New molecular insights based on genetic alterations in gynecological cancers.

Cancer develops due to the accumulation of genetic alterations and new insights in these alterations can lead to a better understanding of cancer biology, diagnosis and therapy. Different characteristics between tumors, depending on the site of origin are well established in terms of genomic landscape, prognosis and response to therapies. Therefore, the site of origin is currently the decision-making factor for therapies. Next-generation sequencing (NGS) has revolutionized the exploration of genetic abnormalities in cancer and led to multiple studies collecting large sets of tumor samples to perform different NGS technologies. These studies showed significant molecular differences between tumors from the same tissue of origin (inter-tumor heterogeneity). The choice of therapy and prediction of prognosis is therefore evolving towards a classification where molecular alterations are gaining more importance. Endometrial Cancer (EC) is one of the cancers that has hugely benefited from these large-scale genomic analyses. Indeed, the Cancer Genome Atlas (TCGA) identified a genome-based classification of ECs with 4 molecular subgroups: (1) ultra-mutated (polymerase epsilon [POLE] mutated), (2) hyper-mutated (microsatellite instability; MSI), (3) copy-number low (non-specific molecular profile; NSMP), and (4) copy-number high (TP53-mutant) subgroups. Two studies have proposed models to classify ECs according to these 4 molecular subgroups, using simplified and more clinically applicable surrogate methodologies. In several hospitals across the world, including our own academic hospital, this classification has recently been implemented in the routine clinical care. The POLE-mutant, MSI and TP53-mutant tumors are characterized by specific molecular characteristics. A major challenge however, is to identify new therapeutic targets and biomarkers for the NSMP subgroup, since this is the largest subgroup containing 40% of all EC patients. In a first part of this manuscript, we aimed at refining the classification of this subgroup and identifying new therapeutic targets by profiling somatic copy-number aberrations (SCNAs). Both in the discovery and validation cohort, 1q32.1 amplifications were associated with a significantly worse prognosis in NSMP ECs. Interestingly, a new molecular classification including 1q32.1 amplifications improved risk prediction of recurrence. MDM4 gene expression strongly correlated with 1q32.1 amplification and silencing MDM4 inhibited cell growth in cell lines carrying 1q32.1 amplification, but not in those without MDM4 amplification. Vice versa, increasing MDM4 expression in non-amplified cell lines stimulated cell proliferation. 1q32.1 amplification was thus identified as a prognostic marker for poorly-characterized NSMP ECs, refining the molecular classification of this subgroup. Moreover, MDM4 was functionally validated as a potential oncogenic driver in the 1q32.1 region. Apart from the heterogeneity in tumors with the tissue of origin, next generation sequencing also revealed a very heterogeneous genetic profile of tumor cells within the same patient (intra-tumor heterogeneity). This has implications for predictive or prognostic biomarker strategies, where a previously undetected tumor subclone may influence therapeutic outcome. Interestingly, this intra-tumor heterogeneity can be used to reconstruct phylogenetic trees that reflect the evolution of metastatic tumors throughout the body. In a second part of this manuscript, we used SCNA data to construct the 'tree of life' of ovarian cancer cells to unravel the metastatic history of patients with bilateral high-grade serous ovarian cancer (HGSOC). This is particularly relevant for this cancer type, since the high mortality of HGSOC is mainly caused by the high percentage (>70%) of patients with metastatic spread within the peritoneal cavity at primary diagnosis. Despite the high prevalence and poor prognosis of HGSOC, the dynamics of metastatic cells remains largely unexplored. Phylogenetic reconstruction of 6 patients including 81 samples revealed a passive dissemination mechanism within the peritoneal cavity carried by the physiological clock-wise movement of peritoneal fluid. Moreover, this study is the first to unravel the dynamics between the two ovaries in patients with bilateral HGSOC. When the tumor initiates at the left ovary, tumor cells disseminate early to the right ovary and later to distant organs. In patients with the primary tumor at the right ovary, tumor cells first disseminate to distant organs and later to the left ovary. Finally, potential therapies need to be tested in pre-clinical models. However, most pre-clinical studies use cell lines that are cultured long term in vitro. This results in the selection of tumor cells with in vitro growth advantage that inadequately reflects the true intra-tumor heterogeneity. Moreover, the amount of cell lines is limited, resulting in a poor representation of the inter-tumor heterogeneity. Therefore, patient-derived tumor xenograft (PDTX) models, established by implanting surgically removed tumor tissue from different patients directly in immunocompromised mice, are expected to fill the gap between preclinical and clinical research. Since there is no in vitro growth, these PDTX models better reflect the true biology the disease. Therefore, in the last part of this manuscript, we characterized a panel of EC PDTX models, both histologically and genetically, to assess their value as pre-clinical models. Collectively, these data showed that such models closely resemble the original tumors in the patients in terms of tissue architecture and genomic features. Moreover, these models have a strong potential for both fundamental and translational studies.status: publishe