Atopic conditions and brain tumor risk in children and adolescents--an international case-control study (CEFALO)

BACKGROUND A number of epidemiological studies indicate an inverse association between atopy and brain tumors in adults, particularly gliomas. We investigated the association between atopic disorders and intracranial brain tumors in children and adolescents, using international collaborative CEFALO data. PATIENTS AND METHODS CEFALO is a population-based case-control study conducted in Denmark, Norway, Sweden, and Switzerland, including all children and adolescents in the age range 7-19 years diagnosed with a primary brain tumor between 2004 and 2008. Two controls per case were randomly selected from population registers matched on age, sex, and geographic region. Information about atopic conditions and potential confounders was collected through personal interviews. RESULTS In total, 352 cases (83%) and 646 controls (71%) participated in the study. For all brain tumors combined, there was no association between ever having had an atopic disorder and brain tumor risk [odds ratio 1.03; 95% confidence interval (CI) 0.70-1.34]. The OR was 0.76 (95% CI 0.53-1.11) for a current atopic condition (in the year before diagnosis) and 1.22 (95% CI 0.86-1.74) for an atopic condition in the past. Similar results were observed for glioma. CONCLUSIONS There was no association between atopic conditions and risk of all brain tumors combined or of glioma in particular. Stratification on current or past atopic conditions suggested the possibility of reverse causality, but may also the result of random variation because of small numbers in subgroups. In addition, an ongoing tumor treatment may affect the manifestation of atopic conditions, which could possibly affect recall when reporting about a history of atopic diseases. Only a few studies on atopic conditions and pediatric brain tumors are currently available, and the evidence is conflicting.

A Hybrid Model for Multimodal Brain Tumor Segmentation

We present a fully automatic segmentation method for multi-modal brain tumor segmentation. The proposed generative-discriminative hybrid model generates initial tissue probabilities, which are used subsequently for enhancing the classi�cation and spatial regularization. The model has been evaluated on the BRATS2013 training set, which includes multimodal MRI images from patients with high- and low-grade gliomas. Our method is capable of segmenting the image into healthy (GM, WM, CSF) and pathological tissue (necrotic, enhancing and non-enhancing tumor, edema). We achieved state-of-the-art performance (Dice mean values of 0.69 and 0.8 for tumor subcompartments and complete tumor respectively) within a reasonable timeframe (4 to 15 minutes).

Unique structures in a tumor herpesvirus revealed by cryo-electron tomography and microscopy.

Gammaherpesviruses, including the human pathogens Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, are causative agents of lymphomas and other malignancies. The structural characterization of these viruses has been limited due to difficulties in obtaining adequate amount of virion particles. Here we report the first three-dimensional structural characterization of a whole gammaherpesvirus virion by an emerging integrated approach of cryo-electron tomography combined with single-particle cryo-electron microscopy, using murine gammaherpesvirus-68 (MHV-68) as a model system. We found that the MHV-68 virion consists of distinctive envelope and tegument compartments, and a highly conserved nucleocapsid. Two layers of tegument are identified: an inner tegument layer tethered to the underlying capsid and an outer, flexible tegument layer conforming to the overlying, pleomorphic envelope, consistent with the sequential viral tegumentation process inside host cells. Surprisingly, comparison of the MHV-68 virion and capsid reconstructions shows that the interactions between the capsid and inner tegument proteins are completely different from those observed in alpha and betaherpesviruses. These observations support the notion that the inner layer tegument across different subfamilies of herpesviruses has evolved significantly to confer specific characteristics related to viral-host interactions, in contrast to a highly conserved capsid for genome encapsidation and protection.

