Endometrial cancer has been the most widespread gynecologic cancer in the United States of America and its incidence and disease-related mortality are constantly increasing. Latest medical studies have shown that early-stage endometrial cancer has been correlated to a promising five-year relative survival rate of 96%. As for the patients affected by distant metastases, the survival rate continues to be around 18%(1). According to World Health Organization (WHO), the prevalence of the endometrial cancer in Romania is 14.4/100,000 per year, with a rate of mortality of 1.7/100,000 women(2). Regarding the age group at diagnosis, the peak of incidence occurs in the age group of 60-64 years old(2).
Investigating immunomarkers involved in endometrial oncogenesis could help in the early detection and treatment of this condition, with a direct impact on prognosis, increasing life expectancy and quality of life. Although the early cases benefit from surgical management, there is currently no effective treatment for patients with recurrent or advanced disease(1,3).
A classification of endometrial cancer subtypes was proposed by Bokhman in 1983(4). It is known that there are at least two major types of tumors: type I tumors, which are mostly endometrioid carcinomas, account for about 80% of endometrial cancers and are generally associated with endometrial hyperplasia and metabolic syndrome; type II tumors, that can be serous carcinomas, clear-cell carcinomas or, most often, squamous cell carcinomas, and frequently develop in the atrophic endometrium(5). Histologically, type I tumors are predominantly well differentiated to moderately differentiated endometrioid tumors, at least 90% expressing moderate to high levels of estrogen receptors, and they have a better prognosis(6). In contrast, type II endometrial cancers are not associated with hyperestrogenism or endometrial hyperplasia and, histologically, these tumors are poorly differentiated(7). Most type II tumors have p53 mutations, but they almost never show microsatellite instability or mutations in the RAS and PTEN genes, as opposed to type I tumors(8).
The Cancer Genome Atlas (TCGA) conducted an extensive endometrial cancer molecular study. During this thorough research, 373 cases of endometrial cancers were classified into four clusters regarding the endometrial carcinoma: microsatellite instability (MSI) assays, exome sequencing, integrated whole-genome sequencing, proteomics and copy number analyses. Histologically, there were identified 373 endometrial cancers: 13 mixed histology cases, 53 serous and 307 endometrioid. The four clusters were categorized as: 1) copy-number high (predominantly serous histology, which represents 26%); 2) copy-number low (microsatellite stable [MSS], which represents 65%); 3) hypermutated/MSI (microsatellite instability-high [MSI-H], which represents 30%) and 4) ultramutated/polymerase e (POLE) – mutated, which represents 7%(9). The progression-free survival correlates with cluster, with POLE tumors offering prognosis that is significantly better than that in the other three groups and copy-number high tumor presenting the lowest survival rate(10,11).
Microsatellites are repetitive deoxyribonucleic acid (DNA) sequences with unknown functions in the genome that are stable throughout a person’s life. Instability involves a fluctuation in the length of the microsatellite and the genes that repair replication errors do not function normally. It occurs in approximately 15% of colorectal cancer cases, but also in other types of cancer, such as endometrial, glioma, melanoma, ovarian, prostate, pancreatic or brain cancer(12-14).
MSI is involved in the oncogenic mechanism of the endometrial cancer. It is characterized by anomalies in the number of microsatellites and occurs as a consequence of inactivating the DNA replication anomaly repair system(15). Genome instability changes have been detected in over 20% of endometrioid endometrial cancers(16). This entails the appearance of structural mutations of the genes that encode proteins from the structure of DNA that is damaged due to cell divisions.
Microsatellite instability testing
The instability test involves firstly the extraction of genomic DNA from the tumor, as well as from normal tissue; then, a polymerase chain reaction (PCR) test is performed, where capillary electrophoresis is used for the identification of five microsatellites: BAT25, BAT26, D2S123, D5S346 and D17S250. A tumor is considered to have a high degree of microsatellite instability when the instability is observed in over 30-40% of the microsatellites analyzed (at least two of the five microsatellites analyzed). When instability is not noticeable in any of the microsatellites, the tumor is considered a stable microsatellite tumor and, if one of the five microsatellites has instability less than 30-40% of those analyzed, it is a tumor with low microsatellite instability(17).
Determining microsatellite instability is important for identifying patients with a high risk of hereditary cancer associated with Lynch syndrome, for the prognosis of patients with endometrial cancer and for the response to immunotherapy. Some studies have tried to reveal that the presence of MSI could have a beneficial effect in endometrial cancer with programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) immunotherapy(18). Thus, in the group of the analyzed patients, 28.2% had loss of mismatch repair (MMR) protein expression and there was no statistically significant relationship between MSI status and age (p=0.193), the International Federation of Gynecology and Obstetrics (FIGO) stage (p=0.508), the histological grade (p=0.097), the depth of myometrial invasion (p=0.494) or pelvic lymph node metastases (p=0.139)(19). Also, in the MSI group, a higher expression rate of PD-L1 and PD-1 was demonstrated (p=0.008), compared to the stable microsatellite group (p=0.001)(19).
