Abstract
Objective
The efficacy of pembrolizumab in patients with microsatellite instability (MSI)-high cancers has been reported; however, the differences in efficacy according to the subtypes of MSI-high endometrial cancers (ECs) remain unclear. MSI-high ECs are classified into at least 3 groups based on their molecular characteristics: MLH1 hypermethylated, Lynch-like syndrome (LLS)-associated, and Lynch syndrome (LS)-associated cancers. This study aimed to investigate whether the efficacy of pembrolizumab differs among these 3 groups, and if so, whether EPM2AIP1 immunohistochemistry (IHC), which correlates with MLH1 promoter methylation, can be used to rule out MLH1 methylation cases.
Methods
This study included 12 patients with MSI-high EC who received pembrolizumab treatment. Patients were categorized into 3 groups based on MLH1 methylation analysis and the Amsterdam Criteria: MLH1 hypermethylated (sporadic [SP]), LLS-associated, and LS-associated. Patients’ medical records were retrospectively reviewed, and the efficacy of treatment was evaluated based on the response rate using the Response Evaluation Criteria in Solid Tumors version 1.1.
Results
The overall response rate was 75% (3/4) in the SP group, while it was 100% including one complete response patient in the LLS-associated and the LS-associated group, respectively. The sensitivity and positive predictive value of EPM2AIP1 IHC for MLH1 methylation were 100% and 66.7%, respectively.
Conclusion
Pembrolizumab may be more effective in LLS and LS-associated groups. EPM2AIP1 IHC was less predictive than MLH1 methylation analysis; however, it may be useful for ruling out MLH1 methylation cases due to its high sensitivity. Further studies are needed to determine whether EPM2AIP1 IHC can predict pembrolizumab efficacy.
Keywords: Microsatellite Instability, Pembrolizumab, Endometrial Cancer, Ovarian Cancer, Lynch Syndrome
Synopsis
The difference in pembrolizumab efficacy among microsatellite instability (MSI)-high endometrial cancers was investigated. Pembrolizumab may be more effective in Lynch-like syndrome-associated and Lynch syndrome-associated MSI-high cancer. EPM2AIP1 immunohistochemistry may be useful for ruling out MLH1 methylation cases due to its high sensitivity.
INTRODUCTION
Pembrolizumab is a humanized monoclonal anti-programmed cell death 1 (PD-1) antibody that has demonstrated clinical benefits in patients with previously treated unresectable or metastatic microsatellite instability (MSI)-high or deficient mismatch repair (dMMR) non-colorectal cancer (CRC), with an overall response rate (ORR) of 34.3% [1]. Pembrolizumab has also demonstrated clinical activity with an ORR of 48% in patients with advanced (EC) [2]. Furthermore, the accumulated data on immunotherapy in patients with uterine cancer showed that the addition of immunotherapy to chemotherapy significantly improved overall survival (OS) along with progression-free survival (PFS) not only with dMMR but also with proficient MMR regarding in patients with EC compared to chemotherapy alone [3].
In recent studies, MSI-high ECs are divided into at least 3 groups based on molecular characteristics: MLH1 hypermethylated EC (sporadic [SP]), Lynch-like syndrome (LLS)-associated EC, and Lynch syndrome (LS)-associated EC [3]. Many ECs with dMMR are due to MLH1 promoter hypermethylation, the frequency of which is reported to be 39%–76% in ECs with dMMR; these are called SP [4,5,6,7]. In contrast, LS is an autosomal dominant hereditary condition caused by germline mutations in MMR genes. Patients with dMMR ECs without MLH1 hypermethylation and no pathogenic germline gene mutations associated with MMR are classified as LLS. The primary cause of LLS may be biallelic somatic MMR gene inactivation [8,9,10]. LLS refers to individuals with a spectrum of LS tumors who lack both MLH1 promoter methylation and germline MMR gene mutations. The tumors result from heterogeneous undetected germline mutations or mosaicism [8,11,12]. Patients with LLS and their first-degree relatives have an intermediate risk of cancer between those with LS and the general population, indicating that special screening and surveillance strategies are needed for these patients and relatives [13].
