Estimation of the tissue and serum levels of IL-35 in Mycosis fungoides: a case-control study.

Mycosis fungoides (MF) is the most common primary cutaneous T-cell lymphoma (CTCL) with its etiology not yet fully understood. Interleukin (IL)-35 is an inhibitory cytokine that belongs to the IL-12 family. Elevated IL-35 in the plasma and the tumor microenvironment increases tumorigenesis and indicates poor prognosis in different types of malignancies. The objective of this study is to estimate the expression levels of IL-35 in tissue and serum of MF patients versus healthy controls. This case-control study included 35 patients with patch, plaque, and tumor MF as well as 30 healthy controls. Patients were fully assessed, and serum samples and lesional skin biopsies were taken prior to starting treatment. The IL-35 levels were measured in both serum and tissue biopsies by ELISA technique. Both tissue and serum IL-35 levels were significantly higher in MF patients than in controls (P < 0.001) and tissue IL-35 was significantly higher than serum IL-35 in MF patients (P < 0.001). Tissue IL-35 was significantly higher in female patients and patients with recurrent MF compared to male patients and those without recurrent disease (P < 0.001). Since both tissue and serum IL-35 levels are increased in MF, IL-35 is suggested to have a possible role in MF pathogenesis. IL-35 can be a useful diagnostic marker for MF. Tissue IL-35 can also be an indicator of disease recurrence.

Determination of immunophenotypic aberrancies provides better assessment of peripheral blood involvement by mycosis fungoides/Sézary syndrome than quantification of CD26- or CD7- CD4+ T-cells.

BACKGROUND: Blood involvement by mycosis fungoides (MF)/Sézary syndrome (SS) influences prognosis and therapeutic decisions. MF/SS blood stage is currently determined by absolute CD4 + CD26- or CD4 + CD7-cell counts, which quantification method may overestimate MF/SS by including CD26- or CD7- normal CD4+ T-cells, or underestimate disease burden when MF/SS cells show incomplete loss of CD26 and/or CD7. Recently, through the standardization effort led by the International Clinical Cytometry Society (ICCS), recommendation was made to quantify MF/SS by enumerating immunophenotypically aberrant CD4+ T-cells, rather than CD26- or CD7- in isolation. METHODS: We compared these two quantitation methods in 309 MF/SS patients who had blood samples analyzed by flow cytometry immunophenotyping (FCI) over a 1-year period. RESULTS: Using the European Organization of Research and Treatment of Cancer (EORTC)/International Society for Cutaneous Lymphomas (ISCL) criteria, 221 (71.5%) patients had a blood stage corresponding to B0, 57 (18.4%) to B1, and 31 (10%) to B2. By FCI analysis, a total of 62 patients (20.0%) were found positive for MF/SS. Among EORTC B0 patients, 11/221 (5%) were positive by FCI (false negatives), and among EORTC Stage B1 patients, 35/57 (61%) were negative by FCI (false positives). Regarding patients positive for MF/SS cells by FCI, there was an overall excellent correlation (r = .999, p < .001) between the EORTC/ISCL method and FCI method; however, four (6.5%) patients would have an altered B stage between B0 and B1. CONCLUSION: The MF/SS cell quantification method using immunophenotypic aberrancies, as recommended by the ICCS, allows to distinguish MF/SS cells from background benign T-cells and enables for more accurate staging, especially among patients currently being considered to have B0 and B1 stage diseases.

Flow cytometric evaluation of peripheral blood for suspected Sézary syndrome or mycosis fungoides: International guidelines for assay characteristics.

A peripheral blood flow cytometric assay for Sézary syndrome (SS) or circulating mycosis fungoides (MF) cells must be able to reliably identify, characterize, and enumerate T-cells with an immunophenotype that differs from non-neoplastic T-cells. Although it is also important to distinguish SS and MF from other subtypes of T-cell neoplasm, this usually requires information in addition to the immunophenotype, such as clinical and morphologic features. This article outlines the approach recommended by an international group with experience and expertise in this area. The following key points are discussed: (a) At a minimum, a flow cytometric assay for SS and MF should include the following six antibodies: CD3, CD4, CD7, CD8, CD26, and CD45. (b) An analysis template must reliably detect abnormal T-cells, even when they lack staining for CD3 or CD45, or demonstrate a phenotype that is not characteristic of normal T-cells. (c) Gating strategies to identify abnormal T-cells should be based on the identification of subsets with distinctly homogenous immunophenotypic properties that are different from those expected for normal T-cells. (d) The blood concentration of abnormal cells, based on any immunophenotypic abnormalities indicative of MF or SS, should be calculated by either direct enumeration or a dual-platform method, and reported.

