Expansion of myeloid-derived suppressor cells in chronic obstructive pulmonary disease and lung cancer: potential link between inflammation and cancer.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a risk factor for lung cancer (LC). Myeloid-derived suppressor cells (MDSCs) down-regulate the T cell receptor ζ chain (TCR ζ) through L-arginine deprivation and lead to T cell dysfunction and deficient antitumor immunity. We hypothesized that abnormally high levels of MDSCs in COPD patients may alter tumor immunosurveillance. METHODS: We compared the proportion of circulating MDSCs (Lin-HLA-DR-/CD33+/CD11b+) (by flow cytometry), arginase I (ARG I) serum levels (by ELISA), and expression levels of TCR ζ on circulating lymphocytes (by flow cytometry) in 28 patients with LC, 62 subjects with COPD, 41 patients with both LC and COPD, 40 smokers with normal spirometry and 33 non-smoking controls. T cell proliferation assays were performed in a subgroup of participants (CFSE dilution protocol). RESULTS: We found that: (1) circulating MDSCs were up-regulated in COPD and LC patients (with and without COPD); (2) MDSCs expansion was associated with TCR ζ down-regulation in the three groups; (3) in LC patients, these findings were independent of COPD and tobacco smoking exposure; (4) TCR ζ down-regulation correlates with T cell hyporesponsiveness in COPD and LC patients. CONCLUSIONS: These results suggest that tumor immunosurveillance might be impaired in COPD and may contribute to the increased risk of LC reported in these patients.

[Immunotherapy of Bronchogenic Carcinoma and Its Perspectives]. / Imunoterapie u bronchogenního karcinomu a její perspektivy.

Immunotherapy is becoming another possible alternative in the treatment of lung cancer. It is a completely different method of treating cancer which is not directed to the tumor itself, but to the immune system. Surface antigens present on tumor cells may be an effective and specific therapeutic targets and strategies based on antibodies inhibiting immune check-points significantly improves the antitumor immune response. Monoclonal antibody blocking CTLA 4 (cytotoxic T-lymphocyte antigen) and PD 1 receptor (protein programmed cell death) and its ligand PD L1 showed clinical efficacy and nivolumab (antiPD 1) was approved in 2nd line treatment squamous nonsmall cell lung cancer.

Molecular subtyping reveals immune alterations associated with progression of bronchial premalignant lesions.

Bronchial premalignant lesions (PMLs) are precursors of lung squamous cell carcinoma, but have variable outcome, and we lack tools to identify and treat PMLs at risk for progression to cancer. Here we report the identification of four molecular subtypes of PMLs with distinct differences in epithelial and immune processes based on RNA-Seq profiling of endobronchial biopsies from high-risk smokers. The Proliferative subtype is enriched with bronchial dysplasia and exhibits up-regulation of metabolic and cell cycle pathways. A Proliferative subtype-associated gene signature identifies subjects with Proliferative PMLs from normal-appearing uninvolved large airway brushings with high specificity. In progressive/persistent Proliferative lesions expression of interferon signaling and antigen processing/presentation pathways decrease and immunofluorescence indicates a depletion of innate and adaptive immune cells compared with regressive lesions. Molecular biomarkers measured in PMLs or the uninvolved airway can enhance histopathological grading and suggest immunoprevention strategies for intercepting the progression of PMLs to lung cancer.

Immunoreactive forms of circulating parathyroid hormone in primary and ectopic hyperparathyroidism.

The immunoreactive forms of parathyroid hormone (iPTH) in the plasma of six patients with primary, adenomatous hyperparathyroidism and six patients with ectopic hyperparathyroidism due to non-parathyroid cancer were compared by using gel filtration on columns of Bio-Gel P-150 and radioimmunoassay of iPTH in eluted fractions after concentration. We found much less (p<0.001) small (mol wt<9,500) COOH-terminal fragments of iPTH in plasma samples from ectopic hyperparathyroid patients (0.52+/-0.13 ng eq/ml) than in samples from primary hyperparathyroid patients (3.70+/-1.15 ng eq/ml). The quantity of iPTH eluting with or before native bovine PTH [1-84] was the same in both syndromes (ectopic hyperparathyroidism, 0.82+/-0.22 ng eq/ml; primary hyperparathyroidism, 0.73+/-0.09 ng eq/ml), and these values correlated positively with plasma calcium concentration (ectopic hyperparathyroidism, r=0.908; primary hyperparathyroidism, r=0.919). In both syndromes, plasma samples had an iPTH component that eluted well before PTH [1-84] (mol wt 9,500), but this component was present in much larger quantities in three patients with ectopic hyperparathyroidism. We conclude that (a) the decreased quantity of biologically inactive COOH-terminal fragments of iPTH circulating in ectopic hyperparathyroidism accounts for the previously reported relatively lower total serum iPTH values in this syndrome as compared with primary hyperparathyroidism (Riggs et al. 1971. J. Clin. Invest. 50: 2079); (b) there appears to be sufficient iPTH with presumed biologic activity to account for the hypercalcemia in both syndromes; (c) a large PTH component, not previously recognized in plasma, is present in both ectopic and primary hyperparathyroidism and may exist as the predominant immunoreactive form of the hormone in some patients with ectopic hyperparathyroidism.

