In our cohort, approximately 1/10 endometrial cancer was a multiple classifier. The clinicopathological characteristics of MMRd-p53abn were intermediate between those of p53abn and MMRd for histotype, grade, and MMR proteins expression. POLEmut-p53abn resembled POLEmut in clinicopathological, molecular characteristics, and oncologic outcomes. Subclonal p53 expression was more frequent in multiple classifiers than in p53abn single classifiers.

This is the first attempt to compare clinicopathological and molecular characteristics between single and multiple classifiers in a cohort of consecutive patients. With the exception of León-Castillo et al, who analyzed 137 multiple classifiers and will be the focus of our discussion, other studies identified only 12 or fewer cases.16 23

León-Castillo et al described 64 MMRd-p53abn as predominantly endometrioid, high-grade, early-stage, without lymphovascular space invasion, and with a high proportion of MSH6±MSH2 or single PMS2 loss. They also revealed a higher frequency of multiple TP53 mutations and subclonal p53abn staining compared with single-classifier p53abn. For genomic features and survival outcomes, MMRd-p53abn was more similar to single-classifier MMRd than to p53abn. The 5-year recurrence-free survival of stage I MMRd-p53abn was significantly different from that of p53abn (92.2% vs 70.8%). However, they did not compare MMRd-p53abn survival with MMRd. These findings are consistent with our results, but we also found a difference in grade and histology between MMRd-p53abn and MMRd, which should be further analyzed in subsequent studies.

Our results on POLEmut-p53abn are in agreement with those of Leon-Castillo et al. They described 31 cases as mostly endometrioid, early-stage, high-grade, without lymphovascular space invasion, and characterized by p53 subclonality and multiple TP53 mutations. POLEmut-p53abn clustered mainly with POLEmut rather than p53abn. Accordingly, 5-year recurrence-free survival was significantly different from p53abn when the analysis was limited to stage I disease (94.1% vs 70.8%). Similarly, our subset of POLEmut-p53abn showed an excellent prognosis and a high similarity to POLEmut in terms of clinicopathological and molecular characteristics.

Leon-Castillo et al also described 14 POLEmut-MMRd with a probably pathogenic POLE mutation, which had a 5-year recurrence-free survival of 92.3%. In addition, 12 endometrial cancers belonged to POLEmut-MMRd-p53abn, which were predominantly early-stage endometrioid, but two were classified as mixed histology. All patients showed p53 subclonality, and most cases clustered with POLEmut. In our cohort only two POLEmut-MMRd and three POLEmut-MMRd-p53abn were found. Therefore, we cannot draw any conclusions about these rare groups.

The high frequency of multiple classifiers found in our cohort is probably the result of the methodology used for molecular analyses.34 The combination of TP53 sequencing and p53 staining has increased the detection of multiple classifiers with p53 abnormalities, as occurred in other studies included in the review (figure 1 and table 3). Indeed, in our study, sequencing allowed the detection of an additional 18.9% of multiple classifiers that had a normal immunohistochemistry for p53.

TP53 mutation is probably a non-driver mutation in multiple classifier, caused by the POLE mutation and its ultramutated phenotype.17 In fact, one in three POLEmut-p53abn showed subclonal p53 expression, potentially indicating a later occurrence of TP53 mutation in tumor development, unable to affect the immunohistochemical pattern ubiquitously and resulting in a subclonal pattern.17 18 Similarly, TP53 mutations found in MMRd-p53abn are probably non-driver. However, based on our results, TP53 mutations seemed to influence the tumor characteristics of MMRd-p53abn more than those of POLEmut-p53abn. Furthermore, discordance between p53 immunohistochemistry and TP53 mutations is higher in multiple (32.4%) than in single classifiers (9.8%). Our findings are consistent with the results of Vermij et al who reported that 22/32 cases with discordance between p53 immunohistochemistry and TP53 gene status were either POLEmut or MMRd.18

Although a subclonal mutation is one of the possible causes of discordance between TP53 sequencing and p53 immunostaining, we acknowledge additional different explanations, such as mutations in non-sequenced exons, post-translational mechanism independent of the TP53 gene sequence, misinterpretation by the operator.35

This is one of the largest datasets on multiple classifiers based on consecutive patients, making the estimation of incidence reliable. The use of next-generation sequencing and microsatellite instability testing, combined with staining for p53 and MMR protein, is also a strength of the study, as it allowed the detection of otherwise missed multiple classifiers. However, further studies are needed to define the usefulness of this approach, as there is no strong evidence of its prognostic impact. The limited number of cases and short follow-up are obvious limitations (the last patients were treated in December 2022). Hence, survival data should be interpreted with caution.

Hierarchical molecular algorithms, meaning that testing is stopped as soon as an abnormality is detected, prevents the identification of multiple classifiers. In addition, the use of the original ProMisE algorithm, which starts with the assessment of the MMR protein, hinders the identification of POLEmut-MMRd. Alternatively, to identify all multiple classifiers, a simultaneous approach performing all tests should be used. The algorithm proposed by our group in a previous work can help to reduce the cost of the simultaneous approach without missing multiple classifiers, at least in early-stage endometrial cancer.15

Evidence on Lynch syndrome in multiple classifiers is currently limited. Hence, screening should be recommended in MMRd patients, regardless of other molecular features.

Collaborative studies engaging research teams from around the world still need to address the prognostic role of multiple classifiers, since current evidence is based on a small number of patients.

Although the combined approach of TP53 sequencing/p53 immunohistochemistry may be highly informative from a scientific point of view, further evidence is needed to support its worldwide applicability. Given the routine inclusion of TP53 gene in a sequencing panel for POLE analysis, it is crucial to understand how to manage TP53 mutant endometrial cancers expressing a normal p53 protein.