Subclonal p53 immunostaining in the diagnosis of endometrial carcinoma molecular subtype

The significance of subclonal expression of p53 (abrupt transition from wild‐type to mutant‐pattern staining) is not well understood, and the arbitrary diagnostic cut‐off of 10% between NSMP and p53abn molecular subtypes of endometrial carcinoma (EC) has not been critically assessed. Our aim was to characterise subclonal p53 and discrepant p53 expression/TP53 sequencing results in EC and assess their clinical significance.


Introduction
In the landmark 2013 study from The Cancer Genome Atlas (TCGA), four genomically distinct and prognostically significant endometrial carcinoma (EC) molecular subtypes were described: (i) POLE ultramutated, (ii) microsatellite instability hypermutated, (iii) copy-number low and (iv) copy-number high. 1 Pragmatic clinically applicable strategies were subsequently developed for diagnosis of the EC molecular subtype; these use targeted sequencing for pathogenic POLE mutations together with immunostaining for mismatch repair (MMR) and p53 to stratify cases into Address for correspondence: J Huvila, Department of Pathology, Turku University Hospital, University of Turku, D5020, Medisiina D, 5. Floor Kiinamyllynkatu 10, FIN-20520, Turku, Finland.e-mail: jutta.huvila@utu.fi3][4][5][6][7] This approach has been validated and confirmed in multiple cohorts [2][3][4][5][6][7] and was endorsed by the World Health Organisation (WHO) in 2020, 8 and molecular subtyping is now integrated into prospective clinical trials to assess its ability to predict response to therapies. 9Given what is known about the prognostic [2][3][4][5][6][7] and predictive 10,11 significance of EC molecular subtypes, implementation into routine clinical care, alongside traditional histopathological parameters, is progressing.][14] One remaining challenge in molecular classification has been the interpretation and clinical implications of subclonal p53 staining.6][17] WHO defines abnormal p53 staining as diffuse strong nuclear expression, complete absence of nuclear staining or cytoplasmic expression, 8 and 80% of positive tumour cells is often used as a cut-off.For subclonal mutantpattern p53 staining, an arbitrary cut-off of ≥10% of tumour cells showing the abnormal staining pattern has been most commonly used to classify a tumour as p53abn, 16,17 but the clinical significance of this cut-off is unknown.There is known to be a low rate of discordance between p53 IHC/TP53 sequencing with limited data to guide treatment decisions in these discrepant cases. 16,18Our aim was to investigate the frequency, molecular basis and clinical significance of subclonal p53 immunostaining in EC and to correlate p53 staining patterns with TP53 mutational analysis.

C A S E S E L E C T I O N
This study was approved by the University of British Columbia Institutional Research Ethics Board.Two cohorts were evaluated.The first was a national cohort of ECs diagnosed/treated in a single calendar year (2016) from the institutional archives of 29 participating Canadian centres. 19,20There were 957 cases of 1357 in this study, where immunostaining for both p53 and MMR (PMS2 and MSH6) was performed centrally at Vancouver General Hospital and interpreted by the study pathologists.A second set of 10 cases with subclonal p53 staining (diagnosed/treated from 2012 to 2018) was provided by one of the authors (M.K.) from a single institution to use, together with 11 cases from the first cohort, for the microdissection studies.Clinicopathological and outcomes data were collected as described previously. 19,20ases (hysterectomy and/or biopsy specimens) were reviewed by pathologists at participating sites for the selection of best EC tumour block for molecular analyses.

H I S T O M O R P H O L O G I C A L C L A S S I F I C A T I O N
Histomorphological classification was based on surgical pathology reports, as described by Thompson et al., 20 with central review conducted only in selected cases, as described below.

