br Fig Mutational signatures and homologous
Fig. 2. Mutational signatures and homologous recombination DNA repair defects in endometrial cancers of copy-number high (serous-like) molecular subtype. Mutational signatures of 60 endometrioid and serous endometrial carcinomas of copy-number high (serous-like) molecular subtype from The Cancer Genome Atlas (TCGA), sorted according to the proportion of mutational signatures 3 and 1. Mutational signatures are color-coded according to the legend on the bottom. Information on the primary and secondary signatures, total number of somatic mutations, large-scale state transition (LST) scores, bi-allelic homologous recombination DNA repair (HRD) gene mutations, histology and FIGO grade for endometrioid and mixed endometrial carcinomas are provided for each case on the right.
cancers (Supplementary Fig. S4). These data suggest that despite the high prevalence of TP53 mutations and high levels of copy number alterations in copy-number high (serous-like) ECs, the DNA repair defects and mutational processes involved in the tumorigenesis of the majority of copy-number high (serous-like) ECs may differ from those of HGSOCs and basal-like breast cancers.
3.4. Mutational signatures of uterine carcinosarcoma
Uterine carcinosarcomas are aggressive biphasic neoplasms with high-grade malignant epithelial and mesenchymal elements. We sought to define whether the mutational processes underlying uterine carcinosarcomas would differ from those of endometrioid and serous carcinomas. Mutational signature analysis of the 57 uterine carcinosar-comas from TCGA  revealed a Chloramphenicol aging-related signature 1 in 77% (44/57) of cases (Fig. 4). In addition, 4/57 (7%) uterine carcinosar-comas had a dominant mutational signature 3 associated with defective HRD, and 12/57 (21%) had a secondary signature 3; however, none of these harbored bi-allelic genomic alterations affecting HR-related genes . One uterine carcinosarcoma had a dominant POLE-related signature 10 and a P286R POLE hotspot EDM, and six cases had a dom-inant defective DNA MMR signature (signatures 6 and 15; Fig. 4). No as-sociations between histologic type (e.g. homologous/heterologous), histologic classification (e.g. endometrioid/serous), percentage of carci-noma/sarcoma present in the frozen tissue subjected to WES and the mutational signatures were found (Fig. 4).
Taken together, uterine carcinosarcomas not only harbor mutations found in both endometrioid and serous carcinomas, but also the muta-tional processes that shaped the genomes of uterine carcinosarcomas were similar to those found in endometrioid and serous carcinomas of copy-number low (endometrioid) and copy-number high (serous-like) subtypes.
3.5. Mutational signatures in the progression from primary to metastatic EC
To define whether the mutational spectra of ECs change with the progression from primary to metastatic disease, we retrieved WES data from 26 primary ECs (19 endometrioid, three serous, three carcino-sarcomas and one clear cell carcinoma) and 35 matched metastases from Gibson et al. . We performed mutational signature analysis of i) all mutations in a given primary tumor/metastasis, ii) mutations
shared between primary tumors and metastases, iii) mutations private to the primary tumor or iv) mutations private to the metastasis of a given case. We reasoned that these private mutations may stem from mutational processes playing a role in tumor maintenance and progres-sion. When assessing all mutations present in a given tumor/metastasis, we observed changes in the dominant mutational signature from pri-mary tumor to metastasis in eight of the 26 ECs (31%; Fig. 5); these in-cluded changes from a dominant aging-related signature 1 in the primary to a dominant DNA MMR-related signature 6 in the metastasis (e.g. EC10, EC27) or changes from a dominant POLE-related signature 10 in the primary to dominant signatures 1 and 6 in two and one metasta-ses of the same case, respectively (EC28; Fig. 5).
Signature analysis of shared vs private mutations was performed in the 13 cases with ≥20 shared/private mutations (n = 12 endometrioid, n = 1 clear cell histology), which revealed that distinct DNA repair mechanisms might play a role in the progression from primary EC to metastatic disease in a subset of cases. In the V411L POLE-mutant EC17, the mutations shared between the primary tumor and metastasis had a dominant POLE signature 10, whereas the dominant mutational processes underpinning the primary tumor and metastasis itself were MSI-related (signatures 6/15; Fig. 6). In this case EC17, a somatic MLH1 missense mutation not associated with loss of heterozygosity (LOH) of the wild-type allele was found in the shared mutations (root), whereas two additional MLH1, two MSH6 and one MSH2 muta-tions were found in the primary tumor only and additional MLH1 and MSH2 mutations in the metastasis only, suggesting that the somatic DNA MMR gene mutations in this tumor may be secondary to the ultramutator POLE EDM [30,32]. Furthermore, we observed a dominant aging mutational process (signature 1) in the shared mutations of EC4 and EC6 but the mutations private to the primary and matched metasta-tic tumors had dominant MSI-related signatures (signatures 6/15/20) and an increase in the fraction of small indels (Fig. 6, Supplementary Fig. S5). No genetic alterations in DNA MMR genes restricted to the mu-tations unique to the primary or metastatic tumors were found in these two cases; it is plausible that the MSI and shift in mutational signature may be due to methylation of MLH1 in these cases, a common event in endometrioid ECs . EC7, EC13, EC23 and EC34 displayed a dominant MSI signature 6 and a secondary signature 1 when assessing the shared mutations; in these cases, an even further increase in the proportion of different MSI signatures (6/15/20) and in the fraction of small indels was observed in the mutations private in the primary tumor and the metastases (Fig. 6, Supplementary Fig. S5).