Optimising primary molecular profiling in non-small cell lung cancer.

INTRODUCTION: Molecular profiling of NSCLC is essential for optimising treatment decisions, but often incomplete. We assessed the efficacy of protocolised molecular profiling in the current standard-of-care (SoC) in a prospective observational study in the Netherlands and measured the effect of providing standardised diagnostic procedures. We also explored the potential of plasma-based molecular profiling in the primary diagnostic setting. METHODS: This multi-centre prospective study was designed to explore the performance of current clinical practice during the run-in phase using local SoC tissue profiling procedures. The subsequent phase was designed to investigate the extent to which comprehensive molecular profiling (CMP) can be maximized by protocolising tumour profiling. Successful molecular profiling was defined as completion of at least EGFR and ALK testing. Additionally, PD-L1 tumour proportions scores were explored. Lastly, the additional value of centralised plasma-based testing for EGFR and KRAS mutations using droplet digital PCR was evaluated. RESULTS: Total accrual was 878 patients, 22.0% had squamous cell carcinoma and 78.0% had non-squamous NSCLC. Stage I-III was seen in 54.0%, stage IV in 46.0%. Profiling of EGFR and ALK was performed in 69.9% of 136 patients included in the run-in phase, significantly more than real-world data estimates of 55% (p<0.001). Protocolised molecular profiling increased the rate to 77.0% (p = 0.049). EGFR and ALK profiling rates increased from 77.9% to 82.1% in non-squamous NSCLC and from 43.8% to 57.5% in squamous NSCLC. Plasma-based testing was feasible in 98.4% and identified oncogenic driver mutations in 7.1% of patients for whom tissue profiling was unfeasible. CONCLUSION: This study shows a high success rate of tissue-based molecular profiling that was significantly improved by a protocolised approach. Tissue-based profiling remains unfeasible for a substantial proportion of patients. Combined analysis of tumour tissue and circulating tumour DNA is a promising approach to allow adequate molecular profiling of more patients.

A hampered chemoattractant-induced cytoskeletal rearrangement in granulocytes of patients with unexplained severe chronic and relapsing infections of the upper and lower airways. In vitro restoration by G-CSF exposure.

Granulocytes play a major role in host defense against bacterial infections. Severe inborn defects in granulocyte function are associated with fulminant bacterial infections in early childhood. Subtle disturbances in granulocyte function might contribute to an enhanced susceptibility to bacterial infections in adulthood. We investigated chemoattractant (N-formyl-methionyl-leucyl-phenylalanine, fMLP and casein) induced cytoskeletal rearrangements (polarization) of blood granulocytes in 77 adults with chronic and recurrent therapy-resistant infections of the upper and lower airways. These infections could not be explained by B- and/or T-cell defects or local anatomic abnormalities. Besides polarization, chemotaxis of blood granulocytes was measured in 33 patients, as well as granulocyte superoxide production in eight patients. The chemoattractant-induced cytoskeletal rearrangement in patient blood granulocytes was significantly lower as compared to healthy control values with both fMLP and casein as stimuli. About two-thirds of the patients showed a defective polarization response to fMLP. Granulocyte colony-stimulating factor (G-CSF) when added in vitro corrected the defective polarization responses; responses in the normal range were not enhanced. The chemotactic motility of patient blood granulocytes was also slightly, but significantly lowered. However, it did not correlate to the lowered polarization. Granulocyte superoxide production was comparable in patients and in healthy controls. Our data thus show that subtle abnormalities in chemoattractant-induced cytoskeletal and motile function of blood granulocytes are frequent in patients with severe therapy-refractory bacterial infections of the upper and lower airways.

Multicentre phase II study of gemcitabine and cisplatin in malignant pleural mesothelioma.

UNLABELLED: Malignant pleural mesothelioma is a notoriously chemoresistant tumour. However, a recent single institution study showed an impressive activity of gemcitabine and cisplatin. Our aim is to investigate the efficacy and toxicity of a gemcitabine and cisplatin combination in selected and chemo-naive patients with histologically proven malignant pleural mesothelioma. METHOD: Gemcitabine 1250 mg m(-2) was administered on day 1 and day 8 and cisplatin 80 mg m(-2) was administered on day 1 in a 3-week cycle with a maximum of six cycles. Response and toxicity evaluations were performed according to WHO and NCIC-CTC criteria. Pathology and radiology were centrally reviewed. Results show that in 25 evaluable patients, four PR were observed (ORR 16%, 95% CI 1-31%). Responses of seven patients were unevaluable. No unexpected toxicity occurred. Time to progression was 6 months (5-7 months) with a median survival from registration of 9.6 months (95% CI 8-12 months). In conclusion this trial excludes with 90% power a response rate of greater than 30% in patients with malignant pleural mesothelioma using a combination of gemcitabine and cisplatin at the proposed dose and schedule.

Glucocorticoids hamper the ex vivo maturation of lung dendritic cells from their low autofluorescent precursors in the human bronchoalveolar lavage: decreases in allostimulatory capacity and expression of CD80 and CD86.

