Abstract PD3-1: Evaluation and clinical impact of intra-tumor heterogeneity (ITH) in primary HER2-overexpressing breast cancers (HER2+BC) treated with adjuvant trastuzumab and chemotherapy (CT)
Authors: Loi Sherene , Savas Peter , Lonnstedt Ingrid , Fumagalli Debora , Caramia Franco , Li Jason , Salgado Roberto , Rowan Andrew , Andre Fabrice , Denkert Carsten , Neven Patrick , Loibl Sibylle , Sotiriou Christos , Swanton Charles , Speed Terence P 
Published: May 2015

Abstract
Background: Despite the success of trastuzumab, when added to CT, for the treatment of women with primary HER2+ BC, diversity in clinical responses and survival are still observed. ITH or the presence of subclonal populations is theorized to be a cause of drug resistance. In this study, we investigated if ITH that could be estimated from a single sample taken at primary surgery from patients with newly-diagnosed HER2+ disease and if ITH was associated with poorer clinical outcomes despite adjuvant trastuzumab-based treatment.
Methods: Fifty-two frozen tumor and matched germline samples were taken from patients diagnosed with primary HER2+BC and treated with adjuvant trastuzumab and CT with a median follow-up of 6.3yrs (range 1.5-13). There were 10 (19%) invasive relapses, 7 (13%) distant relapses and 5 (9.6%) deaths. We performed tumor and germline whole exome sequencing (WES), tumor and germline copy number (CN) data profiling (Affymetrix SNP6.0), tumor gene expression (Affymetrix U133 2.0Plus). Variants were called with MuTect, CNs were called using GISTIC. We developed a novel bioinformatics method that integrated WES variant allele fractions, purity estimates and SNP data to delineate the presence of single or multiple genetically distinct subclones. We confirmed our estimated tumor ploidy using flow cytometry on 9 samples. We investigated the association between the presence of ITH and survival using multivariate Cox regression analyses.
Results: We observed in HER2+ BCs the presence of substantial chromosomal instability, numerous CN alterations and aneuploidy. With regards to their genomic architecture, 76.9% (40/52) displayed the presence of at least one aneuploid subclone, where the median ploidy was 3.3 (range:1.7-6.3) copies. Multiple distinct subclones or the presence of ITH could be clearly detected in 35/52 (67.3%) samples, 8/52 (15.4%) had evidence of only a single clone, whilst in 9/52 (17.3%) samples their genomic structure could not be definitively determined. The existence of ITH was significantly associated with invasive relapse after adjustment (HR:4.38;95%CI:1.61-7.14;p=0.002) with estrogen receptor and nodal status also remaining significant in the Cox model–in contrast, the absence of ITH was associated with excellent outcomes (no distant relapses). Other surrogates of genomic instability (gene expression signature [CIN70] and SNP signature [Genomic Instability Index]) were not significantly associated with survival in this dataset.
As exploratory analyses, potential genetic drivers of ITH were investigated. PIK3CA (n=13,p=0.008), MED1 (n=3,p=0.03) and IKZF1 (n=3,p=0.03) mutations, amplifications in 8q22.2 (n=7,p=0.008) as well as deletion in 1q24.2 (n=5, p=0.006) were associated with the presence of a single clone, whereas we did not find any genetic drivers with significant associations with ITH.
Conclusions: We show for the first time that the presence of ITH in primary HER2+ tumors is associated with worse outcomes despite adjuvant trastuzumab and CT. These findings should be evaluated in larger cohorts.
Citation Format: Sherene Loi, Peter Savas, Ingrid Lonnstedt, Debora Fumagalli, Franco Caramia, Jason Li, Roberto Salgado, Andrew Rowan, Fabrice Andre, Carsten Denkert, Patrick Neven, Sibylle Loibl, Christos Sotiriou, Charles Swanton, Terence P Speed. Evaluation and clinical impact of intra-tumor heterogeneity (ITH) in primary HER2-overexpressing breast cancers (HER2+BC) treated with adjuvant trastuzumab and chemotherapy (CT) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD3-1.