Kinetics of human myeloid-derived suppressor cells after blood draw.

BACKGROUND: Human myeloid-derived suppressor cells (MDSC) have been described as a group of immature myeloid cells which exert immunosuppressive action by inhibiting function of T lymphocytes. While there is a huge scientific interest to study these cells in multiple human diseases, the methodological approach varies substantially between published studies. This is problematic as human MDSC seem to be a sensible cell type concerning not only cryopreservation but also time point after blood draw. To date data on delayed blood processing influencing cell numbers and phenotype is missing. We therefore evaluated the kinetics of granulocytic MDSC (gMDSC) and monocytic MDSC (mMDSC) frequencies after blood draw in order to determine the best time point for analysis of this recently defined cell type. METHODS: In this study, we isolated peripheral blood mononuclear cells (PBMC) of patients with HIV infection or solid tumors directly after blood draw. We then analyzed the frequencies of gMDSC and mMDSC 2, 4 and 6 h after blood draw and after an overnight rest by FACS analysis using the standard phenotypic markers. In addition, part of the cells was frozen directly after PBMC preparation and was measured after thawing. RESULTS: gMDSC levels showed no significant difference using fresh PBMC over time with a limitation for the overnight sample. However they were massively diminished after freezing (p = 0.0001 for all subjects). In contrast, frequencies of fresh mMDSC varied over time with no difference between time point 2 and 4 h but a significantly reduction after 6 h and overnight rest (p = 0.0005 and p = 0.005 respectively). Freezing of PBMC decreased the yield of mMDSC reaching statistical significance (p = 0.04). For both MDSC subgroups, FACS analysis became more difficult over time due to less sharp divisions between populations. CONCLUSIONS: According to our data human MDSC need to be studied on fresh PBMC. gMDSC can be studied with delay, mMDSC however should be studied no later than 4 h after blood draw. These results are crucial as an increasing number of clinical trials aim at analyzing MDSC nowadays and the logistics of blood processing implies delayed sample processing in some cases.

Response to chemotherapy, reexposure to crizotinib and treatment with a novel ALK inhibitor in a patient with acquired crizotinib resistance.

The treatment of advanced non-small cell lung cancer (NSCLC) has dramatically changed over the last decade. It has developed from an unspecific approach based on platinum doublet chemotherapy to a personalized, molecularly targeted therapy. Crizotinib is a new tyrosine kinase inhibitor approved for the treatment of NSCLC with gene rearrangement of EML4 and ALK. Despite good initial responses, patients treated with crizotinib relapse after an average of 10 months. In this case report, we present a patient with acquired crizotinib resistance whose adenocarcinoma responded to a second course of crizotinib following a drug holiday and chemotherapy with pemetrexed. This is the second case report to suggest that retreatment with crizotinib is an option for patients with initial benefit from ALK inhibition.

Lack of significant elevation of myeloid-derived suppressor cells in peripheral blood of chronically hepatitis C virus-infected individuals.

Myeloid-derived suppressor cells (MDSC) are immature myeloid cells with immunosuppressive function. Compared to the level in healthy controls (HC), no elevation of MDSC in chronic hepatitis C (cHEP-C) patients was found, and there was no difference in MDSC based on genotype or viral load (P > 0.25). Moreover, MDSC of cHEP-C patients inhibited CD8 T cell function as efficiently as MDSC of HC did. Since we detected neither quantitative nor qualitative differences in MDSC of cHEP-C patients relative to those of HC, we postulate that MDSC in peripheral blood are most likely not significant regarding immune dysfunction in cHEP-C.

Preselection based on clinical characteristics in German non-small-cell lung cancer patients screened for EML4-ALK translocation.

BACKGROUND: The advent of multiple molecular targets in advanced non-small-cell lung cancer (NSCLC) has brought new treatments, but also new logistic and technical considerations, to the clinician. The small size of endoscopic biopsies and the increasing number of relevant but uncommon markers has increased the need for rational approaches to molecular testing. We present the results of clinical preselection before EML4-ALK testing in a German NSCLC cohort. METHODS: Patients with stage IV NSCLC were included. Clinicians were encouraged to consider screening epidermal growth factor receptor wild-type adenocarcinoma patients with a limited smoking history, relatively young age, or who had benefited from chemotherapy for a relatively long period. Break-apart fluorescence in situ hybridization using archived paraffin tissue was performed in a central facility. RESULTS: From April 2010 to September 2011 we included 61 patients: mean age 56.6 years, 41% women, 90% adenocarcinoma, 5% large-cell, and 5% squamous cell cancers. Only three patients had activating epidermal growth factor receptor mutations; 16.4% of patients were positive for EML4-ALK fusion. The anaplastic lymphoma kinase (ALK)-positive patients included 60% women, tended to be younger, had smoked less, and had received significantly more systemic therapy, on average 3.7 lines of treatment over 3 years, before ALK-testing compared with the ALK-negative patients. Long periods of progression-free survival were experienced by ALK-positive patients treated with pemetrexed, vinorelbine, or cetuximab. CONCLUSIONS: EML4-ALK fusion is uncommon, reported in about 5% of NSCLC patients; however, clinical preselection increased the yield of testing to 16.4%. EML4-ALK positive patients seem to have distinct clinical features and show long responses to a number of systemic therapies.

