A previously unknown Argonaute 2 variant positively modulates the viability of melanoma cells.

In malignant melanoma, a highly aggressive form of skin cancer, many microRNAs are aberrantly expressed contributing to tumorigenesis and progression. Further, deregulation of microRNA processing enzymes, like the miRNA-binding protein Argonaute 2, significantly impacts microRNA function. This study characterizes a novel splice variant of Argonaut 2, AGO2-ex1/3. AGO2-ex1/3 is substantially expressed in different melanoma cell lines and patient-derived tissue samples. It is a mature mRNA, which is translated into an N-terminally truncated Argonaute 2 protein form. Molecular dynamics simulations show that the PAZ, MID, and PIWI domain largely retain their structure in AGO2-ex1/3 and that the truncation of the N-terminus leads to an increased interdomain flexibility. Expression of AGO2-ex1/3 provides a survival advantage for melanoma cells while the knockdown causes significantly reduced proliferation and increases apoptosis. RNA-sequencing revealed that in cells lacking AGO2-ex1/3 expression many miRNA target genes are deregulated, implicating a considerable role of AGO2-ex1/3 for miRNA function. This study inaugurates insights into an important role of a so far unknown splice variant of Argonaute 2 for the miRNA pathway as well as the mechanisms which drive growth and survival of melanoma cells. This knowledge provides the basis for potential new promising therapeutic targets focusing on small RNA-mediated gene regulation in melanoma.

Knockdown of Lamin B1 and the Corresponding Lamin B Receptor Leads to Changes in Heterochromatin State and Senescence Induction in Malignant Melanoma.

Modifications in nuclear structures of cells are implicated in several diseases including cancer. They result in changes in nuclear activity, structural dynamics and cell signalling. However, the role of the nuclear lamina and related proteins in malignant melanoma is still unknown. Its molecular characterisation might lead to a deeper understanding and the development of new therapy approaches. In this study, we analysed the functional effects of dysregulated nuclear lamin B1 (LMNB1) and its nuclear receptor (LBR). According to their cellular localisation and function, we revealed that these genes are crucially involved in nuclear processes like chromatin organisation. RNA sequencing and differential gene expression analysis after knockdown of LMNB1 and LBR revealed their implication in important cellular processes driving ER stress leading to senescence and changes in chromatin state, which were also experimentally validated. We determined that melanoma cells need both molecules independently to prevent senescence. Hence, downregulation of both molecules in a BRAFV600E melanocytic senescence model as well as in etoposide-treated melanoma cells indicates both as potential senescence markers in melanoma. Our findings suggest that LMNB1 and LBR influence senescence and affect nuclear processes like chromatin condensation and thus are functionally relevant for melanoma progression.

HDAC2 Is Involved in the Regulation of BRN3A in Melanocytes and Melanoma.

The neural crest transcription factor BRN3A is essential for the proliferation and survival of melanoma cells. It is frequently expressed in melanoma but not in normal melanocytes or benign nevi. The mechanisms underlying the aberrant expression of BRN3A are unknown. Here, we investigated the epigenetic regulation of BRN3A in melanocytes and melanoma cell lines treated with DNA methyltransferase (DNMT), histone acetyltransferase (HAT), and histone deacetylase (HDAC) inhibitors. DNMT and HAT inhibition did not significantly alter BRN3A expression levels, whereas panHDAC inhibition by trichostatin A led to increased expression. Treatment with the isoform-specific HDAC inhibitor mocetinostat, but not with PCI-34051, also increased BRN3A expression levels, suggesting that class I HDACs HDAC1, HDAC2, and HDAC3, and class IV HDAC11, were involved in the regulation of BRN3A expression. Transient silencing of HDACs 1, 2, 3, and 11 by siRNAs revealed that, specifically, HDAC2 inhibition was able to increase BRN3A expression. ChIP-Seq analysis uncovered that HDAC2 inhibition specifically increased H3K27ac levels at a distal enhancer region of the BRN3A gene. Altogether, our data suggest that HDAC2 is a key epigenetic regulator of BRN3A in melanocytes and melanoma cells. These results highlight the importance of epigenetic mechanisms in regulating melanoma oncogenes.

