Flow cytometric analysis of tumour infiltrating lymphocytes in breast cancer

In 31 patients with carcinoma of the breast the phenotype and activation status of tumour infiltrating lymphocytes (TILs) was analysed by flow cytometry. The predominant cells, in all patients, were T lymphocytes and in the majority of cases CD8+ (cytotoxic/suppressor) T lymphocytes were present in greater numbers than CD4+ (helper) T lymphocytes. There was no relationship between the degree of lymphocytic infiltration and either tumour stage or grade but there appeared to be an inverse correlation with the levels of oestrogen receptor (ER) in the tumour (P less than 0.01). Both populations of T cells had significantly higher numbers of cells carrying HLA DR (class II major histocompatibility antigen) than the equivalent populations in peripheral blood from the same patient group (P less than 0.001). The transferrin receptor was found on similar numbers of CD8+ T cells in peripheral blood and among the tumour infiltrating lymphocytes while more of the CD4+ T cells infiltrating the tumour were found to carry this receptor (P = 0.034). The Tac (CD 25) antigen was also on similar numbers of CD8+ T cells from both peripheral blood and the tumour but was on fewer of the CD4+ T cells in the tumour with respect to peripheral blood (P = 0.029). In both TILs and blood lymphocytes, the Tac antigen was consistently present on greater numbers of CD4+ T lymphocytes than on the CD8+ T lymphocytes (P less than 0.001) and as this is a component of the interleukin 2 (IL-2) receptor this may be of relevance to the use of IL-2 in TIL cancer therapy.     

Prognostic and predictive value of tumor infiltrating lymphocytes in early breast cancer

It is well recognized that the immune system plays an essential role in tumor defense. The presence of tumor-infiltrating lymphocytes reflects an individual immunological response. In early breast cancer, the presence of TILs is associated with a more favorable outcome and response to therapy. In this review, we describe how TILs are assessed. Also, we discuss their role as prognostic and predictive biomarker in the neoadjuvant and adjuvant settings as well as in residual disease. Moreover, we discuss the possible implementation of TILs in daily clinical practice as well as in future clinical trials in order to fine tune prognosis and improve treatments.     

Characterisation of tumour infiltrating lymphocytes and correlations with immunological surface molecules in colorectal cancer

Using flow cytometry, we studied the phenotype of tumour infiltrating lymphocytes (TILs) in 41 enzymatically dissociated colorectal cancers and compared this to the expression of HLA class I and II and CD80 on tumour cells. We studied the possible enzymatic damage to various surface markers after enzymatic dissociation. The reproducibility of flow cytometric determinations obtained from TILs was good (kappa value: 0.79). The median CD4⁺/CD8⁺ ratio was 2.2. Approximately 43–45% (median of cells in each tumour) of both the CD4⁺- and the CD8⁺-TILs expressed HLA class II; 14.2% of the CD4⁺-TILs expressed CD25 and none of the CD8⁺-TILs expressed CD25. CD3⁻/CD16⁺/CD56⁺-TILs were very infrequent. Expression of HLA class II did not correlate with any lymphocyte surface markers. Since TILs are “turned off” rather than stimulated when tumour cells express HLA class II but not CD80, the lack of correlations could be due to anergy.     

Tumor infiltrating lymphocytes in ovarian cancer

The accumulation of tumor infiltrating lymphocytes (TILs) in ovarian cancer is prognostic for increased survival while increases in immunosuppressive regulatory T-cells (Tregs) are associated with poor outcomes. Approaches that bolster tumor-reactive TILs may limit tumor progression. However, identifying tumor-reactive TILs in ovarian cancer has been challenging, though adoptive TIL therapy in patients has been encouraging. Other forms of TIL immunomodulation remain under investigation including Treg depletion, antibody-based checkpoint modification, activation and amplification using dendritic cells, antigen presenting cells or IL-2 cytokine culture, adjuvant cytokine injections, and gene-engineered T-cells. Many approaches to TIL manipulation inhibit ovarian cancer progression in preclinical or clinical studies as monotherapy. Here, we review the impact of TILs in ovarian cancer and attempts to mobilize TILs to halt tumor progression. We conclude that effective TIL therapy for ovarian cancer is at the brink of translation and optimal TIL activity may require combined methodologies to deliver clinically-relevant treatment.     

