/or ulcerative EM-like lesions, especially if immunomodulatory therapy is being considered.

PCR Real time Mismatch Amplification Mutation Assay (MAMA Real Time PCR) for evaluation of TNF-α promoter gene polymorphism -308 G/A in patients with psoriasis.

BACKGROUND: Psoriasis is a common chronic inflammatory disease, the plaques are infiltrated by leukocytes producing high levels of proinflammatory cytokines and TNF-α. Single-nucleotide polymorphisms within the gene promoters have been shown to affect gene expression. The -308 G/A polymorphism could affect TNF synthesis at transcriptional level. The present study develops a MAMA Real Time PCR assay, in order to identify homozygosis or heterozygosis for TNF-α -308 G/A polymorphism. METHODS: Seventy patients with psoriasis and 235 controls were considered for the development of the real time PCR assay. Whole blood was processed for nucleic acid extraction. RESULTS: A percentage of 36.17% controls and 38.6% patients were heterozygosis, considering Amplification-refractory mutation system (ARMS)-PCR assay while 23% and 22.85% were heterozygosis using Mismatch Amplification Mutation Assay (MAMA)-PCR. On the contrary, 1.3% and 1.4% were homozygosis A, while 75.7% and 75.75% presented homozygosis G, taking into account the MAMA-PCR results. The two assays were significantly different (P=0.0004 at χ2 Test), but MAMA-PCR showed a better performance for TNF-α -308 G/A gene polymorphism investigation. CONCLUSIONS: Further studies are needed for a better comprehension of the role of this polymorphism, such as MAMA real time PCR assays development for other players in cellular immune response.

Human Endogenous Retrovirus Expression in Primary Cutaneous T-Cell Lymphomas.

BACKGROUND: Mycosis fungoides (MF) and Sézary syndrome (SS) are the most frequent cutaneous T-cell lymphomas (CTCL). Human endogenous retroviruses (HERVs) were reverse transcribed and integrated into primate chromosomal DNA, becoming noninfectious, although various stimuli may reactivate them. HERV expression seems to be impaired in several human diseases but limited data regarding CTCL are available. OBJECTIVE: To evaluate the endogenous retroviral transcription profile in CTCL and their expression among disease clinical stages. METHODS: Peripheral blood mononuclear cells from 42 MF/SS patients were analyzed. Total RNA was extracted and amplified with reverse transcription polymerase chain reaction. Results were compared with those obtained in a cohort of 20 healthy donors. RESULTS: HERVs were significantly overexpressed in MF/SS patients compared with healthy donors. No differences were found between early and advanced CTCL stages. CONCLUSION: HERVs can act as promoters in MF/SS pathogenesis. It remains to link HERV hyperexpression to the outcome in CTCL patients.

Improved Anti-Tumoral Therapeutic Efficacy of 4-Hydroxynonenal Incorporated in Novel Lipid Nanocapsules in 2D and 3D Models.

4-hydroxynonenal (HNE), a lipid peroxidation product, is a promising anti-neoplastic drug due to its remarkable anti-cancer activities. However, this possibility has not been explored, because the delivery of HNE is very challenging as a result of its low solubility and its poor stability. This study intentionally designed a new type of lipid nanocapsules specifically for HNE delivery. They consist of a medium chain triglyceride liquid oil core surrounded by a polymer shell. A ß-cyclodextrin-poly(4-acryloylmorpholine) conjugate was selected as the shell component. HNE-loaded nanocapsules were about 350 nm in size with a negative surface charge. They were stable for two years when stored in suspensions at 4 degrees C. In vitro experiments showed that HNE was released from the nanocapsules at a considerable rate. Nanocapsule uptake into cells was evaluated using a fluorescent formulation that revealed rapid internalisation. Cytotoxicity studies demonstrated the safety of the formulation. Enhanced anti-tumoral activity against various cell lines, depending on increased HNE stability, was obtained by using HNE-loaded nanocapsules. In particular, we have demonstrated an increase in anti-proliferative, pro-apoptotic and differentiative activity in several tumour cell lines from different tissues. Moreover, we evaluated the effects of these new nanocapsules on a three-dimensional human reconstructed model of skin melanoma. Interestingly, the encouraging results obtained with topical administration on the epidermal surface could open new perspectives in melanoma treatments.

Melanoma of the lower extremities: foot site is an independent risk factor for clinical outcome.