Molecular Changes Induced in Melanoma by Cell Culturing in 3D Alginate Hydrogels.

Alginate hydrogels have been used as a biomaterial for 3D culturing for several years. Here, gene expression patterns in melanoma cells cultivated in 3D alginate are compared to 2D cultures. It is well-known that 2D cell culture is not resembling the complex in vivo situation well. However, the use of very intricate 3D models does not allow performing high-throughput screening and analysis is highly complex. 3D cell culture strategies in hydrogels will better mimic the in vivo situation while they maintain feasibility for large-scale analysis. As alginate is an easy-to-use material and due to its favorable properties, it is commonly applied as a bioink component in the growing field of cell encapsulation and biofabrication. Yet, only a little information about the transcriptome in 3D cultures in hydrogels like alginate is available. In this study, changes in the transcriptome based on RNA-Seq data by cultivating melanoma cells in 3D alginate are analyzed and reveal marked changes compared to cells cultured on usual 2D tissue culture plastic. Deregulated genes represent valuable cues to signaling pathways and molecules affected by the culture method. Using this as a model system for tumor cell plasticity and heterogeneity, EGR1 is determined to play an important role in melanoma progression.

C-Jun drives melanoma progression in PTEN wild type melanoma cells.

Due to the critical impact of active AP-1 transcription factors in melanoma, it is important to define their target genes and to identify and ultimately inhibit oncogenic signals. Here we mapped the genome-wide occupancy of the AP-1 family member c-Jun in different melanoma cells and correlated AP-1 binding with transcriptome data to detect genes in melanoma regulated by c-Jun. Our analysis shows that c-Jun supports the malignant phenotype by deregulating genes in cancer-relevant signaling pathways, such as mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) pathways. Moreover, we demonstrate that the importance of c-Jun depends on melanoma stage and mutation status of the tumor suppressor PTEN. Our study reveals that activation of c-Jun overrules the tumor suppressive effect of PTEN in early melanoma development. These findings help to understand the relevance of c-Jun within cancer pathways in different melanoma cell types, especially in relation to MAPK and PI3K pathways, which are commonly deregulated in melanomas. Consequently, targeting c-Jun in PTEN+ melanoma cells may represent a promising therapeutic strategy to inhibit survival of melanoma cells to prevent the development of a metastatic phenotype.

Dendritic Cells Are Dispensable for T Cell Priming and Control of Acute Lymphocytic Choriomeningitis Virus Infection.

Dendritic cells (DCs) are considered to be the major APCs with potent activity for priming of naive CD4 and CD8 T cells. However, T cell priming can also be achieved by other APCs including macrophages, B cells, or even nonhematopoietic cell types. Systemic low-dose infection of mice with lymphocytic choriomeningitis virus (LCMV) results in massive expansion of virus-specific CD4 and CD8 T cells. To determine the role of DCs as APCs and source of type I IFNs in this infection model, we used ΔDC mice in which DCs are constitutively ablated because of expression of the diphtheria toxin α subunit within developing DCs. ΔDC mice showed lower serum concentrations of IFN-ß and IL-12p40, but normal IFN-α levels during the first days postinfection. No differences were found for proliferation of transferred TCR-transgenic cells during the early phase of infection, suggesting that T cell priming occurred with the same efficiency in wild-type and ΔDC mice. Expansion and cytokine expression of endogenous LCMV-specific T cells was comparable between wild-type and ΔDC mice during primary infection and upon rechallenge of memory mice. In both strains of infected mice the viral load was reduced below the limit of detection with the same kinetic. Further, germinal center formation and LCMV-specific Ab responses were not impaired in ΔDC mice. This indicates that DCs are dispensable as APCs for protective immunity against LCMV infection.

Repeated Radionuclide therapy in metastatic paraganglioma leading to the highest reported cumulative activity of 131I-MIBG.

