Open Access, Peer-reviewed
pISSN 2005-0380   eISSN 2005-0399
    J Gynecol Oncol. 2023 Jul;34(4):e53. English.
    Published online Mar 10, 2023.
    https://doi.org/10.3802/jgo.2023.34.e53
    © 2023. Asian Society of Gynecologic Oncology, Korean Society of Gynecologic Oncology, and Japan Society of Gynecologic Oncology
    Original Article

    PTEN mutation predicts unfavorable fertility preserving treatment outcome in the young patients with endometrioid endometrial cancer and atypical hyperplasia

    Yu Xue,1,* Youting Dong,2,* Yaochen Lou,1 Qiaoying Lv,1 Weiwei Shan,1 Chao Wang,1 and Xiaojun Chen1
      • 1Department of Gynecology, Obstetrics and Gynecology Hospital, Fudan University, Shanghai, China.
      • 2Shanghai Medical college, Fudan University, Shanghai, China.
    • Correspondence to Xiaojun Chen. Department of Gynecology, Obstetrics and Gynecology Hospital, Fudan University, 128 Shenyang Road, Yangpu District, Shanghai 200092, China. Email: xiaojunchen2013@sina.com

      *Yu Xue and Youting Dong contributed equally to this work and should be considered co-first authors.
    Received October 26, 2022; Revised January 06, 2023; Accepted February 24, 2023.

    This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Abstract

    Objective

    This study aimed to investigate the impact of molecular classification and PTEN, KRAS and PIK3CA gene mutation on the outcome of fertility-preserving treatment in the patients with endometrioid endometrial cancer (EEC) and endometrial atypical hyperplasia (EAH).

    Methods

    This is a single-center retrospective study. A total of 135 patients with EEC and EAH receiving fertility-preserving treatment and molecular classification were reviewed. The distribution of the four types of molecular classification was described. The impact of non-specific molecular profile (NSMP), mismatch repair-deficiency (MMRd), and PTEN, KRAS and PIK3CA gene mutation on the outcome of fertility-preserving treatment was analyzed.

    Results

    Of the patients analyzed, 86.7% (117/136) were classified as having NSMP; 14 (10.4%), MMRd; 1 (0.7%), POLEmut EAH; and 3 (2.2%), p53abn EEC. The patients having NSMP and MMRd achieved similar 16-, 32-, and 48-week complete response rates. The patients harboring tier I and tier II PTEN mutations (PTENmut-Clin) achieved lower cumulative 32-week CR rates than those with PTEN-others (without PTENmut-Clin) (22/47, 46.8% vs. 50/74, 67.6%; p=0.023; odds ratio=0.422; 95% confidence interval [CI]=0.199–0.896). Insulin-resistance (hazard ratio [HR]=0.435; 95% CI=0.269–0.702; p=0.001) and PTENmut-Clin (HR=0.535; 95% CI=0.324–0.885; p=0.015) were independent negative predictors for lower 32-week CR rates.

    Conclusion

    PTENmut-Clin is an independent risk factor for unfavorable fertility-preserving treatment outcomes in the patients with EEC and EAH. The patients with MMRd receiving fertility-preserving treatment achieved outcomes similar to those of the patients with NSMP. The molecular profiles might guide fertility-preserving treatment in the prognosis and clinical decisions.

    Synopsis

    PTEN mutation predicts lower 32-week CR rate in the patients with EEC/EAH receiving fertility-preserving treatment.

    Keywords
    Endometrial Cancer; Endometrial Atypical Hyperplasia; Fertility-Preserving Treatment; Molecular Classification; PTEN

    INTRODUCTION

    The incidence of endometrial cancer is increasing world-wide with the spread of obesity and unhealthy life style [1]. Five to thirty percent of endometrial cancers are diagnosed at the age younger than 50 years, and over half of affected patients are nulliparous [2]. The increasing need for fertility-preserving treatment in young patients with endometrioid endometrial cancer (EEC) or its precursor lesion, endometrial atypical hyperplasia (EAH), has drawn increasing attention. Highly efficient progestins are widely accepted as the main regimen for fertility-preserving treatments in these patients. However, approximately 20%–30% of patients are not sensitive to progestins and have to receive other regimens after long-term progestin treatment [3, 4, 5]. Higher body mass index (BMI) [3, 6] and lower estrogen receptor (ER) or progesterone receptor (PR) expression [7] in endometrial lesions have been suggested to predict poorer fertility-preserving treatment outcomes in patients with EEC and EAH. However, additional predictive factors are needed to precisely identify patients who are not sensitive to progestin treatment and thus help doctors select the appropriate treatment for these patients.

