Role of Mast Cell, Ki-67 and Bcl-2 in the Diagnosis of Mycosis Fungoides

doi:10.21203/rs.3.rs-3401395/v1

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Role of Mast Cell, Ki-67 and Bcl-2 in the Diagnosis of Mycosis Fungoides

https://doi.org/10.21203/rs.3.rs-3401395/v1

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Diagnosis of early-stage MF, there are molecular studies that include Bcl-2 and ki-67, but there is no study showing that mast cells can be used both in the diagnosis of early-stage MF and in the etiopathogenesis of MF. In our study, we aimed to show that Ki-67, Bcl-2 and mast cell staining, which can be easily obtained in every laboratory, are helpful markers in the diagnosis of early-stage MF.

Methods:

A total of 81 cases, including 27 Mycosis Fungoides, 27 benign inflammatory dermatoses (psoriasis, lichen planus, eczema) and 27 patients without any disease, were included in the study retrospectively. Ki-67, Bcl-2 and mast cell counts were made under a light microscope with stained slides.

Results:

Mast cells were found to be significantly higher in cases of mycosis fungoides compared to cases of benign inflammatory dermatosis (p>0.001). In mycosis fungoides cases, ki-67, Bcl-2 and mast cells were significantly higher compared to the control group (p>0.001). In benign inflammatory dermatosis cases, ki-67 and Bcl-2 were found to be significantly higher than the control group (p>0.001).

Conclusion:

MF lesions are initially included in the differential diagnosis of many diseases including eczema, psoriasis, and lichen planus. In this case, histopathological examination is used in order to reach a clear diagnosis. Ki-67, Bcl-2 and mast cells were found significantly higher in MF patients compared to the control group. However, no difference was found between MF and benign inflammatory dermatoses in ki-67 and Bcl-2 staining.

Mycosis fungoides

mast cell

Bcl-2

ki-67

Mycosis fungoides (MF) is a variant that occurs at an average age of 55–60 years and is more common in men than in women, constituting more than half of all cutaneous T-cell lymphomas (1, 2). The incidence of MF is estimated to be 5.6 per million (1). Genetic, environmental and infectious causes are blamed for the etiology (3). Detailed physical examination, skin biopsy, blood tests, radiological evaluation and lymph node sampling play an important role in MF staging(4). Histopathology shows papillary dermal lymphoid infiltrate and epidermotropism (± pautrier microabscess) of this infiltrate without spongiosis in the epithelium. Lymphocytes may contain a perinuclear halo, and show variable pleomorphism, nuclear contour irregularity, and hyperchromasia(5).

In the TNMB staging system first proposed by the International Cancer Institute in 1979 for MF, the area covered by the lesions, lymph node, and visceral involvement are taken into account. Accordingly, it is divided into 4 stages as I, II, III, IV. However, a more comprehensive staging was needed to determine the prognostic factor in parallel with the developments in molecular biology, immunohistochemistry and imaging methods. Therefore, the International Cutaneous Lymphoma Society revised this classification in 2007(4). According to the first proposed staging system, early-stage MF includes stages I and IIA(6). The diagnosis of early-stage MF is difficult both clinically and histopathologically, since it is similar to benign inflammatory dermatoses such as psoriasis or eczema(7). Although CD3, CD4, CD8, CD5 and CD7 immunohistochemical markers are used in addition to histopathological findings in cases with suspected MF diagnosis, it is difficult to diagnose especially in early-stage cases (5). Clinical, histopathological, molecular biological and immunopathological methods are included in the diagnosis of early-stage MF. Accordingly, persistent and/or progressive patches and thin plaques are the main criteria, while lesions localized to sunless areas, size/shape variability and poikiloderma are additional criteria. The main criteria in histopathological findings are superficial lymphoid infiltration, additional criteria are epidermotropism without spongiosis and lymphoid atypia. In both clinical and histopathological criteria, 2 points are given for the main criterion and 2 additional criteria, 1 point is given for the main criterion and 1 additional criterion. The molecular biological criterion is the rearrangement of the clonal TCR gene, and its presence is given 1 point. In immunopathological criteria, < 50% of T cells are CD2(+), CD3(+) and/or CD5(+), if < 10% of T cells are CD7(+), CD2(+), CD3(+) 1 point is given for 1 or more of the epidermis/dermis mismatch criteria in terms of CD5(+) or CD7(+).

According to this scoring, it is sufficient to get 4 points from the combination of the criteria for the diagnosis of MF(8). In addition to the existing criteria in the diagnosis of early-stage MF, there are molecular studies including apoptosis inhibitors such as Bcl-2 and the role of proliferation markers such as ki-67(9).

