Biomarker characterization in endometrial cancer in Italy: first survey data analysis

This is an open access journal distributed in accordance with the CC-BY-NC-ND (Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International) license: the work can be used by mentioning the author and the license, but only for non-commercial purposes and only in the original version. For further information: https://creativecommons. org/licenses/by-nc-nd/4.0/deed.en Summary Objective. Endometrial carcinoma (EC) is the most common gynecological malignant disease in high income countries. The 2020 edition of the World Health Organization (WHO) Classification of Tumors of the Female Genital Tract underlines the important clinical implications of the new integrated histo-molecular classification system, in order to correctly define the specific prognostic risk group. This survey analysis will focus on the most commonly adopted immunohistochemical and molecular biomarkers used in daily clinical characterization of a diagnosed endometrial carcinoma in Italian labs.Methods. An evaluation questionnaire was distributed to 41 Italian pathology laboratories. Normal habits in EC evaluation, especially regarding mismatch repair status (MMR) and microsatellite instability (MSI), were collected. A summary and a descriptive statistical analysis were used to show the current practice of each laboratory. Results. The analysis of MMR status by immunohistochemistry (IHC) is carried out on the majority of all EC samples. The most frequent strategy for the analysis of MMR status in EC is IHC of four proteins (PMS2, MSH6, MSH2, MLH1). MSI analysis by molecular method in endometrial cancer is rarer and more restricted to some circumstances. Hypermethylation of the MLH1 promoter by methylation-specific PCR and pyrosequencing was analyzed in case of negative expression of MLH1/PMS2. Also, the analysis of p53 in EC is performed in the majority of cases. POLE mutational profiling is adopted only in a limited number of laboratories. Fifty-five percent of Italian laboratories refer to national/international guidelines when analyzing biomarkers in EC (among those, 45% use the ESGO Guidelines, 18% ASCO-CAP, 18% AIOM, 14% WHO, 5% British Association of Gynaecological Pathologist, 5% ESMO, 5% NCCN). Conclusions. Adoption of guidelines and standardization of pre-analytical and analytical procedures are effective tools for adequate EC prognostic risk stratification and high quality standard of care.

# Introduction
In 2013, The Cancer Genome Atlas (TCGA) Research Network discovered four molecular subtypes of ECgenomic architecture based, with each group having distinct clinical outcomes: POLE/ultra-mutated, MSI/ hypermutated, copy-number low/endometrioid and copy-number high/serous-like 1 . Following this discovery, the Leiden/PORTEC group and the Vancouver/ProM-isE group validated a more pragmatic, cost-effective and clinically applicable molecular classification, using surrogate markers to recapitulate TCGA subtypes. MMR and p53 immunohistochemical assessment has been used as surrogate of molecular testing of MSI and somatic copy-number alteration (SCNA), yielding four molecular subtypes: POLE-mutated, MMR-deficient (MMRd), p53-abnormal (abn) and no specific molecular profile (NSMP) [bib_ref] Confirmation of ProM-isE: a simple, genomics-based clinical classifier for endometrial cancer, Talhouk [/bib_ref] [bib_ref] Final validation of the ProMisE molecular classifier for endometrial carcinoma in a..., Kommoss [/bib_ref] [bib_ref] Improved Risk Assessment by Integrating Molecular and Clinicopathological Factors in Early-stage Endometrial..., Stelloo [/bib_ref]. This surrogate marker approach has been demonstrated to be prognostically informative in low-, intermediate-, and high-risk EC. Among high-grade ECs, the POLE-mutated group shows an excellent prognosis, while the p53-abn group shows the poorest prognosis [bib_ref] ESGO/ESTRO/ ESP guidelines for the management of patients with endometrial carcinoma, Concin [/bib_ref] [bib_ref] New Pathological and Clinical Insights in Endometrial Cancer in View of the..., Santoro [/bib_ref] [bib_ref] Clear cell endometrial carcinomas with mismatch repair deficiency have a favorable prognosis:..., Travaglino [/bib_ref] [bib_ref] TCGA molecular subgroups of endometrial carcinoma in ovarian endometrioid carcinoma: a quantitative..., D&apos; Alessandris [/bib_ref]. According to the 2020 5th edition of the WHO Female Genital Tumours, the EC molecular classification adds new information to the conventional morphologic features and should be integrated in the standard pathologic report. In view of the important prognostic and therapeutic implications, the 2020 ESGO/ESTRO/ ESP guidelines for the EC management defined new prognostic risk groups, integrating molecular markers [bib_ref] ESGO/ESTRO/ ESP guidelines for the management of patients with endometrial carcinoma, Concin [/bib_ref]. Today, morphology represents the framework in which molecular analysis should be interpreted [bib_ref] The emerging role of molecular pathology in directing the systemic treatment of..., Jamieson [/bib_ref] [bib_ref] The leading role of pathology in assessing the somatic molecular alterations of..., Matias-Guiu [/bib_ref] [bib_ref] Molecular diagnostics in pathology: time for a nextgeneration pathologist?, Fassan [/bib_ref] [bib_ref] The role of the pathologist in the next-generation era of tumor molecular..., Angerilli [/bib_ref]. Since the great importance given to this new new morpho-molecular pathologic approach, we decided to perform a survey among 41 Italian laboratories in order to investigate differences and similarities in the current pathologic practice regarding biomarker analysis in EC.