Dendritic cells (DCs) were prepared from human bronchoalveolar lavage (BAL) cells. We previously reported that, in particular, the CD1a fraction of the low autofluorescent (LAF) cells contains the precursors for DCs: after overnight culture, 40% of the LAF cells change into functionally and phenotypically prototypic dendritic/veiled cells. There are, as yet, no data on the modulatory effects of glucocorticoids (GC) on the maturation and function of such DCs isolated from the human lung. Functional tests (allogeneic mixed lymphocyte reaction: allo-MLR) were therefore performed with CD1a+ LAF cells at different stimulator-to-T-cell ratios and after preincubation with different dexamethasone (DEX) concentrations. DEX caused suppression of the T-cell stimulatory capacity of CD1a+ LAF cells, which was dose-dependent, and more evident at the higher stimulator-to-T-cell ratios. Here, we also show that CD80 and CD86 are normally expressed at low levels on CD1a+ LAF cell-derived DCs compared to other DC populations. This low-level expression of costimulatory molecules is discussed here in relation to the previously reported low-level expression of CD80 (and CD86) on lung DCs in experimental animals. This appears to play a role in a predominant Th2 cell stimulating potential of DC from the lung environment. DEX exposure of CD1a+ LAF cells prevented the upregulation of even this low-level expression of CD80 and CD86. The veiled/dendritic morphology and the expression of other relevant cell surface markers and adhesion molecules was not affected by DEX exposure. It is concluded that DEX hampers the maturation of CD1a+ LAF cells into active lung DCs.

CD1a+ and CD1a- accessory cells from human bronchoalveolar lavage differ in allostimulatory potential and cytokine production.

Recently, we described the isolation through fluorescent-activated cell sorting (FACS) of low autofluorescent (LAF) cells from human bronchoalveolar lavage (BAL). These LAF cells displayed an immunophenotype comparable with that of dendritic cells (DC), and showed a high potency to stimulate naive T cells. In the study reported here we investigated the capability of LAF cells to produce interleukin-1 (IL-1), IL-6, and tumor necrosis factor alpha (TNF-alpha), and the role of these cytokines in allogeneic T-cell stimulation by LAF cells. Lipopolysaccharide (LPS)-stimulated LAF cells released biologically active IL-1, IL-6, and TNF, and also showed intracellular immunoreactivity for IL-1, IL-6, and TNF-alpha. A neutralizing antibody against IL-1 slightly but statistically significantly (P < 0.05, Wilcoxon's test) inhibited the ability of the LAF cells to stimulate allogeneic T-cell proliferation (89% of stimulation in the absence of the antibody). Neutralizing antibodies against IL-6 and TNF-alpha had no effect. An antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF) also interfered with the accessory function of the LAF cells (79% of stimulation in the absence of the antibody, P < 0.05). We also investigated whether subsets of LAF cells (i.e., positive or negative for CD1a and purified by FACS sorting) differed in T-cell stimulatory capacity and in the ability to produce IL-1, IL-6, TNF-alpha, and S100. CD1a+ LAF cells were positive for and produced S100, CD1a- LAF cells were negative in this respect. The CD1a+ subset exhibited a clearly higher and very strong accessory capability as compared with the CD1a- subset. Despite this, CD1a+ LAF cells were poor producers of IL-1, IL-6, and TNF-alpha. The neutralizing antibody to IL-1, however, inhibited the ability of CD1a+ cells to stimulate allogeneic T-cell proliferation (43% of stimulation in the absence of the antibody, P < 0.01). Anti-IL-6 and alpha-GM-CSF had no effects. CD1a- LAF cells were potent producers of IL-1, IL-6, and TNF-alpha, and antibodies to IL-1, IL-6, and GM-CSF strongly interfered with their weaker accessory capability. In conclusion, two different subsets of LAF cells could be identified on the basis of accessory capability and cytokine profile. CD1a+ LAF cells (S100+; very potent T-cell stimulators, poor cytokine producers) are the "Langerhans cells" of the lung. CD1a- LAF cells (S100-; lower T-cell stimulatory capability, potent producers of IL-1, IL-6, and TNF-alpha) displayed a marker pattern intermediate between that of monocytes and monocyte-derived DC.

Increased numbers of dendritic cells in the bronchial mucosa of atopic asthmatic patients: downregulation by inhaled corticosteroids.