131I-MIBG therapy for neuroendocrine tumours may be dose limited. The common range of applied cumulative activities is 10-40 GBq. We report the uneventful cumulative administration of 111 GBq (= 3 Ci) 131I-MIBG in a patient with metastatic paraganglioma. Ten courses of 131I-MIBG therapy were given within six years, accomplishing symptomatic, hormonal and tumour responses with no serious adverse effects. Chemotherapy with cisplatin/vinblastine/dacarbazine was the final treatment modality with temporary control of disease, but eventually the patient died of progression. The observed cumulative activity of 131I-MIBG represents the highest value reported to our knowledge, and even though 12.6 GBq of 90Y-DOTATOC were added intermediately, no associated relevant bone marrow, hepatic or other toxicity were observed. In an individual attempt to palliate metastatic disease high cumulative activity alone should not preclude the patient from repeat treatment.

Diagnostic performance of whole body dual modality 18F-FDG PET/CT imaging for N- and M-staging of malignant melanoma: experience with 250 consecutive patients.

PURPOSE: To assess the diagnostic performance of positron emission tomography/computed tomography (PET/CT) using (18)F-fluorodeoxyglucose (FDG) for N- and M-staging of cutaneous melanoma. PATIENTS AND METHODS: This is a retrospective and blinded study of 250 consecutive patients (105 women, 145 men; age 58 +/- 16 years) who underwent FDG-PET/CT for staging of cutaneous melanoma at different time points in the course of disease. Whole-body FDG-PET/CT was performed 101 +/- 21 minutes postinjection of 371 +/- 41 MBq FDG. Diagnostic accuracy for N- and M-staging was determined for CT alone, PET alone, and PET/CT. RESULTS: PET/CT detected significantly more visceral and nonvisceral metastases than PET alone and CT alone (98.7%, 88.8%, and 69.7%, respectively). PET/CT imaging thus provided significantly more accurate interpretations regarding overall N- and M-staging than PET alone and CT alone. Overall N- and M-stage was correctly determined by PET/CT in 243 of 250 patients (97.2%; 95% CI, 95.2% to 99.4%) compared with 232 patients (92.8%; 95% CI, 89.6% to 96.0%) by PET, and 197 patients (78.8%; 95% CI, 73.7% to 83.9%) by CT. All differences were significant. Accuracy of PET/CT was significantly higher than that of PET and CT for M-staging (0.98 v 0.93 and 0.84) and significantly higher than that of CT for N-Staging (0.98 v 0.86). Change of treatment according to PET/CT findings occurred in 121 patients (48.4%). CONCLUSION: The diagnostic performance of FDG-PET/CT for N- and M-staging of melanoma patients suggests its use for whole-body tumor staging, especially for detection or exclusion of distant metastases.

PET recognition of pulmonary metastases on PET/CT imaging: impact of attenuation-corrected and non-attenuation-corrected PET images.

PURPOSE: The aims of this study were to assess the performance of FDG PET at PET/CT imaging for the detection of pulmonary metastases and to evaluate differences in lesion detectability on attenuation-corrected (AC) and non-attenuation corrected (NAC) PET images. METHODS: The institutional PET/CT database was searched for patients with pulmonary metastases of 3-60 mm in diameter. Ninety-two patients with 438 metastases to the lungs were included in the study. The primary tumours were 33 malignant melanomas, 12 carcinomas of unknown primary, 11 colorectal carcinomas, eight differentiated thyroid carcinomas, seven aggressive non-Hodgkin's lymphomas, six head and neck cancers, three breast cancers, two prostate cancers and ten others. Lesion detectability was visually compared between PET and CT and between AC and NAC PET images using a five-point scale. RESULTS: Of the 438 pulmonary metastases, 174 were detected with FDG PET (39.7%), six of them on NAC images only (not significant). Visual scores were higher on NAC images in 41.4% and equal in 54.6% of lesions. The sensitivity of FDG PET increased significantly from 0.405 for metastases of 5-7 mm in diameter to 0.784 for lesions of 8-10 mm and to 0.935 for lesions measuring 11-29 mm in diameter. No metastases smaller than 5 mm in diameter were seen on PET images. CONCLUSION: FDG PET/CT is useful for the assessment of pulmonary metastases. The frequency of lesion detection is similar for AC and NAC PET images. A reduced sensitivity of FDG PET has to be considered for lesions smaller than 11 mm in diameter.

