Evaluation of E-Cadherin Expression in Patients with Pemphigus Vulgaris via Immunohistochemistry

Pemphigus is a group of autoimmune bullous diseases that can afect the skin and mucous membranes, and it is vital to recognize the exact pathogenesis of this disease. Tis study aimed to investigate the role of E-cadherin in the pathogenesis of pemphigus vulgaris (PV) and compare the expression of E-cadherin in the lesions of PV patients with that in healthy individuals’ skin. Tirty tissue samples from histopathologically confrmed PV patients as the case group and 30 skin samples from healthy individuals as the control group were evaluated for E-cadherin expression via the immunohistochemical method. Data analysis was performed using SPSS software version 25; chi-squared and Fisher’s exact tests were used to examine the relationship between qualitative variables. Immunohistochemical staining revealed decreased E-cadherin expression in the basal and suprabasal layers of the epidermis of PV patients compared with healthy individuals ( P < 0 . 001). E-cadherin expression was 1+ in 53.3% of patients, 2+ in 40% of patients, and 3+ in only one (3.3%) patient. On the other hand, the expression of E-cadherin in other layers of the epidermis was 1+ in one patient, 2+ in fve patients (25%), and 3+ in 14 patients (70%). Also, the expression of E-cadherin in all layers of the epidermis was 3+ in all controls. E-cadherin expression in the basal and suprabasal layers of the epidermis appears to be lower in patients with PV compared with controls. Terefore, E-cadherin immunohistochemical staining helps diagnose PV along with other diagnostic methods. Moreover, these fndings may shed light on the role of E-cadherin as a potential target for disease treatment aiming at disease stabilization. However, more studies are needed to clarify this issue.

Cadherins are calcium-dependent intramembrane proteins that fulfll a vital role in intercellular adhesion [14,15].Tese proteins are divided into classical, proto, desmosomal, and atypical types, and E-cadherin is one of the classical cadherins expressed in the epidermis [14].E-cadherin appears to be an immunological target for pemphigus autoantibodies as part of the adhesion proteins of desmosomes [16].Some studies have demonstrated that the level of anti-E-cadherin antibodies in the serum of patients with pemphigus is increased [16].
Many studies have investigated the role of desmogleins in the pathogenesis of PV [4,[13][14][15][16][17][18][19][20], but only one study has examined the immunohistochemical expression of Ecadherin in PV lesions [21].Terefore, we assessed the role of E-cadherin in PV pathogenesis and compared the expression of E-cadherin in the lesions of these patients with that in healthy individuals' skin.

Patients and Methods
Tis study was approved by the Ethics Committee of our institute.All patients provided informed consent.

Subjects.
In this study, we included 30 new cases of PV who were referred to our teaching dermatology clinic in 2019, did not sufer from other systemic and cutaneous diseases, had no history of medication usage, and were diagnosed by a dermatologist and confrmed by a dermatopathologist as the case group.In the control group, we included skin samples from 30 healthy individuals who did not sufer from systemic and cutaneous diseases, had no history of medication usage, and had undergone cosmetic surgery for reasons such as abdominoplasty and mammoplasty.Tese individuals did not have any cutaneous or systemic diseases and were all over 16 years old.Disease severity was assessed according to the pemphigus vulgaris activity score (PVAS), which considers the type, number, and distribution of skin and mucosal lesions and the presence or absence of the Nikolsky sign.Te total score ranges from 0 to 18.

Histopathological Examination. Serial 4 μm cut sections
were obtained from formalin-fxed parafn-embedded blocks.Hematoxylin and eosin (H&E) staining was performed on the sections, and an experienced dermatopathologist performed the histopathologic evaluation.

Immunohistochemical Staining. Immunohistochemical
staining of E-cadherin was performed using a mouse antihuman E-cadherin monoclonal antibody (Clone HECD-1; Master Diagnostica, Spain).From parafn blocks, 4-μm pieces were placed on a charged slide at 60 °C overnight, then the samples were deparafnized, rehydrated, and under heat conditions (95 °C, pH: 8, boiled in EDTA bufer for 20 min) were exposed to the retrieval epitope.Te specimens were washed with distilled water and cooled to room temperature for 20 minutes.Endogenous peroxidase was blocked by utilizing a peroxidase solution for 10 minutes.
Te primary antibodies were incubated for 20 minutes and washed with phosphate-bufered saline and immunohistochemistry (IHC) wash bufer.Secondary antibodies were added and left for 15 minutes, followed by horseradish peroxidase for 30 minutes.Antibodies were detected with DAB (3-3-diaminobenzidine) for two minutes and then washed with distilled water.Slide mounting was performed after hematoxylin staining.
Cells of the surface epithelium of the skin, mucosa, and skin appendages were examined for E-cadherin expression.Te intensity and pattern of E-cadherin expression in these areas were calculated.E-cadherin expression intensity was defned as 0 (loss of expression), 1+ (weak, incomplete membranous expression), 2+ (moderate to strong, incomplete membranous expression), or 3+ (moderate to strong, complete membranous expression).Also, the distribution of nonexpressed areas of E-cadherin was defned as focal, patchy, or difuse (Figures 1(a)-1(e)).