Intrapulmonary IFN-beta gene therapy using an adenoviral vector is highly effective in a murine orthotopic model of bronchogenic adenocarcinoma of the lung.

Given previous work showing that an adenoviral vector expressing IFN-beta (Ad.IFNbeta) was highly effective in eradicating i.p. mesothelioma tumors, the antitumor efficacy of this agent was evaluated in an orthotopic model of bronchogenic adenocarcinoma of the lung. These transgenic mice have a conditionally expressed, oncogenic K-rasG12D allele that can be activated by intratracheal administration of an adenovirus expressing Cre recombinase (Ad.Cre). K-rasG12D mutant mice were given Ad.Cre intranasally to activate the oncogene. Mice were then given 10(9) plaque-forming units of a control vector (Ad.LacZ) or Ad.IFNbeta intranasally 3 and 4 weeks later, a time when lung tumors had been established. Cells derived from K-ras-mutated lung tumors were also grown in the flanks of mice to study mechanisms of therapeutic responses. In two separate experiments, untreated tumor-bearing mice all died by day 57 (median survival, 49 days). Ad.LacZ-treated mice all died by day 71 (median survival, 65 days). In contrast, 90% to 100% of mice treated with Ad.IFNbeta were long-term survivors (>120 days; P < 0.001). In addition, immunity to re-challenge with tumor cells was induced. In vitro and flank tumor studies showed that Ad.IFNbeta induced direct tumor cell killing and that depleting natural killer or CD8+ T cells, but not CD4+ T cells, with antibodies attenuated the effect of Ad.IFNbeta. These studies, showing remarkable antitumor activity in this orthotopic lung cancer model, provide strong preclinical support for a trial of Ad.IFNbeta to treat human non-small cell lung cancer.

Demonstration of antibodies in patients' sera, directed against nonspecific cross-reacting antigen.

Nonspecific cross-reacting antigen (NCA), which strongly cross-reacts with carcinoembryonic antigen, was demonstrated to be an autoantigen. Antibodies directed against NCA were shown in different groups of patients, but high titers (greater than 1/64) were found only in cancer patients. A correlation between tumor mass, antigen load, and antibody titer apparently existed. Sera obtained from patients preoperatively and postoperatively differed significantly (P greater than 0.01) in the sense that titers were high only in sera sampled after the patients were treated. Nevertheless, the formation of these antibodies cannot be considered a cancer-specific phenomenon because of their existence also in patients with nonmalignant diseases.

Partial characterization of a fetal lung antigen associated with human bronchogenic carcinoma.

A human lung tumor-associated fetal antigen (LTFA) has been partially isolated and characterized. The antigen that differs in several immunochemical parameters from previously described lung cancer antigens was shared by fetal lung and liver tissue. The neoantigen migrated in immunoelectrophoresis as an alpha2-beta globulin, had an average molecular size of 7S, and was soluble in 50% saturated ammonium sulfate. Whereas LTFA was insensitive to both DNase and RNase treatment, its antigenicity was completely abolished by pronase. The biologic significance of this antigen and its possible clinical use were discussed.

Prolonged survival in bronchogenic carcinoma associated with HL-A antigens W-19 and HL-A5: a preliminary report.

