HER2 overexpression in urothelial carcinoma with GATA3 and PPARG copy number gains

Abstract HER2, encoded by the ERBB2 gene, is an important druggable driver of human cancer gaining increasing importance as a therapeutic target in urothelial carcinoma (UC). The genomic underpinnings of HER2 overexpression in ERBB2 nonamplified UC are poorly defined. To address this knowledge gap, we investigated 172 UC tumors from patients treated at the University of California San Francisco, using immunohistochemistry and next-generation sequencing. We found that GATA3 and PPARG copy number gains individually predicted HER2 protein expression independently of ERBB2 amplification. To validate these findings, we interrogated the Memorial Sloan Kettering/The Cancer Genome Atlas (MSK/TCGA) dataset and found that GATA3 and PPARG copy number gains individually predicted ERBB2 mRNA expression independently of ERBB2 amplification. Our findings reveal a potential link between the luminal marker HER2 and the key transcription factors GATA3 and PPARG in UC and highlight the utility of examining GATA3 and PPARG copy number states to identify UC tumors that overexpress HER2 in the absence of ERBB2 amplification. In summary, we found that an increase in copy number of GATA3 and PPARG was independently associated with higher ERBB2 expression in patient samples of UC. This finding provides a potential explanation for HER2 overexpression in UC tumors without ERBB2 amplification and a way to identify these tumors for HER2-targeted therapies.

Urothelial carcinoma (UC) is an important cause of cancer morbidity and mortality worldwide.HER2 (encoded by ERBB2) is an oncogenic driver frequently altered in multiple cancer types, including breast cancer and malignancies of the gastrointestinal tract, 1 where HER2-targeted therapies have improved patient outcomes and have received regulatory approvals.ERBB2 is amplified in approximately 7% of UC tumors with correspondingly high HER2 expression on immunohistochemistry (IHC). 2 A recent study reported HER2 positivity (IHC 3+ or 2+ with ERBB2 amplification by fluorescent in situ hybridization) in 15% of UC tumors, while approximately 50% of tumors showed some degree of HER2 expression (IHC ≥ 1+). 2 Ongoing clinical trials are investigating the efficacy of HER2-targeted antibody-drug conjugates (ADCs) in UC with promising results, using HER2 protein expression as the relevant biomarker. 3,4This has led to the recent FDA approval of trastuzumab deruxtecan for patients with unresectable or metastatic HER2-positive (IHC 3+) solid tumors, including UC.However, aside from ERBB2 amplification, 2 the genetic basis of HER2 overexpression in UC is not well understood.
To define the genomic profiles of HER2-overexpressing UC, we first examined ERBB2 alterations (Figure 1B) and found that ERBB2 amplification was present in 12/30 (40.0%) of HER2 IHC 3+ versus 4/142 (2.8%) of HER2 IHC 0/1+/2+ tumors (Figure 1C; p = 1.1 × 10 −7 ).Next, to investigate the gene dosage effect for other cancer-related genes (genes included on the UCSF500 panel other than ERBB2) on HER2 expression, we evaluated a logistic regression model to predict HER2 IHC 3+ (vs IHC 0/1+/2+) status while adjusting ERBB2 amplification status as a covariate (Supplementary Methods).We were able to test 500 and 524 cancer-related genes for amplification and deletion, respectively (Supplementary Tables S1 and S2).We identified 46 gene amplifications and 32 gene deletions associated with HER2 IHC 3+ status with a nominal P < .05 and searched for their overlaps with the top 200 most frequently amplified and deleted genes, respectively, in the Memorial Sloan Kettering/The Cancer Genome Atlas (MSK/TCGA) MIBC dataset (Supplementary Table S3). 6his analysis identified 2 genes, GATA3 and PPARG, both encoding key transcription factors (TFs) in urothelial development and carcinogenesis 7 and frequently amplified in UC, 8 that were enriched in HER2 IHC 3+ tumors (Supplementary Table S1; Figure 1D, 1E).Among the 500 genes tested for amplification, GATA3 was the second (100th percentile) and PPARG the 45th (91st percentile) most significant gene independently associated with HER2 IHC 3+ status in the UCSF dataset (Supplementary Table S1).The remaining 44 genes with amplifications and all 32 genes with deletions were not among the top 200 most frequently amplified and deleted genes in the MSK/TCGA dataset, respectively (Supplementary Figure S2A, S2B).
To assess the clinical utility of examining gene dosage of GATA3 and PPARG for identifying HER2-overexpressing UC tumors, we first analyzed the UCSF cohort.While 12 of the 30 (40.0%) HER2 IHC 3+ tumors had ERBB2 amplification (Figure 1C), 8 more HER2 IHC 3+ tumors (8/30; 26.7%) without ERBB2 amplification were identified by examining the copy number of GATA3 and PPARG, including 5 with GATA3 amplification alone, one with PPARG amplification alone, and 2 with both GATA3 and PPARG amplifications (Figure 2A; Supplementary Figure S8).We next evaluated the MSK/TCGA cohort, where ERBB2 mRNA levels were used as a surrogate for HER2 expression due to a lack of HER2 IHC data.We found that assessing gene dosage of GATA3 and PPARG in addition to ERBB2 consistently identified more tumors overexpressing ERBB2 mRNA (Figure 2B).For example, among MIBC tumors in the top quartile of ERBB2 mRNA expression, 20.3% displayed ERBB2 amplification, but analyzing for GATA3 or PPARG amplification identified an additional 24.3% of tumors overexpressing ERBB2 mRNA.Thus 44.6% of all MIBC tumors in the top quartile of ERBB2 mRNA expression harbored amplification involving at least one of ERBB2, GATA3, and PPARG.This relationship was consistent across all levels of ERBB2 mRNA expression (Figure 2B).
In summary, we reported HER2 overexpression in UC tumors with GATA3 and PPARG copy number gains and demonstrated the clinical utility of examining these 2 genes for identifying additional UC tumors overexpressing HER2, particularly when ERBB2 amplification is not detected.In addition to providing a potential explanation for HER2 overexpression in non-ERBB2 amplified UC tumors, our findings emphasize the need to look beyond ERBB2 amplification when using a tumor DNA-based assay to help assess HER2 expression status in UC, an important biomarker for novel HER2-targeted ADCs.

Figure 1 .
Figure 1.GATA3 and PPARG copy number gains are independently associated with increased ERBB2 expression in urothelial carcinoma.(A) Distribution of HER2 IHC scores of 172 urothelial carcinoma (UC) samples in the UCSF cohort.(B) ERBB2 genetic alterations detected on UCSF500, the institutional cancer gene panel.CNV, copy number variant; SNV, single nucleotide variant.(C) ERBB2 amplifications are enriched in HER2 IHC 3+ UC tumors in the UCSF cohort.(D, E) GATA3 and PPARG amplifications are each enriched in HER2 IHC3+ UC tumors in the UCSF cohort.P values were calculated using Fisher's exact test comparing the proportions of events (ie, GATA3 and PPARG amplifications, respectively) in HER2 IHC 3+ versus 0/1+/2+ samples.(F, G) GATA3 and PPARG gains or amplifications (as defined by the study) are each enriched in muscle-invasive bladder cancer (MIBC) tumors with high ERBB2 mRNA expression (>4th quartile vs ≤4th quartile) in the MSK/TCGA cohort.