Oligoclonal T lymphocytes infiltrating human lung cancer tissues.

To clarify the nature of tumor-infiltrating lymphocytes (TILs), we investigated the possible clonality of the T cells in TILs freshly isolated from human primary lung cancer tissues by assessing the rearrangement pattern of the T-cell receptor (TCR) gene beta locus using Southern blotting. First, in phenotypic analysis, TILs represented different populations among corresponding peripheral blood lymphocytes (PBLs) with an increased proportion of CD20+ (B) cells as well as a decreased proportion of CD16+ (natural killer) cells, and a variable CD4/CD8 ratio. Considering the central role of T cells in immune responses, we analyzed TCR beta gene rearrangement patterns in TILs and corresponding PBLs from 12 patients. In 10 of the 12 cases, TILs showed one or more TCR gene rearrangement bands with a predominance of the C beta 2 gene, in which 2 types of common rearranged band were observed among the cases with different clinical profiles in terms of histological types and disease stage, with bands at about 9.5 kb in 7 and at 11.5 kb in 8 patients. On the other hand, predominant rearranged bands were hardly detected in corresponding PBLs except in 2 cases. From these results, we conclude that TILs in lung cancer tissues frequently contain oligoclonal T-cell populations, which were probably sensitized by relatively common antigens at the tumor sites.     

Flow cytometric analysis of tumour infiltrating lymphocyte activation and tumour cell MHC class I and II expression in breast cancer patients.

The primary tumour cells and tumour infiltrating lymphocytes (TILs) of 31 breast cancer patients have been analysed by dual colour flow cytometry to determine whether the phenotype and/or activation status of the TILs bears any relationship to the expression of MHC antigens on the tumour cells. The phenotype and activation status of 5000 TILs were studied using Mabs to CD4, CD8, HLA DR, CD25 (the low affinity inducible IL-2 receptor) and the transferrin receptor and related to Class I and Class II MHC expression on 5000 primary tumour cells. On the tumour cells, Class I MHC expression ranged from 1-74%, averaging 12.9%. HLA DR expression ranged from 1-69% averaging 14.3%. When the phenotypic proportions of the lymphocytic infiltrate were analysed there was found to be a correlation between tumour expression of Class I MHC and the proportion of both CD4+ (P less than 0.05) and CD8+ (P less than 0.02) T cells within the tumour. No such relationship was found with the MHC Class II antigen. When TIL activation markers were analysed, the percentage of CD8+ TILs positive for HLA DR expression correlated strongly with the expression of Class I (P less than 0.001) and Class II (P less than 0.001) antigens on the tumour cells. The percentage of CD4+ TILs positive for HLA DR expression also correlated significantly, but less strongly with the expression of Class I (P less than 0.01) and Class II (P less than 0.02) antigen expression on the tumour cells. The percentage of CD4+ TILs positive for CD25 expression correlated with both Class I (P less than 0.05) and Class II (P less than 0.03) expression on the tumour cells while the percentage of CD8+ TILs positive for CD25 did not. The percentage of TILs bearing the transferrin receptor showed no measurable correlation with the expression of either class of MHC antigen on the tumour. The data suggest that MHC expression on the tumour cells has a selective effect on the response capacity of different parts of the immune system.     

LAK1 antigen defines two distinct subsets among human tumour infiltrating lymphocytes.