The HL-A antigens were determined retrospectively in a group of 14 surgically cured bronchogenic carcinoma patients and prospectively in another group of 100 untreated patients. In the retrospective group, the frequencies of antigens W-19 and HL-A5 were significantly increased when compared with the noncancer control and the prospective lung cancer populations. In the latter group, 60% of the patients with W-19 and 58% with HL-A5 survived without evidence of tumor for at least 1 year after treatment compared with 15% of patients with neither of these antigens, P less than 0.01 and 0.005, respectively. These comparisons were for adenocarcinoma and squamous carcinoma. The patient groups for oat cell and undifferentiated carcinoma were too small for valid statistical comparisons. This preliminary study suggests that the presence of HL-A antigens W-19 and HL-A5 confers resistance to dissemination of bronchogenic carcinoma.

Assessment of immune responses to tumors using cryostat sections of bronchogenic carcinoma.

Thirty patients with lung cancer were studied using the leukocyte migration-inhibition assay. Cryostat sections of autologous and homologous carcinoma tissues and normal lung were used as antigens. Although no inhibition of migration was seen using autologous cancer tissues, seven of 30 patients (23%) demonstrated inhibition of leukocyte migration in response to homologous cancer sections. No association with clinical stage was appreciated. Seven patients demonstrated enhanced migration to homologous malignant tissues; six of these are still alive more than 18 months after testing. The extent of lymphocytic infiltration of tumors was estimated as a possible reflection of host immune response or tumor antigenicity. Patients whose tumors were prominently infiltrated by lymphocytes had a significantly better prognosis than did those whose tumors showed lesser degrees of infiltration. The degree to which a tumor is infiltrated by the host's lymphocytes appears to correlate with survival. Whether or not this correlation is independent of tumor cell type remains to be determined.

Microcytotoxicity assays of tumor immunity in patients with bronchogenic carcinoma correlated with clinical status.

Peripheral blood lymphocytes from patients with bronchogenic carcinoma were tested in microcytotoxicity assays against cultured bronchogenic cancer cells, other types of tumor cells, and skin fibroblasts. Lymphocytes from patients who were postresection with no clinical evidence of residual or recurrent tumor were more frequently toxic against bronchogenic carcinoma than were lymphocytes from normal donors or from patients with clinically evident disease. Lymphocytes from patients with minimal or no tumor were more frequently toxic against bronchogenic cancer than against skin fibroblasts. Serum samples from a few patients rendered lymphocytes toxic for bronchogenic cancer cells, but this serum activity could not be correlated with the patient's clinical status.

Analysis of human small cell lung cancer differentiation antigens using a panel of rat monoclonal antibodies.

The antigen expression of human small cell lung cancer (SCLC) was studied using a panel of 21 independent rat monoclonal antibodies. The panel was selected by isolating hybridomas producing antibodies reactive with two SCLC lines but not with autologous B-lymphoblastoid lines. The antibodies were then tested in radiobinding assays against a panel of 17 SCLC lines, 13 non-small cell lung cancer lines, 6 SCLC necropsy specimens, 13 neuroectodermal lines (melanomas, neuroblastomas, glioblastomas), 15 other human lines, the glycolipid extracts of SCLC, human meconium, and human red blood cells. Using immunohistochemical assays, 14 of the antibodies were tested against normal lung, liver, and kidney, and lung cancer biopsies and xenografts. These analyses revealed the following: (a) SCLC elicited predominantly immunoglobulin M antibodies despite hyperimmunization; (b) the 21 antibodies displayed distinct binding and immunohistochemical phenotypes, indicating that they recognized many different epitopes; (c) 14 of the 21 antibodies reacted with glycolipid determinants; (d) the 21 determinants were expressed on over 80% of SCLC cell lines, necropsy samples, and xenografts; (e) the determinants were also expressed on normal adult bronchial epithelium, proximal tubules of adult kidney, and in a few instances on other normal cell types; (f) the antigens were expressed less frequently on nonsmall cell lung cancer samples but did not clearly distinguish SCLC from non-small cell lung cancer; (g) biochemical and morphological variants of SCLC exhibiting more malignant and undifferentiated behavior and containing greatly amplified c-myconcogenes failed to express several determinants or expressed them at lower levels; (h) and finally, while many human cell lines failed to express the antigens including human melanoma and glioblastoma lines, human neuroblastoma lines frequently did express the SCLC antigens. These detailed studies utilizing a panel of distinct monoclonal antibodies define a series of antigens on the surface of the majority of SCLC undescribed previously.

Altered HLA class I expression in non-small cell lung cancer is independent of c-myc activation.

