Analysis of prognostic factors for endometrial cancer, taking into account the insulin-like growth factor IGF-1,2, its activation pathway and related genes in the context of the involvement of Forkhead box and FGFR2 proteins in the treatment of endometrial cancer

Entry

Endometrial cancer (EC) is one of the earliest described cancers, with a history dating back to ancient times. The first records indicating the existence of this disease date back to ancient Egypt, and further mentions can be found in the works of Hippocrates [1]. The first scientifically confirmed descriptions (EC) date back to the 18th century, described by the English physician Richard Mead. [1]. In recent years, molecular and genetic research on endometrial cancer has brought new discoveries, enabling the identification of key changes in the genetic code and molecular mechanisms that play an important role in the development and progression of this disease. These new insights open up perspectives for the development of targeted therapies that focus on the specific genetic and molecular changes characteristic of this disease [2].

Despite progress in diagnosis and treatment, endometrial cancer still remains one of the most common cancers in women around the world. The increase in incidence is visible mainly due to the increase in the number of postmenopausal women and the obesity epidemic. The incidence of EC in the world varies significantly, both at the level of continents and individual countries. About 60% of cases of endometrial cancer are reported in countries with developed economies, which are characterized by the so-called Western lifestyle. The highest incidence rates of this type of cancer are observed in North American countries, Europe and Australia [3]. The average level of prevalence is typical for the countries of East Asia, Southeast Asia, Latin America, South America, Australia and New Zealand. The lowest rates are recorded in Asian and African countries [4]. Mortality due to this cancer, similarly to its morbidity, is the highest in the economically developed countries of North America and Europe, and the lowest in the underdeveloped countries of Africa [5].

In 2020, 19.3 million new cases of cancer were recorded worldwide, 10 million of which resulted in death. In the case of endometrial cancer, 417,367 are new cases, of which 97,370 resulted in death [5]. Analyzing the data of the National Cancer Registry, National Institute of Oncology in Poland for 2020, we observe, in accordance with the standardized mortality rate (with the ESP2013 model), that endometrial cancer occurred in 9 out of 100 women. It was diagnosed in 7.4% of women, with a rate of deaths directly attributable to the disease of 4%. Studies in Poland show different morbidity and mortality rates depending on the age group. In the group of middle-aged and oldest women, the proportions of cases and deaths caused by EC ranged from 9% to 3% and 7% to 4%. These data emphasize the need for further research and preventive activities aimed at women in older age groups [6].

More than 90% of endometrial cancer cases are diagnosed in women of perimenopausal age, i.e. between 55 and 70 years of age. Premenopausal cases constitute 10-15% of all cases, which is related to the influence of long-term anovulatory cycles (e.g. polycystic ovary syndrome - PCOS) [7]. Approximately 3% of EC cases are associated with germline mutations in the MMR genes (MLH1, PMS2, MSH2 and MSH6) responsible for Lynch syndrome. The risk of developing the disease before the age of 40 is no more than 4% of all cases, and 70% of patients are childless at the time of diagnosis [8]. Risk factors include: hyperestrogenism (estrogen therapy without gestagens), estrogen-secreting tumors, obesity, lack of offspring, infertility (especially in PCOS), and improper nutrition [9]. Other factors include earlier menarche, later menopause, components of the metabolic syndrome, genetic predisposition (especially Lynch syndrome), tamoxifen use, and CYP450 gene polymorphisms. Protective factors include: shorter reproductive period, having many children, hormone replacement therapy, intrauterine devices or oral contraception, as well as healthy eating habits and smoking [7].

Objective of the work

The aim of the study was to explore the prognostic factors of endometrial cancer, with particular emphasis on IGF-1,2 and the activation pathway, and to investigate the role of Forkhead box and FGFR2 proteins in key cellular processes related to the development of this cancer.

Material and methods

The study included 306 Caucasian women diagnosed with confirmed endometrial cancer at the WSZZ in Kielce between 2005 and 2017. The patients were operated on in two facilities, i.e. ŚCO and WSZZ in Kielce. The patients underwent total hysterectomy with pelvic lymphadenectomy, computed tomography of the abdomen and chest, typical blood tests and endoscopic examinations. Each case was rediagnosed according to the eighth edition of the TNM classification.

The study included a cohort of 103 white patients, aged 48 to 99 years, with various types of endometrial cancer, full clinical history, hospitalized at WSZZ in Kielce in 2005-2017. All patients received typical surgical treatment without prior radiochemotherapy. This approach allowed for a reliable comparative analysis of tumor characteristics in terms of treatment and unchanged molecular profile.

Results

The results of the analysis showed that the dominant group in the studied cohort were women aged 61-80, representing 62% of the total. As many as 97% of them were overweight or obese (BMI > 30). The mean overall survival (OS) was 8.23 years. Recurrence-free survival (RFS) was observed in only six units, with an average duration of 4.6 years.

