The Functional Deficiency of B Lymphocytes in Patients with Lung Cancer is Due to Inadequate T-Cell Help and Excessive Suppression by T and Non-T Cells

The proliferative and plaque-forming cell (PFC) responses of unseparated mononuclear cells (MNC) and B- and T-cell-enriched populations of cells were analyzed in 15 patients with lung cancer to determine the mechanisms involved in the functional abnormality of their B cells. The PFC responses of the MNC of the patient group were significantly lower than those of normal controls. In addition, the enriched B cells of several patients showed a further decrease in their PFC responses after coculture with autologous Tcells compared with their respective MNC responses. The proliferative response against phytohemagglutinin (PHA) was also lower in many of the patients after similar cocultures. Cocultures of patients' B cells with T cells from normal controls significantly enhanced the PFC responses in 7 patients. In most of the normal controls, B lymphocytes showed a significant decrease in their PFC responses after coculture with the patients' T cells. Although the percentages of total T cells, T-helper, and B cells were within the normal range, the number of suppressor T cells was significantly higher in the patient group. These results indicate that a combination of insufficient T-cell help, excessive suppression by both T and non-T cells, and a possible intrinsic B-cell abnormality are responsible for the B-cell functional deficiency observed in patients with lung cancer.     

T-cell subsets defined by monoclonal antibodies in monoclonal gammopathy of undetermined significance (MGUS)

Peripheral T lymphocytes from 31 patients with monoclonal gammopathy of undetermined significance (MGUS), and from a group of controls of the same age range, were stained using monoclonal antibodies of the OKT series. The absolute number and the percentage of OKT3+ cells did not differ in patients compared with the controls. The percentage and absolute number of T-cell subsets with helper/inducer OKT4+ and suppressor/cytotoxic OKT8+ phenotype were not different from those of the controls, thus the OKT4/OKT8 ratio in the patients with MGUS was normal (1.60 versus 1.57 in normal controls). These results suggest that MGUS is a B-cell disorder without imbalance of peripheral T-cell subsets unlike B-cell malignancies such as multiple myeloma and B-cell chronic lymphocytic leukemia.     

Differential methylation of a short CpG-rich sequence within exon 1 of TCF21 gene: a promising cancer biomarker assay.

Detection of cancer cells at early stages could potentially increase survival rates in cancer patients. Aberrant promoter hypermethylation is a major mechanism for silencing tumor suppressor genes in many kinds of human cancers. A recent report from our laboratory described the use of quantitative methylation-specific PCR assays for discriminating patients with lung cancer from those without lung cancer using lung biopsies as well as sputum samples. TCF21 is known to be essential for differentiation of epithelial cells adjacent to mesenchyme. Using restriction landmark genomic scanning, a recent study identified TCF21 as candidate tumor suppressor at 6q23-q24 that is epigenetically inactivated in lung and head and neck cancers. Using DNA sequencing technique, we narrowed down a short CpG-rich segment (eight specific CpG sites in the CpG island within exon 1) of the TCF21 gene, which was unmethylated in normal lung epithelial cells but predominantly methylated in lung cancer cell lines. We specifically targeted this short CpG-rich sequence and developed a quantitative methylation-specific PCR assay suitable for high-throughput analysis. We showed the usefulness of this assay in discriminating patients with lung cancer from those without lung cancer using biopsies and sputum samples. We further showed similar applications with multiple other malignancies. Our assay might have important implications in early detection and surveillance of multiple malignancies.     

Clinical and molecular responses in lung cancer patients receiving Romidepsin.