Both lymphokine activated killer (LAK) cells and specific cytotoxic T lymphocytes appear to play a role in tumour immunity. Tumour infiltrating lymphocytes (TIL) which display a CD56+ phenotype (both CD3+ and CD3-) are also likely to possess anti-tumour activity. We have previously described a 120 kDa surface antigen, termed LAK1, expressed on a subset of human peripheral blood lymphocytes (20-50%) with both NK and LAK activity. The aim of the present study was to determine whether LAK1 antigen is able to distinguish among TIL two populations of effector cells displaying either specific or non MHC-restricted (NK/LAK) activity. We showed that about 25% of freshly derived TIL were weakly stained with anti-LAK1 monoclonal antibody and most of them were also CD3+ CD56-. After culture in recombinant interleukin-2 the majority of TIL were CD3+ CD56- and the percentage of LAK1+ cells increased up to 50%. Among cloned TIL, only those lacking LAK1 antigen displayed a specific cytotoxicity against the autologous tumour, whereas the non-lytic clones were able to produce both tumour necrosis factor and gamma-interferon. Moreover, when TIL from a renal cell carcinoma were fractionated into LAK1- and LAK1+ populations, the specific lytic activity was mainly evident when LAK1- lymphocytes were used as effector cells. Conversely, LAK activity was confined to the LAK1+ subset.     

浸润性T淋巴细胞及Th细胞因子在不同类型乳腺癌中的表达

目的研究浸润性T淋巴细胞和Th细胞因子在不同类型乳腺癌中表达的意义。方法利用免疫组织化学链霉亲和素生物素法（labelled streptavidin-biotin method,LSAB）方法检测61例浸润性微乳头状癌（invasive micropapillary carcinoma,IMPC）、71例浸润性导管癌（invasive ductal carcinoma,IDC）和27例髓样癌（medullary carcinoma,MC）3种乳腺癌中浸润T淋巴细胞亚群分布以及Th细胞因子的表达。T淋巴细胞组间比较采用Kruskal-Wallis Test,组内比较采用Witcoxon Signed Ranks Test;组间Th细胞因子的比较采用x^2检验。结果CD3、CD8和CD4均表达于淋巴细胞胞膜。CD3^＋T细胞在3组间差异无统计学意义（P=0．735）。CD8^＋T和CD4^＋T细胞在3组间差异均有统计学意义（P=0．000,P=0．012）。Th细胞因子主要表达于肿瘤细胞和淋巴细胞的胞质。IL-2表达在IMPC组最低（25／61,41．0％）,其次是IDC组（33／71,46．5％）,最高是MC组（20／27,74．1％）,3组间差异有统计学意义（P=0．014）;IFN-γ的表达类似于IL-2,IMPC组最低（39／61,63．9％）,其次是IDC组（57／71,80．3％）,最高是MC组（24／27,88．9％）,3组间差异有统计学意义（P=0．019）;IL-4的表达在IDC组最高（42／71,59．2％）,其次是IMPC组（19／61,31．1％）,最低是MC组（5／27,18．5％）,3组间差异有统计学意义（P=0．000）;IL-10的阳性率在IMPC组是87．8％（47／61）,在IDC组是87．3％（62／71）,在MC组是88．9％（24／27）,3组间差异无统计学意义（P〉0．050）。结论浸润性T淋巴细胞和Th细胞因子在不同类型乳腺癌中表达不同。     

CD8+淋巴细胞浸润对可手术乳腺癌预后的影响

目的 探讨CD8+淋巴细胞浸润与乳腺癌临床病理特征及预后的关系。方法 回顾性分析2000年1月至2008年12月中山大学肿瘤防治中心收治的681例可手术切除的乳腺癌患者的临床资料,用组织芯片免疫组化法检测乳腺癌原发灶中CD8+淋巴细胞浸润情况,分析其与乳腺癌临床病理特征及预后的关系。结果42.0%（286/681）的乳腺癌间质存在CD8+淋巴细胞高浸润。CD8+淋巴细胞浸润与乳腺癌患者的年龄、月经情况、肿瘤大小、淋巴结转移、病理分期、脉管癌栓、组织学分级、激素受体表达、HER-2表达有关（P＜0.05）。全组患者的5年无病生存率和5年生存率分别为79.2%和87.6%;其中CD8+淋巴细胞高浸润者与低浸润者的5年无病生存率分别为83.2% 和68.8%,5年生存率分别为89.5%和82.2%,差异均有统计学意义（P＜0.05）。Cox多因素分析显示,CD8+淋巴细胞浸润是影响患者无病生存和总生存的独立预后因素。结论 CD8+淋巴细胞浸润可作为预测可手术乳腺癌预后的一个指标。     

Suppressor cell activity of lymphocytes infiltrating human lung and breast tumours.

