Abstract
Background/Aim: Peritumoral ectopic lymphoid-like structures and tertiary lymphoid structures (TLS), have been identified in various cancers. However, evidence for the role of TLS in endometrial cancer (EC) is lacking. We found a cluster of peritumoral lymphocytes with band-like structures (PLB) in the forefront of EC and analyzed their association with the clinical outcome. Materials and Methods: This was a single-center, retrospective cohort study. We evaluated peritumoral lymphoid cells using conventional hematoxylin-eosin and immunohistochemical staining by semi-quantitative digital analysis. Results: A total of 85 cases were included and the presence of PLB was examined. A strong correlation was observed between the density of PLB and progression-free survival (very low, low vs. intermediate, high; HR=0.22; 95%CI=0.093–0.52; p<0.001) and overall survival (very low, low vs. intermediate, high; HR=0.259; 95%CI=0.091–0.73; p=0.011). Conclusion: PLB in type II EC show a strong association with fatal outcome.
Endometrial cancer (EC) is one of the most common cancers of the female genital tract, and its incidence is globally increasing. EC can be classified into two major types, type I and type II, based on histology, and differs in clinical and histopathological profiles. Type I ECs are grade 1 or 2 endometrioid carcinomas that are considered to be estrogen-related, in which approximately 80-90% of ECs are classified (1). They are usually not very aggressive and they do not spread to other tissues quickly. Type II ECs are estrogen independent, with high-grade histologies including grade 3 endometrioid carcinoma, serous carcinoma, and clear cell carcinoma. Only 10% of ECs are classified as type II; however, they are associated with a poor prognosis: approximately 50% recurrence and 5-year overall survival (OS) rate of 35% (2, 3). Treatment options for advanced or recurrence cases are limited; therefore, novel efficacious therapies are needed especially for those with type II EC (4). The underlying tumor biology can be considered the main cause of the heterogeneity of the outcome among patients. Over the last few years, the tumor microenvironment has gained increasing attention, and immune escape is considered the hallmark. Immune checkpoint inhibitors have shown promise for the treatment of various cancers. For EC, the safety and antitumor activity of pembrolizumab, an anti–programmed death 1 monoclonal antibody, in patients with programmed death ligand 1-positive advanced EC have been evaluated (5). Immune checkpoint blockade with therapeutic antibodies has already been reported.
Tertiary lymphoid structures (TLS), which are ectopic lymphoid aggregates present in chronically inflamed tissue, can be confirmed in the tumor stroma. TLS are believed to promote and maintain inflammation and an anti-tumor response similar to secondary lymphoid organs (6). A number of studies have reported that tumor-associated TLS consist of T cells, B cells, mature dendric cells, high endothelial venules (HEV), and chemokines forming germinal center-like patterns in stroma near the tumor that lead to the development of specific humoral and cell-mediated immune responses, which sustain long-term immunity and a favorable clinical outcome for patients (7-16).
Recently, the presence of TLS in the tumor microenvironment has been reported to be associated with a favorable prognosis, and that a higher density of lymphoid accumulation is associated with higher levels of T-cell infiltration in different tumors (9-12, 17), which suggests the presence of TLS in tumoral tissues and that they may be a site of immune induction against cancer. However, it remains unclear whether this association is present in other types of cancer, and evidence on the local immune response in EC remains insufficient.
Therefore, we aimed to investigate the local inflammatory microenvironment in type II EC specimens. We examined whether there is an association between the presence of TLS and beneficial outcomes in EC patients. TLS has never been investigated regarding type II EC, and thus far, no studies have been performed investigating the putative correlation with the clinical outcome. Furthermore, we evaluated a cluster of lymphocytes in the forefront of the tumor in the stroma forming band-like structures, which we called peritumoral lymphoid band-like structures (PLB), as compared with TLS.
Materials and Methods
Study design and patient population. This was a single-center, observational, retrospective cohort study. The study population included all patients with histologically confirmed UICC stage I to IV type II EC that had been surgically removed or biopsied between January 2002 and January 2016, at Nagoya University Hospital. Staging was performed according to the classification suggested by UICC (7th Edition, 2010) at the initial diagnosis. Resected tissues were verified by board-certified pathologists. We evaluated only those patients who had received a valid subsequent analysis of their primary tumor tissue samples.
At the completion of the study, the minimal clinical follow-up was 48 months for the last patient included in the cohort. All patients were routinely examined every 3 months during the first 3 years after surgery. Each of these visits involved detailed medical history-taking, physical examination, and a full laboratory investigation. If patients experienced several relapses in the course of their disease, the time until the first relapse was taken into account. Patients who never reached a disease-free state were defined as having a progression-free survival of 0 months. Baseline and outcome data, recurrence, and progression-free and overall survival were retrospectively extracted from the patients’ clinical records. The primary purpose of this study was to define the association between the peritumoral immune microenvironment and prognosis of type II EC. This research was approved by the ethics committee of the Nagoya University School of Medicine (approval number 2017-0053).
