RNA sequencing as a confirmatory assay and its impact on patient care in multiple cancer types.

Pashtoon Murtaza Kasi Weill Cornell Medicine, Englander Institute of Precision Medicine, New York Presbyterian Hospital, New York, NY info_outline Pashtoon Murtaza Kasi, Sofia Smirnova, Vladislav Nikulin, Jessica H. Brown, Nava Almog, Anna Ogloblina, Clinton Yam, Julio Antonio Peguero, Devesh Mahesh Pandya, Daniel Kerr, Nathan Fowler

Authors Pashtoon Murtaza Kasi Weill Cornell Medicine, Englander Institute of Precision Medicine, New York Presbyterian Hospital, New York, NY info_outline Pashtoon Murtaza Kasi, Sofia Smirnova, Vladislav Nikulin, Jessica H. Brown, Nava Almog, Anna Ogloblina, Clinton Yam, Julio Antonio Peguero, Devesh Mahesh Pandya, Daniel Kerr, Nathan Fowler Organizations Weill Cornell Medicine, Englander Institute of Precision Medicine, New York Presbyterian Hospital, New York, NY, BostonGene, Corp., Waltham, MA, University of Texas MD Anderson Cancer Center, Houston, TX, Oncology Consultants, Houston, TX, Advanced Cancer Treatment Centers: Cancer Centers of South FL, Palm Springs, FL Abstract Disclosures Research Funding No funding received None. Background: Increasing evidence shows the ability of RNA sequencing (RNA-seq) to detect expression of multiple biomarkers with improved sensitivity and scalability, offering a more objective and reproducible interpretation of data compared to pathological assessment methods by immunohistochemistry (IHC). Further, ongoing research demonstrating strong correlation between RNA-seq gene expression values and IHC values suggests the utility of RNA-seq as an objective diagnostic approach. Here, we highlight 4 cases where discordance in expression levels measured by IHC and RNA-seq resulted in re-evaluation of IHC values and a subsequent change in the clinical course. Methods: All patients underwent RNA and DNA sequencing of tumor tissue as part of a standard-of-care workflow. Sequencing was performed and reported with the BostonGene Tumor Portrait test, utilizing whole-exome sequencing (WES) and transcriptome sequencing data for tumor analysis. Gene expression levels were evaluated by comparison to a reference cohort of patients with a similar diagnosis. Results: In one case, a 53-year-old female was diagnosed with metastatic cholangiocarcinoma. While IHC of her biopsy reported positive HER2 (+3) expression, RNA-seq revealed low expression. A repeated IHC assay confirmed negative HER2 expression, leading to a change in therapy options. Another 61-year-old female was diagnosed with triple negative breast cancer (TNBC) (IHC: ER 0%, PR 0%, HER2 1+). Integrated WES and RNA-seq analysis detected HER2 amplification (+8 copies), ERBB2-activating mutation, high ERBB2 (HER2) expression and a HER2-enriched PAM50 subtype. IHC was repeated, now showing positive HER2 (+3) expression. In order to target HER2, systemic treatment was changed to a combination containing trastuzumab and pertuzumab, resulting in pathological complete response. In the third case of an 85-year-old female with relapsed TNBC, IHC demonstrated 1% PDL-1 staining. In contrast, RNA-seq detected high PD-L1 expression leading the treating physician to repeat IHC. Staining showed high PDL-1 (CPS50), prompting a change in therapy to an anti-PDL-1 (pembrolizumab) therapy. Lastly, while IHC of a 21-year-old female diagnosed with diffuse large B-cell lymphoma (DLBCL) showed negative CD30, subsequent RNA-seq detected relatively high CD30 expression that prompted a second IHC evaluation confirming positive CD30 expression. Eventually, the diagnosis was changed to primary mediastinal B-cell lymphoma and DA-EPOCH was selected as the therapy. Conclusions: RNA-seq is emerging as an objective tool to evaluate key diagnostic and targetable events in multiple cancer types. The use of RNA-seq can also add value as an objective measurement to confirm critical IHC findings, potentially changing clinical care.