The traditional binary algorithm for classifying endometrial carcinoma (EC) continues to be mirrored in international clinical guidelines, wherein low-grade endometrioid EC’s are separated from high-grade and nonendometrioid histotypes 1 2–4
In 2013 the Cancer Genome Atlas (TCGA) demonstrated that ECs can be segregated into 4 different molecular clusters, as determined by integration of genomic, transcriptomic, and proteomic analysis: (i) ultramutated, characterized by hotspot mutations in DNA polymerase epsilon (POLE ) and a markedly increased transversion mutation frequency; (ii) hypermutated, characterized by microsatellite instability and a 10-fold higher mutation rate than microsatellite stable tumors; (iii) copy number-high, composed of serous-like EC with extensive somatic copy number alterations and low tumor mutational burden; and (iv) copy number-low, composed largely of endometrioid, microsatellite stable tumors with relatively low somatic copy number alterations 5
The validated Pro active M olecular Ris k Classifier for E ndometrial Cancer (ProMisE) demonstrated that by using immunohistochemistry (IHC) for p53, the protein product of the oncogene TP53 , and mismatch repair proteins (MMR), and sequencing the exonuclease domain of the enzyme DNA polymerase epsilon (POLE ), 4 molecular groups can be identified that replicate those described by TCGA 6–8 9 POLE mutation and MMR deficiency result in high mutational states that can cause secondary or passenger mutations, in each other and in TP53 , that do not influence prognosis in the same way as driver mutations. In other words, EC showing abnormal p53 IHC results or harboring TP53 mutations in the presence of a pathogenic POLE mutation or MMR defect, show similar clinical behaviour as POLE mut or mismatch repair deficient (MMRd) EC, respectively 9
Initially the ProMisE algorithm first identified those that were MMRd EC, followed by those with mutations in the exonuclease domain of POLE (POLEmut EC), and finally classification of the remainder according to p53 IHC results. Having MMRd as the first step allowed all EC patients to be screened for Lynch syndrome. Since the validation of ProMisE, further research has led to refinement of the original algorithm. Firstly, it has been shown that only specific pathogenic POLE mutations are associated with the “ultramutator” phenotype that defines the outstandingly good prognosis of this class of EC 10,11 POLE mutations with MMRd show the same clinical behavior as POLE mut ECs 10 POLE mutation should be the first step in the algorithm.
The revised algorithm incorporating this new information has been included in the 2020 World Health Organization Classification of Female Genital Tumours 12 POLE mut EC to be identified first as those harboring pathogenic POLE mutations, followed by MMRd EC, with the remainder classified as No Specific Molecular Profile (NSMP) EC or p53abn EC on the basis of normal versus abnormal p53 IHC. With superior reproducibility to conventional histologic classification, as well as better prognostication and prediction of treatment response, the new molecular classification has been incorporated into clinical guidelines developed by the National Comprehensive Cancer Network 13 14 POLE mutational status currently presents a major barrier to widespread adoption.
In this study, we proposed and retrospectively evaluated the prognostic value of a clinically driven, selective ProMisE classifier (ProMisE-S), whereby MMR and p53 IHC are evaluated on all EC’s, and POLE mutational analysis is only assessed in patients in whom adjuvant treatment may be considered (Figure 1 ). For the purposes of this study, EC’s fulfilling all of the following criteria: grade 1 or 2, endometrioid histology, p53 normal, MMR proficient, stage IA and no lymphovascular space invasion (LVSI), were labeled “very low-risk” with no further testing, and POLE status was only evaluated in the remaining tumors. In addition, we compared this more pragmatic molecular classification of EC (ProMisE-S) with universal testing using the ProMisE algorithm, the present gold standard. Finally, we compared the allocation of EC patients into risk groups as defined without and with molecular classification by the recent ESGO/ESTRO/ESP guidelines.FIG. 1: Selected testing protocol. On diagnostic evaluation, the restricted testing protocol would require the evaluation of histotype, grade, MMR, and p53 IHC status to determine who should receive full ProMisE molecular characterization. Postsurgically, patients with >stage IA and with any LVSI would also receive full molecular characterization. IHC indicates immunohistochemistry; MMR, mismatch repair; LVSI, lymphovascular space invasion.
