Background: The role of the T-regulatory cells (Tregs) marker forkhead box Protein
3 (FOXP3) in mycoses fungoides (MF) pathogenesis is unclear and the results of
previous studies are inconclusive.
Objective: We aimed at ascertaining the possibility that FOXP3 expression may
serve to predict MF stage and response to therapy.
Patients and methods: Immunohistochemistry staining for FOXP3 was performed
on 30 skin biopsies from patients with MF, and FOXP3 expression level was quantitatively
graded. Disease stage, progression, and response to treatment were determined
based on clinical and imaging evidence, and association with FOXP3 expression
was assessed.
Results: FOXP3 expression in the dermis correlated with poor response to treatment
(P=0.047). A negative non-significant relationship between epidermal FOXP3
expression and clinical stage severity was observed (P=0.17).
Conclusions: Dermal FOXP3 expression in MF lesions could be used to predict response
to treatment in patients with MF

Ellenbogen, Eran

Eytan, Ori

Gat, Andrea

Geller, Shamir

Goldberg, Ilan

Perelman, Max

Rozenblat, Mati

Slutsky Bank, Evgenya

Sprecher, Eli

English

Hrčak - Portal of scientific journals of Croatia

ACTA DERMATOVENEROLOGICA CROATICAActa Dermatovenerol Croat                              2021;29(2):67-71                          ORIGINAL SCIENTIFIC ARTICLE67FOXP3 Predicts Response to Treatment in Mycosis  FungoidesMax Perelman1*, Mati Rozenblat2*, Eran Ellenbogen1, Shamir Geller1,4, Evgenya Slutsky Bank1, Ori Eytan1, Andrea Gat3, Eli Sprecher1,4,  Ilan Goldberg1,41Division of Dermatology, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel;  2Department of Dermatology, Ha’emek Medical Center, Afula, Israel; 3Department  of Pathology, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel ; 4Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, IsraelCorresponding author: Ilan Goldberg, MDDepartment of DermatologySourasky Medical Center6 Weizmann St.Tel-AvivIsraelIlangoldberg1@gmail.comReceived: January 2, 2021Accepted: June 15, 2021*Max Perelman and Mati Rozenblat are equally contributing first authors.ABSTRACTBackground: The role of the T-regulatory cells (Tregs) marker forkhead box Protein 3 (FOXP3) in mycoses fungoides (MF) pathogenesis is unclear and the results of previous studies are inconclusive.Objective: We aimed at ascertaining the possibility that FOXP3 expression may serve to predict MF stage and response to therapy.Patients and methods: Immunohistochemistry staining for FOXP3 was performed on 30 skin biopsies from patients with MF, and FOXP3 expression level was quanti-tatively graded. Disease stage, progression, and response to treatment were deter-mined based on clinical and imaging evidence, and association with FOXP3 expres-sion was assessed.Results: FOXP3 expression in the dermis correlated with poor response to treat-ment (P=0.047). A negative non-significant relationship between epidermal FOXP3 expression and clinical stage severity was observed (P=0.17).Conclusions: Dermal FOXP3 expression in MF lesions could be used to predict re-sponse to treatment in patients with MF. KEY WORDS: MF, mycosis fungoides, CTCL, cutaneous T-cell lymphomas, Tregs, T regulatory cells, FOXP3, forkhead box P3, prognostic factorINTRODUCTIONMycosis fungoides (MF) is a primary cutaneous lymphoma that is characterized by uncontrolled growth of T-cells and is the most common skin lym-phoma. MF has an overall favorable prognosis; how-ever, a small subset of patients can present with a more severe disease progression leading to advanced stages and even death. Prognostic tools capable of predicting MF progres-sion include age, sex, functional status, type of lesions (patch, plaque, tumor), body surface area involved, lymph node involvement, extra-cutaneous disease, transformation to large cell disease and laboratory findings such as elevated C-reactive protein (CRP), lactate dehydrogenase (LDH), erythrocyte sedimenta-tion rate (ESR), clusterin, and β2 microglobulin (1-6). The pathophysiological mechanisms responsible for progression of early-stage MF to tumor and ad-vanced stages remain elusive. 