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The expression of SP263 in muscle invasive bladder carcinoma and its relationship with clinicopathological features


 Department of Pathology, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China

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Date of Submission06-Feb-2022
Date of Decision27-Feb-2022
Date of Acceptance08-May-2022
Date of Web Publication18-Oct-2022
 

   Abstract 


Context: The expression of programmed cell death ligand1 (PDL1) is a research hotspot of immunotherapy. The treatment targeted for its expression has shown effectiveness in many tumors. Objective: The aim of the study was to determine PD-L1 expression in urothelial carcinoma (UC) and to compare the PD-L1 expression in muscle invasive bladder carcinoma (MIBC) and upper urinary tract urothelial carcinoma (UTUC). The predictive value of CD8+ tumor-infiltrating lymphocyte (TIL) density for the diagnosis of PD-L1 positivity and the association between CD8+ TIL density and prognosis in MIBC were also explored. Materials and Methods: Immunohistochemistry (IHC) staining for PD-L1 (SP263), CK5/6, CK20, CD44, and p53 was carried out using a 3D Histech digital scanner to scan and determine CD8+ TIL density. Results: 122 patients received radical cystectomy, and the overall PD-L1 positivity was 34.43% (42/122). PD-L1 positivity in whole sections was higher than in tissue micro-array (TMA) (all P < 0.05). If multiple lesions were detected simultaneously, the number of patients with positive results increased from 42 to 49. The areas under the curve (AUCs) of CD8+ TIL density for the diagnosis of PD-L1 positivity were 0.739, 0.713, and 0.826. Univariate cox regression analysis demonstrated that high CD8+ TIL density and CD8highPDL1+ were protective factors of overall survival (OS), and multivariate cox analyses showed that only CD8+ TIL density was an independent prognostic factor for OS. For UTUC, the overall PD-L1 expression was 40.0% (16/40). Conclusions: Our study results emphasize the importance of detecting PD-L1 expression in multiple tumor lesions from the same patient. In MIBC, CD8+ TIL density could be used as a prognostic marker for predicting the status of PD-L1 expression.

Keywords: Bladder carcinoma, CD8, immunotherapy, PD-L1


How to cite this URL:
Bai Y, Wu X, Weng M, Han Q, Xu L, Chang C, Teng X. The expression of SP263 in muscle invasive bladder carcinoma and its relationship with clinicopathological features. Indian J Pathol Microbiol [Epub ahead of print] [cited 2022 Dec 7]. Available from: https://www.ijpmonline.org/preprintarticle.asp?id=358849





   Introduction Top


Urothelial carcinoma (UC) is the eighth most common cause of deaths due to malignancy in men in the United States.[1] Compared to UCs of the bladder (UCBs), UCs of the upper tract (UTUCs) have an aggressive clinical behavior and a more advanced stage.[2] Approximately, 15–25% of UBCs are muscle invasion bladder cancer (MIBC), which are more aggressive and are the main cause of patient mortality.[3]

Recently, the immune checkpoint inhibitors (ICIs) targeting the programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1) have demonstrated survival benefits in treatment of UC.[4] Another potential biomarker apart from PD-L1 is the tumor-infiltrating lymphocyte (TIL) that plays an important role in identifying potential patients who may benefit from ICIs.[5] Therefore, in this study, we evaluated the expression patterns of PD-L1 in MIBC and explored the heterogeneity of PD-L1 expression. Because there are significant differences in the prevalence of common genomic alternations existing in UTUC and UCB,[6] PD-L1 expression between MIBC and UTUC was also evaluated.


   Materials and Methods Top


Patient selection

UCs diagnosed from January 2016 to September 2018 were enrolled. One hundred and twenty-two cases with MIBC and forty cases with UTUC were whole tumor sections; besides, a representative paraffin block of 252 cases with MIBC were selected for construction of TMAs. Tumor-node-metastasis staging was performed according to the current World Health Organization classification of 2016. The study was approved by our hospital institutional review board. Yes, obtained. The date of the approval was August 29, 2020.