Lynch syndrome (hereditary non-polyposis colorectal cancer)
Lynch syndrome is an autosomal dominant syndrome that results from mutations in the MLH1, MSH2, MSH6 and PMS2 genes, being one of the most common syndromes that predisposes to hereditary cancer(20). The risk of cancer and the clinical phenotype vary depending on the mutation. With the introduction of immunotherapy for advanced disease, the treatment of tumors associated with Lynch syndrome has changed(17).
The carriers of these mutations have a 40-60% risk of developing endometrial cancer and less than 5% of endometrial cancers are attributed to Lynch syndrome, mostly family cases occurring in perimenopause(21,22). In affected women, the risk of endometrial cancer exceeds that of colorectal cancer, and annual screening is recommended from the age of 30 years old by endometrial biopsy(23). The diagnosis of Lynch syndrome can be established based on the Amsterdam clinical criteria or by genetic testing(24). According to the Amsterdam clinical criteria, Lynch syndrome occurs in at least two successive generations up to 50 years(25).
New molecular classification studies describe tumors with microsatellite instability as hypermutated tumors(9). Tumors in this group are characterized by microsatellite instability that is caused by methylation of the MLH1 promoter(24). These tumors have a high degree of phosphoinositide 3-kinase catalytic subunit a (PIK3CA) expression and a low level of PTEN expression(26). KRAS and FGFR 2 mutations are more common in MSI tumors(27). Tumors with a low degree of differentiation (G3) show a microsatellite instability with a higher frequency than tumors with a high degree of differentiation (G1)(28).
Researchers identified seven different MMR genes which cause the appearance of non-polyposis colorectal cancer: hMLH1 on band 3p22; hMSH2 and hMSH6 on band 2p16; hPMS1 on band 3p32; hPMS2 on band 7q22; hMSH3 on band 5q14.1; EXO1 on band1q43(1,15,29). In endometrial cancer, isolated loss of immunohistochemical expression of a single gene or combinations of several genes may occur(30,31).
The role of MMRs is to eliminate the base-base pairing errors and the insertion-deletion loops that occur as a result of DNA replication errors (DNA polymerase). The result of altered function of these genes is the appearance of nucleotide substitutions (G-T), the deletion or insertion of short repetitive units (such as CA) in the composition of microsatellites(32). In endometrial cancer, the following genes are analyzed: MLH1, MSH2, MSH6 and PMS2(33). The loss of nuclear positivity of PMS2 (DNA mismatch repair proteins) indicates an increased probability that tumor proliferation will present high microsatellite instability (MSI-H) and Lynch syndrome(12-14). According to some studies, epigenetic changes of the MLH1 locus may be a pathway to tumorigenesis rather than inherited mutations(15).
Impact on therapy
POLE-mutated and MSI endometrial cancers are associated with high numbers of tumor-infiltrating lymphocytes and with high neoantigen loads, which suggest that these patients would have a good response to immunotherapy, in particular with programmed cell death protein 1 (PD-1)/programmed death-ligand (PD-L1) checkpoint inhibitors(34,35).
Immune checkpoint inhibitors, such as anti-PD-ligand 1 and anti-PD-1 antibodies, fall into the area of interest of new anticancer drugs. Lymphocytes (CD8+) have been shown to infiltrate the tumor in endometrial cancer patients with MSI(19). Immunohistochemical studies have detailed PD-1 and PD-L1 expression levels (40-80% in endometrioid, 10-68% in serous, and 23-69% in clear cell subtypes, respectively) in endometrial carcinomas, representing the highest expression among gynecologic cancers(36,37). This combination of increased mutational load, tumor-infiltrating lymphocytes and PD-1/PD-L1 expression makes endometrial cancer an ideal target for immunotherapeutic interventions(34).
Lenvatinib is an oral multikinase inhibitor that targets vascular endothelial growth factor receptors 1-3, platelet-derived growth factor receptor-a, fibroblast growth factor receptors 1-4, RET and KIT(38,39).
The combination of lenvatinib and pembrolizumab is now under study in two ongoing phase III trials: lenvatinib with pembrolizumab versus doxorubicin or weekly paclitaxel in advanced endometrial cancer previously treated with platinum-based therapy and first-line lenvatinib with pembrolizumab versus carboplatin and paclitaxel chemotherapy in advanced endometrial cancer(40). The combination of immune checkpoint inhibitors with lenvatinib has been evaluated in preclinical mouse xenograft studies(41). In these studies, lenvatinib plus PD-1/PD-L1 signal inhibitors had more potent antitumor activity than either agent alone(41,42).
In the United States of America, Food and Drug Administration approved the use of pembrolizumab, which is an anti-PD-1 monoclonal antibody, in cases of patients with MSI endometrial cancer who do not have another option for treatment or have relapsed or progressed after a previous treatment(9).
