Genomic Analysis of Metastatic Breast Cancer: Exome sequencing of primary tumor, metastasis and circulating tumor DNA

Stephanie Kavan

Research output: ThesisPh.D. thesis

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Breast cancer is the most common invasive malignancy and the leading cause of cancer-related deaths among women. One of the biggest challenges in handling breast cancer is the extensive inter-and intra-tumoral heterogeneity resulting from a natural or therapeutic selection. Data obtained from various genomic profiling studies on primary tumors and matched metastases suggested different models of evolutionary patterns that differ in timing of metastasis-enabling genomic alterations and the degree of genomic concordance between progression states. Characterizing the evolutionary landscape of breast tumors can provide a biological understanding of tumor progression from primary cancers to dissemination and may be necessary for directing effective treatments. Currently, tissue biopsy is considered the gold standard for diagnosis and treatment guidance in breast cancer, although it may insufficiently represent the entire genomic landscape of a tumor. Multiple limitations of this technique have led to the proposal of liquid biopsies as an attractive complementary tool to tissue biopsies. In the form of circulating tumor DNA (ctDNA), liquid biopsy could potentially capture the inter-and intra-tumoral heterogeneity present in metastatic breast cancer and, through serial blood draws, track the clonal evolution of the cancer genome.

Study I is a literature review focused on intratumor and temporal genetic heterogeneity in breast cancer, emphasizing studies tracking clonal evolution by global analysis in multiple progression steps from the same patient. Additionally, we discussed the potential of plasma ctDNA compared to tissue biopsies from primary tumors and metastases for a complete overview of the molecular tumor landscape. Considering the low number of papers published in this part using global analysis, we also included studies using targeted sequencing approaches. Finally, we compared studies focusing on the relevance of genetic heterogeneity and clonal evolution in the clinical setting and discussed plasma circulating tumor DNA as a powerful real-time approach for monitoring the clonal landscape of cancer during treatment and recurrence.

Breast Cancer is a spatial and temporal dynamic disease where differently evolving genetic clones are responsible for progression and clinical outcome. Still, the impact of systemic treatment on clonal evolution and tumor heterogeneity is poorly understood. In Study II, we ought to map the repertoire of genetic alterations in systemically untreated breast cancer patients with de novo metastatic disease. We analyzed wholeexome sequencing data from the paired primary tumor and metastatic samples from three breast cancer patients who had not received systemic therapy yet. Point mutations, copy number alterations, potential driver genes, and mutational cancer cell fractions were identified using state-of-the-art bioinformatics methods. Genomic differences were observed between primary tumor and metastatic lesion, showing a high level of genetic divergence. All three patients followed the parallel progression model, with early monoclonal dissemination from the primary tumor followed by separate clonal evolution. Interestingly, metastasis-specific mutations involved genes EP300, APOBEC3B, KDM5C, ASXL1, and EPCAM, all associated with cancer migration and progression. These results were supported by pathway analysis, showing cancer-driving pathways are mainly significant in stem mutations present in both primary tumor and metastasis. Notably, affected pathways reflected the patient’s molecular subtype and metastasis location. Lastly, alterations specific to the metastasis affected driver genes involved in collagen formation, muscle contraction, and nuclear envelope depolymerizations supporting metastatic tumor cell migration, invasion, and genomic instability. 

The described patterns of evolution and the polyclonal nature of breast cancer have clinical consequences and should be considered during patient diagnosis and treatment selection. Current studies focusing on the relevance of clonal evolution in the clinical setting elucidate the role of liquid biopsy as a noninvasive biomarker for monitoring clonal progression and response to treatment. In the clinical setting, circulating tumor DNA may constitute ideal support for tumor biopsies to characterize the genetic landscape of metastatic disease. This might improve longitudinal monitoring of disease dynamics and treatment effectiveness to detect any residual tumor after resection, relapse, or metastasis within a particular patient.

In Study III, we performed copy number profiling and somatic mutation detection based on whole-exome sequencing of primary tumors, distant metastasis, and plasma circulating tumor DNA from eight metastatic breast cancer patients. Our data showed diverse patterns of tumor evolution. Although linear evolution with late dissemination of metastatic cells was detected in some cases, we mainly observed parallel evolution with early dissemination from primary tumors to distant sites. Comparing tissue biopsies with plasma samples, we detected variants mirroring primary tumor and/or metastasis, depending on the period between the progression steps. The old mutations from the early tumor clone dominate in plasma, followed by metastasis-specific mutations. However, different patterns were observed. The genomic discordance between the various stages of tumor evolution emphasizes the importance of molecular profiling of metastatic tissue and the possibilities of liquid biopsies for real-time tracking of tumor dynamics.
Original languageEnglish
Awarding Institution
  • University of Southern Denmark
  • Thomassen, Mads, Principal supervisor
  • Kruse, Torben Arvid, Co-supervisor
  • Hildebrandt, Malene Grubbe, Co-supervisor
  • Jylling, Anne Marie Bak, Co-supervisor
Date of defence13. Jun 2022
Publication statusPublished - 31. May 2022


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