    The emergence of molecular classification of endometrial cancer has shed light on the precise fertility-preserving treatment for patients with EEC and EAH. Patients having p53 abnormal (p53abn) EEC have poor prognosis when receiving conservative treatment [8]. Preservation fecundity may be possible in patients with POLE-mutated (POLEmut) EEC despite the poor pathological appearance [8, 9]. EECs with non-specific molecular profile (NSMP) and mismatch repair-deficiency (MMRd) might be the most appropriate types for fertility-preserving treatment [10]. However, there have been few studies investigating the impact of molecular classification on fertility preservation in EEC patients. It is also not known whether molecular classification could also be used in EAH patients. Chung et al. [10] first analyzed molecular classification in patients with EEC receiving fertility-preserving treatment and reported that patients having MMRd might have a worse response to hormone treatment than those having NSMP. More studies are needed to further explore the distribution of molecular classification and their impact on fertility preservation in patients with EEC and EAH.

    It is well understood that somatic mutations in other oncogenes, such as PTEN, KRAS, and PIK3CA, are common in patients with well-differentiated EEC eligible for fertility-preserving treatment. Whether mutation of these genes has any impact on fertility preservation is also worth investigation.

    In this single-center retrospective study, we described the distribution of the four types of molecular classifications. The impact of MMRd and NSMP, as well as PTEN, KRAS, and PIK3CA gene mutations on the outcome of fertility-preserving treatment, including the complete response (CR) rate, 1-year cumulative recurrence rate, 1-year cumulative pregnancy rate, and cumulative live birth rate, was also analyzed.

    MATERIALS AND METHODS

    1. Study population

    Consecutive patients with EEC or EAH who received fertility-preserving treatment at the Obstetrics and Gynecology Hospital of Fudan University (Ob & Gyn Hospital) between April 3, 2017 and July 14, 2021 were retrospectively screened. The study was approved by the Institutional Review Board of the Ob&Gyn Hospital (approval no. 2021-230-X1). All patients were fully informed of the benefits and risks of fertility-preserving treatment, and provided written informed consent thereafter. Their permission to publication was obtained.

    All screened patients underwent standardized evaluation and treatment protocols. Clinicopathological and follow-up data were prospectively recorded during treatment and follow-up. All patients were pathologically diagnosed via endometrial biopsy through dilation and curettage with or without hysteroscopy. All specimens were reviewed by two experienced gynecologic pathologists (Dr. Wang Y and Dr. Zhou X) according to the World Health Organization (WHO) pathological classification (female genital tumors) [11]. In cases of discrepancy, a panel discussion held at the Department of Pathology to reach the consensus.

    Eligible patients in this study should meet the following criteria: 1) histologically proven EAH or well-differentiated EEC; 2) no signs of extrauterine involvement on enhanced pelvic magnetic resonance imaging, enhanced abdominal computed tomography, and ultrasound; 3) age of <45 years; 4) strong willingness to preserve fertility; 5) no contraindications for progestin treatment or pregnancy; 6) non-pregnancy; and 7) molecular classification of the endometrial lesion obtained before treatment initiation. A total of 916 patients, who were primarily diagnosed with EEC or EAH, were screened. Seven hundred and eighty-one patients were excluded because of lack of molecular classification analysis results. Finally, 135 patients (82 with EEC and 53 with EAH) were included in this study.

    2. Conservative treatment and evaluation

    All patients received fertility-preserving treatment within one month after diagnosis, including 108 patients (80.0%) treated with daily oral megestrol acetate (MA, 160 mg/day, GuoHai Biological Pharmaceutical Co., LTD, Qingdai, China), 2 patients (1.5%) with daily oral medroxyprogesterone acetate (250 mg/day, Silian Pharmaceutical Co., Ltd., Beijing, China), 11 patients (8.1%) with levonorgestrel-releasing intrauterine system (LNG-IUS, Bayer Oy, Turku, Finland), 8 patients (5.9%) with LNG-IUS plus daily oral MA, and 6 patients (4.4%) with intramuscular injection of gonadotropin-releasing hormone agonist (GnRH-a) (3.75 mg/4 weeks, IpsenPharmaBiotech, Signes, France) plus daily oral letrozole (2.5 mg/day, Hengrui Pharmaceuticals Co., Ltd., Lianyungang, China). Some patients also received daily oral metformin (1,500 mg/day, Shanghai Sine Pharmaceutical Laboratories Co., Ltd., Shanghai, China) at their attending discretion. Hysteroscopic evaluation and lesion resection or endometrium biopsy were performed before medication and every three months during treatment to evaluate the treatment response. Considering the possible delay to have hysteroscopies, the cumulative CR rates within 16-, 32-, and 48-week were used to describe the results for the first, second, and third time for endometrium evaluation after receiving fertility-preserving treatment.

