Immunohistochemical Study of PDL-1 Expression in Urinary Bladder Carcinoma

Conclusion and recommendations

Our study highlighted the important role of PDL1 in urinary bladder carcinogenesis, development of LN metastases. Among the clinicopathological parameters investigated, we found a positive significant association with higher tumor grades, advanced stage and LN spread. The results suggest that PDL1 expression can be beneficial for detection of UBC patient group with poor prognostic features. PDL1 also have an important mechanism in the development of LN spread. A high concordance rate was detected between primary tumor and their LN metastases regarding PDL1 expression explaining the role of PDL1 the metastatic ability in cells of tumor.

A large-scale study is recommended to validate the previous findings and to confirm the important role of PDL1 in UBC.

More studies are indicated to investigate the relationship between PDL1expression in relation to different organ metastasis and UBC primary tumor. Such studies with complete clinicopathological follow up data would help the follow up/survival analysis to identify patients with aggressive tumors to select cases that can benefit from PDL1 targeted therapy.

Additional research was needed to investigate the relationship between PDL1 and its related marker family in UBC.

More research is strongly recommended for investigating target therapy against PDL1 positive tumors of the studied cases.

Introduction

Bladder cancer is considered the tenth most common cancer in our world, with an increasing occurance yearly^[1]. UBC is common malignancy in males in Egypt which ranking the second popular malignancy, constituting about 30% of whole malignancies^[2].

The environmental and toxic factors that are filtered through the kidneys into the urine irritate the transitional cells lining of the urinary system, especially the urinary bladder^[3]. Expectedly, 90% of urinary bladder cancers arise from these urothelial cells, however, 10% of cases are squamous cell carcinomas ^[2]. The

prognosis of urothelial carcinoma (UC) depends on the staging of the tumor. Localized forms have the best prognosis; however, the survival rate drops significantly with muscular invasion ^[2].

The recent research of a new era of immunomodulation therapy in UBC led to approval of new immunotherapeutic treatments. With an initial studies and promising results of drugs used as anti-programmed cell death-1 (anti-PD-1) and anti-programmed cell death ligand-1 (anti-PDL-1) the coming attempts are used for validation of the previous triumph to consolidate the initial gains ^[4].

Programmed death ligand 1 (PDL-1) is an immunoglobulin-like type I transmembrane glycoprotein. PDL-1 is one of the B7 family, which includes seven ligands essential for the second signal of T-lymphocytes. The B7 family proteins are expressed mainly in immune cells B/T lymphocytes, dendritic cells, and mono-cytes^[5]. We suggest making more studies to assess the PDL-1 status and prognostic benefits among UBC Egyptian cases.

Materials and Methods

Case selection

 Our study comprised 50 randomly chosen cases of urinary bladder carcinoma. These cases were obtained from radical cystectomy specimens, the cases were collected from the archives of pathological laboratory of Minia University Hospitals (in the period between January 2018 and December 2021), Oncology Center of Minia and Hospital of Kasr ElAini (between June 2018 and September 2020). The cases included; 30 cases of urothelial carcinoma and 20 cases of squamous cell carcinoma. The grades of differentiation ranged between low and high concerning TCC cases and grades I, II and III as regard SCC cases. Among the fifty selected cases, twelve cases of primary UBC and metastatic lymph nodes have been examined for PDL1 expression.

The available clinicopathological data included patients' age of patient, sex, macroscopic pattern of tumor, tumor type,   tumor grade,   tumor stage, lymph node positivity, and evidence of bilharzial cystitis and presence of insitu component, necrosis of tumor, lymphvascular invasion, perineural invasion and lymphocytic infiltration.

Five µm sections were made and stained with hematoxylin stain and eosin stain for revising the histological findings of all cases and examined according to WHO criteria [7] for confirming type of tumor, grade of tumor and stage.

Immunohistochemistry

Primary antibody

PDL-1

 PDL-1 which is a rabbit anti-human mono-clonal AB for immunohistochemistry. The primary AB is used for qualitative recognition of AG in FFPE tissue specimens. The antibody produces membranous and/or cytoplasmic expression, IgG1 subclass, Synthetic peptide derived from a region of PDL-1 protein, antibody 100 ul concentrate for IHC (Cat. No. 400100295). According to the manufacturer data sheet.

Scoring of immunostaining and cut off point:

  PDL-1 expression was mainly membranous. Counting of 10 high power fields per section for each case was performed. In UBC PDL1 expression is made by using CPS, with counting the number of positively stained tumor cells .also stained lymphocytes and macrophages divided by the whole number of non-necrotic malignant cells x100. The pdl1 expression was divided into 2 types: high expression and low expression. Low expression and negative cases were considered as one type. The specimen is considered to have positive PDL-1 expression if CPS ≥ 10^[8].

