Multi-factor Evaluation of Tumor-inltrating Lymphocytes in Laryngeal Squamous Cell Carcinoma and its Prognostic Value

Background: Laryngeal squamous cell carcinoma (LSCC) is a heterogeneous disease. In clinical practice, patients with similar clinicopathological characteristics often show different outcomes. This study evaluated the levels of primary LSCC intratumoral inltrating lymphocytes (iTILs), tumor-inltrating lymphocyte volume (TILV), frontier tumor-inltrating lymphocytes (fTILs), and their relations to the patient's clinical outcome. Materials and methods: According to the 2017 study of the International TILs Working Group, hematoxyline and eosin-stained slides from 412 patients were evaluated for their morphology of tumor immune inltration status. Results: Kaplan-Meier analysis showed that high levels of iTILs, TILV, and fTILs were signicantly correlated with OS (all P<0.05). Cox regression model analysis showed that high levels of iTILs, TILV, and fTILs were independently associated with better OS (all P<0.05). Conclusion: Local inammatory markers in patients with laryngeal squamous cell carcinoma, especially the levels of iTILs, TILV, and fTILs, are reliable prognostic factors.


Introduction
Laryngeal squamous cell carcinoma (LSCC) is one of the most common malignant tumors of the head and neck. At present, the treatment strategy of LSCC includes a combination of CO 2 laser-assisted oral surgery, open surgery, oral robotic surgery, radiotherapy, and chemotherapy [1]. However, despite the development of improved strategies and more accurate treatments, the 5-year survival rate of laryngeal squamous cell carcinoma has decreased from 66-63% unfortunately in the past 40 years [2]. In recent years, oncology research on LSCC has been focusing on the tumor biology, especially for the advanced LSCC, to nd prognostic markers and potential therapeutic targets [3][4][5][6].
Tumor-in ltrating lymphocytes (TILs) are a heterogeneous group of lymphocytes that are found in the tumor microenvironment. Mainly T lymphocytes, TILs participate in the formation of tumor immune microenvironment locally and the body's anti-tumor immune response. Current studies have found that TILs are responsible for the microenvironment composition and effects in various types of malignant tumors, such as head and neck tumors, melanoma, breast cancer, bladder cancer, urothelial tumors, ovarian cancer, colorectal cancer, kidney cancer, prostate cancer, and lung cancer [7][8][9][10]. It has also been reported that the immune response of the tumor cell matrix has important prognostic and predictive signi cance [8]. The dysfunction of the immune system is a key factor in the occurrence and development of LSCC, and immune checkpoints are an important mechanism for tumor immune escape [11]. According to these studies, the presence of TILs is an important biomarker for predicting cervical lymph node metastasis in LSCC. It is therefore necessary to determine the pathological markers that predict survival and recurrence in order to optimize the treatment and reduce potentially preventable adverse effects on patients. However, there is no research up to date that determines the relationship between TIL and the prognosis of LSCC.
The TIL-related parameters evaluated are the following: the intratumoral in ltrating lymphocyte (iTILs), tumor-in ltrating lymphocyte volume (TILV), and the frontier tumor-in ltrating lymphocytes (fTILs). The iTIL score is de ned as the percentage of tumor islands occupied by lymphocytes. TILV=% stroma in tumor×% stroma iTILs. Frontier TILs (fTILs) are de ned as the percentage of in ltrating lymphocytes in the tissues before tumor invasion. The International Immunological Biomarker Working Group used H&Estained sections to evaluate TILs of solid tumors in 2017 and developed a standardized method for the microscopic detection of iTILs in H&E-stained sections [12]. These standards are repeatable and applied to daily practice [13]. However, the evaluation of the iTILs does not involve the tumor-stroma ratio and the percentage of tumor-in ltrating lymphocytes in the invasion front, which is one-sided for the evaluation of the local immune status of the tumor. To our knowledge, this study is the rst to evaluate the relation between lymphocyte in ltration in different parts of the tumor and the prognosis and recurrence of LSCC after surgery. We rst proposed the concept of TILV and fTILs at LSCC.
Although the histological evaluation of tumor-in ltrating lymphocytes in our H&E-stained samples did not reveal different subpopulations of lymphocytes, it might still be a useful biomarker for evaluating tumor behavior. This method is advantageous in several ways. It is cost-effective and does not require expensive or speci c tools or antibodies. At the same time, it is also easy to incorporate into standard pathology reports. In our study, we conducted a detailed assessment of the real-time immune status in the three-dimensional tumor structure, and explored the relation between the TILs (iTILs, TILV and fTILs) and the LSCC outcomes overall survival (OS) and recurrence-free survival (RFS).