The pathogenesis of MF is largely unknown, but persistent antigen stimulation, exaggerated expression of SRC kinases, abnormal regulation of cytokine receptor signaling, dysregulation of apoptotic pathways, and epigenetic changes have been implicated (10).

Mast cells (MC) are bone marrow-derived hematopoietic cells that play an important role not only in the immune response but also in the tumor microenvironment with protumorogenic and antitumorogenic functions. Although the studies on this subject are very few, in one of the other cutaneous T-cell lymphomas, higher mast cells were detected in MF compared to their subgroups. In another study, it was shown that mast cells are associated with protumorogenic effect and microvessel density in primary cutaneous B and T cell lymphomas. In addition, according to this staging, significantly higher mast cell counts were detected in stages IA and IB compared to stages IIA and IIB (11, 12).

Ki-67 is a cell proliferation marker. It is routinely used in pathology to differentiate benign and malignant lesions, for grading in some tumors, and as a prognostic factor in some tumors. It has been reported to be an objective prognostic indicator in various malignant lymphomas, but few data are available for MF. Ki-67 expression in MF has been shown to be associated with the clinical stage, but it is not known whether this is an independent prognostic indicator (13).

Bcl-2 is a protein that suppresses apoptosis. Its expression has been observed in a variety of normal tissues and lymphomas, but Bcl-2 expression has minimal differential diagnostic value in distinguishing lymphomas from reactive T cell infiltrates. In some of the studies in the literature, it was reported that Bcl-2 is highly expressed in high-grade tumors of the prostate, while it was emphasized that there was no correlation between Bcl-2 and tumor grades in some (14).

Early-stage MF often mimics lesions such as psoriasiform, eczematous or lichenoid lesions, so diagnosis at an early-stage is difficult for both dermatologists and pathologists (15). In contrast to extensive Bcl-2 expression in both early and advanced MF skin lesions, abundant expression of Ki-67 indicates advanced MF lesions (9).

In this study, we tried to use the cell proliferation marker KI-67, apoptosis inhibitor Bcl-2, and toluidine blue staining for mast cell detection in Mycosis Fungoides, benign and normal tissue samples, which are not routinely used in the diagnosis of MF but can help in the diagnosis. We aimed to evaluate the role of mast cell, 67, Bcl2, which can be easily seen in the diagnosis of early MF, in differentiating it from other dermatoses that pose diagnostic difficulties in the early-stages.

Patient

Ethics committee approval was obtained for the study. The cases diagnosed as Mycosis Fungoides, benign inflammatory dermatosis (psoriasis, lichen planus, eczema) followed in Necmettin Erbakan University Meram Faculty of Medicine Department of Dermatology and without any disease were analyzed retrospectively. Twenty-seven Mycosis Fungoides, 27 benign inflammatory dermatoses (psoriasis, lichen planus, eczema) and 27 people without any disease, a total of 81 cases were included in the study.

Immunohistochemistry

For immunohistochemical and histochemical staining, 3-micron sections were taken from the archives of cases diagnosed with MF, inflammatory dermatosis cases, and paraffin blocks of the control group on positively charged slides. Slides cut for immunohistochemical study were incubated for 30 minutes in EDTA buffer at 97 degrees. Then, immunohistochemical Ki-67 (DAKO-Clone-MIB-1) and BCL-2 (DAKO-Clone 124) stains were applied in the DAKO OMNIS device. After the slides cut for the histochemical Toluidine Blue were deparaffinized, manual staining was applied.

Stained slides were evaluated under a light microscope (Olympus BX 46) by a single pathologist.

Ki-67; Nuclearly positively stained cells were given as a percentage of a hot spot.

Cells staining nuclear and cytoplasmic positive with Bcl-2 were evaluated as percentage.

Mast cell counts were performed randomly in 3 different (x400 magnification fields) in the upper dermis of toluidine blue stained preparations.

The staining patterns of control group, lichen planus, psoriasis, eczematous dermatitis and mycosis fungoides preparations with H&E, ki-67, bcl-2 and toluidine blue are shown in Figs. 1–5.

Statistical Method

Data were analyzed with SPSS 24.0 computer program. Descriptive statistics were shown as mean ± standard deviation and numbers and percentages. For intergroup comparison, Kruskal-Wallis analysis of variance was used to compare more than two groups. The Mann-Whitney U test with Bon-Ferroni correction was used to find the group from which the difference originated. In all analyses, p < 0.05 was accepted as the significance level. The results were shown with the help of tables. Sample Mycosis fungoides, benign inflammatory dermatosis, 3 groups as the control group, 27 cases in each group, were calculated using the GPower 3.1.9.7 program and the formula of 80% power, 5% margin of error and medium effect size, comparing 3 groups.

