Amplicon Structure in Early and Advanced HER2 Positive Tumors

Introduction– ERBB2 amplification is a prognostic factor for aggressive breast tumors and a predictive marker for prolonged survival of breast and gastric cancer patients treated with ERBB2 inhibitors.

Three amplicon structures (AS) were described in tumor amplified DNA: inverted duplication (ID), tandem repeat (TR) and double minute. An ERBB2 amplicon with an ID is described in the breast cancer cell line HCC1954 model as well as in eight breast cancer patients. In other tumors, a TR linked by an inversion to 17q21.3, associated with a BRCA1 loss is found. In this work, we describe the ERBB2 AS of 40 HER2+ tumors and the clinical course of the patients.

Material and methods –

A retrospective single institution study of patients with HER2+ tumors. A qualified pathologist identified Formaline - fixed paraffin embedded tumor tissues, the tissue tumor was marked and the DNA extracted using the QIAamp DNA FFPE tissue kit. We analyzed the DNA samples using Digital Droplet PCR (Bio-Rad). Values of six copies or more were considered positive. We prepared libraries using the NEBNext Ultra DNA Library Preparation Kit for Illumina and the Multiplex Oligos Kit for indexes, using 50-100 ng of genomic DNA input for each sample. We denatured the libraries and loaded them on the Illumina Nextseq 500 platform. Paired end sequencing was performed using the Nextseq 500 High Output v2 kit (75 cycles). We performed alignment of short paired-end sequences to the reference genome hg19 using BWA-ALN algorithm. We removed potential PCR duplicates reads using the MarkDuplicates tool from Picard. Structural variations were detected and analyzed using Control FREEC and BreakDancer algorithms.

Results and discussion - We find that a single segment ID is the AS in the majority of HER2+ tumors with a defined AS. In addition, in early stage cancer the ERBB2 amplicon is composed of a single segment while in advanced stage cancer, from several different segments. This result verifies the classic model of gene amplification described by McClintock and found in HCC1954 .

Conclusion - We propose that in early stage HER2+ tumors the AS is a single segment ID, in advanced disease an amplicon with ID AS composed of several segments is common. In addition, we found that characterizing HER2+ tumors using WGS could help identify if a tumor is a recurrent tumor or second primary tumor and identify co-amplified oncogenes. In the future, this approach may aid in patients care.