Analysis of TLS and PLB maturation stages. Formalin-fixed, paraffin-embedded tumor blocks were used to prepare 3-μm-thick serial sections that were used for hematoxylin-eosin (HE) staining and immunohistochemistry. Whole-slide scans were performed at high magnification fields (HMF) imaged at 200×. An unstained slide was used to generate the spectral profile of autofluorescence in EC tissues, and single stained slides were used to generate the spectral profiles of the used fluorophores. Images of all stained tumor slides were acquired with a Zeiss Axioplan 2 microscope (Carl Zeiss Microscopy, Jena, Thüringen, Germany) and recorded with the software system ZEN DESK (Carl Zeiss Microscopy, White Plains, NY, USA).
The peritumoral region was defined as the immediate adjacent non-tumoral tissue within a 7-mm radius of the tumor front. The absolute number of positively stained peritumoral cells was counted using ImageJ count (National Institutes of Health, Bethesda, MD, USA). In each maturation stage, we counted a proportion from all TLS within each patient. The spatial distribution of the immune infiltrate was assessed at low and high magnification at the very front of tumor invasion, and the sections were screened for the presence of TLS based on the cluster of lymphocyte morphologic features (TLS: present vs. absent). Band-like aggregates of lymphatic cells were defined as PLB, whereas perivascular lymphatic aggregates were excluded. PLB were primarily identified based on the HE-morphological appearance. The PLB density estimate was derived from the tissue block with the highest PLB density in the invasive front. We defined PLB density as the number of lymphocytes per HMF of the tumor invasive front in peritumoral regions. Lymphocytes were counted semi-quantitatively in each HMF in the invasive front in peritumoral areas of the entire tissue section. More than 201 peritumoral lymphocytes/HMF were classified as high PLB, 101-200 as intermediate PLB, 6-100 as low PLB, and 0-5 as very low PLB. PLB was dichotomized into two categories (very low, low vs. intermediate, high) for various analyses.
Immunohistochemical staining. Immunohistochemical (IHC) staining of 3-μm-thick sections of formalin-fixed, paraffin-embedded tissue samples was performed. They were deparaffinized using a graded alcohol series described in standard protocols. Subsequently heat-induced antigen retrieval was carried out by incubating in Dako Target Retrieval Solution (×10), EDTA pH 9 for either 10 min or 20 min at 95° C using a water bath. Sections were incubated in blocking buffer (10% fetal bovine serum) before adding primary antibodies followed by secondary antibodies.
All samples were stained with antibodies against the marker protein, and the following antibodies were used for evaluation of tumor stroma cells: CD4 (SP35 antibody, Spring Bioscience, Pleasanton, CA, USA, M3350, 1:100), CD8 (C8/144B, DAKO, Santa Clara, CA, USA, M7103,1:100), CD68 (KPI antibody, Thermo Fisher Scientific, Waltham, MA, USA, 14-0688,1:500), CD20 (L26 antibody, Nichirei Biosciences, Tokyo, Japan, 422741,1:100), CD33 (SP266 antibody, Abcam, Cambridge, UK, ab199432,1:100), Fascin (EP5902 antibody, Abcam, ab126772,1:500), PNAd (MECA-79 antibody, Biolegend, San Diego, CA, USA, 120801,1:200), FOXP3 (236/E7 antibody, Abcam, ab20034, 1:50), IL-17(IL-17 antibody, Proteintech, Rosemont, IL, USA, 130821A, 1:100), IL-27(IL-27 antibody, Proteintech, 661641IG, 1:100). Species-specific secondary antibodies in conjunction with Histofine Simple Stain MAX PO anti-rabbit polymer (Nichirei Biosciences) were used for immunohistochemistry, and goat anti-rabbit IgG (Alexa Fluor® 488, Abcam), goat anti-rabbit IgG (Alexa Fluor® 568, Abcam, goat anti-mouse IgG (Alexa Fluor® 568, Thermo Fisher Scientific), and goat anti-rat IgG (Alexa Fluor® 568, Thermo Fisher Scientific) were used for CD4, IL-17, IL-27, FOXP3, and PNAd immunofluorescence, respectively.
We used specimens that were not incubated with the primary antibody as a negative control, and bovine serum albumin was used instead of the primary and secondary antibodies on the tissue samples.
