Mismatch repair status in Endometrioid type of Endometrial Carcinoma: association with clinicopathological parameters

Shorouk E. Mwafy, Noha El-Anwar, Asmaa M. Eid

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IJCBR Editor, Prof. Mohamed Labib Salem, PhD Professor of Immunology Faculty of Science, Tanta Universiy, Egypt

INTERNATIONAL JOURNAL OF CANCER AND BIOMEDICAL RESEARCH (IJCBR)

RESEARCH ARTICLE

Mismatch repair status in Endometrioid type of Endometrial Carcinoma: association with clinicopathological parameters

Shorouk E. Mwafy, Noha El-Anwar, Asmaa Mustafa Eid Pathology Department, Faculty of Medicine, , Egypt

Introduction: Endometrial carcinoma [EC], particularly the most predominant endometrioid type [EEC] is a major contributor to cancer burden globally, and its molecular classification has gained much importance recently. Aim: This study aimed to determine the immunohistochemical expression of mismatch repair proteins 'MLH1 and MSH2' in relation to clinicopathologic parameters in EEC and to characterize clinicopathologic features of mismatch repair protein (MMRP) deficient EEC. Material and Methods: The current work was carried out on 80 cases of EEC retrieved [with clinical data] from the Department of Pathology, Faculty of Medicine, Tanta University in the period from June 2018 to December 2019. H&E staining and immunohistochemical staining with MLH and MSH2 were done for each

case. Results: 29 (36.3%) carcinomas showed abnormal MMRP expression (11 cases showed isolated MLH1 deficiency (37.93%), 10 cases showed isolated MSH2 Article history

deficiency (34.48%), and 8 cases (27.59) showed a combined loss of both proteins), Received: July 10, 2020 whereas the remaining 51 (63.7%) of cases demonstrated normal MLH1/MSH2 Revised: August 3, 2020 immunoreactivity (MMRP intact). MLH1, MSH2 expression, and MMRP status were Accepted: August 30, 2020 closely related to some clinicopathologic features (patient’s age, histopathological tumor grade, and tumor stage) with a statistically significant relation. Conclusions: A Correspondence to: subset of endometrioid type EC demonstrates MMRP defect; the MMRP deficient Shorouk Ezz Eldin Mwafy, MD EEC often displays adverse clinicopathological parameters as poorly differentiated Pathology Department , or undifferentiated histology, an advanced stage with young age at presentation. Faculty of Medicine , Tanta University, Egypt Keywords: Endometrioid type endometrial cancer; mismatch repair proteins; MLH1 Tel.: +201028460400 and MSH2 E-mail: [email protected] Editor-in-Chief: Prof. M.L. Salem, PhD - Article DOI: 10.21608/JCBR.2020.35282.1053

INTRODUCTION The Mismatch repair (MMR) system is a strand– Endometrial carcinoma is the most common specific DNA repair mechanism that functions to gynecologic malignancy in the world in which maintain genomic integrity by correcting base incidence is rapidly increasing (Ferlay et al, substitution as well as small insertion–deletion 2019). Endometrial cancer arises from the inner mismatches. Such mismatches are generated by layer (endometrium) of the uterus that errors during DNA replication in tandem repeats represents approximately 90% of uterine known as microsatellites. Several MMR genes malignancies (Ritterhouse and Howitt, 2016). are identified, but four only are of most clinical Endometrial carcinoma is classically classified interest [MLH1, MSH2, MSH6, and PMS2] into two types. Type I tumors, estrogen–related, (McAlpine et al, 2018). characterized by endometrioid histology and Tumor DNA can be classified as microsatellite– favorable prognosis. On the contrary, type II stable (MSS), low-level MSI (MSI–L) and high- tumors, estrogen–unrelated, are characterized level MSI (MSI–H). Mismatch repair deficiencies by non-endometrioid histology and poor can result from i) an inherited cancer syndrome prognosis (Alvarez et al, 2012). Recently, the (e.g., Lynch syndrome), ii) acquired/somatic genetic and molecular basis of EC classification mutations or iii) epigenetic events e.g. has gained much importance; with methylation of one of the genes involved in microsatellite instability (MSI) being a major mismatch DNA repair (Hegde et al, 2014). MMR cornerstone.