I M M U N O H I S T O C H E M I S T R Y
Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE)-associated immunomarkers (p53, MSH6 and MSH2) were assessed on 4-lm thick whole sections cut and stained in the clinical laboratory at Vancouver General Hospital, using methods previously described. 19,20Interpretation of immunohistochemistry (IHC) results was as follows: MMR IHC was recorded as intact or deficient; p53 IHC staining was classified as wild-type or abnormal (i.e.overexpression, null or cytoplasmic staining).The presence of any degree of subclonal p53 staining, defined as an abrupt transition between normal and mutant-pattern staining, was documented, including the % tumour cells on slides showing mutant-pattern staining.To ensure that true mutant-pattern p53 staining was present, a minimum size cut-off of a region or regions of contiguous tumour cells comprising at least 0.5% of the tumour cells on the slide was used for this study.
POLE mutation testing was performed by targeted next-generation sequencing (NGS) using methods previously described. 19,20POLEmut assignment was limited to a list of 11 agreed-upon pathogenic mutations. 21TP53 mutational assessment was also undertaken in all cases.In cases showing subclonal staining, directed sequencing was performed on DNA extracted from cores or tumour scraped from slides in the areas with divergent immunostaining, i.e. wildtype and mutant-pattern staining, where tissue was available.To ensure sufficient representative tissue for NGS, the targeted sampling was limited to cases with >5% of mutant-pattern subclonal staining.All TP53 mutation calls were cross-referenced against publicly available databases (ClinVar, available at https:// www.ncbi.nlm.nih.gov/clinvar/ and COSMIC, available at https://cancer.sanger.ac.uk/cosmic). 22,23P53 mutational status for subclonal-stained cases underwent confirmatory sequencing with Sanger sequencing.
All ECs were classified into one of four molecular subtypes using the ProMisE and WHO endorsed algorithm, as reported previously. 19,20If abnormal p53 subclonal staining was present in <10% of tumour cells they were considered to be p53 wild-type for purposes of molecular subtype diagnosis 10 ; that is, if there was <10% mutant-pattern p53 staining in a POLE wild-type and MMR proficient tumour, it was considered NSMP.Cases with more than one molecular feature were classified in accordance with the segregation order and rationale described by Le on-Castillo et al. 17

S T A T I S T I C A L M E T H O D S
Univariable associations between molecular subtype and clinicopathological variables were evaluated as previously described. 19,20Univariable associations between presence of subclonal staining and molecular subtype was undertaken.Agreement for TP53 mutational status assessment between p53 immunostaining and NGS was evaluated in all cases and considering p53abn and NSMP ECs only (i.e.excluding the POLEmut and MMRd ECs).Kaplan-Meier analyses were used to assess the univariable effect of subclonal staining (uniform p53abn versus p53abn with subclonal staining of ≥10% versus p53 subclonal staining of <10%).

C O H O R T D E S C R I P T I O N
The clinical and pathological characteristics of the 957 patients with endometrial cancer from the national cohort are shown in in Table 1.

S U B C L O N A L P 5 3 I H C S T A I N I N G
Subclonal p53 staining (of any extent) was observed in 4.0% (38 of 957) of tumours, with 23 of 38 showing mutant-pattern p53 staining in ≥10% of tumour cells (Table 1, Table S1).The most common pattern of subclonal mutant-pattern staining was overexpression (29 of 38).The remaining nine tumours showing subclonal p53 staining included four with more than one mutant-pattern of p53 expression, two with subclonal-type p53 expression and three with subclonal cytoplasmic expression (Figure 1, Table 2).

A S S O C I A T I O N O F S U B C L O N A L I H C S T A I N I N G A N D M U L T I P L E C L A S S I F I E R E C
Multiple classifier ECs, defined as tumours with more than one molecular feature, were observed in 3.0% (29 of 957) of cases and subclonal p53 expression, with ≥10% of tumour cells showing mutant-pattern p53 staining, was observed in 52% (12 of 23) of those multiple classifier ECs with mutant-pattern p53 staining (Table 3).Note that for the purposes of this analysis p53abn status was determined based on p53 immunostaining results.Using NGS to define multiple classifier tumours we would have identified 19 additional POLEmut EC with TP53 mutations and 28 additional MMRd EC with TP53 mutations (any VAF, Figure S1A).Those cases with discrepant p53 immunostaining and TP53 sequencing results are considered separately below.