1.Patients
A total of 412 cases were retrospectively analyzed. These patients were all diagnosed pathologically and underwent laryngectomy at the Department of Head and Neck Nasopharyngeal Surgery, A liated Tumor Hospital of Harbin Medical University or the Head and Neck Department of Tumor Hospital of Chinese Academy of Medical Sciences between December 2011 and December 2014. This study was reviewed and approved by the ethics committees of the two institutions, and proceeded in accordance with the principles of the Declaration of Helsinki and its amendments. All participants provided informed consents to participate in the study. The clinical data (sex, age, BMI, history of drinking and smoking, tumor location, differentiation, TNM, T-stage and N-stage,) and follow-up information (clinical outcome and survival time) were collected through from the electronic medical records. The prescribed inclusion criteria are as follows: 1) LSCC con rmed by histopathology; 2) No history of anti-cancer treatment; 3) Complete clinical, laboratory, imaging, and follow-up data; 4) The remaining para n-xed tissue is su cient, and the structure is clear; 5) Has at least one slice to assess the edge of tumor invasion; 6) No history of other malignant tumors and no distant metastasis. In this study, a total of 412 patients with laryngeal squamous cell carcinoma were enrolled. There were 2 to 3 pathological tissue slices in each case, and a total of 1112 pathological tissue slices were reviewed. The samples were reviewed by two pathologists in a double-blind manner, and the patients were staged according to the eighth edition of the American Joint Committee on Cancer (AJCC) staging system. Table 1 lists the main clinicopathological characteristics of the patients.

Experimental methods
2.1 Tumor tissue sampling and laryngeal cancer tissue wax block preparation 1) Surgically excised laryngeal tissue specimens are cut and xed with 10% formalin solution; 2) 3*3*0.5 cm tissue blocks are cut from the laryngeal cancer tissue and placed in a tissue embedding box and placed in 10% formalin solution; 3) Laryngeal cancer tissue blocks are dehydrated by gradient alcohol of low concentration to high concentration; 4) Laryngeal cancer tissue blocks are soaked in xylene to remove alcohol transparent tissue; 5) Laryngeal cancer tissue blocks are embedded in para n to make laryngeal cancer Tissue wax blocks.
2.2 Preparation of white slices of laryngeal cancer tissue 1) Slice the laryngeal cancer tissue wax blocks with a microtome 4 µm in thickness; 2) Place the slices in 30°C water and atten with a glass slide; 3) Bake the slides at 72°C for 1-2 hours.

Hematoxylin-Eosin staining (H&E staining)
1) White slices of laryngeal cancer tissue are depara nized in xylene solution; 2) White slices are hydrated with high to low concentration gradient alcohol; 3) White slices are stained with hematoxylin 4) After washing, the sections are placed in hydrochloric acid alcohol to return to blue and differentiated in the differentiation solution; 5) After the sections are rinsed, they are dehydrated in low to high concentration gradient alcohol; 6) The sections are stained in alcohol and eosin; 7) The sections are placed Dehydrate in pure alcohol; 8) place the slices in xylene to be transparent; 9) use neutral resin to seal the slices after drying.

TIL histological scoring in laryngeal cancer tissues
We evaluated TIL-related parameters according to the scoring method introduced by the International Immuno-Tumor Biomarker Working Group recently [14] . The evaluation of iTILs does not include any stromal areas that are not directly related to the tumor. In addition, areas of brosis or central necrosis are not included in the iTILs assessment. The percentage of iTILs was evaluated in 2 areas of each sample (the front and the center of the tumor invasion). The TIL working group guidelines recommend "Don't focus on hot spots" 1.2. Therefore, the average value of TIL in the region should be used when reporting iTILs and fTILs. We evaluated at least ve regions to assess the average value of TIL. As recommended, we used the whole untrimmed tumor sections. Each case in our study had at least one representative section (4 -5 µm). Low-quality tumor sections, such as tumor sections without tumor-stroma interface, were excluded.

Follow-up methods
The demographic, clinicopathological and treatment data of each patient were extracted from the electronic medical record system. The demographic data and clinicopathological characteristics of the patients were collected from the database of two institutions/hospitals. All patients who met the inclusion criteria were followed up by a combination of inpatient case review and telephone through January, 2020. The median follow-up time was 59.9 months (range: 1.9-83.2 months). The median overall survival time was 68.1 months (95% CI: 65.6-70.5 months). The primary outcome was overall survival (OS) from diagnosis to death and the second outcome was recurrence-free survival (RFS) from cancer diagnosis to disease recurrence or metastasis or cancer speci c death, whichever came rst.