METHODS
Cohorts and Data
We retrospectively analyzed data on cohorts from Vancouver (Canada) and Tuebingen (Germany) previously described in the development 6 7 8 POLE pathogenic mutations, followed by assessment of MMR status by IHC, and finally considering the p53 IHC result. The second approach considered, termed ProMisE-S, aimed to fully subtype a targeted subset and identify a “very low-risk” group that could safely forego POLE testing (low-grade endometrioid EC, normal MMR and p53 IHC, stage IA, negative for LVSI). This approach could potentially be applied preoperatively at first, by identifying patients as “likely low-risk” based on evaluation of histotype, grade, and p53 and MMR IHC on diagnostic biopsy specimens (Fig. 1 ). Consideration of postsurgical pathology findings would result in additional patients requiring full molecular classification; after surgery, EC’s with normal p53 and MMR IHC who were stage IA with no LVSI, and remained endometrioid grade 1 or 2 would be considered “very low-risk” and would not require POLE assessment. We estimated the “likely low-risk” group based on histotype, grade, and MMR and p53 IHC, and the “very low-risk group” based on all histopathologic findings, ie including stage and LVSI.
New ESGO/ESTRO/ESP guidelines have proposed 2 definitions of prognostic risk-groups for postsurgical decision-making 14
Statistical Methods
We used cross tabulation to compare the new WHO-endorsed refined ProMisE to the previously validated molecular classification algorithm, as well as to a targeted ProMisE-S. We characterized clinicopathologic characteristics of the preoperative ProMisE-S group using descriptive statistics. Univariable associations of individual clinicopathologic parameters with ProMisE-S preoperatively were compared using 1 way analysis of variance for continuous data (age at surgery, body mass index) and the χ2 test for categorical data [stage, grade, histologic subtype at diagnosis, LVSI, myometrial invasion, and lymph node status], and the ESMO (2020) risk group assignment, with and without molecular classification.
We considered univariable and multivariable survival of the different ProMisE variations to address the association with overall survival (OS), disease‐specific survival (DSS), and progression‐free survival (PFS). We assessed the significance of Kaplan-Meir survival curves using log rank tests in univariable survivals. For multivariable models we used cox proportional hazard, adjusting for age, body mass index, grade, histotype, and adjuvant treatment (any or none). We reported hazard ratios with corresponding 95% confidence intervals and likelihood ratio test P values. We used the Firth penalized maximum‐likelihood bias‐reduction method to estimate the hazard ratios (indicated by a superscript F) when the number of censored cases exceeded 80%, and the corresponding confidence intervals were obtained using the profile likelihood.
We used the R software for statistical computing 15
RESULTS
Data from 912 patients were used to evaluate the refined WHO-endorsed ProMisE and a simplified, targeted ProMisE-S algorithm. These data were obtained from previously published cohorts of which 460 were from Vancouver, Canada and 452 were from Tuebingen, Germany. Reclassification from the previously validated algorithm to the refined WHO endorsed ProMisE classifier was possible in 899 patients which we used for the remaining analyses. The change in the ordering of the classification tree left 13 patients unclassifiable; these were patients previously identified as MMRd but for whom we did not have confirmation of a pathogenic POLE mutation. Reclassification of the remaining 899 resulted in very few (7/899) tumors changing to a different molecular group: 2 changed from MMRd to POLE mut, 4 from POLE mut to NSMP, and 1 from POLE mut to p53abn (Table 1 ). In the first 2, the deficiency in MMR is considered secondary to the pathogenic POLE mutation, and in the latter 5 tumors, the POLE mutations were nonpathogenic.
