CK5/6 and GATA3 Defined Phenotypes of Muscle-Invasive Bladder Cancer: Impact in Adjuvant Chemotherapy and Molecular Subtyping of Negative Cases

Abstract

Introduction and ObjectiveIdentifying patients that benefit from cisplatin-based adjuvant chemotherapy is a major issue in the management of muscle-invasive bladder cancer (MIBC). The purpose of this study is to correlate “luminal” and “basal” type protein expression with histological subtypes, to investigate the prognostic impact on survival after adjuvant chemotherapy and to define molecular consensus subtypes of “double negative” patients (i.e., without expression of CK5/6 or GATA3).Materials and MethodsWe performed immunohistochemical (IHC) analysis of CK5/6 and GATA3 for surrogate molecular subtyping in 181 MIBC samples. The mRNA expression profiles for molecular consensus classification were determined in CK5/6 and GATA3 (double) negative cases using a transcriptome panel with 19.398 mRNA targets (HTG Molecular Diagnostics). Data of 110 patients undergoing radical cystectomy were available for survival analysis.ResultsThe expression of CK5/6 correlated with squamous histological subtype (96%) and expression of GATA3 was associated with micropapillary histology (100%). In the multivariate Cox-regression model, patients receiving adjuvant chemotherapy had a significant survival benefit (hazard ratio [HR]: 0.19 95% confidence interval [CI]: 0.1–0.4, p < 0.001) and double-negative cases had decreased OS (HR: 4.07; 95% CI: 1.5–10.9, p = 0.005). Double negative cases were classified as NE-like (30%), stroma-rich (30%), and Ba/Sq (40%) consensus molecular subtypes and displaying different histological subtypes.ConclusionImmunohistochemical-based classification was associated with histological subtypes of urothelial MIBC. IHC markers like CK5/6 and GATA3 that are used in pathological routine could help to identify patients with basal and luminal tumor characteristics. However, a two-sided classification system might not sufficiently reflect the heterogeneity of bladder cancer to make treatment decisions. Especially the group of IHC-double negative cases, as further analyzed by mRNA expression profiling, are a heterogeneous group with different implications for therapy.

Histomolekulare Klassifikation des Urothelkarzinoms der Harnblase

“…Als externes (ext.) Testset wurden MIUCs aus einer publizierten UC-Kohorte [ 11 ] des Dr. Senckenbergischen Instituts für Pathologie (SIP), Universitätsklinikum Frankfurt, Senckenberg Biobank, ausgewählt.…”

“…Für das ext. SIP-Testset wurden 103 von 181 MIUCs mit durchgeführter RNA-Extraktion, die von einer radikalen Zystektomie stammen, berücksichtigt [ 11 ]. Innerhalb der histologischen Schnitte wie auch innerhalb später erstellter Annotationen der SIP-Fälle (siehe Abb.…”

Optimizing identification of consensus molecular subtypes in muscle-invasive bladder cancer: a comparison of two sequencing methods and gene sets using FFPE specimens

“…Samples were stained for CK5/6 (Clone: D5/16 B4; Dako /Agilent, Santa Clara, CA, USA) and GATA3 (Clone: L50-823; Cell Marque, Rocklin, CA, USA) as described before [ 19 ].…”

“…The mRNA expression was determined using the HTP (HTG Molecular Diagnostics, Tuscon, AZ, USA) as describes before [ 19 , 26 ]. Briefly, target capture was done by the HTG EdgeSeq chemistry with nuclease protection probes on a 96-well plate.…”

Tumor-associated macrophages and Tregs influence and represent immune cell infiltration of muscle-invasive bladder cancer and predict prognosis

“…The construction of the tissue microarray (TMA) has been described before [ 21 ]. In brief, tissue cores (1 mm) of representative tumor areas were transferred to a new block using the TMA Grandmaster (3DHISTECH, Budapest, Hungary).…”