Evaluating tumor-infiltrating lymphocytes in hepatocellular carcinoma using hematoxylin and eosinstained tumor sections

INTRODUCTION

Cancer incidence and mortality are rapidly growing globally. Hepatocellular carcinoma (HCC) is one of the most common primary malignancies of the liver, representing the third leading cause of cancer-related deaths worldwide[1]. HCC is associated with chronic inflammation and fibrosis arising from different etiologies,including hepatitis B and C and alcoholic and non-alcoholic fatty liver diseases[2]. The stromal component of tumors consists of fibroblasts, endothelial cells, and various immune cells. Together, these cells play a critical role in tumor development and response to treatment.

Many different methods have demonstrated the prognostic effect of tumor infiltrating lymphocytes (TILs) in HCC[3]. For instance, the densities of tumor-infiltrating T cells and B cells are correlated with superior survival in HCC patients[4], and patients with high-grade HCC of the predominant immune-high subtype had significantly better prognosis[5]. Different methods of assessing TILs have various pre-analytical,analytical, and post-analytical challenges. For example, semi-quantitative hematoxylin and eosin (H&E)-based scores suffer from low precision and poor interobserver reproducibility due to lack of guidance, while digital quantification of immunohistochemical (IHC)-stained sections may have varied results due to inaccurate measurement of the test variable without controlled calibration.

牛结核病的防治采用的是综合性措施，及时对疫情进行有效处理，加强预防控制，净化周围环境以减少该病的传染。

Univariate and multivariate survival analyses were performed using Cox regression model. A non-pairedtest was conducted to compare the clinicopathological parameters of the immune subtypes. All statistical analyses were performed using GraphPad Prism 7 software.< 0.05 was considered statistically significant and allvalues were two-sided. The statistical methods of this study were reviewed by Xin-xin Xu from Huadong Hospital.

Accumulating evidence suggests that lymphocytic infiltration in tumor tissues can be assessed as a significant parameter by evaluating H&E-stained tumor sections[9],which achieved good consistency and reproducibility in pathologists, including pathology resident trainees[10]. The criteria have been assessed in many different solid tumors, including lung, colon, upper gastrointestinal tract, head and neck, genitourinary tract, gynecological organs, mesothelioma, melanoma, and primary brain tumors[11]. However, evaluating of infiltrating lymphocytes in H&E slides of HCC has rarely been studied.

The present study aimed to assess the prognostic effect and the clinicopathological correlation of TILs evaluated in H&E sections of HCC patients.

MATERIALS AND METHODS

Patients and samples

HCC samples that met the following criteria were enrolled in the present study: (1)Patients who underwent liver resection for the first time from January 2015 to December 2017 in the Department of Liver Surgery, Zhong Shan Hospital, Fudan University, China; (2) Liver resection samples diagnosed as HCC by a pathologist; and(3) Complete clinicopathological data and disease-progression information. Patients who received therapy in addition to antiviruses were excluded,transarterial chemoembolization, ablation, bland embolization, radioembolization, chemotherapy,and immunotherapy.

将采回的新鲜小花清风藤茎藤按照生长年限(1年生、多年生)进行分类，“多年生”是指已生长2～4 a。用枝剪将茎藤剪成长20 cm左右的插穗。插穗要求具有3个以上的节，植物学下端剪成斜面状，上端剪平，保留顶端1个节的叶片。

三是强化现场执行的工作制度建设。现场执行是裁执分离的最后一道环节，同时也是风险最高的环节。现场执行时参与单位与人员众多，除了行政系统相关单位工作人员，还有当事人、见证人，相关公证、施工企业、代履行单位及人员，可能还涉及到供电、供水、供气等公用事业单位人员，必要时还应邀请法院派员现场监督。这些单位与人员在现场的各自履职、相互协作配合，离不开完善的工作机制安排。此外，在现场物品、设施设备、建筑物、构筑物处置，突发性事件管控，证据的固定等方面，均需要明确的工作流程与制度予以规范和保障，防范法律风险，保障人身与财产安全。

The study was approved by the Human Ethics Institutional Review Board of Huadong Hospital, Fudan University (approval number 2019K119), and informed consent was waived by the Review Board because of the retrospective nature of the study.

H&E staining of tumor tissue

HCC patients with high infiltrating lymphocytes tend to have a lower recurrence rate and less MVI. The evaluation of TILs in H&E-stained specimens could be a prognostic parameter for HCC.

According to the architectural growth patterns[12], distinctive and easily recognizable histological features were defined with a predominant (> 50%)architectural pattern. HCC was divided into microtrabecular/pseudoglandular,macrotrabecular, compact, and lymphoepithelioma-like subtypes[13]. The macrotrabecular subtype is classified as a predominant trabecular architectural pattern which is more than six cells thick[14].

