Prognostic impact of count of extratumoral lymphatic permeation in lung adenocarcinoma and its relation to the immune microenvironment

Abstract Extratumoral lymphatic permeation (ly‐ext) has been reported as an independent poor prognostic factor for lung adenocarcinoma, but whether or not the number of ly‐ext foci is associated with prognosis and its relationship to the immune microenvironment is unclear. We counted the number of ly‐ext foci on pathological slides from patients with completely resected lung adenocarcinoma with ly‐ext, and divided them into two groups: a group with a high number of ly‐ext foci (ly‐ext high) and one with a low number of ly‐ext foci (ly‐ext low). Among the patients with ly‐ext, only a high number of ly‐ext foci was an independent poor prognostic factor. The 3‐year recurrence‐free survival (RFS) rate of the ly‐ext high group was significantly lower than that of the ly‐ext low group (14.7% vs. 50.0%, P < 0.01). Then, we analyzed the immune microenvironment of pT1 lung adenocarcinoma with ly‐ext (13 cases of ly‐ext high and 11 cases of ly‐ext low tumor) by immunohistochemistry using antibodies for stem cell markers (aldehyde dehydrogenase 1 A1 and CD44), tumor‐promoting mucin (MUC1), tumor‐infiltrating lymphocytes (CD4, CD8, FOXP3, and CD79a), and tumor‐associated macrophages (CD204). The number of CD8+ TILs within the primary lesion was significantly lower and the number of FOXP3+ TILs within the primary lesion was significantly higher in the ly‐ext high group (P < 0.05 and P < 0.01, respectively). Our results indicated that a high number of ly‐ext foci was an independent poor prognostic factor. Moreover, tumors with high numbers of ly‐ext foci had a more immunosuppressive microenvironment.


| INTRODUC TI ON
Lung cancer is one of the most lethal cancers worldwide, and adenocarcinoma is the most common histological type of lung cancer.
Surgery is still the most effective treatment for locally advanced NSCLC, but some patients who undergo complete resection experience relapse. There have been various studies on the clinicopathological factors associated with postoperative recurrence of NSCLC. [1][2][3][4][5] Many studies have highlighted the prognostic importance of lymphovascular invasion. 6 In lung cancer, vascular invasion and lymphatic permeation are assessed only by its presence or absence, and their location and quantity have not been noted in the current TNM classification.
We previously reported that lymphatic permeation of lung cancer has a different prognostic impact depending on whether it is inside or outside the primary lesion. Saijo et al. 7 reported that the RFS time of patients with ly-ext was significantly shorter than that of patients with intratumoral lymphatic permeation. Matsumura et al. 8 found that the rates of OS and RFS of patients with T1 and T2 tumors with ly-ext were comparable to those of patients with T3 tumors. However, the impact of the number of ly-ext foci on prognosis was unclear.
Cancer tissue comprises not only cancer cells but also different kinds of stromal cells, such as TILs or TAMs, which are commonly found in association with cancer cells. TAMs promote cancer progression by producing cytokines involved in angiogenesis, matrix remodeling, tumorigenesis, and immunosuppression. 9 TILs regulate cancer progression by playing two conflicting roles: suppressing tumor growth and promoting tumor progression. Recently, regulatory T cells in cancer stroma were reported to enable cancer cells to escape host immune surveillance in several cancers by inhibiting cytotoxic T cells and inducing an immunosuppressive environment. 10 In this study, we examined whether the number of ly-ext foci is a prognostic factor among patients with ly-ext. Furthermore, we aimed to identify the differences in the immune microenvironment associated with high or low number of ly-ext foci.

| Patients
We retrospectively reviewed a total of 1865 patients who underwent lung resection for primary lung adenocarcinoma between January 2010 and December 2017 in our hospital. Patients who had undergone incomplete resection, sublobar resection, and preoperative therapies, including chemotherapy or radiotherapy, were excluded from this study. The remaining 1458 patients were finally enrolled in this study. A flowchart of the case selection is shown in Figure S1. This study was approved by the Institutional Review Board of the National Cancer Center (approval numbers 2020-453) and informed consent was obtained from all patients and conformed to the provisions of the Declaration of Helsinki.

