Prediction of response to pemetrexed in non-small-cell lung cancer with immunohistochemical phenotyping based on gene expression profiles.

BACKGROUND: Palliative pemetrexed-based chemotherapy remains a standard of care treatment for the majority of patients with advanced non-squamous non-small-cell lung cancer (NSCLC). Currently, no predictive markers for pemetrexed treatment are available. METHODS: Resected tumour samples from pemetrexed-naïve NSCLC patients were collected. Gene expression profiling with respect to predicted sensitivity to pemetrexed classified predicted responders (60%) and non-responders (40%) based on differentially expressed genes encoding for pemetrexed target enzymes. Genes showing a strong correlation with these target genes were selected for measurement of corresponding protein expressions by immunohistochemical (IHC) staining. A semi-quantitative IHC scoring method was applied to construct a prediction model for response to pemetrexed. A retrospective cohort of patients with advanced NSCLC treated with first-line pemetrexed-based chemotherapy was used for external validation. RESULTS: From ninety-one patients resected tumour samples were collected. The majority of patients had early or locally advanced NSCLC (96.3%). Gene expression profiling revealed five markers, which mRNA levels strongly correlated to pemetrexed target genes mRNA levels: TPX2, CPA3, EZH2, MCM2 and TOP2A. Of 63 (69%) patients IHC staining scores of these markers were obtained, which significantly differed between predicted non-responders and responders (P < 0.05). The optimized prediction model included EZH2 (OR = 0.56, 95% CI 0.35-0.90) and TPX2 (OR = 0.55, 95% CI 0.30-1.01). The model had a sensitivity of 86.8%, specificity of 63.6% and showed a good ability to distinct between responders and non-responders (C-index 0.86). In the external study population (N = 23) the majority of patients had metastatic NSCLC (95.7%). Partial response (PR) was established in 26.1%. The sensitivity decreased drastically to 33.3%, with a specificity of 82.4% and a C-index of 0.73. CONCLUSIONS: Using external validation this prediction model with IHC staining of target enzyme correlated markers showed a good discrimination, but lacked sensitivity. The role of IHC markers as response predictors for pemetrexed in clinical practice remains questionable.

Tumor-associated macrophages in thoracic malignancies.

Tumor-associated macrophages (TAMs) can be abundantly present in numerous cancer types. Under influence of various stimuli in the tumor microenvironment TAMs develop into a tumor-inhibitory (M1) or tumor-promoting (M2) phenotype. Recently, the role of TAMs in tumor biology and their prognostic value in cancer has become a major topic of interest. In this review we will discuss the importance of TAMs in the pathogenesis and clinical outcome of lung cancer and mesothelioma patients. In addition, the potential of TAMs as therapeutic targets will be discussed.

New roads open up for implementing immunotherapy in mesothelioma.

Treatment options for malignant mesothelioma are limited, and the results with conventional therapies have been rather disappointing to this date. Chemotherapy is the only evidence-based treatment for mesothelioma patients in good clinical condition, with an increase in median survival of only 2 months. Therefore, there is urgent need for a different approach to battle this malignancy. As chronic inflammation precedes mesothelioma, the immune system plays a key role in the initiation of this type of tumour. Also, many immunological cell types can be found within the tumour at different stages of the disease. However, mesothelioma cells can evade the surveillance capacity of the immune system. They build a protective tumour microenvironment to harness themselves against the immune system's attacks, in which they even abuse immune cells to act against the antitumour immune response. In our opinion, modulating the immune system simultaneously with the targeting of mesothelioma tumour cells might prove to be a superior treatment. However, this strategy is challenging since the tumour microenvironment possesses numerous forms of defence strategies. In this paper, we will discuss the interplay between immunological cells that can either inhibit or stimulate tumour growth and the challenges associated with immunotherapy. We will provide possible strategies and discuss opportunities to overcome these problems.

Zoledronic acid impairs myeloid differentiation to tumour-associated macrophages in mesothelioma.

BACKGROUND: Suppressive immune cells present in tumour microenvironments are known to augment tumour growth and hamper efficacy of antitumour therapies. The amino-bisphosphonate Zoledronic acid (ZA) is considered as an antitumour agent, as recent studies showed that ZA prolongs disease-free survival in cancer patients. The exact mechanism is a topic of debate; it has been suggested that ZA targets tumour-associated macrophages (TAMs). METHODS: We investigate the role of ZA on the myeloid differentiation to TAMs in murine mesothelioma in vivo and in vitro. Mice were intraperitoneally inoculated with a lethal dose of mesothelioma tumour cells and treated with ZA to determine the effects on myeloid differentiation and survival. RESULTS: We show that ZA impaired myeloid differentiation. Inhibition of myeloid differentiation led to a reduction in TAMs, but the number of immature myeloid cells with myeloid-derived suppressor cell (MDSC) characteristics was increased. In addition, ZA affects the phenotype of macrophages leading to reduced level of TAM-associated cytokines in the tumour microenvironment. No improvement of survival was observed. CONCLUSION: We conclude that ZA leads to a reduction in macrophages and impairs polarisation towards an M2 phenotype, but this was associated with an increase in the number of immature myeloid cells, which might diminish the effects of ZA on survival.