Data analysis
We rst divided the patients into two groups according to the optimal cut-off point of each iTIL, TILV and fTILs level, which was determined by Receiver operating characteristic (ROC) curves with overall survival status as the dependent variable (0, alive; 1, death). We reported means and standard deviations or counts and frequencies for continuous or categorical variables, respectively. Differences in continuous and categorical covariates between groups were compared with Student's t tests and chi-square (χ2) tests, respectively.
We then conducted univariate and multivariate Cox regression analyses and reported hazard ratios (HRs) and 95% con dence intervals (CIs) to assess the association between iTILs, TILV and fTILs and the prognosis of laryngocarcinoma patients. The likelihood ratio backward stepwise selection was used for the multivariate Cox regression analysis. Kaplan-Meier curves and log-rank tests were then conducted to compare the OS and RFS rates between groups. Two-sided statistical signi cance was de ned as P < 0.05. ROC analyses were performed with MedCalc version 12.6.1.0, and all other statistical analyses were performed with SPSS Statistics version 23.0 (IBM, Inc., USA).
A total of 336 men (81.6%) and 76 women (18.4%) were eligible for this study. Most subjects (72.3%) had a current or past history of smoking. As shown in Table 1, 52.2% of patients had supraglottic squamous cell carcinoma, and 47.8% of patients had glottal laryngeal squamous cell carcinoma. Most patients (65.3%) had localized early tumors (T1 or T2), most (59.0%) being moderately or poorly differentiated.

Survival analysis based on tumor in ammation markers
In our study, 330 patients had higher iTILs (80.1%, Figure 2A), 82 patients had lower iTILs (19.9%, Figure  2B). The 5-year OS rate was signi cantly higher in the high iTILs group (75.76%) than in the low iTILs group (59.76%, p < 0.05, Figure 3A). When the patients were strati ed into high TILV group (137 patients or 33.3%, Figure 2C) and low TILV group (275 patients or 66.7%, Figure 2D), the 5-year OS rate is signi cantly higher in the high TILV group (81.20%) than in the low TILC group (68.36%, p < 0.05, Figure  3B). Finally, we strati ed the patients again, according to fTILs. 240 patients had higher fTILs (58.3%, Figure 2E), and 172 patients had lower fTILs (41.7%%, Figure 2F). The 5-year OS rate of the high fTILs group (77.50%) was signi cantly higher than that of the low fTILs group (65.70%, P<0.05, Figure 3C). Therefore, the levels of iTILs, TILV, and fTILs are related to the patient's survival. Furthermore, we analyzed the levels of iTILs, TILV, and fTILs and the recurrence of the disease and found no signi cant correlation (P>0.05, Figure 2.) 3. Single-and multiple-factor analyses Clinicopathological parameters, including the levels of iTILs, TILV and fTILs, and OS and RFS, were subjected to univariate multivariate analyses to determine independent predictors of OS and RFS in LSCC patients. BMI<24, history of drinking, low levels of differentiation, supraglottic carcinoma, high T/N stage or TNM, and low iTILs/TILV/fTILs levels were identi ed as predictors of poor prognosis (Table 1). These factors are determined by the single-factor analysis. Next, we established multiple linear regression models to observe the main effects of iTILs, TILV, and fTILs. age, alcohol consumption, and T4 tumor stage showed statistical signi cance in these three models. More importantly, the three linear regression models showed that high iTILs (P=0.002, HR: 0.518, 95%CI 0.341-0.785), high TILV (P=0.026, HR: 0.604, 95%CI: 0.387-0.943) and high FTILs (P=0.011, HR: 0.605, 95%CI: 0.410-0.892) were signi cantly correlated with better OS (Table 2). Therefore, we believe that high levels of iTILs, TILV, and fTILs are independent predictors of good prognosis. In the Cox regression model analysis to determine the statistically signi cant factors related to RFS, the relationship between the three factors and the recurrence was not found to be statistically signi cant, in either single-factor or multivariate analyses (