Of the 81 patients who participated in the study, 45 were female. Sixteen of the MF cases, 16 of the benign inflammatory dermatosis cases and 14 of the control group were male. The mean age of mycosis fungoides cases was 53.8 ± 13.4, the mean age of benign inflammatory dermatosis cases was 42.8 ± 18.3, and the mean age of the control group was 48.8 ± 18.8. Biopsies from mycosis fungoides cases were taken from the trunk in 12, the extremities in 13, the head and neck in 1, and the genital area in 1 patient. Mycosis fungoides was evaluated according to the WHO/EORTC staging system. Twenty cases were determined as Stage IA and 7 cases as Stage IB. A comparison of demographic data of mycosis fungoides group, benign inflammatory dermatosis group and control group is given in Table 1.

Table 1

Demographic characteristics
	Group 1 (MF)	Group 2 (benign inflammatory dermatosis)	Group 3 (control)
Age(Mean ± SD)	53,8 ± 13,4	42,8 ± 18,3	48,8 ± 18,8
Gender(n %)
Male	16 (%59,3)	16(%59,3)	14(%51,9)
Female	11 (%40,7)	11(%40,7)	13(%48,1)
biopsy site
Body	12	8
extremity	13	13
head-neck	1	3
Genital area	1	2

In mycosis fungoides stage IA cases, ki-67% 3%, Bcl-2 30%, mast cell 7%, ki-67 5%, Bcl-2 30%, mast cell 6% in stage IB cases. The percentage of ki-67, Bcl-2 and mast cells in both stages did not differ statistically (p > 0.05). The percentages of ki-67, Bcl-2 and mast cells according to mycosis Fungoides stages are summarized in Table 2.

Table 2

Percentages of ki-67, Bcl-2 and mast cells by Mycosis Fungoides stages
	Ia Median(min-max)	Ib Median(min-max)	P
Ki-67	3(2–15)	5(3–5)	0,533
Bcl-2	30(10–80)	30(25–40)	0,85
Mast cell	7(3–31)	6(4–14)	0,72

The percentages in mycosis fungoides cases were ki-67%3, Bcl-2 30%, mast cell 7%, in benign inflammatory dermatosis cases ki-67 5%, Bcl-2 25%, mast cell 3%, in the control group − 67%. 1, Bcl-2 10%, mast cell 2%(Table 3)

Table 3

Percentages of ki-67, Bcl-2 and mast cells in all group
	Gorup 1 (MF) Median(min-max)	Group 2 (psoriasis, lichen, eczema) Median(min-max)	Gorup 3 (control) Median(min-max)	P
Ki-67	3(2–15)	5(1–20)	1(0–2)	< 0,001
Bcl-2	30(10–80)	25(10–80)	10(5–40)	< 0,001
Mast cell	7(3–31)	3(0–11)	2(0–6)	< 0,001

Mast cells were found to be significantly higher in mycosis fungoides cases compared to benign inflammatory dermatosis cases (p > 0.001). Ki-67, Bcl-2 and mast cells were significantly higher in mycosis fungoides cases compared to the control group (p > 0.001). Ki-67 and Bcl-2 were found to be significantly higher in benign inflammatory dermatosis cases compared to the control group (p > 0.001).

In MF cases, there was no difference between the percentages of Ki-67, Bcl-2 and mast cells according to the biopsy site.

In this study, different from classical diagnostic staining with mast cell, Bcl-2 and ki-67 immunohistochemical staining in immunohistologically stained tissue sections of mycosis fungoides, benign inflammatory dermatosis and control groups in the literature, both in the diagnosis of early-stage MF and in the differential diagnosis of early-stage MF from other dermatoses. This is the first study known to have diagnostic and predictive staining.

Consistent with the literature, the mean age of the MF cases included in the study was 53.8 and male cases were in the majority (16, 17, 18).

Dummer et al. In the study conducted on Bcl-2, widespread expression of Bcl-2 was found in both early and advanced MF patients(9). Weshahy et al. Bcl-2 levels were not found to be associated with the clinical stage in the study conducted by (19). In our study, however, no statistical difference was found in mycosis fungoides cases in terms of Bcl-2 expression according to the stage. Bal et al. In the study conducted by Bcl-2, it was observed that the expression of Bcl-2 in the dermal lymphocytes of chronic inflammatory dermatosis cases such as psoriasis and lichen planus was increased compared to the control group. They stated that increased Bcl-2 expression contributes to cytokine release, which is involved in the pathogenesis of both psoriasis and lichen planus(20). In our study, in accordance with the literature, a significant difference was found in the benign inflammatory dermatosis group in terms of Bcl-2 compared to the control group. Benign inflammatory dermatosis group consists of proliferative diseases. This explains the statistical significance of Bcl-2, an antiapoptotic marker, between the benign inflammatory group and the control group. Talaat et al. The expression of Bcl-2 in MF, eczema and control groups was investigated. Accordingly, while an intense Bcl-2 expression was observed in the stage-independent MF group, only basal keratinocytes Bcl-2 staining was observed in eczema and control groups (21).

