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CLONAL AND CELLULAR HETEROGENEITY OF BREAST CANCER AND ITS DYNAMIC EVOLUTION WITH TREATMENT

Periodic Reporting for period 2 - CLONCELLBREAST (CLONAL AND CELLULAR HETEROGENEITY OF BREAST CANCER AND ITS DYNAMIC EVOLUTION WITH TREATMENT)

Reporting period: 2018-07-01 to 2019-12-31

Breast cancer remains one of the leading causes of cancer death in women. One of the greatest challenges is that breast cancer is a heterogeneous group of 11 diseases defined by genomic profiling. In addition, each tumour is composed of clones and clonal evolution underpins the successive acquisition of the hallmarks of cancer, including metastasis and resistance to therapy. Furthermore tumours display biologically and clinically relevant cellular heterogeneity: immune system, vasculature, and stroma. This cellular heterogeneity both shapes and is shaped by the malignant compartment and modulates response to therapy.
This project is using longitudinal studies to unravel the clonal and cellular heterogeneity of breast cancer and its dynamic evolution with treatment. The overall goal is to provide a systems level view of evolving clonal and cellular architectures in space and time along the clinical continuum of breast cancers in the clinic, leading to the discovery of new biological and clinical paradigms which will transform our understanding of the disease.
The overall approach is to capture the evolution of clonal and cellular heterogeneity of breast cancers in space and time using unique clinical cohorts where samples (biopsies and blood/plasma) are available spanning the whole disease continuum: early breast cancer surgically treated with curative intent, neo-adjuvant therapy, and matched relapse/metastasis. The 4 aims of the project are:
1. Characterization of the clonal and cellular heterogeneity of primary tumours from the 11 genomic driver-based breast cancer subtypes (ICs)
2. Comparative characterization of the clonal and cellular heterogeneity of matched pairs of primary and metastatic cancers
3. Characterization of the clonal and epigenetic evolution across therapy courses
4. Characterization of the immune response across therapy courses
The project was activated on October 1st 2016 and in these 3 years we have made significant progress across all the aims:
1. Characterization of the clonal and cellular heterogeneity of primary tumours from the 10 (11) genomic driver-based breast cancer subtypes (ICs)
We have optimised a single cell DNA copy number profiling protocol where we are able to shallow whole-genome sequence a single nuclei for under £10. We have processed 177 tumours of which 104 tumours yielded a minimum of 100 informative tumour nuclei. Of the different Integrative Clusters (IC), we have achieved the minimum 10 tumours per IC for 5 out of the 11 IC and are currently processing tumours to fulfil the remaining IC. We have generated an analysis pipeline that performs QC to remove poor quality and non-tumour nuclei, followed by absolute copy number calls and segmentation. The data so far has confirmed our original hypothesis: the clonal architecture of the different ICs is distinct and appears to be prototypical of each IC. This result is a major breakthrough and once data analysis is completed this will result in a major manuscript.
We have optimised an Imaging Mass Cytometry protocol to perform TME profiling of the same tumours. We have also completed exome sequencing and RNA sequencing of all the same tumours. This will allow a directr comparison of clonal architecture and TME.
2. Comparative characterization of the clonal and cellular heterogeneity of matched pairs of primary and metastatic cancers
We have collected a series of matched primary and metastatic fresh frozen samples (to date >25 pairs) and whole exome sequencing and RNA sequencing of these has been completed. Crucially for 10 of these cases we have generated patient-derived xenografts and will be able to study clonal dynamics of both the patient and matched explant models that are serially passaged in the laboratory.
3. Characterization of the clonal and epigenetic evolution across therapy courses
We have completed exome sequencing and RNA sequencing of all the samples from TransNEO (168 cases) and from ARTEMIS (123 cases). Data analysis for the baseline pre-treatment core biopsy has been completed and a first manuscript describing predictive biomarkers is under preparation. The main predictive biomarkers identified are: tumour mutation burden, predicted neo-antigen load, BRCA/APOBEC mutation signatures, mutations in TPO53 and PIK3CA, HLA-LOH, increased proliferation and immune activation expression signatures, higher HLA-I expression and lower expression of CYP.
The most important feature of these studies is the availability of serial biopsies (TransNEO: 116 patients; ARTEMIS: 91 patients) allowing the study of clonal dynamics. Analysis of this data is currently ongoing, but preliminary outputs reveal different patterns of clonal dynamics correlated with sensitivity/resistance. This finding indicates that clonal architectures are modulated by therapy and this modulation profoundly impacts response to treatment.
Uniquely we have generated xenograft models from both primary tumours, metastases and core biopsies and we sue these in co-clinical trials. The advantage of these models is that samples are no longer limiting allowing extensive single cell analysis. We have now characterized 50 of these xenograft models using both single cell mass cytometry (cyTOF) and imaging mass cytometry (which is the tissue section equivalent). A manuscript has been submitted describing the mass cytometry and genomics characterization of the models. Ongoing co-clinical trials from models derived from core biopsies have confirmed that remarkably the response in xenopatients mimics the clinical response in the matched human patient donor. We are now characterizing the clonal and cellular dynamics of matched explants/human samples using a combination of whole genome/exome sequencing, bulk and single cell RNA sequencing. These studies have already yielded preliminary data that indicate that resistance
The project is on course to deliver what is set out to do:
1- Characterize the prototypical clonal architectures and ecosystems of breast cancer genomic subtypes.
2- Unravel the genomic architecture of metastatic progression.
3- Characterize the clonal and TME dynamics across therapy courses and how this will inform our understating of drug sensitivity/resistance.
4- Unravel at single cell level the genomic and tissue architecture of human breast cancer.
5- Define how tumour explant models mimic the disease in the human host.
Schematic of TransNEO trial