HIF1a expression in bronchial biopsies correlates with tumor microvascular saturation determined using optical spectroscopy.

Tumor hypoxia is generally considered to be related to aggressive behaviour of a tumor. As in lung cancer direct determination of oxygenation is difficult, hypoxia-related proteins have been studied. A number of studies on these proteins show different results and the usefulness of these protein expressions remains questionable. In this article, we relate one of these hypoxia-related proteins (hypoxia-inducible factor, HIF1a) to a direct in vivo spectroscopic measurement of tumor blood saturation performed during bronchoscopy. Seventeen samples from malignancies and non-malignant tissues were studied. Microvascular saturation levels in the no malignancy group equalled 87+/-11.5% (range 71-100%) and in the malignant group 43+/-21% (range 6-63%). This difference was statistically significant (p<0.0002). There was a significant difference in the spectroscopically determined saturations between the biopsies with negative expression of HIF1a and the biopsies with positive expression of HIF1a (p<0.005). From these data, it can be concluded that HIF1a expression is related to a low microvascular blood saturation as determined in vivo by optical spectroscopy. This study may lead to a better acceptance of the usage of different techniques to establish hypoxia in order to study the effect of hypoxia on therapeutic interventions and prognosis of lung cancer.

Mesothelioma environment comprises cytokines and T-regulatory cells that suppress immune responses.

Malignant mesothelioma is a cancer with dismal prognosis. The objective of the present study was to address the role of the immune system, tumour micro-environment and potential immunosuppression in mesothelioma. Expression profiles of 80 cytokines were determined in the supernatant of mesothelioma cell lines and the original patient's pleural effusion. Influx of immune effector cells was detected by immunohistochemistry. Angiogenin, vascular endothelial growth factor, transforming growth factor-beta, epithelial neutrophil-activating protein-78 and several other proteins involved in immune suppression, angiogenesis and plasma extravasation could be detected in both supernatant and pleural effusion. Surrounding stroma and/or infiltrating cells were the most likely source of hepatocyte growth factor, macrophage inflammatory protein (MIP)-1delta, MIP-3alpha, neutrophil-activating peptide-2, and pulmonary and activation-regulated chemokine that can cause leukocyte infiltration and activation. There was a massive influx of CD4+ and CD8+ T-lymphocytes and macrophages, but not of dendritic cells, in human mesothelioma biopsies. It was further demonstrated that human mesothelioma tissue contained significant amounts of Foxp3+CD4+CD25+ regulatory T-cells. When these CD25+ regulatory T-cells were depleted in an in vivo mouse model, survival increased. Mesothelioma is infiltrated by immune effector cells but also contains cytokines and regulatory T-cells that suppress an efficient immune response. Immunotherapy of mesothelioma might be more effective when combined with drugs that eliminate or control regulatory T-cells.

Effects of fluticasone propionate in COPD patients with bronchial hyperresponsiveness.

BACKGROUND: Treatment of chronic obstructive pulmonary disease (COPD) with inhaled corticosteroids does not appear to be as effective as similar treatment of asthma. It seems that only certain subgroups of patients with COPD benefit from steroid treatment. A study was undertaken to examine whether inhaled fluticasone propionate (FP) had an effect on lung function and on indices of inflammation in a subgroup of COPD patients with bronchial hyperresponsiveness (BHR). METHODS: Twenty three patients with COPD were studied. Patients had to be persistent current smokers between 40 and 70 years of age. Non-specific BHR was defined as a PC(20) for histamine of

A model system for optimising the selection of membrane antigen-specific human antibodies on intact cells using phage antibody display technology.

The functional expression of human antibody fragments on the surface of filamentous bacteriophage, and selection of phage antibodies (PhAbs) with antigens, has provided a powerful tool for generating novel antibodies. Applications of phage antibody display technology have increased over the past decade. Successful isolation of phage antibodies has been reported mostly using purified antigens. Isolation has proven to be more complicated with complex mixtures of antigens, such as intact cells. A given cell type contains thousands of different epitopes, each capable in theory of binding phage antibodies. Often antigens are not known or cannot be purified without disrupting their conformational integrity. To overcome problems involving phage antibody selections on intact cells, we have developed an experimental model system that allows for optimisation and comparison of various selection strategies. The model system comprises labelling of intact cells with the fluorescently labelled phospholipid fluorescein-DHPE. Upon incubation, this phospholipid is readily incorporated in the membrane of any cell type. Labelling intensity is regulated by varying the phospholipid concentration. After optimisation of key steps in the selection procedure, we were able to isolate fluorescein-DHPE specific phage from a synthetic library using intact cells. This model system can be applied to any cell type and we demonstrate that it can be used to efficiently compare and optimise selection strategies.