Intraepithelial Lymphocytes are Indicators of Better Prognosis in Surgically Resected Endometrioid-Type Endometrial Carcinomas at Early and Advanced Stages

doi:10.21203/rs.3.rs-944317/v1

Background: Tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) may be useful prognostic indicators in endometrial cancer. However, standardized assessment methods and the prognostic roles of these cells in different stage groups are unclear.

Methods: Formalin-fixed paraffin-embedded tissue samples of 107 endometrioid-type endometrial carcinomas (EECs) comprising 60 stage IB and 47 stage IIIC or IVB cases were evaluated. CD3⁺ TILs, CD8⁺ TILs, CD68⁺ TAMs, and CD163⁺ TAMs were detected by immunohistochemistry, and their densities were evaluated by semiquantitative and quantitative methods. TILs within tumor epithelial cell nests (E-TILs) and those within the stroma at the invasive front (S-TILs) were evaluated separately for CD3⁺ and CD8⁺ cells. The “TIL score” was defined as the sum of semiquantitative scores of CD3⁺E-TILs, CD3⁺ S-TILs, CD8⁺ E-TILs, and CD8⁺S-TILs. For TAMs, the area of CD68⁺ and CD163⁺ cells in the invasive margin were semiquantitatively and quantitatively evaluated. Clinicopathological and prognostic implications of TILs and TAMs in stage IB and IIIC/IVB EECs were examined by Cox univariate and multivariate analyses.

Results: By Cox univariate analyses, semiquantitatively low CD3⁺ E-TILs, low CD8⁺ E-TILs, and low “TIL score” were significantly correlated with worse prognosis in stage IB patients (P = 0.011, 0.040, and 0.039, respectively). Likewise, low CD3⁺ E-TILs and low CD8⁺ E-TILs, by both semiquantitative (P = 0.011 and 0.0051) and quantitative evaluations (P < 0.0001, and P = 0.0015) and low “TIL score” (P = 0.020) were significantly correlated with worse prognosis in stage IIIC/IVB patients. By Cox multivariate analyses, semiquantitatively low CD3⁺ E-TILs in stage IB (P = 0.030) and semiquantitatively low CD3⁺ E-TILs or “TIL score” in stage IIIC/IVB (P = 0.022 and 0.049, respectively) were independent worse prognostic factors. CD68⁺ or CD163⁺ TAMs were not correlated with prognosis in any patients.

Conclusions: Semiquantitatively low E-TILs, possibly representing low degree of TILs, are correlated with prognosis in both early and advanced stage patients with EEC. In particular, CD3⁺ E-TILs and CD8⁺ E-TILs are potentially useful prognostic markers in patients with EEC regardless of the stage.

Cancer Biology

endometrial cancer

endometrioid carcinoma

tumor-infiltrating lymphocyte

tumor-associated macrophage

DNA mismatch repair deficiency

Endometrial cancer (EC) is one of most common gynecological malignancies; in 2018, there were 382,069 new cases and 89,929 deaths worldwide [1]. The incidence of EC in Asian women is increasing, particularly in postmenopausal women [2], with 11,120 novel cases in 2017 and 2,601 deaths reported in Japan in 2018 [3, 4]. In EC, endometrioid-type endometrial carcinoma (EEC) is the most prevalent histological type with excellent clinical outcomes in the earlier stage diseases, but the outcomes worsen in more advanced stages. For example, 5-year overall survival (OS) rates were reported to be 95.7% and 89.3% in International Federation of Gynecology and Obstetrics (FIGO) stage I and II patients but 81.9% and 37.2% in FIGO stage III and IV patients, respectively, in Japan [3].

Recently, tumor immune cells, such as tumor-infiltrating lymphocytes (TILs) and tumor-associate macrophages (TAMs), have been shown to be of prognostic significance in EC [5]: a larger number of TILs, detected immunohistochemically as CD3⁺ and CD8⁺ T cells, were associated with better prognosis [5–8]. Most previous studies [5] have investigated TILs within tumor epithelial cell nests (epithelial TILs, E-TILs) and those within the stroma at the invasive front (stromal TILs, S-TILs). In EC, CD8⁺ E-TILs showed prognostic implications [5–10]; but the prognostic roles of CD3⁺ E-TILs, CD3⁺ S-TILs and CD8⁺ S-TILs remain controversial [5, 7–9].

On the other hand, in colorectal cancer, a powerful prognostication tool known as the “Immunoscore” was developed [11, 12]. The Immunoscore is a digital image analysis system for evaluating the immunohistochemical density of CD3⁺ and CD8⁺ lymphocytes in the center and invasive margin of the tumor. A similar approach for EC may be useful, as EC and colorectal cancer have common histology and major prognostic features, including depth of invasion, lymph node metastasis, and lymphovascular invasion.

TAMs have also been examined by many study groups. There are two types of effector macrophages, type I (M1) and type II (M2). M1 macrophages play tumor-suppressive roles including killing of pathogens and tumor cells, whereas M2 macrophages have tumor-promoting roles by inhibiting inflammation, suppressing T-cell function, and promoting angiogenesis [13, 14]. TAMs mainly consist of the M2 type, and abundant M2 infiltration in cancer tissue was reported to indicate poor prognosis, although there are equivocal data among the reports and tumor types [5, 15–17].

In previous studies of TILs and TAMs in EC, most patients were in early stages, i.e., I and II, and all histological types were included. Few separate analyses of TILs in earlier and advanced stages in EC have been performed. The FIGO stage classification is not perfect for predicting clinical outcomes: in patients with stage I EC, the 5-year survival rate is > 90%, but deeper myometrial invasion, grade 3, and vascular invasion are shown to be factors of high risk[18]. Identifying additional powerful prognostic indicators would be important for more appropriate management of stage I patients. The identification of the better prognosis group for patients with advanced stage ECs could also be of clinical value.

