Abstract
Background/Aim: Cancers with a microsatellite instability-high (MSI-H) or deficient mismatch repair (dMMR) status respond to immune checkpoint inhibition (ICI). Regardless of the tumor type, MSI-H/dMMR status is a reliable biomarker for ICI responsiveness. This study aimed at determining the MSI-H status in precursor lesions to esophageal adenocarcinoma (EAC) such as Barrett’s esophagus (BE) and BE with either low-grade dysplasia (LGD) or high-grade dysplasia (HGD). Patients and Methods: We performed immunohistochemical staining (IHC) for PMS2, MSH6, PD1, and PD-L1. Results: All cases of BE (50), LGD (48), and HGD (50) had intact PMS2 and MSH6 nuclear expression; were negative for PD1; and had a PD-L1 combined positive score (CPS) score <1. One EAC case (2%) was negative for PMS2 nuclear expression. One HGD case (2%) and two EAC cases (4%) were PD1 positive (CPS score <1 applied to PD1). One EAC case (2%) had a CPS score >1, and one EAC case (2%) was MSI-H. MSI-H tumors usually show PD-L1 expression, although the MSI-H EAC in this study had a PD-L1 CPS score of <1. Conclusion: Further studies investigating EAC and its precursor lesions for PD1, PD-L1, and dMMR status may be informative regarding the immunogenicity of the evolution of EAC.
Gastroesophageal reflux disease (GERD), which affects nearly 60 million adults in the USA, is a risk factor for development of Barrett’s esophagus (BE), affecting nearly 700,000 adults in the USA (1). The current U.S. criteria for diagnosing BE require the histologic identification of goblet cell intestinal metaplasia (1, 2). The prevalence of BE in the USA, as estimated from a simulation model confirmed by Surveillance, Epidemiology, and End Results (SEER) data, is 5.6% (5.49-5.70%) (2). BE is a risk factor for the development of low-grade dysplasia (LGD), high-grade dysplasia (HGD), and esophageal adenocarcinoma (EAC) (3-5). The 5-year survival rate of EAC patients is still less than 20% (6).
The use of molecularly targeted agents is under investigation in several clinical trials for the treatment EAC (7, 8). However, there are currently targeted therapeutic options that have been approved by the Food and Drug Administration (FDA), which include the monoclonal antibody trastuzumab for patients who have HER2-positive gastroesophageal cancer and have not previously received any treatment (9-12). In addition, according to the results of the REGARD trial the monoclonal antibody ramucirumab, targeting the VEGF/VEGFR2 pathway and inhibiting tumor blood vessel growth, was approved by the FDA for a subset of patients with advanced gastro-esophageal junction (GEJ) adenocarcinoma, after prior treatment with fluoropyrimidine- or platinum-based chemotherapy (13).
Another therapeutic strategy that has been deservedly investigated is immune checkpoint inhibition (ICI) via inhibition of programmed cell death protein 1 (PD-1) on activated T cells, which interacts with programmed cell death ligand 1 or 2 (PD-L1 or PD-L2), and subsequently inhibits T-cell activation (8-10). In tumors with increased immunogenicity, over-expression of PD-L1 is an evolutionary strategy employed by cancer cells to inhibit T-cell immunogenic response (10, 11). Studies that utilize ICI to target tumors with an immunogenic phenotype have had promising results. Specifically, investigation of immunogenic tumors exhibiting microsatellite instability-high (MSI-H) has shown that the deficient mismatch repair (dMMR) status has therapy response implications, to the point that, the FDA approved pembrolizumab for metastatic dMMR solid tumors agnostic of anatomic site of origin (9-12). PD-L1 protein expression assayed by immunohistochemistry (IHC) is also a valuable biomarker predictive of responsiveness to ICI (Nivolumab and Pembrolizumab), as a combined positive score (CPS) ≥1 for PD-L1 has been an FDA-approved test [PD-L1 IHC 22C3 pharmDx (Dako North America Inc., Santa Clara, CA, USA)] for several cancer types, including GEJ carcinomas that are not candidates for surgical resection or definitive chemoradiation based on the results of the KEYNOTE-590 study (14). In addition to the MMR status and PD-L1 expression, there are other biologic markers that have shown correlation with tumor immunogenicity such as tumor mutational burden (TMB), tumor-infiltrating lymphocytes (TILs), peritumoral lymphocytes, and/or tumor-adjacent lymph node–like structures, deficient DNA damage response/repair (dDDR) and the immunoscore, which is a digital pathology-based assay for the quantification of immune cells such as CD3+ and CD8+ lymphocytes (15-17).
