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Original research
β-catenin and PD-L1 expression in mismatch repair deficient endometrial carcinomas
  1. Margaret Rowe1,
  2. Rahul Krishnan2,
  3. Anne Mills3 and
  4. Kari Ring2
  1. 1University of Virginia School of Medicine, Charlottesville, Virginia, USA
  2. 2Obstetrics & Gynecology, University of Virginia Health System, Charlottesville, Virginia, USA
  3. 3Pathology, University of Virginia Health System, Charlottesville, Virginia, USA
  1. Correspondence to Dr Kari Ring, Division of Gynecologic Oncology, Obstetrics & Gynecology Dept., University of Virginia, Charlottesville, Virginia, United States; kel7j{at}virginia.edu

Abstract

Introduction Predictors of non-response in mismatch repair deficiency cancers are poorly understood. Upregulation of the canonical Wnt pathway has been associated with decreased immune cell infiltration in many cancer types. The relationship between Wnt/β-catenin pathway activation and the programmed death-ligand 1 axis in endometrial cancer remains poorly characterized. This study evaluates β-catenin expression in a well characterized cohort of endometrial cancers by mismatch repair status and programmed death-ligand 1 expression.

Methods Whole sections of formalin-fixed, paraffin embedded tissue from 23 Lynch syndrome-associated carcinomas, 20 mutL homolog-1 (MLH1) promoter hypermethylated carcinomas, and 19 mismatch repair intact carcinomas were evaluated. Immunohistochemistry staining for β-catenin and programmed death-ligand 1 was performed on all cases. Programmed death-ligand 1 expression was scored in both the tumor and the peri-tumoral immune compartment. Tumor staining was classified as positive when membranous (programmed death-ligand 1) staining was present in ≥1% of tumor cells. Immune stromal staining was scored as positive when ≥5% of peritumoral and intratumoral immune cells (including lymphocytes and macrophages) showed reactivity.

Results Six tumors (6/62, 9.7%) demonstrated nuclear expression of β-catenin (4 were Lynch syndrome-associated, 1 was MLH1 methylated, 1 was mismatch repair intact). The majority of tumors with nuclear β-catenin expression demonstrated concomitant tumoral programmed death-ligand 1 expression (5/6, 83.3%) and were more likely to demonstrate tumoral programmed death-ligand 1 expression compared to tumors without nuclear β-catenin expression (83.3% vs 39.3%, p=0.04). Both tumoral and immune cell expression of programmed death-ligand 1 was statistically significantly associated with mismatch repair deficient tumors.

Discussion Tumors demonstrating nuclear β-catenin expression were more likely to express tumoral programmed death-ligand 1 staining than tumors without nuclear β-catenin expression. Nuclear β-catenin expression could be a potential predictive biomarker for non-response to immune checkpoint inhibition in mismatch repair deficient tumors. Nuclear β-catenin expression status should be considered as a translational endpoint in future clinical trials of immune checkpoint inhibition in endometrial cancer.

  • endometrial neoplasms
  • lynch syndrome II
  • uterine neoplasms
  • pathology

https://doi.org/10.1136/ijgc-2020-001239

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    HIGHLIGHTS

    • Wnt/β-catenin pathway activation is associated with decreased immune cell infiltration in many cancers.

    • Endometrial cancers with β-catenin pathway activation were more likely to express tumoral programmed death-ligand 1.

    • Wnt/β-catenin activation could predict non-response to checkpoint inhibition in endometrial cancer.