In the present study, we immunohistochemically examined TILs and TAMs in surgically resected ECs to reveal their clinical roles in early and advanced stage groups. Both semiquantitative and quantitative approaches were used to evaluate TILs and TAMs to reveal their optimal measurements. Because EC contains various histological types with different biological properties, we focused on EEC.

This was a retrospective study performed in a single institute.

Ethics approval and consent to participate

This study was performed in accordance with the Declaration of Helsinki and was approved by the institutional review board of National Defense Medical College (registration number: 2516).Written informed concent was obtained from all patients.

Patients

Of the 556 patients with EC treated by the primary surgery including by hysterectomy at the National Defense Medical College Hospital between 1990 and 2014, 464 patients were diagnosed with EEC. Of these, 73, 45, and 17 patients were in stage IB, IIIC, and IVB, respectively, according to FIGO 2008. Archival paraffin-embedded tumor blocks or clinical data were not available for 24 patients (12 IB, 7 IIIC, and 5 IVB), and another four patients were excluded following histological review because of non-EEC histology (1 IB carcinosarcoma and 1 IIIC and 2 IVB serous carcinomas). Finally, a total of 107 cases was enrolled in this study: 60 IB patients in the early stage group and 37 IIIC and 10 IVB patients in the advanced stage group.

All patients were of the Asian race. No patients had undergone radiation or chemotherapy before surgical therapy. Ninety patients were administered adjuvant chemotherapy and/or radiation, whereas the other 17 patients, comprising 14 stage IB, 2 stage IIIC, and 1 stage IVB patients, were not treated with additional adjuvant therapies for various reasons.

The clinicopathological characteristics of the patients are summarized in Table 1. The median follow-up periods for patients other than those who died from EEC were 7.3 years (range, 1.2–23.3 years) in the stage IB group and 6.5 years (range, 0.25–19.3 years) in the stage IIIC/IVB group. In the IB group, 12 patients (20%) suffered from recurrence, and 5 (8.3%) died from EEC. In IIIC/IVB group, recurrence or progression occurred in 28 patients (60%), and 15 patients (32%) died from EEC.

Immunohistochemistry

Among the 107 EECs, one representative paraffin-embedded primary tumor block was selected. The blocks were cut into 4-µm-thick sections and subjected to immunohistochemistry. The antibodies used were rabbit polyclonal anti-CD3 (DAKO/Agilent, Santa Clara, CA, USA), mouse monoclonal anti-CD8 (C8/144B, Nichirei, Tokyo, Japan), mouse monoclonal anti-CD68 (PG-M1, DAKO/Agilent), mouse monoclonal anti-CD163 (10D6, Leica, Wetzlar, Germany), mouse monoclonal anti-MLH-1 (ES05, DAKO/Agilent), mouse monoclonal anti-MSH-2 (FE11, DAKO/Agilent), rabbit monoclonal anti-MSH-6 (EPR3945, GeneTex, Los Angeles, CA, USA), and rabbit monoclonal anti-PMS-2 (EP51, DAKO/Agilent).

After appropriate antigen retrieval, endogenous peroxidase was blocked with 0.3% hydrogen peroxidase in methanol, and nonspecific staining was blocked with skim milk. The slides were incubated with primary antibodies at 4℃ overnight and, then with biotinylated secondary antibody (DAKO REAL Envision) at room temperature for 30 min, followed by staining with 3,3-diaminobenzidine (Muto Pure Chemicals, Tokyo, Japan) and counterstained with Mayer’s hematoxylin.

A tumor was judged as DNA mismatch repair (MMR)-deficient if the expression of one or more of the MLH-1, MSH-2, MSH-6, and PMS-2 protein expressions were completely negative. Otherwise, MMR was designated as proficient.

Two independent observers (T.K-S. and H.T.) counted the number of positively immunostained TILs and TAMs in tumor tissue without prior knowledge of clinical information. Any discrepancies between the two observers were resolved by discussion.

TILs

E-TILs and S-TILs identified by the presence of CD3 and CD8 were evaluated using both semiquantitative and quantitative methods.

Semiquantitative evaluations of E-TILs, S-TILs, and “TIL score” Under low to intermediate magnification using a BX-70 microscope (Olympus, Tokyo, Japan), the density of E-TILs and S-TILs was classified as low (score 0), intermediate (score 1), or high (score 2). To evaluate the E-TILs densities, we also referred to the average number of immunopositive lymphocytes on the tumor cell nests per high-power field (HPF) (x400): E-TILs were considered as high, intermediate, and low when >10, 3–10, and <3 immunopositive lymphocytes were observed, respectively (Figure 1A–1C). High, intermediate, and low S-TILs densities were determined when infiltration of immunopositive lymphocytes was observed in over half length (band-like), focal to less than half the length (10 to <50%), and sparse or absent (<10%), respectively, of the invasive front stromal area (Figure 1D–1F).

As observed for the “Immunoscore” in colorectal cancer [11,12], a combination of evaluations of CD3⁺ TILs and CD8⁺ TILs may be a more powerful prognostic indicator than solitary evaluation. Therefore, a simplified “TIL score” comprised the sum of four semiquantitative scores was assigned to all cases, i.e., CD3⁺ E-TILs, CD3⁺ S-TILs, CD8⁺ E-TILs, and CD8⁺ S-TILs, with the scores ranging from 0 to 8. For each, low, intermediate, and high TILs were assigned as score 0, 1, and 2, respectively.