Statistical analysis

      Analysis of statistics was conducted using (IBM SPSS software version 25). Data were compiled and used to determine the means ± standard deviations (SDs), range and median of various features. The Fischer’s exact and Chi- square test was used to compare categorical features. Wilcoxon test and McNamar test were used for comparing between PDL-1 expression in primary tumor and lymph node metastasis. P value of ˂ 0.05 was considered significant.

Results

Positive significant associations were found between PDL1 expression and tumor grade, stage, type muscle invasion and lymph node status (p. = 0.005, 0.005, 0.001, 0.045, 0.045 respectively).  No significant relation was found between PDL1 expression and other variables.

Discussion

The study found that PDL-1 immunostaining is localized in the cytoplasmic memberane. As regards tumor type, in UC cases 22 out of 30 (73.3%) showed high PDL-1expression but in SCC cases only 12 out of 20 (60%) showed high expression with significant association with tumour type, being significantly higher in TCC cases (p. = 0.012). Like our results, Pichler et al., 2017 found that high PDL-1 expression on UBC was seen more in patients

with UC than patients with SCC (46.2% vs. 20.8%; p=0.002)^[15]. 

Concerning tumor grade, a Positive association was found between PDL-1 expression and grade of tumor. Twenty two out

of 25 high grade UC cases (88%) showed high PDL-1expression, while UC  cases of low grade showed –ve/low expression (p value.= 0.005). As regards Squamous cell carcinoma, positive

high PDL-1 expression was detected in eight cases out of 12 (66.6%) in grade II SCC, while all grade I TCC cases showed –ve/low expression, however all cases (100%) of grade III SCC showed high PDL-1expression (p. = 0.005). This was compatible with the results of Nakanishi et al., 2007, who found that specimens from tumor of higher WHO grade or primary tumor classifications had significantly higher percentages of tumor-associated PDL-1expression ^[16]. 

Regarding muscle invasion, 14/14 (100%) of Non muscle invasive bladder carcinoma cases showed –ve/low expression and no cases showed high pdl1 expression. In muscle invasive bladder carcinoma cases (2/36) (5.5%) showed low expression while the rest of cases (34/36) (94.5%) had high expression, with significant association between high PDL-1expression and muscular invasion (p. = 0.001). These results go with the results of Gupta and his colleagues, 2022, who found that (92%) of muscle invasive bladder carcinoma showed high PDL-1 expression.^[17].

Concerning tumor stage, we detect a significant association between stage of tumor and PDL-1 expression (p. = 0.045). In cases with stage T1 all showed –ve/low PDL-1expression (100%) as compared to cases with T2 stage where only 28.5% of them showed –ve/low expression while 71.5% of cases had high expression. However, in cases with T3 stage, (3/19) (15.8%) showed –ve/low expression, while 16 cases (74.2%) showed high expression. Finally, the three cases of T4 stage showed high expression (100%) (p. = 0.045). This was compatible with the results of Kim et al., 2020, who found that the PDL-1 expression of tumor-infiltrating immune cells (ICs) was significantly correlated with higher pathologic tumor stage^[17]. While Zavalishina et al., 2018 who discovered scoring significantly higher (all P < 0.04) among cases with T2 stage (13/45 or 28.9%) and stage T ≥ 3 (4/10 or 40%) disease compared with the proportion with cases with T1 stage (5/45 or 11.1%) ^[18]. 

Significant positive associations were found between PDL-1 expression and LN metastasis (p. <0.001). Only 12 cases had lymph node spread, of them 4 cases (33.3%) had –ve/ low PDL-1 expression while the remaining eight cases (66.7%) had high PDL-1 expression. As regards the 38 cases without LN metastasis, (12 / 38) (31.6%) showed -ve/ low PDL-1 expression whereas 26 cases (68.4%) showed high PDL-1 expression   (p. ˂0.001). This was compatible with the results of Holah, 2022 PD-L1 immunohistochemical reactivity in tumor cells was associated significantly with occurance of nodal metastasis (P = 0.028)^[16].

There was no significant associations between PDL-1 expression and age, gender, macroscopic pattern of tumour, bilharziasis, insitu component, necrosis of tumor, lumphvascular invasion, perineural invasion or lymphocytic infiltration (p. = 0.581, 0.157, 0.130, 0.330, 0.434, 0.511, 0.151, 0.978, 0.768 respectively). This was different from a study done by Holah, 2022, who found that PD-L1 immuno-histochemical reactivity in tumor cells was significantly related with occurrence of lymphvascular invasion (P = 0.019). This difference may be attributed to the different studied type of population^[17].