CD8+ Tumour-Infiltrating Lymphocytes and Tumour Microenvironment Immune Types as Biomarkers for Immunotherapy in Sinonasal Intestinal-Type Adenocarcinoma

Background. Intestinal-type adenocarcinoma (ITAC) is a rare tumour occurring in the ethmoid sinus. Recent years have brought advances in endoscopic surgery and precision radiotherapy; however, five-year overall survival has not improved and remains at 35–80%, depending on tumour stage and histology. Therefore, there is a need for new therapeutic options. Methods. We evaluated CD8+ tumour-infiltrating lymphocytes (TILs) and tumour microenvironment immune type (TMIT, combining CD8+ TILs and PD-L1) as predictive biomarkers for immunotherapy in a series of 133 ITAC. All results were correlated to clinical and follow-up data. Results. The presence of intratumoural CD8+ TILs was low in 57% of cases and high in 8% of cases. Tumoural PD-L1 positivity was observed in 26% of cases. CD8+ TILs and TMIT correlated with the histological subtype of ITAC and with better overall survival. The presence of stromal PD-L1-positive macrophages was related to intratumoural CD8+ TILs. PD-L1 expression on tumour cells or macrophages did not show prognostic value. Conclusions. TMIT classification did not have additional prognostic value over CD8+ TILs alone. The modest percentage of CD8high/PD-L1pos cases indicates that ITAC is a lowly immunogenic tumour type. Nevertheless, a proportion of ITAC, especially the papillary and colonic subtypes, could benefit from therapy with immune checkpoint inhibitors.


Introduction
Intestinal-type adenocarcinoma (ITAC) is a rare tumour, with an approximate incidence of less than one case per 100,000 inhabitants annually, accounting for 10-20% of all tumours in the sinonasal area, although these proportions vary geographically [1][2][3]. ITAC is etiologically related to professional exposure to organic dust, mainly wood or leather [4][5][6]. Due to the lack of specific symptoms at early stages, patients are often diagnosed at advanced stages. ITAC can be classified into four subtypes, i.e., papillary, colonic, solid and mucinous, the latter two having a worse clinical behavior [7].
CD8 + TILs were scored as 0%, 1-10% and >10% of the cells present in the stromal or in the intratumoural compartment. Staining intensity was similar in all tumours. Staining for PD-L1 was considered positive when >5% of the tumour cells showed membranous and/or cytoplasmic staining. TMIT subtypes were defined by the combination of intratumoural CD8 + TIL presence (CD8 low , either 0% or 1-10%, and CD8 high , >10%) and PD-L1-stained tumour cells (negative, <5% and positive, >5%). TMIT type I was defined as CD8 high /PD-L1 pos , type II as CD8 low /PD-L1 neg , type III as CD8 low /PD-L1 pos and type IV as CD8 high /PD-L1 neg . In the stromal compartment, we observed PD-L1-stained macrophages, which were scored as either absent or present.

Statistical Analysis
The Chi 2 test was used to test possible associations between CD8 + TILs, PD-L1 expression, TMIT types and various clinicopathological factors. Kaplan-Meier curves were plotted to assess the relations of CD8 + TILs and TMITs to overall survival, using the log-rank-test; p-values < 0.05 were considered to indicate statistical significance. Multivariate Cox regression analysis was performed for factors possibly related to survival. Statistical analysis was carried out with the use of SPSS Base, version 15.0 and SPSS Advanced models, version 15.0 (SPSS Inc., Chicago, IL, USA) software.

Tumour Stage and Histological Subtype Are Related to Longer Survival
During the period of follow-up, 63 of 133 patients (48%) developed local recurrence, and 13 (10%) distant metastasis. Sixty of 133 patients (45%) remained alive, and the median disease-free time was 12 months (range 1-96 months). ( Table 1). The overall five-year survival was 50%, the main causes of death being local recurrence and intracranial invasion; however, 20 patients died during the postoperative period or due to intercurrent causes. Overall survival (Figure 1) was significantly related to disease stage (log-rank 50.498; p = 0.000) and histological subtype (log-rank 15.085; p = 0.002).

Statistical Analysis
The Chi 2 test was used to test possible associations between CD8 + TILs, PD-L1 expression, TMIT types and various clinicopathological factors. Kaplan-Meier curves were plotted to assess the relations of CD8 + TILs and TMITs to overall survival, using the log-rank-test; p-values < 0.05 were considered to indicate statistical significance. Multivariate Cox regression analysis was performed for factors possibly related to survival. Statistical analysis was carried out with the use of SPSS Base, version 15.0 and SPSS Advanced models, version 15.0 (SPSS Inc., Chicago, IL, USA) software.