TABLE 1 -
Comparison of molecular classification results obtained from the previously validated ProMisE with the refined WHO endorsed molecular classification
Refined ProMisE, n (%)
POLEmut
MMRd
NSMP/p53wt
p53abn
Total, n (%)
Overall
Pop based
Overall
Pop based
Overall
Pop Based
Overall
Pop based
Overall
Pop based
ProMisE
POLEmut
77 (97.5)
39 (97.5 )
4 (0.9)
2 (0.9 )
1 (0.6)
1 (1.8 )
82 (9.1)
42 (9.5 )
MMRd
2 (2.5)
1 (2.5 )
226 (100)
120 (100 )
228 (25.4)
121 (27.4 )
NSMP/p53wt
424 (99.1)
225 (99.1 )
424 (47.2)
225 (50.9 )
p53abn
165 (99.4)
54 (98.2 )
165 (18.4)
54 (12.2 )
Total
79 (8.8% )
40
(
9%
)
226 (25.1 )
120
(
27.1
)
428 (47.6 )
227
(
51.4
)
166 (18.5 )
55
(
12.4
)
899 (100)
442 (100 )
Bold values indicates the population-based cohort.
MMRd indicates mismatch repair deficient; ProMisE, Pro active M olecular Ris k Classifier for E ndometrial Cancer.
Of the 899 patients, 45% were deemed “likely low-risk” based on parameters available at the time of biopsy diagnosis (MMR and p53 IHC, histotype and grade). As expected, the proportion of “likely low-risk” patients was higher in the population-based German cohort (55%; Table 2 ), since the Vancouver cohort was biased toward high-risk patients reflective of tertiary referral center practice.
TABLE 2 -
Comparison of molecular classification results obtained from a restricted molecular testing protocol versus testing every patient
Refined ProMisE (all patients), n (%)
POLEmut
MMRd
NSMP/p53wt
p53abn
Total
Overall
Pop based
Overall
Pop based
Overall
Pop based
Overall
Pop based
Overall
Pop based
Restricted (preoperative) ProMisE-S
Likely low-risk
47 (59.5)
33 (82.5)
358 (83.6)
210 (92.5)
405 (45.1)
243 (55)
POLEmut
32 (40.5)
7 (17.5)
32 (3.6)
7 (1.6)
MMRd
226 (100)
120 (100)
226 (25.1)
120 (27.1)
NSMP/p53wt
70 (16.4)
17 (7.5)
70 (7.8)
17 (3.8)
p53abn
166 (100)
55 (100)
166 (18.5)
55 (12.4)
Restricted (postsurgical) ProMisE-S
Very low-risk
34 (43)
26 (65 )
237 (55.4)
140 (61.7 )
271 (30.1)
166 (37.6 )
POLEmut
45 (57)
14 (35 )
45 (5)
14 (3.2 )
MMRd
226 (100)
120 (100 )
226 (25.1)
120 (27.1 )
NSMP/p53wt
191 (44.6)
87 (38.3 )
191 (21.2)
87 (19.7 )
p53abn
166 (100)
55 (100 )
166 (18.5)
55 (12.4 )
Total
79 (8.8 )
40
(
9
)
226 (25.1 )
120
(
27.1
)
428 (47.6 )
227
(
51.4
)
166 (18.5 )
55
(
12.4
)
899 (100)
442 (100 )
Bold values indicates the population-based cohort.
MMRd indicates mismatch repair deficient; ProMisE, Pro active M olecular Ris k Classifier for E ndometrial Cancer.
As shown in Table 3 , at the time of biopsy we would have identified 15 p53abn EC’s which were grade 1/2 with endometrioid histology; following surgery 10 of these patients were stage IA, 2 were stage IB and 3 were stage III. Within the “likely low-risk” group, 130/405 (32%) EC’s would need to undergo POLE testing after surgery because they were stage IB or higher (27%) and/or showing presence of LVSI (8%). Of these, 12/130 (9%) would be identified as POLE mut EC, and the remainder classified as NSMP EC. Patients classified postsurgically as “very low-risk’” made up 30% of the full cohort and 38% of population-based cohort (Table 2 ).
