Marlies E. Heuvers

Arginase-1 mRNA expression correlates with myeloid-derived suppressor cell levels in peripheral blood of NSCLC patients

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature and progenitor myeloid cells with immunosuppressive activity that are increased in cancer patients. Until now, the characterization of MDSC in humans was very challenging. The aim of this study was to determine the characterization and optimal assessment of MDSC and to investigate their presence and function in blood of advanced-stage NSCLC patients. We determined MDSC and lymphocyte populations in peripheral blood mononuclear cells (PBMC) samples of 185 treatment-naïve NSCLC patients and 20 healthy controls (HC). NSCLC patients had an increased population of PMN-MDSC compared to HC (p < 0.0001). Frequencies of CD4(+) and CD8(+) T-cells were significantly decreased in NSCLC patients (p < 0.0001 and p = 0.05). We found that PMN-MDSC were able to suppress T-cell proliferation in vitro. qRT-PCR showed that arginase-1 (Arg-1) mRNA is mainly expressed by MDSC and that the level of Arg-1 in PBMC correlates with the frequency of MDSC in PBMC (Spearmans rho: 0.797). There were significant differences in MDSC and lymphocyte populations between NSCLC patients and HC. We found that MDSC frequencies are stable up to six hours at room temperature after blood was drawn and that cryopreservation leads to a strong decrease of MDSC in PBMC. We show that Arg-1 mRNA expression is a valuable method to determine the levels of MDSC in peripheral blood of cancer patients. This method is therefore a useful alternative for the complex flowcytometric analysis in large multicenter patient studies.

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 systems 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.

Patient-tailored modulation of the immune system may revolutionize future lung cancer treatment

Cancer research has devoted most of its energy over the past decades on unraveling the control mechanisms within tumor cells that govern its behavior. From this we know that the onset of cancer is the result of cumulative genetic mutations and epigenetic alterations in tumor cells leading to an unregulated cell cycle, unlimited replicative potential and the possibility for tissue invasion and metastasis. Until recently it was often thought that tumors are more or less undetected or tolerated by the patient’s immune system causing the neoplastic cells to divide and spread without resistance. However, it is without any doubt that the tumor environment contains a wide variety of recruited host immune cells. These tumor infiltrating immune cells influence anti-tumor responses in opposing ways and emerges as a critical regulator of tumor growth. Here we provide a summary of the relevant immunological cell types and their complex and dynamic roles within an established tumor microenvironment. For this, we focus on both the systemic compartment as well as the local presence within the tumor microenvironment of late-stage non-small cell lung cancer (NSCLC), admitting that this multifaceted cellular composition will be different from earlier stages of the disease, between NSCLC patients. Understanding the paradoxical role that the immune system plays in cancer and increasing options for their modulation may alter the odds in favor of a more effective anti-tumor immune response. We predict that the future standard of care of lung cancer will involve patient-tailor-made combination therapies that associate (traditional) chemotherapeutic drugs and biologicals with immune modulating agents and in this way complement the therapeutic armamentarium for this disease.

Immunoglobulin-like transcript 3 is expressed by myeloid-derived suppressor cells and correlates with survival in patients with non-small cell lung cancer

Myeloid-derived suppressor cells (MDSCs) play an important role in immune suppression and accumulate under pathologic conditions such as cancer and chronic inflammation. They comprise a heterogeneous population of immature myeloid cells that exert their immunosuppressive function via a variety of mechanisms. Immunoglobulin-like transcript 3 (ILT3) is a receptor containing immunoreceptor tyrosine-based inhibition motifs (ITIMs) that can be expressed on antigen-presenting cells and is an important regulator of dendritic cell tolerance. ILT3 exists in a membrane-bound and a soluble form and can interact with a yet unidentified ligand on T cells and thereby induce T-cell anergy, regulatory T cells, or T suppressor cells. In this study, we analyzed freshly isolated peripheral blood mononuclear cells (PBMCs) of 105 patients with non-small cell lung cancer and 20 healthy controls and demonstrated for the first time that ILT3 is expressed on MDSCs. We show that increased levels of circulating MDSCs correlate with reduced survival. On the basis of ILT3 cell surface expression, an ILT3low and ILT3high population of polymorphonuclear (PMN)-MDSCs could be distinguished. Interestingly, in line with the immunosuppressive function of ILT3 on dendritic cells, patients with an increased proportion of PMN-MDSCs and an increased fraction of the ILT3high subset had a shorter median survival than patients with elevated PMN-MDSC and a smaller ILT3high fraction. No correlation between the ILT3high subset and other immune variables was found. ILT3 expressed on MDSCs might reflect a previously unknown mechanism by which this cell population induces immune suppression and could therefore be an attractive target for immune intervention.