    The treatment response was categorized as follows: 1) CR, defined as no endometrial lesion; 2) partial response, pathological improvement compared with original diagnosis; 3) stable disease (SD), persistence of disease as originally diagnosed; 4) progressive disease (PD), any appearance of disease with a higher degree, such as recognition of EEC in patients with EAH, or grade II–III EEC, myometrial invasion, and extrauterine lesions in patients with EEC.

    For the patients who achieved CR, the same regimen was administered for another two to three months for consolidation, and another hysteroscopy was performed three months after the first diagnosis of CR for confirmation. Definitive hysterectomy was recommended when PD was detected at any time during treatment, when SD was detected after seven months of treatment, or when CR was not achieved after ten months of treatment. Alternative treatment was suggested after multidisciplinary discussion when affected patients insisted on fertility preservation.

    Assisted reproduction technology under close surveillance was suggested for the patients who achieved CR and desired pregnancy. Progestin-based maintenance therapy, such as the use of oral contraceptive pills or LNG-IUS, was recommended for those without a recent pregnancy plan. All patients were followed up until the latest pathological results were obtained before December 2021.

    3. Data collection

    Clinical data, including height, weight, blood pressure, waistline and hip circumference were recorded at the first diagnosis. Blood samples were collected before treatment initiation and analyzed in the laboratory of the Ob&Gyn Hospital, and fasting blood glucose (FBG), fasting insulin (FINS), sex hormone, blood lipid, CA-125, and HE-4 levels were evaluated. The BMI (weight [kg]/height2 [m2]) and homeostasis model assessment-insulin resistance (HOMA-IR) index (FBG level [mmol/L] × FINS level [μU/mL]/22.5) were calculated. Obesity [12] was defined as a BMI of ≥ 30 kg/m2, while insulin-resistance [13] was defined as an HOMA-IR index of ≥ 2.95. Metabolic syndrome was defined according to the literature [14, 15].

    4. Molecular classification

    Molecular classification was applied according to the diagnostic algorithm for the integrated histomolecular endometrial carcinoma classification (WHO classification of female genital tumors, 5th edition) [11].

    5. Gene sequencing

    Genomic DNA was extracted from endometrial lesions identified by pathologists using formalin-fixed paraffin-embedded (FFPE) tissue slides. The DNA was sequenced using two sets of panels at different time intervals. Before March 1, 2021, endometrial samples from 96 cases (71.1%) were tested using next-generation sequencing (NGS) with a panel (Shanghai Biotechnology Co., Shanghai, China) designed to detect the coding region and splice sites with a total range of 76610 bp covering 13 genes (PTEN, CTNNB1, PIK3CA, POLE, KRAS, FGFR2, PIK3R1, CHD4, TP53, RNF43, POLD1, SOX17, and ERBB2). Paired-end sequencing (2×150 bp) was performed on an Illumina® NovaSeqTM 6000 System (Illumina Trading (Shanghai) Co., Ltd., Shanghai, China), with an average sequencing depth of 1000x.

    Samples from 39 patients (28.9%) after March 1, 2021 were analyzed via NGS using ECG10®-Panel (Xiamen SpaceGen Co., Xiamen, China) to detect mutations in 10 genes. The coding region and the exon/intron junctions of approximately 20 bp were tested for POLE, MLH1, MSH2, MSH6, PMS2, PTEN, EPCAM, and TP53, while exons 2, 3, and 4 of KRAS and exons 10, 14, and 21 of PIK3CA were assessed for mutations, to identify single nucleotide variants and indels (Illumina Trading (Shanghai) Co., Ltd.). The sequencing depth was up to 5000x, with a sensitivity to identify variants of mutation frequency as low as 1%.

    The clinical significance level of mutations was comprehensively evaluated according to ClinVar, ANNOVAR, InterVar, CancerVar (version 1.1), and Catalogue of Somatic Mutation in Cancer (COSMIC), combined with population data evaluation and in silico predictions. The IARC TP53 Database was also used for TP53 mutations. POLE exonuclease domain mutations were evaluated according to the 11 mutations reported by literature [16]. The Integrative Genomics Viewer (IGV© Broad Institute and the Regents of the University of California, Version 2.11.4) was used for variant review. Somatic variants are classified into four tiers after the joint consensus recommendation of the AMP/ASCO/CAP [17]. Only those in tier I and tier II were considered meaningful mutations in this study.