Quantitative evaluation of E-TILs and S-TILs For E-TILs, three representative HPFs (x400) were selected, photomicrographs were acquired as digital images, and from the images, the average numbers of CD3⁺ TILs and CD8⁺ TILs per HPF (0.238 mm²) on the tumor cell nests were counted separately. For S-TILs, five representative HPFs in the invasive front were selected because S-TILs tended to be distributed more heterogeneously than E-TILs, and the average number of CD3⁺ TILs and CD8⁺ TILs in the stroma per HPF was counted separately.

TAMs

We semiquantitatively evaluated the density of CD68^＋ and CD163^＋ cells in the invasive fronts of the tumor with low- to intermediate-power magnification and classified the density as high, intermediate, or low as described for S-TILs (Figure 2A–2C). For quantitative evaluation, three representative intermediate-power fields (x200) were selected, and the area ratios (%) of CD68⁺ and CD163⁺ cells to the stroma were computed using an all-in-one fluorescence microscope BZ-X-700 (Keyence, Osaka, Japan) (Figure 2D, 2E). Imaging analysis of the area ratio was employed because manual counting of TAMs was difficult.

Setting cut-off values

Using all 107 cases, receiver operating characteristic (ROC) curves were drawn to determine the optimal cut-off values of quantitative TILs and TAMs by correlating their levels with recurrence or progression.

Statistical analysis

Recurrence-free survival (RFS) was considered as the period from the day of curative surgery until the day of documented recurrence in stage IB and IIIC patients; progression-free survival (PFS) was the period from the day of non-curative surgery until the day of documented progression of disease in stage IVB patients. Univariate and multivariate analyses were performed by the Cox’s proportional hazard general linear model. RFS and PFS curves were drawn using the Kaplan-Meier method and compared by the log-rank test. The clinicopathological implications of TILs and TAMs were analyzed by the chi-squared test, Fisher exact test, or Wilcoxon rank sum test. P values of <0.05 were considered as statistically significant. All statistical analyses were performed with JMP Pro 14 for Windows (SAS Institute, Inc., Cary, NC, USA).

Cut-off values for quantitative TIL and TAM evaluation

From the ROC curves for the 107 patients, the optimal cut-off values for CD3⁺ E-TILs, CD8⁺ E-TILs, CD3⁺ S-TILs, CD8⁺ S-TILs, CD68⁺ TAMs, and CD163⁺ TAMs were 3.0/HPF, 3.7/HPF, 59.8/HPF, 56.4/HPF, 0.78%, and 3.56%, respectively (Supplementary table 1). These values were employed as thresholds for quantitative evaluations, and the TIL and TAM statuses were two-tiered according to the thresholds.

Clinicopathological significance of TILs in the 60 stage IB EECs

According to semiquantitative evaluations, the densities of CD3⁺ E-TILs, CD8⁺ E-TILs, CD3⁺ S-TILs, and CD8⁺ S-TILs were high or intermediate in 37 (62%), 34 (57%), 36 (60%), and 33 (55%) of cases, respectively, in stage IB EECs (Table 2). Correlations were observed between CD8⁺ E-TILs and vascular invasion (P = 0.025), and between CD8⁺ E-TILs and MMR deficiency (P = 0.0097), and between CD3⁺ S-TILs and lymphatic invasion (P = 0.035), and between CD8⁺ S-TILs and grade (P = 0.014). The “TIL score” was not correlated with any clinicopathological parameters.

According to quantitative evaluations, CD3⁺ E-TILs, CD8⁺ E-TILs, CD3⁺ S-TILs, and CD8⁺ S-TILs were high in 36 (60%), 26 (43%), 37 (62%), and 18 (30%) of the 60 stage IB EECs, respectively (Table 3). Correlations were detected between CD3⁺ E-TILs and CD8⁺ E-TILs and the grade (P = 0.035 and 0.0040, respectively) and between CD8⁺ S-TILs and the grade (P = 0.049) and vascular invasion (P = 0.015).

Clinicopathological significance of TILs in the 47 stage IIIC/IVB EECs

Through semiquantitative evaluations, high/intermediate CD3⁺ E-TILs, CD8⁺ E-TILs, CD3⁺ S-TILs, and CD8⁺ S-TILs in stage IIIC/IVB EECs were detected in 24 (51%), 23 (49%), 26 (55%), and 20 (43%) of cases, respectively (Table 4). High/intermediate CD3⁺ E-TILs and CD8⁺-E-TILs analyzed in semiquantitative evaluations were inversely correlated with vascular invasion (P = 0.041 and 0.019, respectively). The “TIL score” was not correlated with any clinicopathological parameters.

Through quantitative evaluations, CD3⁺ E-TILs, CD8⁺-E-TILs, CD3⁺ S-TILs, and CD8⁺ S-TILs were found to be high in 22 (47%), 15 (32%), 28 (60%), and 12 (26%) of cases, respectively (Table 5). High CD3⁺ E-TILs, CD8⁺ E-TILs, and CD8⁺ S-TILs analysed by quantitative evaluation were inversely correlated with vascular invasion (P = 0.0087, 0.026, and 0.042, respectively), with the former two inversely correlated with lymphatic invasion (P = 0.0069 and 0.0094, respectively).

MMR protein statuses were not associated with TILs or the “TIL score”.

Prognostic significance of TILs and TAMs in the 60 stage IB EECs

In Cox univariate analyses, MMR proficiency (HR: 5.52, 95% CI: 1.08–100, P = 0.037), semiquantitative low CD3⁺ E-TILs (HR: 4.24, 95% CI: 1.38–15.7, P = 0.011), semiquantitative low CD8⁺ E-TILs (HR: 3.25, 95% CI: 1.06–12.0, P = 0.040), and low “TIL score” (HR: 3.47, 95% CI: 1.06-15.5, P = 0.039) were significant risk factors for recurrence (Figure 3A).

