Tumor infiltrating lymphocytes in gastric cancer:Unraveling complex interactions for precision medicine

This editorial will focus on tumor immunity and the factors that alter the tumor immune micro-environment.The role of tumor infiltrating lymphocytes(TILs)will also be discussed in detail,including the types,mechanism of action,and role.Gastric cancer(GC)often presents in the advanced stage and has various factors predicting the outcomes.The interplay of these factors and their correlation with the TILs is discussed.A literature review revealed high intratumoral TILs associated with higher grade,HER2-,and Helicobacter pylori negativity.Moreover,stromal(ST)TILs correlated with lower grade and lesser recurrence risk in GC.High TILs in ST and invasive border also correlated with mismatch repair deficiency status.Further characterization of the CD3+,CD8+,and other cells is also warranted.In the future,this complex correlation of cancer cells with the immune system can be explored for therapeutic avenues.

The Tumor Infiltrating Lymphocytes (TILs): Did We Find the Missed Piece of the Huge Puzzle?

Tumor infiltrating lymphocytes (TILs) are used in evaluating the prognosis and determining treatment of different types of cancer with variable degrees of success. The usage of checkpoint inhibitor immunotherapy as a treatment variety for cancer and Adoptive cell therapy is associated with many complications, severe side effects and high expenses. Recently, in a limited number of metastatic GIT and breast cancers, the identification of T-cell specific against so-called tumor neo-antigens and Adoptive transfer of those lymphocytes resulted in some improvement. In 2020, Detection of a T cell receptor (TCR) in a T cell clone that recognized and killed most human cancer cell lines in vitro via the monomorphic MHC class I-related protein MR1, offers an opportunity for pan-cancer therapy Twenty three years earlier, Moist Heat was used successfully to activate a whole different and new immune response that was able to detect genetic mutation in the affected cancer cells and cured many cases of squamous and basal cell carcinomas. In this commentary review, we aimed to revise the literature for updates of TILs usage in cancer prognosis and treatment.

AR、SKP2、SOX10、PD-L1及TIL表达在三阴性乳腺癌中的意义

目的:探索雄激素受体(androgen receptor,AR)、S期激酶相关蛋白2(S-phase kinase-associated protein 2,SKP2)、性别决定区Y相关的HMG盒含因子10(sry-related HMG box-containing factor 10,SOX10)、程序性死亡配体1(programmed death-ligand 1,PD-L1)及肿瘤浸润性淋巴细胞(tumor infiltrating lymphocyte,TIL)在三阴性乳腺癌(triple negative breast cancer,TNBC)表达与临床病理特征和预后的关系。方法:根据苏木精-伊红染色(hematoxylineosin, HE)染色切片评判109例TNBC瘤巢内TIL的比例,采用Leica Bond-Max全自动免疫组化仪检测TNBC组织中AR、SKP2、SOX10、PD-L1的表达。分析以上各生物指标与临床病理特征间的关系,并采用kaplan-Meier、Log-rank进行生存分析。结果:95例患者获得随访,中位随访时间为48个月,中位无病生存时间(disease-free survival, DFS)为42个月,中位总生存时间(overall survival, OS)48个月。在TNBC中,AR阳性表达与淋巴结转移阴性(P=0.009)、肿瘤最大径<2 cm(P=0.008)相关,TIL高表达与低级别TNBC相关(P=0.007),SKP2阳性表达与神经/脉管侵犯阳性(P=0.011)、高级别TNBC相关(P=0.002),SOX10阳性表达与淋巴结转移阳性(P=0.022)、高级别TNBC(P=0.005)相关,PD-L1阳性表达与淋巴结转移阳性(P=0.020)、神经/脉管侵犯阳性(P=0.006)、高级别TNBC(P=0.042)相关。生存分析显示,SKP2、SOX10阳性表达与更差的DFS(P=0.007、P<0.001)和OS(P=0.013、P<0.001)相关,TIL高表达与更好的DFS(P=0.016)及OS(P=0.004)相关。在生物表志物的联合表达中,AR+/SKP2-、AR+/SOX10-与更好的DFS(P=0.004、P<0.001)及OS(P=0.007、P=0.001)相关,SOX10+/低TIL、PD-L1+/低TIL与更差的DFS(P<0.001、P=0.008)及OS(P=0.001、P=0.002)相关,AR-/低TIL者具有更差的OS(P=0.014)。SKP2(HR=4.143,95%CI为1.578~10.875)、SOX10(HR=7.578,95%CI为2.067~27.782)的阳性表达是影响TNBC患者DFS的独立预后因子,SKP2(HR=3.758,95%CI为1.400~10.084)、SOX10(HR=5.131,95%CI为1.316~20.000)及TIL(HR=0.375,95%CI为0.154~0.917)的阳性表达是TNBC患者OS的独立预后因子(P均<0.05)。结论:在TNBC中,AR阳性、TIL高表达与具有更好预后的临床病理特征相关,SKP2、SOX10和PD-L1与具侵袭性的临床病理特征相关。SKP2、SOX10及TIL表达与TNBC预后相关,提示这些生物指标可能成为TNBC新的预后因子,同时它们也有可能成为潜在的治疗靶点。