Tumour Stage and Histological Subtype Are Related to Longer Survival
During the period of follow-up, 63 of 133 patients (48%) developed local recurrence, and 13 (10%) distant metastasis. Sixty of 133 patients (45%) remained alive, and the median disease-free time was 12 months (range 1-96 months). (Table 1). The overall five-year survival was 50%, the main causes of death being local recurrence and intracranial invasion; however, 20 patients died during the postoperative period or due to intercurrent causes. Overall survival ( Figure 1) was significantly related to disease stage (log-rank 50.498; p = 0.000) and histological subtype (log-rank 15.085; p = 0.002).

The Prognostic Value of CD8 + TILs Is Independent of Tumour Stage and Histological Subtype
Stromal CD8 + TILs were detected in the majority (126/133, 95%), and intratumoural CD8 + TILs in 65% (86/133) of cases. All samples with intratumoural CD8 + TILs also showed stromal CD8 + TILs, reason why we focused our analyses on intratumoural CD8 + TILs only. Complete absence of CD8 + TILs was seen in 35% (47/133) of samples. The presence of intratumoural CD8 + TILs was low in 57% of cases and high in 8% of cases ( Figure 2). All (10/10) CD8 high cases were histologically papillary and colonic, and also among the CD8 low cases these ITAC subtypes were dominant (75%, 57/76). CD8 high cases also appeared to be at a lower tumour stage and developed less recurrence or metastasis compared to CD8 low and CD8 neg tumours (Table 1). Kaplan-Meier analysis showed that CD8 high cases had significantly better overall survival than CD8 low /CD8 neg cases (p = 0.028) ( Figure 3). Using multivariate Cox regression, we found that presence of CD8 + TILs >10% was a positive prognostic factor independent of tumour stage, while histological subtype lost its prognostic value due to its interrelation with CD8 + TILs ( Table 2).

The Prognostic Value of CD8 + TILs Is Independent of Tumour Stage and Histological Subtype
Stromal CD8 + TILs were detected in the majority (126/133, 95%), and intratumoural CD8 + TILs in 65% (86/133) of cases. All samples with intratumoural CD8 + TILs also showed stromal CD8 + TILs, reason why we focused our analyses on intratumoural CD8 + TILs only. Complete absence of CD8 + TILs was seen in 35% (47/133) of samples. The presence of intratumoural CD8 + TILs was low in 57% of cases and high in 8% of cases ( Figure 2). All (10/10) CD8 high cases were histologically papillary and colonic, and also among the CD8 low cases these ITAC subtypes were dominant (75%, 57/76). CD8 high cases also appeared to be at a lower tumour stage and developed less recurrence or metastasis compared to CD8 low and CD8 neg tumours (Table 1). Kaplan-Meier analysis showed that CD8 high cases had significantly better overall survival than CD8 low /CD8 neg cases (p = 0.028) (Figure 3). Using multivariate Cox regression, we found that presence of CD8 + TILs >10% was a positive prognostic factor independent of tumour stage, while histological subtype lost its prognostic value due to its interrelation with CD8 + TILs ( Table 2).

TMIT Types Display Different Clinical Outcomes
The most frequent TMIT was type II, observed in 72% (96/133) of cases, followed by type III in 20% (27/133), type I in 6% (8/133) and type IV in 1% (2/133) of cases. No significant correlation was found between TMIT and any of the clinicopathological characteristics (Table 1). Kaplan-Meier

TMIT Types Display Different Clinical Outcomes
The most frequent TMIT was type II, observed in 72% (96/133) of cases, followed by type III in 20% (27/133), type I in 6% (8/133) and type IV in 1% (2/133) of cases. No significant correlation was found between TMIT and any of the clinicopathological characteristics (Table 1). Kaplan-Meier

TMIT Types Display Different Clinical Outcomes
The most frequent TMIT was type II, observed in 72% (96/133) of cases, followed by type III in 20% (27/133), type I in 6% (8/133) and type IV in 1% (2/133) of cases. No significant correlation was found between TMIT and any of the clinicopathological characteristics (Table 1). Kaplan-Meier analysis showed that type I and IV cases (CD8 high and either PD-L1 pos or PD-L1 neg ) had much better overall survival (LR 9.186, p = 0.027). Analysis of only TMIT II and III cases (CD8 low and either PD-L1 pos or PD-L1 neg ) revealed a worse overall survival for those tumours expressing PD-L1, although not statistically significant (LR 3.593, p = 0.058) (Figure 3).