TABLE 3 -
Clinicopathologic parameters according to a restricted preoperative ProMisE-molecular testing algorithm in all cohorts
Restricted preoperative ProMisE-S protocol
Likely low-risk
POLE mut
MMRd
NSMP/p53wt
p53abn
Total
P
Age, yr
Median (IQR)
62 (54–72)
59 (56–67)
67 (59–74)
64 (56–73)
72 (66–78)
66 (57–74)
<0.001
Missing
0
0
1
2
0
3
BMI
Median (IQR)
29 (24–37)
27 (23–29)
28 (23–33)
25 (22–31)
28 (23–32)
28 (24–34)
<0.001
Missing
32
5
12
9
5
63
FIGO 2009 stage, n (%)
IA
294 (73)
14 (44)
107 (48)
26 (38)
61 (37)
502 (56)
<0.001
IB
68 (17)
14 (44)
57 (25)
14 (21)
20 (12)
172 (19)
II-IV
42 (10)
4 (12)
61 (27)
28 (41)
85 (51)
220 (25)
Missing
2
0
1
2
0
5
Tumor grade, n (%)
Grade 1/2
405 (100)
3 (9)
137 (61)
4 (6)
15 (9)
564 (63)
<0.001
Grade 3
0
29 (91)
89 (39)
66 (94)
151 (91)
335 (37)
Missing
0
0
0
0
0
0
Histologic subtype (reviewed)
Endometrioid
405 (100)
21 (66)
196 (87)
51 (74)
39 (23)
712 (79)
<0.001
Serous
0
3 (9)
10 (4)
11 (16)
108 (65)
132 (15)
Clear cell
0
1 (3)
4 (2)
2 (3)
5 (3)
12 (1)
High grade mixed or other
0
6 (19)
12 (5)
4 (6)
14 (8)
36 (4)
Undifferentiated
0
1 (3)
3 (1)
1 (1)
0 (0)
5 (1)
Missing
0
0
1
1
0
2
LVSI, n (%)
Positive
32 (8)
17 (55)
73 (33)
28 (43)
76 (48)
226 (26)
<0.001
Negative
365 (92)
14 (45)
146 (67)
37 (57)
81 (52)
643 (74)
Missing
8
1
7
5
9
30
Nodal status, n (%)
Positive
9 (2)
2 (6)
21 (10)
9 (13)
36 (23)
77 (9)
<0.001
Negative
266 (73)
25 (81)
166 (79)
40 (60)
102 (66)
599 (73)
Not tested
88 (24)
4 (13)
23 (11)
18 (27)
17 (11)
150 (18)
Missing
42
1
16
3
11
73
Postsurgical treatment, n (%)
None
245 (66)
7 (22)
89 (43)
16 (24)
48 (32)
405 (49)
<0.001
Brachy only
86 (23)
5 (16)
46 (22)
7 (11)
7 (5)
151 (18)
RT+/−brachy
26 (7)
6 (19)
32 (16)
17 (26)
21 (14)
102 (12)
Chemo+/−RT
15 (4)
14 (44)
39 (19)
26 (39)
76 (50)
170 (21)
Missing
33
0
20
4
14
71
Total
405 (45%)
32 (4%)
226 (25%)
70 (8%)
166 (18%)
899
BMI indicates body mass index; IQR, interquartile range; LVSI, lymphovascular space invasion; MMRd, mismatch repair deficient; ProMisE, Pro active M olecular Ris k Classifier for E ndometrial Cancer.
Of note, in the ProMisE-S algorithm, the groups labeled as NSMP and POLE mut correspond to subsets of these original ProMisE categories, that exclude those with the least aggressive clinicopathologic features (Table 2 ).
