上皮生长因子受体与非小细胞肺癌肿瘤免疫及转移的相关关系

目的与方法 采用链霉菌抗生物素蛋白－过氧化物酶连结法（ＬＳＡＢ）免疫组化和图象定量分析技术检测５０例非小细胞型肺癌（其中伴有淋巴结转移者３２例,不伴转移者１８例）上皮生长因子受体（ＥＧＦＲ）的表达,结果：ＥＧＦＲ表达与肿瘤间质淋巴细胞浸润（ＴＩＬ）呈负相关（Ｐ〈０．０１）,ＥＧＦＲ高表达时,ＴＩＬ浸润少,肺癌的转移率高（８３％）ＥＧＦＲ的表达与肺癌的组织类型明显相关（＿Ｐ〈０．０５）,但与肺癌的分     

肿瘤浸润淋巴细胞生物学性状初步研究

本研究发现，掺杂肿瘤细胞的早期ＴＩＬ培养上清液抑制自身淋巴结来源的淋巴细胞增殖作用；后期时，掺杂肿瘤细胞消失（即ＴＩＬ呈对数生长期），ＴＩＬ培养上清液却对自身淋巴结来源的淋巴细胞呈现生长刺激现象。首次发现消化酶对ＴＩＬ这些活力指标有影响。表型与淋巴细胞形态学对照研究时发现，初次分离的ＴＩＬ层细胞中，Ｇｉｅｍ－ｓａ染色的淋巴细胞比例为８３±６％；活细胞镜下计数淋巴细胞占７３±１２％；而ＦＡＣＳ分析ＣＤ３只有２３±８％，ＨＬＡＤＲ３１±２０％；是否属ＴＩＬ前体细胞，还须进一步研究。     

乳腺癌组织蛋白酶D和PSA表达与同侧腋淋巴结转移的关系

目的：探讨乳腺癌中组织蛋白酶Ｄ（ｃａｔｈｅｐｓｉｎＤ，ＣＤ）和前列腺特异性抗原（ＰＳＡ）的表达与同侧腋淋巴结转移的关系。方法：应用ＬＳＡＢ免疫组化法检测ＣＤ和ＰＳＡ在７８例乳腺癌中的表达，其中伴同侧腋淋巴结转移者３６例。结果：（１）７８例乳腺癌ＣＤ阳性表达率４１．０３％（３２／７８），ＰＳＡ阳性表达率４３．５％（３４／７８）；（２）同侧腋淋巴结转移组ＣＤ阳性表达率６９．４４％（２５／３６），无同侧腋淋巴结转移组ＣＤ阳性表达率１６．６７％（７／４２），差异有高度显著性（Ｐ＜０．０１）；（３）同侧腋淋巴结转移组ＰＳＡ阳性表达率２５．０％（９／３６），无同侧腋淋巴结转移组ＰＳＡ阳性表达率５９．５２％（２５／４２），差异有高度显著性（Ｐ＜０．０１）。结论：ＣＤ和ＰＳＡ的表达可作为乳腺癌预后检测的参考指标。ＣＤ表达阳性率与同侧腋淋巴结转移呈正相关，与预后负相关；而ＰＳＡ表达阳性率与同侧腋淋巴结转移呈负相关，与预后正相关     

胃肠道平滑肌肿瘤DNA含量与临床病理的关系

应用流式细胞术对８４例胃肠道平滑肌肿瘤的细胞ＤＮＡ含量及其倍体特征进行定量分析。细胞分裂像为０、１、２～４、５～９和≥１０个／１０ＨＰＦ时，异倍体率分别为０，５０％，７５％，９２．８％和１００％（Ｐ＜０．００５）；细胞异形性为０，＋，＋＋和＋＋＋时，异倍体率分别为３．２％，６０％，９５．８％和１００％（Ｐ＜０．００５）。平滑肌瘤３０例无１例异倍体，平滑肌肉瘤４０例均为异倍体，而潜在恶性平滑肌肿瘤１４例中出现４例异倍体（２５．５７％，Ｐ＜０．０１）。肿瘤大径６ｃｍ以上、恶性生长方式、或肿瘤中心有坏死时，异倍体率分别为６４．３％，７９．５％，６８．３％。异倍体肿瘤病人的５年生存率明显低于二倍体肿瘤病人（Ｐ＜０．００５）。结果提示，细胞分裂像在１个／１０ＨＰＦ以上，出现轻度细胞异形性；肿瘤大径≥６ｃｍ、恶性生长方式、或肿瘤中心有坏死时，ＤＮＡ含量均有显著增高，异倍体肿瘤病人预后明显变差。     

