Abstract P4-05-05: A high-sensitivity molecular profiling approach to spatially characterize archived pre-cancer lesions
Authors: Nachamanson Daniela , Steward Joseph , Kim Taeyong , Yao Huazhen , Jeong Eliza , O'Keefe Thomas , Hirst Gillian L. , Hasteh Farnaz , Jepsen Kristen , Esserman Laura J. , Borowsky Alexander , Harismendy Olivier 
Published: March 2020

Background: The widespread adoption of breast cancer screening has increased the detection of pre-malignant lesions such as ductal carcinoma in situ (DCIS) but has not translated into increased survival rates. There are currently no reliable biomarkers to stratify DCIS into those which would not develop into an aggressive invasive cancer from those that do, and avoid over-treatment. The interaction with the DCIS microenvironment has likely been under-appreciated or not studied in the adequate clinical context. Importantly, the identification of novel biomarkers in DCIS is notoriously difficult. The rare and late recurrence to subsequent breast cancer (10% at 10 years) poses challenges in experimental design that only a few established, retrospective studies can solve. Most tissue specimens in the associated registries are small, fixed and archived in paraffin (FFPE), precluding their characterization via standard genomic assays. Here, we present an approach combining laser capture microdissection (LCM) with state-of-the-art genomic library preparation methods to enable molecular profiling from minute amounts of damaged nucleic acids and demonstrate its validity for the spatial molecular characterization of DCIS specimen. Methods: Targeted DNA sequencing (whole exome and cancer gene panel) was performed on limited dilutions (200, 50, 10,3 ng) of a test DNA from archived breast tissue, or validation DNA from 10 LCM regions from 3 DCIS specimens. Three library preparation strategies were evaluated, included adaptation of classic A-tailing (AT), or alternate single-strand (SS) or blunt-end (BE) adapter ligations. The expression profiling was performed using SMART-3SEQ protocol on 15 LCM regions from 8 DCIS specimen.

Results: The use of an FFPE-optimized hybrid-capture AT protocol resulted in poor coverage performance (7% of targeted base pairs covered at ≥20x) from test DNA inputs lower than 50 ng and suggested the ligation of the sequencing adapter as the initial bottleneck. Alternatively steps using SS or BE adapter ligation from 3 ng of test DNA (~500 cells) increased the library complexity, reducing the fraction of duplicate reads and increasing the fraction of targeted base pairs covered at 20x to 49% and 100% respectively. Artifactual cytosine to tyrosine substitutions, a signature of formalin damage, were restricted to variants of low allelic fraction (&lt;5%). As a result, the use of molecular barcodes, ensemble variant calling and heuristic variant filtering, resulted in 98% precision and 96% recall in variant calling accuracy from 3ng input, when compared to 200 ng input from a fresh frozen mirrored specimen. Similarly, the presence of an ERBB2 amplification was validated in all tested conditions. The validation of the BE approach on 10 LCM regions from 3 DCIS showed decreasing levels of clonal relationships when comparing regions from adjacent sections, then from matching distinct regions, then from distinct specimen. The heterogeneity of TP53 loss of heterozygosity within a specimen was consistent with high-grade. Furthermore, gene expression profiling of 15 regions from 8 archived DCIS specimen differentiated expression profiles of stromal and epithelial regions, within and between specimen, highlighting sets of genes associated with DCIS subtype and the immune make-up of the micro-environment. Conclusion: The proposed approach - from coordinated specimen preparation to mutational and expression profiling - was therefore validated to support the spatial characterization of pre-cancerous lesions and the microenvironment in which they evolve. Compatible with a limited quantity of tissue from archived specimens, the approach sets a new paradigm for the retrospective molecular characterization of specimen with longitudinal follow-up and associated long-term outcome.

Citation Format: Daniela Nachamanson, Joseph Steward, Taeyong Kim, Huazhen Yao, Eliza Jeong, Thomas O'Keefe, Gillian L. Hirst, Farnaz Hasteh, Kristen Jepsen, Laura J. Esserman, Alexander Borowsky, Olivier Harismendy. A high-sensitivity molecular profiling approach to spatially characterize archived pre-cancer lesions [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 P4-05-05.