PURPOSE: Our preclinical experiments indicated that Romidepsin (Depsipeptide FK228; DP) mediates growth arrest and apoptosis in cultured lung cancer cells. A phase II trial was done to examine clinical and molecular responses mediated by this histone deacetylase inhibitor in lung cancer patients. EXPERIMENTAL DESIGN: Nineteen patients with neoplasms refractory to standard therapy received 4-h DP infusions (17.8 mg/m(2)) on days 1 and 7 of a 21-day cycle. Each full course of therapy consisted of two identical 21-day cycles. Plasma DP levels were evaluated by liquid chromatography-mass spectrometry techniques. A variety of molecular end points were assessed in tumor biopsies via immunohistochemistry techniques. Long oligo arrays were used to examine gene expression profiles in laser-captured tumor cells before and after DP exposure, relative to lung cancer cells and adjacent normal bronchial epithelia from patients undergoing pulmonary resections. RESULTS: Nineteen patients were evaluable for toxicity assessment; 18 were evaluable for treatment response. Myelosuppression was dose limiting in one individual. No significant cardiac toxicities were observed. Maximum steady-state plasma DP concentrations ranged from 384 to 1,114 ng/mL. No objective responses were observed. Transient stabilization of disease was noted in nine patients. DP enhanced acetylation of histone H4, increased p21 expression in lung cancer cells, and seemed to shift global gene expression profiles in these cells toward those detected in normal bronchial epithelia. CONCLUSION: Although exhibiting minimal clinical efficacy at this dose and schedule, DP mediates biological effects that may warrant further evaluation of this histone deacetylase inhibitor in combination with novel-targeted agents in lung cancer patients.     

Defects in natural killer cell activity and interferon response in human lung carcinoma and malignant melanoma

The natural killer (NK) activity of peripheral blood mononuclear cells from 25 patients with squamous cell carcinoma of the lung, malignant melanoma, or epitheloid cancers of the gastrointestinal tract was measured by the lysis of 51Cr-labeled K562 target cells. NK activities of many patients with lung cancer or malignant melanoma were decreased relative to normal controls. This abnormality was significantly correlated with advancing stage of disease and the percentage of monocytes in the cell suspensions. Addition of indomethacin or removal of monocytes did not restore depressed NK function to normal levels. Abnormalities of NK function did not appear to be secondary to the presence of mononuclear suppressor cells. The response to interferon was also impaired in patients with advanced disease. The number of effector:target conjugates was normal even in patients with depressed NK function; however, the number of active lytic effectors was decreased. These results imply that the cells which bind tumor targets are present in patients with advanced cancers, but these cells are either immature or functionally inactive.     

Induction of immunity to prostate cancer antigens: results of a clinical trial of vaccination with irradiated autologous prostate tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor using ex vivo gene transfer.

Vaccination with irradiated granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting gene-transduced cancer vaccines induces tumoricidal immune responses. In a Phase I human gene therapy trial, eight immunocompetent prostate cancer (PCA) patients were treated with autologous, GM-CSF-secreting, irradiated tumor vaccines prepared from ex vivo retroviral transduction of surgically harvested cells. Expansion of primary cultures of autologous vaccine cells was successful to meet trial specifications in 8 of 11 cases (73%); the yields of the primary culture cell limited the number of courses of vaccination. Side effects were pruritis, erythema, and swelling at vaccination sites. Vaccine site biopsies manifested infiltrates of dendritic cells and macrophages among prostate tumor vaccine cells. Vaccination activated new T-cell and B-cell immune responses against PCA antigens. T-cell responses, evaluated by assessing delayed-type hypersensitivity (DTH) reactions against untransduced autologous tumor cells, were evident in two of eight patients before vaccination and in seven of eight patients after treatment. Reactive DTH site biopsies manifested infiltrates of effector cells consisting of CD45RO+ T-cells, and degranulating eosinophils consistent with activation of both Th1 and Th2 T-cell responses. A distinctive eosinophilic vasculitis was evident near autologous tumor cells at vaccine sites, and at DTH sites. B-cell responses were also induced. Sera from three of eight vaccinated men contained new antibodies recognizing polypeptides of 26, 31, and 150 kDa in protein extracts from prostate cells. The 150-kDa polypeptide was expressed by LNCaP and PC-3 PCA cells, as well as by normal prostate epithelial cells, but not by prostate stromal cells. No antibodies against prostate-specific antigen were detected. These data suggest that both T-cell and B-cell immune responses to human PCA can be generated by treatment with irradiated, GM-CSF gene-transduced PCA vaccines.     