Schnitzler's disease as an important differential diagnosis of chronic recurrent multifocal osteomyelitis: a case report.

Introduction. At first sight, chronic recurrent multifocal osteomyelitis (CRMO) and Schnitzler's disease are diagnoses of exclusion and can be similar in their manifestation. Methods. In this paper we present the reevaluation of the 13-year-old diagnosis of chronic recurrent osteomyelitis of a 58-year-old man with chronic ostealgia, night sweat, and pruritic urticarial lesions on the extremities and trunk. For further examination, we performed blood analysis, bone and skin biopsies, CT scans, and magnetic resonance imaging. Results. Laboratory findings showed increased inflammation parameters. Magnetic resonance imaging (MRI) revealed a diffuse bone marrow infiltration. A bone and skin biopsy showed a sclerotic bone marrow involvement and a superficial dermal and perivascular infiltrate of neutrophils. Based on these findings, the diagnosis of Schnitzler's disease was made. Conclusion. Here, we want to present Schnitzler's disease as an important differential diagnosis to CRMO in adults presenting with signs suggestive of CRMO.

The hedgehog pathway inhibitor GDC-0449 alters intracellular Ca2+ homeostasis and inhibits cell growth in cisplatin-resistant lung cancer cells.

AIM: Cisplatin resistance is an important issue in lung cancer. We aimed at investigating if the Hedgehog pathway inhibitor GDC-0449 is effective in cisplatin-resistant cells and if it alters intracellular Ca(2+)-homeostasis. MATERIALS AND METHODS: The cytoplasmatic ([Ca(2+)](cyto)) and endoplasmatic ([Ca(2+)])(ER) Ca(2+) concentration of HCC (adeno carcinoma of the lung) and H1339 (small cell lung carcinoma) cells were measured with the calcium indicator dye Fura-2 AM. The expression of the inositol-1,4,5-trisphosphate receptor (IP(3)R) and sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) were analyzed using western blot analysis. RESULTS: GDC-0449 inhibited cell growth in cisplatin-naïve and -resistant cells. In both cell types, GDC-0449 increased [Ca(2+)](cyto) and reduced endoplasmatic [Ca(2+)](ER). Cisplatin failed to considerably alter Ca(2+) homeostasis in resistant cells. The effects of GDC-0449 on intracellular Ca(2+) homeostasis were not mediated by an altered expression of IP(3)R or SERCA. CONCLUSION: GDC-0449 alters intracellular Ca(2+) homeostasis and inhibits cell growth in cisplatin-resistant lung cancer cells.

Altered Ca2+-homeostasis of cisplatin-treated and low level resistant non-small-cell and small-cell lung cancer cells.

BACKGROUND: Chemotherapy often leads to encouraging responses in lung cancer. But, in the course of the treatment, resistance to chemotherapy ultimately limits the life expectancy of the patient. We aimed at investigating if treatment with cisplatin alters the intracellular Ca2+-homeostasis of lung cancer cells and how this may be related to cisplatin resistance. METHODS: The squamous cell lung carcinoma cell line EPLC M1 and the small cell lung cancer cell line H1339 were exposed to cisplatin analogue to the in vivo pharmacokinetics. Changes in the cytoplasmic Ca2+-concentration ([Ca2+]c) were recorded using fluorescence microscopy. Protein expression was quantified using immuno-fluorescence and Western Blot analysis. Changes in gene expression were accomplished by small-interfering (si) RNA techniques. RESULTS: Four "cycles" of cisplatin led to low level resistance in EPLC and H1339 cells. In the low level resistant cell clones, the Ca2+-content of the endoplasmic reticulum (ER) was decreased. In low level resistant EPLC cells, this was correlated with an increased expression of the inositol-1,4,5-trisphosphate receptor (IP3R). Inhibiting the increased expression of IP3R using siRNA, the low level resistance could be reversed. In low level resistant H1339 cells, the decreased Ca2+-content of the ER was correlated with a decreased expression of sarco/endoplasmic reticulum Ca2+-ATPases (SERCA). Decreasing the expression of SERCA in naïve H1339 cells resulted in low level cisplatin resistance. CONCLUSION: We conclude that cisplatin therapy leads to a decreased Ca2+-content of the ER thereby inducing low level resistance. This is caused by upregulation of the IP3R in EPLC and decreased expression of SERCA in H1339 cells.