Title: Intratumour heterogeneity, clonal selection and metastatic propensity in breast cancer

Project Coordinator:
Per Eysten LONNING (Norway) Department of Clinical Science, Faculty of Medicine and Dentistry, University of Bergen

Project Partners:
Fabrice ANDRE (France) Department of Medical Oncology, Institut Gustave Roussy, Villejuif
Christos SOTIRIOU (Belgium) Breast Cancer Translational Research Laboratory J-C Heuson - Université Libre de Bruxelles, Faculty of Medicine, Jules Bordet Institute / Brussels
Dimitris A: MAVROUDIS (Greece) Department of medical oncology, University Hospital of Heraklion, Heraklion

Project Abstract:
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.
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.
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.
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.

Final summary:
Axillary lymph node (ALN) metastases play a central role in the management of BC as they have been shown to be the most important factor predicting distant relapse. One may envision that rare sub-clones in the primary tumour, capable of metastasizing, are disseminated to the axilla where they expand. However, little is known of the genetic alterations that characterize ALNs. In the present project, we hypothesize that ALN metastases are a surrogate of intra-tumor heterogeneity at the primary site and could represent a more representative picture of the alterations characterizing heterogeneous primary BC. By using state of the art next generation sequencing technologies we aim to gather a broader knowledge of the genetic alterations of ALN metastases and matched primary tumours, in order to assess the added value of characterizing ALN in the context of inta-trumor heterogeneity and molecularly targeted therapies.
Results from the project has suffered delays, much due to temporary facility close-downs because of the Covid-19 situation. However, we have completed sample collection and performed high quality DNA extraction from breast cancer belonging to two main subgroups; tumours associated with a high number of lymph metastatic deposits, from which both the primary tumour and corresponding lymph nodes have been processed, and a control group of node negative tumours. In total, samples from 93 patients have been included in the project (49 ALN positive and 44 ALN negative). Multiple regions from the primary tumours have been samples as well as a total of 98 ALNs from the 49 ALN positive patients, mounting to a total of 372 samples. These samples have successfully undergone whole exome sequencing (WES) and as such, all wet-lab analyses are completed.
The generated data is currently undergoing extensive bioinformatics analyses. We envison the results to be of significant importance to our understanding of the prognostic role of lymph node metastases (node numbers) to breast cancer prognosis. If it turns out that this may be related to intra-tumour clonal diversity, as hypothesised, we envision it may pave the way for e better rational understanding the metastatic process in breast cancer in general and, as such, improve therapy and outcome further.

(Project funded under JTC 2014)