PD-1/PD-L1联合CD8+TILs在宫颈癌组织中的表达及临床相关性分析

目的探讨宫颈癌组织中CD8+TILs和PD-1/PD-L1分子表达情况及临床相关性。方法选取蚌埠医学院第附属一医院2016年1月—2020年1月收治的152例宫颈癌患者作为本次研究对象。收集患者基本资料,应用免疫组化染色法对其PD-1/PD-L1阳性、CD8+TILs密度进行检测,按照PD-1/PD-L1表达和CD8+TILs密度情况将152例患者分为A组(n=40)、B组(n=59)、C组(n=43)、D组(n=10)。对PD-1/PD-L1、PD-L1/CD8+TILs密度与宫颈癌临床病理特征关系、CD8+TILs与PD-1/PD-L1的相关性分析情况予以分析,分析不同PD-1/PD-L1表达和CD8+TILs密度宫颈癌患者的生存情况。结果PD-L1和PD-1和阳性表达率65.13%、61.18%;PD-L1阳性表达和肿瘤大小、间质浸润深度、FIGO分期、淋巴结转移、宫旁浸润有密切联系(P<0.05);PD-1阳性表达与宫颈癌临床病理特征无紧密关联(P>0.05);CD8+TILs高密度和低密度分别为82例、70例,Spearman检验分析结果显示,PD-1和PD-L1表达分别与CD8+TILs密度呈正相关(r=0.742,P<0.001)和负相关(r=-0.385,P<0.001);四组在肿瘤大小、FIGO分期、淋巴血管间隙侵犯方面比较差异显著(P<0.05);FIGO分期、分化程度、淋巴血管间隙侵犯、淋巴结转移是影响宫颈鳞癌患者OS的独立因素;PD-L1阳性组的生存时间短于PD-L1阴性组(经Log-Rank检验,χ^(2)=20.986,P=0.000<0.05),CD8+TILs低密度组生存时间短于CD8+TILs高密度组(经Log-Rank检验,χ^(2)=40.788,P=0.000<0.05),D-L1-/CD8+TILs高密度组生存时间均高于其他三组(经Log-Rank检验,χ^(2)=47.145,P=0.000<0.05)。结论PD-L1在宫颈癌中呈高表达,且PD-1和PD-L1表达分别与CD8+TILs密度呈正相关和负相关。PD-1/PD-L1联合CD8+TILs在宫颈癌的预后评估中具有较高的临床应用价值。

Association of tumor budding with clinicopathological features and prognostic value in stage III-IV colorectal cancer