Survival results when comparing the original and refined ProMisE classifiers were very similar, as expected, given that only 7/899 of all EC’s changed classification (Table 1 ). Using the targeted testing with ProMisE-S, a slightly different prognostic trend emerges owing to the exclusion of patients with least aggressive forms of the disease. As would be expected, the “very low-risk” group had excellent OS, DSS and PFS outcomes rivaling outcomes in the POLE mut group, which in turn followed a similar OS and DSS as in the refined ProMisE algorithm, but with slightly inferior PFS (Fig. 2 ). The worst survival remained attributable to p53abn EC. However, we note that NSMP EC had worse survival outcomes relative to MMRd EC, this is likely because in this group, as defined by the ProMisE-S algorithm, the subset with the most favorable features were part of the “very low risk” group. Multivariable analyses which adjust for age, grade, histotype, LVSI, and stage show that all versions of the ProMisE classifier, original, refined as well as selective, remain prognostic, with the order of survival similar to that observed in univariable models (Table 4 ).
FIG. 2: OS (DSS and PFS are provided for the 3 ProMisE algorithms: (i) refined ProMisE, as endorsed by WHO (2020), whereby the assessment of POLE status precedes that of mismatch repair (MMR), (ii) ProMisE-S based on tumor features that would be available at biopsy (histotype, grade and MMR and p53 IHC), and (iii) ProMisE based on hysterectomy findings, incorporating all of the following: histotype, grade, MMR and p53 IHC, stage, and LVSI. DSS indicates disease-specific survivival; OS, overall survival; PFS, progression-free survival; ProMisE, Pro active M olecular Ris k Classifier for E ndometrial Cancer.
TABLE 4 -
Univariable and multivariable hazard ratios of molecular subtypes with p53abn set as reference
Univariable
Multivariable*
# Of events/n
Hazard ratio (95% CI)
LRT, P
# Of events/n
Hazard ratio (95% CI)
LRT, P
Overall survival
ProMisE
NSMP/p53wt
232/912
0.23 (0.16–0.31)F
<0.01
216/877
0.57 (0.36–0.9)F
0.05
POLE mut
0.18 (0.09–0.31)F
0.51 (0.25–0.98)F
MMRd
0.36 (0.26–0.49)F
0.61 (0.4–0.95)F
Refined ProMisE
NSMP/p53wt
229/899
0.23 (0.17–0.31)F
<0.01
213/865
0.58 (0.36–0.91)F
0.06
POLE mut
0.17 (0.08–0.3)F
0.49 (0.23–0.95)F
MMRd
0.37 (0.26–0.51)F
0.62 (0.4–0.96)F
ProMisE-S (postsurgical)
NSMP/p53wt
229/899
0.39 (0.27–0.54)F
<0.01
213/865
0.59 (0.37–0.95)F
0.07
POLE mut
0.19 (0.08–0.38)F
0.4 (0.15–0.87)F
MMRd
0.37 (0.26–0.51)F
0.63 (0.4–0.97)F
Very low-risk
0.12 (0.08–0.18)F
0.63 (0.32–1.22)F
Disease-specific survival
ProMisE
NSMP/p53wt
150/900
0.15 (0.1–0.23)F
<0.01
136/866
0.63 (0.36–1.1)F
<0.01
POLE mut
0.07 (0.02–0.18)F
0.21 (0.06–0.57)F
MMRd
0.31 (0.21–0.46)F
0.71 (0.42–1.17)F
Refined ProMisE
NSMP/p53wt
147/887
0.15 (0.1–0.23)F
<0.01
133/854
0.64 (0.36–1.12)F
0.01
POLE mut
0.07 (0.02–0.18)F
0.23 (0.06–0.62)F
MMRd
0.31 (0.21–0.46)F
0.7 (0.41–1.16)F
ProMisE-S (postsurgical)
NSMP/p53wt
147/887
0.32 (0.21–0.48)F
<0.01
133/854
0.67 (0.38–1.17)F
0.03
POLE mut
0.13 (0.04–0.33)F
0.25 (0.07–0.69)F
MMRd
0.31 (0.21–0.46)F
0.68 (0.4–1.13)F
Very low-risk
0.03 (0.01–0.07)F
0.34 (0.11–0.99)F
Progression-free survival
ProMisE
NSMP/p53wt
152/865
0.16 (0.1–0.23)F
<0.01
141/836
0.49 (0.28–0.86)F
<0.01
POLE mut
0.09 (0.03–0.2)F
0.2 (0.06–0.5)F
MMRd
0.32 (0.22–0.47)F
0.63 (0.38–1.03)F
Refined ProMisE
NSMP/p53wt
150/852
0.16 (0.1–0.23)F
<0.01
139/824
0.48 (0.27–0.84)F
<0.01
POLE mut
