Clonal Evolution of the HER2 L755S Mutation Leads to Acquired HER‐targeted Therapy Resistance That Can Be Reversed by the Irreversible HER1/2 Inhibitor Afatinib
Authors: Xu Xiaowei , Nardone Agostina , Hu Huizhong , Qin Lanfang , Nanda Sarmistha , Mitchell Tamika , Shea Marty , Heiser Laura , Wang Nicholas , Covington Kyle , Chen Edward , Renwick Alexander , Wang Tao , De Angelis Carmine , Contreras Alejandro , Gutierrez Carolina , Fuqua Suzanne , Chamness Gary , Shaw Chad , Wheeler David , Gray Joe , Hilsenbeck Susan , Rimawi Mothaffar , Osborne Kent , Schiff Rachel 
Published: November 2023

BackgroundTargeting HER2 with lapatinib (L), trastuzumab (T), or the LT combination, is effective in HER2+ breast cancer (BC), but acquired resistance commonly occurs. In our 12‐week neoadjuvant trial (TBCRC006) of LT without chemotherapy in HER2+ BC, the overall pathologic complete response rate (pCR) was 27%. To investigate resistance mechanisms our lab developed 9 HER2+ BC cell lines resistant (R) to these drugs (LR/TR/LTR). To discover potential predictive markers/therapeutic targets to circumvent resistance, we completed genomic profiling of the cell line panel and a subset of pre‐treatment baseline specimens from TBCRC006.Materials and methodsParental (P)/LR/TR/LTR lines of 9 HER2+ BC cell line models were profiled with whole exome and RNA sequencing. Mutations detected in R lines but not in P lines of the same model were identified. Single cells of the BT474AZ‐LR line were cloned and their cDNAs were sequenced. Mutation‐specific Q‐PCR was designed to more sensitively quantify mutations. Whole exome sequencing (>100X) and Ion AmpliSeq of 17 baseline tumor/normal pairs from TBCRC006 were performed.ResultsWe found the HER2 L755S mutation in the BT474ATCC‐LTR line and the BT474AZ‐LR line, in which the HER pathway was reactivated to cause resistance. Overexpression of this mutation in the BT474AZ‐P line can induce LR and growth of both resistant lines can be significantly inhibited by HER2‐L755S‐specific siRNA knock‐down, suggesting its role as an acquired L/LT resistance driver in HER2+ BC. Sanger sequencing of BT474AZ‐LR single cell clones found the HER2 L755S mutation in every clone but only in ~30% of the HER2 copies. Using sensitive mutation‐specific Q‐PCR, we found statistically higher levels of HER2 L755S expression in BT474ATCC‐P and BT474AZ‐P compared to other HER2+ BC cell line parentals. These data suggest that this mutation exists subclonally within BT474 parentals and was selected to become the more dominant population in the two resistant lines. The HER1/2 irreversible tyrosine kinase inhibitor (TKI) afatinib (Afa) robustly inhibited growth of both BT474ATCC‐LTR/AZ‐LR cells and BT474AZ‐LR xenografts. Western blots confirmed inhibition of the HER and downstream Akt and MAPK signaling in the LR cells by Afa. Sequencing of TBCRC006 pre‐treatment samples found the HER2 L755S mutation in 1/17 subjects. This patient did not achieve pCR after neoadjuvant LT treatment. The variant was present in 2% of the reads, indicating it as a subclonal event in this patient's baseline tumor.ConclusionAcquired resistance in two of our BT474 LR/LTR lines is due to selection of HER2 L755S subclones present in the parental cell population. The higher HER2 L755S levels detected in BT474 parentals compared with other HER2+ BC parental lines, and detection of its subclonal presence in a pre‐treatment HER2+ BC patient, suggest that sensitive mutation detection methods will be needed to identify patients with potentially actionable HER family mutations in primary tumor. Treating this patient group with an irreversible TKI like Afa may prevent resistance of this subset of HER2+ BC.Support or Funding InformationSupported by: the Specialized Programs of Research Excellence (SPORE) grants P50 CA58183 and CA 186784; the Dan L. Duncan Cancer Center Grant P30CA125123 from the National Cancer Institute; a Stand Up To Cancer Dream Team Translational Research Grant (SU2C‐AACR‐DT0409). The Breast Cancer Research Foundation.