BACKGROUND Tumor budding(TB)has emerged as a promising independent prognostic biomarker in colorectal cancer(CRC).The prognostic role of TB has been extensively studied and currently affects clinical decision making in patients with stage I and II CRC.However,existing prognostic studies on TB in stage III CRC have been confined to small retrospective cohort studies.Consequently,this study investigated the correlation among TB categories,clinicopathological features,and prognosis in stage III-IV CRC to further enhance the precision and individualization of treatment through refined prognostic risk stratification.AIM To analyze the relationship between TB categories and clinicopathological characteristics and assess their prognostic value in stage III-IV CRC to further refine the prognostic risk stratification of stage III-IV CRC.METHODS The clinical data of 547 CRC patients were collected for this retrospective study.Infiltration at the front edge of the tumor buds was counted according to the 2016 International Tumor Budding Consensus Conference guidelines.RESULTS Multivariate Cox proportional hazards regression analysis demonstrated that chemotherapy(P=0.004),clinical stage IV(P<0.001),≥4 regional lymph node metastases(P=0.004),left-sided colonic cancer(P=0.040),and Bd 2-3(P=0.002)were independent prognostic factors in patients with stage III-IV CRC.Moreover,the density of tumor infiltrating lymphocytes was higher in Bd 1 than in Bd 2-3,both in the tumor stroma and its invasive margin.CONCLUSION TB has an independent predictive prognostic value in patients with stage III-IV CRC.It is recommended to complete the TB report of stage III-IV CRC cases in the standardized pathological report to further refine risk stratification.

Ex vivo expansion of tumor-infiltrating lymphocytes from nasopharyngeal carcinoma patients for adoptive immunotherapy

Establishing Epstein-Barr virus (EBV)-specific cytolytic T lymphocytes (EBV-CTLs) from peripheral blood mononuclear cells (PBMCs) for adoptive immunotherapy has been reported in EBV-associated malignancies including Hodgkin's lymphoma and nasopharyngeal carcinoma (NPC). In the current study,we performed ex vivo expansion of tumor-infiltrating lymphocytes (TILs) obtained from NPC biopsy specimens with a rapid expansion protocol using anti-CD3 monoclonal antibody (OKT3), recombinant human interleukin (IL)-2, and irradiated PBMCs from healthy donors to initiate the growth of TILs. Young TIL cultures comprised of more than 90% of CD3+T cells, a variable percentage of CD3+CD8+and CD3+CD4+T cells, and less than 10% of CD3-CD16+natural killer cells, a similar phenotype of EBV-CTL cultures from PBMCs. Interestingly, TIL cultures secreted high levels of the Th1 cytokines, interferon gamma (IFNγ) and tumor necrosis factor-alpha (TNF-α), and low levels of the Th2 cytokines, IL-4 and IL-10. Moreover, young TILs could recognize autologous EBV-transformed B lymphoblast cell lines, but not autologous EBV-negative blast cells or allogeneic EBV-negative tumor cells. Taken together, these data suggest that ex vivo expansion of TILs from NPC biopsy tissue is an appealing alternative method to establish T cell-based immunotherapy for NPC.