0.09 (0.03–0.21)F
0.21 (0.07–0.54)F
MMRd
0.33 (0.22–0.49)F
0.64 (0.38–1.05)F
ProMisE-S (postsurgical)
NSMP/p53wt
150/852
0.3 (0.2–0.46)F
<0.01
139/824
0.51 (0.28–0.89)F
<0.01
POLE mut
0.16 (0.05–0.38)F
0.25 (0.08–0.63)F
MMRd
0.33 (0.22–0.49)F
0.62 (0.37–1.02)F
Very low-risk
0.05 (0.02–0.1)F
0.29 (0.11–0.73)F
* Adjusted for age, grade, histotype, LVSI and stage, F.
F indicates Firth bias correction; LRT, likelihood ratio test; MMRd, mismatch repair deficient; ProMisE, Pro active M olecular Ris k Classifier for E ndometrial Cancer.
We used ESGO/ESTRO/ESP guidelines 14 Table 5 ). Without molecular classification 35/89 would be assigned to a lower risk group representing patients who would potentially be undertreated. Conversely, 54/89 would be assigned to a higher risk group representing patients who would potentially be overtreated. Using the ProMisE-S classification all patients would have been placed within the appropriate ESGO/ESTRO/ESP risk category.
TABLE 5 -
Risk categorization of endometrial carcinoma with and without molecular classification
With refined ProMisE molecular classification, n (%)
Low
Intermediate
High-intermediate
High
Advanced metastatic
Total
Without molecular classification
Low
349 (89.9)
6 (4.6)
3 (1.3)
358 (40.0)
Intermediate
10 (2.6)
117 (90.0)
5 (2.2)
132 (14.7)
High-intermediate
21 (5.4)
0 (0.0)
123 (93.9)
21 (9.3)
165 (18.4)
High
8 (2.1)
7 (5.4)
8 (6.1)
198 (87.2)
221 (24.7)
Advanced metastatic
20 (100.0)
20 (2.2)
Total
388 (43.3)
130 (14.5)
131 (14.6)
227 (25.3)
20 (2.2)
896 (100)
DISCUSSION
The comprehensive genomic analysis of EC published by the TCGA discovered 4 nonoverlapping and prognostically relevant molecular groups 5 7,8,16–18 17,19,20 12,14,21
The benefits of this combined approach are, firstly, superior diagnostic reproducibility between pathologists and laboratories 21 22 4 6,14 23 24 25 POLE mut EC ‘s, account for 6% of EC’s overall 23 11 21,22 18 POLE mut EC patients, or the use of chemotherapy for all p53abn EC with myoinvasion, or a range of targeted therapies 19,20,26
We have previously developed 6 7 8 POLE mutation status, followed by the MMR status and, lastly, the p53 result 12 POLE testing is mandatory for the accurate classification of all patients.
There are major barriers to universal implementation of molecular classification. The prospective evaluation of this classification and its therapeutic implications remain to be seen and are the subject of numerous clinical trials worldwide. In the absence of prospective trial results, however, several clinical implications are already part of routine care, such as the need for referral to Clinical Genetics Services and consideration of immunomodulatory treatments in certain subsets of patients with MMRd EC. On the basis of the current state of knowledge, there are many potential implications for prognosis and clinical management. Financial constraints, a major barrier, require pathologists and laboratories to be looking to refine their diagnosis as much as possible through judicious use of available resources. While MMR and p53 IHC are relatively readily available, access to POLE testing, currently only achievable through gene sequencing and lacking an immunohistochemical surrogate, remains a challenge. Cheaper and accessible testing options for POLE testing are therefore needed, and in addition, there is a need to examine whether testing is necessary on all patients, or whether an amended protocol can diminish the burden of testing.
