Abstract P6-10-21: Evaluation of genomic changes in ductal carcinoma in situ as potential biomarkers of recurrence risk
Authors: Lesche Dorothea , Saunders Hugo , Mahale Sakshi , Zenthoven Magnus , Huges Siobhan , Kader Tanjina , Thio Niko , Miligy Islam M , Pang Jia-Min , Green Andrew R , Rakha Emad A , Campbell Ian G , Fox Stephen B , Gorringe Kylie 
Published: March 2020

Routine breast cancer screening has increased detection of pre-invasive lesions such as ductal carcinoma in situ (DCIS). Currently, DCIS cases are mostly treated with wide local excision combined with adjuvant radiotherapy and/or hormonal therapy, even though only 25% of DCIS treated with surgery alone recur with 50% of recurrences being invasive. At present, prediction of recurrence risk is based on histopathological features and patient characteristics but reliable identification of individual recurrence risk is still challenging. Certain genetic aberrations (i.e. copy number variation, somatic mutations) are associated with breast cancer progression. While most recurrences are assumed to be genetically related to the primary tumour (“clonal recurrence”), the prevalence of ipsilateral independent primary events (“non-clonal recurrence”) after treatment for DCIS is uncertain. If this is a substantial proportion of cases, their presence would compromise attempts to find tumour intrinsic biomarkers of recurrence. To enable the discovery of better recurrence biomarkers, this project initially aims to identify cases with primary DCIS and later clonal ipsilateral recurrence.

Sections from formalin-fixed paraffin-embedded blocks from primary DCIS cases and their subsequent recurrences were obtained from Nottingham City Hospital (UK). Two pathologists reviewed representative sections to identify areas for microdissection. Tumour epithelial cells were needle-microdissected and DNA was isolated. Sequencing libraries were prepared using a) a custom capture panel of 110 breast cancer-related genes or b) NEBNext Ultra II for low-coverage whole-genome sequencing (LC-WGS) depending on DNA quality and quantity. Sequencing was then performed on Illumina NextSeq 500 system with an average coverage of a) 400x and b) 1.3x, respectively. Data was processed by an in-house bioinformatics pipeline to detect and filter for high confidence somatic variants. Copy number profiles were obtained using CopywriteR (panel off-target reads), PureCN (panel on-target reads) and Control-FREEC (LC-WGS). Of 33 patients initially ascertained on the basis of a recurrent breast event, 30 had an ipsilateral recurrent tumour after 4.7 years on average, and 24 primary/recurrence pairs had sufficient data available to determine clonality. Eighteen pairs (75%) were classified as clonal by the following criteria: occurring in similar locations, showing a similar or higher tumour grade, and sharing ≥ 3 copy number breakpoints or likely somatic variants in the genes included in the panel. Six pairs (25%) did not meet this criteria and were classified as non-clonal. Preliminary data analysis shows that invasive recurrences (n = 12) were detected on average 3 years later than DCIS recurrences (n = 6), despite constant surveillance. The most commonly observed somatic mutations occurred in the TP53 and PIK3CA genes. Genetic variants in TP53 and PIK3CA were equally common in primary tumours with clonal and non-clonal recurrence. Frequent copy number alterations involved losses on 16q as well as gains on 1q, 8q and 17q, whereat the 8q gain was less profound. The presence of 25% non-clonal recurrences shows that differentiating between clonal and non-clonal recurrences might be crucial for further recurrence risk biomarker discovery. Thus, this project will proceed with comparing genetic variation in primary DCIS with clonal recurrence to DCIS cases without later recurrence. These samples are currently being obtained and analysed.

Citation Format: Dorothea Lesche, Hugo Saunders, Sakshi Mahale, Magnus Zenthoven, Siobhan Huges, Tanjina Kader, Niko Thio, Islam M Miligy, Jia-Min Pang, Andrew R Green, Emad A Rakha, Ian G Campbell, Stephen B Fox, Kylie Gorringe. Evaluation of genomic changes in ductal carcinoma in situ as potential biomarkers of recurrence risk [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-10-21.