Recent studies have reported that MSI-high ECs have increased immune cell infiltration compared to microsatellite instability-stable ECs and have also reported significant differences in immune response between hereditary and SP origins of MSI [14,15]. MSI-high ECs of hereditary or SP origin respond differently to immune checkpoint inhibitors [14]. However, the difference in efficacy among the 3 groups is unclear.
In colon cancer, the prevalence of BRAF mutations is used clinically as a surrogate marker to determine the somatic or epigenetic inactivation of MLH1; however, no clarified surrogate markers are available for the identification of MLH1 status in ECs [16,17,18]. A recent study reported that EPM2AIP1 immunohistochemistry (IHC) is consistent with MLH1 promoter methylation (94.5% sensitivity and 98.1% positive predictive value) in ECs [19]. EPM2AIP1 and MLH1 are the bi-directional genes on human chromosome 3p22.1 which lie head-to-head within a CpG island [20,21]. EPM2AIP1 and MLH1 share a common promoter whose methylation has been shown to affect both genes. MLH1 promoter methylation has been shown to silence EPM2AIP1 gene expression in vitro and in vivo [20,22]. In EPM2AIP1 IHC, nuclear staining was lost in the tissues with MLH1 promoter methylation [19].
The objective of this study was to assess whether the efficacy of pembrolizumab differs among 3 groups of patients with MSI-high ECs and, if so, to investigate whether EPM2AIP1 IHC can be used to rule out MLH1 methylation cases.
MATERIALS AND METHODS
1. Study population
This study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Ethics Committee of the National Cancer Center Hospital (NCCH) (2014-393). The patients were provided with the option to refuse to participate by providing opt-out consent. Patients with MSI-high EC who were treated with pembrolizumab at the NCCH between April 2019 and December 2022 were included and those with histology of carcinosarcoma were excluded (n=2). We retrospectively reviewed the medical records, and efficacy was assessed using the ORR according to the new Response Evaluation Criteria in Solid Tumors: Revised RECIST guidelines (RECIST ver. 1.1). MLH1 promoter methylation analysis was performed in all patients. Patients with hypermethylated MLH1 were assigned to the SP group. Patients who fulfilled the Amsterdam Criteria and underwent genetic counseling with a recommendation for germline testing were assigned to the LS group. In the LS group, only one patient did not undergo germline testing because of patient preference; the other patients were included in the LLS group (Fig. 1).
Fig. 1. Study flow diagram.
LLS, Lynch-like syndrome; LS, Lynch syndrome; MSI, microsatellite instability; SP, sporadic.
*One patient was not performed germline testing.
2. MLH1 promoter methylation analysis
MLH1 promoter methylation analysis using SALSA MLPA Probemix ME011-D1 Mismatch Repair Genes (MRC Holland, Amsterdam, The Netherlands) was performed and evaluated in all patients according to the manufacturer’s instructions. Tumor DNA was extracted from mapped tumor areas in formalin-fixed paraffin-embedded tissue sections. Samples were classified as positive for MLH1 promoter methylation if the methylation ratio of the probe to the baseline level was above 0.1. If the samples were positive for methylation in the tumor areas, they were also examined in non-tumor areas [5].
3. Pathological diagnoses, immunohistochemical staining, and evaluation
For all patients, EPM2AIP1 IHC was performed (Clone OTI2G3, OriGene Technologies, Dilution, 1:1,000; heat induced antigen retrieval at pH 9; with linker). Staining was considered positive if at least 80% of tumor cells showed strong diffuse nuclear expression, focal or patchy if at least 10% of tumor cells showed some nuclear expression, and negative if less than 10% of tumor cells showed nuclear expression [19].
4. Statistical analysis
The median age was compared using the Kruskal-Wallis test. The χ2 test was used to evaluate the statistical difference in the response rates among the 3 groups. PFS and OS rates were estimated using the Kaplan-Meier method, and survival curves were compared using the log-rank test. Statistical analysis was performed using EZR version 1.54 (Saitama Medical Center, Jichi Medical University, Saitama, Japan) [23].