IJCBR (jcbr.journals.ekb.eg) is published by Egyptian Society of Cancer Research (eacr.tanta.edu.eg) and sponsored by the Egyptian Knowledge Bank (www.ekb.eg) Mwafy et al., 2020

function defect has been detected in peroxidase for 10 min, slides were incubated approximately 20% to 30% of endometrial with the primary antibody for 2h at room cancers (McAlpine et al, 2018). Histologically, temperature. Rabbit polyclonal antibody endometrial carcinoma with MSI or MMRP against MLH1 (Kit no. E17810. Spring defect is mostly of endometrioid type (Köbel et bioscience, USA), and a rabbit polyclonal anti- al, 2017). Although genetic testing is important body against MSH2 (Kit no, E17790. Spring for detection of MSI, loss of expression of bioscience, USA) were used. Then, slides were mismatch repair proteins MLH1, MSH2, MSH6 washed in phosphate-buffered solution (PBS), and PMS2 by immunohistochemistry (IHC) may incubated with the biotinylated secondary be used as a surrogate marker for MSI (Hashmi antibody, followed by avidin–biotinylated et al, 2018). Detection of patients with MSI–high peroxidase complex, and finally, DAB endometrial cancers due to germline mutations (diaminobenzidine tetrachloride) was is important; as these patients are predisposed developed as the chromogen. The sections were to develop a variety of other malignancies, counterstained with hematoxylin, dehydrated especially colorectal carcinoma (Okoye et al, with graded ethanols and xylene and then 2016). Suggested universal screening targeting mounted with a coverslip. the four MMR proteins (MLH1, MSH2, MSH6 Evaluation of immunohistochemical staining and PMS2) is recommended (Goodfellow et al, 2015). The corresponding normal tissue was considered as an internal positive control The association between MMR status and [stromal cells and infiltrating tumor outcome in endometrial carcinoma remains lymphocytes]. Loss of MMR proteins expression unclear. The aim of the current study was to was defined as the absence of nuclear staining evaluate the MMRP status in relation to in tumor cells (less than 10% of cells), with clinicopathological parameters in endometrioid preservation of nuclear staining in adjacent non type of endometrial carcinoma. neoplastic cells (Shikama et al, 2016). Cases with MATERIAL AND METHODS positive IHC staining of MLH1 and MSH2 proteins were regarded as intact MMR protein This retrospective study included 80 specimens (MMRP intact). Cases with negative staining of of endometrial carcinomas of endometrioid one or both MMR proteins were interpreted as subtype. Formalin–fixed paraffin embedded MMRP deficient (Tangjitgamol et al, 2017). blocks were collected from Pathology Department, Faculty of Medicine, Tanta Statistical analysis University in the period from June 2018 to Statistical analysis was performed using December 2019. For each specimen, the block Statistical Package for Social Science (SPSS best representing the tumor was selected. version 23, IBM corp., Armonk, New York, USA). Detailed clinicopathological data of these Categorical variables were expressed as patients were obtained from their medical frequencies and percentages, whereas mean + reports. Endometrioid adenocarcinomas were SD was used to express continuous variables. subclassified into three grades (G1, G2 and G3) Chi-square (X2) test was performed for according to the International Federation of comparing categorical variables. Fisher's exact Gynecology and Obstetrics (FIGO) criteria test was applied when one of the expected (Kurman, 2014). The staging was performed frequencies was ≤ 5. P values of < 0.05* were based on FIGO 2009 criteria (Creasman, 2009). considered statistically significant. Immunohistochemical staining RESULTS Sections were deparaffinized in xylene then 1. Clinicopathological Characteristics rehydrated in a series of ethanol for 5 min each. The clinical and pathologic features of the For antigen retrieval, slides were subsequently included cases are represented in Table 1. Forty- incubated in modified citrate buffer for 40 five (56.3%) of the patients aged ≥ 60 years. 45 minutes. After blocking of endogenous cases were moderately differentiated peroxidase activity with 3% hydrogen carcinomas [grade 2]; representing 56.3% of