68 ACTA DERMATOVENEROLOGICA CROATICAPerleman  et al. Acta Dermatovenerol CroatFOXP3 in mycosis fungoides   2021;29(2):67-71T-regulatory cells (Tregs) may play a major role in the development and progression of both solid and hematologic tumors (7,14). Forkhead box protein 3 (FOXP3) is a major transcription factor and a specific marker of Tregs. Berger et al. showed in vitro that Tregs ability to block anti-tumor immune responses plays a key role in the pathogenesis of cutaneous T-cell lym-phoma (CTCL) (13). Thus, we hypothesized that FOXP3 may predict disease course in patients with MF. Gjerdum et al. found a significantly higher num-ber of FOXP3 positive Tregs in early-patch stage MF in comparison with late-plaque and tumor stages of the disease. They therefore concluded that the existence of such cells predicts a better prognosis (8). In con-trast, Hallermann et al. found an association between FOXP3 expression and a more aggressive disease course in a limited number of patients with large cell transformation (9). Finally, Klemke et al. (10) as well as Fried and Cerroni (11) did not find any difference in the number of FOXP3-positive Tregs between differ-ent MF stages. The latter study evaluated sequential biopsies of patients at various stages of disease. Fried and Cerroni (11) showed that while Tregs percentage increased from 10% in the patch stage to 15% in the tumor stage (in sequential biopsies of one patient), the prognosis was better and the patient remained alive for 11 more years, in contrast to the average survival seen in tumor-stage MF. Herein, we evaluated the predictive value of FOXP3 expression in MF lesions for a number of dis-ease characteristics.PATIENTS AND METHODSStudy population The study was conducted in a series of patients treated at the Cutaneous Lymphoma Clinic at the Tel Aviv Sourasky medical center between 2007 and 2014 according to a protocol reviewed and approved by the institutional ethics committee. We analyzed 30 patients who were diagnosed according to the WHO-ISCL\EORTC classification of cutaneous lymphomas (15). Data were retrieved retrospectively from the patients’ medical records. Patient characteristics re-corded included: demographic data (age, sex) as well as clinical data (age of onset, age at diagnosis, clinical stage according to the EORTC criteria). Stage severity was graded on a scale of 1-3: mild (1) – stages IA-IIA; moderate (2) – stages IIB-IIIB; severe (3) – stages IVA1-IVB.A response to treatment scale was defined, as de-scribed below (Table 1), taking into consideration the following elements: complete or partial remission, time period until reaching remission, elapsed time until disease recurrence, change in clinical stage dur-ing therapy. Immunohistochemistry Paraffin-embedded sections were stained using an anti-FOXP3 specific monoclonal antibody (236\E7 specific anti FOXP3 antibody, Serotec, Kidlington, UK). FOXP3 expression was assessed separately in the dermis and epidermis along a semi quantitative scale Figure 1. Histopathologic findings of MF skin biopsies, showing superficial and deep skin infiltrates of FOXP3-positive T lymphocytes. (a) FOXP3 positive T lymphocyte infiltrates of the dermis with no stain-ing in the epidermis; (b) Combined positive epidermal and dermal FOXP3-positive T lymphocyte infiltrates. (Staining with 236\E7 specific anti FOXP3 antibody, Serotec, Kidlington, UK, original magnification ×10)ACTA DERMATOVENEROLOGICA CROATICA 69Perleman  et al. Acta Dermatovenerol CroatFOXP3 in mycosis fungoides   2021;29(2):67-71ranging 1-5 (Figure 1). Positive cells were counted over the entire lymphoid infiltrate.Statistical methodsCorrelations between FOXP3 expression levels in the dermis and epidermis, disease stage, and re-sponse to treatment were examined using the Spear-man correlation test – using SAS 9.2 for Windows.RESULTSWe studied 30 patients (Table 2) including16 male individuals (53.3%), with an average age of 51.5 years. Age of onset ranged from 5-87 years, and the average time from onset of symptoms to diagnosis was 18.75 months for men and 54.86 months for women. Most patients were diagnosed at an early stage of MF. FOXP3 expression levels in the dermis and epi-dermis of patients with MF was on average 2.23 and 1.43, respectively. Data analysis revealed a statisti-cally significant positive correlation between FOXP3 expression in the dermis and response