    Genes harboring variants in tiers I and II were named as (name of the gene) mut-Clin. Genes with wild-type or variants in tier III or IV were named as (name of the gene) -others.

    6. IHC (immunohistochemistry) analysis

    FFPE tissue specimens were used for IHC staining. The analysis was performed using a Leica Bond Max detection system with the following monoclonal antibodies: MLH1 (DAKO-ES05), PMS2 (DAKO-EPS1), MSH2 (DAKO-FE11), MSH6 (DAKO-EP49), and p53 (DAKO-DO-7). For the MMR proteins, nuclear positivity of more than 5% of the cancer cells was considered to indicate an intact expression. Normal lymphocytes and/or stromal cells were used as internal positive controls. Notably, once there was a doubt about the IHC results of MMR status, a Promega MSI Analysis System (version 1.2) was used on Biosystems 3500 and 3500xL Genetic Analyzer (Thermo Fisher Scientific) to verify the diagnosis. For p53, diffuse and strong nuclear staining (>80% of tumor cell nuclei) was defined as an overexpression pattern, no staining as a complete absence pattern, and weak focal positive staining as a wild-type pattern.

    7. Statistical analysis

    The time to CR was measured from the time point of start of fertility-preserving treatment to the first time point of CR diagnosing. CR rate at a certain time point was measured among the patients who had already achieved CR, or who did not achieved CR but were still in follow-up, excluding those without CR whose follow-up period was shorter than the time point.

    All data were presented descriptively as medians, ranges, frequencies, or proportions. Continuous variables between the two groups were compared using Student’s t-test or the Mann-Whitney U test. Frequency distributions were compared using the χ2 test or Fisher’s exact test. The cumulative CR rates were estimated using the Kaplan-Meier method and compared between the groups using the log-rank or Breslow test. Cox regression analysis was used to estimate the hazard ratios (HRs) for CR. A two-tailed p-value of <0.05 was regarded as significant. All statistical analyses were performed using SPSS for Windows (version 22.0; IBM, Armonk, NY, USA).

    RESULTS

    1. Patient characteristics

    The basic characteristics of the 135 patients are showed in Table 1. At the initial diagnosis, the median age was 32 years (range, 19–45 years), while the median BMI was 25.20 kg/m2 (range, 16.16–47.48 kg/m2). The clinicopathological characteristics of the patients are plotted in Fig. 1. A total of 129 patients (95.6%) received progestin-based treatment as the first-line treatment.

    Table 1
    Characteristics of patients (n=135)

    Fig. 1
    Clinicopathological characteristics spectra across the patients. Time to CR (vertical axis, top panel) was plotted for each case (horizontal axis). Mutations are shown in the middle panel. MSI status and MMR proteins expression were listed below, followed by histology according to each case. Patients were listed according to the molecular classification, and arranged by their time to CR (or the follow-up period if the patient did not achieve CR) based on the histology. The mutations, MSI status, and MMR proteins expression were matched to each case. Three patients had doubtful results of deficient MMR expression, and MSI status analysis was used for verification. Two of them were found to be MSS and the remaining one was found to be MSI-L. These three patients were finally classified as NSMP.
    CR, complete response; EAH, endometrial atypical hyperplasia; EEC, endometrioid endometrial cancer; IHC, immunohistochemistry; ND, not done; NSMP, non-specific molecular profile; MMRd, mismatch repair deficient; MSI, microsatellite instability; MSI-H, microsatellite instability-high; MSI-L, microsatellite instability-low; MMR, mismatch repair; MSS, microsatellite stable; p53abn, p53 abnormal; POLEmut, POLE-mutated.

    According to the molecular classification, 117 patients had NSMP (86.7%); 14 (10.4%), MMRd; 1 (0.7%), POLEmut EAH; and 3 (2.2%), p53abn EEC (Table 1). Detailed information on the 18 patients with MMRd, p53abn, or POLEmut cancer is presented in Table S1. There was no significant difference in the distribution of MMRd or NSMP between the patients with EEC and EAH. The basic characteristics, including age, BMI, obesity, hypertension, diabetes, MS, insulin-resistance, and therapeutic regimen, were comparable between the patients with MMRd and NSMP (Table 2).