History of tuberculosis as an independent prognostic factor for lung cancer survival

INTRODUCTION It is well known that pulmonary tuberculosis is associated with an increased risk of lung cancer. We investigated whether a history of pulmonary tuberculosis is an independent risk factor for lung cancer survival in Caucasian patients. METHODS The data of the prospective population-based cohort of The Rotterdam Study were used. During a mean follow-up time of 18 years, there were 214 incident cases of pathology-proven lung cancer in a source population of 7983 study participants. History of tuberculosis was assessed at baseline by interviewers using standardized questionnaires. Associations of lung cancer survival with the occurrence of pulmonary tuberculosis were assessed using Coxs proportional hazard regression analysis adjusted for age, gender, pack-years, educational level and tumor stage. RESULTS A history of tuberculosis was reported in 13 of the 214 subjects with lung cancer. The survival of patients with lung cancer was significantly shorter in subjects with a history of pulmonary tuberculosis (HR=2.36, CI95%: 1.1-4.9), than in subjects without a history of pulmonary tuberculosis with a mean difference of 311 days. CONCLUSION The presence of a history of pulmonary tuberculosis may be an important prognostic factor in the survival of lung cancer.

Improving lung cancer survival; time to move on.

BackgroundDuring the past decades, numerous efforts have been made to decrease the death rate among lung cancer patients. Nonetheless, the improvement in long-term survival has been limited and lung cancer is still a devastating disease.DiscussionWith this article we would like to point out that survival of lung cancer could be strongly improved by controlling two pivotal prognostic factors: stage and treatment. This is corresponding with recent reports that show a decrease in lung cancer mortality by screening programs. In addition, modulation of the patient’s immune system by immunotherapy either as monotherapy or combined with conventional cancer treatments offers the prospect of tailoring treatments much more precisely and has also been shown to lead to a better response to treatment and overall survival of non-small cell lung cancer patients.SummarySince only small improvements in survival can be expected in advanced disease with the use of conventional therapies, more research should be focused on lung cancer screening programs and patient tailored immunotherapy with or without conventional therapies. If these approaches are clinically combined in a standard multidisciplinary policy we might be able to advance the survival of patients with lung cancer.

Generalizing lung-cancer screening results

Investigators in the Rotterdam Study note that only about 30% of their study population met the entry criteria for the National Lung Screening Trial (NLST). Others at some risk were excluded; whether the 20% reduction in mortality seen in the NLST will transfer to other risk groups is unclear.

Dendritic cell-based immunotherapy in mesothelioma

Mesothelioma is a rare thoracic malignancy with a dismal prognosis. Current treatment options are scarce and clinical outcomes are rather disappointing. Due to the immunogenic nature of mesothelioma, several studies have investigated immunotherapeutic strategies to improve the prognosis of patients with mesothelioma. In the last decade, progress in knowledge of the modulation of the immune system to attack the tumor has been remarkable, but the optimal strategy for immunotherapy has yet to be unraveled. Because of their potent antigen-presenting capacity, dendritic cells are acknowledged as a promising agent in immunotherapeutic approaches in a number of malignancies. This review gives an update and provides a future perspective in which immunotherapy may improve the outcome of mesothelioma therapy.

Abstract 5394: Tumor-induced immunological changes in peripheral blood of untreated stage IV non-squamous NSCLC patients

Background Lung cancer is the most common cause of cancer mortality world wide. The five-year survival rate of stage III and IV non-small cell lung cancer (NSCLC) is poor with 16%. There is growing evidence that the immune system plays a paradoxical role in the development and progression of lung cancer. It influences tumor incidence, tumor growth, response to therapy, and the prognosis of the disease. Mouse models show that myeloid-derived suppressor cells (MDSCs) play a role in these processes. MDSCs are a heterogeneous population of immature myeloid cells and myeloid progenitor cells, which are present in the tumor micro-environment and the peripheral circulation of cancer patients. These cells are able to suppress features of immune responses, like T-cell proliferation and cytokine production. Aim In the present study we want to determine the role of pivotal immunological populations such as CD4+ T-cells, CD8+ T-cells, and MDSCs. Methods Fresh blood of 60 stage IV non-squamous-NSCLC patients and 15 healthy controls (HC) was studied. Mononuclear cells were purified from peripheral blood by density gradient centrifugation and analysed by flowcytometry. MDSCs were characterized as CD11b, CD15 CD33 positive and low expression of HLA-DR and CD16 cells. Suppressive activity of MDSCs was measured by CD3α-chain expression assays, and reactive-oxygen species (ROS) production using flowcytometry. Also T-cell proliferation assays were performed, in which sorted MDSCs were co-cultured with activated CD8-T-cells to investigate whether the MDSCs could inhibit the T-cell proliferation. Results All patients had a significantly increased MDSC population compared to healthy controls (p = 0.0009). Peripheral blood fractions possessed strong immunosuppressive functions, as shown by a high ROS production by MDSC, a decreased CD3α-chain expression in T cells compared to HC. In addition, the CD8+ T-cells showed no proliferation when co-cultured with MDSCs, indicating that MDSCs could block CD8+ -T cell proliferation. The populations of CD4+ T-cells and CD8+ T-cells were significantly decreased in lung cancer patients compared to healthy controls (p = 0.01 and p = 0.0024, respectively). Conclusion Circulating MDSCs are significantly increased, while CD4+ T-cells and CD8+ T-cells are significantly decreased in stage IV non-squamous-NSCLC patients compared to controls. MDSCs suppress the immune system by ROS production, down regulating the CD3α-chain expression and thereby inhibiting T-cell proliferation. This suggests that MDSCs may play an important role in disease progression and overall survival. Therefore, further investigation is warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5394. doi:1538-7445.AM2012-5394

