HetMet

Background and rationale.
Lymph node metastases is the most important parameter predicting recurrence and death in breast cancer. We aim at exploring the relationship between primary tumour heterogeneity, lymph node metastases and circulating tumour cells / plasma DNA in order to assess the correlations between intratumour heterogeneity and risk of relapse.
Hypothesis.
Tumour heterogeneity is a risk factor for lymph node and, subsequent, distant metastases, in breast cancer. We aim at identifying mechanisms explaining such potential correlations, including outlining the time dependency of metastatic clone development during primary tumour evolution.
Aims.
1. To identify the clonal relationships between the different lymph node metastases and different anatomical areas (periphery vs centre) of the same primary tumour.
2. To explore whether there is an association between the presence of primary tumour heterogeneity and the development, as well as the extent of, lymph node metastases.
3. To interrogate whether, the genomic aberrations that are identified in lymph nodes are also identified in matched synchronous plasma circulating tumor DNA and circulating tumour cells.
4. To identify potentially “druggable” genomic aberrations that are responsible for lymphatic and hematogeneous dissemination.
Methods.
We will collect tissue from primary tumours, metastatic nodes, circulating tumour cells and plasma DNA. Mutations will be identified by targeting sequencing (a 500 gene panel) to a depth of 500X. Further, we will perform high density SNP-arrays for all samples, enabling us to assess copy number alterations (CNAs) across the genomes of each sample.
Bioinformatics: Sequencing data will be processed by already established in-house informatics pipelines for mutation calling (based on BWA aligner and GATK mutation caller). Aberrant cell fraction and allele specific copy number assessments based on SNP-array data, will be performed using the ASCAT algoritmh. Heterogeneity within and between samples will be assessed by comparison of fractions of mutated cancer cells and copy number alterations through calculations and comparisons of pairwise Euclidean distance.
Expected results and potential impact:
1. We hope the results generated through the proposed program may outline the impact of primary tumour heterogeneity and lymph node metastases to such an extent that it will allow primary tumour analysis to replace lymph node dissection for diagnostic purposes in breast cancer in the future.
2. The results will add to our understanding of genetic markers characterizing early cell clones harbouring high metastatic propensity. This will aid identification of patients commonly defined as belonging to low-risk groups that will need adjuvant chemotherapy.
3. Finally, identification of genetic “drivers” in the metastatic process should lead to identification of targeted therapy towards cellular clones harbouring such gene defects.