The results of the phase II KEYNOTE-158 trial, published in January 2020, which investigated the clinical efficacy of pembrolizumab monotherapy across 27 advanced MSI-H or mismatch repair deficiency (MMRd) solid tumors, supported the hypothesis that MMRd tumors are sensitive to ICB (immune checkpoint blockade), independent of the primary tumor site(43). There were 49 patients with endometrial cancer included in the study, with a general response level of 57%, including 41% (20 patients) with a partial response and 16% (eight patients) with a complete response(44).
The prognosis of patients with endometrial cancer according to MMR status/MSI
According to The International Society of Gynecological Pathology (ISGyP), testing for MMR status/MSI has been strongly advised. Thus, this recommendation has been applied for the entire spectrum of endometrial carcinoma samples, regardless of age(45). In patients with endometrial carcinoma, testing for MMR status/MSI has proven to be important for the following reasons: pre-screening, to identify patients at higher risk for having Lynch syndrome; diagnostic, as MMRd/MSI is considered a marker for endometrioid-type endometrial carcinoma; prognostic, as identified by TCGA and predictive for possible utility of immune checkpoint inhibitor therapy. Germline mutations of one of the MMR genes MLH1, PMS2, MSH2 and MSH6 are responsible for approximately 10% of MMRd/MSI endometrial carcinomas and for approximately 3% of all endometrial carcinomas(24).
The preferred approach to identifying patients with a higher chance of having Lynch syndrome is by MMR-immunohistochemistry (IHC) on well-preserved tumor tissue. ISGyP guidelines recommend that MMR-IHC be chosen, as it offers valuable pieces of information on the altered gene/protein. Moreover, it has been proven to be an accurate method to evaluate MMR status(45). In order to identify patients at high risk of Lynch syndrome, there are generally used clinical criteria such as Amsterdam II and the Bethesda criteria(46). According to latest medical studies, the use of family history criteria alone fails to diagnose up to 80% of endometrial cancer patients with Lynch syndrome and 24% of Lynch syndrome patients are diagnosed with endometrial cancer after the age of 60(31). Therefore, tumor testing for MMR or MSI status has become a well-established mainstay in endometrial cancer, especially now that MSI status has therapeutic implications(47).
The age of cancer onset in Lynch syndrome varies among specific mutated genes and types of mutations(48). For endometrial carcinoma, the cumulative incidences at 70 years old are: 34% for MLH1 mutation carriers, 51% for MSH2 mutation carriers, 49% for MSH6 mutation carriers, and 24% for PMS2 mutation carriers(49). According to Ryan et al., a constant gynecological monitoring is recommended from the age of 40 for the patients with MSH6 and truncating MLH1 mutations, from the age of 35 for the ones with nontruncating MLH1 mutations, and from the age of 30 for MSH2 mutations(50). As for the patients with Lynch syndrome or hereditary non-polyposis colorectal cancer, Lachiewicz et al. mentioned that the risk of any occult malignancy during prophylactic surgery may increase to 17%(50).
Endometrial cancer patients with MSI-high and/or MMR deficient tumors appear to have better responses to checkpoint inhibitor therapy, but some MSI-high/MMR deficient endometrial cancer patients do not respond well to checkpoint inhibitors(47). Regarding the treatment with checkpoint inhibitors, in the Le et al. study, the objective response rate for endometrial cancer patients reached a remarkable rate of 53%, but approximately 20% of the endometrial cancer patients with MMRd/MSI-high still had progressive disease after the treatment with pembrolizumab(35). After the use of pembrolizumab in clinical practice, carrying out tumor testing in patients with endometrial cancer who have recurrence or progression of disease makes practical sense(47).
MSI and MMR protein assessments already have been evaluated extensively in patients with endometrial cancer. Given the great potential for immunotherapy in patients with endometrial cancer demonstrating MSI-high/MMR deficiency, expanding these assessments for the purpose of therapeutic decision makes good clinical sense(47).
Although ICB monotherapy has resulted in modest activity in advanced endometrial cancer, numerous combination strategies which have the potential to alter the treatment landscape – such as targeted therapies, other immunotherapeutic agents, chemotherapy and radiation therapy – are currently investigated(40). The key for advancing in this field will be the optimal patient selection based on tumor molecular and immunophenotypic properties (tumor mutational burden, MMR status, PD-L1 status and tumor-infiltrating lymphocyte classification). At this time, studies are being conducted in order to improve the activity of immune control point inhibitors by associating them with other chemotherapeutic agents or poly ADP-ribose polymerase inhibitors (PARP inhibitors)(9).
Taking into account the data published in literature up until the present time, we consider that there is a strong evidence to further support the widespread implementation of the molecular screening for all women diagnosed with endometrial cancer. Moreover, once patients are found to be at risk for Lynch syndrome through positive screening, an enhanced effort to counsel them during follow-up (including genetic counseling and testing) is of paramount importance in order to improve their prognosis.
Conflict of interests: The authors declare no conflict of interests.