一种高活力分离肿瘤浸润淋巴细胞方法的建立

肿瘤浸润淋巴细胞（ＴＩＬ）的增殖力、活力、杀伤力是ＴＩＬ临床治疗的基本问题。由于Ｒｏｓｅｎｂｅｒｇ方法存在着许多不足之处，本实验室对其一些重要步骤进行了改进：①用单一冷胶原酶消化肿瘤组织块；②省去影响ＴＩＬ活力的离心操作；③ＴＩＬ培养４８ｈ内除去掺杂在ＴＩＬ中的肿瘤细胞等。由于除去了影响ＴＩＬ活力的抑制因素，ＴＩＬ的增殖率、生存率、活力、杀伤力明显增加。有８０％肿瘤组织的ＴＩＬ经扩增可超过１×１０     

肺鳞癌肿瘤浸润淋巴细胞的体外扩增,抗瘤活性及表型分析

对８例人原发性肺鳞癌的肿瘤浸润淋巴细胞（ＴＩＬ）进行分离培养并检测了ＴＩＬ的抗瘤活性及细胞表型，结果表明８例ＴＩＬ的平均扩增倍数为４３０；ＴＩＬ在１０～３０天之间显示出高水平的非特异性杀伤活性；１０天～时对Ａ１及Ｄａｕｄｉ细胞的杀伤百分率分别为７９．２％和４３．４％，２０天～时分别为５１．７％和２７．９％。非特异杀伤活性在３０天以后降至较低水平，所进行的几次实验表明肺鳞癌ＴＩＬ对自体癌细胞的杀伤活     

胃癌中骨桥蛋白与OCT2的表达及意义

目的探讨骨桥蛋白（osteopontin，OPN）及转录因子OCT2在胃癌中的表达及其与临床病理参数及预后的关系。方法应用组织微阵列技术和免疫组化SP法检测99例胃癌组织中OPN蛋白与OCT2蛋白的表达情况。结果99例胃癌中OPN蛋白及OCT2蛋白的阳性表达率分别为60．61％、54．55％；OPN蛋白表达与肿瘤大小、浸润深度、脉管侵犯、淋巴结转移及远处转移呈正相关；OCT2蛋白表达也与肿瘤大小、浸润深度、脉管侵犯、淋巴结转移及远处转移呈正相关；OPN蛋白表达与OCT2蛋白表达呈正相关；OPN及OCT2蛋白阳性表达病例的平均生存时间和5年生存率均明显低于阴性表达的病例。结论OPN及OCT2蛋白表达可预测胃癌浸润和转移，是指导临床治疗及评估预后的有价指标。     

p53过表达与胃癌患者淋巴结转移的关系

应用Ｓ－Ｐ免疫组化方法，研究ｐ~（５３）在８８例原发性胃癌患者的表达变化与肿瘤细胞增殖和浸润深度及淋巴结转移的关系。发现ｐ~（５３）阳性染色率为４８．９％，ｐ~（５３）阳性染色与肿瘤浸润深度及癌细胞增殖活性呈正相关，ｐ~（５３）阳性肿瘤的淋巴结转移率（９３％）明显高于ｐ~（５３）阴性的肿瘤（６０％，Ｐ＜０．０５）。提示ｐ~（５３）过表达在胃癌淋巴结转移和肿瘤浸润及增殖中起重要作用。     

乳腺癌中p53基因丢失与p53 mRNA高表达相关性研究

为探讨乳腺癌组织中ｐ５３基因丢失与ｐ５３ ｍＲＮＡ表达的相关性，应用Ｓｏｕｔｈｅｒｎ杂交及反转录－定量ＰＣＲ技术对４７例乳腺癌组织进行了检测。结果发现：ｐ５３基因杂合性缺失率为３４．０％；在乳腺正常腺体中有中度ｐ５３ＲＮＡ表达，面 肿瘤组织中有４０．０％，患者伴ｐ５３ ｍＲＮＡ高表达。ｐ５３基因与ｐ５３ ｍＲＮＡ高表达之间呈显著性相关。     