Immunohistochemistry

For PD-L1 immunohistochemistry (IHC) analysis, we used Ventana/SP263 (Ventana Benchmark Ultra, prediluted antibody, pretreatment: CC1 64 min incubation at 100°C). CD8 (4B11, Novocastra), CK5/6 (CK5/6.007, Invitrogen), CK20 (EP23, Invitrogen), CD44 (VFF-7, LabVision), and p53 (D0-7, LabVision) were used according to Envision protocols. The overall PD-L1 positivity was defined as PD-L1 expression on ≥25% of all tumor cells (TCs) and/or immune cells (ICs). All slides stained for CD8 were scanned using a 3D Histech digital scanner. The density of CD8+ TILs = total number of CD8+ TILs/total area of counting area (piece/mm2). CD44 and CK5/6 scores were used as a surrogate for the basal subtype and CK20 for the luminal subtype [Figure 1].[7]
Figure 1: PD-L1 was positive in TCs, and molecular subtype was basal. (a) hematoxylin and eosin (HE) staining for the first case, (b) TCs positive for PD-L1, (c and d) TCs positive for CK5/6 and CD44, and (e) TCs negative for CK20. PD-L1 was positive in ICs, and the molecular type was luminal. (f) HE staining for the second case, (g) ICs positive for PD-L1, (h) TCs positive for CK20, and (i and j) TCs negative for CK5/6 and CD44

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Statistical analyses

Chi-squared test, Kappa statistics, univariate and multivariate cox proportional hazard models, Kaplan–Meier analysis, log-rank testing, and receiver operating characteristic (ROC) curve were used in our study.


   Results Top


Patients' characteristics

The median age of 122 cases with MIBC was 70 (range: 47–90) years. Male patients (106, 86.89%) were 6.6 times as many as female patients. Thirty-three (27.05%) cases were T2 and eighty-nine (72.95%) cases were T3-4. Most patients (93, 76.23%) were N0. For UTUC, the median age was 67.5 (range: 30–86) years. Male patients (25, 62.50%) were 1.6 times as many as female patients. Ten (25.0%) cases were T2, and thirty (75.0%) cases were T3 or T4. Most (33, 82.50%) cases were N0.

PD-L1 expression in UCs

For MIBC whole sections, the overall positive rate of PD-L1 expression was 34.43% (42/122). PD-L1 positivity in TCs and ICs was 26.23% (32/122) and 16.39% (20/122), respectively. Twelve cores were lost during operation, and 240 cores were obtained. PD-L1 positivity was 21.7% (52/240), 16.7% (40/240), and 9.6% (23/240) for overall, TCs, and ICs, respectively. PD-L1 positivity in whole sections was higher than in TMA (all P < 0.05). Among the 40 UTUCs, the PD-L1 expression was 40.0% (16/40), 35.0% (14/40), and 25.0% (10/40) of overall, TCs, and ICs, respectively. There was no significant difference between PD-L1 expression in MIBC whole sections and UTUC.

Among the 122 cases who received radical cystectomy, we performed multiple PD-L1 tests for 65 cases [radical cystectomy, biopsy/transurethral resection of bladder tumor (TURB), and metastasis], and the remaining 57 patients were tested for radical cystectomy alone. According to the specimen type (radical cystectomy, radical cystectomy + biopsy/TURB, radical cystectomy + metastasis, and radical cystectomy + biopsy/TURB + metastasis), PD-L1 positivity by ≥1 time among the four groups was 34.43% (42/122), 37.70% (46/122), 37.70% (46/122), and 40.16% (49/122), respectively. The positive rate only increased by 5.73%; the number of patients with positive results increased from 42 to 49, that is, an increase of 16.7% (7/42).

Comparison of PD-L1 expression in TCs and ICs between primary and metastasis lesions in 122 surgical specimens

A total of 28 patients had both primary and metastatic lesions. The differences in PD-L1 expression between primary and metastasis were identified in 4, 1, and 6 of 28 cases (14.3%, 3.6%, and 21.4%) of the overall, TC, and IC cohorts. The K value for the three cohorts was 0.710, 0.916, and 0.368, respectively.