    Table 2
    Fertility-preserving treatment outcomes between patients of NSMP and MMRd (n=131)

    2. Impact of molecular classification on the fertility-preserving treatment outcomes

    The fertility-preserving treatment outcomes were compared between the patients with NSMP and MMRd (Table 2 and Fig. S1). The patients with EEC with NSMP and MMRd achieved similar 16-, 32-, and 48-week CR rates. The 16-week CR rates were 33.3% (21/63) in the NSMP group and 37.5% (3/8) in the MMRd group (p=1.000; odds ratio [OR]=0.833; 95% confidence interval [CI]=0.181–3.826). The 32-week CR rates were 63.9% (39/61) and 50.0% (4/8) in the NSMP and MMRd groups, respectively (p=0.464; OR=1.773; 95% CI=0.403–7.797). The 48-week CR rates were 87.7% (50/57) and 85.7% (6/7) in the NSMP and MMRd groups, respectively (p=1.000; OR=1.190; 95% CI=0.124–11.408). No difference was found in the 16-, 32-, and 48-week CR rates between the patients with NSMP and MMRd EAH.

    Among the 113 patients with NSMP or MMRd who achieved CR, 18 patients experienced recurrence within one year after achieving CR, including 9 patients (9/62,14.5%) with EEC and 9 patients with EAH (9/51, 17.6%). No significant difference was found between the NSMP and MMRd groups with EEC or EAH in the one-year cumulative recurrence rates (EEC: 19.3% vs. 37.5%; log rank p=0.289; EAH: 31.0% vs. 0.0%; log rank p=0.221), one-year cumulative pregnancy rates (EEC: 36.4% vs. 100.0%; log rank p=0.167; EAH: 19.2% vs. 66.7%; log rank p=0.689), or cumulative life birth rates (EEC: 71.4% vs. 66.7%; log rank p=0.284; EAH: 100.0% vs. 50.0%; log rank p=0.480).

    3. Impact of somatic oncogene mutations on fertility-preserving treatment outcomes

    We then investigated whether specific somatic mutations of other key oncogenes affected the fertility-preserving treatment outcomes. Three commonly mutated oncogenes in EEC, including PTEN, PIK3CA, and KRAS (Table 3, and Table S2), were analyzed.

    Table 3
    Comparison according PTEN variants (n=135)

    Fifty-five patients (40.7%) harbored PTENmut-Clin, among whom 40 were diagnosed with EEC, accounting for 48.8% (40/82) of all patients with EEC; this proportion was significantly higher than that of all patients with EAH (15/53, 28.3%; p=0.018). No significant difference was found in the basic characteristics, including age, BMI, comorbidities, and therapeutic regimen between the patients with PTENmut-Clin and PTEN-others. Among the 135 patients with EEC or EAH, those with PTEN-others had a higher 32-week CR rate than those with PTENmut-Clin (PTEN-others: 50/74, 67.6% vs. PTENmut-Clin: 22/47, 46.8%; p=0.023; OR=0.422; 95% CI=0.199–0.896). The trend of difference in CR rate between patients harboring PTENmut-Clin and PTEN-others in EEC or EAH patients respectively was also found but without a statistical difference, except for the 32-CR rate in patients with EAH (PTENmut-Clin: 4/14, 28.6% vs. PTEN-others: 25/38, 65.8%; p=0.017; OR=0.208; 95% CI=0.055–0.794) (Table S3). The cumulative CR rates were then compared in the patients with EEC or EAH separately using Kaplan-Meier analysis. The analysis showed that the patients with EEC or EAH with PTEN-others had higher CR rates with a shorter treatment duration to achieve CR than those with PTENmut-Clin, but without a significant difference (EEC: median treatment time to CR of 30.14 weeks in those with PTENmut-Clin vs. 22.00 weeks in those with PTEN-others; HR=0.609; 95% CI=0.363–1.021; log rank p=0.056, Breslow p=0.062; EAH: median treatment time to CR of 42.86 weeks in those with PTENmut-Clin vs. 26.29 weeks in those with PTEN-others; HR=0.592; 95% CI=0.317–1.108; log rank p=0.097, Breslow p=0.027) (Fig. 2A-C). The patients with PTENmut-Clin had a higher one-year cumulative recurrence rate after achieving CR than those with PTEN-others (37.8% vs. 16.3%; log rank p=0.027). The trend of difference in cumulative one-year recurrence rate between patients harboring PTENmut-Clin and PTEN-others in EEC or EAH patients respectively was also found but without a statistical difference, which might be due to limited sample size (Table S3). No significant difference was found in the one-year cumulative pregnancy rates (33.0% vs. 57.7%; log rank p=0.854) and cumulative live birth rates (100.0% vs. 52.0%; log rank p=0.113) between the two groups (Table 3).