    Introduction

    Endometrial cancer remains the most common gynecologic malignancy in the USA.1 While surgery with or without adjuvant radiotherapy/chemotherapy is curative for a majority of early stage endometrial cancers, there remains no curative treatment options for advanced stage and recurrent disease despite advances in the treatment of these tumors.2 3 Chemotherapeutic agents exhibit only modest efficacy in these scenarios. Immunotherapy, targeting immune checkpoint receptors such as programmed cell death protein 1, has emerged as a promising new treatment option for endometrial cancers with microsatellite instability. Given high neoantigen loads, lymphocyte infiltration, and expression of immune checkpoint ligands that contribute to immune system evasion, microsatellite stability-high endometrial tumors are candidates for immune checkpoint blockade therapy.4

    Programmed death-ligand 1 expression has been utilized as a predictive biomarker for immune checkpoint blockade in several cancer types such as melanoma5 and non-small cell lung cancer.6 However, to date, programmed death-ligand 1 expression alone has not been shown to be a reliable biomarker for predicting response to immune checkpoint inhibition in endometrial cancer.7 Elucidating the heterogeneity of the tumor immune environment is therefore crucial to understanding which immunotherapeutic interventions can be efficacious in endometrial cancer, including those targeting programmed cell death protein 1 and programmed death-ligand 1.

    Upregulation of the highly conserved Wnt/β-catenin pathway has recently been associated with decreased immune cell infiltration across a number of human cancers, suggesting that somatic mutational gain may be linked to an immune exclusion phenotype in many tumors.8–10 Dysregulation of the Wnt/β-catenin pathway in endometrioid adenocarcinomas of the uterus is commonly caused by a mutation in exon 3 of the gene encoding β-catenin, CTNNB1.11–13 Endometrioid endometrial carcinomas with CTNNB1 mutations have recently been associated with significantly worse recurrence-free survival and overall survival in low grade and early stage subset of patients.12 14 15 Wnt pathway activation leads to β-catenin transportation and accumulation within the nucleus, where it binds to transcription factors T cell factor/lymphoid enhancer factor-1 (TCF/Lef1) and mediates transcription of Wnt genes. Accumulated nuclear β-catenin can be visualized via immunohistochemistry and it is estimated that between 11–38% of endometrial cancers show nuclear β-catenin expression and is associated with CTNNB1 mutations.14 16 17

    The relationship between Wnt/β-catenin pathway activation and programmed death-ligand 1 expression in high-frequency microsatellite instability/mismatch repair deficient endometrial cancers remains poorly understood, and a better understanding of this relationship may potentially aid in determining its use as a biomarker for treatment response. Our objective was to evaluate the relationship between nuclear β-catenin expression and programmed death-ligand 1 expression in a well characterized cohort of endometrial cancers by microsatellite instability status.

    Methods

    Case Selection

    Following institutional review board approval, cases of endometrial cancer treated at our institution with available tissue were identified retrospectively using a departmental database. Beginning with the most recent cases, endometrioid adenocarcinomas were included if the patient was 18 years of age, received treatment at the University of Virginia, and had tumor pathology samples available for analysis. Whole sections of formalin-fixed, paraffin embedded tissue from 23 Lynch syndrome-associated carcinomas, 20 mutL homolog-1 (MLH1) promoter hypermethylated carcinomas, and 19 mismatch repair intact carcinomas were evaluated. Numbers were based on the maximum available number of cases from the least common subgroup (Lynch syndrome-associated cases). Clinical and pathologic characteristics were obtained from the electronic medical record.

    Mismatch Repair Status

    Mismatch repair status was classified at the time of original diagnosis through the universal Lynch syndrome screening program using immunohistochemistry for the four mismatch repair proteins MLH1 (clone ES05, predilute; Leica Biosystems), PMS2 (clone MRG-28Mab, predilute; Cell Marque), MSH2 (clone 25D12, predilute; Leica), and MSH6 (clone 44Mab, predilute; Cell Marque). The 23 Lynch syndrome-associated carcinomas included tumors from 10 patients with germline testing confirmed Lynch syndrome and tumors from 13 patients with mismatch repair loss suggestive of Lynch syndrome but without germline confirmation. Mismatch status repair losses suggestive of Lynch syndrome were total loss of nuclear expression of both MSH2/MSH6, isolated loss of MSH6, or isolated loss of PMS2. The 20 MLH1 promoter hypermethylated tumors were classified by complete nuclear loss of expression of both MLH1/PMS2 on immunohistochemistry and polymerase chain reaction (PCR) confirmation.

