2331P Interest of next generation sequencing (NGS) for integrated molecular diagnosis of HER2-low breast cancer

Based on HER2 expression using immunohistochemistry (IHC) and HER2 gene amplification using in situ hybridization (ISH), breast cancers (BC) are usually classified, into HER2 positive (IHC 3+ or IHC 2+/ISH+) and HER2 negative (IHC 0, IHC 1+ or IHC 2+/ISH-). Recently, HER2-low breast cancers (HER2-low BC) emerged as a new subtype defined as IHC1+ or IHC2+/ISH- tumors. HER2-low BC represent more than half of all BC. Consistently with the clinical activity of HER2 antibody-directed chemotherapy (ADC) in HER2-low BC, this subtype should be identified to enable early set-up of therapy. Since HER2-low BC identification could be equivocal using conventional IHC and ISH, we evaluated the performance of NGS for integrated diagnostic including HER2 copy number analysis. BC tumor specimens were analyzed using IHC and ISH as performed routinely. NGS was performed using a custom capture based 51-gene panel, including BC molecular target genes such as ESR1, PIK3CA, AKT1, ERBB2, TP53, BRCA1 and BRCA2. Gene mutations as well as copy number variation (CNV) and Microsatellite Instability (MSI) were determined. Thirty-one FFPE BC tumor specimens were classified using IHC and ISH into 11 HER2-positive (IHC2+ ISH+ and IHC3+), 10 HER2-negative (IHC 0) and 10 HER2-low cancers (IHC1+ and IHC2+ ISH-). Using NGS, CNV values for ERBB2 gene were significantly (p<0.001) different between HER2 negative, HER2low and HER2- positive tumors with mean CNV values of 2.0 (SD=0.3), 1.9 (SD=0.3) and 7.8 (SD=6.8), respectively. Using 3.25 as cutoff value for CNV, 90% concordance of HER2 amplification status was achieved between ISH and NGS. Using NGS, additional drug-targetable gene mutations as well as amplifications were detected in 68% (21/31) and 19% (6/31) of the cases, respectively. One case of MSI was detected in a HER2-negative and ISH unamplified case. These results show that in HER2 IHC positive (scores 1+ to 3+) BC, CNV determination using NGS allows the identification of HER2-low status simultaneously with the detection of multiple molecular target genes while sparing tissue samples. This workflow could support molecular board decision between HER2-ADC or other targeted therapies.