    Fig. 2
    Cumulative complete response rates of patients with CR according to PTEN mutations. (A) Cumulative CR rate between patients with or without PTEN variants in tiers I and II. (B, C) Cumulative CR rate between patients with or without PTEN variants in tiers I and II in EEC and EAH, respectively.
    CI, confidence interval; CR, complete response; EAH, endometrial atypical hyperplasia; EEC, endometrioid endometrial cancer; HR, hazard ratio; PTENmut-Clin, PTEN gene harboring variants in tiers I and II; PTEN-others, PTEN gene with wild-type or variants in tier III or IV.

    Twenty-one (15.6%) and fifty patients (32.6%) harbored KRASmut-Clin and PIK3CAmut-Clin, respectively. The basic characteristics and fertility-preserving treatment outcomes are presented in Table S2 and Fig. S2. The patients with KRASmut-Clin were older than those with KRAS-others (p=0.003). No significant differences were found in the cumulative CR rates, one-year cumulative recurrence rates, one-year cumulative pregnancy rates, or cumulative live birth rates between the KRASmut-Clin and KRAS-others groups. The patients with PIK3CA-others had a higher BMI (p=0.044) and proportion of insulin resistance (PIK3CA-others: 52/90, 57.8% vs. PIK3CAmut-Clin: 16/44, 36.4%; p=0.020) than the patients with PIK3CAmut-Clin. The patients with PIK3CAmut-Clin achieved a lower one-year cumulative pregnancy rate than those with PIK3CA-others (9.1% vs. 67.3%; log rank p=0.023). No significant differences were found in the cumulative CR rates or cumulative live birth rates between the PIK3CAmut-Clin and PIK3CA-others group.

    4. Risk factors associated with the 32-week CR rate

    Univariate analysis was used to explore the possible risk factors associated with the cumulative 32-week CR rate. Insulin resistance and PTENmut-Clin were associated with a lower 32-week cumulative CR rate (Fig. 3). In the multivariate analysis, insulin-resistance (HR=0.435; 95% CI=0.269–0.702; p=0.001) and PTENmut-Clin (HR=0.535; 95% CI=0.324–0.885; p=0.015) remained independent negative predictors of the 32-week-CR rate. We then had an analysis on EEC patients only (Fig. S3). According to the multivariate analysis, insulin-resistance (HR=0.497; 95% CI=0.296–0.833; p=0.008) was still the independent negative predictor of the CR rate and there was a trend that those harboring PTENmut-Clin (HR=0.612; 95% CI=0.366–1.026; p=0.063) might had a lower CR rate, though without significant difference.

    Fig. 3
    Risk factors related to 32-week CR rate. Univariate and multivariate cox regression models were used to identify risk factors associated with fertility-preserving treatment outcomes according to 32-week CR rates.
    (name of the gene) mut-Clin, genes harboring variants in tiers I and II; (name of the gene) -others, genes with wild-type or variants in tier III or IV; CI, confidence interval; CR, complete response; EAH, endometrial atypical hyperplasia; EEC, endometrioid endometrial cancer; HR, hazard ratio; IR, insulin-resistance; MS, metastatic syndrome.

    *Missing data for 1 case for IR.

    5. Fertility-preserving treatment outcomes of the patients with p53abn cancer, POLE-mutated cancer, and Lynch syndrome

    Of the three patients with p53abn cancer, two were lost to follow-up after the first visit, and the other developed PD after standard fertility-preserving treatment for 19.43 weeks. Only one patient was classified as having POLEmut cancer; this patient was diagnosed with EAH but took 81.71 weeks to achieve CR. Germline genetic testing was performed in 11/14 patients with MMRd (78.6%), two of whom were diagnosed with Lynch Syndrome and achieved CR, although one experienced recurrence after achieving CR. Detailed information is provided in Table S1.

    DISCUSSION

    Molecular classification has been widely accepted for guiding postsurgical adjuvant treatment in patients with endometrial cancer [18, 19]. However, the distribution of the four types and their impact on the fertility-preserving treatment outcomes remain unclear. Our study showed that most of the patients eligible for fertility-preserving treatment were classified as having NSMP (117/135, 86.7%), which is consistent with the results of previous studies [8, 9, 10, 20, 21]. In our study, 8 EEC patients and 6 EAH patients were classified as having MMRd, accounting for 9.8% and 11.3% in EEC and EAH patients, respectively, the percentage of which was much lower than historical data of 30% [22]. This might be mainly caused by the younger age of our patients than general EEC or EAH patients. A recent Chinese study showed similar findings. In their study, 4 out of 59 patients with EEC or EAH who received fertility-sparing treatment were classified as having MSI-H, accounting for only 6.78% of the patients [21]. Chung et al. [10] showed that MMRd patients accounted for 15.8% of all EC patients receiving fertility preserving treatment. Zakhour et al. [23] showed 7% of patients younger than 55 years of age with endometrial complex atypical hyperplasia or FIGO grade 1 endometrial adenocarcinoma had loss of MMR proteins by immunohistochemistry. We suggest that more MMRd EEC will develop with the increasing of age. Further studies are needed to clarify the distribution of molecular classification in young EEC and EAH patients receiving fertility-preserving treatment.

