Clinicopathologic Evaluation of CD80, CD86, and PD-L1 Expressions with Immunohistochemical Methods in Malignant Melanoma Patients

Objective: Diagnostic and prognostic biomarkers for malignant melanoma are crucial for treatment and for developing targeted therapies. Malignant melanoma is a highly immunogenic tumor, and its regression, treatment, and prognostic evaluation are directly related to escape from immune destruction. Therefore, we aimed to determine the expression levels of CD80, CD86, and PD -L1 in malignant melanoma tissue samples by immunohistochemistry and to investigate the possible relationship between these proteins and the clinicopathological features in this study. Material and Methods: Hematoxylin and eosin staining and immunohistochemical staining for CD80, CD86, and PD-L1 were evaluated for clinical data, survival, prognosis, tumor location, malignant melanoma subtypes, tumor size, and prognostic findings. Results: Higher survival rates were observed in patients with lower PD-L1 staining scores in the tumor. The 5-year survival was higher in patients with CD80-positive and CD86-positive biopsies. Mortality was lower in superficial spreading melanoma and Lentigo maligna melanoma types, whereas staining positivity of CD80 and CD86 was higher. Furthermore, a relationship between clinical stage and Breslow thickness (<2mm/≥2mm), tumor ulceration, lymph node metastasis, and CD80 and CD86 expression was also identified. Conclusion: Our findings suggest that PD-L1, CD80, and CD86 expression are essential in malignant melanoma and could be used as prognostic markers.


INTRODUCTION
Malignant melanoma (MM) is a tumor with high immunogenicity due to tumor antigens (1).Because of this feature of MM, treatment protocols are centered on immunotherapeutic approaches, which include targets such as induction of anti-tumor immune responses, modulation of tumortargeted immune reactions, and inhibition of immune control pathways (1).
Like many other tumors, tumor cells in MM inactivate the immune system through various escape mechanisms (2).The change in co-stimulatory receptors on dendritic cells is one of these escape mechanisms.The two-signal model proposes that activation of naive T cells requires both stimulation of T cell receptor (TCR) by major histocompatibility complex (MHC)-peptide molecules (signal 1) and co-stimulation via co-stimulatory receptors and their corresponding ligands on antigen-presenting cells (APCs) (signal 2).In this pathway, the tumor reduces (downregulates) the number of activating co-stimulatory receptors (CD28, CD40, OX40, CD137) or increases (upregulates) the number of inhibitory surface receptors (LAG-3, CTLA4, B7-H3, PD-1) in dendritic cells (1,3).
Although studies have shown the expression of CD80 and CD86 in tumor cells, the related studies are limited, and their relationship with prognosis is unclear (2).As a result, the goal of this study was to look at the expression rates of CD80, CD86, and PD-L1 on paraffin sections from 80 malignant melanoma cases using immunohistochemistry, as well as to evaluate the possible correlation between these proteins and clinical features like the stage, prognosis, and survival, in order to see if these proteins can be used as prognostic markers and to shed light on new treatment modalities.

MATERIAL and METHODS
Between 2005 and 2015, 80 patients diagnosed with MM were included in this study.The samples, of which 2 were incisional biopsies and 75 were excisional biopsies, were fixed in 10% buffered formalin, embedded in paraffin, cut into 4 mm thick sections, and stained with hematoxylin and eosin.A total of 3 consultation paraffin blocks were cut into 4 mm thick sections and stained with hematoxylin and eosin.In the presence of more than one block, we chose the block with the highest tumor ratio.In addition, demographic data such as age, gender, and clinical findings were retrieved from the files using the hospital automation system.

Immunohistochemical Examination
Four-micrometer-thick sections were cut from each patient's paraffin block.One of these sections was stained with hematoxylin and eosin (HE), and the others were immunohistochemically stained with antibodies against In this study, we developed a method of evaluating and scoring inspired by studies by Flörcken et al (2).Positive staining for CD80 and CD86 in tumor cells was characterized by cytoplasmic and membranous staining (2).PD-L1 was considered as positive when complete or partial linear membranous staining and nuclear staining were observed in tumor cells and tumor-infiltrated lymphocytes.Tonsillar tissue sections were positive controls for all PD-L1, CD80, and CD86 immunostainings (2,15,16).
We scored the staining intensity for CD80, CD86, and PD-L1.A score of 0 indicated no staining, while a score of +1 indicated weak staining.A score of +2 was for medium staining, and a score of +3 was given for intense staining.≤10% positive staining of tumor cells was evaluated as 1 point, 10.1-50% positive staining as 2 points, and 50.1-100% positive staining as 3 points.The staining value was calculated by adding the staining percentage and intensity values.The patients were divided into groups based on their staining scores: those with a score of 0-4 and those with 5-6.Cases in the study were also classified as positive or negative for CD80 and CD86.The immunohistochemical staining results were evaluated based solely on the presence or absence of lymphocytic infiltration; however, no assessments were made regarding whether the infiltration was brisk or non-brisk.