宫颈癌淋巴结转移患者PD-L1与TILs表达水平及其与预后的关系

目的探讨宫颈癌淋巴结转移患者程序性死亡配体-1(PD-L1)、肿瘤浸润淋巴细胞亚群(TILs)表达水平及其与患者预后的关系。方法选取2007年1月至2013年9月在梅州市人民医院诊治并接受宫颈癌根治性切除术后发生淋巴结转移的101例患者作为研究对象。采用免疫组织化学法检测PD-L1、CD4^(+)TILs、CD8^(+)TILs、叉头翼螺旋转录因子3(Foxp3)^(+)TILs表达水平。比较不同PD-L1表达水平患者临床病理特征差异;采用Spearman相关对PD-L1表达水平与TILs密度的相关性进行分析;采用Kaplan-Meier生存分析法分析PD-L1表达水平、TILs密度及密度比值与累计生存率的关系;采用Cox比例风险模型分析患者预后的独立影响因素。结果PD-L1表达于肿瘤细胞的细胞质和(或)细胞膜,阳性表达率为34.65%(35/101);CD4^(+)TILs计数为94(45,190);CD8^(+)TILs计数为63(31,117);FoxP3^(+)TILs计数为6.0(2.5,13.5);FoxP3^(+)TILs/CD8^(+)TILs比值为0.109(0.036,0.193)。PD-L1阳性表达组与PD-L1阴性表达组CD4^(+)TILs、CD8^(+)TILs、FoxP3^(+)TILs密度比较,差异均有统计学意义(P<0.05)。PD-L1表达情况与CD4^(+)TILs、CD8^(+)TILs、FoxP3^(+)TILs密度均呈正相关(r=0.305、0.222、0.222,P=0.002、0.026、0.026)。生存组与死亡组CD4^(+)TILs密度、CD8^(+)TILs密度和PD-L1表达情况比较,差异均有统计学意义(P<0.05)。手术年龄<50岁、PD-L1阳性表达、CD4^(+)TILs高密度、CD8^(+)TILs高密度、FoxP3^(+)TILs/CD8^(+)TILs低比值患者累计生存率均高于手术年龄≥50岁、PD-L1阴性表达、CD4^(+)TILs低密度、CD8^(+)TILs低密度、FoxP3^(+)TILs/CD8^(+)TILs高比值患者,差异均有统计学意义(P<0.05)。多因素Cox回归分析结果显示,PD-L1阳性表达、CD8^(+)TILs高密度是患者预后的独立保护因素(P<0.05)。结论PD-L1、CD4^(+)TILs、CD8^(+)TILs、FoxP3^(+)TILs/CD8^(+)TILs比值与宫颈癌淋巴结转移患者预后均有关,其中PD-L1阳性表达、CD8^(+)TILs高密度是宫颈癌淋巴结转移患者预后的独立保护因素。

肿瘤浸润淋巴细胞(TIL)制剂制备质量管理专家共识

近年来,采用肿瘤浸润淋巴细胞(TIL)进行过继性T细胞疗法(ACT)的新型免疫治疗方式备受关注。TIL是从肿瘤组织中分离出的一种新型的抗肿瘤效应淋巴细胞,具有高效、特异、不良反应小等优点,临床应用潜在治疗价值巨大。国内外已有多家企业布局TIL疗法:2024年,美国FDA加速批准了LN-144,用于治疗PD-1耐药的晚期黑色素瘤患者;国内暂无TIL疗法的上市产品。鉴于TIL细胞的来源、类型、生产工艺等方面异质性较大,以及肿瘤组织的获取与运输要求、纯化TIL细胞获取方法、产品放行检测项目等有关TIL制剂生产安全性和质量方面的国家标准和行业标准均无统一规范,因此,制订针对性的TIL制剂制备标准对于全面保证TIL细胞制剂质量的安全性、有效性和可控性具有积极意义。为加强TIL制剂的质量控制,规范和指导TIL细胞制剂制备的质量管理,我院集合领域内众专家学者,参考国家免疫细胞治疗产品有关文件,结合TIL细胞特点,针对TIL制剂关键工艺步骤对质量控制要素和质量控制技术方法进行规定,特别编撰了《肿瘤浸润淋巴细胞(TIL)制剂制备质量管理专家共识》,以期为国家制定TIL制剂制备管理相关政策提供借鉴和参考。该共识明确了编制背景、TIL制剂生产术语、生产条件、质量控制要求、检测方法,重点阐述了肿瘤组织的采集和运输、TIL细胞的分离和纯化、TIL细胞的激活与扩增、TIL细胞制剂的放行等TIL制剂生产质量管理内容,适用于TIL制剂生产的质量控制。

快速微生物检测方法在肿瘤浸润淋巴细胞(TIL)产品中的应用

目的:采用呼吸信号法和定量聚合酶链反应(QPCR)技术对肿瘤浸润淋巴细胞(TIL)产品的无菌检测进行评估,旨在研究高效、可靠的实验方法,以快速完成免疫细胞产品微生物检测。方法:提取患者外周血或者肿瘤组织中免疫细胞,按照直接接种法、呼吸信号法以及QPCR法,对2020版《中华人民共和国药典》1101中规定的6种菌进行方法适用性验证。结果:结果显示,在<100 CFU·mL^(-1)条件下,TIL6种菌株在呼吸信号法适用性试验中,0.40~1.53 d检测出阳性信号,在QPCR方法检测适用性试验中,4 h内检出阳性信号。TIL中不存在干扰细菌和真菌检出的成分,两种方法专属性、耐用性良好。呼吸信号法检测限最优,QPCR法检测时间最短。结论:呼吸信号法的灵敏度和培养时间优于传统的检测方法,QPCR法将检测时间从传统无菌检测14 d缩短至4 h,两种方法都具有良好的特异性。因此在新鲜或者冻存等不同细胞使用条件下均可以作为快速检测方法的选择,在细胞治疗产品微生物检验中具有良好的应用前景。