转移抑制基因nm23/NDPK与肺癌生物学行为的相关性研究

目的：研究转移抑制基因ｎｍ２３ 表达产物二磷酸核苷激酶（ＮＤＰＫ）在人肺癌中的表达及意义。方法：应用免疫组化ＳＰ法，检测不同病理分级及淋巴结有无转移肺癌组织中ｎｍ２３／ＮＤＰＫ 的表达。结果：８８ 例肺癌ｎｍ２３／ＮＤＰＫ 阳性表达率为８１－８２％ （７２／８８），其中呈高表达率３６－３６ ％（３２／８８） 。高分化肺癌高表达率７１－４３％ （１０／１４），中分化肺癌高表达率４１－３ ％（１９／４６） ，低分化肺癌高表达率１０－７２ ％ （３／２８） 。各组间有显著差异（ Ｐ＜ ０－０５）。而与肺癌有无肺门淋巴结转移无关（ Ｐ＞ ０－０５）。结论：ｎｍ２３／ＮＤＰＫ 的表达与肺癌的分化程度呈负相关，可作为预测肿瘤转移潜能及预后的指标之一。     

Ultrastructure of IL2-stimulated Tumor-infiltrating Lymphocytes Showing Cytolytic Activity Against Tumor Cells

Tumor-infiltrating lymphocytes (TIL) obtained from tumor tissue and pleural effusion of breast carcinoma were cultured with interleukin-2 (IL2) and thus activated. The ultrastructure of TIL stimulated by IL2 to kill various breast carcinoma cells was then investigated. Freshly isolated TIL cultured with autologous tumor cells for 48 h without IL2 were small, round and showed neither binding to nor killing of tumor cells. TIL stimulated to proliferate by IL2 became effector cells and showed cytotoxicity against tumor cells. Ultrastructurally, the effector TIL resembled large granular lymphocytes, and adhered to tumor cells through interdigitation or close apposition of the two plasma membranes accompanied by spot-like close membrane contacts. At the site of each spot-like contact, there was a 5-nm intercellular space. The morphology of the TIL processes did not differ from those of LAK and other CTL or NK cell processes during contact, invagination or the killing of target cells. The granules in TIL were considered to participate in the cytotoxic effect. Phenotypically heterogeneous TIL, CD87CD57- and CD8⁺/CD57⁺, adhered to autologous tumor cells and MCF7 (human breast carcinoma cell line). However, it was unclear which cell or cells acted as the effector for tumor-cell killing. Acta Pathol Jpn 41: 94-105, 1991.     

恶性脑肿瘤浸润淋巴细胞的临床应用研究

用冷酶法对７例脑恶性肿瘤进行肿瘤浸润淋巴细胞分离、培养并用于临床。７例肿瘤的ＴＩＬ扩增倍数均大于刚分离时的５００倍，其中２例大于１０００倍，另２例分别大于２０００及３０００倍；ＴＩＬ的表型检测ＣＤ３、ＣＤ４、ＣＤ８、ＨＬＡ－ＤＲ分别为９７％，３５％，５４％，９７％；电镜观察到ＴＩＬ先靠近肿瘤细胞，伸出触手，接触肿瘤细胞膜上，随后肿瘤细胞破坏、死亡的过程。临床应用ＴＩＬ后随访至今９ｍｏ，７例肿瘤患者     