miR-21调控非小细胞肺癌细胞增殖和凋亡的机制研究

目的 研究非小细胞肺癌(NSCLC)中高表达的miR-21对细胞增殖和凋亡的影响及其调控机制.方法 采用荧光定量聚合酶链反应检测miR-21在人NSCLC组织、癌旁组织和A549细胞系中的表达.通过序列分析预测被miR-21调控的抑癌基因,通过荧光素酶活性检测和Western blot 检测验证miR-21对靶基因表达调控的影响.采用RNA干扰技术抑制miR-21和程序性细胞死亡因子4(PDCD4),以台盼蓝染色和流式细胞术检测A549细胞增殖和凋亡的变化.结果 NSCLC组织和A549细胞中miR-21的表达水平分别为癌旁组织的2.24倍和3.06倍.序列预测和基因表达调控研究表明,miR-21可以在NSCLC中反向调控PDCD4的表达(P＜0.01).荧光素酶活性检测结果显示,共同转入Wt 3'UTR和miR-21会显著抑制A549细胞中的荧光素酶表达(P＜O.001).Western blot检测结果显示,导入反义寡核苷酸抑制miR-21的功能后,PDCD4的表达水平明显上升.抑制miR-21的作用可以显著抑制A549细胞的增殖,并使细胞凋亡率由2.6％上升到10.9％,而抑制PDCD4的表达可以在很大程度上消除miR-21介导的细胞增殖障碍,抑制细胞凋亡.结论 在NSCLC中,抑制抑癌基因PDCD4的表达可能是miR-21介导肿瘤细胞增殖、抵抗凋亡的重要途径之一.     

Relationship between T lymphocyte levels and lymphoproliferative responses to mitogens and alloantigens in lung and breast cancer patients.

Lung and breast cancer patients were studied for relationships between lymphocyte function, as measured by lymphoproliferative (LP)³ assays, and levels of circulating thymus-derived lymphocytes (T lymphocytes) measured by rosette formation with sheep erythrocytes (E rosettes). LP responses to the T lymphocyte mitogens phytohemagglutinin (PHA) and concanavalin-A (Con-A) and to alloan-tigens in one-way mixed leukocyte cultures (MLC) were assessed in a microculture assay. Total T lymphocyte levels were determined using an E rosette assay performed at 4°C and a subpopulation of T lymphocytes with high affinity receptors for E rosettes was measured in a 29°C assay. Using the relative proliferation index (RPI) which compares patient reactivity to normal reactivity, we found that one-third to one-half of the lung and breast cancer patients had depressed LP responses to at least one of the stimulants. In contrast, only 21% of the breast and 25% of the lung cancer patients had low levels of total E-rosette-forming cells (E-RFC) as measured by the 4°C E rosette assay. Using the 29°C E rosette assay, we found that a significantly higher proportion of the patients demonstrated decreased levels of this subpopulation of lymphocytes and in both patient groups the depression noted in functional assays was closely paralleled by the percentage of patients with abnormal levels of high-affinity T lymphocytes. To further examine this apparent relationship between high-affinity E-RFC levels and LP results, the correlation between these measures in individual patients was examined. Regression analysis indicated a significant correlation between mitogen and alloantigen responses in both patient groups as well as between total T lymphocyte and high-affinity T lymphocyte levels. However, there was no significant correlation between LP responses and T lymphocyte levels. Studies in which MLC and PHA reactivities, 4°C and 29°C E rosette levels were determined on the same peripheral blood leukocyte (PBL) sample demonstrated that a number of lung and breast cancer patients with abnormal LP responses had normal total and high-affinity T lymphocyte levels. Patients with a decrease in high-affinity E-RFC but normal levels of total T lymphocytes had a high incidence of depression to PHA (56 and 82% in breast and lung cancer patients respectively) but fewer had depressed MLC reactivity (33 and 71% in breast and lung cancer patients, respectively). In patients with low levels of total and high-affinity T lymphocytes, there was a high incidence of depression in responses to both PHA and MLC. The results of this study indicate that some cancer patients may have depressed LP responses in association with depressed levels of T lymphocytes, but that other patients may have depressed functional reactivity despite normal levels of E-RFC.     