Clinical significance of tumor-infiltrating lymphocytes for gastric cancer in the era of immunology

Immunotherapy has begun to revolutionize cancer treatment, by introducing therapies that target the host immune system instead of the tumor, therapies that possess unique adverse event profiles, and therapies that may cure certain types of cancer. The immune microenvironment of tumors is emerging as the most important means of understanding the relationship between a patient' immune system and their cancer, informing prognosis, and guiding immunotherapy, such as an antibody blockade of immune checkpoints. For some solid tumors, simple quantitation of lymphocyte infiltration would seem to have prognostic significance, suggesting that lymphocyte infiltration is not passive but may actively promote or inhibit tumor growth. For gastric cancers, several studies have provided strong evidence that immune cells contribute to determining prognosis. However, the exact role of immune cells in gastric cancer remains unclear. Therefore, this review focuses on the clinical significance of immune cells, especially tumor-infiltrating lymphocytes, in gastric cancer.

Tumor infiltrating lymphocytes in triple negative breast cancer receiving neoadjuvant chemotherapy

AIM To determine influence of neoadjuvant-chemotherapy(NAC) over tumor-infiltrating-lymphocytes(TIL) intriple-negative-breast-cancer(TNBC).METHODS TILs were evaluated in 98 TNBC cases who came to Instituto Nacional de Enfermedades Neoplasicas from 2005 to 2010. Immunohistochemistry staining for CD3, CD4, CD8 and FOXP3 was performed in tissue microarrays(TMA) sections. Evaluation of H/E in full-face and immunohistochemistry in TMA sections was performed in pre and post-NAC samples. STATA software was used and P value < 0.05 was considered statistically significant. RESULTS Higher TIL evaluated in full-face sections from pre-NAC tumors was associated to pathologic-complete-response(pCR)(P = 0.0251) and outcome(P = 0.0334). TIL evaluated in TMA sections showed low level of agreement with full-face sections(ICC = 0.017-0.20) and was not associated to pCR or outcome. TIL in post-NAC samples were not associated to response or outcome. PostNAC lesions with pC R had similar TIL levels than those without pCR(P = 0.6331). NAC produced a TIL decrease in full-face sections(P < 0.0001). Percentage of TIL subpopulations was correlated with their absolute counts. Higher counts of CD3, CD4, CD8 and FOXP3 in pre-NAC samples had longer disease-free-survival(DFS). Higher counts of CD3 in pre-NAC samples had longer overallsurvival. Higher ratio of CD8/CD4 counts in pre-NAC was associated with pCR. Higher ratio of CD4/FOXP3 counts in pre-NAC was associated with longer DFS. Higher counts of CD4 in post-NAC samples were associated with pCR.CONCLUSION TIL in pre-NAC full-face sections in TNBC are correlated to longer survival. TIL in full-face differ from TMA sections, absolute count and percentage analysis of TIL subpopulation closely related.

TREATMENT OF 23 PATIENTS WITH ADVANCED GASTRIC CANCER BY INTRAVENOUSLY TRANSFER OF AUTOLOGOUS TUMOR-INFILTRATING LYMPHOCYTES COMBINED WITH rIL-2

Tumor-infiltrating lymphocytes (TIL)isolated from metastatic lymph nodes in patients with nonoperable advanced gastric cancer were induced to become LAK-like cytotoxic activrty of TIL after in vitro culture with rlL-2.Twenty-three patients with advanced gastric cancer were treated by intravenously transfer of autologous TIL combined with rlL-2. The tumor forus disappeared (complete remission, CR) in 3 patients (13. 0%) and significantly decreased (partial remission, PR) in 5 patients (21. 7%). Fifteen patients did not respond to the treatment. The amount of soluable IL-2 receptor in serum was significantly decreased after treatment, the cytotoxicity of NK cells and OT test were significantly increased. No significant difference in CD4/CD8 was found between before and after treatment. No serious side effect was obseved in the treatment.