Density of infiltrating lymphocytes

Two general pathologists and one senior pathologist were involved in this study. The density of ILs was determined based on the recommendation by the International Immuno-Oncology Biomarker Working Group[15]: (1) The number of ILs on full sections was scanned at low magnification and evaluated at higher magnification (400×) manually under an optical microscope; (2) ILs were assessed in the areas of the tumor center (TILs), the invasive front (TILs) and on the portal areas of the peritumour 1 cm away from the border (PILs). The “invasive front” (IF) is defined as the region centered on the border separating the host tissue from the malignant nests by 1 mm. Areas with crush artifacts, necrosis, and previous biopsy sites were excluded; and (3) All mononuclear cells, including lymphocytes and plasma cells,were counted (polymorphonuclear leukocytes were excluded from the count of ILs,and neutrophils were recorded separately from the count of ILs).

Immunohistochemistry staining

Programmed cell death-ligand 1 (PD-L1) (SP142) rabbit monoclonal primary antibody(Ventana Medical Systems Inc, Tucson, AZ, United States) was optimized for a fully automated IHC assay on the BenchMark ULTRA (Ventana Medical Systems Inc)staining platform using the OptiView DAB IHC Detection Kit and OptiView Amplification Kit (Ventana Medical Systems Inc)[16]. All the tissues were subjected to PD-L1 (SP142) IHC staining.

The expression of PD-L1 on tumor cells (TCs) was assessed as the proportion of TCs showing membrane staining of any intensity. The expression on TILs was assessed as the proportion of stromal areas occupied by PD-L1-positive TILs of any intensity(approved by the US Food and Drug Administration).

Follow-up

Patients were followed up by ultrasound, computed tomography (CT), or magnetic resonance imaging every 3-6 mo after the resection, with a maximum period of 1063 d.The primary study endpoint was progression-free survival (PFS), which refers to the duration of patient survival without any evidence of the tumor.

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Statistical analyses

Furthermore, the immunoscore proposed by Jerome Galon showed great prognostic power and outperformed the tumor node metastasis classification for disease-free survival, disease-specific survival and overall survival[6,7]. However, the immunoscore requires rigorous pathology and experimental practice for the staining, and deviation from the predefined standardized operating procedure might result in improper quantification[8].

RESULTS

Clinical and pathological factors

Nevertheless, the present study had some limitations. This was a retrospective,single-center study with a small number of patients. Additionally, this method is more challenging to implement in daily practice and has lower inter-observer reproducibility than stromal TILs. The method should be improved upon with further study undertaken and as evidence becomes available. The study lacked immune cell characterization. Understanding the types and function of immune cells as well as different cytokines will provide more insight into tumor immunology and immunotherapy.

Areas with microtrabecular/pseudo-glandular, macrotrabecular, compact, and lymphoepithelioma-like histological architectural patterns were identified in 42.64%,52.94%, 2.45%, and 1.96% of the tumors, respectively (Table 1).

传统的二胡演奏重视独奏能力而忽视合奏能力，导致演奏者在合奏时，只重视个人发挥，缺乏合奏意识。因此在新形势下开展二胡的多元合奏训练，提高团队合作意识，对二胡合奏的演出效果来说是非常必要的。

A total of 42/204 (20.6%) patients experienced tumor recurrence. The univariate analysis indicated that MaVI (= 0.001), MVI (= 0.012), multiple tumors (= 0.008),large tumors (> 10 cm) (= 0.001), absence of a tumor capsule (= 0.026), and the macrotrabecular histological subtype (= 0.001) were independent predictors of PFS (Supplementary Figure 1 and Table 2). MaVI (= 0.009) and absence of a capsule (=0.031) were multivariate analysis predictors of PFS (Table 2).

Immune microenvironment was heterogeneous

In the current study cohort, the number of TILs, TILs, and PILs was 10-1200/high power field (HPF). The ILs showed a great diversity among TILs, TILs, and PILs.Compared to the adjacent non-tumor liver tissues, the tumor microenvironment was found to be relatively inert due to a lower number of TILs(= 0.001). A significantly higher proportion of TILswas observed compared to TILsand PILs (< 0.0001)(Figure 1).

Immunehigh patients had better PFS and a lower rate of MVI

Immune cell densities in the tumor center, invasive front, and peritumor regions were converted into percentiles: 0%-25% was scored as low, and 25%-100% was scored as high. Patients with high TILs, TILs, and PILs had better PFS than those with low TILs, TILs, and PILs (Figure 1). Multivariate analysis, including those variables that appeared statistically significant in the univariable analysis, showed that low TILs(= 0.0495) and PILs (= 0.047) were independent risk factors for PFS in patients with HCC.