Mycosis Fungoides and Its Relationship to Atopy, Serum Total IgE, and Eosinophil Counts.

INTRODUCTION: A recent serologic study and reports of increased serum total IgE (IgE-t) and eosinophil counts have suggested that the prevalence of atopy is more common in patients with mycosis fungoides (MF) than previously recognized. PATIENTS AND METHODS: Patients with clinicopathologic features that were diagnostic and/or consistent with MF and/or the presence or absence of an atopic disorder (eg, allergic rhinitis, asthma, eczematous dermatitis), which was determined by patient history, eosinophil counts, and serum IgE-t obtained at evaluation, were selected from a patient registry. The MF population was divided into those with atypical and typical clinical presentations. We performed matching of controls using age, sex, and race from the 2005 to 2006 National Health Education Survey. RESULTS: A history of allergic rhinitis was recorded for 186 of 728 patients (25.5%) with typical MF and 71 of 229 patients (31%) with atypical MF. However, the prevalence of asthma and eczema was low. The IgE-t and eosinophil counts were higher for patients with typical MF than for controls and for patients with atopic diathesis than for patients without atopy. The IgE-t and eosinophil counts were higher for the patients with advanced-stage MF compared with those for the patients with less-advanced disease for both atopic and nonatopic cohorts. In the Cox model with age and clinical stage as covariates, a history of atopy, increased IgE-t, and blood eosinophilia (> 500 cells/mm3) did not correlate with overall survival. CONCLUSION: The findings from the present study did not reveal a significant association of atopy in patients with MF. However, atopy is a factor in the increased IgE-t and eosinophil counts observed in MF. Another factor is related to the disease stage, including possibly the influence of cytokines secreted by T-helper type 2-polarized neoplastic cells.

Interleukin-33 polymorphisms and serum concentrations in mycosis fungoides.

BACKGROUND: Mycosis fungoides (MF) skin lesions are characterized by low-grade inflammation, which may be sustained by proinflammatory cytokines as probably interleukin-33 (IL-33). We compared serum concentrations of IL-33 and its receptor ST2 and the frequency of selected IL-33 single nucleotide polymorphisms (SNPs) between patients with MF and healthy controls. METHODS: In 88 patients with MF and 66 healthy controls, we analyzed SNPs in the 9894 and 11877 loci of the IL-33 gene. Moreover, we measured serum concentrations of IL-33 and its receptor ST2. RESULTS: There were no statistically significant differences in the frequencies of both IL-33 SNPs between patients and controls. Compared with controls, patients with MF had similar IL-33 serum concentrations (P = 0.71) but significantly increased ST2 concentrations (P < 0.001). Patients in MF-IA stage had significantly lower ST2 serum concentrations than those with the remaining MF stages (P = 0.002). The studied variables were not related to pruritus severity. Patients with the C(+) IL-33 11877 SNP had lower ST2 serum concentrations than patients with the C(-) 11877 SNP (P = 0.043). CONCLUSIONS: It was published before that the knockout of the ST2 gene after injection of IL-33 is associated with a reduced inflammatory reaction in the skin, as well as that IL-33 plays a role in allergic and neoplastic disorders. Concerning the difference in ST2 concentration between control and MF group, and C IL-33 11877 SNP possibly influencing the ST2 concentration, the role of IL-33/ST2 signaling, needs further studies.

Multi-institutional Investigation: Circulating CD4:CD8 ratio is a prognosticator of response to total skin electron beam radiation in mycosis fungoides.