In multivariate analysis including semiquantitative CD3⁺ E-TILs and MMR, only CD3⁺ E-TILs had an independent impact on RFS (P = 0.030) (Figure 3B). When semiquantitative CD8⁺ E-TILs or the “TIL score” was included in multivariate analysis, instead of CD3⁺E-TILs, the independent impact on RFS was not observed (data not shown). RFS curves significantly differed between high/intermediate and low TILs groups for semiquantitative CD3⁺ E-TILs (P = 0.0088) and CD8⁺ E-TILs (P = 0.038) and between the 2-tiered “TIL score” (score 0–3 vs. 4–8) groups (P = 0.044) (Figure 3C–3G).

Quantitative evaluations of the TILs showed no significant differences in the RFS curves although quantitative CD3⁺ and CD8⁺ E-TILs were nearly correlated with RFS (P = 0.071 and 0.10, respectively) (Supplementary Fig. 1).

Prognostic significance of TILs in the 47 stage IIIC/IVB EECs

According to Cox univariate analyses, significant prognostic factors for RFS/PFS included the FIGO stage [HR: 3.93, 95%CI: 1.66–8.65, P = 0.0027], grade 3 (HR: 2.34, 95%CI: 1.09–5.04, P = 0.029), and vascular invasion (HR: 3.72, 95%CI: 1.70–8.76, P = 0.0009). In addition, semiquantitative low CD3⁺ E-TILs (HR: 2.68, 95% CI: 1.26–6.07, P = 0.011), semiquantitative low CD8⁺ E-TILs (HR:3.00, 95% CI: 1.38–6.99, P = 0.0051), semiquantitative low CD8⁺ S-TILs (HR: 2.34, 95%CI: 1.07–5.65, P = 0.033), and low “TIL score” (HR: 2.70, 95% CI: 1.16–7.35, P = 0.020) were also significant indicators of worse prognosis (Figure 4A).

In multivariate analysis including stage, “TIL score”, vascular invasion, and grade, the former three had independent impact on RFS/PFS (P = 0.033, 0.049, and 0.010, respectively) (Figure 4B). When semiquantitative CD3⁺ E-TILs were included in the analysis, instead of the “TIL score”, an independent impact on RFS/PFS was also observed (P = 0.022); however, when semiquantitative CD8⁺ E-TILs or CD8⁺ S-TILs was included in the analysis, an independent impact on RFS/PFS was not observed (data not shown).

Based on the semiquantitative evaluation methods, there were significant differences in the RFS/PFS curves between the high/intermediate and low groups of CD3⁺ E-TILs, CD8⁺ E-TILs, and CD8⁺ S-TILs (P = 0.0089, 0.0043 and 0.035, respectively) (Figure 4C,4D,4F). There was also significant differences in the RFS/PFS curves between the high and low “TIL score” (score 0–3 vs 4–8) groups (P = 0.024) (Figure 4G).

Univariate analyses revealed that quantitatively low CD3⁺ E-TILs (HR: 4.86, 95%CI: 2.14–12.5, P < 0.0001) and quantitatively low CD8⁺ E-TILs (HR: 4.39, 95%CI: 1.69–15.0, P = 0.0015) were also indicative of a significantly worse prognosis (Figure 5A). In Cox multivariate analysis including the advanced stage, quantitatively low CD3⁺ E-TILs, positive vascular invasion, and higher histological grade, the former two were independent risk factors for recurrence/progression (P = 0.0064 and 0.0004, respectively) (Figure 5B). When quantitative CD8⁺ E-TILs were included in the analysis, instead of quantitative CD3⁺ E-TILs, an independent impact on RFS/PFS was also observed (P = 0.0021).

Based on quantitative evaluation, there were significant differences in the RFS/PFS curves between the high and low groups of both CD3⁺ E-TILs and CD8⁺ E-TILs (P < 0.0001 and 0.0026, respectively), whereas this relationship was not observed for CD3⁺ S-TILs or CD8⁺ S-TILs (P = 0.12 and 0.12, respectively) (Figure 5C–5F).

Prognostic significance of TAMs

Semiquantitative or quantitative CD68⁺ or CD163⁺ TAMs were not correlated with any clinicopathological parameters or MMR statuses in the stage IB or stage IIIC/IVB patient groups (Supplementary Figure 2, A-H). They also showed no correlation with RFS/PFS (Supplementary tables 2 and 3).

We examined the prognostic and clinicopathological implications of TILs and TAMs in both patient groups with EEC at early stages (FIGO IB) and advanced stages (FIGO IIIC and IVB). In 60 patients with stage IB disease, semiquantitative low CD3⁺ E-TILs, low CD8⁺ E-TILs, and low “TIL score” were correlated with lower RFS rates. Similarly, in 47 patients with stages IIIC/IVB disease, low CD3⁺ E-TILs and low CD8⁺ E-TILs, evaluated by semiquantitative and quantitative methods, and a low “TIL score” were correlated with a worse prognosis. Based on these results, low E-TILs may be useful as biomarkers for worse clinical outcomes in patients in early stages, whereas high E-TILs were shown to indicate a better prognosis in patients in advanced stages. In contrast, S-TILs, CD68⁺ TAMs, and CD163⁺ TAMs evaluated by all methods were not correlated with the prognosis of patient groups at both early and advanced clinical stages, except for semiquantitative CD8⁺ S-TILs in the IIIC/IVB group.

In semiquantitative evaluation, the density of E-TILs was classified as low, intermediate, and high. On their evaluation, we need introduce the consideration of quantity whether the number of immunopositive lymphocytes within the tumor cell nests was >10 per HPF (x400, approximately 0.238 mm²) or not. With the help of semiquantitative evaluation, both CD8⁺ E-TILs and CD3⁺ E-TILs were strongly correlated with the better prognosis of patients in both early and advanced stages.