In this study we assessed the extent to which an amended ProMisE algorithm (termed ProMisE-S) can reduce the number of EC’s needing POLE mutational testing. We evaluated a selective testing algorithm, based on test accessibility, whereby MMR and p53 IHC would be carried out on all biopsies; with POLE status only assessed after excluding patients deemed “likely low-risk”: grade 1 or 2, endometrioid histology, p53 normal, MMR proficient. Following surgical staging, there would be no clinical benefit from knowing the POLE status of a “very low-risk” group, defined as fulfilling all of the above parameters as well as being stage IA, grade 1 or 2, with absence of LVSI. POLE testing would be recommended in all of the other substage and histotype designations for accurate risk assessment through complete molecular classification, where adjuvant treatment recommendations would differ based on POLE status.
Our findings were, firstly, that universal p53 IHC identified 15/166 (10%) p53abn ECs that were reported as grade 1/2 endometrioid tumours. These constituted <3% (15/564) of all grade 1/2 endometrioid EC in this cohort. We recommend universal p53 IHC in this algorithm to prevent missing any potential high-risk cases. It remains unknown whether p53abn grade 1/2 “endometrioid” EC have a prognosis and response to treatment equivalent to the p53abn EC group as a whole and thus are candidates for adjuvant chemotherapy. We are unable to address this issue satisfactorily based on this series as there are only 15 such cases and no slide review was performed to confirm the morphologic diagnosis; this is, however, and important area for future study. Only if the p53abn grade 1/2 “endometrioid” EC have the poor prognosis of p53abn EC would the expense of universal p53 IHC be justified.
It is also possible that the grade 1/2 EC with mutant pattern p53 staining can be recognized based on their nuclear features, as suggested by Kang et al. 27 18 27
Several studies have previously reported a low proportion of p53 abnormal IHC or TP53 mutation within morphologically low-grade EC with varying frequency 18,27–29 28,29
Our second finding was that using the ProMisE-S protocol at the time of diagnostic biopsy, POLE testing would be indicated on all patients other than those deemed “likely low-risk,” constituting 55% of the total. The rationale for carrying out POLE testing on p53abn and MMRd EC is to exclude cases showing these abnormalities secondary to an underlying POLE mutation, ie to exclude multiple classifiers and validate the p53 and MMR IHC result. The rationale for testing high-grade endometrioid and nonendometrioid cases would be to unmask potentially low-risk EC masquerading morphologically as high-risk. Thirdly, 134/405 (33%) of EC’s considered to be “likely low-risk” based on findings potentially available at the time of biopsy (namely histotype, grade if endometrioid, MMR and p53 IHC results) were found to be stage IB or higher and/or positive for LVSI; in these patients POLE testing would be recommended post-surgery, with possible impact on adjuvant treatment decisions. Overall, the adoption of the targeted ProMisE- algorithm would reduce the proportion of EC’s requiring POLE testing by a minimum of ~40%. In real life this proportion could be higher still for at least 2 reasons: this additional testing could be restricted to those patients in whom this information would truly impact on treatment decision-making, excluding those in whom adjuvant treatment would either be given or withheld, regardless knowledge of POLE mutational status. The second reason relates to the definition of “substantial” LVSI; in the retrospective cohorts studied in this project, LVSI is recorded as present versus absent, while current definitions of extensive or substantial LVSI would exclude cases showing only focal LVSI, as the latter finding does not affect risk categorization 14
We also compared risk categorization based on clinicopathologic parameters without and with molecular classification and found that knowledge of ProMisE results altered the risk category in 10% of all EC’s, with 4% moving to a higher risk group, as a result of universal p53 testing, and 6% moving to a lower risk group, as a result of POLE testing; these results would be unchanged using universal ProMisE or ProMisE-S approaches.
