Indian Journal of Pathology and Microbiology

ORIGINAL ARTICLE
Year
: 2017  |  Volume : 60  |  Issue : 2  |  Page : 172--176

Diagnostic value of immunohistochemistry staining of Bcl-2, CD34, CD20 and CD3 for distinction between discoid lupus erythematosus and lichen planus in the skin


Mazaher Ramezani1, Bita-Sadat Hashemi1, Sedigheh Khazaei1, Mansour Rezaei2, Ali Ebrahimi3, Masoud Sadeghi4,  
1 Molecular Pathology Research Center, Imam Reza University Hospital, Kermanshah University of Medical Sciences, Kermanshah, Iran
2 Department of Biostatistics and Epidemiology, Kermanshah University of Medical Sciences, Kermanshah, Iran
3 Department of Dermatology, Kermanshah University of Medical Sciences, Kermanshah, Iran
4 Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran

Correspondence Address:
Masoud Sadeghi
Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah
Iran

Abstract

Background: Cluster of differentiation (CD) markers is a classification system for monoclonal antibodies against cell surface molecules on leukocytes and antigens from other cells. Aims: The aim of this study is to evaluate immunohistochemical markers in patients with discoid lupus erythematosus (DLE) and lichen planus (LP) and correlation of these markers in two groups and with the normal group in the West of Iran. Settings and Design: Analytical cross-sectional study. Materials and Methods: This study was performed on paraffin blocks of DLE and LP patients with normal group (21, 21 and 16 cases, respectively) between 2009 and 2012. Formalin-fixed, paraffin-embedded tissue sections from each DLE, LP, and normal skin biopsy specimen were cut into 4-μ thick sections and mounted on glass slides. Initial sections were stained with hematoxylin and eosin. Primary antihuman antibodies against CD3, CD20, CD34, and Bcl-2 were applied. Positive control samples for CD3, CD20, and Bcl-2 were received from lymph nodes and for CD34 from dermal microvessels. Statistical Analysis Used: SPSS version 19 (SPSS, Inc., Chicago, USA) and Microsoft Excel 2007. Results: The mean staining for four markers was more significant in DLE and LP groups compared to normal group and for CD34 and CD3 was more significant in LP groups versus DLE group. Conclusions: CD3+, CD34+, Bcl-2+, and CD20+ cells are significantly higher in DLE and LP lesional skins versus normal skin. In addition, there were higher expressions of CD3 and CD34 in LP lesional skin versus DLE lesional skin.



How to cite this article:
Ramezani M, Hashemi BS, Khazaei S, Rezaei M, Ebrahimi A, Sadeghi M. Diagnostic value of immunohistochemistry staining of Bcl-2, CD34, CD20 and CD3 for distinction between discoid lupus erythematosus and lichen planus in the skin.Indian J Pathol Microbiol 2017;60:172-176


How to cite this URL:
Ramezani M, Hashemi BS, Khazaei S, Rezaei M, Ebrahimi A, Sadeghi M. Diagnostic value of immunohistochemistry staining of Bcl-2, CD34, CD20 and CD3 for distinction between discoid lupus erythematosus and lichen planus in the skin. Indian J Pathol Microbiol [serial online] 2017 [cited 2020 Apr 1 ];60:172-176
Available from: http://www.ijpmonline.org/text.asp?2017/60/2/172/208381


Full Text

 Introduction



Discoid lupus erythematosus (DLE) is a chronic connective tissue disease of unknown etiology,[1] but immunologic factors may play an important role in the pathogenesis with peripheral hyperpigmentation most frequently found on the face, scalp, and neck. DLE lesions frequently expand during active inflammation after which they heal with scarring, atrophy, and hyper- or hypo-pigmentation.[2] Lichen planus (LP) is a chronic inflammatory disease of probable immune-based etiology. The pathogenesis of the LP is unclear, but apoptotic changes in epidermal (epithelial) cells have been reported.[3] LP and DLE have distinctive pathology but share histological features of lymphohistiocytic infiltration, vacuolar degeneration of the basal layer, and cytoid bodies. Both diseases, however, have a spectrum of histopathological features, and hence, may at times be difficult to differentiate from each other.[4] The cluster of differentiation (CD) markers is a classification system for monoclonal antibodies against cell surface molecules on leukocytes and antigens from other cells.[5] The monoclonal antibody against CD34 (a transmembrane glycoprotein expressed on endothelial cells and involved in tethering lymphocytes)[6] reacts with the endothelium of arteries and venules and has been found to stain capillary endothelium most intensely because it is highly sensitive for endothelium and produces lowest background staining.[7] CD20 is the most consistently positive marker for B-cells and CD3 is the most consistently positive marker for T-cells.[8] The Bcl-2 gene family plays a significant role in the propagation of cell survival and tissue modeling. Bcl-2 is originally described in follicular lymphomas and is associated with the suppression of cellular apoptosis.[9] Expression of Bcl-2 protein has been studied in a variety of normal tissues and lymphomas.[10]

The aim of this study is to evaluate CD markers (CD3, CD20, and CD34) and Bcl-2 in patients with DLE and LP and the association of these markers in two groups and also with the normal group in the West of Iran and to use immunohistochemistry (IHC) for differentiation between two diseases in difficult cases, especially in scarring alopecia due to DLE and LP.