In the analyzed cohort of patients, the incidence of microsatellite instability was identified at 28%. All tumors expressing MLH-1 without mutations in the analyzed molecular panel were characterized by a high grade of endometrial cancer malignancy (p<0.01). Multivariate analysis revealed significant associations, where MLH-1 correlated with FGFR-2 mutation and led to higher tumor grade (p < 0.01). [10,11].

In the study group, 14 mutations were identified in the genes tested. The most common missense mutations were single nucleotide polymorphisms (SNPs). In many cases, more than one mutation was found.

Statistical analysis did not show a significant relationship between the type of mutation and the stage of cancer advancement, recurrence-free survival (RFS) and overall survival (OS) with a value of (p>0.05) [10].

Multivariate analysis showed a significant relationship between the degree of tumor differentiation and the presence of mutations in the FGFR2 and TP53 genes with a value of (p<0.01). Moreover, FGFR2 mutation was observed more often in cases of more advanced tumors with a value of (p< 0.01). Mutations in the FGFR2 genes showed a strong correlation with the advancement of the cancer [11]. In the examined material, we found 10 cases of G1 endometrial cancer, which had a good follow-up period of up to 15 years, without mutations in the analyzed gene panel. Kaplan-Meier analysis of FGFR-2 genes (HR = 0.40; 95% Cl: 0.142–1.144; p= 0.975) did not show significant results, but the effect of FGFR-2 on overall survival (OS) was more significant than in other cases. In this group, a strong correlation was observed between the stage of tumor advancement, disease progression and mutations in the FGFR2 gene with a value of (p<0.01) [10,11].

The study confirmed the significant value of molecular subgroups in endometrial cancer (EC). The analysis revealed many mutated genes contributing to the development and progression of cancer. The FGFR-2 receptor plays an important role in disease progression through the epithelialmesenchymal transition. Our results revealed other driver mutations that could be compared to the recommended gene panel [10,11].

Discoveries regarding the important role of transcription factors from the forkhead box (FOX) family in the progression of endometrial cancer (EC) led us to conduct a study to investigate the expression of the FOX protein and its impact on the molecular basis of EC and the development process of this cancer. The methodology included the analysis of FOX protein expression levels (FOXA1, FOXA2, FOXP1) in tumor tissue samples of EC patients using advanced molecular, genetic and immunohistochemical techniques [12].

In the study population, it occurred in 24 cases, most often in the FIGO IA stage. Univariate Cox analysis was performed and provided significant results for FOXA1 protein. The log-rank test for FOXA1 was 2.031559 and the p-value was 0.04220. The hazard ratio (HR) for FOXA1 was calculated and was 2.66, with a p-value of 0.033. These results indicate that FOXA1 silencing was associated with worse clinical outcome. A correlation was observed between FOXA1 and FOXP1 protein expression, where the correlation coefficient was R = 0.2872, with a p-value of 0.0041. The analysis of overall survival (OS) depending on FOXP1 expression did not show a significant correlation (p > 0.05). These results indicate that the presence of FOXA1 protein may be an important prognostic factor in endometrial cancer. Its silencing may have a negative impact on the clinical condition of patients. The survival analysis related to FOXP1 did not show a significant relationship, which may indicate that FOXP1 may be less important in predicting clinical outcomes in the studied group of patients [12].

All these results provide important information in the context of understanding the role of FOXA1 and FOXP1 proteins in the progression of endometrial cancer and may have clinical significance in assessing the prognosis and developing therapeutic strategies for patients with this cancer.

The comparison of FOX protein expression with genes showed no expression of FOXP1 protein with the TP53 mutation (correlation coefficient R = 0.31, p-value = 0.0106) and no expression of FOXA1 protein with the KRAS mutation (correlation coefficient R = −0.246, p-value = 0.0446). These results suggest that the lack of FOXP1 expression may be associated with TP53 mutation, while the lack of FOXA1 expression may correlate with KRAS mutation [10-12].

Our study demonstrated the effect of FOXA1 and FOXP1 protein expression on FIGO stage in advanced cancer. The correlation coefficient for FOXP1 was R = 0.2379, with a p-value of 0.018924, while for FOXA1, the correlation coefficient was R = −0.2643, with a p-value of 0.0088. This means that reduced FOXP1 expression may be associated with an advanced stage of the disease, while reduced FOXA1 expression may correlate with a less advanced stage [12].

Multivariate analysis using Cox models that included the studied genes and FOX proteins showed prognostic values for the TP53 genes (HR = 0.12, p-value = 0.011), KRAS (HR = 0.31, p-value = 0.023) and the lack of FOXA1 expression (HR = 0.19, p-value = 0.017). These results suggest that the presence of mutations in TP53 and KRAS, as well as the lack of FOXA1 protein expression, may influence the prognosis of patients with endometrial cancer. Estrogen receptor expression was detected in all FOXP1-positive cases and over 90% of FOXA1-positive cases [12].