Association between Up-regulation of Fas Ligand Expression and Apoptosis of Tumor-infiltrating Lymphocytes in Human Breast Cancer

In order to study the significance of FasL expression in immune escape of breast cancer, FasL protein expression and the number of tumor-infiltrating lymphocytes （TILs） in 40 specimens of breast cancer were detected by immunohistochemitry. The expression of FasL mRNA was measured by in situ hybridization in the consecutive tissue slices of 40 breast cancers respectively. By using terminal deoxynucleotidyl transferase-mediaed dUTP nick end labeling （TUNEL）, apoptotic cells were detected in 40 specimens of breast cancer. The expression of FasL was detected in all 40 specimens to varying degrees. In the consecutive tissue slices, the location of expression of FasL protein corresponded with that of FasL mRNA. In those with FasL extensive expression, the number of TILs was less （P〈0.05）, the apoptotic index （AI） of TILs was higher and the AI of tumor cells was lower （P〈0.01） than those with FasL weak expression respectively. The AI of TILs was correlated with that of tumor cells （r=-0.629, P〈0.01）. In conclusion, breast cancer cells can induce the apoptosis of TILs through the expression of FasL, which can counterattack the immune system. This may be a mechanism of immune evasion in breast cancer.

PREPARATION AND CYTOLYTIC TUMOR ACTIVITY OF OSTEOSARCOMA TUMOR INFILTRATING LYMPHOCYTES IN VITRO

In this study, the isolation, purification and differentiation of tumor-lnflltratlng lymphocytes (TIL) from 6 fresh osteosarcoma specimens were achieved by discontinuous density gradient centrifugation. One specimen of the osteosarcoma TIL were enlarged in IL-2 for long time in vitro, reaching 28 days and their cytolytic activity against different tumor cell lines was Investigated. The experimental results indicated that the preparation of osteosarcoma TIL adopted by the mechanical means was simple, having higher purifity, keeping higher effects on killing NK- sensitive tumor cell lines and NK-insensitive tumor cell lines as well as rapid proliferation in vitro cultured in IL-2.

Immune signature of small bowel adenocarcinoma and the role of tumor microenvironment

In this editorial we comment on the article published“Clinical significance of programmed cell death-ligand expression in small bowel adenocarcinoma is determined by the tumor microenvironment”.Small bowel adenocarcinoma(SBA)is a rare gastrointestinal neoplasm and despite the small intestine's significant surface area,SBA accounts for less than 3%of such tumors.Early detection is challenging and the reason arises from its asymptomatic nature,often leading to late-stage discovery and poor prognosis.Treatment involves platinum-based chemotherapy with a 5-fluorouracil combination,but the lack of effective chemotherapy contributes to a generally poor prognosis.SBAs are linked to genetic disorders and risk factors,including chronic inflammatory conditions.The unique characteristics of the small bowel,such as rapid cell renewal and an active immune system,contributes to the rarity of these tumors as well as the high intratumoral infiltration of immune cells is associated with a favorable prognosis.Programmed cell death-ligand 1(PD-L1)expression varies across different cancers,with potential discrepancies in its prognostic value.Microsatellite instability(MSI)in SBA is associated with a high tumor mutational burden,affecting the prognosis and response to immunotherapy.The presence of PD-L1 and programmed cell death 1,along with tumor-infiltrating lymphocytes,plays a crucial role in the complex microenvironment of SBA and contributes to a more favorable prognosis,especially in the context of high MSI tumors.Stromal tumor-infiltrating lymphocytes are identified as independent prognostic indicators and the association between MSI status and a favorable prognosis,emphasizes the importance of evaluating the immune status of tumors for treatment decisions.

