A multicenter round robin test of PD-L1 expression assessment in urothelial bladder cancer by immunohistochemistry and RT-qPCR with emphasis on prognosis prediction after radical cystectomy

Abstract

BackgroundImmunohistochemical PD-L1 assessment is currently used to identify responders towards checkpoint inhibitors although it is limited by inter-observer effects. Here, we conducted a multi-center round robin test to prove the possibility of assessing the PD-L1 status by gene expression to avoid inter-observer effects.Patients and methodsGene expression of PD-L1 was analyzed in a total of 294 samples (14 cases non-muscle invasive and muscle-invasive bladder cancer; MIBC) in seven centers by a RT-qPCR kit and compared with immunohistochemical scoring of three pathologists (DAKO, 22c3). Both assays were compared towards prognosis prediction in a cohort of 88 patients with MIBC.ResultsPD-L1 gene expression revealed very high inter center correlation (centrally extracted RNA: r = 0.68–0.98, p ≤ 0.0076; locally extracted RNA: r = 0.81–0.98, p ≤ 0.0014). IHC Inter-observer concordance was moderate to substantial for immune cells (IC), fair for combined IC/ tumor cell (TC) (IC: κ = 0.50–0.61; IC + TC: κ = 0.50), and fair for TC scoring (κ = 0.26–0.35). Gene expression assessment resulted in more positive cases (9/14 cases positive vs. 6/14 cases [IHC]) which could be validated in the independent cohort. Positive mRNA status was associated with significantly better overall and disease-specific survival (5-year OS: 50% vs. 26%, p = 0.0042, HR = 0.48; 5 year DSS: 65% vs. 40%, p = 0.012, HR = 0.49). The 1% IHC IC cut-off also revealed significant better OS (5 year OS: 58% vs. 31%, p = 0.036, HR = 0.62).ConclusionGene expression showed very high inter-center agreement. Gene expression assessment also resulted in more positive cases and revealed better prognosis prediction. PD-L1 mRNA expression seems to be a reproducible and robust tool for PD-L1 assessment.

mRNA-Expression of KRT5 and KRT20 Defines Distinct Prognostic Subgroups of Muscle-Invasive Urothelial Bladder Cancer Correlating with Histological Variants

“…RT-qPCR was applied for relative quantification of KRT5 and KRT20 mRNA as well as CALM2 (calmodulin 2; housekeeping gene) expression by using gene-specific TaqMan ® -based assays as described previously [ 21 , 23 ]. CALM2 is a stably expressed gene among breast cancer tumor tissue samples and has been applied successfully to bladder cancer specimens [ 21 , 24 , 25 ].…”

Piwi-like 1 and -2 protein expression levels are prognostic factors for muscle invasive urothelial bladder cancer patients

“…The TMA was prepared as follows: HE slides were scanned (Panoramic P250, 3DHistech, Budapest, Hungary) and annotated using a TMA annotation tool (Caseviewer v2). Four cores (diameter 1 mm; two cores from the invasion margin, two cores from the tumor center) were taken utilizing an automated tissue microarrayer (TMA Grandmaster, 3DHistech, Budapest, Hungary) as described previously 49 , 50 . The research carried out on human subjects is in compliance with the Helsinki Declaration.…”

POFUT1 mRNA expression as an independent prognostic parameter in muscle-invasive bladder cancer

“…qRT-PCR setting: (1) 20 s at 95 °C, (2) 40 cycles of 3 s at 95 °C and of 30 s at 60 °C. Calmodulin2 ( CALM2 ) was measured as a reference gene [ 6 , 7 , 9 ]. The means were calculated from the technical duplicates of each gene.…”

“…Furthermore, owing to the limited knowledge available regarding molecular and biologic variants of the disease, potential therapy responders have not yet been identified. Previous studies have shown that qRT-PCR investigations of formalin-fixed and paraffin-embedded (FFPE) tissue may be applicable to MIBC biomarker research, in particular for the identification of patients at high risk for progression or invasion [ [6] , [7] , [8] , [9] ].…”