BACKGROUND: Dendritic cells (DC) are the most potent antigen-presenting cells (APC) and stimulators of T cells. Dendritic cells are also likely to be essential for the initiation of allergic immune responses in the lung. However, there are not many data on the presence of dendritic cells in the airways of patients with atopic asthma and on the effects of corticosteroid-treatment on such dendritic cells. OBJECTIVE: We investigated the distribution of dendritic cells in the bronchial epithelium and mucosa of 16 non-smoking atopic asthmatic patients and eight healthy control subjects using detailed immunohistochemistry (CD1a, HLA-DR, L25 as markers for dendritic cells). METHODS: Eleven asthmatics were treated for 2.5 years with bronchodilators only and five with bronchodilators and inhaled beclomethasone dipropionate (BDP), 800 micrograms daily. The patients were randomly sampled from a double-blind multicentre study. RESULTS: There were higher numbers of CD1a+ DC (P = 0.003), L25+ DC (P = 0.002) and HLA-DR expression (P = 0.042) in the bronchial mucosa of asthmatic patients compared with healthy controls. After 2.5 years of treatment, we found a significant increase in flow expiratory volume in 1 second (FEV1) (P = 0.009) and a significant decrease in hyperresponsiveness (PC20 histamine) (P = 0.013) in the corticosteroid group (n = 5) compared with the bronchodilator group (n = 11). This clinical improvement in the corticosteroid-treated group was accompanied by significantly lower numbers of CD1a+ DC (P = 0.008), and HLA-DR expression (P = 0.028) in the bronchial mucosa than in the bronchodilator-treated group. CONCLUSION: Our data suggest that dendritic cells are involved in asthmatic inflammation and that corticosteroids may downregulate the number of dendritic.

Dendritic cells and their precursors isolated from human bronchoalveolar lavage: immunocytologic and functional properties.

Human bronchoalveolar lavage (BAL) has been described to contain, besides a large number of alveolar macrophages (AM) (approximately 95%), small numbers of monocyte-like cells (approximately 2%) and dendritic cells (DC) (approximately 0.4%). To separate AM (high autofluorescence) from DC, we used a fluorescence activated cell sorter (FACS) to separate BAL cells into a low autofluorescent (LAF) fraction and a high autofluorescent (HAF) fraction. Immunocytologic and functional properties of these fractions were investigated. The LAF fraction was composed of acid phosphatase (APh)- and RFD9-negative cells, which were strongly positive for HLA-DR, L25, RFD1, and CD68. A portion of these cells expressed CD1a (22%) and My4 (60%). The marker pattern of these cells is reminiscent to that of intraepithelial bronchial DC and to that of blood DC. The majority of the LAF cells had a monocyte-like morphology, but after overnight culture the percentage of LAF cells with long cytoplasmic extensions (DC morphology) was strongly augmented (from 18 to 51%). The HAF fraction contained 100% AM, strongly positive for APh, HLA-DR, CD68, RFD7, and RFD9. In culture, the LAF cells formed clusters with T cells and vigorously stimulated the proliferation of allogeneic T cells and naive (CD45RO-negative) T cells. BAL and LAF cells produced higher responses in nonsmokers than in smokers. In contrast, HAF cells did not form clusters with T cells and did not stimulate allogeneic T cell proliferation. HAF cells even suppressed mitogen-driven T cell proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution and immunophenotype of mononuclear phagocytes and dendritic cells in the human lung.

Mononuclear phagocytes and dendritic cells (DC) play an important role in the immune response in the lung. DC act in the afferent phase of the immune response by presenting antigen to T cells, while macrophages play a role in the efferent phase by exerting phagocytic/cytotoxic functions. We investigated the localization and the marker pattern of these cells in the human lung. Macrophages, identified as large, rounded, acid phosphatase-positive cells, were mainly detected in the alveolar spaces, in the lumen of the bronch(iol)us, and in the bronchoalveolar lavage (BAL). They were positive for major histocompatibility complex (MHC) class II antigens (DR, DQ), CD68, RFD7, RFD9, and partly positive for RFD1. Irregularly shaped cells with a marker pattern comparable to that of blood-derived DC (positive for DR, DQ, L25, RFD1, and CD68) were predominantly observed in the epithelium and subepithelial tissue of the bronch(iol)us and in the bronchus-associated lymphoid tissue. In the epithelium, approximately 30% of these cells were positive for CD1a (OKT6). In the subepithelial tissue, these DC formed characteristic small clusters with T cells. The BAL, the alveolar spaces, and the alveolar walls contained only a small number of DC. These immunohistologic data suggest that the bronch(iol)us is well equipped to initiate immune responses. The high number of macrophages in the alveolar compartment, which have been described to suppress T cell proliferation, together with low numbers of DC, makes the alveolar compartment less suited for mounting an immune response.

Enrichment and characterization of dendritic cells from human bronchoalveolar lavages.

In the present study about 0.3% to 1.6% of human bronchoalveolar lavage (BAL) cells were identified as typical dendritic cells (DC), having an irregular outline, lobulated nucleus, and clear distinguishable acid phosphatase activity or EBM11 (anti-CD68) reactivity in a spot near the nucleus. After DC enrichment, using transient adherence to plastic, FcR-panning, and a density metrizamide gradient, a population containing 7-8% typical DC was obtained. This DC-enriched low density fraction, containing the highest percentages of DC, very strongly induced T cell proliferation in an allogeneic mixed leucocyte reaction (MLR), which was significantly higher than that induced by other partly (un)fractionated BAL cells. These data indicate that DC seem to be the major accessory cells in the BAL fluid, and therefore may be important in the regulation of T cell immune responses in the lung.