|Year : 2020 | Volume
| Issue : 1 | Page : 243-247
Thymocyte selection-associated HMG-box 'thymocyte selection-associated HMG-box' in mycosis fungoides: should it be mandatory in diagnosis?
Mohamed A Gaber1, Asmaa G Abdou2, Hesham N Khaled1, Mona E Kamel3
1 Department of Dermatology, Andrology and STDs, Menoufia University, Shebeen El-Kom, Egypt
2 Department of Pathology, Faculty of Medicine, Menoufia University, Shebeen El-Kom, Egypt
3 Department of Dermatology, Ministry of Health, Alexandria, Egypt
|Date of Submission||09-Jun-2019|
|Date of Decision||09-Jul-2019|
|Date of Acceptance||14-Jul-2019|
|Date of Web Publication||25-Mar-2020|
Mona E Kamel
Source of Support: None, Conflict of Interest: None
To study the immunohistochemical expression of thymocyte selection-associated HMG-box (TOX) as a diagnostic marker in mycosis fungoides (MF).
MF is the most common type of cutaneous T-cell lymphoma. Differentiation of MF from benign inflammatory dermatoses is important to ensure proper management. TOX is a critical regulator of early T-cell development that is considered as a useful marker for MF diagnosis and prognosis.
Patients and methods
This study was carried out on 32 skin biopsies, 20 MF cases, and 12 benign inflammatory dermatosis. The diagnosis was established after clinicopathologic correlation immunohistochemical (IHC) was done in MF cases and benign inflammatory dermatosis for TOX. This was a cross-sectional analytical study.
TOX expression showed significant positive expression in MF cases compared with benign inflammatory dermatoses, with 80% sensitivity and 83% specificity. Also, TOX positivity increased with the progression of the disease in MF cases. There was a significant difference between MF and inflammatory groups as regards TOX expression which was in favor of the malignant group (P = 0.001).
TOX may have a potential role as a molecular diagnostic marker for MF and overexpression of it correlates with increased risk of disease progression and poor prognosis.
Keywords: benign inflammatory dermatosis, cutaneous T-cell lymphoma, marker mycosis fungoides, thymocyte selection-associated HMG-box
|How to cite this article:|
Gaber MA, Abdou AG, Khaled HN, Kamel ME. Thymocyte selection-associated HMG-box 'thymocyte selection-associated HMG-box' in mycosis fungoides: should it be mandatory in diagnosis?. Menoufia Med J 2020;33:243-7
|How to cite this URL:|
Gaber MA, Abdou AG, Khaled HN, Kamel ME. Thymocyte selection-associated HMG-box 'thymocyte selection-associated HMG-box' in mycosis fungoides: should it be mandatory in diagnosis?. Menoufia Med J [serial online] 2020 [cited 2020 Aug 14];33:243-7. Available from: http://www.mmj.eg.net/text.asp?2020/33/1/243/281270
| Introduction|| |
Cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of extranodal non-Hodgkin's lymphomas that are characterized by a cutaneous infiltration of malignant monoclonal T lymphocytes. They typically afflict adults with a median age of 55–60 years. In 1806, Alibert initially described mycosis fungoides (MF) as the infiltration of the skin by lymphocytes. In 1974, Edelson used the term 'cutaneous T-cell lymphomas' (CTCLs) for MF and its leukemic variant, Sézary syndrome, which are the major types of CTCL. Nowadays, the CTCLs, which are characterized by infiltration of malignant monoclonal T lymphocytes in the skin, are considered a heterogeneous group of extranodal non-Hodgkin's lymphomas. MF is the most common type of primary cutaneous lymphoma, a malignant disease initially affecting the skin. MF is characterized by a clonal expansion of atypical CD4+ skin-homing T lymphocytes. MF has an indolent and prolonged clinical course over years or sometimes decades, progressing from patches to more infiltrated plaques and eventually to tumors. In the early stage, MF is mostly limited to the skin, but in advanced cases of MF, malignant lymphocytes may disseminate to lymph nodes, peripheral blood, and visceral organs. The survival rate for MF critically depends on the stages of the disease. The diagnosis of MF is mainly based on an integrated algorithm of clinical and histological criteria. The diagnosis of early-stage MF (patch, and early plaque MF) is challenging even for experienced dermatologists, because of the morphologic and histological similarities of MF to benign inflammatory dermatitis. Thymocyte selection-associated HMG-box (TOX) encodes a high-mobility group family (HMG) domain DNA-binding nuclear protein. TOX is primarily expressed in the thymus and downregulated before CD4+ T cells exit the thymus. TOX mRNA and proteins were poorly expressed in the peripheral lymphoid tissue. TOX is highly expressed in the thymus and in developing T cells, but after exiting the thymus and entering the peripheral tissue, mature CD4+ T cells never again expressed TOX to a significant level. Recent studies have shown that the TOX gene is highly expressed in early-stage MF lesion in comparison to controls. This study aimed to study the immunohistochemical expression of TOX as a diagnostic marker in MF.