Sirolimus-related dyspnoea, airway obstruction and pleural effusion after lung transplantation

Renal insufficiency remains a major side-effect of the treatment with calcineurin inhibitors after organ transplantation and when it appears, switching to a calcineurin inhibitor (CNI)-free regime including sirolimus or everolimus may be a favourable possibility. Sirolimus is a mammalian target of rapamycin (mTOR) inhibitor, which has been reported to exhibit less renal toxicity than calcineurin inhibitors [1,2]. However, sirolimus can have other serious side-effects such as dose-dependent myelosuppression, hypertension, hyperlipidaemia, glucose intolerance, dehiscence of the bronchial anastomosis, interstitial pneumonitis and pulmonary vasculitis [3–6]. In this report, we describe a patient who showed deterioration of lung function, after being switched from tacrolimus to sirolimus. A 47-year-old female, with a smoking history of 20 pack-years, underwent bilateral sequential lung transplantation because of pulmonary emphysema. After transplantation, she was treated with an immunosuppressive regimen including tacrolimus (initial trough levels 15–20 lg/l), mycophenolate and prednisolone combined with induction therapy with daclizumab (1 mg/kg body weight) on days 0 and 10. After discharge, her clinical course was unremarkable without signs of acute or chronic rejection. Her FEV1 increased from 0.40 l (16% predicted, pre-transplantation) to 2.50 l (102% predicted, 9 months post-transplantation). However, a gradual decrease of renal function developed. Two years postoperatively, her serum creatinine had increased to 223 lmol/l and her calculated serum creatinine clearance had dropped to 22 ml/min. At this point, we decided to prescribe sirolimus (initial trough levels 10–15 lg/l) instead of tacrolimus. Her renal function improved significantly, illustrated by a decrease in serum creatinine level to 131 lmol/l (calculated creatinine clearance 40 ml/min) 35 days after the switch. Unfortunately, 21 days after the switch, she presented with shortness of breath. At that time a drop in the FEV1 was seen, with the lowest FEV1 of 53% suggesting bronchiolitis obliterans syndrome (BOS) stage 2. Lung function was obstructive as the Tiffeneau index (FEV1/FVC) decreased from 88% (110% of predicted) to 73% (91% of predicted). Chest X-ray and CT-scan revealed bilateral pleural effusion (see Fig. 1 panels A and D). Furthermore, slight peripheral oedema and proteinuria were observed. We considered five main causes for the decline in FEV1 and appearance of pleural effusion. First, we considered acute rejection because sirolimus is generally regarded as a less powerful immunosuppressive agent than calcineurin inhibitors. Although we could prove neither rejection nor interstitial pneumonitis by biopsy, we decided to give high doses of methylprednisolone (1000 mg/day for three consecutive days). This did not result in significant improvement in lung function and therefore the drop in FEV1 and the pleural effusion were unlikely to be a result of acute rejection. Second, we considered infection. At the time of admission her C-reactive protein (CRP) was 32 mg/l (reference value <9 mg/l). Elevated CRP-levels have been described earlier possibly attributable to sirolimus therapy, although the authors underlined that CRP remains an unspecific marker [6]. She did not have a productive cough and there was no evidence of bronchitis. On the chest X-ray, there were no signs of pneumonia. Her bronchial lavage fluid showed Haemophilus influenzae, but lung function did not improve upon treatment with either amoxycillin or cefuroxime. Therefore, infection was considered an unlikely cause of the drop in FEV1. Unfortunately, no thoracentesis was performed. Third, hypoproteinaemia may be a cause of pleural effusion. However, the proteinuria was not in the nephrotic range (maximal protein loss 4.08 g/l) and lowest serum albumin level was 37 g/l. Fourth, cardiac dysfunction may result in pleural effusion, but in this patient cardiac evaluation was unremarkable. Despite polypragmatic treatment, her FEV1 did not improve and eventually her decline in FEV1, the appearance of bilateral pleural effusion, in combination with the development of proteinuria and peripheral oedema were all regarded as secondary to sirolimus. Therefore, after 63 days of treatment with sirolimus, we decided to reconvert her to tacrolimus. Thirty-five days after this conversion, her FEV1 had increased significantly to 84% (BOS