RESULTS
1. Patient and clinical characteristics
Twelve patients were included in this study. All patients were treated with pembrolizumab for recurrent EC and did not include cases of primary advanced EC. The median age of all patients was 59 (range, 31–80) years. The Eastern Cooperative Oncology Group performance status score was 0, 1, and 2 in 5, 5, and 2 patients, respectively. The histological types of the tumors were endometrioid carcinoma G1/G2 in 8 patients, endometrioid carcinoma G3 in 2 patients, and mixed carcinoma in 2 patients. The median number of cycles of pembrolizumab was 22 (range, 1–47). The patient characteristics of each group are shown in Table 1. The patients were divided into 3 groups: SP (n=4), LLS (n=4), and LS (n=4). The median age was 67 years (range, 57–80) in the SP group, 62.5 years (range, 40–71) in the LLS group, and 61 years (range, 31–70) in the LS group. There was no significant difference between groups (p=0.44). Two patients had both endometrial and ovarian cancer, one in the LLS, the other in the LS group. One patient had a recurrence more than 20 years later, and the stage was unknown owing to a lack of information at the time of initial onset. All patients received at least one chemotherapy regimen before pembrolizumab monotherapy. Regarding the site of recurrence, 5 patients had intra-abdominal lesions and 7 patients had extra-abdominal lesions.
Table 1. Patients’ characteristics.
| Characteristics (n=12) | SP (n=4) | LLS (n=4) | LS (n=4) | |
|---|---|---|---|---|
| Age (range) | 67 (57–80) | 62.5 (40–71) | 61 (31–70) | |
| ECOG PS | ||||
| 0 | 1 | 2 | 2 | |
| 1 | 2 | 2 | 1 | |
| 2 | 1 | 0 | 1 | |
| Histology | ||||
| Endometrioid G1/G2 | 3 | 2 | 3 | |
| Endometrioid G3 | 1 | 1 | 0 | |
| Mixed | 0 | 1 | 1 | |
| FIGO stage | ||||
| I | 0 | 2 | 2 | |
| II | 0 | 0 | 0 | |
| III | 3 | 0 | 1 | |
| IV | 0 | 2 | 1 | |
| Unknown | 1 | 0 | 0 | |
| Prior chemotherapy | ||||
| 1 | 3 | 1 | 2 | |
| 2 | 0 | 3 | 2 | |
| ≥3 | 1 | 0 | 0 | |
| Prior radiotherapy | ||||
| Yes | 3 | 0 | 1 | |
| No | 1 | 4 | 3 | |
| Site of recurrence | ||||
| Intra-abdominal | 1 | 3 | 1 | |
| Extra-abdominal | 3 | 1 | 3 | |
ECOG PS, Eastern Cooperative Oncology Group - performance status; FIGO, International Federation of Gynecology and Obstetrics; LLS, Lynch-like syndrome; LS, Lynch syndrome; SP, sporadic.
2. Response rate and survival outcomes
The ORR in the SP group was 75%, while that in the LLS and LS groups was 100% including one complete response in each group (Table 2) (p=0.34). Furthermore, there were also no responders in the both of 2 patients with carcinosarcoma harboring the character of the SP. PFS and OS are shown in Fig. 2. The median follow-up period was 41 months (range, 0.1–56 months). The median PFS and OS were not reached. When comparing 2 groups (group 1: LLS and LS groups and group 2: SP group), the PFS and OS tended to be longer in the LLS and LS groups than in the SP group (p=0.617, p=0.157); however, the difference was not statistically significant.
Table 2. Response rate.
| Characteristics | SP (n=4) | LLS (n=4) | LS (n=4) | |
|---|---|---|---|---|
| Best response | ||||
| CR | 0 | 1 | 1 | |
| PR | 3 | 3 | 3 | |
| SD | 0 | 0 | 0 | |
| PD | 1 | 0 | 0 | |
| Response rate (%) | 75 | 100 | 100 | |
CR, complete response; LLS, Lynch-like syndrome; LS, Lynch syndrome; PD, progressive disease; PR, partial response; SD, stable disease; SP, sporadic.