肿瘤相关巨噬细胞在晚期卵巢癌组织中的浸润与肿瘤浸润淋巴细胞表型、免疫效能之间的关系

目的:探讨晚期卵巢癌组织中浸润的肿瘤相关巨噬细胞(tumor-associated macrophage,TAM)与肿瘤浸润淋巴细胞(tumor-infiltrating lymphocyte,TIL)表型及免疫效能的关系。方法:免疫组化方法分析175例低分化卵巢癌组织病理切片中TAM分布密度,以中位数为界限将病例分为TAM高密度组和TAM低密度组,对照组为32例良性卵巢病变组织;应用流式细胞术分析TAM高密度组与TAM低密度组中TIL的CD8⁺和CD25⁺表型变化情况;体外扩增培养TIL后取细胞培养上清液,ELISA法分析各组TIL中白细胞介素2(IL-2)、白细胞介素-10(IL-10)、转化生长因子β(TGF-β)和干扰素γ(IFN-γ)细胞因子表达变化。结果:175例低分化卵巢癌组织中TAM平均浸润密度为62.8/高倍镜视野(HP,×400),中位数为53.3/HP,其中TAM高密度组87例,TAM低密度组88例;对照组TAM平均浸润密度10.5/HP(P<0.05)。CD8⁺在TAM高密度组中表达平均值为24%,在TAM低密度组中表达平均值为52%(P<0.05);CD25⁺在TAM高密度组中表达平均值为48%,在TAM低密度组中表达平均值为25%(P<0.05);对照组中CD8⁺和CD25⁺的TIL平均浸润密度为7%, TAM高密度组及TAM低密度组中CD8⁺和CD25⁺的TIL平均浸润密度显著高于对照组(P<0.05)。与TAM低密度组比较,TAM高密度组中TIL的杀伤性细胞因子IL-2和IFN-γ表达明显减少(P<0.05),而抑制性细胞因子IL-10和TGF-β表达明显增加(P<0.05)。结论:高密度TAM浸润的卵巢癌组织中,CD25⁺的TIL表型增多,CD8⁺的TIL表型减少,抑制性细胞因子IL-10和TGF-β表达增加,杀伤性细胞因子IL-2和IFN-γ表达减少,提示TAM浸润密度与TIL表型及免疫效能相关。     

The relationships between PD-L1 expression,CD8+ TILs and clinico-histomorphological parameters in malignant melanomas

IntroductionMalignant melanomas (MM) are often connected with the expression of PD-L1 protein and the presence of tumor-infiltrating lymphocytes (TILs), however, their impact on prognosis remains controversial. Due to their supposed clinical significance and lack of convincing data, we decided to establish the relationships between CD8 + TIL count, PD-L1 level and certain clinical and histopathological parameters in patients with malignant melanoma, especially those associated with unfavorable prognosis.Materials and methodsWe performed immunohistochemistry for PD-L1 and CD8 on 56 formalin-fixed paraffin-embedded specimens from patients with cutaneous and metastatic malignant melanomas. PD-L1 expression levels were determined by immunohistochemistry (clone 28-8) and subsequently the tumor proportion scores (TPS) were evaluated. CD8 + TIL expressions were classified as either grade 0, 1+, 2+ or 3+, based on the density and distribution of the infiltrating lymphocytes.ResultsThe PD-L1 expression was detected in 20 out of 56 cases (35,71 %). The expression of PD-L1 on tumor cells was significantly increased with higher TILs infiltration in the tumor microenvironment (p = 0,038). Lower TIL score corresponds with poor prognostic clinicopathological parameters such as higher number of mitotic figures (p = 0,005), Clark's level (p = 0,007) and Breslow's depth (p = 0,010).ConclusionsOur results suggest a favorable prognostic value for CD8 + TIL infiltration. Moreover, TIL density was strongly correlated and geographically associated to PD-L1 expression. This analysis provides more insight into the role of TIL count and PD-L1 level in MM and their relationship with each other and association with other prognostic indicators.     

Identification of unique murine tumor associated antigens by tumor infiltrating lymphocytes using tumor specific secretion of interferon-γ and tumor necrosis factor

Stimulation of multiple CD8⁺ murine tumor infiltrating lymphocyte (TIL) lines and one TIL clone with the tumor of origin of the TIL induced at least three-fold more secretion of TNF and/or INF-γ than was elicited by other syngeneic, methylcholanthrene (MCA) induced sarcomas. TIL which specifically secreted lymphokines were generated from three different sarcomas. Specific lymphokine secretion was a stable characteristic of the lines over time. IL-2 was necessary for maximal lymphokine secretion by TIL. These investigations demonstrate that lymphokine secretion by CD8⁺ lymphocytes derived from tumor bearing mice can be used to define unique tumor associated antigens on at least three different sarcomas and may be valuable in studies of the biologic nature of these antigens and of the adoptive immunotherapy of cancer.     

Separation of tumor-infiltrating lymphocytes from tumor cells in human solid tumors A comparison between velocity sedimentation and discontinuous density gradients

The separation of viable tumor-infiltrating lymphocytes (TIL) from surgical biopsies of human solid tumors was achieved by velocity sedimentation at unit gravity or by discontinuous density gradients. The two methods were adapted to small volumes and cell numbers not exceeding 1 × 10⁸. The recovery, purity and composition of the TIL-enriched fractions were comparable in the two methods. Density gradients were more rapid, simpler and more practical for preparation under sterile conditions of TIL from clinical material than velocity sedimentation. Lymphocytes in the TIL-enriched fractions obtained by either of the methods were poorly responsive to mitogens. This poor responsiveness is a characteristic of the human TIL and seems to be related to effects exerted by tumor cells.     