| Patients and Methods|| |
This study was carried out on 32 cases previously diagnosed clinically and histologically. These cases were received as marked paraffin blocks. Twenty cases were diagnosed as MF and 12 cases were diagnosed as benign inflammatory dermatosis; three cases of chronic nonspecific dermatitis, three cases of spongiotic dermatitis, three cases of lichen planus, and three cases of psoriasis. These specimens were collected from the Dermatology, Andrology, and STDs Department, Faculty of Medicine, Menoufia University in the period between August 2017 and January 2019. Approval from the Research Ethics Committee, Faculty of Medicine, Menoufia University, was taken beforehand to conduct the study.
Confidentiality of the data: the prepared paraffin blocks were signed with code numbers instead of the name of the patient to maintain privacy of participants and confidentiality of the data. Also, the results of the research were used only in scientific aims.
In this study, the work protocol entailed the following:
- Clinical data: clinical data such as age, sex, and clinical presentation were obtained from the cases' clinical sheets from the Department of Dermatology, Andrology, and STDs, Faculty of Medicine, Menoufia University
- Preparation of cases: two paraffin sections, each 4 μm thick, were cut from each block, one of them was stained by hematoxylin and eosin (H and E) to confirm the pathological diagnosis and the other sections were cut on poly-l-lysine-coated slides for immunostaining procedure (routine H and E staining, immunohistochemical staining using TOX staining).
H and E histopathological evaluation: H and E-stained sections were examined by light microscope to evaluate and verify epidermal and dermal pathological changes.
Immunohistochemical staining: the method used for immunostaining was a streptavidin–biotin-amplified system. The antibody used was rabbit polyclonal antibody, TOX1 antibody (thymocyte selection-associated high-mobility group box antibody) (25 μl) (kit no. ab155768, dilution 1: 500; Abcam Company, Cambridge, UK).
The slides were subjected to deparaffinization and rehydration. Antigen retrieval was performed by boiling in citrate buffer saline (pH 6), followed by cooling at room temperature. Endogenous peroxidase was blocked by incubation with 3% H2O2. The primary antibody (TOX antibody) was incubated overnight at room temperature, and then washed in PBS. The secondary antibody (biotinylated goat anti-polyvalent) was added for 10 min and the section rewashed in BPS and subsequently incubated for another 10 min with streptavidin–peroxidase complex and then washed again in PBS for 5 min.
Thymus tissue was used as a positive control for TOX1.
Replacement of the primary antibody in the staining procedure with a blocking buffer was included as a negative control.
Interpretation of TOX expression: positive TOX expression was detected as a homogeneous brown stain in the nucleus of T lymphocytes. Counting the number of cells with brown nuclear staining comparable to the total number of infiltrating lymphocytes was performed when the percentage was determined.
The percentage of positive TOX was further graded into the following:
Negative (−) (<30%), positive (+) (30–50%), and strongly positive (++) (>50%).
The results were collected, tabulated, and statistically analyzed by the IBM personal computer and statistical package for the social sciences (SPSS version 11; SPSS Inc., Chicago, Illinois, USA). Two types of statistics were done; descriptive statistics using mean and SD and analytical statistics using Student's t-test and χ2-test. A P value of up to 0.05 was considered statistically significant.
| Results|| |
The age of the patients of the MF group ranged between 21 and 67 years with a mean of 48.75 years and a median of 50.5 years. Among the 20 MF patients, nine were men and 11 were women. Regarding the inflammatory group, the age of the patients ranged between 25 and 70 years with a mean of 44.17 years and a median of 43.5 years. Five cases were men and seven cases were women. There were no significant differences between MF and inflammatory groups as regards their age and sex [Table 1].
|Table 1: Comparison of the personal data between mycosis fungoides and inflammatory groups|
Click here to view
Immunohistochemical results of TOX in the studied MF cases: 16 cases showed positive expression and four cases were negative (<30%). The positive cases were divided according to the extent of TOX immunoreactivity into + (30–50% expression) in six cases and ++ (>50% expression) in 10 cases [Figure 1].
|Figure 1: A case of mycosis fungoides showing (++) (>50%) thymocyte selection-associated HMG-box expression in atypical lymphocytic infiltrate of pautrier microabscesses (red arrow) (×400).|
Click here to view
Among the 12 cases of inflammatory dermatoses, six cases were diagnosed clinically and confirmed histopathologically by chronic dermatitis; three cases were chronic nonspecific dermatitis and the others were spongiotic dermatitis. Three cases were lichen planes and three cases were psoriasis.