BACKGROUND AND PURPOSE: A lower proportion of CD8+ tumor infiltrating lymphocytes in mycosis fungoides (MF) patients is associated with worse survival. However, it is not known whether circulating CD4:CD8 ratio is a prognosticator of response to total skin electron beam therapy (TSEBT). METHODS AND MATERIALS: We identified 126 MF patients treated with TSEBT from 2001 to 20014 at two high-volume academic centers. Circulating CD4:CD8 ratio was obtained within 1 week before TSEBT. TSEBT was delivered with 6-9mEV electrons with low (12 Gy) or conventional (≥12 Gy) doses. Treatment response was assessed with the modified Severity Weighted Assessment Tool (mSWAT). Post-treatment mSWAT decrease of ≥75% was classified as near complete response (CR) while mSWAT decrease of <75% was considered partial response (PR). Receiver operating characteristic analysis determined an optimal CD4:CD8 threshold value to predict TSEBT response in the derivation cohort and was applied to an external validation cohort. RESULTS: 71.4% and 28.6% of patients achieved CR and PR after TSEBT. Higher CD4:CD8 ratio predicted poorer response: median CD4:CD8 in patients with PR vs. CR was 4.84 vs. 1.97 (p = 0.002). A threshold CD4:CD8 of 4.42 optimally discriminated in the discovery cohort patients with PR vs. XR (sensitivity 90%, specificity 59%, area under curve (AUC) = 0.71; p = 0.002). Within an independent test cohort (n = 32), 73.9% of patients with CD4:CD8 <4.42 achieved CR vs. 33.3% of those with CD4:CD8 ≥4.42 (p = 0.033). Among all patients with CD4:CD8 <4.42 (n = 73), 74% achieved CR with low-dose TSEBT vs. 93% with conventional dose TSEBT (p = 0.02). On multivariable logistic regression, CD4:CD8 remained a significant independent predictor of TSEBT response in all patients (OR = 0.107, 95% CI 0.395-0.290, p < 0.001). CONCLUSION: Peripheral blood CD4:CD8 ratio was a significant independent predictor of TSEBT response of MF patients as validated in an independent cohort at separate academic center. The potential for CD4:CD8 ratio as a biomarker to inform radiation treatment dosing warrants further investigation.

Prognostic Significance of Serum Copper in Patients With Cutaneous T-cell Lymphoma.

BACKGROUND: Serum copper has been reported to be increased in various cancers, including lymphoma. The purpose of the present study was to investigate the clinical and prognostic importance of serum copper levels in patients with cutaneous T-cell lymphoma (CTCL). PATIENTS AND METHODS: Serum copper was measured in 60 men and 38 women with mycosis fungoides (MF) and 14 men and 3 women with erythrodermic CTCL (6 with Sézary syndrome) consecutively evaluated from July 1980 to June 1985. RESULTS: A greater than normal copper level was present in nearly 20% of patients and was associated with an increased risk of disease progression and shortened disease-specific survival for patients with patch or plaque phase MF, but not for those with tumor phase MF or erythrodermic CTCL. In contrast, the serum lactate dehydrogenase level and neutrophil/lymphocyte ratio were not significantly associated with prognosis in our patient cohort. CONCLUSION: The reason for the association between the high serum copper levels and adverse prognosis is unknown. We hypothesized that interleukin-6 is secreted primarily by non-neoplastic cells at MF skin sites, leading to release of copper by the liver. Local production of interleukin-6 at the lesion sites might conceivably also promote neoplastic cell progression by stimulation of the STAT3 pathway. Further studies on the relationship between activated tumor-associated macrophages, serum copper levels, interleukin-6, or C-reactive protein and prognosis might be informative.

Decreased progranulin expression in Mycosis fungoides: a possible association with the high frequency of skin infections.

Progranulin (PGRN) is a multi-functional protein known to be involved in diverse biological processes, including tumourigenesis, anti-inflammation, and anti-infection. PGRN expression in sera or tissues is elevated in a variety of malignancies and is associated with poor prognosis. However, it remains to be determined whether PGRN is involved in Mycosis fungoides (MF). To investigate the roles of PGRN in MF. Serum PGRN levels were measured in patients with MF and normal controls by enzyme-linked immunosorbent assay. PGRN expression in MF and normal skin was examined by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. Moreover, we analysed correlations between the expression levels of PGRN and antimicrobial peptides in lesional skin. PGRN levels were significantly lower in sera of MF patients than those of normal controls. PGRN mRNA levels in lesional skin of MF were also significantly decreased. Immunohistochemical staining revealed that PGRN was expressed in epidermal keratinocytes of normal controls, however, PGRN expression in epidermal keratinocytes was also weaker in MF skin. Furthermore, significant inverse correlations were identified between PGRN and antimicrobial peptide mRNA expression. These results suggest that low PGRN expression may contribute to the frequent occurrence of skin infections in patients with MF.