    In our analyses, the molecular classification of NSMP or MMRd did not affect the fertility-preserving treatment outcomes. However, other studies have suggested a correlation between MMRd and a poor response to progestin treatment [10, 23]. The reason might lie in the application of hysteroscopic resection, which was routinely performed in our study but not in some previous studies [10, 23]. The results of Raffone et al. [24] are consistent with ours. It was speculated that hysteroscopic resection might detect and remove all possible lesions with direct visualization, thus decreasing the risk of resistance to progestin; this might have led to the result that the patients with EEC and EAH with NSMP and MMRd achieved similar fertility-preserving treatment outcome.

    Our findings suggest that PTENmut-Clin is an independent risk factor for poorer fertility-preserving treatment outcomes. PTEN, a tumor suppressor gene, is involved in the PI3K-PTEN-AKT-mTOR pathway, and its somatic mutation occurs in 69%–80% of EECs [25]. The PI3K pathway could be activated in patients with mutated PTEN, resulting in insensitivity to progestins. Previous studies have also noted a possible association between the loss of PTEN combined with other molecules and a poor response to progestins [26, 27, 28]. Our findings add new evidence on the use of PTENmut-Clin in predicting the fertility-preserving treatment outcomes of patients with EEC and EAH. Although there was no statistical significance in EEC cohorts only, which might be due to small sample size, a trend could still be found that patients harboring PTENmut-Clin might have a worse treatment outcome. Previous studies have shown that obesity [3, 6], larger lesion size (>2 cm) [3], expression of ER/PR [7], comorbidities of polycystic ovarian syndrome [29], and insulin resistance [30] are correlated with poor fertility preserving-treatment outcome in patients with EEC or EAH. Our findings indicate that PTENmut-Clin is a possible risk factor for poor progestin-based fertility-preserving treatment outcomes. Other regimens, such as GnRH-a combined with letrozole, may be considered as the first-line fertility-preserving treatments for such patients.

    In our study, we found that patients with PTENmut-Clin had poor 32-week CR rate and overall cumulative CR rate compared with PTEN-others, but the comparison of the 48-week CR rate between the two groups had no significant difference. This might be caused by the regular usage of hysteroscopic evaluation and lesion removal every three months during treatment. With the repeated lesion removal and the extension of treatment duration, the difference of treatment response between PTENmut-Clin lesion and PTEN-others lesion might be reduced, resulting in the approaching of 48-week CR rate between the two groups.

    Notably, PTENmut-Clin was found both in the patients with EEC and EAH, and the mutation rate was significantly higher in the patients with EEC than that in those with EAH; these findings demonstrate that PTENmut-Clin is an early event in the pathogenesis of endometrioid carcinoma, which is consistent with previous findings [31, 32].

    There was a trend for those harboring KRASmut-Clin to have a lower CR rate, but without a significant difference. According to previous studies, KRAS mutation might be an unfavorable prognostic factor in endometrial cancer cases, predicting not only endometrial tumorigenesis and development [33, 34], but also progressive biological behaviors of invasive proliferation [35] and metastasis [36]. Our analyses also showed that the patients harboring PIK3CAmut-Clin had a lower BMI and were less likely to develop insulin resistance. It has been reported that PIK3CA is involved in cellular and organismal metabolism, which has an impact on insulin responses in the muscle, liver, and fat [37]. However, more studies are required to clarify the relationship between specific oncogenes mutations and the fertility-preserving treatment outcomes in patients with EEC and EAH.

    The experience of fertility-preserving treatment in patients with Lynch Syndrome is limited [38, 39]. In our study, two patients with Lynch syndrome achieved CR using progestin-based therapy, adding to the evidence that Lynch syndrome might not be a contraindication to conservative therapy. However, one of our patients developed recurrence three months after achieving CR, suggesting the need for closer and more careful follow-up in these patients.

    Patients with POLEmut or p53abn EEC are rare to see in those receiving fertility-preserving treatment. In our study, one patient with p53abn EEC developed PD after 27 weeks of treatment. Although the number of cases has remained limited [8, 10], the patients with p53abn EEC all showed aggressive biological behaviors and poor outcomes, indicating that it is not safe to conduct fertility-preserving treatment in these patients. Although patients with POLEmut EEC have an exceptionally good progression-free survival rate after surgery, they can hardly benefit from additional therapies including radiation and chemotherapy [40]. The patient with POLEmut EAH in our study also showed resistance to long-term progestin treatment, wherein complete lesion resection might have played a dominant role. Hence, individualized evaluation and careful observation are needed because the impact of POLEmut EEC on the fertility-preserving treatment outcomes is still unclear.

