Abstract 4861: Hybrid clustering methodologies to distinguish CNAs and/or SNVs that drive subclonal differentiation in samples from a breast cancer patient primary tumor and metastatic lymph nodes
Authors: Champion Mia D. , Francis Princy , Pockaj Barbara A. , Barrett Michael T. 
Published: August 2015

Abstract
Background: Next generation sequencing (NGS) has revealed that the genetic profiles of individual tumors are highly heterogeneous due to their subclonal composition. Tumor heterogeneity has profound clinical implications affecting differences in treatment response and therapeutic resistance.
Methods: An estrogen receptor positive, Her2 normal breast cancer patient underwent definitive surgical treatment with modified radical mastectomy. Multiple samples were obtained from the primary cancer and metastatic lymph nodes. Flow cytometry based methods were used to isolate and classify distinct cell subpopulations according to their ploidy, and samples were processed for whole exome sequencing (WES). Comparative Genomic Hybridization (CGH) methods were used to identify somatic unique copy-number alterations (CNAs). WES-Single Nucleotide Variations (SNVs) were used to infer subclonal phylogenetic relationships using Maximum Parsimony methods and annotated with variation-cluster-barcodes (vc-barcodes) generated using a variation Bayesian mixture model (VBMM) approach that focuses on copy-number neutral sequence variations for subclone identification (SciClone). Our comparative phylogenetic-VBMM clustering method was used to identify CNA and/or SNV drivers that are key in differentiating subclonal populations at multiple tumor sites.
Results: CNA-neutral WES-SNVs from 25 subclonal populations (isolated from 4 distinct sites) clustered into 6 major groups and were used to generate vc-barcodes. A phylogenetic reconstruction of subclonal architecture annotated with vc-barcode information expedited identification of key variations underlying the differentiation of subclones at distinct tumor sites. For example, we identified an amplification event involving the Anaplastic Lymphoma Kinase (ALK) gene that was more frequent in primary biopsy subclones (12/17, ∼71%) in comparison to metastatic subclones (2/6, ∼34%). As the ALK amplification is lost, we find that most of the metastatic subclones also acquire a predicted damaging mutation in this oncogene (4/6, ∼67%). In addition, we identified a potential driver metastatic cell lineage that carries the ALK amplification in the absence of the nonsynonymous mutation.
Conclusions: We developed a hybrid clustering methodology and used it to reconstruct the subclonal architecture in primary and metastatic tumors from a single breast cancer patient. Our methods have identified several CNAs and/or SNVs underlying subclonal differentiation, as well as potential biomarkers of disease progression and indicators of emerging resistance. Application of these methods to larger cohorts and types of tumors should be conducted to ascertain more precise estimates of the predictive accuracy of our processes.
Citation Format: Mia D. Champion, Princy Francis, Barbara A. Pockaj, Michael T. Barrett. Hybrid clustering methodologies to distinguish CNAs and/or SNVs that drive subclonal differentiation in samples from a breast cancer patient primary tumor and metastatic lymph nodes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4861. doi:10.1158/1538-7445.AM2015-4861