 Materials and Methods



Patients

This analytical cross-sectional study was approved by the Kermanshah University Ethics Committee and was performed on paraffin blocks of DLE and LP patients with normal group (21, 21 and 16 cases, respectively) between 2009 and 2012 in Special Clinic and Imam Khomeini Hospital in Kermanshah city, Iran.

Immunohistochemistry preparation

Formalin-fixed, paraffin-embedded tissue sections from each DLE, LP, and normal skin biopsy specimen were cut into 4-μ thick sections and mounted on glass slides. Initial sections were stained with hematoxylin and eosin. The clinical diagnosis of DLE and LP was done by the dermatologist who was blind about the results of histopathology and IHC staining. The histopathology diagnosis was made by dermatopathologist and assistant pathology resident who were blind from clinical diagnosis. The criteria used for histopathologic diagnosis of DLE were hyperkeratosis, follicular plugging, epidermal atrophy, basal layer vacuolization, civatte bodies, pigmentary incontinence, superficial and deep perivascular; periadnexal lymphocytic infiltration and interstitial mucin deposition in the dermis. The criteria used for histopathologic diagnosis of LP were hyperkeratosis, hypergranulosis, irregular acanthosis with saw-toothing, basal layer damage and squamatization, civatte bodies, pigment incontinence, and lichenoid infiltrate of lymphocytes in dermoepidermal junction. The selected cases had most of the criteria. The cases with the discrepancy between dermatologist, dermatopathologist, and assistant were censored from the study. Primary antihuman antibodies against CD3 (DAKO diagnostics, IR503, clone F7.2.38), CD20 (DAKO diagnostics, IR604, clone L26), CD34 (DAKO diagnostics, IR632, clone QBEnd10), and Bcl-2 (DAKO diagnostics, IR614, clone 124) were applied by IHC method. Positive control samples for CD3, CD20, and Bcl-2 were received from lymph nodes and for CD34 from blood vessels (dermal microvessels). The percentage of positive inflammatory cells among total inflammatory cells in the dermis for CD3, CD20, and Bcl-2 was estimated in ×40 and confirmed in ×100 and ×400 separately by the dermatopathologist and the assistant pathology resident and the mean percentage was applied. Two-head microscope (Zeiss Axiostar Plus) was used for the estimation. More than 5 blood vessels stained (brown in the cytoplasm) with the ×40 showed CD34-positive [Figure 1].{Figure 1}

Histopathology analysis

The percentage of stained cells was estimated in scanning field (×40) and classified as negative: 0%, weak: <10%, moderate: 10%–25%, intense: 26%–50%, very intense: >50% positive cells for CD20 or CD3 and classified as negative: <5%, weak: 6%–25%, moderate: 26%–50%, strong: >50% positive cells for Bcl-2. More than 5 of blood vessels stained were positive for CD34 [Figure 2].{Figure 2}

Statistical analysis

For comparisons between variables was used SPSS software version 19 (SPSS Inc., Chicago, USA) by t-test for means and Chi-square test for other values. P< 0.05 was considered statistically significant. The charts were plotted in Microsoft Excel software 2007.

 Results



Age and sex

Twenty-one DLE, 21 LP, and 16 controls were analyzed [Table 1]. Out of 21 DLE patients, 12 (57.1%) were women and 9 (42.9%) were men. The mean age was 35 years (range, 19–53 years). All biopsies of DLE patients were from the face and scalp. In LP patients, 12 (57.1%) were women and 9 (42.9%) were men. The mean age of this group was 40 years (range, 19–61 years) and biopsy site of 20 patients (95.2%) was from the face and scalp; and one biopsy (4.8%) was from the oral mucosa. The controls were selected from safe margins of basal cell carcinoma. Five (31.2%) were women and 11 (68.8%) were men that the mean age was 59 years (range, 43–90 years). The age and sex of normal group, DLE group, and LP group have been shown in [Table 1]. There was no significant correlation between DLE group and LP group (P > 0.05).{Table 1}

Immunohistochemistry

CD34 positivity was present in the endothelial cells of vessels. This marker positivity was viewed by brown created in the cytoplasm with or without membrane staining. The result of CD34 marker staining (average of counting vessels in inflammatory infiltrate with ×40 magnification ± standard deviation) showed higher expression in DLE and LP groups than controls (P < 0.001) [Table 2].{Table 2}

Bcl-2 positivity was viewed by brown created in the nucleus of inflammatory cells (lymphocytes). In each sample, high expression (>25% of cells) considered as positive. There was no significant difference between DLE and LP groups in inflammatory cells (P = 0.44). DLE and LP patients exhibited significant difference in Bcl-2 expression in inflammatory cells compared with the controls (P = 0.001 and P< 0.001, respectively).