ISOLATION AND CULTURE OF TUMOR-INFILTRATING LYMPHOCYTES FROM MOUSE HEPATOMA

By uaing enzyme digestion and Flcoll- Hypaque or Percoll discontinuous density methods, we have successfully obtained tumor-infiltrating lymphocytes (TIL) from mouse hepatoma. When analyzing the purity of TIL after separation. It was found that Percoll was more effective than Flcoll (P<0. 01). TIL could be activated In the presence of recombinant lL-2 (rIL-2) and begin to expand after culturing for 5-7 days, the tumor cells tend to decrease and disappeared after 14 days or so. TIL increased 105-fold over 40 days. Conditioned medium containing supernatant of PHA and rIL- 2 stimulated syngeneic spleen cell culture could promote the expansion of TIL.

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

BACKGROUND Tumor-infiltrating lymphocytes(TILs)constitute a prognostic factor in hepatocellular carcinoma(HCC).However,different methods of assessing TILs have various pre-analytical,analytical,and post-analytical challenges.The evaluation of TILs in hematoxylin and eosin(H&E)-stained tumor sections proposed by the International Immuno-Oncology Biomarker Working Group was demonstrated to be a reproducible,affordable and easily applied method in many tumors.AIM To evaluate the prognostic significance of TILs in H&E-stained slides of HCCs.METHODS This was a retrospective study performed in the hospital.HCC patients who underwent liver resection between 2015 and 2017 in Zhongshan Hospital were enrolled in this study.Patients who experienced recurrence or received therapy in addition to antiviral therapy before surgery at this time were excluded.A total of 204 patients were enrolled in the study.The ILs were counted manually in tumor sections stained with H&E under an optical microscope at 400×.The ILs were assessed separately in the center of the tumor(TILs^(CT)),the invasive front(TILs^(IF)),and peritumor(PILs)areas.Univariate and multivariate survival analyses were performed using a Cox regression model.P<0.05 was considered statistically significant and all P-values were two-sided.RESULTS Among the 204 patients,univariate analysis indicated that macrovascular invasion(MaVI)(P=0.001),microvascular invasion(MVI)(P=0.012),multiple tumors(P=0.008),large tumors(>10 cm)(P=0.001),absence of a tumor capsule(P=0.026),macrotrabecular histological subtype(P=0.001),low density of TILs^(CT)(P=0.039),TILs^(IF)(P=0.014),and PILs(P=0.010)were predictors of progressionfree survival(PFS).Cox multivariate analysis indicated that MaVI(P=0.009),absence of a tumor capsule(P=0.031),low-density of TILs^(IF)(P=0.047)and PILs(P=0.0495)were independent predictors of PFS.A three-category analysis was carried out by combining TILs^(CT),TILs^(IF),and PILs,after which HCCs were classified into immune^(high)[(TILs^(CT))^(high),(TILs^(IF))^(high),and PILs^(high),83 cases],immune^(mod)(tumors other than immune^(high) and immune^(low) subtypes,94 cases),and immune^(low)[(TILs^(CT))^(low),(TILs^(IF))^(low),and PILs^(low),27 cases]subtypes.The immune^(high) subtype had a lower rate of MVI(40.96%)than the immune^(mod)(61.70%,P=0.017)and immune^(low)(66.67%,P=0.020)subtypes.The recurrence rates of the immune^(high),immune^(mod) and immune^(low) subtypes were 10.8%,25.5%and 33.3%,respectively.CONCLUSION 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.