    β-Catenin Immunohistochemistry

    Immunohistochemical staining for β-catenin (Epitomics, E247, 1:500 dilution) was performed on all cases. Nuclear localization of β-catenin immunohistochemical expression in the 62 tumor samples was performed with light microscopy by a trained gynecologic pathologist (AMM) blinded to the clinical and pathologic information. Nuclear β-catenin localization of any extent was classified as positive as prior studies have suggested that greater than half of cases with CTNNB1 mutations show <10% of cells bearing nuclear β-catenin localization.17

    Programmed Death-Ligand 1 Immunohistochemistry

    Immunohistochemical staining for programmed death-ligand 1 (Spring Biosciences, AP142, 1:200 dilution; internally validated against Dako 22C3 clone) was performed in all cases. Programmed death-ligand 1 expression for this tumor set has been previously reported.18 Programmed death-ligand 1 expression was scored in both the tumor and the peri-tumoral immune compartment. Staining was scored manually by AMM. Tumor staining was classified as positive when clear membranous programmed death-ligand 1 staining was present in ≥1% of tumor cells. Staining extent was further characterized in the following subcategories: 1–5%, 6–10%, 11–25%, 26–50%, and >50%. Immune stromal staining was scored as positive when ≥5% of peritumoral and intratumoral immune cells (including lymphocytes and macrophages) showed reactivity and was further subdivided by extent as 5–10%, 11–25%, 26–50%, and >50%.

    Statistics

    Descriptive statistics were calculated for variables of interest. Statistical analyses were performed using Fisher’s exact test for categorical variables and a one-way analysis of variance (ANOVA) with Tukey’s test for multiple comparisons for continuous variables with Statistical Package for the Social Sciences (SPSS) statistics (version 24, Armonk, NY).

    Results

    Case Distribution

    Clinical characteristics by mismatch repair status are shown in Table 1. There were no differences in tumor grade or stage at diagnosis across groups by mismatch status repair status. The mean age of onset of Lynch syndrome-associated tumors (59 years) was younger than the mean age of onset of MLH1 promoter hypermethylated tumors (68 years, p=0.018), but not mismatch status repair intact tumors (64 years, p=0.12). There were no differences in adjuvant therapy, recurrence, or vital status based on mismatch status repair status.

    View this table:
    Table 1

    Clinical characteristics

    β-Catenin Expression

    Six tumors (6/62, 9.7%) demonstrated nuclear expression of β-catenin (Table 2; Figure 1 and online supplementary figure 1). The majority of the entire tumor set showed only normal (membranous) expression of β-catenin (56/62,90.3%, Figure 2). Of the tumors that demonstrated nuclear β-catenin expression, four were Lynch syndrome-associated (4/23, 17.4%), one was MLH1-methylated (1/20, 5%), and one was mismatch status repair intact (1/19, 5.3%) (Online supplementary table 1). Two tumors showed dual MSH2/MSH6 loss on immunohistochemistry and tested negative for germline mutations, one showed isolated MSH6 loss on immunohistochemistry and tested negative for germline mutations, and one tumor showed isolated PMS2 loss on immunohistochemistry and tested positive for a pathogenic variant in PMS2 (c.150delinsAG). Three tumors were stage IA endometrioid tumors and were either grade 1 or 2. One tumor was a stage IIIC1, grade 3 de-differentiated tumor. All three stage IA endometrioid tumors showed positive tumoral programmed death-ligand 1 expression in 1–5% of cells while the stage IIIC1 de-differentiated tumor showed positive tumoral programmed death-ligand 1 expression in 6–10% of cells.