BACKGROUND: Despite the better prognosis of melanomas localized on lower extremities, some studies have suggested that melanomas on the foot are related to a poorer survival and should be considered separately. OBJECTIVE: To review our case series of cutaneous melanomas on the lower extremities and to analyze the clinicopathological association, time course, types of progression, and survival differences. METHODS: We included 1671 patients (stage 0-II) with a cutaneous melanoma on the lower extremities (subungual melanomas were excluded). Of these, 327 were localized on the foot. Multivariate analyses were performed to evaluate disease-specific survival and disease-free interval. RESULTS: Distribution of known prognostic factors and patterns of progression of foot and leg melanoma differ across genders. The foot site was confirmed as a negative independent prognostic factor on disease-specific survival and disease-free interval. CONCLUSION: Foot melanoma could represent a particular subgroup, which could require specific management in the future.

Spiky follicular mycosis fungoides: a clinicopathologic study of 8 cases.

BACKGROUND: The early stages of follicular mycosis fungoides (FMF) have not been described previously in the literature. OBJECTIVE: Our goal was to better categorize the clinicopathologic features of early stages of FMF. METHODS: The clinical notes of patients with a diagnosis of FMF seen during the previous 5 years were reviewed to identify any cases that at presentation had only hyperkeratotic follicular lesions. RESULTS: Eight patients (five male, three female) with a mean age of 55.4 years were enrolled. Noteworthy, FMF was not a clinical consideration in any of these patients initially. Patients presented with disseminated, slightly erythematous, hyperkeratotic, spiky follicular papules which, histopathologically, showed hyperkeratotic columns protruding from follicular plugging in concert with selective infiltration of the infundibular epithelium by atypical, mostly CD4+, lymphocytes. T-cell clonality was demonstrated in four of eight cases. The mean duration of the lesions before diagnosis was 17.1 months. The course was indolent in most of the cases (median follow up: 18 months), whilst progression to overt FMF was noted in two patients. LIMITATIONS: The number of cases is small and follow up relatively short. CONCLUSIONS: Spiky FMF is a deceptive clinicopathologic presentation of FMF that has been poorly described and that can mimic numerous follicular disorders.

Regulatory T cells as well as IL-10 are reduced in the skin of patients with dermatitis herpetiformis.

BACKGROUND: Dermatitis herpetiformis (DH) and celiac disease (CD) are considered as autoimmune diseases that share a defined trigger (gluten) and a common genetic background (HLA-DQ2/DQ8). However, the pathogenesis of DH is not fully understood and no data are available about the immune regulation in such a disease. OBJECTIVE: The aim of this study was to assess if alterations in the pattern of the immune response and, in particular, impairments of regulatory T (Tregs) cells may contribute to the phenotypic differences between DH and CD. METHODS: We investigated the presence of Tregs cell markers, in the skin, the duodenum and the blood of patients with DH by immunohistochemistry, confocal microscopy and flow cytometry. As controls, we included patients with bullous pemphigoid, patients with CD without skin lesions, as well as healthy subjects (HS). RESULTS: In the skin of DH patient, we found a significantly lower proportion of FOXP3(+) Tregs and IL-10(+) cells than in HS (p < 0.001 for both cell populations). In duodenal samples, no differences where found in the proportion of Tregs between patients with DH and patients with CD without skin manifestations. Finally, the frequency of CD25(bright)FOXP3(+) cells within the CD4(+) subset was significantly reduced in CD patients either with or without DH with respect to HS (p = 0.029 and p = 0.017, respectively). CONCLUSIONS: Our findings suggested that a reduction of Tregs may play a major role in the skin, leading to a defective suppressive function and thus to the development of the lesions. By contrast, no differences could be detected about Tregs between patients with DH and patients with CD in the duodenum, suggesting that the mechanisms of the intestinal damage are similar in both diseases.

Blood flow cytometry in Sézary syndrome: new insights on prognostic relevance and immunophenotypic changes during follow-up.

OBJECTIVES: Sézary syndrome (SS) is characterized by erythroderma, generalized lymphadenopathy, and the presence of circulating atypical lymphocytes, which are difficult to identify by morphologic data. METHODS: We revised our series of 107 patients in an attempt to better define the phenotypic aberrancies in blood at diagnosis and the immunophenotypic stability over time detected by flow cytometry. Polymerase chain reaction assay was also used to study CD26/dipeptidyl peptidase IV (DPPIV) gene methylation. RESULTS: The most common aberrancies were represented by the lack of CD26 (96/107) or CD38 (101/107) expression and the presence of a "dim" CD3, CD4, or CD2 population. There was a high variability in CD7 expression. In total, 31% of the patients had phenotypical heterogeneity in CD26 and CD7 expression at diagnosis. The phenotype was stable over time in 73 of 95 patients with available follow-up data, while 22 of 95 patients developed changes in CD26, CD7, or CD2 expression. CD4+CD26- SS showed hypermethylation of the CpG islands for the promoter region of CD26/DPPIV. Multivariate analysis showed that CD26 expression is a favorable prognostic factor (hazard ratio, 2.94; P = .045). CONCLUSIONS: We confirm the relevance of CD26 negativity in SS diagnosis and monitoring. Nevertheless, the presence of rare CD26+ cases suggests that a multiparameter flow cytometry approach should be used. Changes in methylation profile could account for phenotypical heterogeneity.

