Through the comparison and meta-analysis of CIN signatures in multiple EC datasets, our study demonstrated that unfavorable prognostic factors of histopathology and molecular pathology, including poor differentiation, non-EEC, advanced disease, deep MI, advanced age, MSI and CNV-H, usually had aggravated CIN. A favorable prognostic factor, POLE mutation, and an unfavorable prognostic factor, CTNNB1 mutation, did not follow the above trend. The prognostic value of CIN signatures were well established in different adjuvant radiotherapy subgroups without POLE / CTNNB1 mutation and in CTNNB1 mutant patients from OB subgroup. An integrated risk model that combines pathology, CIN signatures and mutations was defined for improved prognostic refinement and better management of Stage Ⅰ EEC.
Most non-EECs are serous and high-grade cancers, which exactly have complex aneuploidies and polyploidy (32), hence CIN showed a consistent change in fields of histopathological type and tumor differentiation of EC (Fig. 1A ~ 1D). At least three potential mechanisms generated by CIN, including the induction of mesenchymal transition, the activation of STING pathway and immune evasion, may contribute to invasion and metastasis (11), which may explain the high CIN25 and CIN70 in Stage Ⅲ & Ⅳ patients and in patients with deep MI or aortic lymph node metastasis (Fig. 1E ~ 1G). Although we cannot verify the CIN status in LVSI-positive patients due to a lack of sufficient pathological information, we speculate that CIN may also increase in LVSI-positive cases since aneuploidy has been correlated with the LVSI of EC (25). Given the propensity for aging somatic cells to generate unstable chromosomes resulting from gene misexpression, telomeric attrition and senescence failure (33–35), the older EC patients were more prone to CIN enrichment (Fig. 1H ~ 1J).
Several well recognized molecular features of EC also had characteristic CIN. One of the final results of CIN is CNV (11). That is why we observed the lowest CIN signature expression in CNV-L and the highest expression in CNV-H (Fig. 2C). As for the moderate exacerbation of CIN in MSI patients, the fact that MSI causes some degree of genomic instability and the tendency for MSI to have aggressive phenotypes are two possible reasons (18–20, 29, 30). From CNV-L to MSI and then to CNV-H, as the CIN gradually becomes serious, the risk of recurrence gradually increases (Fig. 2B). In terms of MSI subtype itself, high CIN signatures were unfavorable prognostic factors (22). These two evidences, combined with the fact that CIN signatures did identify recurrent patients who belonged to different TCGA molecular subtypes in each adjuvant radiotherapy subgroup (Fig. 3B ~ 3F), implies that CNV-L, MSI, and CNV-H may be pooled together for prognosis evaluation by CIN.
Mutation of POLE causes impaired proofreading activity and DNA repair ability, followed by the poor fidelity of DNA replication and severe genomic instability (36, 37). This makes the CIN of POLE-mutant subtype was roughly the same as that of CNV-H (Fig. 2C). Unlike POLE mutation, however, why the mutation of CTNNB1 associated with a more stable chromosome status is not clear (Fig. 2D). Aberrant WNT/CTNNB1 pathway in colon cancer always induces CIN (38, 39), so the complete opposite relationship between CTNNB1 mutation and CIN in EC is confusing and interesting. Considering that patients with unstable chromosomes usually have poor clinical outcomes (26), how the aggravated CIN produces the excellent prognosis of POLE-mutant patients and the alleviated CIN leads to the poor outcomes of CTNNB1-mutant patients is another important issue worthy for further research (Fig. 2D ~ 2F; Tables 2 and 3). Serious CIN allows tumor to have different clonal selections in response to various biological stimuli and environmental stresses. However, this selective advantage also has a fitness cost of CIN, because the extremely excessive instability of chromosomes is not conducive to the stable survival of tumor cell itself (11, 14, 27, 40, 41). For this reason, besides the immune activation triggered by POLE-related mutations (42), severe CIN may contribute to the excellent prognosis of POLE-mutant cases. Similarly, CTNNB1-mutant cases, which benefit from the progression and proliferation caused by the activation of WNT/CTNNB1 signaling (6, 43), may protect cells from the adverse effects of this pathway activation with the help of the alleviated CIN. Although this conjecture is still to be confirmed by molecular biology, it may provide CIN-targeted therapeutic strategies for mutation-specific EC.
From these data and references, the inherent biological connections between CIN and different prognostic factors of EC suggest that CIN may be a common hallmark in the evolution of different clinicopathological and molecular features, which is the root cause for the success of our integrated risk model (Fig. 3 and Fig. 4). From the perspective of risk assessment, CIN signature, on the one hand, properly addressed the problems of heterogeneity and reproducibility in the conventional pathology system by the precise quantification of CIN status, thereby achieving prognostic refinement. “Multiple classifiers” who cannot be stratified by TCGA subtypes also be able to get accurate and reasonable risk assessments. On the other hand, the prognostic refinement achieved by CIN signatures existed in all adjuvant radiotherapy subgroups in the guidelines, which means that CIN may have more universal applications compared to other risk stratification systems such as TCGA subtypes, FGA and Aneuploidy Score. From a therapeutic point of view, high concordance of molecular alterations between curettage samples and hysterectomy specimens from EC suggested the potential for CIN signatures to guide surgical management (24, 44). More importantly, because the accurate risk stratification accomplished by CIN signature presupposed the adjuvant radiotherapy classification based on the guidelines, the treatment recommendations obtained from our integrated risk model may be an intact inheritance of and effective supplement to the indications for postoperative radiotherapy in the guidelines. In summary, the intrinsic relationships between CIN and clinicopathological or molecular features make CIN a bridge that comprehensively integrates histopathology and molecular pathology, which is difficult for other biomarkers to achieve.
To further refine our integrated risk model, we face two outstanding challenges. Firstly, the CIN signature should be optimized on the basis of CIN25 and CIN70. Different adjuvant radiotherapy subgroups in the guidelines have different clinicopathological and molecular features (Table 1), which leads to that the same CIN signature had different capabilities of risk assessment in different adjuvant radiotherapy subgroups. For the same adjuvant radiotherapy subgroup, different CIN signatures also have different risk assessment capabilities. Consequently, we believe that it is necessary to improve respective CIN signatures for the OB, OB & CTNNB1 mutation, VBT and EBRT EEC patients classified by the guidelines to realize the full potential of CIN. Secondly, the relationships between CIN and LVSI or several EC prognostic factors assessed by immunohistochemistry (45), such as L1CAM, ER and PR, remain to be explored. It is unclear whether these features are still independent prognostic factors in our integrated model. We look forward to high-quality retrospective studies with mature long-term follow-up data and large sample size that will meet these two challenges and provide a solid foundation for future clinical applications.