| Evaluation of clinicopathological factors
We reviewed the clinicopathological characteristics of the patients from the available medical records. The following clinicopathological factors were investigated retrospectively to assess their prognostic effect: age, sex, smoking history, pT status, pN status, vascular invasion, lymphatic permeation, pleural invasion, and intrapulmonary metastasis.

| Histological examination
All specimens were fixed with 10% formalin via infusion through the bronchial tree and then embedded in paraffin. The tumors were sliced at approximately 5 mm intervals and serial 4 µm sections were stained with HE. VVG staining was performed to evaluate blood vessel and pleural invasion in all cases. Lymphatic permeation was determined via sections stained with HE based on the following criteria: more than one tumor cell floating in vessels without supporting smooth muscles or elastic fibers within the bronchovascular bundle, subpleural, and intralobular pleural space. The slides were reviewed by two pathologists (TN and GI). We classified lymphatic permeation into the following three categories: ly0, absence of lymphatic permeation; ly-int, presence of intratumoral lymphatic permeation; and ly-ext, presence of extratumoral lymphatic permeation ( Figure S2). Tumors with both ly-int and ly-ext were classified as lyext. Histological typing was based on the 5th revised World Health Organization histological classifications. All the tumors were pathologically staged using the 8th edition of the TNM classification of lung cancer published by the Union for International Cancer Control.

| Counting the number of ly-ext foci
All HE-stained slides were scanned using the Aperio scan system (Leica Biosystems). We counted the total number of ly-ext foci in all slides made from a slice that contained the maximum cut surface of tumor. If there were no ly-ext foci in a slice of the maximum cut surface of the tumor, we searched for ly-ext foci in the slices that were directly above or below that slice. We measured the area outside the tumor where extratumoral lymphatic permeation was located ( Figure   S3). We counted the total number of ly-ext foci and calculated the total number of ly-ext foci per square centimeter in the slice.

| Antibodies and immunohistochemical staining
The primary antibodies used in this study are listed in Table S1. We took 4 μm sections from the blocks and stained them with antibodies using a BenchMark ULTRA automated immunohistochemical slide staining system (Ventana Medical Systems).

| Calculation of immunohistochemical scores
Immunostaining scores of CD44, ALDH1A1, and MUC1 were calculated from the staining-intensity scores and percentages of positively stained cells. The staining intensity scores were as follows:

TA B L E 1
Clinicopathological characteristics according to lymphatic permeation status RFS rates were estimated using the Kaplan-Meier method and compared using the log-rank test. Univariate and multivariate Cox regression analyses were performed to assess the impact of clinicopathological factors on OS and RFS. We divided patients with ly-ext into two groups with the median number of ly-ext foci as a cut-off value, and the impact of the number of ly-ext foci on RFS of patients with ly-ext were assessed. The Mann-Whitney U test was used to evaluate the immunostaining scores. All statistical calculations were performed using JMP Pro software (version 12.2.0, SAS Institute).
Results were considered significant when the P value was <0.05.

| Differences in prognosis between patients with ly-ext and those with ly-int
The median follow-up duration was 60 months (range 1-129 months). During the study period, 269 patients died. Cancer recurrence was detected in 336 patients. The 3-year OS rates for the ly0, ly-int, and ly-ext groups were 92.1%, 83.3%, and 76.1%, respectively. The OS of the ly-ext group was significantly inferior to that of the ly0 group (P < 0.01), but there was no significant difference in the OS of the ly-int and ly-ext groups. The 3-year RFS rates for the ly0, ly-int, and ly-ext groups were 82.3%, 62.8%, and 31.3%, respectively, and the RFS of the ly-ext group was significantly inferior to that of the ly0 group (P < 0.01) and the ly-int group (P < 0.01; Figure S4).

| Clinicopathological characteristics and prognosis of patients with high and low numbers of ly-ext foci
Since the median number of ly-ext foci in ly-ext patients was 5, we classified ly-ext high group as 5 or more than 5 and ly-ext low group as less than 5. Figure 1B-E shows representative HE and anti D2-40 staining of the ly-ext high and low groups.