    Supplemental material

    Supplemental material

    Figure 2
    Figure 2

    The majority of endometrial carcinomas demonstrated normal β-catenin expression with staining restricted to the cell membrane and no evidence of nuclear expression, as demonstrated in this MLH1-hypermethylated, grade 1 endometrial carcinoma (200x; A: hematoxylin and eosin; B: β-catenin).

    View this table:
    Table 2
    Figure 1
    Figure 1

    This MSH6-deficient grade 1 endometrioid carcinoma demonstrated strong nuclear β-catenin expression in a subset of cells, consistent with a CTNNB1 mutation. (200x; A: hematoxylin and eosin; B: β-catenin). Membranous PD-L1 expression was also present in a subset of tumor cells (200x, C: PD-L1).

    Programmed Death-Ligand 1 Expression

    Tumor programmed death-ligand 1 staining of at least 1% of cells was present in 27/62 (43.5%) of the tumor set. Positivity was most common at the infiltrating edge of the tumor. The minority of mismatch status repair-intact tumors showed positive tumoral programmed death-ligand 1 staining (1/19, 5.3%). In contrast, 18/23 (78.3%) of Lynch syndrome-associated tumors and 8/20 (40%) of MLH1 promoter hypermethylated tumors demonstrated tumoral programmed death-ligand 1 staining (p<0.001). Positive tumoral programmed death-ligand 1 staining occurred predominantly between 1–10% of tumor cells for each tumor type (Lynch syndrome-associated 15/23, 65%; MLH1 promoter hypermethylated 6/20, 30%; mismatch status repair-intact 1/19, 5.3%). Three Lynch syndome-associated tumors (3/23, 13.0%) and two MLH1 promoter hypermethylated tumors (2/20, 10%) showed tumoral programmed death-ligand 1 staining >10% of cells.

    Positive immune staining of programmed death-ligand 1 showed a similar pattern of increased positivity in mismatch status repair-deficient tumors. All of the Lynch syndrome-associated tumors (23/23, 100%) and MLH1 promoter hypermethylated tumors (20/20, 100%) demonstrated immune programmed death-ligand 1 staining, while only 13/19 (68.4%) mismatch status repair-intact tumors stained positively for immune programmed death-ligand 1 expression (p=0.001).

    Relationship between β-Catenin and Programmed Death-Ligand 1 Expression

    There were no significant differences in immune programmed death-ligand 1 expression between tumors with nuclear β-catenin expression (Table 3). However, the majority of tumors with nuclear β-catenin expression also demonstrated tumorous programmed death-ligand 1 expression (5/6, 83.3%) and were more likely to demonstrate tumoral programmed death-ligand 1 expression compared with tumors without nuclear β-catenin expression (83.3% vs 39.3%, p=0.04); 22/62 tumors with only membranous β-catenin expression (39.3%) exhibited positive tumoral programmed death-ligand 1 expression.

    View this table:
    Table 3

    Discussion

    This study investigated immunohistochemical expression patterns of programmed death-ligand 1 and Wnt pathway activation via nuclear β-catenin expression in 62 cases of endometrial carcinoma, comparing mismatch repair-deficient, MLH-hypermethylated and mismatch status repair intact tumors. To our knowledge, this study is the first investigation of nuclear β-catenin expression specifically in endometrial carcinoma by mismatch repair status.

    Therapies targeting the programmed cell death protein 1/programmed death-ligand 1 checkpoint axis have shown promise in multiple tumor types such as melanoma and non-small cell lung cancer.18–20 Data on gynecologic cancers7 21 have suggested that single agent checkpoint inhibitor therapy is not universally effective in targeting all cancers that express programmed death-ligand 1. In the KEYNOTE-028 trial, pembrolizumab single agent immunotherapy showed an objective response rate of only 13% among 24 patients in advanced programmed death-ligand 1 expression positive, microsatellite stable endometrial cancers,7 suggesting programmed death-ligand 1 expression alone was not a sufficient predictor of response to immune checkpoint inhibition.