Patient Characteristics and Outcomes of Gastrointestinal Stromal Tumor Patients Undergoing Imatinib Plasma Level Testing

Although gastrointestinal stromal tumor (GIST) is effectively treated with imatinib, there are a number of clinical challenges in the optimal treatment of these patients. The plasma steady-state trough level of imatinib has been proposed to correlate with clinical outcome. Plasma imatinib level may be affected by a number of patient characteristics. Additionally, the ideal plasma trough concentration of imatinib is likely to vary based on the KIT genotype (genotype determines imatinib binding affinity) of the individual patient. Patients’ genotype or plasma imatinib level may influence the type and duration of response that is appreciable by clinical evaluation. The objectives of this study were to determine effects of genotype on the type of response appreciable by current imaging criteria, to determine the distribution of plasma imatinib levels in patients with GIST, to determine factors that correlate with plasma imatinib level, to determine the incremental effects of imatinib dose escalation; and to explore the median plasma levels and outcomes of patients with various KIT mutations. We therefore obtained KIT mutation information and analyzed CT response for size and density measurement of GISTs at baseline and within the first four moths of imatinib treatment. In 126 patients with metastatic/unresectable disease, the KIT genotype of patients’ tumor was significantly associated with unique response characteristics measurable by CT. Furthermore, hepatic and peritoneal metastases differed in their response characteristics. A subgroup of patients with KIT exon 9 mutation, who received higher doses of imatinib and experienced higher trough imatinib levels, experienced improved progression-free survival similar to that of KIT exon 11 patients. Therefore, we have found that imatinib plasma levels were higher in patients with elevated Aspartate amino transferase, were women, were older, or were being treated concomitantly with CYP450 substrate drugs. As expected, CYP450 inducers correlated with a lower plasma imatinib levels in GIST patients. Renal metabolism of imatinib accounts for <10%, so it was not included in the analysis but may affect covariates. Interestingly, there was a trend for low imatinib levels and inferior progression-free survival in patients who had undergone complete gastrectomy. Patients with KIT exon 9 mutation in our cohort received higher imatinib doses, experienced higher trough imatinib levels, and experienced a PFS similar to that of KIT exon 11 patients. In conclusion, imatinib plasma levels are influenced by a number of patient characteristics. The optimal imatinib plasma level for individual patients is not known but is an area of intense investigation. Our study confirms patients with KIT exon 9 mutations benefit from high-dose imatinib and higher trough imatinib levels.

ABERRATIONS OF A PUTATIVE TUMOR SUPPRESSOR GENE SEL1L IN PANCREATIC DUCTAL ADENOCARCINOMA

Introduction: Pancreatic cancer is the fourth leading cause of cancer-related death among males and females in the United States. Sel-1-like (SEL1L) is a putative tumor suppressor gene that is downregulated in a significant proportion of human pancreatic ductal adenocarcinoma (PDAC). It was hypothesized that SEL1L expression could be down-modulated by somatic mutation, loss of heterozygosity (LOH), CpG island hypermethylation and/or aberrantly expressed microRNAs (miRNAs). Material and methods: In 42 PDAC tumors, the SEL1L coding region was amplified using reverse transcription polymerase chain reaction (RT-PCR), and analyzed by agarose gel electrophoresis and sequenced to search for mutations. Using fluorescent fragment analysis, two intragenic microsatellites in the SEL1L gene region were examined to detect LOH in a total of 73 pairs of PDAC tumors and normal-appearing adjacent tissues. Bisulfite DNA sequencing was performed to determine the methylation status of the SEL1L promoter in 41 PDAC tumors and 6 PDAC cell lines. Using real-time quantitative PCR, the expression levels of SEL1L mRNA and 7 aberrantly upregulated miRNAs that potentially target SEL1L were assessed in 42 PDAC tumor and normal pairs. Statistical methods were applied to evaluate the correlation between SEL1L mRNA and the miRNAs. Further the interaction was determined by functional analysis using a molecular biological approach. Results: No mutations were detected in the SEL1L coding region. More than 50% of the samples displayed abnormally alternate or aberrant spliced transcripts of SEL1L. About 14.5% of the tumors displayed LOH at the CAR/CAL microsatellite locus and 10.7% at the RepIN20 microsatellite locus. However, the presence of LOH did not show significant association with SEL1L downregulation. No methylation was observed in the SEL1L promoter. Statistical analysis showed that SEL1L mRNA expression levels significantly and inversely correlated with the expression of hsa-mir-143, hsa-mir-155, and hsa-mir-223. Functional analysis indicated that hsa-mir-155 acted as a suppressor of SEL1L in PL18 and MDAPanc3 PDAC cell lines. Discussion: Evidence from these studies suggested that SEL1L was possibly downregulated by aberrantly upregulated miRNAs in PDAC. Future studies should be directed towards developing a better understanding of the mechanisms for generation of aberrant SEL1L transcripts, and further analysis of miRNAs that may downregulate SEL1L.