TA B L E 2
Univariate and multivariate analysis of clinicopathological factor associated with recurrence-free survival have more patients with lymph node metastasis or solid or micropapillary predominant tumor. In pT1 patients with ly-ext, the ly-ext high group tended to have more patients with vascular invasion (Table S5). Figure 2 shows the OS and RFS curves of patients with high and low numbers of ly-ext foci. There was no significant difference in 3-year OS rate between the ly-ext high and low groups (70.5% vs. 81.4%, P = 0.11; Figure 2A), but the 3-year RFS rate of the ly-ext high group was significantly lower than that of the ly-ext low group (14.7% vs. 50.0%, P < 0.01; Figure 2B). Moreover, there was no significant difference in 3-year OS between ly-ext foci/cm 2 high and low groups ( Figure 2C), but the 3-year RFS of patients with a high number of ly-ext foci/cm 2 was significantly shorter than that of patients with a low number of ly-ext foci/cm 2 ( Figure 2D). Table 3 shows the result of univariate and multivariate Cox regression analyses on RFS of the patients with ly-ext. A high number of ly-ext foci was an independent poor prognostic factor (P < 0.01, HR 2.38, 95% CI 1.44-4.00). When the number of ly-ext foci/cm 2 was used, the number of ly-ext foci/cm 2 was also an independent prognostic factor (Table S6). Even in the analysis limited to stage I patients, the 3-year RFS rate of patients in the ly-ext high group was significantly lower than that of patients in the ly-ext low group (15.4% vs. 75.0%, P < 0.05; Figure S5).

| DISCUSS ION
In the present study, we retrospectively evaluated the prognostic impact of the number of ly-ext foci in patients with completely resected lung adenocarcinoma. The ly-ext high group had a remarkably shorter RFS than the ly-ext low group. In addition, we compared differences in the immune microenvironment between the ly-ext high and low groups and found that the ly-ext high group had a more suppressive immune microenvironment. This is the first study to investigate the prognostic impact of the number of ly-ext foci and the differences in the microenvironment based on the number of ly-ext foci.
In many types of cancers, such as gastric, esophagus, colorectal, hepatic, and breast cancers, the frequency of blood vessel and lymphatic permeation has been reported as an independent prognostic factor. [11][12][13][14][15][16] However, in lung cancer, the prognostic impact  prognostic factor for lung cancer, whereas a high number of stromal FOXP3+ T cells was considered to be a poor prognostic factor. 21,22 In breast cancer, as in our study, a high number of FOXP3+ T cells in In present study, the number of CD204+ TAMs in primary tumor stroma tended to be higher in the ly-ext high group than in the ly-ext low group. TAMs stimulate angiogenesis via expressing factors such as VEGF. TAMs also produce various growth factor and chemokines and contribute to the migration of tumor cells towards vessels. 27 This may be one of the reasons for our finding that the number of CD204+TAMs is associated with the number of ly-ext foci.
In present study, there were no differences in the expression levels of ALDH1 and CD44 in cancer cells between two groups.

Matsumura et al. reported that tumor cells that form multiple small
nests have high expression levels of CD44 and a higher frequency of intrapulmonary metastasis. 28 Kirita et al. showed that low ALDH1 expression in cancer cells is independent predictive factors for LN metastasis. 29 In their study, the expression level of ALDH1 and CD44 in cancer cells in the primary lesion was not a significant predictive factor for LN and intrapulmonary metastasis. Taken together with the results of the present study, the expression of CD44 and ALDH1 in tumor cells in the primary lesion may not be related to the frequency of lymphatic invasion.
There were some limitations in the current study. This was a retrospective study carried out at a single institution. The total number of patients with ly-ext was relatively small and the follow-up period was short.
To our knowledge, this is the first report to examine the association between the number of ly-ext foci and the prognosis, and between the number of ly-ext foci and the immune microenvironment. This study clearly indicates that the frequency of extratumoral lymphatic permeation is an independent prognostic factor, as is its presence, and suggests that tumors with a suppressive immune