    Several challenges exist with utilizing programmed death-ligand 1 expression as a biomarker for immune checkpoint inhibition response. In addition to differences among the various proprietary detection assays available, variability exists among study trials in terms of tissue types analyzed (fresh vs archival).22 The cell types included (tumor alone vs tumor and immune cells) in calculating programmed death-ligand 1 expression can vary22 and can further impact the interpretation of programmed death-ligand 1 expression levels. There is also heterogeneity among studies for cut-off thresholds for what constitutes a positive result, ranging from 1–50% based on percent tumor cells stained.23 In non-small cell lung cancer, tumor cell expression alone is used (tumor proportion score); in contrast, the combined positive score, which measures both tumor and immune cell expression,24 has been utilized in cervical cancer21 where a score of 1% is considered positive. New evidence also suggests that programmed death-ligand 1 expression in tumors is subject to both temporal and spatial variation.25

    The lack of a meaningful clinical response for programmed death-ligand 1 immunotherapies in many settings is thought to be at least partly attributable to the concomitant presence of other immunomodulatory molecules.26 The Wnt/β-catenin pathway has recently come into focus for its potential role in perturbing immune cell infiltration across a number of human cancers.10 12 In our study, β-catenin nuclear expression was present in 9.7% of all tumors (four were Lynch syndrome-associated, one was MLH1 methylated, was mismatch status repair intact). This is less than previous studies where nuclear β-catenin expression rates in endometrial cancer have ranged from 13–44%.11 27 28 Nuclear β-catenin expression was strongly associated with concomitant tumoral programmed death-ligand 1 expression compared with tumors without nuclear β-catenin expression (83.3% vs 39.3%, p=0.04). Nuclear β-catenin expression was, however, not associated with tumor grade or stage. It is likely that our study was not sufficiently powered to detect differences by tumor stage and grade, which have previously been associated with CTNNB1 mutations.14

    Upregulation of the canonical Wnt/β-catenin pathway has been associated with decreased immune cell infiltration in a variety of other malignancies.29–31 A recent analysis of The Cancer Genome Atlas showed mutations of β-catenin signaling molecules were enriched threefold in non-T-cell-inflamed tumors relative to T-cell-inflamed tumors across 14 tumor types including uterine carcinoma.30 Additionally, murine melanoma models have demonstrated that tumor cell-intrinsic β-catenin activation prevented T-cell priming and subsequent infiltration into the tumor microenvironment, resulting in resistance to immune checkpoint blockade therapy.29

    One mechanism of this reduced immune cell infiltration phenomenon appears to be through reduced transcription of key chemokine genes, leading to less Batf3-lineage dendritic cell recruitment and subsequent failure to prime and recruit CD8+ T cells.29 Upregulation of transcriptional repressor ATF3, by β-catenin, has also been observed, which prevents the tumor cell from secreting CCL4, a chemokine that allows antigen-presenting cells to infiltrate the tumor.30 Furthermore, RNA interference of CTNNB1 and β-catenin pathway signaling in syngeneic mouse tumor models has been shown to increase T cell infiltration and increase the susceptibility of tumors to immune checkpoint inhibition via programmed cell death protein 1 and cytotoxic T lymphocyte-associated protein 4 antibodies.32

    The increased rate of concomitant β-catenin nuclear expression with tumor programmed death-ligand 1 in these endometrial cancers raises interesting questions about the implications of nuclear β-catenin co-expression in these tumors. One possibility is that the response to anti-programmed death-ligand 1 therapies in patients with these tumors may be tempered by the presence of Wnt pathway expression. The pattern of nuclear β-catenin expression seen in our study additionally provides evidence for variation within subsets of endometrial cancers with microsatellite instability. Though it did not meet statistical significance, nuclear β-catenin expression was higher among Lynch syndrome-associated cancers, compared with MLH1-hypermethylated tumors as well as mismatch status repair intact tumors. The involved Lynch syndrome tumors harbored isolated PMS2, MSH6 as well as, in one instance, dual mutations of MSH2/MSH6. Identifying clinically distinct subtypes of endometrial cancers defined by CTNNB1 mutations could aid as a potential marker for non-response to immune checkpoint inhibition, providing a potential means to aid immune checkpoint inhibition.