Discussion
As far as we know, this is the largest study in sample size assessing the prognostic value of TILs in LSCC. We analyzed the relationship between TILs and the prognosis/recurrence from multiple aspects. We are also the rst to propose the concepts of TILV and fTILs in LSCC, which are useful for understanding tumor immune status in detail. In addition, our work con rms and extends other studies that have successfully quanti ed TIL levels in tissues and shown the correlations between the TIL levels and the outcomes [15][16][17][18]. Unlike the results of the 120 LSCC cohort study by Want et al [17], neither the Kaplan-Meir curve nor the single-factor multivariate analysis showed signi cant correlations between iTILs/TILV/fTILs and tumor recurrence in our study cohort. Despite the discrepancy, we believe this noncorrelation to be credible, given the large size of our retrospective analysis (412 LSCC cohort). Our ndings should add reliable indicators for the prognosis of LSCC patients, and contribute to future TNM stagings.
Tumor in ltration by chronic in ammatory cells includes lymphocytes, plasma cells, and macrophages [19]. Lymphocytes are the main type of in ltrating immune cells, represented by T cells, B cells, and natural killer cells. TILs are considered to be a manifestation of the host's immune response to tumor cells. Some studies have reported the potential of TILs as prognostic indicators in various human malignancies [20][21][22][23]. Formalin-xed para n-embedded sections can be used to assess tumor immunity from multiple perspectives such as TIL morphology [23], T cell subsets (e.g. CD3(+) or CD8(+)) immune score [24], and immunophenotype reaction [25]. In our study, only three aspects of TILs were evaluated morphologically. The results show that high levels of iTILs, TILV, and fTILs are good prognostic indicators for LSCC. In addition, the morphological evaluation of TILs is simple, can be performed routinely in clinical practice, and can provide better histopathological predictions on top of immune response without additional costs. This prediction is helpful for clinical decision-making. For instance, multimodal treatment is advised for early LSCC cases with low levels of TILs. A recent study demonstrated the important role of immune cells in regulating cancer invasion and metastasis [26]. The immune response is believed to be one of the main factors affecting the clinical outcome of tumors. In fact, tumors of the same clinical stage and/or the same histopathological grade may have very different immune responses [27]. Therefore, the immune heterogeneity of early LSCC can be used to divide patients into low-and highrisk groups, which is essential for the personalized treatment of patients to improve patient prognosis.
The role of immunotherapy in patients with relapsed or metastatic LSCC continues to expand, promising new treatment approaches for potentially curable LSCC patients. The characterization of the immune status in the tumor microenvironment is a key prerequisite for understanding which patients may bene t from immune regulation, and will be very important for the introduction of immunotherapy [28][29][30]. How to e ciently, reliably, and timely measure the immune status in TME is currently unclear. Simple histological methods provide advantages because they are easy to obtain, quickly and quantitatively describe the tumor immune status in real-time. Recent studies have described the correlation between tumor genetics and immune-in ammatory response. This may help pro le patients into different subpopulations for more personalized treatments [31]. Tumor patients with depleted immune cells have different responses to cell reduction therapy [32,33]. Therefore, it is possible to provide different treatment options for patients with the same tumor in clinical and imaging. Evaluating the changes in TME immune cells after chemotherapy and/or immunotherapy may also play an important role in evaluating response or drug selection/delivery. In the multivariate model, we found that the combination of iTILs, TILV, and fTILs can predict prognosis independent of other clinical variables. This nding provides further evidence that the evaluation of iTILs, TILV, and fTILs should be included in the clinicopathological prognosis model of LSCC patients. Similar proposal has been made in breast cancer. Loi et al.'s comprehensive analysis of 2148 patients with the early triple-negative disease showed that TILs increase the prognostic value of known clinical variables [34]. In addition, the International Immuno-oncology Biomarker Breast Cancer Working Group has proposed a standardized method for pathologists to evaluate iTILs in a post-assisted residual disease setting [35]. This standardization will be a necessary step to expand the use of iTILs in LSCC. Although the concept of evaluating TILs in LSCC in relation to clinical outcomes is not new, there are inadequate consistent data to support TILs as reliable prognostic factors [34]. In this study, we thoroughly assessed the local tumor immune microenvironment status and introduced two indicators, TILV, and fTILs for the following reasons: First, iTILs only re ects the content of tumor stroma, which may be incomplete information. On the other hand, TILV considers the percentage of stroma to the overall tumor when calculating iTILs, hence TILV may be more illuminative than iTILs. Secondly, many studies have shown that the frontier of tumor invasion is the part that best represents the real-time immune status of tumors [36][37][38]. Finally, when iTILs alone are not enough to evaluate the local immune status of tumors, TILV, and fTILs may provide additional information. This research will make a further contribution to this debate, providing reliable indicators for the prognosis of LSCC patients, and adding new knowledge for new TNM staging in the future. This study has several limitations. although this study is based on the largest cohort of 412 eligible patients, these analyses still need to be validated in larger patient cohorts. In addition, LSCC is a maledominated disease, hence there is inevitably a signi cant gender bias in our patient cohort. Lastly, this is a retrospective analysis that needs to be veri ed in a prospective study.

Conclusion
In the age of LSCC heterogeneity, tumor immunogenicity, and immunotherapy, our study con rmed that higher levels of TILs are bene cial to the prognosis of patients with laryngeal SCC and proposed two new standards, TILV, and fTILs, that can satisfactorily evaluate the local immune status of the tumor. Our results indicate that not only can iTILs, TILV, and fTILs predict longer OS, they are important independent prognostic factors for the LSCC patients after surgery. Although our study did not detect any signi cant relationship between immune in ltration and relapse, a thorough examination for local in ammatory markers is worthy of consideration for the evaluation of OS in LSCC.

Declarations
Ethics approval and consent to participate The present study was approved by the ethical review committee of A liated Tumor Hospital of Harbin Medical University and Tumor Hospital of Chinese Academy of Medical Sciences.

Consent for publication
Written informed consent for publication was obtained from all participants.

Availability of data and materials
The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.