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cases, stage I was the predominant stage among and isolated loss of MSH2 expression was cases (51.2%); 46 cases (57.5%) showed detected in 10 cases (34.48%); combined loss of lymphovascular invasion, whereas myometrial MLH1/MSH2 was detected in 8 cases (27.59%). invasion [more than 1/2 myometrial thickness] MMRP deficient was found to be significantly was reported in 41 (51.3%) of cases. associated with patients aged <60 years than

the older age group (p=0.001*). MMRP Table 1. Clinicopathological features of the studied cases deficient was also found to be significantly <60 35 (43.7%) Age related with advanced FIGO stage and high ≥60 45 (56.3%) tumor grade (p=0.004*, p<0.001*), Grade I 14 (17.5 %) respectively. No significant association was Grade Grade II 45(56.3 %) Grade III 21(26.2 %) found regarding myometrial invasion and Stage I 41 (51.2%) lymphovascular invasion (p=0.054 and 0.431), Stage II 25 (31.2%) respectively. Stage Stage III 9 (11.2 %) DISCUSSION Stage IV 5 (6.2 %) <1/2 39 (48.7%) Myometrial invasion Type 1 endometrial carcinoma or the so-called >1/2 41 (51.3%) endometrioid type endometrial carcinoma Lymphovascular Positive 46 (57.5%) [EEC] typically arises through the progression of Negative 34(42.5%) invasion a precursor lesion. DNA mismatch repair system helps to prevent tumor progression by 2. Immunohistochemical results correcting errors that occur continuously during The immunohistochemical expression of MMRP DNA replication; and accounts for a in relation to clinicopathological features of the considerable proportion of endometrial cancers patients in this study was illustrated in Table 2 (Wong et al, 2016). Microsatellite instability and Figure 1. (MSI) is a hyper–mutable phenotype caused by defects in DNA mismatch repair due to the Among 80 cases, 29 (36.3%) were considered inactivation of one of mismatch repair genes: MMRP deficient, isolated loss of MLH1 most importantly MLH1, MSH2, MSH6 and expression was detected in 11 cases (37.93%) PMS2 (Coppedè et al, 2014).

Table 2. Expression of MMRP in relation to Clinical and pathological features of EEC cases

MMRP Total p-value Negative Positive 80 29 (36.25%) 51 (63.75%) (100%) Age <60 20 (57.1%) 15 (42.9%) 35 (100%) 0.001* ≥60 9 (20.0%) 36 (80.0%) 45 (100%) Grade Grade I 1 (7.1%) 13 (92.9%) 14 (100%) <0.001* Grade II 11 (24.4%) 34 (75.6%) 45 (100%) Grade III 17 (81 %) 4 (19%) 21(100%) Stage Stage I 8 (19.5%) 33 (80.5%) 41(100%) 0.004* Stage II 11 (44%) 14 (56%) 25(100%) Stage III 7(77.8%) 2 (22.2%) 9 (100%) Stage IV 3(60%) 2 (40%) 5(100%) Myometrial <1/2: 10 (25.6%) 29 (74.4%) 39 (100%) 0.054 invasion >1/2: 19 (46.3%) 22 (53.7%) 41(100%) Lymphovascular Positive 15 (32.6%) 31 (67.4%) 46 (100%) 0.431 invasion Negative 14 (41.2%) 20 (58.8%) 34 (100%)

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A D A A

B E A A

C F A A

Figure 1. Immunohistochemical expression of mismatch repair proteins (MMRP) in endometroid type of endometrial carcinoma showing intact nuclear expression of (A) MLH 1 in grade I EEC(x200), (B) MSH2 in grade II EEC (x200), and (C) MLH1 in grade II EEC (x400). Loss of expression of (D) MSH2 in grade II EEC (x400), (E) MLH1 in grade III (x200) and (F) MLH1 in grade III (x400).

A possible potential genetic background is the Tumor microenvironment suppresses DNA argued phenomena of genetic instability. repair pathways to help genomic instability and Genetic instability is thought to be a stamp of encourage tumor proliferation and survival some human malignancies (Rajagopalan et al, (Tian et al, 2015). In colorectal carcinoma, MSI 2003). Lack of genetic stability occurs frequently may be associated with lower stages despite in many types of cancers, including endometrial poor differentiation (Perea et al, 2014). carcinoma and colorectal carcinoma (Black et al, 2006).