Correlations between clinicopathological parameters and PD-L1 expression

For 122 whole tumor sections, there was a reverse correlation between overall PD-L1 positive and vascular invasion (P = 0.043). The TCs positive for PD-L1 were inversely correlated with vascular invasion and positively correlated with tumor size (P = 0.002 and 0.019, respectively). The ICs positive for PD-L1 were significantly associated with age ≤70 (P = 0.003). In UTUC, the overall PD-L1 expression was higher in females (P = 0.046, data not shown). Compared with the non-basal subtype, the positive rate of PD-L (overall, TCs, or ICs for MIBC; overall and TCs for UTUC) in the basal subtype was higher (P < 0.05). In both MIBC and UTUC, there was no significant difference between abnormal p53 expression (0% or >50% positivity) and wild-type (1–49% positivity) [Table 1].
Table 1: Correlations between clinicopathological parameters and PD-L1 expression

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We validated the presence of two intrinsic molecular subtypes of MIBC. The 362 cases with MIBC were divided into luminal (165, 45.6%) and basal (176, 48.6%) groups. A small subset of double-negative (21, 5.8%) tumors which did not express either luminal or basal immunohistochemical markers were also identified. Based on the molecular subtypes of UCB, 19 (47.5%) and 20 (50%) of UTUC tumors were luminal and basal subtypes, respectively.

Correlations between PD-L1 expression and CD8+ TILs in MIBC

The optimal cutoff value of CD8+ TIL density was 222.9 pieces/mm2 using x-tile software [Figure 2]a and [Figure 2]b. The high positive rate of the overall, TC, and IC PD-L1 expression correlated closely with the strong infiltration of CD8+ TILs (P = 0.002, P = 0.024, and P < 0.001, respectively, [Table 1]). We applied the ROC curve to analyze the positivity of the overall, TC, and IC PD-L1 expression using the continuous CD8+ TIL density. The areas under the curve (AUCs) of CD8+ TIL density were 0.739, 0.713, and 0.826, respectively.
Figure 2: Kaplan–Meier survival curves based on CD8+ TIL density and tumor type. (a and b) X-tile analysis of the CD8+ TIL density. (c) OS estimated using the Kaplan–Meier method for patients with low or high CD8+ TIL density (P = 0.003). (d) OS estimated using the Kaplan–Meier method for patients with different tumor type. (CD8+ TILslow PD-L1- vs CD8+ TIL high PD-L1+ P = 0.008; CD8+ TIL low PD-L1 + vs CD8+ TIL high PD-L1 + P = 0.047; else P for all >0.05)

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Teng et al.[8] proposed that tumors can be classified into type I–type IV. According to this criterion, the four groups in the study were 23.8% (29/122), 39.3% (48/122), 10.7% (13/122), and 26.2% (32/122), respectively.

Survival analysis

For 122 whole tumor sections with MIBC, univariate cox regression analysis demonstrated that at age >70, T3 + T4 were the risk factors of OS, whereas high CD8+ TIL density, without neural infiltration and CD8highPD-L1+ were protective factors of OS. Multivariate cox analyses showed that only CD8+ TIL density was an independent prognostic factor for OS [Table 2]. Kaplan–Meier analysis and the log-rank test demonstrated that the OS of patients with high CD8+ TIL infiltration was significantly longer than low infiltration, and the median OS was 22 and 16 months, respectively (P = 0.003, [Figure 2]c). The survival curves based on Teng's classification are shown in [Figure 2]d. The OS of patients with CD8highPDL1+ (Type I) was longer than patients with CD8low/PD-L1 (Type II, P=0.008) and CD8low/PD-L1+ (Type III, P= 0.047).
Table 2: Univariate and multivariate analyses for OS in 122 cases with MIBC