Alteration of expression or phosphorylation status of tob, a novel tumor suppressor gene product, is an early event in lung cancer

Tob is a member of the Tob/BTG family, a novel class of anti-proliferative proteins. To investigate the involvement of tob as a tumor suppressor gene in human lung cancer, we analyzed the expression of tob mRNA and protein in lung cancer tissue and adjacent normal lung tissue. Immunohistochemical analysis using anti-Tob antibody showed decreased expression of Tob in 72% (31/43) of lung cancer tissues. Furthermore, 95% (19/20) of squamous cell carcinoma patients showed an apparent decrease in Tob in cancer tissues, associated with smoking status. The phosphorylated form of Tob, an inactive form of Tob, was detected in 76% (16/21) of cancer tissues of adenocarcinoma patients, but not in normal alveolar epithelial cells. Either a decrease in Tob expression or an accumulation of phosphorylated Tob was observed from early clinical stages, even in bronchial dysplasia, a premalignant lesion of squamous cell carcinoma. The above findings suggest that the disruption of anti-proliferative Tob plays a distinct part in the early stage of lung carcinogenesis.     

miR-127在肺癌中的表达及功能

 目的 探讨miR-127在肺癌中的表达及对其肺癌细胞生物学特性的影响。方法 收集20例患者的肺癌组织及癌旁组织，通过实时荧光定量PCR检测miR-127在肺癌及癌旁组织中的表达水平。应用mimics转染A549细胞株，采用平板克隆、MTT法检测miR-127 mimics组、空白载体转染组（NC）肺癌细胞的增殖活性。Transwell实验计算肺癌细胞过膜数量，研究miR-127与肺癌细胞迁移作用的关系。结果 miR-127在肺癌组织中的表达显著低于癌旁正常组织（0.55±0.05 vs. 1.45±0.13, P=0.001）。肺癌组织中miR-127的表达随着病理分期恶性程度的增高而降低（H=6.528, P=0.034）。平板克隆及MTT实验结果显示miR-127 mimics组的细胞活性及增殖明显受抑制（P=0.0032; P=0.0045），Transwell实验显示miR-127 mimics组的细胞过膜数量（79±18/视野）明显低于NC组（352±21/视野），差异有统计学意义（P=0.002）。结论 miR-127在肺癌组织中低表达，其在抑制肺癌细胞的增殖和转移过程中发挥重要作用，可作为肺癌的潜在临床诊断标志物。     

Radiosensitive, thymic hormone-sensitive peripheral blood suppressor cell activity in cancer patients

Suppressor cell activity which was radiosensitive in most subjects and thymic hormone sensitive in some was identified in patients with cancer, and compared to simultaneously studied normal controls. Suppressor cell activity was measured in cocultures of normal lymphocytes with patient lymphocytes added in microwells using the blastogenic response to phytohemagglutinin and concanavalin A as the measure of activity. Thirty-five patients (lung cancer, 21; leukemia in remission, seven; and various solid tumors, seven) and an equal number of controls were studied. Suppressor cell activity was identified in 71% of the patients. In approximately 75% of these, the suppressor cell activity was radiosensitive (4000 to 6000 rads). For the phytohemagglutinin response, suppressor cell activity was thymic hormone sensitive in approximately 40% (Thymosin Fraction 5 or thymic humoral factor), and for the concanavalin A response, it was thymic hormone sensitive in about 25% of the cases. There was a significant correlation between the presence of immunodeficiency (defined as a phytohemagglutinin response < 35,000 or a concanavalin A response < 12,000 cpm) and the presence of the suppressor cell activity. The suppressor cell activity was heterogenous relative to its radiosensitivity and thymic hormone sensitivity. Suppressor cell activity was observed in all the patient categories. These results indicate that certain available therapeutic manipulations may have significant effects on suppressor cell activity and should be an important subject for further investigation.     