Dual Targeting of Tumor Angiogenesis and Chemotherapy by Endostatin- Cytosine Deaminase-Uracil PhosphoribosylTransferase

Antiangiogenesis is a promising anti-tumor strategy through inhibition tumor vascularformation to suppress tumor growth. Targeting specific VEGF/R has been showntherapeutic benefits in many cancer types and become a first approvedantiangiogenic modalities by Food and Drug Administration (FDA) in United States.However, interruption of homeostasis in normal tissues that is likely due to theinhibition of VEGF/R signaling pathway induces unfavorable side effects. Moreover,cytostatic nature of antiangiogenic drugs frequently causes less tumor cell specifickilling activity, and cancer cells escaped from cell death induced by these drugseven gain more malignant phenotypes, resulting in tumor invasion and metastasis.To overcome these issues, we developed a novel anti-tumor therapeutic EndoCDfusion protein which linked endostatin (Endo) to cytosine deaminase-uracilvphosphoribosyl transferase (CD). Endo targets unique tumor endothelial cells toprovide tumor-specific antiangiogenesis activity and also carries CD to the localtumor area, where it serves nontoxic prodrug 5-fluorocytosine (5-FC) enzymaticconversion reaction to anti-metabolite chemotherapy drug 5-fluorouracil (5-FU). Wedemonstrated that 5-FU concentration was highly increased in tumor sites, resultingin high level of endothelial cells and tumor cells cytotoxic efficacy. Furthermore,EndoCD/5-FC therapy decreased tumor growth and colorectal liver metastasisincident compared with bevacizumab/5-FU treatment in human breast and colorectalliver metastasis orthotropic animal models. In cardiotoxicity safety profile,EndoCD/5-FC is a contrast to bevacizumab/5-FU; lower risk of cardiotoxicityinduction or heart function failure was found in EndoCD/5-FC treatment thanbevacizumab/5-FU does in mice. EndoCD/5-FC showed more potent therapeuticefficacy with high safety profile and provided stronger tumor invasion or metastasisinhibition than antiangiogenic drugs. Together, EndoCD fusion protein with 5-FCshowed dual tumor targeting activities including antiangiogenesis and tumor localchemotherapy, and it could serve as an alternative option for antiangiogenic therapy.

Malignant solitary fibrous tumor involving the liver

Solitary fibrous tumors are predominantly benign and are most commonly found in the thoracic cavity and pleura; while reports exist in the literature of malignant solitary fibrous tumors and those located in extrathoracic organs, these cases are considered extremely rare. Herein, a case is reported of a malignant solitary fibrous tumor involving the liver that was diagnosed and treated in a 62-year-old woman. The patient presented with complaints of upper abdominal pain and unintentional weight loss. Computed tomography scan of the abdomen revealed a remarkably large mass, measuring 15 cm × 10 cm × 20 cm, which appeared to be unrelated to any particular organ. The intraoperative finding of a wide communication with the left liver suggested hepatic origin, and served as an indicator for tumor resection via left hemihepatectomy. The diagnosis of solitary fibrous tumor and its malignant nature was confirmed by histological and immunohistochemical examination of the resected tissues. Hepatic solitary fibrous tumor is very rare, and surgery remains the mainstay of treatment. Due to limited reports of such tumors in the literature, little can be said about the benefit of adjuvant therapy and prognosis for the rare cases with malignant histological findings.

Generation and characterization of antigenic variants induced by exposure of tumor cells to UV radiation in vitro