CD20 positivity was viewed by brown created in the membrane of inflammatory cells. In each sample, high expression (≥10% of cells) considered as positive. There was no significant difference between DLE and LP groups for CD20 expression in inflammatory cells (P = 0.092), but DLE patients exhibited significant difference with the control group (P = 0.028). Furthermore, there was no significant difference for CD20 expression between LP and the control groups (P = 0.291).

CD3 positivity was viewed by brown created in the membrane of inflammatory cells. In each sample, high expression (≥10% of cells) considered as positive. There was a significant difference between DLE and LP group for CD3 expression in inflammatory cells (P = 0.004) and also was statistically higher in LP and DLE groups than the control group (P < 0.001).

Medical diagnosis

The specificity, sensitivity, positive and negative predictive values; and accuracy of each biomarker for diagnosis of disease have been calculated and summarized in [Table 3]. CD34 exhibited the highest specificity and positive predictive value in diagnosis of LP (90.9% and 95.2%, respectively). Furthermore, CD3 exhibited the highest sensitivity and negative predictive value in diagnosis of LP (95.2% and 87.5%, respectively).{Table 3}

 Discussion



We checked four markers (CD3, CD20, CD34, and Bcl-2) in DLE, LP, and normal groups and compared these markers in three groups. To determine which these markers had more expression in DLE, LP lesional skins, or normal skin.

DLE occurs most frequently in women compared with men [11],[12] that our study confirmed it. One study [13] showed that the mean age of patients with DLE was 30.18 years and in our study, the mean age was 35 years. Furthermore, the prevalence of LP is more in women versus men,[14],[15],[16] similar to our study. LP is the most common among middle-aged adults.[15] The mean age for oral LP (OLP) at the onset of the disease is 55 years.[17] In our study, the mean age of LP group was 40 years. Therefore, the mean age of LP group is probably higher than DLE group, but the female/male ratio is almost similar in two groups.

Two studies [1],[2] demonstrated that compared with normal skin, staining for CD3 and CD20 in DLE lesional skin is significantly higher that our study confirmed it. In one study,[18] 31 interface dermatitis lesions (19 cases of LP and 12 cases of DLE) and 20 specimens of normal skin were examined that the mean counts of CD3+ cells were significantly higher in LP compared with DLE. Alternatively, the density of CD20+ cells was significantly higher in DLE compared with LP. This result was also confirmed by our study, but for CD20, the difference was not statistically significant. A study of Lee et al.[4] demonstrated that T-cells were predominant lymphocytic cells like us, but B-lymphocytes were few or absent in their cases. We found more percentage of B-lymphocytes. In addition, they demonstrated that there are significantly more vessels in the inflammatory infiltrate of LP compared with DLE as identified by the monoclonal antibody CD34. Hussein et al.[19] showed that compared with normal skin, dermal microvessel density was increased in cutaneous LP (mean, 2.50 vs. 1.39). We found similar results with CD34. Recent data suggest that with respect to the expression of Bcl-2 protein, 97% samples were positive in OLP group.[20] Out of 24 cases of OLP in another study, 4 cases (16.67%) were positive for Bcl-2,[9] but Nafarzadeh et al.[3] reported that Bcl-2 was negative for all OLP samples. In our study, Bcl-2 was positive in 90.5% of LP cases. Abdel-Latif et al.[21] showed that expression of Bcl-2 was positive in 28% LP cases, with no significant difference between patients with LP and controls. This controversy between different studies may be due to different techniques. In our study, the mean of staining of CD34, CD20, Bcl-2, and CD3 was more in DLE and LP groups compared to the normal group (P < 0.05). In addition, the mean staining for CD34 and CD3 was more in LP group compared to DLE group.

 Conclusions



Differentiation between DLE and LP is a challenging manner, both clinically and histologically in many patients. We found that CD3+, CD34+, Bcl-2+, and CD20+ cells are significantly higher in DLE and LP lesional skins versus normal skin. Furthermore, there were higher expressions of CD3 and CD34 in LP lesional skin versus DLE lesional skin. This study may be done in future researches on more cases, especially cases with scarring alopecia for DLE and LP groups which differentiation between two groups may sometimes be very difficult.

Acknowledgment

This work was performed in partial fulfillment of the requirements for (Residency of Pathology) of (Bita-Sadat Hashemi), Faculty of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran.

Financial support and sponsorship

This work was supported by the Research Council of Kermanshah University of Medical Sciences (Grant number: 91327).

Conflicts of interest

There are no conflicts of interest.

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