Tc-99m labelled anti CD 66 antigranulocyte antibody imaging for assessment of bone marrow reserve.

Patients with metastatic disease to the bone marrow are at risk of significant hematologic toxicity, if they undergo myelotoxic chemotherapy. Tc-99m labelled Anti CD 66 monoclonal antibody imaging is a useful, noninvasive approach to the assessment of bone marrow reserve under such circumstances.

Anti-CD30-scFv-Fc-IL-2 antibody-cytokine fusion protein that induces resting NK cells to highly efficient cytolysis of Hodgkin's lymphoma derived tumour cells.

The pathogenesis of Hodgkin's disease (HD) is associated with the accumulation of functionally anergic T cells in the near vicinity of the malignant Hodgkin/Reed-Sternberg (H/RS) cell. To stimulate locally the anti-tumour immunity in Hodgkin's disease, we generated an anti-CD30-antibody-interleukin-2 fusion protein (HRS3-scFv-Fc-IL-2) that binds to CD30 constitutively expressed on H/RS cells. The fusion protein is composed of a CD30 binding domain (HRS3-scFv) that is linked via the human IgG hinge-CH2/CH3 domain to human IL-2. The HRS3-scFv-Fc-IL-2 fusion protein is expressed as a 140 kDa homodimer, has binding specificities to both the CD30 antigen and the IL-2 receptor and stimulates proliferation of preactivated T cells in vitro, demonstrating its IL-2 bioactivity. After binding to CD30+ Hodgkin lymphoma cells, HRS3-scFv-Fc-IL-2 moreover induces resting NK cells, but not T cells, to lyse the lymphoma cells with high efficiency. Recruitment of resting NK cells towards a cytolytic immune response against CD30+ lymphoma cells has the potential to build up an effective anti-tumour response despite of Hodgkin's disease associated T-cell anergy and makes the HRS3-scFv-Fc-IL-2 fusion protein suitable for the specific immunotherapy of Hodgkin's lymphoma.

Radioimmunotherapy of non-Hodgkin's lymphoma with 90Y-DOTA humanized anti-CD22 IgG (90Y-Epratuzumab): do tumor targeting and dosimetry predict therapeutic response?

UNLABELLED: A DOTA (1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid)-conjugated, (111)In- and (90)Y-labeled humanized antibody to CD22, epratuzumab, was studied in patients with non-Hodgkin's lymphoma (NHL) to assess biodistribution and tumor targeting, pharmacokinetics, dosimetry, and anti-antibody response. Of particular interest was to evaluate whether pretherapy targeting and tumor dosimetry could predict therapeutic responses. METHODS: Patients received a pretherapy imaging study with (111)In-DOTA-epratuzumab IgG (0.75 mg/kg), followed about 1 wk later with (90)Y-DOTA-epratuzumab starting at a dose level of 0.185 GBq/m(2) (5 mCi/m(2)) in patients who had prior high-dose chemotherapy (group 2), and at 0.370 GBq/m(2) in patients who did not have a prior transplant (group 1), with escalation in 0.185-GBq/m(2) increments. RESULTS: The effective blood half-life for (111)In-DOTA epratuzumab was 36.1 +/- 7.9 h (n = 25) compared with 35.2 +/- 7.0 h for (90)Y-DOTA-epratuzumab (n = 22). The whole-body half-life for (90)Y-DOTA-epratuzumab estimated from (111)In-DOTA-epratuzumab scintigraphy was 58.3 +/- 4.7 h (n = 20), with urine collection confirming the loss of between 2.2% and 15.9% of the injected activity over 3 d (n = 3). One-hundred sixteen of 165 CT-confirmed lesions were visualized with (111)In-DOTA-epratuzumab. Radiation-absorbed doses to liver, lungs, and kidneys averaged 0.55 +/- 0.13 (n = 17), 0.28 +/- 0.06 (n = 17), and 0.38 +/- 0.07 mGy/MBq (n = 10), respectively, with 0.14 +/- 0.02 and 0.23 +/- 0.04 mGy/MBq delivered to the whole-body and red marrow, respectively. Tumor doses (n = 14 lesions in 10 patients) ranged from 1.0 to as much as 83 mGy/MBq for a 0.5-g lesion (median, 7.15 mGy/MBq). Group 2 patients were more likely to experience significant hematologic toxicities, but doses of up to 0.370 GBq/m(2) of (90)Y-DOTA-epratuzumab were tolerated with standard support measures, whereas patients in group 1 tolerated doses of up to 0.740 GBq/m(2) with the potential for further escalation. Anti-tumor effects were seen in both indolent and aggressive NHL. The data also suggest that anti-tumor responses of potentially equal magnitude can occur irrespective of tumor targeting and tumor size. Hence, tumor response did not correlate with the radiation dose delivered or with the tumor being visualized by external imaging. An anti-antibody response to epratuzumab was detected by an enzyme-linked immunosorbent assay in only 2 of 16 patients. CONCLUSION: These results suggest that (90)Y-DOTA-epratuzmab is a promising agent for the treatment of NHL and warrants further study. There was evidence suggesting that in this system, factors other than tumor radiation dose and targeting may be involved in the success of radioimmunotherapy.

