Immunohistochemical Assays for Bladder Cancer Molecular Subtyping: Optimizing Parsimony and Performance of Lund Taxonomy Classifiers

Abstract

Transcriptomic and proteomic profiling classify bladder cancers into luminal and basal molecular subtypes, with controversial prognostic and predictive associations. The complexity of published subtyping algorithms is a major impediment to understanding their biology and validating or refuting their clinical use. Here, we optimize and validate compact algorithms based on the Lund taxonomy, which separates luminal subtypes into urothelial-like (Uro) and genomically unstable (GU). We characterized immunohistochemical expression data from two muscle-invasive bladder cancer cohorts ( n=193, n=76) and developed efficient decision tree subtyping models using 4-fold cross-validation. We demonstrated that a published algorithm using routine assays (GATA3, KRT5, p16) classified basal/luminal subtypes and basal/Uro/GU subtypes with 86%–95% and 67%–86% accuracies, respectively. KRT14 and RB1 are less frequently used in pathology practice but achieved the simplest, most accurate models for basal/luminal and basal/Uro/GU discrimination, with 93%–96% and 85%–86% accuracies, respectively. More complex models with up to eight antibodies performed no better than simpler two- or three-antibody models. We conclude that simple immunohistochemistry classifiers can accurately identify luminal (Uro, GU) and basal subtypes and are appealing options for clinical implementation.

Progression‐associated molecular changes in basal/squamous and sarcomatoid bladder carcinogenesis

“…Histologically, nearly half of Ba/Sq MIBCs harbor squamous divergence and may eventually progress to the aggressive sarcomatoid variant, often composed of tumor cells of spindle morphology [12][13][14]. Tumors of the Ba/Sq phenotype can be accurately identified by the combined overexpression of basal (KRT5, KRT6, KRT14) and underexpression of luminal cell markers (FOXA1, GATA3), including by immunohistochemistry [8,15,16]. They display high EGFR activity, with EGFR dependence in vitro and in vivo [7,17].…”

International Society of Urological Pathology Consensus Conference on Current Issues in Bladder Cancer. Working Group 4

“…However, immunohistochemistry is limited in the number of targets that may be quantified compared with RNA-based methods because there are practical limitations to the number of immunostains that may be performed on a single tissue block. Some of this challenge may be overcome by assigning molecular subtypes based on a smaller number of targets, an approach shown to be feasible given the tremendous collinearity of gene expression in individual subtypes 21,67,68 . Multiplex immunohistochemistry may also be useful, though this is practically limited to only 3 or 4 markers per slide.…”

“…Some of this challenge may be overcome by assigning molecular subtypes based on a smaller number of targets, an approach shown to be feasible given the tremendous collinearity of gene expression in individual subtypes. 21,67,68 Multiplex immunohistochemistry may also be useful, though this is practically limited to only 3 or 4 markers per slide. The data suggest an optimal biomarker strategy will likely include several signatures beyond the molecular subtype and such strategy may be hindered by the limited number of targets evaluable by immunohistochemistry.…”