A G R E E M E N T O F P 5 3 I H C W I T H P R E S E N C E / A B S E N C E O F T P 5 3 M U T A T I O N
We first examined the correlation between p53 immunostaining and TP53 mutational assessment in the 38 tumours showing subclonal expression of p53 by IHC; these results are shown in Table S1.A TP53 mutation was identified in 31 of 38 cases (81.6%) showing subclonal p53 staining.TP53 mutations were identified in 22 of 23 tumours with ≥10% subclonal staining, and in the one discrepant case without mutation detected, 98% of tumour cells showed null p53 staining, histology was serous and there was disease progression at 2.1 years.TP53 mutations were less likely to be identified in tumours with <10% subclonal staining (22 of 23 versus nine of 15 in  ≥10% and <10, respectively).This is an expected result, given that the cores for sequencing for this analysis were not directed; that is, they were taken from representative areas of tumour without intentionally sampling areas with mutant-pattern p53 immunostaining (as was conducted for the subset of cases described below).
In the remaining 919 tumours (excluding tumours with subclonal p53 staining), p53 immunostaining showed an accuracy of 92.6% [95% confidence interval (CI) = 90.7-97.2%]for the presence of a TP53 mutation, with a sensitivity of 68.9% and a specificity of 99.4% (Table 4A).There were 64 tumours in which a TP53 mutation was detected on NGS, but p53 IHC was interpreted as wild-type.Of these, 18 were POLEmut and 25 were MMRd ECs (Figure S1B).There were four tumours in which there was mutant-pattern p53 immunostaining and no TP53 mutation detected on NGS; none of these four were POLEmut or MMRd EC.After excluding all MMRd and POLEmut tumours, the accuracy of p53 immunostaining increased to 95.8% (95% CI = 93.9-97.3%),with a sensitivity of 86.2% and specificity of 99.1% (Table 4B).Forty-three of 68 (63%) tumours with discrepant p53 immunostaining and TP53 NGS results were either MMRd and POLEmut EC, where p53/TP53 abnormalities are considered 'passenger mutations' and are not associated with an adverse prognosis. 17Of the remaining 25 MMR-proficient and  POLE wild-type tumours, there were four with mutant-pattern p53 immunostaining in which no TP53 mutation was detected.Of these, two showed null-type staining (one serous EC and one carcinosarcoma, both stage 3, with one patient dead of disease and the other patient alive and well after 3.5 years of follow-up), and two showed overexpression (one serous and one grade 1 endometrioid EC, both stage 1A, both alive and well after 1.7 and 4.5 years, respectively).Twenty-one EC were interpreted as showing p53 wild-type staining, but a TP53 mutation was detected on NGS.Of these 21 cases, two had very low TP53 variant allele frequency (<5%), one had both low allele frequency and the variant allele could not be independently verified by polymerase chain reaction (PCR) and one had a POLE mutation (Y458C) considered to be pathogenic in colorectal carcinoma, 24 together with three different TP53 mutations and other mutations consistent with ultramutated phenotype.Of the remaining 17 tumours, most were low-grade endometrioid ECs (12 grade 1 and three grade 2), with one high-grade endometrioid, one serous and one clear cell EC.Of these 17 tumours with wild-type p53 immunostaining and TP53 mutation on sequencing, all but two were stage 1, and there were two deaths due to disease within the first 2 years of follow-up (Table S2).

Twenty-one
ECs with subclonal p53 IHC staining underwent directed TP53 mutational analysis by NGS of areas of tumour showing wild-type and mutant-pattern p53 expression.Of these, nine showed at least one TP53 mutation that was present in both the wild-type and mutant-pattern staining regions of the tumour, and these were considered to not be true subclonal mutations (Table 5).The 12 remaining tumours showed TP53 mutations only in the abnormal staining region or there was a very significantly lower variant allele frequency (<10% VAF) in the wild-type staining area, and these were considered to represent true subclonal mutations (Table 5, Figure 2).Of the nine POLE mutated tumours, six had two or more TP53 mutations detected on NGS, and two of the four MMRd EC had two TP53 mutations.

O U T C O M E A N A L Y S I S
Disease-specific survival of the 16 POLE wild-type and MMR proficient tumours, 11 of which had subclonal mutant-pattern staining in ≥10% of tumour cells (p53abn EC) and five had mutant-pattern p53 staining in <10% of tumour cells (NSMP EC), was compared to NSMP (n = 475) and p53abn (n = 145) EC with no subclonal staining (Figure 3).There were no disease-specific deaths in the patients with <10% subclonal p53 immunostaining and four disease-specific deaths among 11 patients with subclonal p53 expression in ≥10% of cells.