Similarly, in semiquantitative evaluation, the density of S-TILs in the invasive front was classified as low, intermediate, or high. This classification is similar to that used by Yamashita et al., who classified the density of CD8⁺ TILs, including both S-TILs and E-TILs, as low (0–30%), moderate (30–60%), and high (>60%), in which moderate/high CD8⁺ TILs were correlated with a higher PFS rate in 141 patients with EC [19]. Using the present evaluation method, we observed a significant relationship of CD8⁺ S-TILs with higher RFS/PFS rates only in the stage IIIC/IVB EEC group but their prognostic impact was weaker than those of CD8⁺/CD3⁺ E-TILs.

In quantitative evaluation, the cut-off values of high TILs varied among studies, ranging from 7.065 to 25 per x200 field (0.785 mm²) for CD8⁺ E-TILs [8-11] and from 13.345 to 35 cells per x200 field for CD3⁺ E-TILs [8,9]. Hendry et al. used 40 lymphocytes per 10 HPFs on haematoxylin and eosin-stained slides, approximately corresponding to 16 cells per x200 field, as the cut-off point for E-TIL evaluation [20]. In the present study, the thresholds for CD8⁺ E-TILs and CD3⁺ E-TILs were set to 14.8 and 12.0 cells per x200 field, respectively. These values were within or near the ranges of the thresholds. Based on these values, these E-TILs were significantly correlated with prognosis in the stage IIIC/IVB group and showed marginal prognostic significance in the stage IB group.

The depth of invasion and status of the invasive front of the primary site are important determinants of clinical outcomes of EC as well as colorectal cancer. The TIL status in the invasive tumor front would be a parameter of strength of in situ adaptive immune reaction at the primary site. The “Immunoscore”, evaluated using a combination of CD3⁺ and CD8⁺ TILs in both the tumor center and invasive front, showed the greatest impact on the risk of recurrence and death among the clinical parameters of colorectal cancer [11,12]. Because our digital pathology environment was incomplete, we employed a surrogate “TIL score” by summing the semiquantitative points of CD3⁺ E-TILs, CD8⁺ E-TILs, CD3⁺ S-TILs, and CD8⁺S-TILs. This “TIL score” was a significant predictor of prognosis in both patients with stage IB and stage IIIC/IVB disease. However, the combination did not appear to dramatically increase the prognostic impact compared with simple measurement of CD3⁺ E-TILs or CD8⁺ E-TILs.

TAMs play important roles in immunity and the tumor microenvironment. CD68 and CD163 have been used as immunohistochemical markers of these cells. Soeda et al. classified TAMs that infiltrated into the cancer nests or stroma along the tumor-myometrial junction (margin TAM) into high and low levels based on a threshold of 20 cells per x200 field (0.785 mm²) and observed the prognostic significance of high-level CD68⁺ TAMs [15]. Kübler et al. and Espinosa et al. also showed that CD163⁺ TAMs were correlated with worse prognosis and/or regional lymph node metastases in patients with EECs [21,22]. Espinosa et al. scored the density of CD163-positive cells as 0, 1, and 2 when the number of cells was ≤20, >20 to ≤50, and >50 per 1-mm diameter core (0.785 mm²) [22], whereas Kübler et al. set the median value of immunostaining as a cut-off point between high and low TAM counts [21].

We examined both CD68⁺ and CD163⁺ cells using two different methods but found no significant correlations of TAM density with clinicopathological parameters or with patient prognosis in early or advanced stage EECs. Although there were no similar results, the present findings may be explained as follows: As the cancer progresses, cancer-associated fibroblasts begin continuously releasing growth factors such as TGF-β (transforming growth factor-β) and SDF-1/CXCL12 (stromal derived factor-1/ C-X-C Motif Chemokine Ligand 12), which chemically attract macrophages and promote M2 polarization [23] and, in parallel, modulate the extracellular matrix. TAMs are preferably attracted to hyaluronan, which is a major component of the extracellular matrix. Depletion of hyaluronan synthase 2 in cancer-associated fibroblasts reduces TAM recruitment and thereby attenuates tumor angiogenesis and lymphangiogenesis [23]. Therefore, even in EECs at advanced stages, the number of TAMs may not be increased compared to in EECs at earlier stages. To clarify the reproducibility of the present results for TAMs in EEC, further studies of a much larger cohort are necessary.

Interestingly, the clinicopathological implications of TILs somewhat differed between the early and advanced stages of EEC. TILs tended to be correlated with lymphatic and vascular invasions in the IB group but were inversely correlated with these invasions in the IIIC/IVB group. The relationship between TILs and MMR deficiency also appeared to differ between early and advanced stage EECs. In the present study, only the stage IB EEC group showed a tendency for a relationship between MMR deficiency and higher CD8⁺ E-TILs and better prognosis. Deficient MMR is detected in approximately 30% of EC cases [24] and was shown to be correlated with CD8⁺ TILs in previous studies [10,25,26]; however, its prognostic significance is controversial [10,25-29]. The relationship of the TIL status with the grade, lymphovascular invasion, and MMR status appeared to significantly differ between EECs in early and advanced stages. Such differences may derive from the status of the tumor microenvironment of the tumor stroma.

Limitations of the present study include the manual evaluation methods and relatively small number of cases. First, quantitative evaluations of TILs were performed using images from several microscopic HPFs. Particularly, because S-TILs showed a relatively heterogeneous distribution, selection bias of the evaluation fields may have affected the results and caused underestimation of the prognostic impact of S-TILs. The conditions appeared similar in the evaluation of TAMs. Second, the numbers of stage IB, IIIC, and IVB cases was small. Specifically, the number of events in stage IB cases may not have strong enough detection power to reveal the prognostic implication of TILs or TAMs. A large-scale study using a simpler, more accurate, and well standardized imaging analysis is required to determine the prognostic roles of these cells in early EECs.