Results of TOX in the inflammatory group: most of the studied cases (83.3%) showed negative expression [Figure 2], while only two cases (16.7%) showed + expression.
|Figure 2: A case of lichen planus showing negative thymocyte selection-associated HMG-box staining (×200).|
Click here to view
There was a significant difference between MF and inflammatory groups as regards TOX expression which was in favor of the malignant group (P = 0.001; [Table 2]. Diagnostic validity of TOX was determined by receiver operating characteristic curve (ROC) analysis that showed 80% sensitivity, 83% specificity, 89% positive predictive values (PPV), 1% negative predictive values (NPV), and 81% accuracy [Figure 3].
|Table 2: Comparison between mycosis fungoides and inflammatory groups as regards thymocyte selection-associated HMG-box expression|
Click here to view
|Figure 3: ROC curve of diagnostic validity of thymocyte selection-associated HMG-box.|
Click here to view
There was no significant relationship between TOX expression (negative vs. positive) and studied parameters (age and sex). However, comparing cases that showed + TOX expression versus those showed ++ showed that advanced clinical phase and stage of MF were in favor of ++ TOX (>50%) compared with + (30–50%) (P = 0.013 and 0.005; [Table 3].
|Table 3: The relation between +versus ++thymocyte selection-associated HMG-box immunohistochemical expression and different clinicopathological data of mycosis fungoides studied cases|
Click here to view
| Discussion|| |
MF is the most common type of CTCL. The diagnosis of MF is clinically and histologically challenging because of the similarities to a group called benign inflammatory dermatosis. The disease progresses slowly for years, evolving from erythematous patches on sun-protected skin to plaques and then to tumors and erythroderma. The clinical course generally remains indolent with disease-specific survival that approaches 90%. Also, the expected outcome and treatment options for patients with MF differ from patients with inflammatory mimics. For these reasons, accurate and early diagnosis of MF is essential. Regarding the age of MF in this study, the mean age of MF presentation was 48.75 ± 3.19 which ranged between 21 and 67 years. This was in agreement with several Pimpinelli et al., Mokhtar et al. and, while Swerdlow et al. showed that most of the MF cases affecting childhood and adolescence. Regarding the sex of MF in this study, women were slightly more than male patients with 1.2 : 1 as the female-to-male ratio. This was in agreement with Mokhtar & Adel, While Pimpinelli et al, reported that the incidence was higher in men. The variability in a number of cases of different studies may explain the difference between age and sex distribution. According to the clinical phase of the studied MF cases, half of the cases was in patch stage (50%) followed by plaque stage (40%) and tumor stage was the least (10%). This was in agreement with others. MF clinically resembles benign skin inflammatory diseases such as chronic dermatitis, psoriasis, and cutaneous reactions to drugs.
