Diagnostic and prognostic implications of a three‐antibody molecular subtyping algorithm for non‐muscle invasive bladder cancer

Abstract

Intrinsic molecular subtypes may explain marked variation between bladder cancer patients in prognosis and response to therapy. Complex testing algorithms and little attention to more prevalent, early-stage (non-muscle invasive) bladder cancers (NMIBCs) have hindered implementation of subtyping in clinical practice. Here, using a three-antibody immunohistochemistry (IHC) algorithm, we identify the diagnostic and prognostic associations of well-validated proteomic features of basal and luminal subtypes in NMIBC. By IHC, we divided 481 NMIBCs into basal (GATA3 À /KRT5 + ) and luminal (GATA3 + /KRT5 variable) subtypes. We further divided the luminal subtype into URO (p16 low), URO-KRT5 + (KRT5 + ), and genomically unstable (GU) (p16 high) subtypes. Expression thresholds were confirmed using unsupervised hierarchical clustering. Subtypes were correlated with pathology and outcomes. All NMIBC cases clustered into the basal/squamous (basal) or one of the three luminal (URO, URO-KRT5 + , and GU) subtypes. Although uncommon in this NMIBC cohort, basal tumors (3%, n = 16) had dramatically higher grade (100%, n = 16, odds ratio [OR] = 13, relative risk = 3.25) and stage, and rapid progression to muscle invasion (median progression-free survival = 35.4 months, p = 0.0001). URO, the most common subtype (46%, n = 220), showed rapid recurrence (median recurrence-free survival [RFS] = 11.5 months, p = 0.039) compared to its GU counterpart (29%, n = 137, median RFS = 16.9 months), even in patients who received intravesical immunotherapy (p = 0.049). URO-KRT5 + tumors (22%, n = 108) were typically low grade (66%, n = 71, OR = 3.7) and recurred slowly (median RFS = 38.7 months). Therefore, a simple immunohistochemical algorithm can identify clinically relevant molecular subtypes of NMIBC. In routine clinical practice, this three-antibody algorithm may help clarify diagnostic dilemmas and optimize surveillance and treatment strategies for patients.

Are We Ready to Implement Molecular Subtyping of Bladder Cancer in Clinical Practice? Part 2: Subtypes and Divergent Differentiation

“…Conversely, other subtyping schemes, including the Lund taxonomy and the recent consensus molecular classification, suggested that the Lum group may be further split into subgroups with peculiar biological and prognostic features [ 13 , 83 , 84 ]. Both Uro and GU express urothelial differentiation markers GATA3 and FOXA1 while lacking basal markers CK5 and CK14 [ 85 , 86 ], yet differ with regard to the mechanisms of cell cycle checkpoint inactivation [ 25 ]. Uro cancers inactivate the cell cycle regulator RB1 indirectly through loss of CDKN2A, which encodes the p16INK4A cyclin-dependent kinase inhibitor [ 87 , 88 ] and may be further split into UroA and UroB.…”

“…Jackson et al reported that the GATA3+/P16−/CK5− Uro NMIBCs had earlier recurrences post-BCG treatment as compared to GATA3+/P16+/CK5− GU tumors [ 25 ]. Deep deletion or loss of chromosome 9p, including the CDKN2A locus, which encodes P16, is a recurrent alteration in early BCs [ 11 , 23 , 37 , 90 ], whereas GU NMIBCs do not show changes in CDKN2A nor in p16 protein expression [ 23 , 37 ].…”

“…Deep deletion or loss of chromosome 9p, including the CDKN2A locus, which encodes P16, is a recurrent alteration in early BCs [ 11 , 23 , 37 , 90 ], whereas GU NMIBCs do not show changes in CDKN2A nor in p16 protein expression [ 23 , 37 ]. This subtype is enriched for increased CD3+-infiltrated tumors, in comparison with Uro tumors, along with higher mutational burden [ 25 , 83 , 91 ], thus suggesting that poor recurrence-free survival (RFS) post-BCG treatment in the latter might be due to a weaker immune response [ 25 ]. Lack of CDKN2A/P16 alterations has been reported to portend response to BCG treatment in NMIBCs [ 25 , 91 ], unlike the poor prognosis described in MIBC cohorts [ 23 , 37 , 92 ], possibly due to the gradual development of further genetic aberrations in higher stage and grade tumors [ 25 , 50 , 83 ].…”

“…According to two studies, eight GD-UCs (7 NMIBCs/1 MIBC) and three NMIBCs were all classified into the GU subtype by their molecular and immunophenotypical signature [ 22 , 25 ]. A small subset of BCs with divergent differentiation (including GD) within the M.D.…”

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

“…For example, the p16 assay used in the studies analyzed here showed a lower dynamic range than an assay used in subsequent work. 31 Thus, implementing this classifier will require normalizing differences in dynamic range between assays and centers, including necessary control tissues.…”