Tumour-infiltrating lymphocytes (TIL) can be isolated from human lung and breast tumours by the stepwise application of velocity and density sedimentations on discontinuous Ficoll-Triosil or bovine serum gradients. The populations obtained showed variable composition with respect to lymphocyte subsets but generally corresponded to that of peripheral blood lymphocytes (PBL) containing primarily T cells (mean, 49% E rosettes; 15% EA rosettes). Comparison of reactivity to PHA in 15 individuals revealed a significant deficit in reactivity in the TIL population compared with blood and lymph-node lymphocytes. TIL showed no natural killer (NK) activity, no response to autologous tumour in mixed lymphocyte target interaction tests (MLTI) and no cytotoxicity against autologous tumour, in agreement with previous findings. In order to investigate this depressed reactivity, we measured the effect of admixture of mitomycin-treated TIL to PBL on PHA and MLTI responsiveness in nine individuals. Eight of these nine showed reduced reactivity to PHA (mean depression of responsiveness, 49%) and five subjects who were responsive to autologous tumour, showed markedly reduced reactivity in the presence of TIL (mean reduction, 65%). In a case of medullary carcinoma of breast it was possible to revover sufficient TIL to allow fractionation on nylon wool columns. Passage through these columns resulted in a higher level of inhibition of PBL reactivity than that seen in unpassed or column-attached lymphocytes. These data show that TIL have suppressive activity which may account for their poor performance in other assays of effector and memory function.     

Chromosomal radiosensitivity in G2-phase lymphocytes identifies breast cancer patients with distinctive tumour characteristics.

A substantial proportion of women with breast cancer exhibit an abnormally high radiosensitivity as measured by the frequency of chromatid breaks induced in G2-phase, PHA stimulated lymphocytes. Chromatid break frequencies were compared for a cohort of previously untreated sporadic breast cancer patients and hospital outpatient controls. In the breast cancer group 46% showed high radiosensitivity compared to 14% of controls (P< 0.001). Comparison of those breast cancer patients with a high G2 radiosensitivity (G2RS) versus those with a low G2RS showed no difference in menopausal status or age but the high G2RS group had on average a lower score on the Nottingham Prognostic Index. Predicted survival in the high G2RS group at 15 years was 55% compared to 36% for the low G2RS group. Furthermore, 81% of tumours from the high G2RS were oestrogen receptor positive compared to 45% from the low G2RS group. Thus high G2RS identifies a sub-population of patients with distinctive tumour characteristics and with a predicted improved prognosis as compared with those in the low G2RS group. Our findings imply that besides influencing risk of breast cancer the genetic factors determining G2 radiosensitivity also influence the tumour characteristics and prognosis in these patients.     

Comparison of tumor‐infiltrating lymphocytes between primary and metastatic tumors in breast cancer patients

The presence of tumor‐infiltrating lymphocytes (TILs) is associated with favorable long‐term outcome in breast cancer. However, little is known about changes in TILs during metastatic progression. To confirm our hypothesis that malignant tumors escape from the host immune system during metastasis, we evaluated the percentage of TILs in paired samples of primary and metastatic breast tumors. We retrospectively identified 25 patients with human epidermal growth factor receptor‐2 (HER2⁺, n = 14) and triple negative (TN, n = 11) early breast cancer diagnosed between 1990 and 2009 at Tokai University Hospital (Isehara, Japan) and who subsequently experienced regional or distant recurrence confirmed by tumor biopsy/resection. Hematoxylin–eosin‐stained slides of these paired samples were evaluated for stromal TILs. Immunohistochemical staining was carried out using primary antibodies against CD4, CD8, Foxp3, programmed cell death ligand 1 (PD‐L1), PD‐L2, and HLA class I for characterizing the TILs and breast tumors. The percentage of TILs in the primary tumors was significantly higher (average 34.6%) than that in metastatic tumors (average 15.7%) (paired t‐test, P = 0.004) and that of CD8⁺ and CD4⁺ T cells significantly decreased from primary to metastatic tumors (paired t‐test, P = 0.008 and P = 0.026, respectively). The PD‐L1, PD‐L2, and HLA class I antibody expression changed from positive to negative and vice versa from the primary to the metastatic tumors. Tumors at first metastatic recurrence in HER2⁺ and TN breast cancers have a lower percentage of TILs and CD8⁺ and CD4⁺ T cells compared to primary tumors, which indicates that immune escape plays a role in tumor progression.     