# Methods
The survey was focused on: - The immunohistochemical and molecular analysis in EC. - The different testing approaches depending on the laboratory setting. - The information about the type of technology used in molecular evaluation. From 2020 to 2021, we selected 41 Italian pathology laboratories (both public or Academic), equally located in all the country, to which a 12-item ques-tionnaire was distributed. Most of the questions were answered by selecting one choice. A summary and descriptive statistical analysis were used to describe the current practice of each laboratory. In accordance with the journal's guidelines, we will provide our data for the reproducibility of this study in other centers if requested.

# Results

# Mmr analysis
How do you perform MMR analysis? Do you routinely use IHC markers or molecular assessment? Most responders performed MMR analysis by IHC. In all, 47.5% of laboratories performed MMR analysis by IHC in all EC samples, and 32.5% in selected cases: morphological ambiguity, suspicion of Lynch Syndrome and early-stage cancers. 20% of centers did not use either IHC or molecular analysis.

# Ihc analysis: antibodies
In EC do you use an IHC panel? If yes, which clones do you prefer? 97% of Italian laboratories testing MMR used an IHC antibody panel and the most common is Ventana with the following clones: MLH1-M1 (69%), MSH2-G219-1129 (69%), MSH6-SP93 (69%), PMS2-A16-4 (69%). Other antibodies were described in. of labs perform their analysis using Leica antibodies, as second choice, with the clones: MLH1-ES05, MSH6-EP49, PMS2-EP51, MSH2-79H11. iHC analysis: platfoRMs What kind of immunohistochemical platform do you routinely use? Regarding platforms, the Ventana platform with Ventana clones was the most commonly adopted by the largest part of Italian laboratories testing MMR. Twelve labs used Ventana BenchMark ULTRA (38%), while in 10 centers (31%) Ventana BenchMark XT was preferred. Leica (Leica BOND-MAX and Leica BOND-III) was chosen in 6 cases (19%), and DAKO platform (Dako Omnis and Dako Link 48+ Leica BOND-MAX) in 5 cases (12%). All (100%) of laboratories used commercial kits of MMR testing. iHC analysis: waiting tiMe How many days does your laboratory take to give you IHC results? Most responders reported IHC results in a rapid turn- around time, within 1-3 days (62.5%). Some laboratories had longer times for analysis from 4-6 days (25%) to 7-10 days (12.5%). used Idylla MSI test (19%), 4 used NGS (15%). The Promega kit was used in 3 cases.

## Mmr analysis italian strategy
The most frequent strategy for MMR analysis in EC was IHC with four proteins (PMS2, MSH6, MSH2, MLH1), accounting for 58% of labs, followed by the MSI analysis alone (14%) or by the combination of IHC analysis and MSI testing (17%).

## Ec: selection of study cases
During the survey, 1096 ECs from the different labs were selected for the typology of MMR assessment: 706 cases were tested IHC (64%) and 123 by molecular analysis (11%), by confirming that in Italy IHC remains the 'first choice test' . In 267 cases no integrative analysis was performed.

## Combination of ihc and molecular analysis
In case of negative MLH1/PMS2 do you perform further analyses? 50% of labs testing MMR IHC did not perform further analyses in case of negative MLH1/PMS2, whereas 53% of hubs performed further molecular investigations. Other strategies included analyses of MLH1 promoter hypermethylation (28%), MLH1 and BRAF p.V600E evaluation (6%) or BRAF p.V600/ MSI + BRAF p.V600 Pyrosequencing/ RT-PCR/ Genetic counseling.

## Mlh1 promoter methylation analysis: methods

## How do you perform it?
The most frequent methods for the analysis of MLH1 promoter hypermethylation are methylation-specific PCR and pyrosequencing, respectively used in 38% and 31% of cases.

## Do you also routinely analyze p53 antibody in ec?
In EC molecular characterization, numerous other prognostic and diagnostic molecules are employed. P53, together with MMR proteins, represents the most studied, with 85% of Italian Labs analyzing it by IHC: an interesting data, when compared to other molecules, such as ER and PR, analyzed only by 10% of Italian labs. A large number of laboratories analyzed more than one biomarker.