Antigenic changes present in nonantigenic tumor cells exposed to UV radiation (UV) in vitro were investigated by addressing the following questions: (1) Are antigenic variants (AV) produced that are rejected in normal but not immunosuppressed mice? (2) Does generation of AV depend upon intrinsic properties of the cells exposed or result from the action of UV? (3) Is antigenic modification induced by UV due to increased histocompatibility antigen expression? (4) Do AV crossreact immunologically with parental tumor or with other AV? and (5) Is the UV-associated common antigen expressed on UV-induced tumors present on UV-irradiated tumor cells? AV were generated at different frequencies following in vitro UV irradiation of a spontaneous murine fibrosarcoma (51% of cell lines tested), a murine melanoma (56%), and two melanoma clones (100% and 11%). This indicated that the percentage of AV produced is an intrinsic property of the cell line exposed. The increased antigenicity did not correlate with an increased expression of class I histocompatibility antigens. Immunological experiments demonstrated that the AV and parental cells shared a determinant that was susceptible to immune recognition, but incapable of inducing immunity. In contrast, the AV were noncrossreactive, suggesting that variant-specific antigens were also expressed. Finally, the AV were recognized by UV-induced suppressor cells, indicating that the UV-associated common antigen expressed by UV-induced tumors was also present. This investigation provides new information on the susceptibility of tumors to antigenic modification by UV and on the relationship between tumor antigens and neoplastic transformation. Furthermore, it suggests an immunological approach for cancer therapy. ^

Long range physical map of the Wilms' tumor - aniridia region on human chromosome 11

Nephroblastoma or Wilms' tumor is a pediatric renal malignancy that is the most frequently occurring childhood solid tumor. Approximately 1-2% of children with Wilms' tumor also present with aniridia, a congenital absence of all or part of the iris of the eye. These children also have high rates of genitourinary anomalies and mental retardation resulting in what is called the WAGR (Wilms' tumor, aniridia, genitourinary anomaly, mental retardation) syndrome. Cytogenetic analysis of metaphase chromosomes from these patients revealed a consistent deletion of band P13 on chromosome 11. These observations suggest close physical linkage between the disease-related loci, and further imply that development of each phenotype results from the loss of normal gene function.^ The objective of this work is to understand the molecular events at chromosome band 11p13 that are essential to the development of sporadic Wilms' tumor and sporadic aniridia. Two human/hamster somatic cell hybrids have been used to identify sixteen independent DNA probes that map to this segment of the human genome. These newly identified DNA probes and four previously reported probes (CAT, FSHB, D11S16, and HBVIS) have been used to subdivide 11p13 into five intervals defined by overlapping constitutional deletions from several WAGR patients. A long-range physical map of 11p13 has been constructed using each of these probes in Southern blot analysis of genomic DNA after digestion with infrequently cutting restriction enzymes and pulse-field gel electrophoresis. This map, established primarily with MluI and NotI, spans approximately 13 $\times$ 10$\sp{6}$ bp and encompasses deletion and translocation breakpoints associated with genitourinary anomalies, aniridia, and sporadic Wilms' tumor. This complete physical map of human chromosome band 11p13 enables us to localize the genes for sporadic Wilms' tumor and sporadic aniridia to a small number of specific NotI fragments. ^

Inherited and noninherited sources of variation in metastasis and the growth rate of tumors: Implications for tumor evolution and therapy

I have undertaken measurements of the genetic (or inherited) and nongenetic (or noninherited) components of the variability of metastasis formation and tumor diameter doubling time in more than 100 metastatic lines from each of three murine tumors (sarcoma SANH, sarcoma SA4020, and hepatocarcinoma HCA-I) syngeneic to C3Hf/Kam mice. These lines were isolated twice from lung metastases and analysed immediately thereafter to obtain the variance to spontaneous lung metastasis and tumor diameter doubling time. Additional studies utilized cells obtained from within 4 passages of isolation. Under the assumption that no genetic differences in metastasis formation or diameter doubling time existed among the cells of a given line, the variance within a line would estimate nongenetic variation. The variability derived from differences between lines would represent genetic origin. The estimates of the genetic contribution to the variation of metastasis and tumor diameter doubling time were significantly greater than zero, but only in the metastatic lines of tumor SANH was genetic variation the major source of metastatic variability (contributing 53% of the variability). In the tumor cell lines of SA4020 and HCA-I, however, the contribution of nongenetic factors predominated over genetic factors in the variability of the number of metastasis and tumor diameter doubling time. A number of other parameters examined, such as DNA content, karyotype, and selection and variance analysis with passage in vivo, indicated that genetic differences existed within the cell lines and that these differences were probably created by genetic instability. The mean metastatic propensity of the lines may have increased somewhat during their isolation and isotransplantation, but the variance was only slightly affected, if at all. Analysis of the DNA profiles of the metastatic lines of SA4020 and HCA-I revealed differences between these lines and their primary parent tumors, but not among the SANH lines and their parent tumor. Furthermore, there was a direct correlation between the extent of genetic influence on metastasis formation and the ability of the tumor cells to develop resistance to cisplatinum. Thus although nongenetic factors might predominate in contributing to metastasis formation, it is probably genetic variation and genetic instability that cause the progression of tumor cells to a more metastatic phenotype and leads to the emergence of drug resistance. ^