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   Discussion Top


Our results demonstrated equal PD-L1 expression on TC compared with that previously reported in the literature ranging from 14 to 28%.[9] Besides, approximately, 32.7% of invasive UTUC demonstrated positive PD-L1 expression,[10] which is also similar to our results. Although a close link between Lynch syndrome and UTUC has been recognized, most of the UTUCs were sporadic. We did not find any significant difference between PD-L1 expression in UTUC and MIBC. The dynamic temporally and spatially heterogeneous expression of PD-L1 has been described.[11] PD-L1 positivity in whole slides was higher than in TMA, which reflects that PD-L1 staining is heterogeneous in MIBC. If multiple lesions were detected simultaneously, the number of positive patients increased by 7 cases. Therefore, PD-L1 expression in surgical specimens cannot be replaced by TMA, which indicates that for inoperable patients, multiple PD-L1 detection is recommended to obtain maximum benefit from immunotherapy.

Our study demonstrated positive PD-L1 expression overall and in TCs, revealing a tendency to occur in the absence of vascular invasion. It was reported that a lower expression of PD-L1 was coupled with an increase in lymphatics and blood vessels[12] and associated with renal vein tumor thrombus in clear cell renal cell carcinoma.[13] This may be due to the fact that the vascular density of the tumor is low when the TCs were PD-L1-positive. Meanwhile, it was demonstrated that there was a positive relationship between PD-L1 and CD8+ TILs count, suggesting that PD-L1 expression plays a role in cytotoxic cell activation. As the three AUC values were greater than 0.7, the CD8+ TIL density is of significance for predicting the positivity of PD-L1.

The molecular subtype in UTUC is less studied. Although it was reported that UTUC belongs to the luminal type, our study shows that the incidence of two molecular subtypes was almost identical.[14] The incidence of double-negative MIBC in our results is consistent with the literature, ranging from 4–16%.[15] For type II and type III, blocking the expression of PD-L1 alone cannot cause the killing effect of T cells to the TCs. Bringing the lymphocytes into the tumors should be considered. It is useful to evaluate the expression of PD-L1 and CD8+ TIL numbers for ICIs in MIBC.

There was discrepancy between the prognostic significance and PD-L1 expression in UC.[16] PD-L1 positivity was associated with better clinical outcomes among patients with high-grade organ-confined UTUC, and others failed to detect an association between PD-L1 expression and prognosis.[16] Our study did not reveal the association between the PD-L1 expression with the disease-free survival (DFS) or OS, whereas there may be a tendency that TCs and ICs positive for PD-L1 may be associated with longer DFS (P = 0.077) and OS (P = 0.077), respectively (data not shown).


   Conclusion Top


In summary, our study results emphasize the need for detection of PD-L1 expression in multiple tumor lesions from the same patient. In MIBC CD8+ TILs, density could be used as a prognostic marker and also a biomarker for predicting the status of PD-L1 expression.

Acknowledgements

The study was published as a meeting abstract in Modern Pathology (March, 2020).

Financial support and sponsorship

The study was supported by Zhejiang Provincial Research Center for Cancer Intelligent Diagnosis and Molecular Technology, China (JBZX-202003).

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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López JI, Pulido R, Lawrie CH, Angulo JC. Loss of PD-L1 (SP-142) expression characterizes renal vein tumor thrombus microenvironment in clear cell renal cell carcinoma. Ann Diagn Pathol 2018;34:89-93.  Back to cited text no. 13
    
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Balar AV, Galsky MD, Rosenberg JE, Powles T, Petrylak DP, Bellmunt J, et al. Atezolizumab as first-line treatment in cisplatin-ineligible patients with locally advanced and metastatic urothelial carcinoma: A single-arm, multicentre, phase 2 trial. Lancet 2017;389:67-76.  Back to cited text no. 14
    
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Miyama Y, Morikawa T, Miyakawa J, Koyama Y, Kawai T, Kume H, et al. The prognostic value of PD-L1 expression in upper tract urothelial carcinoma varies according to platelet count. Cancer Med 2018;7:4330-8.  Back to cited text no. 16
    

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Correspondence Address:
Xiaodong Teng,
Department of Pathology, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou - 310 003
China
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijpm.ijpm_142_22



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