    Molecular testing has been applied in EEC patients, but not regularly used in EAH patients. EAH is the precursor lesion of EEC, and they share similar features in many perspectives. In our study, similar trends of treatment response were found in patients with EEC and EAH when having the same molecular profiles, where the molecular classification of NSMP and MMRd had no impact on the treatment outcomes and PTENmut-Clin might lead to lower CR rate with long treatment duration. However, more prospective data are required to confirm the role of molecular testing in EAH and EEC patients.

    Our study analyzed the prognostic value of molecular profiles in a relatively large sample of patients with EAH or well-differentiated EEC receiving fertility-preserving treatment, with all information regarding their clinicopathological features collected prospectively. The limitations lie in the retrospective design and use of a single institution database, arousing possible bias in selecting patients. More prospective studies are required to validate the impact of specific molecular profiles on the fertility-preserving treatment.

    In this study, we found that PTENmut-Clin was an independent risk factor for unfavorable fertility-preserving treatment outcomes in the patients with EEC and EAH, with a lower 32-week CR rate. The patients with MMRd achieved outcomes similar to those of the patients with NSMP, in whom hysteroscopic endometrial lesion resection was performed. The molecular profiles might be helpful in guiding clinical decision-making for patients with EEC and EAH receiving fertility-preserving treatment.

    In summary, molecular analysis is an important approach in guiding fertility-preserving treatment in patients with EEC or EAH. It is suggested that all patients who are candidates for conservative therapy should undergo somatic variant analysis with a panel that includes PTEN to identify the molecular classification and other molecular profiles to predict treatment efficacy.

    SUPPLEMENTARY MATERIALS

    Table S1

    Characteristics and fertility-preserving treatment outcomes of patients with POLEmut (n=1), MMRd (n=14), and p53abn (n=3)

    Click here to view.(42K, xls)

    Table S2

    Comparison according to KRAS and PIK3CA variants in tiers I and II (n=135)

    Click here to view.(51K, xls)

    Table S3

    Fertility-preserving treatment outcomes according to PTEN variants in patients with EEC or EAH

    Click here to view.(51K, xls)

    Fig. S1

    Cumulative complete response rates of patients with CR according to molecular classification. (A) Cumulative CR rate between patients of NSMP and MMRd. (B, C) Cumulative CR rate between patients of NSMP and MMRd in EEC and EAH, respectively.

    Click here to view.(451K, ppt)

    Fig. S2

    Cumulative response rates according to pathogenic or likely pathogenic mutations in KRAS or PIK3CA. A, B, C, Cumulative CR rate of patients with or without KRAS variants in tiers I and II in total patients and in patients with EEC or EAH, respectively. D, E, F, Cumulative CR rate of patients with or without PIK3CA variants in tiers I and II in total patients and in patients with EEC or EAH, respectively.

    Click here to view.(751K, ppt)

    Fig. S3

    Risk factors related to CR rate in EEC patients. Univariate and multivariate cox regression models were used to identify risk factors associated with fertility preserving treatment outcomes according to CR rates in patients with EEC.

    Click here to view.(204K, ppt)

    Notes

    Conflict of Interest:No potential conflict of interest relevant to this article was reported.

    Author Contributions:

    • Conceptualization: W.C., C.X.

    • Data curation: X.Y., D.Y., L.Y., L.Q., S.W.

    • Formal analysis: X.Y., D.Y.

    • Investigation: X.Y., D.Y., L.Y., L.Q., S.W.

    • Methodology: X.Y., W.C., C.X.

    • Project administration: W.C., C.X.

    • Supervision: L.Y., L.Q., S.W., C.X.

    • Writing - original draft: X.Y., D.Y.

    • Writing - review & editing: C.X.

    ACKNOWLEDGEMENTS

    We gratefully acknowledge our funding source that supported this project, and we thank Dr. Wang Y and Dr. Zhou X, who work in the department of pathology in the Obstetrics and Gynecology Hospital of Fudan University, for their assistance.

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    MeSH Terms
    Classification
    Endometrial Neoplasms*
    European Union
    Female
    Fertility*
    Humans
    Hyperplasia*
    Prognosis
    Retrospective Studies
    Risk Factors
    Treatment Outcome*
    Metrics
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