We studied the expression of major histocompatibility complex class I antigens in 59 bronchogenic carcinomas, as well as in pneumocytes and epithelial respiratory cells distant from the tumor. We observed in all cases that normal lung tissue expressed major histocompatibility complex class I antigens, while this expression was completely lost in 16 tumors (27%). The defect in HLA gene expression affected both heavy chain and beta 2-microglobulin, as demonstrated by the null reactivity with the monoclonal antibodies GRH1, W6/32, and HC10. Selective underexpression was detected in 1 tumor for HLA-A locus antigens and in 3 tumors for HLA-B locus antigens. Southern blot analyses of major histocompatibility complex class I genes were performed in 20 tumor tissue specimens and 6 cell lines. No class I gene rearrangements were detected using HLA coding and locus specific noncoding probes. We also used the Southern blot method to investigate the possible relationship between c-myc amplification and HLA class I antigens in non-small cell lung cancers and detected no apparent amplification in 20 tumor tissue specimens (5 negative for HLA class I antigens) and 6 cell lines (3 with decreased expression). Northern blot analysis revealed no relationship between c-myc mRNA levels and specific mRNA for HLA-A and HLA-B antigens in cell lines with imbalanced HLA-A or HLA-B expression.

Phenotype and function of natural killer cells in patients with bronchogenic carcinoma.

Decreased peripheral blood natural killer (NK) cell lytic activity may be associated with tumor presence. We evaluated peripheral blood NK lytic activity in 38 patients with bronchogenic carcinoma and compared this to ten normal volunteers of comparable age. The patients with carcinoma had significantly (P less than 0.001) less NK activity (20 +/- 17 lytic units at 25% specific lysis)/10(6) peripheral blood lymphocytes) against the K562 tumor target compared to normal (69 +/- 9, SD). NK subpopulations can be defined phenotypically using CD56, CD16, and CD3 monoclonal antibodies and express differing degrees of lytic activity. NK cells from patients with carcinoma had the same absolute number of CD56+ cells and percentage of CD56+CD16+ NK cells (the most lytic subpopulation). However, patients with carcinoma had significantly (P less than 0.001) more CD56+CD16-CD3- cells in their overall NK population. This is of note, since this subpopulation is the least lytic. Patient NK cells bound to tumor cells as effectively as those from normal volunteers; however, the maximum rate of kill of patient NK cells was significantly (P less than 0.001) less. Thus, decreased NK lytic activity in patients with carcinoma was not due to decreased numbers or proportion of NK cells in peripheral blood or to defective tumor cell binding, but rather to the large CD56+CD16-CD3-NK subpopulation which is characterized by minimal lytic activity. The relation of this NK cell population with the presence of carcinoma is currently under investigation.

Immunohistochemical study with monoclonal antibodies on immune response in human lung cancers.

Fifty consecutively resected lungs in the National Cancer Center of Japan were studied immunohistochemically using monoclonal antibodies (B1, B2, Leu4, Leu3a, Leu2a, Leu7, OKT6, OKM1, OKI1, OKT9) with special reference to infiltrating lymphocytes and histiocytes. The histological types of these cases were: 14 squamous cell carcinomas; 23 adenocarcinomas; four adenosquamous carcinomas; three large cell carcinomas; one small cell carcinoma with areas of large cell component; one carcinoid; and four nonneoplastic focal lesions. A large number of lymphocytes and histiocytes was present in the tumor stroma, especially in papillary adenocarcinoma, in which close association between increased OKT6-positive T-zone histiocytes (Langerhans' cells and their precursors) and proliferation of Leu3a-positive helper-inducer T-lymphocytes was conspicuous. B1-positive primary follicles were occasionally associated with proliferation of Leu3a-positive T-lymphocytes. These features correlated to regional lymph node reaction. Leu7-positive natural killer cells were present sparsely, and there was no suggestion of any significant contact with either neoplastic cells or inflammatory cells.

Expression of HLA class I and II antigens in bronchogenic carcinomas: its relationship to cellular DNA content and clinical-pathological parameters.

