Difference in the distribution of tumor‐infiltrating CD8+ T cells and FOXP3+ T cells between micronodular thymoma with lymphoid stroma and micronodular thymic carcinoma with lymphoid stroma

Micronodular thymoma with lymphoid stroma (MNT) is a rare thymic epithelial neoplasm subtype characterized by a micronodular tumor cell growth pattern and abundant lymphoid stroma. Micronodular thymic carcinoma with lymphoid stroma (MNCA) is considered as a malignant counterpart of MNT and exhibits a growth pattern similar to that of MNT but has histologic features reminiscent of thymic squamous cell carcinoma, such as cytologic atypia and CD5 and CD117 immunoexpression. Although both MNT and MNCA are characterized by abundant lymphoid stroma, it remains unknown whether there are differences in infiltrating lymphocytes between MNT and MNCA. We analyzed the immune microenvironment profile in eight MNT and three MNCA cases. The cell density of CD8‐positive T cells was significantly higher in MNT than in MNCA, whereas that of FOXP3‐positive T cells was significantly higher in MNCA than in MNT. There was no significant difference in the cell density of programmed death protein 1‐positive T cells and programmed death ligand 1 expression between the MNT and MNCA cases. Our findings indicated that the immune microenvironment of MNCA differed from that of MNT and, compared with the T‐cell profile of MNT, that of MNCA was more suppressive to patients′ antitumor immune response.


INTRODUCTION
Thymic epithelial neoplasms are classified into thymoma (types A, AB, B1, B2 and B3), thymic squamous cell carcinoma (SCC), and other rare subtypes in the World Health Organization (WHO) Classification of Tumors of the Lung, Pleura, Thymus and Heart 4th Edition. 1 Micronodular thymoma with lymphoid stroma (MNT) is a rare subtype that accounts for 1-5% of all thymic epithelial neoplasms characterized by a micronodular tumor cell growth pattern and abundant lymphoid stroma with germinal centers. [2][3][4][5][6] Micronodular thymic carcinoma with lymphoid stroma (MNCA) is an extremely rare subtype of thymic epithelial neoplasm, and only 20 cases have been reported so far in previous English publications. 4 MNCA is also characterized by micronodular cell proliferation and abundant lymphoid stroma, similar to MNT. However, in contrast to MNT, MNCA has thymic SCC-like features, such as cytologic atypia, increased mitotic activity, and CD5 and CD117 (c-kit) expression. 2,[4][5][6] In one study, 25% of MNCA cases had an adjacent MNT that transitioned from the MNCA. 4 Although these findings suggest that MNCA is a malignant counterpart of MNT, 4,5 the clinical features of MNCA and the relationship between MNT and MNCA are largely unknown due to the extreme rarity of MNCA.
Lymphoid stroma is generally associated with host immune response to tumor antigens. 7,8 The lymphoid stroma of MNT has a larger number of Langerhans cells and mature dendritic cells than type A and AB thymomas, suggesting the presence of a specific tumor immune microenvironment in MNT. 7 In recent years, a large number of studies have investigated the tumor immune microenvironment, including tumor-infiltrating lymphocytes (TILs), such as CD8 (+) cytotoxic T cells, FOXP3 (+) regulatory T cells, programmed death protein 1 (PD-1) (+) T cells, and the programmed death ligand 1 (PD-L1) expression in tumor cells. [9][10][11] While previous studies have explored TILs and PD-L1 expression in typical thymoma and thymic SCC cases, no studies have focused on TILs and PD-L1 expression in MNT and MNCA.
This study aimed to investigate the immune microenvironment, including the distribution and cell density of CD8 (+) T cells, FOXP3 (+) T cells and PD-1 (+) T cells and PD-L1 expression in tumor cells from patients with MNT and MNCA.

Patients and tissue specimens
We retrieved eight MNT and three MNCA cases, all of which were surgically resected at Seirei Mikatahara Hospital, Nagoya University Hospital and Japanese Red Cross Nagoya First Hospital in Japan between 2006 and 2019. The histologic diagnosis was reconfirmed by two board-certified pathologists (HY and TI) based on the WHO criteria. 2 To distinguish between MNT and MNCA, we examined the presence of nuclear atypia, the mitotic counts of tumor cells, and CD5 and CD117 expression using immunohistochemistry. For the comparative analysis, data and tissue specimens of 21 patients with common types of thymoma (four with type A, six with type AB, five with type B1, four with type B2 and two with type B3) and three patients with thymic SCC were also retrieved. The extracted patient data included patient age, sex, surgical procedure, Masaoka stage, 1 TNM stage based on the TNM Classification of Malignant Tumours 8th Edition of the UICC, 12 and clinical outcomes. The clinical courses of two cases (Cases 2 and 11) were previously published as case reports. 13,14 This study was conducted in accordance with the recommendations of the Declaration of Helsinki and was approved by the ethics committees of the participating hospitals.
Ten high-power fields (HPF, ×400) of the tumor nest and peri-tumoral lymphoid stroma were randomly selected for MNT and MNCA, and photographs including both tumor nest and peri-tumoral lymphoid stroma were taken. The cell densities of the infiltrating CD8 (+) T cells, FOXP3 (+) T cells and PD-1 (+) T cells (per mm 2 ) in the tumor nest and peritumoral lymphoid stroma in each photograph were measured and the average was calculated ( Supplementary Fig. S1). Lymphoid follicles were excluded from the photograph to evaluate the stroma in the identical histomorphologic settings. The TIL densities in the tumor nest and peri-tumoral lymphoid stroma were analyzed separately to avoid bias due to the difference in densities of TILs in these areas.
The CD8/FOXP3 ratio of the tumor nest and peri-tumoral lymphoid stroma in each case was defined as the density of CD8 (+) T cells divided by the density of FOXP3 (+) T cells.
The PD-L1 positivity rate was defined as the percentage resulting from dividing the number of PD-L1 (+) tumor cells by the number of all tumor cells in each photograph. Two pathologists independently calculated and averaged the PD-L1 positivity rate in three different HPFs for each tumor.