Differentiating MF from these benign conditions is difficult, largely due to lack of well-defined molecular markers in the clinical setting. Although CD2, CD3, CD5, and CD7 deficiency is included in the International Society for Cutaneous Lymphomas criteria to define early MF, the loss of CD2, CD3, and/or CD5 in T cells is only 10% sensitive, despite its 100% specificity. CD7 deficiency is about 40% sensitive and 80% specific in general. Therefore, better markers with higher sensitivity and specificity are needed. TOX encodes a high-mobility group family (HMG) domain DNA-binding nuclear protein. TOX is primarily expressed in the thymus and downregulated before CD4+ T cells exit the thymus. TOX mRNA and proteins were poorly expressed in the peripheral lymphoid tissue. In recent years, the TOX gene has been proved to be aberrantly expressed in various tumors, such as lung cancer, breast cancer, and leukemia. TOX is considered a critical regulator of early T-cell development, strictly regulated in thymocyte differentiation during the transition from CD4 to CD4+ T cells. Once this mission is completed, it became suppressed. TOX expression in MF was analyzed in several studies by Zhang and colleagues, Nielsen et al. and. According to the current study, TOX was significantly higher in MF compared with inflammatory mimics as the following: we found that most of the cases (16/20) showed positive TOX compared with only two cases of the inflammatory group. This was a great suggestion in its potential role as a diagnostic marker for MF. This was demonstrated by an Area under the ROC Curve (AUC) value of 0.817 with a sensitivity of 80% and specificity of 83%. The sensitivity and specificity were different from other studies like Zhang et al. and Morimura et al., their results showed 100% specificity and 62% sensitivity. reported a lower specificity value (75%) and higher sensitivity value (90.3%). Several cases, the cut off values, and different methodology cause these changes, but all studies including this one suggest the role of TOX as a diagnostic marker for MF cases. Different methodology in the examination of TOX expression was used by Yu and Li. This study examined TOX expression by immunohistochemistry, real-time RT-PCR, and western blotting. The same results were given by the three tests. According to this study, TOX expression was increased with the progression of MF from patch stage to plaque stage to tumor stage. This was in agreement with several studies Yu and Li and Nielsen et al.. According to this study, there was a significant relation between TOX expression MF studied cases and staging of the disease from stage I to III. This was in agreement with Yu and Li in MF.
| Conclusion|| |
This results demonstrate that TOX may have a potential role as a molecular diagnostic marker for MF and overexpression of it is associated with poor prognosis and disease progression from patch to plaque to tumor stage, so TOX can be also a promising prognostic marker.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Bagherani N, Smoller BR. An overview of cutaneous T cell lymphomas. F1000Res 2016; 5
Rodd AL, Ververis K, Karagiannis TC. Current and emerging therapeutics for cutaneous T-cell lymphoma: histone deacetylase inhibitors. Lymphoma 2011; 2012
Yu X, Li Z. TOX gene: a novel target for human cancer gene therapy. Am J Cancer Res 2015; 5
Olsen E, Vonderheid E, Pimpinelli N, Willemze R, Kim Y. Revision to the staging and classification of mycosis fungicides and Sezary syndrome: a proposal of the international Society of cutaneous lymphoma (ISCL) and the cutaneous lymphoma task force of the European Organization of Research and Treatment of Cancer (EORTC). Blood 2007; 110
Wilkinson B, Chen JY, Han P, Rufner KM, Goularte OD. TOX: an HMG box protein implicated in the regulation of thymocyte selection. Nat Immunol 2002; 3
Aliahmad P, Seksenyan A, Kaye J. The many roles of TOX in the immune system. Curr Opin Immunol 2012; 24
Zhang Y, Wang Y, Yu R, Huang Y, Su M. Molecular markers of early-stage mycosis fungoides J Invest Dermatol 2012; 132:1698–1706.
Nielsen PR, Eriksen JO, Bzorek M, Gniadecki R, Fogh H, Ødum N, et al
. TOX expression in patients with mycosis fungoides-a potential diagnostic marker? Eur J Cancer 2018; 101
Cerroni L, Gatter K, Kerl H. Mycosis fungicides. In: Cerroni L, Gatter K, Kerl H, editors. Skin lymphoma
. The illustrated guide
. Third edition. Oxford, UK: Wiely-Blackwell. 2009. pp. 11–56.
Willemze R, Jaffe ES, Burg G, Cerroni L, Berti E, Swerdlow SH, et al.
WHO – EORTC classification for cutaneous lymphomas. Blood 2005; 105
Pimpinelli N, Olsen EA, Santucci M, Vonderheid E, Haeffner AC, Stevens S et al
. Defining early mycosis fungicides. J Am Acad Dermatol 2005; 53
Mokhtar N, Gouda I, Adel I. Cutaneous lymphomas. In: Lymphoma. 1st
Ed, Cairo, Egypt. 2007. p. 46–54.
Jenni D, Karpova MB, Seifert B, Golling P, Cozzio A. Primary cutaneous lymphoma: two-decade comparison in a population of 263 cases from a Swiss tertiary referral centre. Br J Dermatol 2011; 164
Swerdlow A, Campo E, Harris NL, Pileri S, Stein H, Jaffe ES. World Health Organization classification of tumors of cutaneous T-cell lymphoma hematopoietic and lymphoid tissue
edition. Lyon: IARC. 2008.p. 439.
Chung W, Kwabi-Addo B, Ittmann M, Jelinek J, Shen L. Identification of novel tumor markers in prostate, colon and breast cancer by unbiased methylation profiling. PLoS One 2008; 3
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]