Statistical
Analysis.We used the statistical package for the social sciences (SPSS) software (version 25.0, Armonk, NY: IBM Corp., USA) for data analysis.Quantitative variables were described using means and standard deviations, while qualitative variables were described using frequencies and percentages.Fisher's exact test was used to compare qualitative data.P < 0.05 was regarded as statistically signifcant.

Results
In this study, 30 patients with PV were studied as the case group and 30 healthy individuals as the control group.Te mean age was 46.27 ± 12.14 years in the PV group and 41.47 ± 11.95 years in the control group.In the PV group, 8 (26.7%) participants were males and 22 (73.3%) were females; in the control group, 1 (3.3%) was male and 29 (96.7%) were females.Te female predominance in the latter group was expected as the controls were selected from individuals who had undergone mammoplasty or abdominoplasty.Pemphigus vulgaris was only cutaneous in 6.7%, only mucosal in 10%, and mucocutaneous in 83.3%.Te mean disease severity in this group based on the PVAS was 7.40 ± 3.76 (min � 1.5 and max � 16).
E-cadherin expression in the basal and suprabasal layers of the epidermis was +1 in 53.3%, +2 in 40% of patients, and +3 in only one patient (3.3%).On the other hand, E-cadherin expression in other layers of the epidermis was +1 in one patient, +2 in fve patients (25%), and +3 in 14 patients (70%).Te expression of E-cadherin in all layers of the epidermis in all controls was +3 (Figure 1).Te nonstained distribution in basal and suprabasal layers and other epidermal layers was mostly difuse (69% and 25%, respectively) (Table 1).Te results of immunohistochemical staining showed a decrease in the expression of Ecadherin in the basal and suprabasal layers of the epidermis of PV patients compared with healthy individuals (P < 0.001).

Discussion
Our study represents only the second investigation on the immunohistochemical expression of E-cadherin in tissue samples of patients with PV [21].We found decreased E-cadherin expression in the basal and suprabasal layers of the epidermis in all but one patient.On the other hand, in most patients (70%), no decrease in E-cadherin expression was observed in other layers of the epidermis.Te distribution of lack of staining in the basal and suprabasal layers and other epidermal layers was mostly difuse.Also, no decrease in E-cadherin expression in the epidermal layers was seen in all controls.Tese fndings indicate a signifcant decrease in E-cadherin expression in the basal and suprabasal layers of patients with PV compared with controls.Tis study also showed no signifcant relationship between Ecadherin in the basal/suprabasal layers of the epidermis and the PV severity and type.Mignogna et al. used immunohistochemistry to examine the expression of catenins as part of the cadherin/catenin protein complex (such as E-cadherin and B-catenin) in 7 patients with diferent stages of oral PV and 18 healthy individuals.In the controls, the intensity of staining gradually decreased from the spinosum layers to the keratinized layers of the epithelium, while in patients with PV, loss of expression of these molecules was observed, especially in areas with severe acantholysis.Tis decrease in expression was not associated with the severity of mucosal involvement or symptoms [21].Tus, this decrease in expression in patients and its lack of relationship with the disease's severity were consistent with our study's results, but the gradual decrease in expression in the upper epithelial mucosa of healthy individuals was not consistent with the results of the current study.Since the samples of our control group were taken from the epidermis, this could be the probable source of this diference.
Tsang et al. used confocal microscopy to show a lack or decrease in E-cadherin expression in cells adjacent to blisters [23] which is consistent with the fndings of this study.
According to the abovementioned studies and the results of this study, it seems that, compared with healthy individuals, patients with PV have deceased tissue E-cadherin expression in the basal and suprabasal layers, independent of disease severity and type.However, more studies with more samples are needed to confrm the aforementioned fndings.
Te lack of mucosal samples in the control group and the small number of eligible patients were the key limitations of this study, which should be addressed in future works.

Conclusion
In patients with PV, E-cadherin expression in the basal and suprabasal layers of the epidermis was signifcantly lower compared with the control group.Hence, E-cadherin may fulfll a role in the pathogenesis of PV.Further studies are needed to confrm this issue and evaluate the usefulness of E-cadherin immunohistochemical staining in diagnosing the disease and the role of E-cadherin as a potential target for disease treatment.

Figure 1 :
Figure 1: (a) Normal skin showing strong (3+) membraneous staining of E-cadherin (×400).(b) Suprabasal blister with acantholysis.Te blister foor shows weak (2+) staining and the blister roof has a normal 3+ staining pattern (×400).(c) Suprabasal blister of pemphigus vulgaris with loss of E-cadherin expression in the foor of the blister in some parts and 1+ staining in the blister roof (×400).(d) Follicular epithelium in a case of pemphigus vulgaris shows suprabasal weak (1+) E-cadherin expression and 2+ membranous staining in upper layers (same as surface epithelium in this case), ×400.(e) Oral mucosa biopsy in a case of pemphigus shows 1+ basal and suprabasal staining of Ecadherin and 2+ staining in the upper layers of the epithelium (×400).

Table 1 :
Frequency and percentage of qualitative variables in the pemphigus vulgaris (PV) and control groups.