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MSI is corresponding to the loss of MMRP deficiency was significantly associated immunohistochemistry (IHC) staining of one of with postmenopausal status. Ring et al, 2013 the mismatch repair genes; most frequently and Ruiz et al, 2014 found no significant MSH2 and MLH1 (De la Chapelle and Hampel, association between age and MMRP defect. The 2010). Genetic analysis of MSI status is great differences encountered between various expensive and requires long turnout time and reports and the current studymay be explained specialized laboratories. Therefore, recently, it by including other histopathological tumor cannot be performed routinely on various variants. carcinomas. Immunohistochemical analysis of Concerning the relationship of MMRP and other MLH1 and MSH2 protein expression clinicopathological features; the current study representing a rapid, easier, less costly, as well observed significant or highly significant results as sensitive and specific alternative method for with inverse relationship; regarding both tumor the detection of tumors of the mutator grade and stage. Abnormal MMRP is phenotype, and it could be performed by characterized by a high proportion of poorly histopathology laboratories as a routine differentiated tumors and advanced stage at diagnostic test (Tangjitgamol et al, 2017). diagnosis; regarding grading of EEC, MMRP The present study investigated the defect increase in frequencies with increasing immunohistochemical expression of mismatch grades as follows 7.1% vs. 24.4% vs. 81.0% in repair proteins (MLH1 and MSH2) in EEC; grade I, II and III, respectively, P<0.001*. abnormal MMR proteins (MMRP) expression Regarding myometrial invasion and was detected in 29 (36.3%) of the included lymphovascular invasion, there was no cases. Previous reports investigating the significant relation between MMR status and expression of MMR proteins using IHC have them. Previous studies reported that the MMRP shown that approximately 16% to 45% of defect EC had more aggressive features as grade endometrial cancer had MMRP deficient status III cancer, deep myometrial invasion, more (Grzankowski et al, 2012 and Kato et al, 2015). frequent LVI, and more advanced stage (Ring et In a study by Long et al, 2014, approximately al, 2013 and Shia et al, 2013). A close study to 23.7% of EC patients showed abnormal the current work done by An et al, 2007, which expression of MMRP by IHC, also, Buchanan et included only the endometrioid subtype, al, 2014 reported a rate of IHC abnormality in showed an increase in the frequencies of MSI– 29% of their cases. The different proportions high phenotype in higher histologic grade (13% encountered between various reports may be vs. 21% vs. 50% in histologic grade I, II, and III, due to contribution of other features: for respectively. The MSI–high phenotype was example, age, history of other cancers in related to the presence of lymphovascular individuals and cancers in the family which are invasion, deep myometrial invasion, and the associated with higher genetic risk, higher clinical stages. Hashmi et al, 2019, histopathological tumor types and grades of reported that MMRP defect was related to high differentiation, and even screening techniques FIGO stage, but no relation with tumor grade. used. There was a similarity in the frequency of Another study by Tangjitgamol et al, 2017 found MMRP deficient status in the present study and that early stage, more endometrioid histology, the previous reports, suggesting no ethnic and lower grade tumor were associated with difference of frequency in MMRP among the MMRP deficiency. Kim et al, 2018 pointed to a studied endometrial cancers. contradictory data as they reported that MMRP defect was associated with a higher histologic In the current work, the percentage of MMRP grade (G2–3), yet a lower FIGO stage (I–II).On deficient tumors was associated significantly the other hand, Ruiz et al, 2014 did not find a with young aged patients (<60) as p-value correlation between MMRP and =0.001. Similar results were observed by histopathological grade, myometrial invasion, Shikama et al, 2016 and Tangjitgamol et al, lymphovascular invasion or clinical stage. 2017; who reported increased rates of MMRP defects among young EC patients. In contrast to A significance of MSI testing is the therapeutic their data, Kim et al, 2018 demonstrated that benefit of various targeted therapies (including

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