Use of a corrected standardized uptake value based on the lesion size on CT permits accurate characterization of lung nodules on FDG-PET.

The purpose of this study was to determine the actual standardized uptake value (SUV) by using the lesion size from computer tomography (CT) scan to correct for resolution and partial volume effects in positron emission tomography (PET) imaging. This retrospective study included 47 patients with lung lesions seen on CT scan whose diagnoses were confirmed by biopsy or by follow up CT scan when the PET result was considered negative for malignancy. Each lesion's FDG uptake was quantified by the SUV using two methods: by measuring the maximum voxel SUV (maxSUV) and by using the lesion's size on CT to calculate the actual SUV (corSUV). Among small lesions (2.0 cm or smaller on CT scan), ten were benign and 17 were malignant. The average maxSUV was 1.43+/-0.77 and 3.02+/-1.74 for benign and malignant lesions respectively. When using an SUV of 2.0 as the cutoff to differentiate benignity and malignancy, the sensitivity, specificity, and accuracy were 65%, 70%, and 67% respectively. When an SUV of 2.5 was used for cutoff, the sensitivity, specificity, and accuracy were 47%, 80%, and 59% respectively. The average corSUV was 1.65+/-1.09 and 5.28+/-2.71 for benign and malignant lesions respectively. Whether an SUV of either 2.0 or 2.5 was used for cutoff, the sensitivity, specificity, and accuracy remained 94%, 70%, and 85% respectively. The only malignant lesion that was falsely considered benign with both methods was a bronchioalveolar carcinoma which did not reveal any elevated uptake of fluorine-18 fluorodeoxyglucose (FDG). Of the large lesions (more than 2.0 cm and less than 6.0 cm), one was benign and 19 were malignant and the corSUV technique did not significantly change the accuracy. It is concluded that measuring the SUV by using the CT size to correct for resolution and partial volume effects offers potential value in differentiating malignant from benign lesions in this population. This approach appears to improve the accuracy of FDG-PET for optimal characterization of small lung nodules.

Phase I radioimmunotherapy trial with iodine-131--labeled humanized MN-14 anti-carcinoembryonic antigen monoclonal antibody in patients with metastatic gastrointestinal and colorectal cancer.