Discussion
In the new era of EC molecular classification, p53 IHC results and/or TP53 mutation status is one of the critical molecular stratification features to assign molecular subtype, and impacts patient management.
Having guidance on what should be called abnormal is now of great importance.According to the EC molecular classification algorithm, 8 p53 status is the last step in molecular assignment characterising EC patients without POLE mutations or MMRd to either p53abn or NSMP (assessed by either IHC or NGS).p53abn EC represent the most aggressive ECs, making up only 10%-15% of all ECs, but accounting for more than 50% of EC related mortality.Recent retrospective data have shown that identification of p53abn EC selects a group of patients who significantly benefit from adjuvant chemotherapy used in addition to radiotherapy. 10,11The ESGO-ESTRO-ESP (2021) and ESMO (2022) EC guidelines now recommend that all p53abn ECs with myometrial invasion be considered high-risk and be treated with chemotherapy with or without radiotherapy, regardless of the stage, grade or histotype.In contrast, the majority of NSMP EC have an excellent prognosis, especially when low-grade and oestrogen receptorpositive, and treatment de-escalation can now be considered. 25,2653 status can also have an effect on histotype assignment, as detection of mutant-pattern p53 staining favours high-grade diagnosis such as serous carcinoma or carcinosarcoma, although we now know that only 50% of p53abn ECs are serous carcinomas.20,27 p53abn ECs are observed across a range of histotypes, including low-grade endometrioid ECs.
Subclonal p53 IHC staining is uncommon (4.0% of ECs with any extent, 2.4% with mutant-pattern p53 staining in ≥10% of tumour cells and only 1.1% of ECs after excluding 'multiple classifier' ECs).This is in line with previous work, although a higher frequency of subclonal staining was also seen in high-risk EC cohorts when lower cut-off points, such as 5% or 12 consecutive cells, were used. 16,18,282023 The Authors.Histopathology published by John Wiley & Sons Ltd., Histopathology, 83, 880-890.When present, it is most commonly seen in POLEmut or MMRd EC ('multiple classifier' ECs), where subclonal p53 staining does not impact the molecular subtype diagnosis, and is thought to represent passenger mutations acquired during tumour progression in these ultramutated and hypermutated EC molecular  subtypes. 17Only 16 of 957 (1.7%) of EC in this large cohort showed subclonal p53 mutant-pattern staining in the presence of wild-type POLE and proficient MMR, and in only 11 (11 of 957, 1.1%) of these was there subclonal staining in ≥10% of tumour cells.Because of the small number of cases with subclonal p53 staining, we were unable to adequately perform a critical determination of an optimal cut-off that is clinically relevant.It is reassuring, however, that in this series the arbitrary cut-off of 10% currently in use suffices to identify all disease-specific deaths.
In almost half of tumours (42.9%) with subclonal mutant-pattern staining for p53, the underlying TP53 mutation is more widespread in the tumour than is appreciated based on the p53 immunostaining.The mechanism for wild-type staining in TP53 mutated tumour cells was not explored, but could include variably altered regulation of p53 protein expression in tumour regions with TP53 mutations, areas where there has not been loss of heterozygosity and deletion of the normal TP53 allele, suboptimal fixation of tumour tissue or sampling issues, and merits further interrogation.
At this time, either p53 immunostaining or sequencing TP53 can be used for molecular subtype diagnosis.Comparison of the two methods is critical, as there is insufficient evidence at present that they are equivalent.The accuracy of p53 IHC for predicting TP53 mutations was 92.3% (sensitivity 68.8%) when all molecular subtypes were evaluated, and this improved to 95.8% (sensitivity 84.9%) when POLEmut and MMRd ECs were excluded from the analysis.This is similar to previous studies showing an overall accuracy of 91%-92% in all ECs, improving to 95% after excluding MMRd and POLEmut EC. 10,18 In our series, only five tumours (including the one case with 98% null p53 staining) that had abnormal p53 immunostaining showed no TP53 mutation on NGS.Three of five were of histotypes associated with p53abn molecular subtype (such as serous and carcinosarcoma) and three of five showed null p53 staining.False-negative results on NGS have been reported, but are fortunately rare and are often related to null type p53 staining commonly associated with large TP53 deletions, which can remain undetected with NGS.If a POLE wild-type, MMRproficient tumour has features suggestive of p53abn EC but without a TP53 mutation on NGS, consideration should be given to performing p53 immunostaining.In 17 tumours a convincing TP53 mutation was detected on NGS (VAF range = 5%-91%) but IHC showed wild-type staining.Most of these were low-grade endometrioid carcinomas, with two patients dying from disease within 2 years of treatment.Some of these TP53 mutations are uncommon or of uncertain clinical significance, and together with the infrequent disease related events (recurrence and or disease-related death) in this group it is possible that sequencing may be too sensitive and IHC is a better surrogate marker of this aggressive molecular subtype.The significance of a TP53 mutation in a POLE wild-type and MMR-proficient EC with wildtype p53 staining is, however, uncertain at this time and a larger series of such ECs with outcomes is needed to understand the clinical significance of this molecular phenotype.In a recent study of 164 ECs that underwent molecular classification, there was no discordance in p53 IHC versus TP53 sequencing on NGS, suggesting that with advances in p53 IHC interpretation and attention to proper fixation of tumour tissue, this discordance is exceptionally rare. 29In cases with discrepant NGS and IHC results, careful assessment of both TP53 mutation and IHC should be performed, as either can give false negative results, and abnormality of either can be used to molecularly classify as p53abn molecular subtype.
Variant allele frequency is dependent upon tumour cell percentage submitted for NGS and cannot as such be used to define a 'subclonal' TP53 mutation.With increasing use of NGS, which is required to determine POLE status, discordant p53 IHC and TP53 NGS may be encountered more often and consensus on how to manage these discordant cases clinically will be important.Inconsistent molecular subtype assignment in these discordant cases could result in a significant difference in treatment recommendations, such as adjuvant chemoradiation if assigned p53abn versus surgery alone if assigned as NSMP EC.
In this series 3.0% of ECs were so-called 'multiple classifier' EC, having more than one molecular feature.It is noteworthy that most of the multiple classifier EC showed subclonal p53 staining.This follows from the observation, noted above, that most cases of EC with subclonal staining (22 of 38) were associated with a pathogenic mutation in POLE or MMR deficiency, thus the presence of subclonal p53 staining should prompt assessment of MMR expression and POLE sequencing, if not already performed.
Strengths of this study include the large sample size, with cases drawn from a wide range of clinical practice settings.It is the first study, to our knowledge, to critically assess the clinical and pathological correlates of subclonal p53 expression in EC and the currently used, but arbitrary, cut-off of 10% subclonal p53 expression as a diagnostic cut-off between NSMP and p53abn molecular subtypes of EC.Whole section immunostaining in an accredited diagnostic laboratory was used to assess p53 expression.Weaknesses include the relatively small number of tumours with subclonal p53 expression, despite the large initial sample size, which precluded a rigorous assessment of the cut-off between NSMP and p53abn.Another weakness was non-standardised specimen handling and fixation, which could have impacted the immunostaining results.
In light of our work and others, our recommendations are that (i) cases with any extent of subclonal p53 staining should be tested for POLE mutations and MMR.Additionally, in POLE wild-type and MMRproficient tumours, (ii) ≥10% of subclonal mutantpattern p53 staining, defined as abrupt change from wild-type to mutant-pattern staining, justifies classification as p53abn, noting the presence (and extent) of subclonal staining, (iii) <10% of mutant-pattern staining in a NSMP should be commented upon in the report but the tumour is not classified as p53abn, and (iv) if subclonal p53 is identified on a biopsy specimen it should be repeated on a representative hysterectomy slide and that staining one representative slide is sufficient.
In conclusion, we demonstrate that subclonal mutant-pattern p53 immunostaining outside the context of MMRd or POLEmut EC, where it does not impact molecular subtype diagnosis, is rare and that the current cut-off of ≥10% mutant-pattern staining to diagnose p53abn EC serves to identify those patients with more aggressive disease.