Statistical analysis
Student's t-tests were used to compare the cell densities of TILs (CD8 (+) T cells, FOXP3 (+) T cells and PD-1 (+) T cells) and the CD8/FOXP3 ratio between the MNT and MNCA cases and also to compare the PD-L1 positivity rates across the MNT, MNCA, typical thymoma and thymic SCC cases. P-values of < 0.05 were considered statistically significant.

Clinical features
The clinical findings are summarized in Table 1  Difference in TILs between MNT and MNCA 455 MNT and one with MNCA were classified as having Stage II disease. According to TNM classification 12 , all patients with MNT and MNCA were classified as having Stage I disease. In the follow-up period (13-120 months, median 58.7 months), one MNT patient showed local recurrence about 10 years after partial resection.

Histological findings
The representative histological and immunohistochemical features are shown in Fig. 1. Both MNT and MNCA showed micronodular tumor cell proliferation separated by abundant lymphoid stroma with germinal centers (Fig. 1a,e). The tumor nests in MNT comprised spindle-or oval-shaped epithelial tumor cells with uniform nuclei containing indistinct nucleoli (Fig. 1b). The mitotic counts ranged from 0 to 2 per HPF. Immunohistochemically, the tumor cells were negative for CD117 (Fig. 1c) and CD5 (Fig. 1d). On the other hand, the tumor cells of MNCA had round, vesicular, and larger nuclei than those of MNT (Fig. 1f) and had mitotic counts ranging from 3 to 6 per HPF. The tumor cells were diffusely positive for CD117 (Fig. 1g) in all MNCA cases. Two cases were diffusely positive for CD5, whereas one case showed partial positivity (Fig. 1h).
Distribution and cell density of infiltrating CD8 (+), FOXP3 (+) and PD-1 (+) T cells Representative immunohistochemical features are shown in Fig. 2, and the results are summarized in Table 2 and Fig. 3.
In the normal thymic tissue, CD8 (+) T cells were observed in both the cortex and medulla. Conversely, FOXP3 (+) T cells and PD-1 (+) cells were mainly observed in the medulla, with small amounts in the cortex (Supplementary Fig. S2). These findings are consistent with those of previously published studies. 15,16 We compared the cell densities of CD8 (+) T cells and FOXP3 (+) T cells in the tumor nest and peri-tumoral lymphoid stroma between MNT and MNCA cases. The cell density of CD8 (+) T cells was higher in MNT than in MNCA in both the tumor nest (P = 0.009) and peri-tumoral lymphoid stroma (P = 0.033) (Figs. 2a,b,3a,b; Table 2). FOXP3 (+) T cells in the tumor nest and peri-tumoral lymphoid stroma was significantly higher in MNCA than MNT (tumor nest, P < 0.001; peri-tumoral lymphoid stroma, P < 0.001) (Figs. 2c,d,3c,d; Table 2). As a result, the CD8/FOXP3 ratio was significantly higher in MNT cases than in MNCA cases in both the tumor nest (P = 0.041) and peri-tumoral lymphoid stroma (P = 0.046) (Fig. 3e,f; Table 2).
Next, we compared the distribution and cell density of PD-1 (+) T cells in the tumor nest and peri-tumoral lymphoid stroma in the MNT and MNCA cases. In both tumors, PD-1 (+) T cells were observed predominantly in the germinal centers, but not    in the tumor nests, and were scattered in the peri-tumoral lymphoid stroma (Fig. 2e,f). The cell density of PD-1 (+) T cells in the peri-tumoral lymphoid stroma was not significantly different between the MNT and MNCA cases (P = 0.295) ( Fig. 3g; Table 2).