The ProMisE-S algorithm thus provides a pragmatic way forward to introduce molecular classification into routine diagnostic practice. It saves on unnecessary, POLE testing within the very low-risk group. It improves on the alternative of restricting molecular classification only on high-grade and high-risk EC by ensuring, through universal p53 IHC, that no EC’s belonging to the worst clinical category of p53abn EC are missed. It ensures that EC’s showing more than one of the key molecular changes, that is “multiple classifiers,” are accurately categorized. Overall the data directly supports ESGO/ESTRO/ESP recommendation of omitting POLE mutation analysis in low/intermediate risk EC, by providing objective histopathologic criteria. At present there are several possible approaches to molecular testing in EC, summarized in Table 6 . We present the results of retrospective evaluation of strategy 4 to demonstrate one economical approach.
TABLE 6 -
Possible approaches to move forward with molecular testing, depending on resources available; these in turn will be subject to changes in test costs and further refinement of molecular groups
Approach
Description
Comment
1. Continue with standard histologic classification (status quo )
Based on pathology and histomorphology alone
2. Universal MMR and p53 IHC (or low threshold for p53 IHC in all morphologically ambiguous cases), and accurate histologic diagnosis; no POLE testing
MMR/MSI testing is universally recommended by numerous national and international clinical management guidelines, and pathologists should have a low threshold for carrying out p53 IHC. This will increase diagnostic sensitivity for high-risk EC
POLEmut EC accounts for 12% of high-risk EC and 3%–5% of EC show multiple classifiers; these cases are potentially over-treated in the absence of POLE testing, estimated to be 6% of all cases based on our results
3. Universal MMR and p53 IHC and complete molecular classification (including POLE testing) of intermediate -high-risk cases in whom treatment modulation on the basis of molecular category is considered (Fig. 2 )
Cases classified as intermediate or high risk are currently offered additional radiation and/or chemotherapy. In some cases the decision to offer or withhold additional treatment will not be affected by the molecular classifier, ie these will be offered or will not be offered treatment regardless of molecular class. These can be classified based on given information. This will further narrow down the need for NGS testing to only those in whom treatment modulation will be considered based on NGS results: this may be due to patient age, comorbidities, patient/clinician choice or other factors
4. Universal MMR and p53 IHC and complete molecular classification of all cases other than very low-risk EC (Fig. 2 )
A majority of EC cases fall into a low-risk category and do not require adjuvant treatment. As long as universal p53 IHC is carried out to identify any potential high-risk cases that could be misclassified by histology alone
Based on our results this will diminish the POLE testing burden by ~40%
5. Universal ProMisE testing (Fig. 1 )
If resources permit testing could be carried out on all cases though a combination of POLE sequencing with MMR and p53 IHC
6. Universal NGS panel testing
In some health care settings and going forward it may be preferable and more cost-effective to carry out universal panel testing on all solid tumors, such that POLE mutational analysis, microsatellite instability testing and TP53 mutational analysis are included and molecular subtype can be assigned based on the panel results
This would need to be validated and compared with conventional ProMisE algorithm that had been developed according to Institute of Medicine guidelines through discovery, confirmation and validation phases that used POLE NGS and IHC for MMR and p53 proteins
EC indicates endometrial carcinoma; IHC, protein immunohistochemistry; MMR, mismatch repair; MSI, microsatellite instability; NGS, next-generation sequencing.
CONCLUSIONS
In conclusion, adoption of molecular testing has tremendous potential to provide reproducible diagnosis, inform prognosis, and impact treatment decisions. In the absence of unlimited resources for molecular testing in all EC, we present clinically relevant alternative strategies for selective testing that appear to be safe and informative. We also demonstrate the extent to which testing can be restricted while allowing accurate categorization into ESGO/ESTRO/ESP risk groups.
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