In recent years, changes have been observed in the molecular subgroup of EC. We recorded over 90% concordance in coexpression with ER and FOXA1. Linear and combined statistics demonstrated the prognostic value of FOXA1. The results of our study confirmed previous data and added new information regarding correlations with key genes worsening prognosis. The Notch pathway is believed to be a new target for cancer therapy. It is clearly observed in neuroendocrine and lymphoid tumors with delta-ligand 3 (DLL3) [12].

The clinical impact of Forkhead protein box FOXP1 is still unclear. In our study, we did not observe the importance of FOXP1 in EC carcinogenesis. As with FOXA1, we noticed a close association with ER and AR (androgen receptor was not examined). The above-mentioned link between pathways, especially Notch, is at the center of attention in modern oncology. [12].

Groundbreaking research on endometrial cancer prognostic factors with an emphasis on insulin-like growth factor IGF-1,2, its activation pathway, and associated genes is essential to deepen our understanding of this type of cancer. Forkhead box proteins and FGFR2 are associated with many cellular processes, including proliferation, regulation of apoptosis and angiogenesis, which are crucial for cancer development and progression. Additionally, the Epithelial-Mesenchymal Transition (EMT) process is an important element in the process of cancer infiltration and metastasis [11].

Discussion

EC risk stratification systems are based on conventional TNM staging principles, such as depth of myometrial invasion, lymph node metastasis, histology, and tumor grade. Genomic factors are not yet in standard clinical use for assessing prognosis. The subtypes proposed by Bokhman in 1983 are still widely accepted and used. The Cancer Genome Atlas provides modern insight into the EC pathway and tumor biology [13]. The most important clinical aspect is the correct separation of EC by survival. The traditional three-stage FIGO classification divides EC into three stages (G1, G2 and G3). The FIGO binary grading system was proposed nearly 20 years ago and is consistent with gastrointestinal pathology [13]. For reproducible classification, FIGO grade 3 endometrioid, serous, and clear cell carcinoma will be considered “high-grade endometrial carcinoma” (HGEC) and G1-G2 endometrioid carcinoma will be considered low-grade endometrioid carcinoma (LGEC). G3 cancers are highly heterogeneous because they occur in every genomic category and are least represented in the low-copy group. [13,14] FGFR-2 plays an important role in the dynamics of cancer development through the epithelial-mesenchymal transition [15].

In recent years, a change in the molecular subset of EC has been observed. The report by Cruz et al. presents the relationship between FOX protein and beta-catenin in an experimental model of breast cancer. FOXA1 expression has been shown to be positively associated with estrogen receptor (ER)+ breast cancer [16]. In our study, we reported over 90% concordance in coexpression with ER and FOXA1, thus confirming the protective effect of ER/FOXA1 coexpression. These results confirmed previous data and added new information regarding correlations with key genes, thus worsening the prognosis. The clinical significance of the FOXP1 forkhead protein remains unclear. We did not observe its significance in EC [10]. Due to the key role of FOXA in tumorigenesis and hormone-dependent cancers, numerous efforts have been made to target FOXA for anticancer therapies. There is still no specific treatment strategy. [10]. In this situation, many authors believe that the Notch pathway is a new target for cancer therapy. This is clearly observed in neuroendocrine and lymphoid tumors with delta-ligand 3 (DLL3) [17].

Recent studies have shown that FGFR-2 mutation is the causative factor of EMT in EC [18]. Our results regarding EMT aspects confirmed a direct negative impact on OS. Interestingly, the hallmark tumor feature of lymphovascular invasion (LVI) was observed more frequently in high-grade TP53 mutation-positive tumors, especially in EMT [11]. Our results confirm that EMT features are useful for prediction, but only LVI achieved predictive validity in the multivariate Cox model. The FGFR2 mutation in this area is still unclear. Our results confirmed previous data on RFS and OS, observing shorter RFS and ACSS times without any final impact on OS. [11]. We observed that loss of tumor cell integrity along with vimentin expression clearly promotes EMT, and there is a close correlation between EMT and stage and overall OS. The present study indirectly confirms the association between transcription factors and abnormal gene function, as we have recently published [11].

Recent work by Chen et al. presents the clinical implications of EMT regulation. The presented in vitro and in vivo preclinical studies demonstrated that isoliquiritigenin (ISL) effectively inhibits endometrial cancer cell migration and reduces HEC-1A-LUC tumor metastasis in nude mice by inhibiting the TGF-β/Smad signaling pathway. These findings will likely lead to further studies to highlight the potential of ISL in the treatment of endometrial cancer metastasis [19].

In summary, the goals of this research are important for improving disease prognosis and treatment outcomes for patients with endometrial cancer, and may also contribute to expanding our knowledge of the biology of this cancer.

Conclusions