The clinical management of breast cancer patients is complicated by the high genetic heterogeneity of this disease, which makes the standardization of treatments, the prediction of prognosis and therapy response, and the development of personalized therapies difficult. Nevertheless, the advent of high-throughput genomics screenings based on microarray or next-generation sequencing (NGS) technologies has greatly enhanced our understanding of the genomic landscapes underlying breast cancer development and progression. Such discoveries are now allowing clinicians to tailor therapies based on the molecular subtype of the tumour (luminal, basal and HER2). NGS studies have also started to provide insights into the range of molecular profiles of tumour cells from the same tumour, and have shown that in some breast cancers a high level of intratumoral genetic heterogeneity exists. The findings from these studies support a scenario in which breast tumours can be either: i) monogenomic, comprised of a single clonal cell population; ii) or polygenomic, composed of several related clonal subpopulations. The co-existence of different cancer driver genetic lesions in polygenomic tumours might contribute to treatment failure in some cases, as relapse could be driven by the expansion of a subpopulation of cells intrinsically resistant to the therapy. Importantly, cancer genetic heterogeneity has been recapitulated in experimental settings using cancer stem cells (CSCs) xenografted in mouse models. We hypothesized that the mutational events that drive the onset and progression of breast tumours lie within the CSC compartment. To explore this possibility, we analysed and compared the mutational profiles of a primary breast tumour and its matched mammospheres (source of CSC-derived population), patient-derived xenograft (PDX) and PDX-derived mammospheres using Whole Exome Sequencing (WES). We setup a NGS approach to look for rare mutations in the primary tumour that may be present in the CSC compartment using low amounts of DNA input. We optimised an experimental protocol in which the genomic DNA (gDNA) of each sample was subjected to Whole Genome Amplification (WGA) prior to performing WES. This enabled us to obtain a sufficient amount of DNA (≥ 3 μg) to perform WES. We also introduced a filtering step in our analysis, based on the Xenome software, for PDX-derived samples to eliminate possible contamination from murine DNA. Our study allowed us to characterize the genetic profiles of CSCs and to identify cancer-relevant mutations that could drive breast cancer onset and progression. We identified 15 candidate driver mutations in 11 genes that were enriched, in terms of mutation frequency, within primary tumour-derived mammospheres and the PDX. Together with these mutations, we identified 4 mutations in 4 genes, not enriched, but shared among all analysed samples, which likely represent “founder” mutations. Based on our results, we will now endeavour to determine the clinical relevance of the candidate driver mutations identified in our study by determining their prevalence in independent patient cohorts. Having optimised the protocol for NGS of matched primary tumour, PDX and mammosphere populations, we will also extend our mutational analysis to additional breast tumours for the identification of more driver mutations and for the deconvolution of intratumoral genetic heterogeneity of breast cancer. Understanding the driving mutational forces of breast tumours and relative mechanisms involved is paramount for the development of more effective therapeutic strategies.

A NOVEL APPROACH FOR THE IDENTIFICATION OF CANDIDATE DRIVER LESIONS IN BREAST CANCER BASED ON THE COMPARISON OF THE MUTATIONAL PROFILES OF A PRIMARY TUMOUR AND ITS MATCHED MAMMOSPHERES AND XENOGRAFT / V. Melocchi ; added supervisors: S. Pece, F. Bianchi ; supervisor: P. P. Di Fiore. DIPARTIMENTO DI ONCOLOGIA ED EMATO-ONCOLOGIA, 2017 Mar 02. 28. ciclo, Anno Accademico 2016. [10.13130/v-melocchi_phd2017-03-02].

A NOVEL APPROACH FOR THE IDENTIFICATION OF CANDIDATE DRIVER LESIONS IN BREAST CANCER BASED ON THE COMPARISON OF THE MUTATIONAL PROFILES OF A PRIMARY TUMOUR AND ITS MATCHED MAMMOSPHERES AND XENOGRAFT.