Infectious events and associated risk factors in mycosis fungoides/Sézary syndrome: a retrospective cohort study.

BACKGROUND: Infections are one of the major causes of death in patients with advanced-stage mycosis fungoides (MF) or Sézary syndrome (SS). However, few recent data are available on the characteristics and risk factors of these infectious events. OBJECTIVES: To describe infectious events occurring in a cohort of patients with MF/SS, and to identify associated clinical and biological risk factors. METHODS: A retrospective cohort study was performed to investigate infectious events and associated factors in patients diagnosed with MF (stage IB and beyond) or SS followed from May 2011 to May 2016 at the University Hospital of Bordeaux, France. RESULTS: Seventy-one patients with complete follow-up were included. Eighty infectious events were recorded in 40 patients, including 28 skin and soft tissue infections and 25 cases of pneumonia. Opportunistic infections, which are usually associated with depleted cell-mediated immunity, were scarce (9%). In multivariate analysis, cardiac, renal or lung comorbidities [odds ratio (OR) 7·2, 95% confidence interval (CI) 3·3-15·9; P = 0·002], SS (OR 8·8, 95% CI 7·7-10·2; P = 0·037) and lymphocyte count < 0·5 × 109 cells L-1 (OR 6·4, 95% CI 1·5-27·4; P = 0·004) were significantly associated with a higher risk of infection. CONCLUSIONS: Opportunistic germs were rarely recorded, but their incidence was probably prevented by adequate prophylaxis (ongoing in 28% of patients). As in patients living with AIDS, pneumonias were frequent. On the other hand, bacterial cutaneous infections represent a specific pattern in patients with MF/SS. Patients with chronic organ failure, lymphocytopenia and SS should be considered as being at high risk for infectious events. Pneumococcal vaccination should be systematically recommended, and prophylaxis with co-trimoxazole and valaciclovir when the CD4 count is < 0·2 × 109 cells L-1 .

Mycosis Fungoides and Vitamin D Status: Analyses of Serum 25-Hydroxyvitamin D Levels and Single Nucleotide Polymorphisms in the Vitamin D Receptor Gene.

Various types of cancer, including melanoma and non-melanoma skin cancer, are associated with vitamin D receptor (VDR) polymorphisms. However, few studies have addressed VDR polymorphisms in patients with mycosis fungoides (MF), and previous studies have reported conflicting results. Aim of this case-control study was to assess the correlation between VDR single nucleotide polymorphisms (SNPs) Cdx2, Fok1, Apa1, Bsm1, and Taq1 and MF. Venous blood samples were collected from 41 patients with MF and 59 age- and sex-matched healthy controls. VDR genotypes of both groups were analyzed. Serum vitamin D levels of patients with MF were also analysed among varying stages and VDR genotypes. Vitamin D levels were significantly low (<30 ng/mL) in 87.9% of the patients (P<0.001). No associations were found between Apa1, Cdx2, Fok1, and Bsm1 SNPs and MF. However, Taq1 polymorphisms were higher in the healthy control group (P<0.001). Our study supports the claim that vitamin D deficiency is common in patients with MF. On the other hand, our findings suggest that Taq1 polymorphisms may be associated with decreased susceptibility to MF. Therefore, VDRs may have complex and heterogeneous effects on the pathogenesis of MF.

Blood classification and blood response criteria in mycosis fungoides and Sézary syndrome using flow cytometry: recommendations from the EORTC cutaneous lymphoma task force.

