Abstract P4-01-19: Liquid biopsy and re-biopsy: Tracking mutational trajectories in HER2+ breast cancer patients undergoing T-DM1 treatment
Authors: Allegretti M , Giordani E , Casini B , Romania P , Gasparro S , Russillo M , Gallo E , Buglioni S , Pescarmona E , Cognetti F , Ciliberto G , Giacomini P , Fabi A 
Published: May 2019

Abstract
Background: The antibody-drug conjugate Trastuzumab emtansine (T-DM1, Kadcyla®) is standard of care in HER2+ breast cancer patients on clinical progression after Trastuzumab/Pertuzumab and taxanes. Despite considerable clinical benefits, most patients rapidly develop progressive disease due to adaptive resistance, the molecular bases of which remain largely unknown and/or controversial. Next generation sequencing (NGS) and digital PCR (dPCR) applied to serial biological samples obtained through liquid biopsy (LB) and re-biopsy (RB) offer a unique opportunity to intercept mutational trajectories and uncover molecular patterns linked to primary as well as adaptive resistance.
Materials and methods: Tumor tissues (n=14), either from primary or metastatic lesions, and plasma samples (n=99) were collected, upon informed consent, from 9 breast cancer patients undergoing T-DM1 administration. Tissue (tDNA) and circulating tumor DNA (ctDNA) were extracted by the QIAmp DNA FFPE and CNA kits (Qiagen), respectively, and analyzed by ultra-deep sequencing and dPCR (IonTorrent S5 and QuantStudio 3D, LifeTechnologies) with commercial 400-gene panel and custom-designed dPCR assays. Genomic data were correlated with clinical imaging (CT/PET).
Results: Six out 9 (66.7%) patients experienced progression within 1 year of treatment (mean 192±97 days), whereas the remaining 3 were stable at the last follow up (&gt; 400 days). No correlation was found between outcome and HER2, ER or PR status in the latest available (prior to T-DM1) archival tissue, in which NGS revealed several pre-existing mutations, including some associated with resistance to ERBB2 blockade. LB analysis detected increases in both baseline and de novo occurring aberrations in 5/6 (83.4%) relapsing patients. As compared to clinical imaging, progression disease was anticipated by an average lead time of 1.9 months (range 0.7-2.8). Surprisingly, the sixth relapsing patient underwent rapid progression (3 months) in spite of decreased PIK3CA p.E545K in blood, further confirmed in the re-biopsy, thus suggesting heterogeneous response to T-DM1 across multiple cancer cell populations. Of note, we observed progressive accumulation of ERBB2 p.L755S (associated with Lapatinib resistance) in multiple biopsies of serial metastatic foci from a patient over 18 years of multimodal ERBB2 blockade. However, a single administration of T-DM1 resulted in ultra-fast (within few weeks) clearance of ERBB2 p.L755S ctDNA, and stabilization of two distinct 'bystander' TP53 ctDNAs (p.R273H and p.S241T).
Conclusions: Non-invasive LB monitoring of a small cohort of T-DM1-treated patients provides proof of principle of intersecting mutational trajectories, anticipation and classification of resistance, as well as de novo appearance/clearance of resistance mutations. Thus, LB and RB may hint at disease evolution and successive lines of medical treatment.
This work was supported by AIRC (Nuvenia Fellowship to MA, IG 19052 to PG), EU commission (grant #633937 – ULTRAPLACAD), and Regina Elena National Cancer Institute intramural funding.
Citation Format: Allegretti M, Giordani E, Casini B, Romania P, Gasparro S, Russillo M, Gallo E, Buglioni S, Pescarmona E, Cognetti F, Ciliberto G, Giacomini P, Fabi A. Liquid biopsy and re-biopsy: Tracking mutational trajectories in HER2+ breast cancer patients undergoing T-DM1 treatment [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-19.