V. Melocchi
2017

Abstract

The clinical management of breast cancer patients is complicated by the high genetic heterogeneity of this disease, which makes the standardization of treatments, the prediction of prognosis and therapy response, and the development of personalized therapies difficult. Nevertheless, the advent of high-throughput genomics screenings based on microarray or next-generation sequencing (NGS) technologies has greatly enhanced our understanding of the genomic landscapes underlying breast cancer development and progression. Such discoveries are now allowing clinicians to tailor therapies based on the molecular subtype of the tumour (luminal, basal and HER2). NGS studies have also started to provide insights into the range of molecular profiles of tumour cells from the same tumour, and have shown that in some breast cancers a high level of intratumoral genetic heterogeneity exists. The findings from these studies support a scenario in which breast tumours can be either: i) monogenomic, comprised of a single clonal cell population; ii) or polygenomic, composed of several related clonal subpopulations. The co-existence of different cancer driver genetic lesions in polygenomic tumours might contribute to treatment failure in some cases, as relapse could be driven by the expansion of a subpopulation of cells intrinsically resistant to the therapy. Importantly, cancer genetic heterogeneity has been recapitulated in experimental settings using cancer stem cells (CSCs) xenografted in mouse models. We hypothesized that the mutational events that drive the onset and progression of breast tumours lie within the CSC compartment. To explore this possibility, we analysed and compared the mutational profiles of a primary breast tumour and its matched mammospheres (source of CSC-derived population), patient-derived xenograft (PDX) and PDX-derived mammospheres using Whole Exome Sequencing (WES). We setup a NGS approach to look for rare mutations in the primary tumour that may be present in the CSC compartment using low amounts of DNA input. We optimised an experimental protocol in which the genomic DNA (gDNA) of each sample was subjected to Whole Genome Amplification (WGA) prior to performing WES. This enabled us to obtain a sufficient amount of DNA (≥ 3 μg) to perform WES. We also introduced a filtering step in our analysis, based on the Xenome software, for PDX-derived samples to eliminate possible contamination from murine DNA. Our study allowed us to characterize the genetic profiles of CSCs and to identify cancer-relevant mutations that could drive breast cancer onset and progression. We identified 15 candidate driver mutations in 11 genes that were enriched, in terms of mutation frequency, within primary tumour-derived mammospheres and the PDX. Together with these mutations, we identified 4 mutations in 4 genes, not enriched, but shared among all analysed samples, which likely represent “founder” mutations. Based on our results, we will now endeavour to determine the clinical relevance of the candidate driver mutations identified in our study by determining their prevalence in independent patient cohorts. Having optimised the protocol for NGS of matched primary tumour, PDX and mammosphere populations, we will also extend our mutational analysis to additional breast tumours for the identification of more driver mutations and for the deconvolution of intratumoral genetic heterogeneity of breast cancer. Understanding the driving mutational forces of breast tumours and relative mechanisms involved is paramount for the development of more effective therapeutic strategies.
2-mar-2017
Settore MED/04 - Patologia Generale
Next-generation sequencing; breast cancer; cancer stem cells; xenograft; whole exome sequencing
DI FIORE, PIER PAOLO
PECE, SALVATORE
Doctoral Thesis
A NOVEL APPROACH FOR THE IDENTIFICATION OF CANDIDATE DRIVER LESIONS IN BREAST CANCER BASED ON THE COMPARISON OF THE MUTATIONAL PROFILES OF A PRIMARY TUMOUR AND ITS MATCHED MAMMOSPHERES AND XENOGRAFT / V. Melocchi ; added supervisors: S. Pece, F. Bianchi ; supervisor: P. P. Di Fiore. DIPARTIMENTO DI ONCOLOGIA ED EMATO-ONCOLOGIA, 2017 Mar 02. 28. ciclo, Anno Accademico 2016. [10.13130/v-melocchi_phd2017-03-02].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/468649
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