Initial studies have demonstrated inter-study variability regarding the rates of PD-L1 and (MSI-H) status in EAC and the precursor lesions from which EAC arises (18-21).
This study was performed to address the unmet need to systematically characterize the prevalence of these key immune biomarkers in EAC and established precursors. In this study, we determined the prevalence of PD-L1 expression in combination with MMR expression status in BE, LGD, HGD and EAC to assess potential response to ICI.
Patients and Methods
This study was approved by the Moffitt Cancer Center Institutional Review Board (IRB). The clinical records, and hematoxylin and eosin (H&E) stained sections selected from endoscopic mucosal resection or esophagogastrostomy resection specimens, containing BE, BE-LDG, BE-HGD, and EAC were reviewed.
This study focused on 425 patients of which 198 were selected. Specimens from these patients included: 50 cases of BE, 48 cases of BE with LGD (BE-LGD), 50 cases of BE with HGD (BE-HGD), and 50 cases of invasive EAC. When available, samples of normal gastroesophageal mucosa (NM), were also selected. These tissues were used to build a Barrett’s esophagus - tissue microarray (BE-TMA).
The H&E slides were evaluated by two pathologists (M.S. and D.C.) to confirm the pathologic diagnosis, while overt areas of BE, BE-LGD, BE-HGD, and EAC were annotated. The formalin-fixed paraffin-embedded (FFPE) tissue blocks corresponding to the H&E slides of the 198 cases were used to produce the BE-TMA.
For analysis of MMR protein expression by IHC, 4 μm TMA sections were pretreated and stained using the Benchmark Ultra Ventana platform (Roche Diagnostics, Indianapolis, IN, USA). MMR status was evaluated using “ready to use” (RTU), monoclonal antibodies against PMS2 [Clone EPR3947; Ventana/Roche (Basel, Switzerland)], MSH2 [Clone G219-1129; Ventana/Roche (Basel, Switzerland)] and MLH1 [Clone M1; Ventana/Roche (Basel, Switzerland)], and MSH6 (EPR3945; Nordic Biosite, Taby, Sweden) diluted 1:50. IHC was denoted as negative when all tumor cells showed loss of nuclear staining. Surrounding stromal cells and tumor-initiating cells (TICs) served as internal controls for each TMA core. For immunohistochemical analyses of PD-1 and PD-L1, 4 μm tissue sections were pre-treated using the DAKO PT link system (DAKO; Glostrup, Copenhagen, Denmark) and stained in an Autostainer Plus (DAKO) with the anti PD-1 antibody NAT105 (Abcam, Cambridge, MA, USA) diluted 1:50 and the anti PD-L1 (E1L3N; Cell Signaling Technology, Inc. (CST), Danvers, MA, USA) diluted 1:200. The scoring of these stains was performed according to the FDA approved combined positive score (CPS) criteria (22).
Results
Patients’ characteristics are summarized in Table I. The cases of EAC included 47 men and 3 women. The tumor size ranged between 0.2 and 7.3 cm (median, 2.1 cm). Five (5) tumors were well differentiated, 23 were moderately differentiated, and 22 were poorly differentiated. In addition, 26 cases were stage I, 14 cases were stage II, 9 cases were stage III, and 1 case was stage IV. Ten cases had received preoperative adjuvant radio chemotherapy. The tumor stage was assigned according to the 8th edition of the UICC/AJCC TNM classification.
Clinical and pathological findings (adopted from reference 38).
All cases of BE [50], LGD [48], and HGD [50] had an intact nuclear expression of PMS2 and MSH6, were negative for PD1, and had a PD-L1 CPS score <1. One case of EAC (2%) had a loss of nuclear expression of PMS2. One case of HGD (2%) and two cases of EAC (4%) were PD1 positive (CPS score <1 applied to PD1). One case of EAC (2%) had a CPS score >1, and one case of EAC (2%) was MSI-H (Figure 1).
Immunohistochemical results. All cases had an intact nuclear expression of mismatch repair proteins, MSH6 and PMS2. (A) Intact MSH6 protein expression in EAC; (B) A case of EAC exhibiting PDL1 positivity with CPS>1.
Discussion
In this study, an anti-PD-L1 antibody (E1L3N), was used, which has been shown in a recent study to be comparable to the three clones (22C3, 28.8 and SP142) used in the FDA-approved assays for reporting PD-L1 expression by IHC (23).