Statistical analysis. Progression-free survival (PFS) was defined as the time between primary surgery for the tumor to evidence of recurrence at any organ site. OS was defined as the period between the primary surgery for the tumor until death from any cause. Follow-up was defined as the time from the day of surgery until recurrence or death. Survival curves were estimated using Kaplan–Meier methods and significant differences between the groups of patients were evaluated using the log-rank test. Significant variables (p-values<0.05) in the univariate analysis were added to a multivariate Cox regression model.
All statistical analyses were performed using IBM SPSS Statistics (Windows version 24.0, IBM, Chicago, IL, USA).
Results
Patient characteristics. We first searched for the presence of TLS and PLB in paraffin-embedded sections of 40 type I ECs with more than 1/2 myometrium tumor invasion. We could only find 1 patient with TLS and 3 patients with PLB (data not shown). One hundred patients with histologically verified type II ECs were enrolled, but 7 were excluded due to incomplete clinical data, insufficient amounts of paraffin-embedded tissue, or inadequate tissue, and 8 tumors involved serosa at the forefront of the tumor that could not be observed, resulting in a final study population of 85 patients (Figure 1).
Flowchart of subjects included in this study.
The median age at the time of primary surgery was 61 years (range=36-84 years). As for tumor markers, CA125 median was 22 (5-1655) IU/ml and CA19-9 median was 18 (1-2606) ng/ml. Thirty-seven patients were Stage I (43.5%), 7 Stage II (8.2%), 29 Stage III (34.1%), and 12 Stage IV (14.1%). Histologic subtypes included 58 (68.2%) grade 3 endometrioid carcinomas, 13(15.3%) serous carcinomas, 9 (10.6%) clear cell carcinomas, and 5 (5.9%) mixed tumors. All patients underwent surgical therapy, total abdominal hysterectomy with bilateral salpingoophorectomy was performed in all 85 patients and among them, 70 patients (82.4%) with lymphadenectomy (pelvic with or without para-aorta). Seventy-five patients (88.2%) had no residual intra-abdominal tumor after the primary operation, 5 (5.9%) with a ≤1 cm postoperative intra-abdominal residual tumor and 5 (5.9%) with a >1 cm postoperative intra-abdominal residual tumor. Fifty patients (58.8%) were negative for lymphovascular space invasion and 35 (41.2%) were positive. Malignant ascites was observed in 36 patients (42.4%). After a follow-up, 32 patients (37.6%) showed vaginal (8; 9.4%), intraperitoneal (17; 20.0%), and other-site (7; 8.2%) recurrence, and 19 patients (22.4%) had died (Table I).
Patients’ clinicopathological characteristics.
TLS and PLB in invasive margin of type II EC tissues. Eighty-five patients with more than 1/2 myometrium tumor invasion from the primary surgery were included in this study, and 10 of them had TLS and 59 had PLB. We classified the PLB shape of type II EC in the fourth grade by HE staining (Figure 2).
Peritumoral lymphoid band-like structure in the invasive margin of type II endometrial cancer tissue and their grading. There are peritumoral lymphoid band-like structures (PLB, ►) surrounding the cancer tissue (*) in a low-power view. Representative HE-stained images of PLB grading. A: very low, B: low, C: intermediate, and D: high. Original magnification: ×50, the image at each top-right corner is ×200 magnification.
Semi-quantitative evaluation of peritumoral lymphocytes in type II EC. Type II EC patients were also classified according to the density of peritumoral lymphoid cells. It was associated with the PLB grade and PLB density (Figure 3).
Semi-quantitative evaluation of peritumoral lymphocytes in type II endometrial cancer (EC). Type II EC patients were classified according to the density of peritumoral lymphoid cells, using the following cut-offs: very low: 0-5 cells/HMF, low: 6-100 cells HMF, intermediate: 101-200 cells/HMF, high: ≥201 cells/HMF. Stained cells were counted semi-quantitatively (×200) in the peritumoral areas.