Figure 2 .
Figure 2. Selected cases that underwent directed sampling for next generation sequencing of TP53.Images depict post-core p53 IHC-stained slides; mutant-pattern and wild-type areas sampled for NGS are indicated by orange and green, respectively.A, A POLEmut-p53abn double classifier EC with 20% of tumour cells showing mutation-pattern p53 staining.Two mutations in TP53 were present in the abnormal staining region and a third mutation was present in the wild-type staining region (ID 256).B, A p53abn EC with 60% of tumour cells showing mutant-pattern p53 staining.A TP53 mutation was present in both the abnormal and wild-type-stained areas (ID 378).C, An MMRd-p53abn double classifier EC with 50% of tumour cells showing mutant-pattern p53 staining.Two TP53 mutations were observed in both the abnormal and wild-type-stained areas (ID 333).D, A p53abn EC in which 75% of tumour cells show mutant-pattern p53 staining.A TP53 mutation was present in both the abnormal (homozygous) and wild-type (heterozygous) staining areas (ID 271).E, A POLEmut-p53abn double classifier EC in which 50% of tumour cells show mutant-pattern p53 staining.A TP53 mutation was observed in both the abnormal and wild-type staining areas (ID 439).F, An NSMP molecular subtype EC in which 8% of tumour cells show mutant-pattern p53 staining.A TP53 mutation was observed in the abnormal staining area only (ID 330).

Table 1 .
Characteristics for a national cohort of molecularly subtyped ECs where IHC staining for both p53 and MMRd were performed and reviewed centrally

Table 2 .
Distribution of different subclonal p53 staining patterns across molecular subtypes

Table 3 .
Multiple classifier ECs (i.e. more than one molecular feature)

Table 5 .
Sequencing results obtained via directed sampling of areas with mutant pattern and wild-type pattern p53 staining +, Homozygous; À, heterozygous.† Interpretation of the nature of the TP53 mutations.True subclonal = mutation not present or present at significantly lower (<10%) VAF (probable contaminant) than in the mutated area frequency in the area of wild-type staining pattern of p53 IHC.Not true subclonal = same TP53 mutation is present in both the IHC wild-type p53 and p53 abnormal components with at least 10% of the VAF.Ó 2023 The Authors.Histopathology published by John Wiley & Sons Ltd., Histopathology, 83, 880-890.