We studied the presence of HLA class I antigens in 115 samples of bronchogenic carcinomas (66 frozen and 49 formalin-fixed and paraffin-embedded specimens) by the immunophosphatase alkaline and immunoperoxidase methods with antibodies against major histocompatibility complex antigens. We also studied HLA class II antigens on the 66 frozen tumor samples. Nonneoplastic lung tissue was also analyzed for purposes of comparison. Pneumocytes and epithelial respiratory cells expressed HLA class I and II antigens. The expression of class I antigens was totally lost in 29 tumors (25%). The defect in HLA gene expression affected both heavy chain and beta 2-microglobulin, as demonstrated by the null reactivity with specific antibodies. In 2 cases of 66 studied in cryostatic section, the selective loss of A locus was observed, and in three cases selective loss of B locus was detected. The expression of class I antigens was compared with clinical-pathological parameters such as histological type, degree of differentiation, and tumor stage, as well as tumoral ploidy. The absence of expression of HLA class I molecules was significantly associated with poorly differentiated and undifferentiated tumors (P less than 0.0001) and with aneuploid tumors (P less than 0.001), suggesting that some lung tumors may escape immune surveillance and become biologically more aggressive. Class II antigens were expressed in 13 cases of 66 studied (18%) in frozen specimens, and a clear relationship was observed with well-differentiated tumors (P less than 0.05).

Herpes zoster and small cell bronchogenic carcinoma.

In nine of 74 (12 per cent) consecutive, previously untreated patients with small cell bronchogenic carcinoma receiving combination chemotherapy herpes zoster developed. This is the highest frequenzy reported for this viral infection in patients with nonlymphoproliferative solid tumors. Cutaneous dissemination developed in six of the nine patients, but visceral involvement did not occur. The major difference between the patients with herpes zoster and those without was the superior duration of median survival for the infected patients. No relationship could be established between the development of herpes zoster and the extent of neoplastic disease, prior radiotherapy, treatment with specific chemotherapeutic agents or corticosteroids, cutaneous anergy or granulocytopenia. Serum specimens obtained from six of the nine patients prior to their infection demonstrated the preexistence of varicella zoster antibodies. As more effective and intensive chemotherapy prolongs the survival of patients with solid tumors, it is possible that the frequency of herpes zoster infection may approach that observed in patients with lymphoproliferative malignancies.

T-cell receptor biases and clonal proliferations in blood and pleural effusions of patients with lung cancer.

We sought evidence that pulmonary carcinomas mediate a cellular immunologic response by analyzing T-cell antigen receptor beta-chain variable gene (TCRBV) repertoires of lymphocytes from peripheral blood (PBL) and malignant pleural effusions (PEL) of five lung cancer patients. Expression levels of 27 TCRBV were quantitated by multiprobe RNase protection assay (RPA), and clonal expansions were identified by sequence enrichment nuclease assay (SENA) and junctional region sequencing. Abnormal TCRBV expansions were identified in all subjects by RPA (mean 6.9 +/- 1.7/patient), and their number closely correlated with elapsed time since initial diagnosis (r = 0.97). SENA, performed in specimens from three patients, confirmed the presence of mono or oligoclonality in 48% of abnormal RPA expansions, and further identified T-cell clones among TCRBV with normal expression levels. The majority of clonal expansions were among PEL, and were nearly equally divided between CD4 and CD8. These data show that T-cell repertoires of lung cancer patients are characterized by marked abnormalities and frequent clonal expansions, most likely representing responses to unique, tumor-specific antigens (TSA). Moreover, this process appears exaggerated among PEL, further suggesting that malignant effusions include local proliferations of tumor reactive T cells. These findings imply the presence of lung cancer TSA capable of eliciting cellular immune responses and raise the possibility that selective immunotherapies can ultimately be developed.

Bronchogenic carcinoma: immunologic aspects.

Evidence that host immunologic function may influence the behavior of lung cancer is accumulating. Non-small-cell lung cancers are heavily infiltrated by host lymphocytes. The fact that monoclonal antibodies have been developed against lung cancer cells implies that such cells express surface antigens and are therefore vulnerable to immune recognition. Failure of the host defense mechanism to control tumor growth may involve (1) reduced natural killer cell activity, (2) inadequate lymphokine-activated killer cell function, or (3) tumor secretion of immunomodulating factors. Basic immunologic research studies of lung cancer are increasing the potential for clinical applications. New monoclonal antibodies have improved both the sensitivity and the specificity of immunohistopathologic analyses of pulmonary specimens. Links between immune function and prognosis in patients with lung cancer have been established. Finally, initial results from protocols that have used tumor-infiltrating lymphocytes, interleukin 2, and tumor vaccines suggest that immunobiologic treatment modalities may be increasingly applicable in patients with lung cancer.