Statistical Evaluation
Descriptive findings were presented as number and percentage distributions for categorical variables, and mean±standard deviation for continuous variables.
The Pearson Chi-square test was used to compare categorical variables.For survival analysis, survival probabilities were first estimated with the Kaplan-Meier method and a log-rank test was performed to see if there was a difference between variable levels in terms of survival probabilities.
The University Institute of Health Sciences, Medical Statistics Consultancy Center used SPSS Package Program v20 to conduct the statistical analysis for the study (IBM, USA).Statistical significance was defined as p values less than 0.05.

Clinicopathological Characteristics
The median age of the patients was 57 years (range: 25-89 years).Among the study group, the death rate was 46.8% (n=22) in males, and 33.3% (n=11) in females, with no statistically significant difference between the two genders (p=0.256).Based on the World Health Organization's classification of malignant melanoma, we identified 18 melanocytic tumors in intermittently sun-exposed skin (Superficial spreading melanoma), six melanocytic tumors in chronically sun-exposed skin (5 Lentigo maligna melanoma +1 desmoplastic melanoma), 20 melanoma arising at sun-shielded sites or without known etiological associations with UV radiation exposure (19 acral lentiginous melanoma and 1 balloon cell melanoma), and 36 nodular melanoma (17).
The patients in the study had a median tumor size of 1.78 cm in diameter (min: 0.2 cm, max: 7.6 cm), with 12.5% (n=10) having tumors smaller than or equal to 0.6 cm in diameter and 87.5% (n=70) having tumors larger than 0.6 cm in diameter.On the other hand, the patients in the study were divided into two groups based on reticular dermis invasion, Clark I-II, and Clark III-IV-V, with the Clark level I-II group accounting for 11.3 % of all patients (n=9).The Clark level III-IV-V group accounted for 88.7% (n=71).The mean Breslow thickness in all patients was 3.6.±3.24 mm: 37.5% (n=30) having a Breslow thickness of <2 mm, and 62.5% (n=50) having a Breslow thickness of ≥2 mm.Ulceration was found in 37.5 % of the patients (n=30).Regarding growth phases, 7.5 % of patients (n=6) showed only the radial growth phase, while 92.5 % (n=74) showed both the vertical/radial and vertical growth phases.Lymphocytic infiltration was found in 58.8% (n=47) of the patient samples examined.There was evidence of neurotropism in 23.8% of the cases (n=19).Regression was seen in 18.7% (n=15) of the patients, while lymph node metastasis was seen in 20% (n=16

Expression of PD-L1
As previously described, PD-L1 staining was assessed on tumor cells and tumor-infiltrating lymphocytes (2).Tumor cells had a low staining score (28/80)  Patients with low PD-L1 staining in the tumor had a significantly higher 5-year survival (Figure 2).However, no significant relationship existed between the PD-L1 staining levels in the lymphocytes and the 5-year survival (Figure 3).
Table I summarizes the relationships between PD-L1 expression levels and the clinical profiles of the patients.
Table III shows the correlations between CD80 expression levels and the clinical patient profiles.
When CD80 expression was examined, it was significantly higher in the superficial spreading melanoma and lentigo maligna melanoma subtypes than in other subtypes.
CD80 staining scores were higher in superficial spreading melanoma and lentigo maligna melanoma subtypes when evaluating CD80 staining scores.
CD80 expression (staining intensity) and staining scores were higher in patients with Breslow thickness below 2 mm.
The positivity for CD80 expression was higher in patients who did not have tumor ulceration.
CD80 expression was found to be higher in cases of regression.CD80 expression was higher in patients who did not have lymph node metastasis.CD80 expression and staining scores were higher in clinical stage 1-2 cases.Cases with positive CD80 expression had a significantly higher 5-year survival rate (Figures 5 and 6).
Table IV shows the correlations between CD86 expression levels and the clinical profiles of the patients.When CD86 expression was examined, it was significantly higher in superficial spreading melanoma and lentigo maligna melanoma than in other subtypes.
The simultaneous presence of CD80 and CD86 positivity or negativity was found to be significantly higher (there was concurrent CD86 expression in cases with CD80 expression or absent CD86 expression in cases without CD80 expression).

