Background: Recent groundbreaking work has shown that patients with lower levels of HER2 expression (HER2-low) may benefit from treatment with trastuzumab deruxtecan—an HER2 antibody-drug conjugate FDA-approved for treatment for HER2-positive (HER2+) patients
and thus can represent a new molecular subtype. In fact, this HER2-low patient population is
enriched with luminal disease but is clinically heterogeneous and outcomes have therefore not
been extensively characterized due to the lack of annotated multimodal real-world data (RWD).
We used Tempus RWD to identify unique HER2-low subtypes using RNA sequencing and
compare outcomes across subtypes.

Methods: We retrospectively analyzed 1,545 breast cancer samples from the Tempus database
tested with the Tempus xT assay that includes whole-exome capture RNA-seq. Only tumors with
known HER2 status determined via immunohistochemistry [IHC] and/or FISH were included. A
HER2 RNA gene signature was developed by comparing HER2- (n=464, IHC 0+) and HER2+
(n=161, IHC 3+ or IHC 2+ and FISH+) patients—controlling for HR status—to identify genes
associated with HER2 over-expression. This HER2 signature was used to stratify independent
HER2-low samples (n=920, determined by IHC 1+ or IHC2+ and FISH-) via hierarchical
clustering. Treatment use in this cohort was not assessed. Clusters were subsequently
assessed according to clinical, demographic, and molecular factors including PAM50 molecular
subtype classification of the RNA signatures. Real-world progression-free survival (rwPFS) was evaluated based on progression and death captured through manual expert abstraction for a
subset of stage 4 patients (n=336) and estimated via Kaplan-Meier analysis.

Results: HER2-low patients were clustered according to our HER2 expression signature
identifying three distinct molecular clusters (Table 1). Stage and demographic distributions
(race, ethnicity, age) were similar across clusters. Of note, cluster 3 (n=186, 20% of the
HER2-low population) was significantly enriched in hormone receptor negative (HR–) patients
(p<1e-5) and had lower ERBB2 RNA expression (p<1e-5). Interestingly, molecular
characterization using PAM50 demonstrated that cluster 3 was predominantly a basal-like
subtype, whereas luminal-like samples dominated cluster 1 and 2, and cluster 2 had the largest
composition of HER2-like samples (Table 1). Strikingly, cluster 3 stage 4 patients (n=57) had a
median rwPFS that was significantly shorter (>12 months) than cluster 1 and 2 stage 4 patients
(n=279, HR>1.66, p<2e-2, Table 1).

Conclusions: Tempus multimodal RWD reveals that HER2-low breast cancers are comprised
of distinct molecular subtypes. In a preliminary analysis, a cluster of HER2-low, predominantly
basal-like patients demonstrated dramatically worse outcomes than other clusters. These data
further emphasize the importance of using RNA expression to fully characterize clinically
relevant subpopulations. Further prospective studies are urgently needed to assess treatment
response in this heterogenous emerging HER2-low distinct population.

TABLE: Description of HER2-low molecular subtypes identified

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