This trial was conducted to determine the pharmacokinetics, dosimetry, dose-limiting toxicity, and the maximum tolerated dose of iodine-131 humanized MN-14 immunoglobulin G (131I-hMN-14 IgG), a humanized complementary-determining region-grafted anti-carcinoembryonic antigen monoclonal antibody, in metastatic gastrointestinal and colorectal cancer patients. Patients were divided into 2 groups: group A consisted of patients who had prior external beam radiation therapy (n = 8), and group B included patients who had received standard chemotherapy (n = 13). All patients received a diagnostic infusion of 131I-hMN-14 IgG (approximately 8.0 mCi, 15 mg/m2) to study the pharmacokinetics, biodistribution, and dosimetry. One week later, 17 of 21 patients received infusional therapy of escalating radioactive doses of 131I-hMN-14 IgG. Blood pharmacokinetics and quantitative imaging were performed again after the therapeutic dose. Radiation-absorbed doses to normal organs and tumors were determined by MIRDOSE-3 algorithms. The primary dose-limiting toxicity was hematologic toxicity at 40 mCi/m2. The blood half-life (n = 20) was identical for the diagnostic and therapy infusions. The mean red marrow dose was 2.2 +/- 2.4 cGy/mCi. The mean tumor radiation dose (n = 8) was 24.2 +/- 22.6 cGy/mCi. Tumor targeting was seen in most large metastatic lesions. No objective responses were seen in these heavily pretreated and mostly advanced patients. In conclusion, 131I-hMN-14 IgG has good targeting, good tumor to normal organs radiation absorbed ratios, and an acceptable toxicity profile in advanced metastatic gastrointestinal and colorectal cancer patients.

Breast cancer imaging with PET and SPECT agents: an in vivo comparison.

UNLABELLED: Several radiopharmaceuticals and imaging techniques are proposed for breast cancer imaging. Since limited data are available of the uptake of SPECT and PET radiopharmaceuticals in malignant breast tumors and their metastases the aim of this study was to compare the uptake values and to correlate these data with imaging findings. METHODS: We have studied the uptake of F-18 FDG, Tc-99m MIBI and Tc-99m (V)DMSA in 31 tumors using immunosuppressed rats implanted with HH-16 clone 4 mammary tumor cells. Tumor gamma camera and PET imaging was performed to gain biokinetic data and uptake values by ROI-analysis. RESULTS: Tumor uptake was highest for F-18 FDG > Tc-99m (V)DMSA > Tc-99m MIBI. The uptake ratios (tumor to muscle) correlated well with the ratios calculated by ROI-analysis determined by imaging. CONCLUSIONS: In this in-vivo model, F-18 FDG revealed the best uptake and imaging properties and may be the radiopharmaceutical of choice for routine breast cancer imaging.

Dual time point 18F-FDG PET for the evaluation of pulmonary nodules.

UNLABELLED: 18F-FDG PET has reached widespread application in the assessment of pulmonary nodules. This study compares the diagnostic accuracy of standard 18F-FDG PET scanning with those of dual time point 18F-FDG PET scanning. METHODS: Thirty-six patients (21 women, 15 men; mean age, 67 y; range, 36-88 y) with 38 known or suspected malignant pulmonary nodules underwent PET of the thorax at 2 time points: scan 1 at 70 min (range, 56-110 min) and scan 2 at 123 min (range, 100-163 min) after the intravenous injection of 2.5 MBq 18F-FDG per kilogram of body weight. All scanning was performed on a dedicated C-PET scanner. The mean interval between the scans was 56 min (range, 49-64 min). Regions of interest were overlaid onto each fully corrected image in the areas of the radiographically known lung densities. The standardized uptake values (SUVs) were calculated for both time points. RESULTS: Surgical pathology and follow-up revealed 19 patients with 20 malignant tumors, whereas 16 patients had benign lesions. The tumor SUVs (mean +/- SD) were 3.66 +/- 1.95 (scan 1) and 4.43 +/- 2.43 (scan 2) (20.5% +/- 8.1% increase; P < 0.01). Four of 20 malignant tumors had SUVs of <2.5 on scan 1 (range, 1.12-1.69). Benign lesions had SUVs of 1.14 +/- 0.64 (scan 1) and 1.11 +/- 0.70 (scan 2) (P = not significant). Standard PET scanning (single time point) with a threshold SUV of 2.5 (at time point 1) reached a sensitivity of 80% and a specificity of 94%; dual time point scanning with a threshold value of 10% increase between scan 1 and scan 2 reached a sensitivity of 100% with a specificity of 89%. CONCLUSION: Dual time point 18F-FDG PET results in a very high sensitivity and specificity for detection of malignant lung tumors.