Fig. 2. Kaplan-Meier curves. (A) PFS, (B) OS of all patients, and (C) PFS and (D) OS according to 2 groups (group 1, LLS and LS groups; group 2, SP group).
LLS, Lynch-like syndrome; LS, Lynch syndrome; OS, overall survival; PFS, progression-free survival.
3. Accuracy of EPM2AIP1 IHC
EPM2AIP1 nuclear staining was negative in 6 patients, focal/patchy in 3 patients, and positive in 3 patients. Four of the 6 negative patients (66.7%) had MLH1 hypermethylation by MLH1 methylation analysis (Table 3, Fig. 3). The sensitivity, specificity, and positive predictive value of EPM2AIP1 IHC for detecting MLH1 methylation were 100%, 75%, and 66.7%, respectively.
Table 3. EPM2AIP1 IHC result.
| EPM2AIP1 IHC | No. of MLH1 methylation | ||
|---|---|---|---|
| Positive | Negative | Total | |
| Negative | 4 | 2 | 6 |
| Focal/Patchy | 0 | 3 | 3 |
| Positive | 0 | 3 | 3 |
| Total | 4 | 8 | 12 |
IHC, immunohistochemistry.
Fig. 3. EPM2AIP1 IHC. (A) MLH1 promoter methylation (positive control), (B) EPM2AIP1 retained, (C) EPM2AIP1 lost, (D) EPM2AIP1 lost.
DISCUSSION
We aimed to clarify the difference in the efficacy of pembrolizumab among 3 groups of patients with MSI-high ECs and the usability of EPM2AIP1 IHC to predict the efficacy of pembrolizumab. Our results showed that the ORR was higher in the LLS and LS groups and the OS was also tended to be longer in these groups than in the SP group. In addition, EPM2AIP1 IHC had a high sensitivity for detecting MLH1 methylation.
Our results support the possibility that LLS- and LS-related ECs respond better to pembrolizumab. In patients with CRC, BRAF testing can exclude the MLH1 hypermethylated group from the MSI-high population, and BRAF V600E IHC can be used as a surrogate marker [24,25,26]. Unlike the reports on patients with MSI-high-related ECs, no statistical difference was observed in the ORR for nivolumab and ipilimumab based on the BRAF mutation status of patients with CRC in a phase II trial (CheckMate 142) [27]. Previous studies on ECs have shown a difference in the enrichment of the tumor microenvironment with immunologically active populations between MSI-high with LS-related and MSIhigh with SP ECs [14,15,28]. LS-related ECs have increased numbers of CD8+ cytotoxic T lymphocytes in the stroma and reduced numbers of CD68+ macrophages in the stromal and tumor compartments compared to SP ECs [28]; however, it is not yet clear how these differences affect responses to immunotherapies. A recent study showed that the frequency of PD-1+CD8+ T cells in the tumor microenvironment can predict the clinical efficacy of PD-1 blockade therapies [29]. Differences in the tumor microenvironment may influence the differences in efficacy. In another report, LS-related ECs were significantly more likely to demonstrate tumor cell expression of programmed death-ligand 1 (PD-L1) positive when compared with SP ECs [15,30]. These differences may be responsible for differences in efficacy; however, our study did not examine the expression of PD-L1. Whether there is a difference between LLS-related and LS-related ECs remains unclear. Further investigation is required to confirm this difference.
The positive predictive value of EPM2AIP1 in our study was lower than that reported previously. There are several reasons for this discrepancy. Methylation analysis methods differed from those in the previous study. The polymerase chain reaction method was used in a previous study [19], whereas the MLPA method was used in our study [31].
Differences in patient backgrounds may also be a cause. In a previous study, most cases were methylation-positive, and a small number of cases were LLS or LS [19]. This study included approximately equal numbers of patients. A lower prior probability of methylation positivity led to a lower positive predictive value. Several studies have described the heterogeneous methylation of MLH1 [32,33,34], intra-tumor heterogeneity may also cause inaccurate results of EPM2AIP1 IHC for detecting MLH1 methylation.