Semiquantitative E-TILs were suggested as representative of TILs correlated with prognosis in both early and advanced stage patients with EEC. Particularly, CD3⁺ E-TILs and CD8⁺ E-TILs were shown to be useful markers of better prognosis in patients with EEC, regardless of the stage.

CI: confidence interval

CTL: cytotoxic T lymphocyte

EC: endometrial cancer

EEC: endometrioid carcinoma of the endometrium, endometrioid-type endometrial carcinoma

E-TILs: epithelial tumor-infiltrating lymphocytes

FIGO: International Federation of Gynecology and Obstetrics

HPF:high-power field

HR: hazard ratio

MMR: mismatch repair

PFS: progression-free survival

RFS: recurrence-free survival

ROC: receiver operating characteristic

SD: standard deviation

S-TILs: stromal tumor-infiltrating lymphocytes

TAMs: tumor-associated macrophages

TILs: tumor-infiltrating lymphocytes

Ethics approval and consent to participate

This study was performed in accordance with the Declaration of Helsinki and was approved by the institutional review board of National Defense Medical College (registration number: 2516). All patients agreed to participate in this study, and written informed consent was obtained from all these patients.

Consent for publication

Not applicable.

Availability of data and materials

Datasets used and/or analyzed during this study are available from the corresponding author on reasonable request.

Competing interests

The author(s) declare that they have no competing interests.

Funding

Not applicable.

Authors’ contributions

TK-S, and HT performed the planning, acquisition of data, analysis of data, and writing of the manuscript. MM, and MT acquired clinical data, KM and SM acquired pathological data, and YY conducted data analysis. KS substantively revised the draft. All authors substantively revised the draft. All authors read and approved the final manuscript.

Acknowledgements

The authors would like to thank Ms. Ai Matsumoto, Ms. Chinami Onuma, and Ms. Ru Hokari for their excellent technical assistance.

Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68(6):394–424.
Lortet-Tieulent J, Ferlay J, Bray F, Jemal A. International patterns and trends in endometrial cancer incidence, 1978–2013. J Natl Cancer Inst. 2018;110(4):354–61.
Japan Society of Obstetrics and Gynecology. Patient annual report for 2017. Acta Obstet Gynaecol Jpn. 2019;71(5):669–724.
The editorial board of the cancer statistics in Japan. Cancer Statistics in Japan 2018. Tokyo: The Foundation for Promotion of Cancer Research; 2019. p. 62.
Guo F, Dong Y, Tan Q, Kong J, Yu B. Tissue infiltrating immune cells as prognostic biomarkers in endometrial cancer: A meta-analysis. Dis Markers. 2020;2020:1805764.
Kondratiev S, Sabo E, Yakirevich E, Lavie O, Resnick MB. Intratumoral CD8 + T lymphocytes as a prognostic factor of survival in endometrial carcinoma. Clin Cancer Res. 2004;10(13):4450–6.
de Jong RA, Leffers N, Boezen HM, ten Hoor KA, van der Zee AG, Hollema H, et al. Presence of tumor-infiltrating lymphocytes is an independent prognostic factor in type I and II endometrial cancer. Gynecol Oncol. 2009;114(1):105–10.
Čermáková P, Melichar B, Tomšová M, Zoul Z, Kalábová H, Spaček J, et al. Prognostic significance of CD3 + tumor-infiltrating lymphocytes in patients with endometrial carcinoma. Anticancer Res. 2014;34(10):5555–61.
Ino K, Yamamoto E, Shibata K, Kajiyama H, Yoshida N, Terauchi M, et al. Inverse correlation between tumoral indoleamine 2,3-dioxygenase expression and tumor-infiltrating lymphocytes in endometrial cancer: its association with disease progression and survival. Clin Cancer Res. 2008;14(8):2310–7.
Suemori T, Susumu N, Iwata T, Banno K, Yamagami W, Hirasawa A, et al. Intratumoral CD8 + lymphocyte infiltration as a prognostic factor and its relationship with cyclooxygenase 2 expression and microsatellite instability in endometrial cancer. Int J Gynecol Cancer. 2015;25(7):1165–72.
Galon J, Mlecnik B, Bindea G, Angell HK, Berger A, Lagorce C, et al. Towards the introduction of the 'Immunoscore' in the classification of malignant tumors. J Pathol. 2014;232(2):199–209.
Pagès F, Mlecnik B, Marliot F, Bindea G, Ou FS, Bifulco C, et al. International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study. Lancet. 2018;391(10135):2128–39.
Mills CD. M1 and M2 macrophages: oracles of health and disease. Crit Rev Immunol. 2012;32(6):463–88.
Allavena P, Sica A, Solinas G, Porta C, Mantovani A. The inflammatory micro-environment in tumor progression: the role of tumor-associated macrophages. Crit Rev Oncol Hematol. 2008;66(1):1–9.
Soeda S, Nakamura N, Ozeki T, Nishiyama H, Hojo H, Yamada H, et al. Tumor-associated macrophages correlate with vascular space invasion and myometrial invasion in endometrial carcinoma. Gynecol Oncol. 2008;109(1):122–8.
Takanami I, Takeuchi K, Kodaira S. Tumor-associated macrophage infiltration in pulmonary adenocarcinoma: association with angiogenesis and poor prognosis. Oncology. 1999;57(2):138–42.
Forssell J, Oberg A, Henriksson ML, Stenling R, Jung A, Palmqvist R. High macrophage infiltration along the tumor front correlates with improved survival in colon cancer. Clin Cancer Res. 2007;13(5):1472–9.
Ambros RA, Kurman RJ. Combined assessment of vascular and myometrial invasion as a model to predict prognosis in stage I endometrioid adenocarcinoma of the uterine corpus. Cancer. 1992;69(6):1424–31.
Yamashita H, Nakayama K, Ishikawa M, Nakamura K, Ishibashi T, Sanuki K, et al. Microsatellite instability is a biomarker for immune checkpoint inhibitors in endometrial cancer. Oncotarget. 2018;9(5):5652–64.
Hendry S, Salgado R, Gevaert T, Russell PA, John T, Thapa B, et al. Assessing tumor-infiltrating lymphocytes in solid tumors: A practical review for pathologists and proposal for a standardized method from the international immuno-oncology biomarkers working group: Part 2: TILs in melanoma, gastrointestinal tract carcinomas, non-small cell lung carcinoma and mesothelioma, endometrial and ovarian carcinomas, squamous cell carcinoma of the head and neck, genitourinary carcinomas, and primary brain tumors. Adv Anat Pathol. 2017;24(6):311–35.
Kübler K, Ayub TH, Weber SK, Zivanovic O, Abramian A, Keyver-Paik MD, et al. Prognostic significance of tumor-associated macrophages in endometrial adenocarcinoma. Gynecol Oncol. 2014;135(2):176–83.
Espinosa I, José Carnicer M, Catasus L, Canet B, D'Angelo E, Zannoni GF, et al. Myometrial invasion and lymph node metastasis in endometrioid carcinomas: tumor-associated macrophages, microvessel density, and HIF1A have a crucial role. Am J Surg Pathol. 2010;34(11):1708–14.
Zheng X, Turkowski K, Mora J, Brüne B, Seeger W, Weigert A, et al. Redirecting tumor-associated macrophages to become tumoricidal effectors as a novel strategy for cancer therapy. Oncotarget. 2017;8(29):48436–52.
Ferguson SE, Aronson M, Pollett A, Eiriksson LR, Oza AM, Gallinger S, et al. Performance characteristics of screening strategies for Lynch syndrome in unselected women with newly diagnosed endometrial cancer who have undergone universal germline mutation testing. Cancer. 2014;120(24):3932–9.
Shia J, Black D, Hummer AJ, Boyd J, Soslow RA. Routinely assessed morphological features correlate with microsatellite instability status in endometrial cancer. Hum Pathol. 2008;39(1):116–25.
Talhouk A, Derocher H, Schmidt P, Leung S, Milne K, Gilks CB, et al. Molecular subtype not immune response drives outcomes in endometrial carcinoma. Clin Cancer Res. 2019;25(8):2537–48.
Nelson GS, Pink A, Lee S, Han G, Morris D, Ogilvie T, et al. MMR deficiency is common in high-grade endometrioid carcinomas and is associated with an unfavorable outcome. Gynecol Oncol. 2013;131(2):309–14.
Diaz-Padilla I, Romero N, Amir E, Matias-Guiu X, Vilar E, Muggia F, et al. Mismatch repair status and clinical outcome in endometrial cancer: a systematic review and meta-analysis. Crit Rev Oncol Hematol. 2013;88(1):154–67.
Ruiz I, Martín-Arruti M, Lopez-Lopez E, Garcia-Orad A. Lack of association between deficient mismatch repair expression and outcome in endometrial carcinomas of the endometrioid type. Gynecol Oncol. 2014;134(1):20–3.

