Abstract 1031: Specific gene alterations of HER2 positive single circulating tumor cell (CTC) compared to autologous leukocytes in metastatic breast cancer (MBC)
Authors: Zhang Qiang , Gerratana Lorenzo , Ji Zhe , Wang Xinkun , D’Amico Paolo , Singhal Seema , Zhang Youbin , Davis Andrew A. , Shah Ami N. , Gradishar William 
Published: April 2023

Abstract
Introduction: Monitoring of CTC in MBC showed ability to predict treatment resistance and outcome. HER2 is one of the most important tumor markers which is associated with rapid cell division and the metastasis and prognosis of MBC in the clinic. More recently, we reported single CTC gene mutation is a key point to distinguish metastasis capability in MBC (2022 AACR and 2022 ASCO). Herein, we report specific gene alterations in HER2+ single CTC compared to autologous leukocytes in MBC.
Methods: Whole blood sample (7.5ml/each) was collected from MBC patient before therapy. CTC enumeration was performed in FDA approved CellSearch™ System and then the single HER2+ CTC and leukocytes were isolated by using DEPArrayTM System (Menarini). The single cell DNA was isolated and then the initial library was prepared by using Ampli1™ Whole Genome Amplification, Ampli1™ QC Kit (Menarini) and AMPure XP kit (Beckman Coulter). The exome capture was performed by TruSeq DNA Exome kit (illumine). The sequencing was prepared by NextSeq 500 mid output V2.5 kit and was performed on the NextSeq 500 (Illumina).
Results: We identified 107 CTCs including 55 HER2+ CTCs and 14 CTC-clusters. Autologous single HER2+ CTC (HER2+CK+CD45−, Group 1), HER2+CK−CD45+ leukocyte (Group 2) and HER2−CK−CD45+ leukocyte (Group 3) were isolated and sequenced respectively. The sequencing data was processed following the GATK pipeline and annotated using SnpEff. There were 486,119 counts (56.69%) for intron variants, 175,819 counts (20.51%) for intergenic variants, 70,334 counts (8.20%) for exon variants, 50,370 counts (5.87%) for downstream genes, 45,915 counts (5.36%) for upstream genes and others (3.37%) in HER2+ CTC. Meanwhile, there were 71,848 (8.76%) and 0 counts for exon variants found in Group 2 and Group 3 respectively. There were 79 gene variants ((SNP and Ins-Del) identified to have the highest impact effect (≥20) on HER2+ CTC chromosome, when there were 85 and 0 highest impact gene variants were identified in Group 2 and Group 3 respectively. The specific gene alterations in Group 1 compared to Group 2 includes CCNA2, FOLH1, BRD4, SAMHD1, CYP17A1, IDE, HPGDS and CTNNB1 Among the top 50 high impact gene variants, there were 26 genes alteration sites are same in both Group 1 and Group 2, including FECH, HDAC8, CYP11B2, TTN(6 sites), RAN, CD207, HK1, CASP1(2 sites), BRCA1, PIK3CG (3 sites), APEX1(4 sites), KIF11, SIRT5 (2 sites), XYLB and CHKA.
Conclusion: Genomic characterization of HER2 positive single CTCs elucidated specific gene alterations associated with disease metastasis compared to autologous leukocytes in MBC. The newfound HER2+CK−CD45+ cells with gene alterations maybe a new kind of cancer transformed leukocytes which need further validation. Specific gene alterations will help to develop novel drugs aimed at the eradication of CTCs using molecularly driven therapies for disease metastasis.
Citation Format: Qiang Zhang, Lorenzo Gerratana, Zhe Ji, Xinkun Wang, Paolo D’Amico, Seema Singhal, Youbin Zhang, Andrew A. Davis, Ami N. Shah, William Gradishar. Specific gene alterations of HER2 positive single circulating tumor cell (CTC) compared to autologous leukocytes in metastatic breast cancer (MBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1031.