Her2 alterations in muscle-invasive bladder cancer: Patient selection beyond protein expression for targeted therapy

Abstract

Although the introduction of novel targeted agents has improved patient outcomes in several human cancers, no such advance has been achieved in muscle-invasive bladder cancer (MIBC). However, recent sequencing efforts have begun to dissect the complex genomic landscape of MIBC, revealing distinct molecular subtypes and offering hope for implementation of targeted therapies. Her2 (ERBB2) is one of the most established therapeutic targets in breast and gastric cancer but agents targeting Her2 have not yet demonstrated anti-tumor activity in MIBC. Through an integrated analysis of 127 patients from three centers, we identified alterations of Her2 at the DNA, RNA and protein level, and demonstrate that Her2 relevance as a tumor driver likely may vary even within ERBB2 amplified cases. Importantly, tumors with a luminal molecular subtype have a significantly higher rate of Her2 alterations than those of the basal subtype, suggesting that Her2 activity is also associated with subtype status. Although some of our findings present rare events in bladder cancer, our study suggests that comprehensively assessing Her2 status in the context of tumor molecular subtype may help select MIBC patients most likely to respond to Her2 targeted therapy.

Figure 1. Micrographs of ERBB2 FISH and IHC in bladder cancer samples.

Micrographs show representative bladder cancer samples without (A) and with (B) ERBB2 amplification. ERBB2 yields a red signal while the chromosome 17 centromere is stained with green. Amplification is defined as a gene to centromere ratio ≥2.0 or copy number ≥6.0. Scale bar represents 10 μm. Representative immunohistochemical stains demonstrate Her2 negative (C, score 0) and strongly positive bladder cancers (D, score 3+). Scale bar represents 200 μm.

Figure 2. Relationship between ERBB2 gene status and Her2 expression.

(A) Bar plot demonstrating that ERBB2 amplified MIBC (determined by FISH) was enriched among the samples with high ERBB2 mRNA expression in our NAC cohort (lr, reference level [rl]: ERBB2 amplification status: normal). (B) ERBB2 amplified MIBC were similarly enriched among MIBC with a high mRNA expression in the TCGA bladder cohort. Somatic SNVs (indicated by arrows) were evenly distributed between cases with different mRNA expression (lr, mRNA expression vs. CNV, rl: hemizygous deletion). (C) Stacked bar plot showing that a significantly higher proportion of tumors with a Her2 protein expression score of 3+ harbored ERBB2 amplification by FISH, than tumors with scores of 0–2 (Fisher’s test). (D) Barplot showing Her2 protein expression in the TCGA bladder cohort, indicating significantly higher protein expression in patients with ERBB2 amplification. SNVs are indicated by arrows (lr, protein expression vs. CNV, rl: hemizygous deletion).

Figure 3. Single nucleotide variants (SNV) in ERBB2 and relationship to amplification and expression.

(A) Schematic of the ERBB2 coding region showing all somatic missense ERBB2 mutations detected in the TCGA bladder cohort. Twenty of 45 detected SNVs were located in the Furin-like domain. (B) Barplot showing the ERBB2 mutant allele frequency in four TCGA cases with both a mutation and concomitant ERBB2 amplification. Note that in 3/4 samples the mutant allele frequency was greater than 85%, suggesting that the SNV is present on all tumor amplicons and occurred prior to gene amplification. (C) Boxplot showing that among the 45 TCGA cases with ERBB2 mutations, mRNA expression levels were dependent on copy number status (lr, rl: hemizygous deletion). (D) Boxplot demonstrating that cases with Her2 extracellular domain mutations had a significant lower protein expression/detection than samples with intracellular domain mutations (lr, rl: extracellular SNVs). (E) Whole genome copy number profiles from two cases in our NAC cohort with ERBB2 amplification. Note that Case 1 (upper panel) appears to harbor isolated ERBB2 amplification, while Case 2 (lower panel) exhibits multiple focal amplifications, including in other well-known oncogenes.

Figure 4. Relationship between Her2 alterations and TCGA clusters.

(A) In the TCGA bladder cohort, ERBB2 copy number was significantly higher in luminal (cluster I and II) compared to basal tumors (cluster III and IV), but was similar between cluster I and II (Fisher’s test). (B) Consistent with our NAC dataset, ERBB2 mRNA expression in the TCGA bladder cohort was higher in cluster I and II than in cluster III and IV (lr, rl: cluster I). (C) Her2 protein expression, determined by RPPA was significantly higher in cluster I when compared to all other clusters (lr, rl: cluster I).

Figure 5. Potential algorithm for molecularly stratifying MIBC patients for Her2 targeted therapy.