Table 1 Clinicopathological features of endometrioid-type endometrial carcinoma patients (n = 107)
Parameter	Number of cases (%)
	Total		Stage IB		Stage IIIC/IVB
	(n = 107)		(n = 60)		(n = 47)
Age (years)
mean ± SD [range]	62.7 ± 11.0	[33-87]	65.2± 10.7	[33-87]	59.4± 10.7	[38-84]
≤ 50	11	(10)	3	(5)	8	(17)
> 50	96	(90)	57	(95)	39	(83)
Histological grade
G1	40	(37)	28	(47)	12	(26)
G2	38	(36)	22	(37)	16	(34)
G3	29	(27)	10	(17)	19	(40)
Lymphatic invasion
Positive	74	(69)	33	(55)	41	(87)
Negative	33	(31)	27	(45)	6	(13)
Vascular invasion
Positive	44	(41)	19	(32)	25	(53)
Negative	63	(59)	41	(68)	22	(47)
Lymph node metastasis
Positive	42	(39)	0	(0)	42	(89)
Negative	65	(61)	60	(100)	5	(11)
Surgical therapy
Total hysterectomy + BSO	104	(97)	59	(98)	45	(96)
Total hysterectomy + LSO	1	(1)	1	(2)	0	(0)
Supracervical hysterectomy+ BSO	1	(1)	0	(0)	1	(2)
Supracervical hysterectomy + LSO	1	(1)	0	(0)	1	(2)
Adjuvant therapy
Chemotherapy	79	(74)	43	(72)	36	(77)
Radiation	4	(4)	2	(3)	2	(4)
Radiation followed by chemotherapy	7	(6)	1	(2)	6	(13)
Not done	17	(16)	14	(22)	3	(6)
BSO, Bilateral salpingo-oophorectomy; LSO, Left salpingo-oophorectomy; SD, Standard deviation

Table 3 Correlations of quantitatively high TILs with clinicopathological parameters in stage IB endometrioid-type endometrial carcinoma (n = 60)