Correlation between class I antigen expression and the ability to generate tumour infiltrating lymphocytes from bladder tumour biopsies.

Analysis of tissue sections from transurethrally resected bladder tumours using anti-CD3 antibody showed the presence of T lymphocytes in intra-epithelial layers in eight of 12 cases investigated. In a larger group of patients, Tumour Infiltrating Lymphocyte (TIL) growth was established from six of 19 cases using Interleukin-2 (IL-2) and conditioned medium (CM) and resulted in the expansion of TILs up to 100-fold. TILs from these individuals were phenotyped with W6/32 (anti-HLA-A,B,C), HB55 (anti-DR) and anti-CD3 antibodies using FAC sorter. The mean +/- s.d. frequency of positive staining with these antibodies were 96.7 +/- 4.0%, 87.5 +/- 10.0% and 82.5 +/- 7.8% respectively, indicating the activated nature of these T cells. The cytotoxic activity of these TILs against Daudi (ie, LAK activity) cell line at 25/1 E/T ratios varied from 26.3 +/- 3.2 to 62.8 +/- 5.2%. In one case where TILs and autologous tumour cell line were established, cytotoxicity studies showed low level of cytotoxicity against the autologous tumour cells (15.8 +/- 1.6%) compared with 62.8 +/- 5.2% against Daudi. Staining of tumour sections from these 19 individuals with W6/32 and BBM.1 revealed positive staining in six of six that developed TILs but only six of 13 (46%) cases, whose tumour failed to grow TILs (P less than 0.02, Fisher exact test). These results are indicative of the presence of IL-2 passageable T cells in bladder cancer biopsy and demonstrate that the successful expansion of these cells correlates with the normal expression of class I antigens on the tumour cells.     

Characterization of tumor infiltrating lymphocytes in paired primary and metastatic renal cell carcinoma specimens

Renal cell carcinoma (RCC) is one of the most chemo- and radio-resistant malignancies, with poor associated patient survival if the disease metastasizes. With recent advances in immunotherapy, particularly with PD-1/PD-L1 blockade, outcomes are improving, but a substantial subset of patients does not respond to the new agents. Identifying such patients and improving the therapeutic ratio has been a challenge, although much effort has been made to study PD-1/PD-L1 status in pre-treatment tumor. However, tumor infiltrating lymphocyte (TIL) content might also be predictive of response, and our goal was to characterize TIL content and PD-L1 expression in RCC tumors from various anatomic sites. Utilizing a quantitative immunofluorescence technique, TIL subsets were examined in matched primary and metastatic specimens. In metastatic specimens, we found an association between low CD8+ to Foxp3+ T-cell ratios and high levels of PD-L1. High PD-L1-expressing metastases were also found to be associated with tumors that were high in both CD4+ and Foxp3+ T-cell content. Taken together these results provide the basis for combining agents that target the PD-1/PD-L1 pathway with agonist of immune activation, particularly in treating RCC metastases with unfavorable tumor characteristics and microenvironment. In addition, CD8+ TIL density and CD8:Foxp3 T-cell ratio were higher in primary than metastatic specimens, supporting the need to assess distant sites for predictive biomarkers when treating disseminated disease.     

The role of infiltrating lymphocytes in the neo-adjuvant treatment of women with HER2-positive breast cancer

Breast Cancer Research and Treatment - Pre-treatment tumour-associated lymphocytes (TILs) and stromal lymphocytes (SLs) are independent predictive markers of future pathological complete response...