Characterization of the cytochrome P-450 drug metabolism system of LS174T human colon tumor cell line

The human colon tumor cell line, LS174T, has been shown to have four major components of the drug metabolizing system; cytochrome b$\sb5$ reductase, cytochrome b$\sb5$, cytochrome P450 reductase and cytochrome P450, by activity measurements, spectral studies and antibody cross-reactivity. Cytochrome P450IA1 is induced by benzanthracene in these cells as shown by activity with the specific substrate, ethoxyresorufin, cross-reactivity with rabbit antibodies to rat IA1, and by a hybridizing band on a Northern blot to a rat IA1 probe.^ Further, this system has proven responsive to various inducers and conditions of growth. The enzyme activities were found stable over limited cell passages with control values of 0.03 and 0.13 $\mu$mol/min/mg protein for NADPH and NADH cytochrome c (cyt c) reducing activity, 0.05 nmol cyt b$\sb5$ per milligram and 0.013 nmol cytochrome P450 per milligram of microsomal protein. Phenobarbital/hydrocortisone treatment showed a consistent, but not always significant increase in the NADPH and NADH cyt c reducing activity and benzanthracene treatment an increase in the NADH cyt c reducing activity. Delta-aminolevulinic acid (0.5mM) caused a significant decrease in the specific activity of all enzyme contents and activities tested.^ Finally, the cytochrome b$\sb5$ to cytochrome P450, by the coordinate induction of the cytochrome b$\sb5$ pathway by P450 inducers, by the high ratio of NADH to NADPH ethoxycoumarin deethylase activity in uninduced cell microsomes, and by the increase in NADH and NADPH ethoxycoumarin deethylase activity when the microsomes were treated with potassium cyanide, a desaturase inhibitor. ^

Host factors in metastasis: Direct lysis of bloodborne metastatic tumor cells by murine vascular endothelial cells

During the process of cancer metastasis, the majority of circulating tumor cells arrest in microcapillary beds and then rapidly die. To study whether vascular endothelial cells can directly lyse tumor cells, we isolated vascular endothelial cells by perfusion of lungs from immunocompetent or nude mice. The cells were grown in culture, and then cloned and characterized. Cloned endothelial cells were incubated with several lymphokines and cytokines. Cells incubated with IFN-$\gamma$ and TNF lysed a variety of tumor cells with different metastatic potential. Mouse skin and lung fibroblasts treated with the same cytokines did not. Endothelial cell mediated tumor cell lysis was not due to different binding ability of tumor cells to cytokine treated and untreated endothelial monolayers. Kinetic studies demonstrated that the continuous presence of cytokines in the tumor-endothelial cocultures was necessary to produce maximal lysis of tumor cells. Target cell lysis was not due to the direct effects of IFN-$\gamma$ or TNF, since vascular endothelial cells isolated from the lung of nude mice lysed human melanoma cells that are sensitive or resistant to TNF. Cytokine treated endothelial cells produced a high level of nitric oxide, which is known to be cytotoxic to a variety of target cells. The level of nitric oxide production was directly correlated with the degree of tumor cell lysis. A specific inhibitor of nitric oxide synthesis(N$\sp{\rm G}$-monomethyl-L-arginine), completely inhibited production of nitric oxide and tumor cell lysis. Treatment of cytokine activated endothelial cells with dexamethasone also inhibited tumor cell lysis. This inhibition was independent of tumor-endothelial adhesion but correlated with inhibition of nitric oxide production. Collectively, these results suggest that vascular endothelial cells can directly destory tumor emboli and thus play an active role in the pathogenesis of cancer metastasis. ^