After integrating TILs, TILs, and PILs, we divided HCCs into three-category analysis: (1) Immunesubtype [(TILs), (TILs), and PILs, 83 cases]; (2)Immunesubtype (tumours other than Immuneand Immunesubtypes, 94 cases);(3) Immunesubtype [(TILs), (TILs), and PILs, 27 cases]. The H&E images of the three immune subtypes are illustrated in Figure 2.

A higher number of the immunesubtype (46.1%) HCCs was noted compared to the immunesubtype (40.7%), while 13.2% of the HCCs were immunesubtype.Recurrent disease was identified in 10.8% of the immunepatients compared to the 25.5% of the immunepatients and 33.3% of the immunepatients (= 0.0153). Theimmunesubtype had a lower rate of MVI (40.96%) than the immune(61.70%;=0.017) and immune(66.67%;= 0.020) subtypes. A large number of patients had neutrophils in the microenvironment of the immunand immunesubtypes compared with the immunesubtype (Figure 3).

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Regarding other parameters, including MaVI, multiple tumors, tumor diameter,capsule, differentiation, histological subtype, and lymphoid follicle, PD-L1 (SP142)expression did not exhibit a significant difference between the three groups (Table 3).

Patients with neutrophils or tertiary lymphoid structures among the TILs had a low recurrence rate

Neutrophils and tertiary lymphoid structures (TLSs) were distinguished in the tumor microenvironment on H&E-stained slides. Therefore, we recorded the presence and density of these inflammatory cells. Patients with neutrophils among the TILs exhibited a tendency for decreased recurrence, albeit without a significant difference.The patients with TLSs in the microenvironment did not show any recurrence after a follow-up of 37-791 d.

High PD-L1 (SP142) expression on TILs was associated with better PFS

PD-L1 (SP142) was expressed on TCs in 80 patients and TILs in 200 patients. Patients with a higher expression of PD-L1 (SP142) on TILs (> 5%) had a lower recurrence rate than those with lower expression (Figure 4). The greater the number of TILs, the higher the level of PD-L1 (SP142) expression on the TILs. However, the expression of PD-L1 (SP142) on TCs was not associated with PFS or TILs in our cohort. Additionally,we observed the expression of PD-L1 (SP142) on neutrophils; however, the proportion of neutrophils in TILs was not significantly associated with the expression of PD-L1(SP142).

We performed the IHC assay of (SP142), (28-8), and (E1L3N) in the other cohort of HCC patients; (SP142) is a more robust PD-L1 staining reagent than (28-8) and(E1L3N) in both tumors and immune cells of HCC, while (28-8) and (E1L3N) have similar staining effect in tumor cells. Therefore, we chose (SP142) as the major reagent analyzed in this study (Supplementary Figure 2).

DISCUSSION

This study revealed that the density of infiltrating lymphocytes in H&E-stained tissues can predict the recurrence of HCC. The International Immuno-Oncology Biomarker working Group proposed that TILs should be reported separately for the stromal compartment (= % stromal TILs) and the tumor cell compartment (= % intra-tumoral TILs). The stroma of classical HCC is composed of sinusoid-like blood spaces lined by a single layer of endothelial cells, which sometimes show varying degrees of dilatation or may be difficult to recognize owing to compression by tumor cells[17]. Most classical HCCs do not induce a desmoplastic stroma, therefore the method of stromal TILs is not suitable for HCC assessment. The method of intra-tumoral TILs with tumor cell area for the denominator is hard to accomplish manually, as visual estimation is subjective and TILs are manifested as infiltrating nests in tumor area in our study;meanwhile in daily practice most pathologists will report discrete estimates, forexample 13.5% will be rounded to 15%, which will result in underestimation of the difference. Therefore, we tried to distinguish the immune subtypes of HCC by recording the densities of infiltrating lymphocytes in the tumor center, invasive front and peritumor. However, this method is admittedly challenging, and inter-observer reproducibility requires particular attention. The method showed a prognostic effect for HCC recurrence and might be helpful to select patients with the highest likelihood of responding to immunotherapeutic agents.