In our study, a significant difference was found in the expression of Bcl-2 in the MF group compared to the control group, in line with the literature. There was no statistically significant difference in Bcl-2 expression in the MF group compared to the benign inflammatory dermatosis group. Bcl-2 expression was found to be increased in both MF and benign inflammatory dermatosis groups, but Bcl-2 was not found to be a helpful marker in distinguishing the two groups from each other.

Dummer et al. showed that the expression of ki-67 in advanced lymphomas increased in line with the stage(9). Zohyd et al. in a study on Ki-67 proliferation were found to be significantly higher in advanced MF compared to early MF and parapsoriasis or other inflammatory dermatoses (22). In our study, there was no statistical difference in terms of ki-67 expression according to the stage in MF cases, but since advanced-stage MF cases were not included, a comparison of early-stage with advanced-stage could not be made. Bovenschen et al. In a study investigating the relationship between ki-67 expression in psoriasis, lichen planus and atopic dermatitis cases, the highest staining with ki-67 was detected in psoriasis (23). In our study, the ki-67 staining was statistically significantly higher in mycosis fungoides and benign inflammatory dermatosis cases compared to the control group. However, there was no statistically significant difference in Ki-67 expression in the MF group compared to the benign inflammatory dermatosis group. According to this result, it was determined that the expression of ki-67, a proliferation marker, was high in proliferative lesions such as MF and benign inflammatory dermatosis, but ki-67 was not an auxiliary marker in differentiating MF patients from benign inflammatory dermatoses.

In a study investigating the relationship between biopsy sites and MF in the literature, it was reported that patients with lesions localized in the lower extremities showed longer survival than patients with lesions localized in the upper extremities, head and neck, and trunk. However, there were no studies investigating the relationship between ki-67, Bcl-2 and mast cell and MF biopsy sites. In our study, no statistically significant difference was found between biopsy sites and ki-67, Bcl-2 and mast cells in the MF group (24).

Eder et al. found significantly higher mast cell counts in MF stage IA and IB compared to stage IIA and IIB(11). In our study, no significant difference was found between stage IA and IB in terms of mast cell count.

Rabenhorst et al. In a study by MF, mast cells were significantly increased in patients with primary cutaneous lymphoma, including MF, compared to normal skin and inflammatory cutaneous diseases (25). In our study, the number of mast cells was found to be statistically significantly higher in MF cases compared to benign inflammatory dermatoses and the control group. This result suggests that mast cells play an important role in the differential diagnosis of MF.

Mycosis fungoides is included in the differential diagnosis of many dermatological diseases. Lesions of MF may initially be similar to papulosquamous diseases with additional eczema, psoriasis, and lichen planus, thus making the diagnosis of early-stage MF difficult. In this case, histopathological examination is beneficial in order to reach a clear diagnosis. In our study, we think that Ki-67, Bcl-2 and mast cell staining, which can be easily obtained in every laboratory, may be helpful in cases where CD staining, which is included in the diagnosis of early-stage MF, is not available. Ki-67, Bcl-2 and mast cells were found to be significantly higher in MF patients compared to the control group. However, no difference was found between MF and benign inflammatory dermatoses in ki-67 and Bcl-2 staining. Therefore, it is recommended to take control biopsies and evaluate for early-stage MF in repeated follow-ups of cases with significantly higher ki-67, mast cell population and Bcl-2 staining in the differential diagnosis. In addition, our study is the first known study showing that mast cells can be used in the diagnosis of early-stage MF and in the etiopathogenesis of MF. In addition, no relationship was found between biopsy sites and ki-67, Bcl-2 and mast cells in MF cases.

Since advanced-stage MF cases were not included, a comparison of early-stage with advanced-stage could not be made. And the study should be done in large case groups.

Funding: This study was no funded

Conflict of Interest: The authors declare that they have no conflict of interest.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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No competing interests reported.

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Role of Mast Cell, Ki-67 and Bcl-2 in the Diagnosis of Mycosis Fungoides

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Abstract

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INTRODUCTION

MATERIAL-METHOD

RESULTS

DISCUSSION

CONCLUSION

LIMITATIONS

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