PD-L1-Expressing Tumour Cells and Macrophages Co-Occur with CD8 + TILs
By analysing PD-L1 staining for the evaluation of TMIT, we found 26% (35/133) of cases with positivity in patches of tumour cells, and this was positively correlated (p = 0.000) with intratumoural presence of CD8 + TILs (Table 3). We also observed a notable PD-L1 positivity on macrophages, particularly in the stromal compartment (Figure 4), again correlated (p = 0.001) with intratumoural presence of CD8 + TILs (Table 3). Mucinous ITAC displayed a notable lower presence of PD-L1-positive macrophages compared to papillary, colonic and solid ITAC, corresponding, respectively, to 3% and 18-30%. We found no other correlations between the presence of PD-L1-positive macrophages and clinical and follow-up data (Table S1). Table 3. Correlations between CD8 + TILs and PD-L1-expressing tumour cells and macrophages. analysis showed that type I and IV cases (CD8 high and either PD-L1 pos or PD-L1 neg ) had much better overall survival (LR 9.186, p = 0.027). Analysis of only TMIT II and III cases (CD8 low and either PD-L1 pos or PD-L1 neg ) revealed a worse overall survival for those tumours expressing PD-L1, although not statistically significant (LR 3.593, p = 0.058) (Figure 3).

PD-L1-Expressing Tumour Cells and Macrophages Co-Occur with CD8 + TILs
By analysing PD-L1 staining for the evaluation of TMIT, we found 26% (35/133) of cases with positivity in patches of tumour cells, and this was positively correlated (p = 0.000) with intratumoural presence of CD8 + TILs (Table 3). We also observed a notable PD-L1 positivity on macrophages, particularly in the stromal compartment (Figure 4), again correlated (p = 0.001) with intratumoural presence of CD8 + TILs (Table 3). Mucinous ITAC displayed a notable lower presence of PD-L1positive macrophages compared to papillary, colonic and solid ITAC, corresponding, respectively, to 3% and 18-30%. We found no other correlations between the presence of PD-L1-positive macrophages and clinical and follow-up data (Table S1).

Discussion
Patients with ITAC in advanced stages generally have a poor prognosis, with limited therapeutic options. In this study, we analysed 133 ITAC for the presence of CD8 + TILs and TMIT, combining the presence of CD8 + TILs and the expression of PD-L1 on tumour cells as a possible predictive marker for treatment with PD-1/PD-L1 immune checkpoint inhibitors. In addition, we investigated their possible prognostic value. To the best of our knowledge, this is the first study analysing CD8 + TILs and TMIT as indicators for immunotherapy in sinonasal ITAC.
We found 65% (86/133) of cases with intratumoural CD8 + TILs, all of which also showing these lymphocytes in the stromal compartment. Kaplan-Meier survival analysis revealed no difference between cases with absence and low presence of CD8 + TILs; however, all cases with a high presence of CD8 + TILs showed significantly better overall survival (p = 0.028), in multivariate analysis, independently of tumour stage and histological subtype. There are no studies on ITAC to compare our results with; however, in other sinonasal tumour types such as SNSCC and olfactory neuroblastoma (ONB), CD8 + TILs have also been found to correlate to better survival [18,19]. The