Our current mycosis fungoides (MF) and Sézary Syndrome (SS) staging system includes blood-classification from B0-B2 for patch/plaque/tumour or erythroderma based on manual Sézary counts but results from our EORTC survey confirm these are rarely performed in patch/plaque/tumour MF, and there is a trend towards using flow cytometry to measure blood-class. Accurately assigning blood-class effects overall stage and the 'global response' used to measure treatment responses in MF/SS and hence impacts management. The EORTC Cutaneous Lymphoma Task Force Committee have reviewed the literature and held a Workshop (June 2017) to agree a definition of blood-class according to flow cytometry. No large study comparing blood-class as defined by Sézary count with flow cytometry has been performed in MF/SS. The definition of blood-class by flow cytometry varies between publications. Low-level blood involvement occurs in patch/plaque/tumour much less than erythroderma (p < 0.001). The prognostic relevance of blood involvement (B1 or B2) in patch/plaque/tumour is not known. Studies have not shown a statistically worse difference in prognosis in erythrodermic MF patients with low-level blood involvement (IIIB) versus those without (IIIA), but Sezary patients who by definition have a leukaemic blood picture (staged IVA1 or higher) have a worse prognosis. For consistency flow, definition for blood-class must be an objective measurement. We propose absolute counts of either CD4+CD7-or CD4+CD26-where B0<250/µL, B1 = 250/µl-<1000/µL and B2≥1000/µL plus a T-cell blood clone. Fluctuations between B0 and B1 should not be considered in the treatment response criteria until further prognostic information is known.

Increased interleukin-36γ expression in skin and sera of patients with atopic dermatitis and mycosis fungoides/Sézary syndrome.

Interleukin (IL)-36γ is expressed by keratinocytes and functions as a key initiator of inflammation in the skin. IL-36γ expression is enhanced by tumor necrosis factor-α and IL-17A, having a strong association with psoriasis. In this study, we examined the role of IL-36γ in atopic dermatitis (AD) and mycosis fungoides (MF)/Sézary syndrome (SS). Serum levels of IL-36γ in AD patients and MF/SS patients were elevated compared with those of healthy controls. Importantly, serum IL-36γ levels in AD patients positively correlated with Eczema Area and Severity Index and those of MF/SS patients positively correlated with serum soluble IL-2 receptor levels. IL-36γ mRNA levels in AD skin and MF/SS skin were significantly higher than those of normal skin. IL-36γ mRNA levels in MF/SS skin positively correlated with IL-17A mRNA levels. Immunohistochemical staining revealed that IL-36γ was highly expressed in keratinocytes in lesional skin of AD and MF/SS. Taken together, our study demonstrated that IL-36γ expression was increased in sera and skin of patients with AD and MF/SS as was reported in psoriatic patients.

Serum vascular endothelial growth factor A levels reflect itch severity in mycosis fungoides and Sézary syndrome.

Angiogenesis is an important step to support progression of malignancies, including mycosis fungoides (MF) and Sézary syndrome (SS). Vascular endothelial growth factor (VEGF)-A, a key player in angiogenesis, is secreted by tumor cells of MF/SS and its expression levels in lesional skin correlated with disease severity. In this study, we examined serum VEGF-A levels in MF/SS patients. Serum VEGF-A levels were elevated in patients with erythrodermic MF/SS and the levels decreased after treatment. Importantly, serum VEGF-A levels positively correlated with markers for pruritus. We also found that VEGF-A upregulated mRNA expression of thymic stromal lymphopoietin by keratinocytes. Taken together, our study suggests that VEGF-A can promote progression and pruritus in MF/SS. Inhibition of VEGF-A signaling can be a therapeutic strategy for patients with erythrodermic MF/SS.

CD4+CD26- lymphocytes are useful to assess blood involvement and define B ratings in cutaneous T cell lymphoma.

Bernengo et al. reported that >30% CD4+CD26- lymphocytes detect blood involvement in patients with mycosis fungoides (MF) and Sézary syndrome. In addition, the ISCL/EORTC suggested that this threshold might serve as a criterion for the B2 blood rating for staging. In this manuscript, we report our experience with measurement of CD4+CD26- and CD4+CD7- cells, Sézary cell counts, and aberrant T cells with diminished expression of CD2, CD3, or CD5 antigens. CD4+CD26- ≥30% occurred in 15 of 373 (4.0%) patients with MF, 33 of 47 (70%) patients with erythrodermic cutaneous T cell lymphoma (ECTCL) and 2 of 54 (4%) patients with inflammatory skin diseases. CD4+CD26- measurements provided a more reliable assessment of neoplastic cell numbers in the blood than Sézary cell or CD4+CD7- percentages. CD4+CD26- measurements may be used to define B ratings for staging with B2 defined as CD4+CD26- ≥ 1000/µL, plus clonality or phenotypically abnormal cells.