Parameter	Total	Number of cases (%)
			High
			CD3⁺		P	CD8⁺		P	CD3⁺		P	CD8⁺		P
			E-TILs		P	E-TILs		P	S-TILs		P	S-TILs		P
Age
≤ 50	3		1	(33)	0.55	1	(33)	1.00	0	(0)	0.051	0	(0)	0.54
> 50	57		35	(61)	0.55	25	(44)	1.00	37	(65)	0.051	18	(32)	0.54
Histological grade
G1	28		12	(43)	0.035	6	(21)	.0.0040	14	(50)	0.22	5	(18)	0.049
G2	22		16	(73)		13	(59)		16	(73)		7	(32)
G3	10		8	(80)		7	(70)		7	(70)		6	(60)
Lymphatic invasion
Positive	33		22	(67)	0.29	17	(52)	0.20	20	(61)	1.00	13	(39)	0.096
Negative	27		14	(52)	0.29	9	(33)	0.20	17	(63)	1.00	5	(19)	0.096
Vascular invasion
Positive	19		14	(73)	0.17	12	(63)	0.050	13	(68)	0.57	10	(53)	0.015
Negative	41		22	(54)	0.17	14	(34)	0.050	24	(59)	0.57	8	(20)	0.015
MMR protein
Deficient	18		14	(78)	0.087	11	(61)	0.091	12	(67)	0.77	7	(39)	0.36
Proficient	42		22	(52)	0.087	15	(36)	0.091	25	(60)	0.77	11	(26)	0.36
Total	60		36			26			37			18

E-TILs, Epithelial tumor-infiltrating lymphocytes; MMR, Mismatch repair; S-TILs, Stromal tumor-infiltrating lymphocytes

Table 5

Correlations of quantitatively high TILs with clinicopathological parameters in stage IIIC/IVB endometrioid-type endometrial carcinoma (n = 47)

Parameter	Total	Number of patients (%)
		Quantitatively high TILs
		CD3⁺		P	CD8⁺		P	CD3⁺		P	CD8⁺		P
		E-TILs		P	E-TILs		P	S-TILs		P	S-TILs		P
Age
≤ 50	8	2	(25)	0.25	1	(13)	0.40	3	(38)	0.23	2	(25)	1.00
> 50	39	20	(51)	0.25	14	(36)	0.40	25	(64)	0.23	10	(26)	1.00
Stage
IIIC	37	18	(49)	0.72	11	(30)	0.70	23	(62)	0.49	10	(27)	1.00
IVB	10	4	(40)	0.72	4	(40)	0.70	5	(50)	0.49	2	(20)	1.00
Histological grade
G1	12	7	(58)	0.54	6	(50)	0.075	8	(67)	0.83	3	(25)	0.99
G2	16	6	(38)		2	(13)		9	(56)		4	(25)
G3	19	9	(47)		7	(37)		11	(58)		5	(26)
Lymphatic invasion
Positive	41	16	(39)	0.0069	10	(24)	0.0094	23	(56)	0.37	10	(24)	0.63
Negative	6	6	(100)	0.0069	5	(83)	0.0094	5	(83)	0.37	2	(33)	0.63
Vascular invasion
Positive	25	7	(28)	0.0087	4	(16)	0.026	12	(48)	0.14	3	(12)	0.042
Negative	22	15	(68)	0.0087	11	(50)	0.026	16	(73)	0.14	9	(41)	0.042
Lymph node metastasis
Positive	42	20	(48)	1.00	13	(31)	1.00	25	(60)	1.00	11	(26)	1.00
Negative	5	2	(40)	1.00	2	(40)	1.00	3	(69)	1.00	1	(20)	1.00
MMR protein
Deficient	18	9	(50)	0.77	7	(39)	0.52	13	(72)	0.22	5	(28)	1.00
Proficient	29	13	(45)	0.77	8	(28)	0.52	15	(52)	0.22	7	(24)	1.00
Total	47	22			15			28			12
E-TILs, Epithelial tumor-infiltrating lymphocytes; MMR, Mismatch repair; S-TILs, Stromal tumor-infiltrating lymphocytes

No competing interests reported.

S1.jpg
Supplementary Fig. 1 Recurrence-free survival curves of 60 stage IB endometrioid-type endometrial carcinoma. Curves were stratified by (A) quantitative CD3+ epithelial tumor-infiltrating lymphocytes (E-TILs), (B) CD8+ E-TILs, (C) quantitative CD3+ stromal TILs (S-TILs) and (D) CD8+ S-TILs. Curves were not significantly different.
S2.png
Supplementary Fig. 2 Survival analyses using semiquantative and quantative TAMs in endometrioid-type endometrial carcinoma. Recurrence-free survival (RFS) curves of 60 patients with stage IB EEC stratified by (A) semiquantitative CD68+ tumor associated macrophages (TAMs), (B) semiquantitative CD163+ TAMs, (C) quantitative CD68+ TAMs, and (D) quantitative CD163+ TAMs. Curves were not significantly different. RFS/progression-free survival (PFS) curves of 47 patients with stage IIIC/IVB EEC stratified by (E) semiquantitative CD68+ TAMs, (F) semiquantitative CD163+ TAMs, (G) quantitative CD68+ TAMs and (H) quantitative CD163+ TAMs. Curves were not significantly different.
Supplementarytables.docx

Intraepithelial Lymphocytes are Indicators of Better Prognosis in Surgically Resected Endometrioid-Type Endometrial Carcinomas at Early and Advanced Stages

Status:

Journal Publication

Version 1

Abstract

Figures

Background

Methods

Ethics approval and consent to participate

Patients

Immunohistochemistry

TILs

TAMs

Setting cut-off values

Statistical analysis

Results

Cut-off values for quantitative TIL and TAM evaluation

Clinicopathological significance of TILs in the 60 stage IB EECs

Clinicopathological significance of TILs in the 47 stage IIIC/IVB EECs

Prognostic significance of TILs and TAMs in the 60 stage IB EECs

Prognostic significance of TILs in the 47 stage IIIC/IVB EECs

Prognostic significance of TAMs

Discussion

Conclusion

Abbreviations

Declarations

Ethics approval and consent to participate

Consent for publication

Availability of data and materials

Competing interests

Funding

Authors’ contributions

Acknowledgements

References

Tables

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1