# Discussion

## Summary of main results
The present study represents the first survey analysis addressed to 41 Italian labs, focusing on the most commonly adopted immunohistochemical and molecular biomarkers in daily clinical characterization of a diagnosed EC. The first emerging data regards the IHC that still turns out as the standard ancillary tool in diagnostic pathology. The advantages of the IHC-based approaches: - the ready availability; - its inexpensive nature; - rapid turnaround time;
- rapid identification of the defective protein; - correlation with morphology;
- the need of only a limited amount of tissue;
- feasibility for all types of FFPE specimens (small biopsies and/or surgical samples); - the propensity to IHC external quality assurance schemes. Moreover, the simultaneous presence of two or three molecular signatures has been identified in about 3% of EC cases, the so-called "multiple classifier": MMRd/ p53abn; POLEmut/p53abn; MMRd/POLEmut/p53abn; MMRd/POLEmut. In these latter subgroups, the clinical outcome is strictly related to the driver molecular subtype; in detail, pathogenetic POLE mutations prevail over the other signatures, conferring a good prognosis; on the other hand, the MMRd signature prevails over the p53abn signature. From our survey data, complete molecular characterization of EC, including POLE panel testing, is restricted only to 3 labs, where POLE analysis is performed on physician demand or in histologically selected cases.

## Results in the context of published literature
EC is the sixth most frequent cancer in postmenopausal women worldwide and the most prevalent cause of mortality in the developed countries, with 382,096 new cases and 89,929 deaths reported in 2018 [bib_ref] Cancer statistics, 2020, Siegel [/bib_ref]. Despite recent advances in molecular pathology and in therapeutical regimens, the number of new cases is expected to further increase, owing to the increase in obesity rates and the aging population [bib_ref] Worldwide burden of cancer attributable to diabetes and high body-mass index: a..., Pearson-Stuttard [/bib_ref]. To date, patients with stage III and IV cancers still experienced low 5-year OS rates, ranging from 57-66% and 20-26%, respectively, compared to early stage disease (stage I or II), with a 5-year overall survival ranging from 74-97% [bib_ref] Phenotype of POLE-mutated endometrial cancer, Imboden [/bib_ref].

# Strengths and weaknesses
Our study represent the first survey analysis that focused on the most commonly adopted immunohistochemical and molecular analysis in daily clinical characterization of EC in Italian labs. This study has been replicated in Spain and UK in order to compare the practices between different countries. Although complete histo-molecular classification of EC is highly recommended by the International guidelines, in Italy the complete panel, including POLE analysis, is adopted only by a minority of labs, because this molecular test is not still reimbursed by the National Health Service. Thus, it may be possible to restrict POLE sequencing to low-risk EC showing abnormal or subclonal p53 staining, and omitted in advanced (stage III-IV) ECs since adjuvant therapy is always performed regardless of molecular classification.

## Implications for practice and future research
Adoption of guidelines and standardization of pre-analytical and analytical procedure are effective tools for correct prognostic risk stratification. Therefore, it could be useful to build a network of certified centers for EC histo-molecular diagnosis and treatment, which can provide high quality standard of care, and offer patients the specific skills, experience, optimal levels of organization, and dedication.

# Conclusions
From our Italian survey, the IHC based method for the biomarker analysis in EC emerges as the preferred and adopted diagnostic tool by the majority of investigated labs. It appears manageable in clinical practice, providing high diagnostic accuracy and high inter-observer concordance only when grounded on validated optimized laboratory protocols, together with correct pathological interpretation, according to the well established guidelines [bib_ref] Interpretation of P53 immunohistochemistry in endometrial carcinomas: toward increased reproducibility, Kobel [/bib_ref] [bib_ref] p53 immunohistochemistry is an accurate surrogate for TP53 mutational analysis in endometrial..., Singh [/bib_ref]. The most common prognostic assessment strategy in EC in Italy includes the analysis of MMR by IHC in all samples, followed by the analysis of MLH1 promoter hypermethylation and/or BRAF mutation and the analysis of other biomarkers such as p53.
In this way, we are able to identify MMR deficient or proficient ECs, p53 mutant or p53 wild-type ECs and multiple classifiers such as MMR deficient/p53 mutant ECs.
In the precision medicine era, optimizing and ensuring high histopathological diagnostic quality is essential to improve the management and outcomes of EC patients. The pathology report still remains a major component of patient multi-disciplinary management and the quality of the histo-molecular approach can contribute to avoid suboptimal treatment, guide genetic investigations and address women to adequate therapies.
[fig] Figure 1: (A) MMRd by IHC in endometrial carcinoma: when? (B) MMRd by IHC in endometrial carcinoma: which antibodies? (C) MMRd by IHC in endometrial carcinoma: which platforms? (D) MMRd by IHC in endometrial carcinoma: TAT [/fig]
[fig] Figure 2: (A) MSI in endometrial carcinoma: when? (B) MSI in endometrial cancer: how? A B [/fig]
[fig] Figure 3: (A) MMRd/MSI analysis strategy. (B) MMR assessment in 1096 ECs from different Italian labs. [/fig]
[fig] Figure 4: (A) MLH1 promoter hypermethylation in EC: which technology? (B) Other biomarkers in endometrial carcinoma. [/fig]
[fig] Figure 5: International/national guidelines in EC. [/fig]