to treatment score (r=0.36, P=0.047) (Figure 2). Epidermal FOXP3 expression was not associated with response to treatment. We found that the higher the FOXP3 level in the dermis, the longer it took to achieve remission, with relapse occurring earlier. Furthermore, we iden-tified a non-significant trend for negative correlation between FOXP3 expression in the epidermis and stage severity (r=-0.25, P=0.17) and a non-significant correlation with the clinical stage (P=0.66). No signifi-cant correlation was found between dermal FOXP3 expression levels and stage severity or clinical stage at the time of diagnosis.Figure 2. Correlation between dermal FOXP3 expres-sion and response to treatment score.Table 1. Response to treatment scaleComplete Response (CR) and no relapse:1 Did not progress to a more advanced stage, reached CR within 3 months, no relapse2 Did not progress to a more advanced stage, reached CR within 3-12 months, no relapse3 Did not progress to a more advanced stage, reached CR within more than 12 months, no relapseComplete Response (CR), with relapse after more than 12 months:4 Did not progress to a more advanced stage, reached CR within 3 months, relapse after more than 12 months5 Did not progress to a more advanced stage, reached CR within 3-12 months, relapse after more than 12 months6 Did not progress to a more advanced stage, reached CR within more than 12 months, relapse after more than 12 monthsComplete Response (CR), with relapse after 3-12 months:7 Did not progress to a more advanced stage, reached CR within 3 months, relapse after 3-12 months8 Did not progress to a more advanced stage, reached CR within 3-12 months, relapse after 3-12 months9 Did not progress to a more advanced stage, reached CR within more than 12 months, relapse after 3-12 monthsComplete Response (CR), with relapse after less than 3 months:10 Did not progress to a more advanced stage, reached CR within 3 months, with relapse after less than 3 months11 Did not progress to a more advanced stage, reached CR within 3-12 months, with relapse after less than 3 months12 Did not progress to a more advanced stage, reached CR within more than 12 months, with relapse after less than 3 monthsDid not reach CR13 Did not progress to a more advanced stage, reached PR only14 Progressed to a more advanced stage (PD), but not to large cell transformation or to Sézary syndrome15 Progressed to a more advanced stage, to Sézary syndrome, or to large-cell transformation16 Sézary syndrome was the initial diagnosis17 Died from MF/SS, or from related complicationsAbbreviations: CR – complete response; PR – partial response; PD – progressive disease70 ACTA DERMATOVENEROLOGICA CROATICADISCUSSIONPrevious studies which assessed the prognos-tic value of FOXP3 staining in skin biopsies yielded contradictory results (9,11). In the present study, we detected a positive and statistically significant corre-lation between FOXP3 expression in the dermis and response to treatment score. Higher levels of FOXP3 in the dermis predicted a more severe course of the disease and poorer response to therapy, including longer periods for achieving remission and higher chance for recurrence and shorter remission. Further-more, a negative non-significant correlation between FOXP3 expression level in the epidermis and stage severity was found. The small number of patients with severe stage disease was a limitation of this study. Our conclusions are valid for patients with early stage disease, but no firm conclusions can be drawn from the expression of FOXP3 in patients with advanced stage MF. There could be several explanations for the con-tradicting results in the different studies which in-vestigated the expression of FOXP3 in MF, including: varying antibody specificity (9); the fact that neo-plastic and reactive cells could not be separated in these studies (FOXP3-positive malignant cells seem to loose expression of the protein during disease progression (9)); the fact that several types of Tregs have been recognized with different effect on disease Case num SexSymptoms duration prior to diagnosis (y)Age at disease presentationAge at diagnosisFOXP3 epidermisFOXP3 dermisStage by EORTCStage severityResponse to treatment score1 F 1 81 82 0 2 IIB Moderate 142 F 2 18 