Exposure to anti‐PD‐1 causes functional differences in tumor‐infiltrating lymphocytes in rare solid tumors

The pervasive use of therapeutic antibodies targeting programmed cell death protein 1 (PD‐1) to boost anti‐tumor immunity has positioned this approach to become the standard‐of‐care for some solid tumor malignancies. However, little is known as to how blockade of PD‐1 may alter the function or phenotype of tumor‐infiltrating lymphocytes (TIL). We used our ongoing Phase II clinical trial of pembrolizumab for patients with rare solid tumors from various types (NCT02721732) with matched core biopsies taken at baseline and after initial dose of anti‐PD‐1 (15–21 days post‐dose) to elucidate this question. One fresh core needle biopsy was used to propagate TIL ex vivo to analyze phenotype and function using flow cytometry in both CD8⁺ and CD4⁺ TIL populations. An enriched CTLA‐4 expression in the CD4⁺ TIL population was observed in TIL expanded from the on‐treatment samples compared to TIL expanded from the matched baseline (n = 22, p = 0.0007) but was not observed in patients who experienced tumor regression. Impact on functionality was evaluated by measuring secretion of 65 soluble factors by expanded TIL from 26 patients at baseline and on‐treatment. The CD8⁺ TIL population demonstrated a diminished cytokine secretion profile post‐pembrolizumab. Overall, our study assesses the ramifications of one dose of anti‐PD‐1 on TIL in rare solid tumor types.     

恶性肿瘤胸腹水中TIL生物学活性研究

从卵巢癌腹水和肺癌胸水中分离出肿瘤浸润淋巴细胞（ＴＩＬ），用ｒＩＬ－２诱导培养，观察其增殖活性。同时观察了ＰＪ－ＣＷ、ＰＯＧＰ、ＰＨＡ等对其杀伤活性的调节作用。结果表明，在培养５～１０天时ＴＩＬ增殖较快，其表面产生的ＣＤ３、ＣＤ４、ＣＤ８、Ｔａｃ等抗原，随培养时间的延长，表达量增加，尤以ＣＤ８增加较明显，ＣＤ４／ＣＤ８比值逐降。其表面产生的ＨＬＡ－ＤＲ、ＨＬＡ－ＡＢＣ抗原，在培养１５天内有增加表现，以后下降。其杀伤活性在培养５～１０天较高，其后活性下降。在培养系统中加入ＰＪ－ＣＷ、ＰＯＧＰ、ＰＨＡ等后，培养５天时，ＴＩＬ杀伤活性分别为８２．７０％，８５．５０％，８４．３５％（对照组为内保持较高的杀伤活性。     

Characterization of immune responses in gastric cancer patients: a possible impact of H. pylori to polarize a tumor-specific type 1 response?

Recently, we were able to show that Helicobacter pylori-positive gastric cancer (GC) patients have a significantly better survival after the complete resection of their tumor compared to H. pylori-negative GC patients. H. pylori is known to polarize an immune response towards a type 1 cytokine profile and tumor-specific type 1 cytokine responses are associated with protection from tumor challenge and T-cell-mediated tumor regression. Therefore, we hypothesized that the improved survival in H. pylori-positive patients may be secondary to the induction of a GC-specific type 1 T cell response. To characterize the anti-tumor immune response in GC patients we analyzed tumor-infiltrating lymphocytes (TIL) isolated from primary tumors. The CD3+ T cell population contained 50% CD4+ (range 0.4-81%) and 39% CD8+ cells (range 22-53%). The number of B cells (CD19+, P = 0.03) was significantly increased and the number of T cells (CD3+, P = 0.02) significantly decreased in intestinal compared to diffuse type of tumors. Four tumor cell lines were established from primary GCs and three from lymph node metastases. T cell cultures were established from isolated TIL from four H. pylori-positive and one H. pylori-negative GC patients and tested for tumor-specific cytokine secretion. Eight of ten T cell cultures derived from H. pylori-positive patients secreted both IFN-gamma and IL-5 after restimulation with autologous tumor cells. The only tumor-specific TIL line expressing a dominant IL-5 response was derived from an H. pylori-negative patient.