Timed copy number changes and chromothriptic events in sample pairs

Chromosome regions significantly enriched for chromothriptic events and breast cancer drivers statistically overrepresented in such regions

Supplementary Figure S1. Effects of anti-estrogens on cell proliferation in MCF-7 #Wt, #TamR and #FulvR cells.

Supplementary Figure S6. Immunohistochemical stainings of ERRalpha in primary ERalpha-positive breast carcinomas on a tissue microarray (n=1041).

Overall chromothripsis prevalence (DKFZ-HIPO17, WES), link with clinical outcome and chromothripsis 
occurrence per chromosome for each breast cancer subtype

AbstractChromothripsis is a form of genome instability by which a presumably single catastrophic event generates extensive genomic rearrangements of one or a few chromosomes. Widely assumed to be an early event in tumor development, this phenomenon plays a prominent role in tumor onset. In this study, an analysis of chromothripsis in 252 human breast cancers from two patient cohorts (149 metastatic breast cancers, 63 untreated primary tumors, 29 local relapses, and 11 longitudinal pairs) using whole-genome and whole-exome sequencing reveals that chromothripsis affects a substantial proportion of human breast cancers, with a prevalence over 60% in a cohort of metastatic cases and 25% in a cohort comprising predominantly luminal breast cancers. In the vast majority of cases, multiple chromosomes per tumor were affected, with most chromothriptic events on chromosomes 11 and 17 including, among other significantly altered drivers, CCND1, ERBB2, CDK12, and BRCA1. Importantly, chromothripsis generated recurrent fusions that drove tumor development. Chromothripsis-related rearrangements were linked with univocal mutational signatures, with clusters of point mutations due to kataegis in close proximity to the genomic breakpoints and with the activation of specific signaling pathways. Analyzing the temporal order of events in tumors with and without chromothripsis as well as longitudinal analysis of chromothriptic patterns in tumor pairs offered important insights into the role of chromothriptic chromosomes in tumor evolution.Significance:These findings identify chromothripsis as a major driving event in human breast cancer.

Supplementary Figure S2. Chromatin immunoprecipitation (ChIP).

Description of the patient cohorts and available data

Copy-number plots and rainfall plots for the 11 patients with two tumors from the DKFZ-HIPO17 cohort

Chromothripsis scoring by visual and algorithm-based approaches

Chromosome regions with significant differences in copy-number gains and losses between cases with and without chromothripsis

Supplementary Material and Methods, Figure Legends S1-6. Supplementary Material and Methods, Figure Legends S1-6.

Cancer-associated fibroblasts (CAFs) are a diverse cell population within the tumour microenvironment, where they have critical effects on tumour evolution and patient prognosis. To define CAF phenotypes, we analyse a single-cell RNA sequencing (scRNA-seq) dataset of over 16,000 stromal cells from tumours of 14 breast cancer patients, based on which we define and functionally annotate nine CAF phenotypes and one class of pericytes. We validate this classification system in four additional cancer types and use highly multiplexed imaging mass cytometry on matched breast cancer samples to confirm our defined CAF phenotypes at the protein level and to analyse their spatial distribution within tumours. This general CAF classification scheme will allow comparison of CAF phenotypes across studies, facilitate analysis of their functional roles, and potentially guide development of new treatment strategies in the future.

Supplementary Figure S3. ChIP-Seq validation by qRT-PCR.

Legends of Supplementary Figures and Tables

Supplementary Figure S4. AIB1 in tamoxifen- and fulvestrant-resistant breast cancer cells.

Differentially expressed genes between cases with and without chromothripsis

Supplementary Figure S5. Co-administration of anti-estrogens upon lentiviral-mediated ERRα knockdown.

Copy-number differences between cases with and without chromothripsis in the CATCH cohort

Unsupervised clustering analyses of RNAseq data for ER+, HER- liver metastases of the CATCH cohort (a) and luminal tumors from the DKFZ-HIPO17 cohort (b)