Immune response to regressor and progressor tumor variants

Most skin cancers induced in mice by Ultraviolet (UV) radiation express highly immunogenic Tumor specific transplantation antigens (TSTAs) and thus exhibit a regressor phenotype. In this study, I have used cloned genes encoding tumor antigens and oncogenes in conjunction with DNA transfection technique to isolate and characterize regressor variants from progressor tumors and vice versa. The purpose of this study was (1) to determine whether the product of a cloned gene (216) from UV-1591 tumor, which encodes a novel MHC class I antigen can function as a tumor rejection antigen when expressed on unrelated, nonantigenic, murine tumor cells or whether its function is restricted to UV-induced tumors, and (2) to determine the processes by which progressor variants derived from a regressor UV-2240 cell line by transfection with an activated Ha-ras oncogene escape the immune defenses of the normal immunocompetent host.^ To answer the first question, a spontaneously transformed, nonimmunogenic cell line (10T-1) was cotransfected with DNA from p216 and pSV2-neo plasmids. Results demonstrate that the product of a cloned TSTA gene from a UV-induced murine tumor is capable of functioning as a tumor rejection antigen when expressed on unrelated, nonantigenic tumor cells. In addition, these results indicate that this approach could be used to augment the immune response against poorly antigenic tumors.^ To answer the second question, progressor variants were isolated from a highly antigenic UV radiation-induced C3H mouse regressor fibrosarcoma cell line, UV-2240, by transfection with an activated Ha-ras oncogene. Subcutaneous injection of Ha-ras-transfected UV-2240 cells into immunocompetent C3H mice produced tumors in 4 of 36 animals. In addition, the Ha-ras-induced progressor variants produced experimental lung metastasis in both normal C3H and nude mice, although they induced more lung nodules in nude mice than in normal C3H mice. Results indicate that the progressor phenotype of the Ha-ras-induced tumor variants is not due to loss of TSTAs or MHC class I antigens. This implies that some tumors can escape the immune defenses of the normal immunocompetent host by mechanisms other than the loss of TSTAs and MHC class I antigens. (Abstract shortened with permission of author.) ^

Tumor cell-associated epitopes recognized by lymphokine-activated killers (LAK)

Interleukin-2 activated lymphocytes, designated lymphokine-activated killers (LAK), acquire the unique capacity to express potent cytologic activity against a broad spectrum of abnormal and/or transformed NK-sensitive and NK-resistant target cells while sparing normal cell types. Investigations into the target spectra exhibited by cloned effector cells indicate that LAK cells express a polyspecific recognition mechanism that identifies an undefined class of cell surface-associated molecules expressed on susceptible targets. This report extends our previous investigations into the biochemical nature of these molecules by characterizing the functional role of two tumor cell-surface-associated epitopes implicated in conferring target cells with susceptibility to LAK-mediated cytotoxicity. The first moiety is implicated in the formation of effector/target cell conjugates. This binding ligand is preferentially expressed on tumor cells relative to LAK-resistant PBL target cells, sensitive to trypsin treatment, resistant to functional inactivation by heat- and detergent-induced conformational changes, and does not require N-linked glycosylation to maintain binding activity. In contrast, a carbohydrate-associated epitope represents the second tumor-associated molecule required for target cell susceptibility to LAK cells. Specifically, N-linked glyoprotein synthesis represents an absolute requirement for post-trypsin recovery of target cell susceptibility. The minimal N-linked oligosaccharide residue capable of restoring this second signal has been identified as a high mannose structure. Although ultimately required for tumor cell susceptibility, as measured in $\sp{51}$Cr-release assays, this N-glycan-associated molecule is not required for the differential tumor cell binding expressed by LAK cells. Furthermore, N-glycan expression is not adequate in itself to confer target cell susceptibility. Additional categories of cell surface components have been investigated, including O-linked oligosaccharides, and glycosaminoglycans, without identifying additional moieties relevant to target cell recognition. Collectively, these data suggest that tumor cell recognition by LAK cells is dependent on cell surface presentation of two epitopes: a trypsin-sensitive molecule that participates in the initial conjugate formation and an N-glycan-associated moiety that is involved in a post-binding event required for target cell killing. ^