IMMUNOLOGIC CHARACTER OF TUMOR INFILTRATING LYMPHOCYTES IN OVARIAN CARCINOMA

Objective: To study immunologic character of tumor-infiltrating lymphocytes (TIL) on postin vitro expansion in ovarian carcinoma, and evaluate the prospects by adopting TIL treatment of ovarian carcinoma at an advanced stage. Methods: Cellular phenotype changes in TIL were analyzed by flow cytometry. By means of molecular biology and immunologic methods, ability to secrete cytokines and anti-tumor activities of in TIL was studied. Results: Difference of cellular phenotypes in TIL was probably related to the type, feature and resource of the tumor. TIL obtained from phoroplast and parenchyma was dominant in CD3+CD4+. TIL obtained from tumor tissues, around microvessels and ascitic fluid was dominant in CD3+CD8 Concentration of rIL-2in vitro played a significant role in immunologic character of TIL. By means of rIL-2 expansionin vitro, TIL has apparently been improved in competence of secreting some cytokines, such as IL-2, TNF-α, IFN-γ, and anti-tumor activities. The activated TIL was more stimulated by further adding anti-CD3 or PHA (suitable concentration), which significantly increased its ability to secrete cytokines. Treatment with TIL+CTX or TIL+ rIL-2, could apparently improve phenotypes in peripheral blood of patients, with definitive effects. Conclusion: Immunologic activities of TILin vitro are apparently improved by rIL2 expansion. Regression of tumor, by means of infusion TIL, is not largely attributed to direct cytotoxicity to tumor cells, but indirectly and partly augmenting cellular activities and abilities of immunomodulation in patients with ovarian carcinoma being dependent on secreting multiple cytokines.

Separation and Expansion of Tumor Infiltrating Lymphocytes of Digestive System in Vitro and Their Cytotoxicity

Primary tumor tissues in the digestive system were harvested form 15 patients. By mincing,enzymatic digestion and gradient density separation, sufficient TILs(>5×106) were obtained from 13 of 15 (88.7%) patients in vitro in the presence of 500 μ/ml of recombinant interleukin-2 and 5% fetal calf serum after one month culture. 92.3% (12/13) of TILs proliferated well in vitro (92.3%). TILs expanded from 102-folds to 103-folds after being cultured for one month. CD25+ cell of the most fresh TILs was more than that of peripheral blood lymphocytes. CD25+ cells of TILs during 4th week of the culture was significantly greater (P<0.01) than that of fresh TILs. CD4+/CD8+ ratio was decreased during four culture weeks because of increase of CD8 cells. By using modified colorimetric MTT assay for measuring activity of TILs against various tumor cells the results showed that cytotoxicity of gastro-intestinal TILs autologous tumor cells is greater than on the other tumor cells.

恶性胸/腹水肿瘤浸润淋巴细胞(TIL)体外选择性CD8+T细胞扩增的培养体系建立及功能表型分析

目的探讨体外恶性胸/腹水中肿瘤浸润淋巴细胞(TIL)大量培养扩增方法并初步研究其分子表型及功能特征.方法无菌操作条件下抽取恶性肿瘤患者胸/腹水,密度梯度离心法分离淋巴细胞,采用γ干扰素(IFN-γ)、抗CD3多克隆抗体OKT3和白细胞介素2(IL-2)为基础的联合方案扩增TIL,并记录其细胞形态及扩增速度.采用流式细胞术动态检测扩增淋巴细胞的分子表型T淋巴细胞亚群中淋巴细胞激活信号分子F7(SLAMF7)、CD45RO、颗粒酶B(GZMB)、程序性死亡蛋白1(PD-1)、CD57阳性细胞比例,CCK-8法检测其对肿瘤的杀伤能力.结果在本培养方案中,TIL细胞培养至第26天状态良好,第30天开始增殖速率下降.CD4^(-)CD8^(+)T细胞及CD8^(+)CD56^(+)T细胞比例随培养时间的延长逐渐提高,CD4^(+)CD8^(-)T细胞的比例逐渐下降,CD4^(+)CD25^(+)T细胞始终保持低水平状态.与扩增前相比,SLAMF7、CD45RO、GZMB、PD-1阳性细胞比例均有显著提高,耗竭性T细胞标志CD57表达比例也逐渐升高.TIL源性扩增性T细胞杀伤肿瘤细胞能力明显强于外周血单个核细胞(PBMC)源性,当效靶比为10∶1时杀伤能力最强,且杀伤能力在肿瘤细胞类型间存在明显差异.结论建立了从癌性胸/腹水中大量扩增TIL的培养方案,方法简便易行、效率高、杀伤肿瘤能力强,其效应细胞主要为CD8^(+)T细胞,表型处于活化状态.