Suppressor T cells in B-cell chronic lymphocytic leukaemia: relationship to clinical stage

In 32 patients with B-cell chronic lymphatic leukaemia (CLL), OKT8+ suppressor T cells were increased in relative (mean 54 +/- 14%; normal mean 34 +/- 6%) and absolute (1.8 +/- 1.6 X 10(9)/1; normal range 0.3-0.6 X 10(9)/1) numbers. OKT4+ helper cells were reduced in relative (mean 53 +/- 15%; normal mean 65 +/- 7%) but not absolute (1.7 +/- 1.4 X 10(9)/1; normal range 0.6-1.4 X 10(9)/1) numbers. Essentially identical results were obtained in treated and untreated patients. There was no significant association between T-cell subset numbers and clinical stage, whether assessed by the Rai classification or the more recent Binet system, although the OKT4+/8+ ratio was slightly lower in advanced disease. The study suggests that the immunoparesis so characteristic of CLL may be attributable to increased suppressor T-cell activity.     

Defective suppressor cell activity in cancer patients: a defect in immune regulation

Although suppressor cells appear to be involved in the normal regulatory mechanism of the lymphoid system, they are also considered to have a role in the immunosuppression of certain malignancies. Suppressor activity of lymphocytes can be reproducibly measured by use of the mixed lymphocyte culture-mitogen interaction (MLC-M) in which the stimulating cell either is in the basal state or has been induced by Concanavalin A. This yields a quantitative measure of resting suppressor cell activity as well as the maximum generation of suppressor activity as induced by Con A. This test was performed, using autologous and allogenic cell combinations in a group of 13 cancer patients and 18 normal controls. Normal lymphocytes activated by Con A in 48-hour lymphocyte cultures significantly decreased the mitogenic response of lymphocytes from healthy, male donors to phytohemagglutinin (PHA), Con A, and Pokeweed mitogen (PWM) in both autologous and homologous systems. In contrast, Con A activated lymphocytes from cancer patients demonstrated diminished suppressor activity compared with controls in autologous (P less than 0.01), and allogenic (P less than 0.005) systems. There was a correlation between the degree of immunosuppression and suppressor cell activation: i.e., the patients most depressed generally had the lowest suppressor cell activation. Untreated lymphocytes from cancer patients also exerted suppressive effects on normal lymphocyte responses, suggesting an increased resting level of suppressor cells. These data suggest that in addition to having depressed cellular immune responses, cancer patients frequently have reduced capability to generate suppressor cell activity, which implies a generalized defect in this aspect of immune regulation.     

Copy number gains on 5p15, 6p11-q11, 7p12, and 8q24 are rare in sputum cells of individuals at high risk of lung cancer

Lung cancer specimens display recurrent copy number aberrations in distinguished chromosomal regions as compared with normal lung cells. Such alterations have been utilized in design of fluorescence in situ hybridization (FISH) probe sets in attempts to improve the cytological diagnosis of lung cancer. One of such probe sets, LAVysion, detects copy number changes in the centromeric region of chromosome 6 (CEP6), and regions 5p15, 8q24, and 7p12, often gained in lung cancer. METHODS: We evaluated the feasibility of the LAVysion multi-color probe set in detection of individuals at high risk of lung cancer by applying the FISH probe set on smears prepared of induced sputa obtained from 20 lung cancer patients, 43 asbestos-exposed workers, 21 heavy tobacco smokers, and 15 healthy never-smokers. The hybridized sputum smears were examined using fluorescence microscopy and the number of signals in epithelial cells was examined throughout the hybridized area. Additionally, we review here the previous studies using LAVysion probe set. RESULTS: The FISH probe set was slightly more sensitive than cytology alone in detecting lung cancer. No significant differences in copy number gain were found between high-risk individuals and healthy never-smokers. The proportions of individuals with copy number gains in sputa among the lung cancer patients, asbestos-exposed workers, tobacco smokers, and never-smokers were 50, 20, 12, and 27%, respectively, when three or more cells with a copy number gain detected by at least two different probes was used as the cut-off point. In comparison, the sensitivity of cytology in detecting lung cancer was 44%. In the lung cancer patients the number of abnormal cells by FISH correlated well with the cytologic atypia class (Spearman rank correlation coefficient 0.77, p<0.01). Using multivariant variance analysis, gains in CEP6, 5p15, 8q24 and 7p12 were not associated with smoking or asbestos exposure. CONCLUSIONS: FISH did not significantly exceed the sensitivity of sputum cytology in finding lung cancers. Significant differences were not found between sputa of asbestos-exposed individuals, heavy-smokers and never-smokers. More sensitive methods are needed for the follow-up of populations at high risk of contracting lung cancer.     