20 2 3 IA Mild 23 M 0 12 12 1 3 IA Mild 34 M 1 55 56 3 4 IIA Mild 135 F 0 81 81 2 3 IIIB Moderate 176 F 0 50 50 1 1 IA Mild 27 M 2 66 68 1 3 IIIA Moderate 138 F 3 37 40 2 3 IA Mild 139 F 0 54 53 2 3 IA Mild 1310 F 0 58 58 1 5 IIIA Moderate 1311 M 1 55 56 3 2 IA Mild 512 F 23 63 86 2 2 IB Mild 1113 F 21 5 26 1 1 IIA Mild 1314 M 1 54 55 2 2 IIB Moderate 615 F 4 52 56 0 1 IVA1 Severe 1516 M 2 30 32 1 1 IA Mild 817 M 4 55 59 1 1 IA Mild 218 M 3 37 40 2 3 IA Mild 519 M 0 54 54 1 1 IVA1 Severe 1520 M 1 64 65 0 1 IA Mild 121 F 8 9 17 1 4 IB Mild 1322 F 1 47 48 0 0 IA Mild 123 M 0 53 53 3 1 IB Mild 1124 F 1 18 19 0 1 IA Mild 225 M 3 86 89 1 2 IB Mild 326 M 3 44 47 1 1 IIA Mild 1127 M 2 63 65 1 3 IA Mild 528 M 1 47 48 2 0 IA Mild 829 M 1 23 24 5 5 IB Mild 1130 F 0 87 87 1 5 IVA1 Severe 15Table 2. Demographic and clinical features of the cohortPerleman  et al. Acta Dermatovenerol CroatFOXP3 in mycosis fungoides   2021;29(2):67-71ACTA DERMATOVENEROLOGICA CROATICA 71prognosis (8-11): (a) suppressor Tregs; (b) malignant Tregs; (c) direct tumor-killing Tregs; and (d) incompe-tent Tregs (16). Tumor-killing Tregs and incompetent Tregs are associated with better prognosis, while the opposite is true for suppressor Tregs and malignant Tregs. FOXP3 promoter demethylation may underlie the role of malignant Tregs in MF (17). In summary, FOXP3 expression in skin biopsies of patients with MF might serve as an adjunct prognos-tic tool. The present study suggests the need for con-firmatory larger and prospective studies using series of sequential biopsies. References:1.  Diamandidou E, Colome M, Fayad L, Duvic M, Kurzrock R. Prognostic factor analysis in myco-sis fungoides/Sezary syndrome. J Am Acad Der-matol. 1999;40:914-24.2.  Agar NS, Wedgeworth E, Crichton. S, Mitchell TJ, Cox M, Ferreira S, et al. Survival outcomes and prognostic factors in mycosis fungoides/Sezary syndrome: validation of the revised International Society for Cutaneous Lymphomas/European Or-ganisation for Research and Treatment of Cancer staging proposal. J Clin Oncol. 2010;28:4730-9.3.  Tobisawa S, Honma M, Ishida-Zamamoto A, Saijo Y, Iizuka H. Prognostic factors in 105 Japanese ca-ses of mycosis fungoides and Sezary syndrome: clusterin expression as a novel prognostic factor. J Dermatol Sci. 2013;71:160-6.4.  Hallermann C, Niermann C, Fischer RJ, Schulze HJ. Erythrocyte sedimentation rate as an indepen-dent prognostic factor in mycosis fungoides. Br J Dermatol. 2012;166:873-4.5.  Wilcox RA. Cutaneous T-cell lymphoma: 2016 up-date on diagnosis, risk-stratification, and manage-ment. Am J Hematol. 2016;91:151-65.6.  Jawed SI, Myskowski PL, Horwitz S, Moskowitz A, Querfeld C. Primary cutaneous T-cell lymphoma (mycosis fungoides and Sezary syndrome): part II. 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Profiles of Foxp3+ regulatory T cells in eczematous dermati-tis, psoriasis vulgaris and mycosis fungoides. Br J Dermatol. 2008;158:1256-63.13. Berger CL, Tigelaar R, Cohen J, Mariwalla K, Tinh J, Wang N, et al. Cutaneous T-cell lymphoma: ma-lignant proliferation of T-regulatory cells. Blood. 2005;105:1640-7.14. Marshall NA, Christie LE, Munro LR, Culligan DJ, Johnston PW, Barker RN, et al. Immunosuppres-sive regulatory T cells are abundant in the reac-tive lymphocytes of Hodgkin lymphoma. Blood. 2004;103:1755-62.15. Olsen E, Vonderheid E, Pimpinelli N, Willemze R, Kim Y, Knobler K, et al. Revisions to the staging and classification of mycosis fungoides and Se-zary syndrome: a proposal of the International Society for Cutaneous Lymphomas (ISCL) and the cutaneous lymphoma task force of the European Organization of Research and Treatment of Can-cer (EORTC). Blood. 2007;110:1713-22.16. Wang J, Ke XY. The four types of Tregs in malignant lymphomas. J Hematol Oncol. 2011;4:50.17. Heid JB, Schmidt A, Oberle N, Goerdt S, Krammer PH, Suri-Payer E, et al. FOXP3+CD25- tumor cells with regulatory function in Sezary syndrome. J In-vest Dermatol. 2009;129:2875-85.Perleman  et al. Acta Dermatovenerol CroatFOXP3 in mycosis fungoides   2021;29(2):67-71

FOXP3 Predicts Response to Treatment in Mycosis Fungoid

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FOXP3 Predicts Response to Treatment in Mycosis Fungoid

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