    Recent research has illustrated the potential of combination therapies in settings where immune checkpoint inhibition monotherapy has been less effective. In a recent phase 2 study,33 the use of the multikinase inhibitor lenvatinib in combination with pembrolizumab produced an objective response rate in 25 (47%) of 53 patients with primarily advanced microsatellite stable endometrial cancer, a higher response rate than seen with pembrolizumab or lenvatinib alone.7 34 Compared with the results seen with pembrolizumab single agent treatment in the KEYNOTE-028 trial, these findings illustrate the complex interplay of various signaling pathways present within the tumor microenvironment and the need for identifying new biomarkers that predict response to immune checkpoint inhibition.

    The scope of our study was exploratory in nature; ultimately these findings will need to be explored and validated in translational studies. Future clinical trials of immune checkpoint inhibition in endometrial cancer should incorporate CTNNB1 mutation status either via sequencing or nuclear β-catenin expression as a translational endpoint. If validated, inhibition of β-catenin may represent an additional strategy for improving the response rate to immune checkpoint blockade treatments in select patients. At present, no trials exist targeting β-catenin directly; given the numerous downstream genes regulated by Wnt/β-catenin signaling, a therapy that directly and effectively targets β-catenin in tumor cells without toxicity towards normal cells remains elusive.35

    The limitations of this study include its small sample size, which limited the ability to detect meaningful survival differences or association with tumor grade and stage. Second, we utilized β-catenin immunohistochemical staining in place of CTNNB1 sequencing. CTNNB1 sequencing was not performed for confirmation of immunohistochemistry in samples. While prior studies have shown that nuclear β-catenin localization by immunohistochemistry has a strong association with CTNNB1 exon 3 gene mutations,16 17 it is not a perfect surrogate and not all mutations may have been detected. There are also currently no standardized criteria for interpreting β-catenin immunostaining. In addition, while implications of CTNNB1 exon 3 mutations have been studied in the literature, the same does not exist for β-catenin immunostaining in the setting of endometrial cancer. Nevertheless, the utility of immunohistochemical staining in these settings is valuable given its potential to aid in economical molecular analysis of individual tumors.

    In summary, our study has demonstrated that nuclear β-catenin is commonly co-expressed with programmed death-ligand 1 in mismatch repair deficient endometrial carcinomas. Given its association with immune cell function perturbation in the tumor microenvironment, the role of nuclear β-catenin expression in programmed death-ligand 1 expressing endometrial cancers should be further explored. Further studies are needed to elucidate the association between β-catenin expression and any possible clinical implications as a potential biomarker for response to checkpoint inhibition.

    Acknowledgments

    The authors would like to acknowledge the University of Virginia Biorepository and Tissue Research Facility in the performance of immunohistochemistry staining.

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    Footnotes

    • Correction notice Since the online publication of this article, the title was updated to include the abbreviation 'PD-L1'

    • Contributors All authors (MR, RK, AM and KR) substantially contributed to study design, acquisition, and analysis of data collected. All authors (MR, RK, AM and KR) drafted and/or provided critical revisions to the manuscript. All authors (MR, RK, AM and KR) have approved the final manuscript and are accountable for all aspects of the work submitted.

    • Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.

    • Competing interests None declared.

    • Patient consent for publication Not required.

    • Provenance and peer review Not commissioned; externally peer reviewed.

    • Data availability statement Data are available upon reasonable request. De-identified research data available upon request. Requests can be sent to Anne Mills, MD (AMM7R@hscmail.mcc.virginia.edu).