PD-L1 expression in tumor cells
We compared the tumor cell PD-L1 expression in thymic epithelial neoplasms including MNT, MNCA, typical thymoma and thymic SCC (Fig. 4). The PD-L1 expression levels were significantly higher in type B1-3 thymoma and thymic SCC than in type A/AB thymoma, while no significant differences were observed between type A/AB thymoma and MNT, and between thymic SCC and MNCA (Fig. 4c). The PD-L1 positivity rate tended to be higher in the MNCA cases (33.6%, range: 3.3-89.1%) than in the MNT cases (5.6%, range: 0-16.1%), but the difference was not significant (P = 0.112) ( Fig. 4c; Supplementary Table S1). The PD-L1 positivity rate in MNCA varied widely between cases. The clinical characteristics of cases in our cohort were compared with those noted in a previous meta-analysis (Table 3). 4 Neither metastasis nor tumor death has been reported in MNT patients in the previous studies and in our study. 2,4 In contrast, one tumor-related death was reported among eight MNCA patients in the previous studies, 4 whereas no case exhibited metastasis or tumor-related death in our cohort. One possible reason for this difference could be the tumor stage. Most cases in our study were diagnosed at an early stage compared with cases in the previous studies (Table 3). 4,5 Although MNCA was suggested to be a malignant counterpart of MNT, it remains inconclusive whether MNCA is associated with a worse prognosis than MNT because of the scarcity of MNCA cases. 4,5 In this study, the cell density of CD8 (+) T cells was higher in MNT, while the cell density of FOXP3 (+) T cells was higher in MNCA and consequently, the CD8/FOXP3 ratio was remarkably higher in MNT than in MNCA (Fig. 3). This tendency was observed in both the tumor nest and peritumoral lymphoid stroma. CD8 (+) T cells are the major effector cells responsible for inducing apoptosis in the tumor immune microenvironment. 9,17 In various malignant tumors, including esophageal carcinoma and colorectal carcinoma, a high degree of CD8 (+) T cell infiltration was shown to correlate with better prognoses. [18][19][20][21][22][23][24][25][26][27] FOXP3 is a biomarker of regulatory T cells, which suppress the function of CD8 (+) cytotoxic T cells and reduce the level of antitumor immune activity. 11,28-30 As a result, a higher degree of FOXP3 (+) T cell infiltration correlates with a worse prognosis in various malignant tumors including hepatocellular carcinoma and breast carcinoma. 22,23,[31][32][33] These results suggest that antitumor immunoactivity is higher in MNT than in MNCA. In typical thymomas and thymic SCC, type B thymoma and thymic SCC presented a larger number of FOXP3 (+) T cells than did type A or AB thymoma, 34 and the prognosis of type B thymoma and thymic SCC was worse than that of type A or AB thymoma in previous studies. 1 In our study, the cell density of FOXP3 (+) T cells was significantly higher in MNCA than in MNT. This tendency may support the hypothesis that MNCA is the malignant counterpart of MNT. However, this hypothesis is largely based on the cytomorphologic and immunohistochemical features of MNCA, not on prognostic data from a large cohort. Our results suggested that MNCA may have a specific immune microenvironment that evades immunity, despite similar histomorphological features of the lymphoid stroma.
PD-L1 binds to PD-1 on CD8 (+) T cells and suppresses tumor immune response. 35 Several studies focusing on PD-L1 expression in tumor cells of thymic epithelial neoplasms have shown that the PD-L1-positivity rate in the tumor cells tends to be higher in type B thymoma and thymic SCC cases than in type A or AB thymoma cases. 34,[36][37][38][39][40][41][42][43] The results of PD-L1 expression in typical thymoma and thymic SCC cases of the present study are similar to those obtained in previous studies.   Table S1). PD-1 is expressed not only on regulatory T cells but also on exhausted cytotoxic T cells, B cells, monocytes, natural killer T cells and dendritic cells. 44 The PD-1 expression in cell populations other than regulatory T cells may be another reason for the different distribution of FOXP3 (+) cells and PD-1 (+) cells. Consequently, these results did not show a significant difference in the level of PD-1/PD-L1-related immune tolerance between the two diseases.
The relationship between PD-L1 expression and prognosis of thymoma and thymic carcinoma remains unclear, and the use of immune checkpoint inhibitors (ICIs) for treating thymoma or thymic SCC has not yet been established. 34,[36][37][38][39][41][42][43]45 In a review of ICI therapy for thymoma and thymic SCC patients, a high expression of PD-L1 was associated with more effective response, and the incidence of side effects was higher in thymoma. 46 In our study, a high expression of PD-L1 was observed in one case of MNCA. Although no tumor recurrence or tumor-related death was recorded for this patient, ICI therapy for patients with recurrent or unresectable MNCA with high PD-L1 expression requires further investigation.
In conclusion, this study revealed that the cell density of CD8 (+) T cells in MNT was significantly higher than that in MNCA, while the cell density of FOXP3 (+) T cells in MNCA was significantly higher than that in MNT. These results suggest that antitumor immunoreactivity is lower in MNCA than in MNT.   Difference in TILs between MNT and MNCA 461

SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section.