HCC is characterized by immune tolerance and comprises numerous infiltrated immune cells, a large number of suppressive molecules, complex proinflammatory/immunoregulatory signaling and intricate interactions between different components. The immune microenvironment in HCC plays a key role in HCC progression and recurrence[18]. The immune system plays a dual role in cancer: It can not only suppress tumor growth by destroying cancer cells or inhibiting their outgrowth but also promote tumor progression either by selecting tumor cells that are more fit to survive in an immunocompetent host or by establishing conditions within the tumor microenvironment that facilitate tumor outgrowth[19]. Regulatory T cells and myeloid-derived suppressor cells are two major types of immunosuppressive leukocyte populations that play key roles in inhibiting host-protective antitumor responses. Tumor infiltration by IFN-γ-producing Th1 CD4+ T cells and CD8+ T cells and the presence of cytokines such as IFN-γ and TNF-α that promote tumor control have been associated with an improved prognosis for patients with many different cancers[20]. Therefore, tumor-promoting inflammation and protective tumor immunity are dynamically interconnected. Many different approaches are used to assess the immune infiltrate in tumors with highly variable requirements, costs and complexity[21-23]. TILs assessment of H&E sections has shown clinical validity as a prognostic marker in invasive breast carcinoma and is reproducible, affordable and widely available[24].

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Neutrophils and TLSs were associated with lower recurrence in the present study.The bulk of the clinical evidence assessing neutrophil to lymphocyte ratios (NLRs)mostly supports the notion that neutrophils promote, rather than inhibit, cancer progression[25]. In comparison with NLR, the prognostic and predictive power of intratumoral neutrophils is murkier and more variable, and positive (gastric cancer),negative (renal cancer and melanoma) or no (lung cancer) correlation with patient outcome has been observed in different studies. However, experimental studies have highlighted multifaceted and sometimes opposing roles of neutrophils in cancer[26].Analysis of the current literature shows that the presence of TLSs is associated with a favorable clinical outcome for cancer patients, regardless of the approach used to quantify TLSs and the stage of the disease[27]. Researchers have indicated that TLSs represent a privileged area for the recruitment of lymphocytes into tumors and the generation of central memory T and B cells that circulate and limit cancer progression[28].

Different immunotherapeutic modalities have been used to treat HCC, including diverse vaccine platforms, adoptive T-cell therapy, cytokines, gene therapy and monoclonal antibodies that target immune checkpoint molecules[29]. The importance of lymphocytes has been highlighted in many studies, wherein increasing infiltration of tumors with lymphocytes has been associated with enhanced response to cytotoxic treatment and prognosis in cancer patients[30]. HCC immunogenicity is indicated by the presence of tumor-infiltrating lymphocytes and an evident reduction in relapse rates after resection and transplantation in patients with dense lymphocytic infiltration.

A total of 204 patients were included in the present study, 91.67% of the patients were hepatitis B virus infected. Macrovascular invasion (MaVI) was presented in 21(10.29%) tumors, while microvascular invasion (MVI) was observed in 110 (53.92%)tumors. A total of 156 patients had a single tumor and 117 tumors were capsulated.Cirrhosis was observed in 171 (83.82%) tumors (Table 1).

CONCLUSION

HCC patients with high infiltrating lymphocytes tend to have a lower recurrence rate and less microvascular invasion. The evaluation of TILs in H&E-stained specimens could be a prognostic parameter for HCC.

Based on this research, low density of TILs(= 0.039), TILs(= 0.014), and PILs (= 0.010) were independent predictors of progression-free survival (PFS). The immunesubtype [(TILs), (TILs), and PILs, 83 cases] had a lower rate of microvascular invasion (MVI) (40.96%) than the immune(tumors other than immuneand immunesubtypes, 94 cases) (61.70%,= 0.017) and immune[(TILs), (TILs), and PILs, 27 cases] (66.67%,= 0.020) subtypes. The recurrence rates of the immune, immuneand immunesubtypes were 10.8%,25.5% and 33.3%, respectively.

This study proposed that the density of TILs in HCC tissues can predict the recurrence of the patient. The method of evaluating TILs in H&E-stained specimens may also be meaningful in HCC.

根据三个典型年的汛期逐日最高水位Z′m，计算频率并绘制频率曲线，取频率p为10%、50%、90%所对应的水位为高、中、低水位，分别为51.40 m、51.10 m、50.80 m。由此得出王家会站的高水期为大于等于51.40 m，中水期为 51.10～51.40 m，低水期为 50.80～51.10 m，枯水期为小于50.80 m。对照历年实测大断面比较图和平均河底高程变化图，据此分析的水位级代表性较差。

Increasing multicenter research to validate and improve this method should be implemented in the future.

The authors thanks all the colleagues for their help in this study. Min Du carried out the study, Yu-Meng Cai made genuine contributions to the data collection, Yu-Lei Yin and Li Xiao helped in data analysis and modification of manuscript, Yuan Ji endorsed the data and conclusions.