EPM2AIP1 IHC is less predictive than MLH1 methylation analysis, but it may become a useful surrogate marker for MLH1 methylation because it takes less time and is less expensive. Further studies are needed to determine whether EPM2AIP1 IHC is a predictor of pembrolizumab efficacy, but EPM2AIP1 IHC may be useful in identifying patients with poor response to pembrolizumab. In addition, the possibility that these patients may benefit from additional lenvatinib has been reported, and more cases need to be accumulated [35].
This study had some limitations. Previous research found that only 30%–40% of patients with LS meet the Amsterdam Criteria [36,37,38]. We did not conduct a germline variant analysis for all patients, which means that some patients who should have been categorized as having LS may have been included in the group classified as having LLS. Among the 4 patients identified in our study, germline mutations were confirmed in 3 patients as indicative of LS, however, only one patient refused to receive germline testing. In addition, most patients in this study received pembrolizumab for up to 2 years if treatment could be continued. Although there is currently no clear evidence regarding the optimal duration of treatment, there is a possibility that prolonged treatment beyond 2 years may improve prognosis in patients who have responses to treatment [39]. More data are needed on this point. Furthermore, patient background histology and previous treatment between the 3 groups may have affected the treatment outcomes, but this was not investigated due to the small number of patients in this study.
It has been suggested that pembrolizumab may be more effective in the LLS and the LS groups. Although MLH1 methylation is a more accurate predictor than EPM2AIP1 IHC analysis, it may be useful for ruling out MLH1 methylation cases due to its high sensitivity. However, further research is required to determine whether EPM2AIP1 IHC can accurately predict the efficacy of pembrolizumab.
ACKNOWLEDGEMENTS
The authors thank Mineko Ushiama, Maiko Matsuda, Yoko Shimada, and other physicians and staff members at the National Cancer Center Hospital for their assistance and support.
Footnotes
Funding: This work was supported by the National Cancer Center Research and Development Fund (2023-J-2, NCC Biobank, and NCC Core Facility).
Presentation: This study was presented at the 38th Annual Meeting of the Korean Society of Gynecologic Oncology.
Conflict of Interest: Tadaaki Nishikawa reports receiving Grants to his institution from Daiichi-Sankyo, and AstraZeneca. He also reports receiving honoraria for speakers’ bureaus and manuscript writing from AstraZeneca, Chugai, Takeda, MSD, Eisai, Taiho, Roche Diagnostics and Sanofi.
Kan Yonemori reports receiving grants or contracts from Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc. (Rahway, NJ, USA), Daiichi-Sankyo, AstraZeneca, Taiho, Pfizer, Novartis, Takeda, Chugai, Ono, Seattle Genetics, Eisai, Eli Lilly, Genmab, Boehringer Ingelheim, Kyowa Hakko Kirin, Nihon Kayaku, Sanofi and Haihe. He also reports receiving honoraria for lectures, presentations, and speakers’ bureaus for Pfizer, Eisai, AstraZeneca, Eli lilly, Takeda, Chugai, MSD, FujiFilm Pharma, Bayer, Asteras, Boehringer Ingelheim, Daiichi Sankyo, PDR Pharma, Sanofi.
- Conceptualization: O.R., N.T.
- Data curation: O.R., Y.H., S.K., N.T.
- Formal analysis: O.R., N.T.
- Funding acquisition: S.K., Y.K.
- Investigation: O.R., Y.H., S.K., N.T.
- Methodology: O.R., S.K., S.T.
- Project administration: S.T., N.T.
- Resources: O.R., Y.H., S.K., S.T., K.T., Y.K., N.T.
- Supervision: Y.H., S.K., S.T., K.T., Y.K., N.T.
- Validation: S.T., K.T., Y.K.
- Visualization: O.R., Y.H., S.K.
- Writing - original draft: O.R.
- Writing - review & editing: Y.H., S.K., S.T., K.T., Y.K., N.T.
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