Association of PLB with clinical outcome in type II EC. In univariable competing risk analysis, the UICC stage (I, II vs. III, IV) (HR=7.700; 95%CI=2.939-20.170; p<0.001), CA125 (>35 IU/ml) (HR=2.579; 95%CI=1.255-5.299; p<0.001), malignant ascites (negative vs. positive) (HR=3.779; 95%CI=1.763-8.099; p<0.001), and PLB (very low, low vs. intermediate, high) (HR=0.198; 95%CI=2.58-11.46; p<0.001) showed a strong trend of an association with the recurrence risk. In multivariable competing risk analysis of the recurrence risk, UICC stage (I, II vs. III, IV) (HR=7.131; 95%CI=1.958-25.960; p=0.003), CA125 (>35 U/ml) (HR=3.823; 95%CI=1.471-9.938; p=0.006), histology (endometrioid carcinoma grade 3 vs. clear cell, serous, mix) (HR=2.986; 95%CI=1.313-6.787; p=0.009), and PLB (very low, low vs. intermediate, high) (HR=0.220; 95%CI=0.093-0.519; p<0.001) showed a strong trend for an association (Table II). The cumulative risk of death was similar to that of recurrence. In univariable competing risk analysis of death, UICC stage (I, II vs. III, IV) (HR=10.160, 95%CI=2.330-44.310; p=0.002), malignant ascites (negative vs. positive) (HR=4.190; 95%CI=1.491-11.780; p=0.007), LVSI (negative vs. positive) (HR=3.146; 95%CI=1.173-8.437; p=0.023), and PLB (very low, low vs. intermediate, high) (HR=0.207; 95%CI=0.0805-0.537; p=0.001) showed a strong trend for an association. In multivariable competing risk analysis, age (<60 vs. ≥60 years) (HR=3.484; 95%CI=1.094-11.090; p=0.037), UICC stage (I, II vs. III, IV) (HR=6.250; 95%CI=1.061-37.00; p=0.043), and PLB (very low, low vs. intermediate, high) (HR=0.259; 95%CI=0.0913-0.734; p=0.011) showed a strong trend for an association with survival outcomes (Table III).
Univariate and multivariate analyses of prognostic factors associated with progression-free survival in patients with type II endometrial carcinoma.
Univariate and multivariate analyses of prognostic factors associated with overall survival in patients with type II endometrial carcinoma.
The impact of the PLB density on outcome parameters at the invasive margin was significantly correlated with a decreased PFS (very low, low vs. intermediate, high; HR=0.220; 95%CI=0.093-0.519; p<0.001) and with improved OS (very low, low vs. intermediate, high; HR=0.259; 95%CI=0.091-0.734; p<0.001) (Figure 4).
Kaplan–Meier survival curves showing a comparison of progression-free survival (A) and overall survival (B) between high-density (intermediate or high) and low-density (very low or low) groups of peritumoral lymphoid band-like structure.
We have shown through both uni- and multi-variate analyses that if present, the density of PLB is closely associated with both recurrence and survival.
IHC analysis of TLS and PLB. Next, we investigated the type of lymphocytes and cytokines present in TLS and PLB of type II EC from IHC analysis. Both TLS and PLB contained limited levels of CD8+ T cells, CD38+ lymphocytes, CD68+ macrophages, and fascin and high densities of CD4+ T and CD20+ B cells. They were both composed of CD4+ T cells, CD8+ T cells, CD38+ lymphocytes, CD68+ macrophages and fascin located in the outer layer and most CD20+ B cells were observed in the inner layer (Figure 5).
Histopathologic and immunohistochemical findings of tertiary lymphoid structures and peritumoral lymphoid band-like structure in type II endometrial cancer tissue. A) Representative HE-stained images of tertiary lymphoid structures (TLS, the square with the solid line) and peritumoral lymphoid band-like structures (PLB) (the square with the dotted line) in type II endometrial cancer tissue. Original magnification: left panel, ×20; right panels, ×200. B and C) Immunostaining images of TLS (B) or PLB (C) for CD4, CD8, CD20, CD38, CD68, and fascin. Original magnification: ×200.
Immunofluorescence (IF) showed the characteristics of the cellular and structural components of PLB. A rich network of CD4+ lymphocytes was detected in PLB. FOXP3 and IL-17 were detected at low levels and IL-27 was absent. A rich network of PNAd, a marker for high endothelial venules, which in lymph nodes constitutively recruits B and T cells from the circulation, was observed in the tumor and a few were placed around PLB (Figure 6). These structures varied in size and the degree of cell density; however, the location of each type of lymphocyte in PLB morphologically resembles that of TLS.
Immunofluorescence images of PLB of type II endometrial cancer stained for the indicated markers. The tumor border is shown with a dotted line. A rich network of CD4+ (A) lymphocytes was detected in peritumoral lymphoid band-like structures (PLB). Low levels of FoxP3 (B) and IL-17 (C) were detected but no IL-27 (D). A rich network of PNAd (E) could be seen in the tumor and a few surrounding PLB.
Discussion
Ectopic lymphoid aggregates are reported to be present in chronically inflamed tissue, infection, auto-immune disease, and the tumor stroma. This structure is generally called TLS and resembles secondary lymphoid organs in its shape and role, which is to promote and maintain the immune response (6). In this study, we found ectopic lymphoid aggregates in the stroma forefront of EC, which formed band-like structures instead of follicle structures like TLS. The presence of TLS in the tumor microenvironment is considered to be associated with the prognosis in other tumors, though TLS has been observed in only a few cases of EC (9-12).