Expression of PD1 was negative in all cases of BE-LGD and BE, while 1 case (2%) of BE-HGD and 2 EAC cases (4%) were PD1 positive. One EAC case (2%) was found to be statistically indicative of PD-L1 with a CPS score of greater than 1.
A recent study by Fassan et al. reported that premalignant esophageal (BE, BE-LGD, BE-HGD), gastric lesions and adenocarcinomas of the GEJ showed PD-L1 expression, as defined by a CPS≥1, in a significant proportion of cases (48 out of 155; 31%) as well as dMMR in 3.1% of esophageal lesions (24). Other studies investigating dMMR have demonstrated variability regarding dMMR status in Barrett’s related pre-neoplastic lesions. For instance, a recent study of dMMR status in BE reported that 0.8% of BE cases showed a loss of MLH1 and MSH2 (19). Another study of 70 patients reported that 81.8% of patients with EAC, 32.4% of patients with BE and dysplasia, as well as 50% of patients with BE had loss of MLH1 expression; while EAC, BE-LGD and BE-HGD did not show loss of MSH2 and MSH6 expression, and 18.2% BE showed loss of MSH2 and MSH6 expression (25).
In light of the inter-study variability found between previous studies in the literature, the contrast of the relatively high prevalence in positive PD-L1 and dMMR status in these studies, when compared with the lower prevalence in positive PD-L1 and dMMR status in the esophageal pre-neoplastic lesions of this study, suggests that there would be benefit in evaluating an larger diverse cohort to determine the exact prevalence in positive PD-L1 and dMMR status in Barrett’s related pre-neoplastic lesions. Moreover, investigation of other genomic alterations within Barrett’s related pre-neoplastic lesions would enrich the current understanding the evolution of EAC complementing the studies characterizing Barrett’s related pre-neoplastic lesions to date (26-32).
The limitations of this study are primarily based on the interpretation of PD-L1/MMR expression that is limited due to the relatively small size of the TMA cores evaluated, which restricted the amount of tissue available for evaluations, in comparison to the amount of tissue typically available in the specimens from which the TMA cores were derived. Adequate size of the tissue for evaluation is a factor that can mitigate the heterogeneity observed in PD-L1/MMR expression. Given that PD-L1 expression has been shown to be heterogenous in multiple studies, including those demonstrating significant differences in PD-L1 expression between surgically resected specimens and matched biopsies, a confounding factor limiting interpretation of PD-L1 expression is that BE, BE-LGD and BE-HGD tissues were overwhelmingly solely available as biopsies (33, 34).
Investigation of ICI responsiveness in EAC has deservedly warranted a number of studies including notably the KEYNOTE-590 study, the findings of which were supportive for the FDA approval of pembrolizumab for EAC or gastroesophageal (GEJ) carcinoma (14, 35, 36). It is understandable that given the challenge of retrospective studies for pre-neoplastic lesions of EAC, there are limited studies investigating ICI responsiveness of precursor lesions to EAC, which consequently limits the data available regarding these precursor lesions.
Given that the studies available confirm that ICI is effective in the context of dysplasia (37), additional studies corroborating ICI effectiveness in dysplasia have profound implications for potential therapeutic strategies, as there are many clinical scenarios wherein concomitant dysplasia may exist separately from a discrete EAC. Thus, precursor lesions (BE, BE-LGD, BE-HGD) of EAC have the potential to be an early target, or a concomitant target with respect to a contemporaneous EAC, which could have clinical implications for PD-L1 expression and MMRd status, although further studies in a larger population should be performed. Given that regression of HGD of the cervix after ICI has been reported, the concept of prophylaxis to future EAC is not an unprecedented concept, although future studies should confirm that there is clear benefit for this approach.
Further studies evaluating the expression of MMR proteins and PD-L1/PD-1 in BE, BE-LGD, BE-HGD, and EAC, using larger endoscopic mucosal resection specimens would facilitate the characterization of the overall prevalence of PD-L1 expression and dMMR status in these lesions. Continued investigation will assist in identifying patients who may be candidates for ICI.
Acknowledgements
The project described was supported by Departmental funds of the Department of Pathology, H. Lee Moffitt Cancer Center.
Footnotes
Authors’ Contributions
Mohammad Shahid and Zachary Mayer drafted the manuscript, Linda Mora and Aejaz Nasir revised the original draft, James Saller extensively revised the draft and formatted the references, Domenico Coppola planned, originated and supervised the study and provided the pictures.
Conflicts of Interest
The Authors have no conflicts of interest to declare in relation to this study.
- Received October 25, 2021.
- Revision received December 23, 2021.
- Accepted January 4, 2022.
- Copyright © 2022, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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