Discussion
Patients with ITAC in advanced stages generally have a poor prognosis, with limited therapeutic options. In this study, we analysed 133 ITAC for the presence of CD8 + TILs and TMIT, combining the presence of CD8 + TILs and the expression of PD-L1 on tumour cells as a possible predictive marker for treatment with PD-1/PD-L1 immune checkpoint inhibitors. In addition, we investigated their possible prognostic value. To the best of our knowledge, this is the first study analysing CD8 + TILs and TMIT as indicators for immunotherapy in sinonasal ITAC.
We found 65% (86/133) of cases with intratumoural CD8 + TILs, all of which also showing these lymphocytes in the stromal compartment. Kaplan-Meier survival analysis revealed no difference between cases with absence and low presence of CD8 + TILs; however, all cases with a high presence of CD8 + TILs showed significantly better overall survival (p = 0.028), in multivariate analysis, independently of tumour stage and histological subtype. There are no studies on ITAC to compare our results with; however, in other sinonasal tumour types such as SNSCC and olfactory neuroblastoma (ONB), CD8 + TILs have also been found to correlate to better survival [18,19]. The same has been reported in head and neck cancer [26,27], lung adenocarcinoma [28,29] and most other studied cancers. This confirms the protective, cytotoxic anti-tumour function of CD8 + TILs.
Our evaluation of TMIT classification showed that type I and IV cases (CD8 high and eitherPD-L1 + or PD-L1 -) were exclusively papillary and colonic and had much better overall survival. This indicates that the more important component of TMIT is an elevated presence of CD8 + TILs, while tumoural PD-L1 expression does not have additional prognostic value. Nevertheless, among the CD8 low cases, those expressing PD-L1 showed a tendency toward a worse survival (Figure 3). This confirms previous studies on PD-L1 in sinonasal tumours of different histotypes [17,18]. Also for lung adenocarcinomas, a tumour type similar to ITAC, most studies report a worse survival in PD-L1-positive cases [29,30], Vaccines 2020, 8, 202 7 of 10 which may be explained by PD-L1 immunosuppressive effect, which prevents cytolysis by activated CD8 + TILs.
We observed PD-L1-and CD68-positive macrophages in the stromal compartment in 17% (23/133) of cases, which significantly co-occurred with CD8 + TILs. Infiltration of macrophages in solid tumours has been associated with poor prognosis, promoting angiogenesis and invasion, and suppressing antitumour immunity [31]. These anti-inflammatory and pro-tumoural properties are mostly ascribed to M2 (CD68 + and CD163 + /CD204+) macrophages, whereas M1 macrophages (CD68 + and HLA-DR + ) would be pro-inflammatory [32,33]. However, this distinction is not generally accepted, and both M1 and M2 macrophage infiltrations have also been associated with favourable clinical outcome, perhaps through the recruitment of CD8 + TILs [34][35][36]. In our study, the association of CD68-positive macrophages with CD8 + TILs and a prolonged overall survival appears to support an antitumour role for both types of infiltrating lymphocytes.
Immune checkpoint inhibitors are an emerging and hopeful therapeutic option for a variety of tumours, and several clinical trials are showing good efficacy and tolerance to PD-1 checkpoint inhibitors in locally advanced HNSCC [14][15][16]. However, clinical responses are seen in only 13-22% of patients, underlining the need for predictive biomarkers. The presence of tumour-activated CD8 + TILs is an essential requirement for the treatment with immune checkpoint inhibitors, while PD-L1 expression has been shown to be associated with improved outcomes upon immunotherapy [24]. These two factors are united in the TMIT classification, where type I (CD8 high /PD-L1+) and, to a lesser extent, type IV (CD8 high /PD-L1-) tumours are predicted to present the best response [22][23][24]. Our analysis revealed 8/133 (6%) of ITAC to be TMIT I, and 1.5% TMIT IV and indicated that ITAC is a lowly immunogenic cancer, with solid and mucinous subtypes even less immunogenic. This contrasts with known strong immunogenic tumours such as skin melanoma, renal cell and bladder cancer or head and neck and lung SCC, where TMIT I characterizes 40-50% of cases [21]. On the other hand, ITAC is comparable to adenocarcinomas of other organs such as lung, colon and stomach, with reports of 12-14%, 19% and 23% TMIT I, respectively [21,28,37,38].
A limitation of using TMIT as a predictor of response to therapy is the lack of consensus on the scoring and cut-off values of CD8 + TILs and PD-L1. Indeed, the proportions of cases among the TMIT types vary considerably between studies on the same types of cancer. In addition, the exact localization of TILs, either inside the tumour or in the stroma, can be important. Moreover, the studied series of tumours may be heterogeneous, for example, squamous cell carcinoma and adenocarcinoma subtypes in non-small cell lung cancer. Similarly, ITAC includes different histological subtypes, and we found significant differences among them. Among the mucinous tumours, none were TMIT I or IV, and PD-L1-positive macrophages were virtually absent. This suggests that this subtype is even less immunogenic than papillary and colonic ITAC. Another possible limitation of our study is the large period of time over which the samples were collected. To address this, we compared cases treated before and after the year 2003, the point in time when endoscopic surgery was introduced in our hospital, and we found the same distribution of CD8 + TILs and TMIT types and the same association with overall survival. Apart from CD8 + TILs, the tumour microenvironment includes many more types of immune cells (myeloid-derived suppressor cells, regulatory T cells, dendritic cells, macrophages, natural killer cells, B cells) that may also be related to prognosis or play a role in the response to immune therapy [32]. However, from all studies, the strongest relation to clinical outcome and therapeutic response are found for CD8 + TILs [23,39]. Finally, aside from CD8 + TILs, PD-L1 expression and TMIT, there are other biomarkers that may predict the efficacy of anti-PD1 immunotherapy, such as tumour mutational burden, lactate dehydrogenase concentration in serum and pro-inflammatory interferon gene signature [24].

Conclusions
The main aim of this study was to evaluate possible predictors of response to immunotherapy. Our results demonstrate that intratumoural CD8 + TILs are present in up to 65% of ITAC. Moreover, Vaccines 2020, 8, 202 8 of 10 a high number of CD8 + TILs and TMIT types I and IV were associated with longer overall survival. TMIT classification did not have additional prognostic value over CD8+ TILs alone. The modest percentage of TMIT type I CD8 high /PD-L1 pos cases indicate that ITAC is a lowly immunogenic tumour type. Nevertheless, a subgroup of patients may benefit from anti-PD-1 agents already approved in HNSCC.