Eruptive dermal clear cell desmo-plastic mesenchymal tumors with perivascular myoid differentiation in a young boy. A clinical, histopathologic, immunohistochemical and electron microscopy study of 17 lesions.

Clear cell tumors of the skin are observed in a wide variety of benign and malignant conditions with different histogenesis, sharing the presence of cells with abundant clear cytoplasm. Herein, we report the clinicopathologic features of a healthy young patient affected by asymptomatic, eruptive and disseminated, benign clear cell dermal tumors since early infancy. Neither family history nor genetic testing and counseling provided further useful information. The lesions were mostly confined to the face and lower left extremity with pink teleangiectatic papules and small nodules. Over a 4-year period, a total of 16 different cutaneous lesions were biopsied and histopathologic and immunohistochemical studies carried out; an additional lesion was also removed for electron microscopy examination. Histopathology evidenced multiple perivascular growths of spindle to oval and round cells intermingled with clear/granular cells throughout the dermis, with prominent desmoplasia and numerous capillary-like vessels with focal hemangiopericytoma-like features. Immunohistochemical neoplastic cells were uniformly positive for h-caldesmon and focally smooth muscle α-actin and CD13 indicating myoid differentiation whereas the consistent diffuse cytoplasmic staining for lysosome antigen, such as CD68PG-M1 and NKI/C3 along with the ultrastructural findings supported the view of a lysosome-mediated apoptotic process. The differential diagnosis with other clear cell cutaneous neoplasms is discussed.

The inclusion complex of 4-hydroxynonenal with a polymeric derivative of ß-cyclodextrin enhances the antitumoral efficacy of the aldehyde in several tumor cell lines and in a three-dimensional human melanoma model.

4-Hydroxynonenal (HNE) is the most studied end product of the lipoperoxidation process, by virtue of its relevant biological activity. The antiproliferative and proapoptotic effects of HNE have been widely demonstrated in a great variety of tumor cell types in vitro. Thus, it might represent a promising new molecule in anticancer therapy strategies. However, the extreme reactivity of this aldehyde, as well as its insolubility in water, a limiting factor for drug bioavailability, and its rapid degradation by specific enzymes represent major obstacles to its possible in vivo application. Various strategies can used to overcome these problems. One of the most attractive strategies is the use of nanovehicles, because loading drugs into nanosized structures enhances their stability and solubility, thus improving their bioavailability and their antitumoral effectiveness. Several natural or synthetic polymers have been used to synthesize nanosized structures and, among them, ß-cyclodextrin (ßCD) polymers are playing a very important role in drug formulation by virtue of the ability of ßCD to form inclusion compounds with a wide range of solid and liquid molecules by molecular complexation. Moreover, several ßCD derivatives have been designed to improve their physicochemical properties and inclusion capacities. Here we report that the inclusion complex of HNE with a derivative of ßCD, the ßCD-poly(4-acryloylmorpholine) conjugate (PACM-ßCD), enhances the aldehyde stability. Moreover, the inclusion of HNE in PACM-ßCD potentiates its antitumor effects in several tumor cell lines and in a more complex system, such as a human reconstructed skin carrying melanoma tumor cells.

Functional and phenotypical impairment of polymorphonuclear cells in atopic dermatitis: an additional cause for the known susceptibility to infections?.

BACKGROUND: Atopic dermatitis (AD) patients present an high susceptibility to infections. The phagocytic activity of polymorphonuclear granulocytes (PMNs) is mediated by the interactions between Toll-like receptors (TLRs) and pathogen-associated molecular patterns. OBJECTIVE: To investigate functional activity and phenotype of PMNs in AD patients. METHODS: In vitro PMN phagocytosis and intracellular killing towards Klebsiella pneumoniae were evaluated in 24 AD patients; flow cytometry was applied to analyze PMN phenotype. RESULTS: PMNs from AD patients displayed both reduced phagocytic activity and intracellular killing against K. pneumoniae than healthy subjects (HS). CD11b, CD66b, TLR2, TLR4 and TLR5 median fluorescence intensity (MFI) on PMN membrane were significantly higher in AD patients than in HS. CONCLUSION: PMN functional impairment in AD patients could represent an additional cause of skin infections, coupled with other known defects in the innate immune system. The increased MFI of adhesion molecules and TLRs is rather a consequence of the increased skin barrier permeability to bacterial molecules capable of stimulating immunological reactions.