Cellular immunity in neoplasia. Antigen and mitogen responses in patients with bronchiogenic carcinoma.

Cellular immune responses of patients with histologically confirmed lung carcinoma were assessed in vivo using cutaneous response and in vitro with a microlymphocyte blastogenic transformation (LBT) assay. In addition, correlation of the cutaneous response with the migration inhibitory factor (MIF) assay and LBT response was examined. The results indicated that cutaneous responses seen in patients with cancer of the lung were consistently lower than similar responses in normal controls (p less than 0.001). Similarily, the percentage of positive cutaneous responses seen with patients included in this study was lower than the frequencies reported by others. Stimulation of cells from lung cancer patients by PHA-M was also depressed when compared to similar lymphocytic responses in normal volunteers (p less than 0.001). The correlation between cutaneous response to tuberculin and the in vitro assays was high. The few instances of disparity demonstrate the need to utilize more than one assay in evaluating cellular immune functions. These data would support the work of others that indicate a depression of cellular immunity in advanced malignancy.     

Lymphocyte subsets in pulmonary venous and arterial blood of lung cancer patients

Lymphocyte subsets in pulmonary venous blood (PVB) smears from 42 patients with lung cancer were immunocytochemically determined. In four patients, pulmonary arterial blood (PAB) smears were also studied for comparison with PVB. Seven healthy donor peripheral blood (PB) smears were used as controls. The percentage of T cells, helper/inducer T (Th) cells and B cells were significantly lower (P less than 0.01) than in normal controls but those of suppressor/cytotoxic T (Ts) cells, natural killer (NK) cells (P less than 0.01) and S100+ small lymphoid cells (P less than 0.05) were higher. This resulted in a decrease in the Th:Ts value in cancer patients (1.46 vs. 2.28 for normal controls; P less than 0.01). The Th and Ts value of PVB from patients in pathological Stages III and IV was lower than from those in Stages I and II because of the increase in Ts cells in the former (P less than 0.05). S100+ small lymphoid cells were increased in cancer patients, especially in those with adenocarcinoma. The present study demonstrates immunoregulation abnormalities in cancer bearing hosts, the results correlating well with the stage of the cancer. Determining lymphocyte subset alterations in PVB did not, however, enable us to detect the changes associated with local immune responses against cancer.     

Suppressor cells and survival of patients with advanced gastric cancer

The association of suppressor cells with survival of patients with gastric cancer was investigated. Phytohemagglutinin (PHA)-induced lymphocyte response and the presence of nonspecific suppressor cells were assessed in patients with different stages of gastric cancer. The presence of suppressor cells was determined by their ability to inhibit the PHA response of normal peripheral blood mononuclear leukocytes. Depression of the PHA response was related to the stage of disease and was also associated with the presence of suppressor cells. Of 245 patients tested, 76 (31%) had suppressor cells. Adherent, nonspecific esterase-positive cells (presumably, monocytes) accounted for the suppression in most cancer patients. The occurrence of suppressor cells and the tumor load were related because the incidence of detectable suppressor cells decreased after surgery in patients with resectable tumor but increased in patients undergoing palliative surgery. In patients with advanced disease who had a generally poor prognosis, the occurrence of suppressor cells was associated with a significantly increased survival. Hence the common view that a depressed lymphocyte response correlates with a poor clinical outcome may not be valid in all types of cancer.