Mutations targeting the coagulation pathway are enriched in brain metastases

Scientific Reports, Jul 2017

Brain metastases (BMs) are the most common malignancy of the central nervous system. Recently it has been demonstrated that plasminogen activator inhibitor serpins promote brain metastatic colonization, suggesting that mutations in serpins or other members of the coagulation cascade can provide critical advantages during BM formation. We performed whole-exome sequencing on matched samples of breast cancer and BMs and found mutations in the coagulation pathway genes in 5 out of 10 BM samples. We then investigated the mutational status of 33 genes belonging to the coagulation cascade in a panel of 29 BMs and we identified 56 Single Nucleotide Variants (SNVs). The frequency of gene mutations of the pathway was significantly higher in BMs than in primary tumours, and SERPINI1 was the most frequently mutated gene in BMs. These findings provide direction in the development of new strategies for the treatment of BMs.

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Mutations targeting the coagulation pathway are enriched in brain metastases

Abstract Brain metastases (BMs) are the most common malignancy of the central nervous system. Recently it has been demonstrated that plasminogen activator inhibitor serpins promote brain metastatic colonization, suggesting that mutations in serpins or other members of the coagulation cascade can provide critical advantages during BM formation. We performed whole-exome sequencing on matched samples of breast cancer and BMs and found mutations in the coagulation pathway genes in 5 out of 10 BM samples. We then investigated the mutational status of 33 genes belonging to the coagulation cascade in a panel of 29 BMs and we identified 56 Single Nucleotide Variants (SNVs). The frequency of gene mutations of the pathway was significantly higher in BMs than in primary tumours, and SERPINI1 was the most frequently mutated gene in BMs. These findings provide direction in the development of new strategies for the treatment of BMs. Introduction Brain metastases (BMs) are the most common malignancy of the central nervous system, and their incidence is dramatically increasing due to the improvements in overall survival of primary tumour patients and failure of systemic chemotherapy to reach the central nervous system1. Mainly, BMs derive from primary tumours originating in the breast, lung, skin, gastrointestinal tract and kidney2, 3. Breast cancer (BC) is a heterogeneous disease that remains the second leading cause of death among women worldwide4: up to 16% of patients with metastatic BC develop symptomatic BMs during the course of their disease, and another 10% of patients have asymptomatic brain involvement in post-mortem autopsies5, 6. Because of improvements in the treatment of patients with metastatic breast cancer, the development of brain metastases has become a major impediment to improved life expectancy and quality of life for many breast cancer patients. The median survival time is very short, ranging from 2 to 25 months despite treatment7,8,9,10. In addition, the intra-tumour and inter-tumour heterogeneity of BC influences the clinical course of the disease and the response to treatment. Although some gene expression signatures have been previously associated with metastasis to the brain11, 12, the driver mutations that lead to the formation of BM are still largely unknown. Although the frequency of these mutations is fairly low, the metastatic spread is sustained by gene mutations conferring selective growth advantage to target cells13,14,15. Large-scale genome sequencing of human tumours found little evidence for specific metastasis-associated mutations other than mutations in classic initiator oncogenes (driver genes) that are also present in primary tumours and enriched in metastatic lesions16. We therefore searched for metastasis-associated mutations in genes that belong to the same pathway but do not fall in recognized oncogenes. Results and Discussion All the surgical specimens employed in this study were collected from consenting patients at Istituto Neurologico “Carlo Besta”, Milan, Italy, European Institute of Oncology, Milan, Italy and Erasmus University Medical Center, Rotterdam, The Netherlands. We performed whole-exome sequencing (WES) (Illumina Hiseq. 2000 platform with 101 bp paired-end reads) of primary breast tumour and BMs from 10 patients (Supplementary Table S1a). For 7 patients we could perform WES of the matched normal brain tissue while for 3 patients (S_2, S_4 and S_9) the normal sample was unavailable. To identify metastasis-specific mutations, we compared each metastasis sample to the corresponding tumour DNA and to the normal DNA, when available. We identified somatic mutations, including single nucleotide variants (SNVs, using MuTect17) and small insertions and deletions (indels, using Indelocator), present in each primary BC and in the corresponding metastasis. Since we analysed somatic mutations in multiple samples for the same patient, to increase sensitivity in mutation-calling we merged the mutations identified in each sample (i.e. SNVs and indels called in the metastasis sample and tumour sample compared to the matched normal sample), and we counted the number of alternative and reference reads supporting the mutations in all the samples. Duplicated reads were not considered in this calculation. In addition, we selected variants that were present only in the metastasis, or with a variant allele frequency (VAF) significantly higher in the metastasis compared to the matched primary tumour (Fisher’s exact test corrected for FDR, values in Supplementary Table S2). The availability of normal samples allowed us to assess which somatic mutations were present in both primary and metastasis samples at higher frequency in the metastasis compared to the primary sample. Considering non-synonymous SNVs occurring with an allelic frequency of at least 5% (corresponding to a fraction of mutated cells of 10% or more, assuming the majority of mutations to be heterozygous), we i (...truncated)


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Cristina Richichi, Lorenzo Fornasari, Giorgio E. M. Melloni, Paola Brescia, Monica Patanè, Massimiliano Del Bene, Dana A. M. Mustafa, Johan M. Kros, Bianca Pollo, Giancarlo Pruneri, Angela Sciandivasci, Elisabetta Munzone, Francesco DiMeco, Pier Giuseppe Pelicci, Laura Riva, Giuliana Pelicci. Mutations targeting the coagulation pathway are enriched in brain metastases, Scientific Reports, 2017, Issue: 7, DOI: 10.1038/s41598-017-06811-x