Abstract 3120: Genetic heterogeneity profiling for HER2 positive single circulating tumor cell (CTC) in metastatic breast cancer (MBC) by application of DEPArrayTM System and single cell DNA sequencing for HER2 positive single circulating tumor cell (CTC) in metastatic breast cancer (MBC) by application DEPArrayTM system and single cell DNA sequencing
Authors: Zhang Qiang , Gerratana Lorenzo , Pivarski Kara L. , Wang Xinkun , Ji Zhe , Stroup Emily , Vagia Elena Vagia , Davis Andrew Adam Davis A. Adam , Shah Ami , Kerby Katy L. , Flaum Lisa Flaum , Wehbe Firas , Zhang Youbin , Qiang Wenan , Behdad Amir , Gradishar William , Platanias Leonidas , Cristofanilli Massimo 
Published: September 2020

Abstract
Introduction: Although CTCs display the same spatial and temporal heterogeneity as the primary tumor, they represent a privileged window to disclose mechanisms of metastases. We previously reported that overexpression of HER2 in CTCs is associated with worse prognosis in MBC (2018 AACR #5195). Herein, we report a new finding of heterogeneity profiling for HER2 positive CTC by using a new approach for single CTC isolation and sequencing.
Methods: Whole blood sample (7.5ml/each) was collected from stage III/IV MBC patients before therapy. CTC enumeration were performed in FDA approved CellSearch™ System by targeting the EpCAM antigen for capturing CTCs which were then stained by Anti-CK-PE, DAPI, anti-CD45-APC and anti-HER2-FITC. The single HER2+ CTCs were isolated by using DEPArrayTM System (Menarini). The single cell DNA was isolated and the initial library was prepared by SMARTer® PicoPLEX® Gold Single Cell DNA-Seq Kit, and the exome capture was performed by Twist Human Core Exome EF Multiplex Complete Kit. The sequencing was prepared by NextSeq 500 mid output V2.5 kit and was performed on the NextSeq 500 (Illumina). It was a paired end run, 75×75 bps run with dual indexing.
Results: We identified 208 CTCs by CellSearch™ system, including 114 HER2+ CTCs and 4 CTC-clusters. Single HER2+ CTC was isolated and sequenced. The sequencing data was processed following the GATK pipeline and annotated using SnpEff. There were 362,015 counts (58.23%) for intron variants, 104,011 counts (16.73%) for intergenic variants, 56,678 counts (9.19%) for exon variants, 41,133 counts (6.67%) for downstream genes, 33,903 counts (5.45%) for upstream genes and others (3.73%). There were 44 genes (64 sites) variants were identified to have highest impact effect (≥20) on HER2+ CTC chromosome, among which TP53, PIK3CA, ESR1, PIK3R1 variants matched the most recent genomic characterization report for MBC tissue (Bertucci F. Nature 2019) when the XYLB, CYP17A1, CA2 and CAD variants were firstly reported. More important, although overexpression of PTP1B (PTPN1) was implicated in the development of MBC, our evidences firstly demonstrated that PTPN1 mutation is the most important variant (≥20) in CTCs for MBC. Furthermore, we also found that ESR1 mutation in CTC correlated with ESR1 status in ctDNA. Except for the high impact variants, there were 42 genes (66 sites) and 83 genes (136 sites) have medium (10-19) and significant (5-9) impact variants respectively.
Conclusion: Genomic characterization of HER2 positive single CTCs will elucidate new specific gene alterations (e.g. PTPN1) associated with disease metastasis, which will improve understanding of this critical process in MBC and lead to the development of novel drugs aimed at their eradication using molecularly driven therapies for this deadly condition.
Citation Format: Qiang Zhang, Lorenzo Gerratana, Kara L. Pivarski, Xinkun Wang, Zhe Ji, Emily Stroup, Elena Vagia Vagia, Andrew Adam Davis A. Adam Davis, Ami Shah, Katy L. Kerby, Lisa Flaum Flaum, Firas Wehbe, Youbin Zhang, Wenan Qiang, Amir Behdad, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. Genetic heterogeneity profiling for HER2 positive single circulating tumor cell (CTC) in metastatic breast cancer (MBC) by application of DEPArrayTM System and single cell DNA sequencing for HER2 positive single circulating tumor cell (CTC) in metastatic breast cancer (MBC) by application DEPArrayTM system and single cell DNA sequencing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3120.