The shape of the ectopic lymphoid structure in EC was different compared with that of previously reported TLS, and in this report, we called it PLB. The peritumoral lymphoid band markedly varied in type II ECs. Many type I ECs lacked PLB, conversely it was expressed in many type II ECs. The presence of PLB and its density were strongly correlated with PFS and OS in type II EC, with a high density of PLB being associated with longer PFS and OS than a lower density or no PLB. This is interesting due to the fact that type I EC is typically associated with a better prognosis compared with type II EC, though very few cases showed PLB in type I EC. The reason for type I EC lacking PLB but showing a better prognosis than type II EC might be that type I EC is not as aggressive as type II, and does not involve local immunity. This is just speculative, and other reasons may exist. However, elucidating these mechanisms may lead to a better understanding of tumor immunity and the identification of new therapeutic targets.
Previous reports have shown that TLS have a follicular shape with T-cell areas surrounding B-cell areas. In T-cell areas of TLS, T cells, mature dendritic cells, HEV, and IL-17 were found, while in B-cell areas, B cells and follicular dendritic cells were found (18).
IL-17 is considered critical for initiating the formation of TLS and identifying the Th17 cells that promote the formation of ectopic B-cell follicle clusters. It introduces the mechanism that mainly acts on a wide range of cells such as fibroblasts, epithelial cells, vascular endothelial cells, and macrophages to induce inflammatory cytokines such as IL-6 and TNF-α, chemokines, and neutrophil migration (18-20). On the other hand, IL-27 negatively regulates the development of TLS and blocks Th17 (18). Many studies have clearly shown that tumor-associated TLS and their components are involved in the outcomes of malignant tumor patients with an important role of HEV, which induce lymphocytes and cytokines to maintain TLS (9, 10, 21). Based on IHC and IF figures, PLB includes these components, which resemble the composition of reported TLS. However, the formation and role of local immunity in the cancer stroma and TLS are complex, and further investigation of PLB is necessary to clarify their association.
Immune interactions between immunological molecules remain unclear in the development of EC, and according to one theory, immune escape mechanisms might be similar to those of the maternal-fetal interface (22). Progression of cancer is influenced by immunoediting and similar process such as invasion and angiogenesis, can be seen between the mother and the fetus during gestation (23-25). In pregnancy and labor, the fetal–maternal immune properties require changes in immune tolerance (26-29). On the other hand, immune tolerance of a tumor is essentially different to that of the fetal-maternal interface and is uncontrolled. In this way, the endometrium is unique regarding the focal immune system and may affect the existence of PLB.
This study has limitations such as the fact that the structure of PLB and Treg mechanisms have not been fully investigated, and the relationship with TIL remains unclear. There is also a limitation regarding the method of measuring the concentration of PLB. In this study, we measured it using image software, but we consider that it should be verified (30, 31). In addition, regarding its definition, PLB of the visual field of HMF (×200) at the tip of the tumor’s muscular invasion was confirmed, but a method to examine the entire muscular invasion tip of the tumor should have been developed for more precise evaluation of the local immunity. To understand the interplay with the immune system more comprehensively, we are planning to carry out genetic profiling of PLB by micro dissecting the tumor and the peritumoral section. However, our study suggests that the presence of unique band-like aggregates of lymphocytes at the tumor forefront in EC may be associated with the prognosis. An effect of chimeric antigen receptor (CAR) -T therapy using lymphocytes from TIL has been shown, and we consider that elucidation of PLB may lead to the development of a similar treatment method (32).
An improved understanding of the role of the immune response within tumors may help guide treatment decisions and provide insights that lead to more effective therapeutic approaches. In the future, further investigation of the mechanism of PLB induction in the uterus may allow identification of an immunological approach that can control the cancer microenvironment, which may lead to the development of practical treatments for cancer.
Acknowledgements
This work was supported by JSPS KAKENHI Grant Number 19K09778.
Footnotes
Authors’ Contributions
YS and SS had the original idea for the study, analyzed and interpreted data, did statistical analyses, and wrote the manuscript. MU and SH participated in data collection. NY and HK helped to design the study and performed collection and assembly of data. HK coordinated the study over the entire period and participated in editing and proofreading.
Conflicts of Interest
The Authors have no potential conflicts of interest in relation to this study.
- Received November 25, 2020.
- Revision received December 8, 2020.
- Accepted December 14, 2020.
- Copyright© 2021, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