DISCUSSION
Malignant melanoma is a highly immunogenic tumor and its progression, treatment, and prognosis are directly related to this feature (1).Antagonistic monoclonal antibodies against CTLA-4 and PD-1 have been introduced for immune checkpoint blockade.These agents' anti-tumor CD86 expression and a higher CD86 staining intensity score were found in patients with a Breslow thickness of less than 2mm.
CD86 expression was significantly higher in tumors that did not have ulceration.The expression of CD86 was higher in patients who did not have lymph node metastasis.
CD86 expression and CD86 staining scores were significantly higher in pathological Stages 1-2 cases.
While patients with positive CD86 expression had a significantly higher 5-year survival rate, there was no correlation between staining score and survival (Figure 7,8).   of the CD80/CD86-CD28 interaction on immune modulation are also known.
Our retrospective clinical study investigated the association between CD80, CD86, and PD-L1 expression, tumor characteristics, prognostic factors, and survival in cutaneous malignant melanoma.
In the patients enrolled in our study, no significant relationship was found between the tumor size and Clark level, as well as the CD80, CD86, and PD-L1 expression levels.However, it has been suggested that the immune response to the tumor effects the vertical growth phase rather than the radial growth phase because the mortality rate is high in patients with a Breslow thickness of 2 mm or more, and the expression of CD80 and CD86 is significantly lower in these cases.
activities have been demonstrated in Phase I, II, and III studies, with CTLA-4 and PD-L1 standing out as the primary molecules targeted by immunotherapy modalities results (2).
Although most studies on the inhibitory or stimulatory effects of signal-2 in the formation of the immune response focus on the two molecules mentioned above, the results        PD-L1 expression in tumor cells and lymphocytes was associated with a poor prognosis in a study evaluating the expression of CD80, CD86, and PD-L1 in both the tumor and lymphocytes in renal cell tumor cases in the literature, whereas CD80 and CD86 expressions were not correlated with the prognosis (2).In support of these reports, publications indicate that CD80 and CD86 expression in melanoma patients does not effect the prognosis (42).
CD80 and CD86 expression on the cell surface was significantly positive in cases with Breslow thickness less than 2mm, no ulceration on the tumor surface, clinical stage I-II, and no lymph node metastasis in our study.Furthermore, CD80 and CD86 positivity was found to indicate a favorable prognosis.
When analyzed, only CD80 positivity and the presence of regression had a significant relationship.The failure to see any significant relationship for CD86 positivity is likely due to a small sample size.
Histologic subtype analysis revealed that CD80 and CD86 expression was significantly higher in superficial spreading melanoma and lentigo maligna melanoma.
Based on our findings, we recommend using PD-L1, expressed in tumor tissue, as a prognostic marker in cases of malignant melanoma, with a high PD-L1 staining score in the tumor indicating a poor prognosis and determining the indications for the patient's clinical management and immunotherapy.CD80 and CD86 expressions and high staining scores were statistically significant predictors of a good prognosis in our study's survival analysis.As a result, CD80 and CD86 immunohistochemical markers predict the prognosis of malignant melanoma cases.

Conflict of Interest
No conflict of interest.There was no statistically significant relationship between PD-L1 staining intensity and Breslow thickness.However, the Breslow thickness was significantly higher in patients with strong PD-L1 expression compared to those with weak expression in Hino and colleagues' study (15).In addition, PD-L1 expression was also associated with vertical growth pattern, Clark level status, and lymph node metastasis but not with age, sex, or histologic subtype in the same study (15).

Funding
Hino and colleagues found no link between ulceration and PD-L1 expression, and Massi and colleagues found ulceration to be a poor predictor of survival.Their findings were consistent with ours (15,16).
Similar to previous research suggesting that PD-L1 in tumor cells can be used as a poor prognostic marker based on the presence or absence of staining, we found that strong staining of PD-L1 in tumor tissue indicated poor prognosis and survival.However, this was not found with PD-L1 expression in TIL.There is little evidence in the literature to support the use of CD80 and CD86 immunohistochemistry in tumor immunity and prognosis.While most rodent tumors have been reported to lack tumor expression of CD80 and CD86 (37,38), molecular, immunohistochemical, and flow cytometry studies have revealed the presence of CD80 and CD86 in tumor cells in some human tumor cells (39)(40)(41).

Figure 3 :
Figure 3: Survival plot according to PD-L1 staining scores in lymphocytes.
34/80) in 42.5% of the patients and a high staining score (46/80) in 57.5% of the patients.Figure1A-Dshows examples of low and high staining scores in tumors and TIL.PD-L1 expression and staining score were high in clinical stage 3-4 cases.
in 35% of the patients and a high staining score (52/80) in 65% of the patients, while tumor-infiltrating lymphocytes (TIL) had a low staining score (

Table I :
The correlations between PD-L1 expression level and clinical patient profiles.
*Pearson-chi square test

Table III :
The correlations between CD80 expression level and clinical patient profiles.
*Pearson-chi square test

Table IV :
The correlations between CD86 expression level and clinical patient profiles.
*Pearson-chi square test