Transcriptional Analysis of Immunohistochemically Defined Subgroups of Non-Muscle-Invasive Papillary High-Grade Upper Tract Urothelial Carcinoma

Abstract

Immunohistochemical (IHC) staining for CK5/6 and CK20 was reported to be correlated with the prognosis of early urothelial carcinoma in a way contrary to that of advanced tumors for unknown reasons. We aimed to characterize the gene expression profiles of subgroups of non-muscle-invasive papillary high-grade upper tract urothelial carcinoma (UTUC) classified by CK5/6 and CK20 expression levels: group 1 (CK5/6-high/CK20-low), group 2 (CK5/6-high/CK20-high), and group 3 (CK5/6-low/CK20-high). Expression of group 3 was predictive of worse prognosis of non-muscle-invasive papillary high-grade UTUC. Transcriptional analysis revealed 308 differentially expressed genes across the subgroups. Functional analyses of the genes identified cell adhesion as a common process differentially enriched in group 3 compared to the other groups, which could explain its high-risk phenotype. Late cell cycle/proliferation signatures were also enriched in group 3 and in some of the other groups, which may be used as a prognostic biomarker complementary to CK5/6 and CK20. Group 2, characterized by low levels of genes associated with mitogen-activated protein kinase and tumor necrosis factor signaling pathways, was hypothesized to represent the least cancerous subtype considering its normal urothelium-like IHC pattern. This study would facilitate the application of easily accessible prognostic biomarkers in practice.

CK14 Expression Identifies a Basal/Squamous-Like Type of Papillary Non-Muscle-Invasive Upper Tract Urothelial Carcinoma

“…Network and functional enrichment analyses as well as Ki-67 IHC staining demonstrated that cellular growth and proliferative functions are particularly upregulated in CK14positive papillary NMIUTUC, in accordance with previously reported high-risk phenotypes of NMIBC (13) and NMIUTUC (18) 50], which were upregulated and downregulated, respectively, in CK14-positive tumors. Dysregulation of these genes modulates proliferation, survival, migration, EMT, or metastasis of urothelial carcinoma, and other malignancies, leading to poor prognoses of these tumors.…”

“…Different studies further support the association between luminal-like type and either advanced pathological states or short survival durations in NMIBC using CK5/6 (KRT5/KRT6) and CK20 (KRT20) as surrogate protein or mRNA markers (14)(15)(16). In line with these results when studying the urinary bladder, we previously reported that papillary non-muscle-invasive upper tract urothelial carcinoma (NMIUTUC) with luminal-like, CK5/6-negative/CK20-positive, or CD44-negative/CK20positive, immunophenotypes had distinctly poor prognoses that were probably associated with altered cell adhesion and late cell cycle/proliferation functions (17,18).…”

“…Of the subclasses, a common subtype, named the BASQ subtype, harbors a high level of basal-type genes, squamous-like features, and an aggressive phenotype (10). Although the BASQ subtype is a good foundation for MIBC evaluation, the applicability of BASQ markers (CK14, CK5/6, GATA3, FOXA1) to NMIUTUC or NMIBC has been questioned by us and others (13,14,(16)(17)(18). For instance, CK5/6-negative/CK20-positive luminal-like papillary NMIUTUC had worse survival and was enriched with motility-and proliferation-associated genes (17,18).…”

“…Although the BASQ subtype is a good foundation for MIBC evaluation, the applicability of BASQ markers (CK14, CK5/6, GATA3, FOXA1) to NMIUTUC or NMIBC has been questioned by us and others (13,14,(16)(17)(18). For instance, CK5/6-negative/CK20-positive luminal-like papillary NMIUTUC had worse survival and was enriched with motility-and proliferation-associated genes (17,18). In the same way, "class 2" NMIBC defined by Hedegaard and coworkers (13) was characterized by luminal-like genetic attributes, including low KRT5, KRT6, and CD44 expression and high GATA3, FOXA1, and KRT20 expression, but it had far worse survival than the other types of NMIBC.…”

“…GEP analysis in the context of CK14 protein expression was determined from RNA sequencing and CK14 IHC staining data from independent high-grade papillary NMIUTUC specimens, which were generated previously (18). In short, surgically resected fresh tumors were cut in half: one half was subjected to RNA sequencing using a HiSeq 2500 platform (Illumina, San Diego, CA, US), and the other half was embedded in paraffin blocks for evaluation of CK14 immunoexpression.…”