Repeat Stereotactic Radiosurgery for Acoustic Neuromas.

Purpose
To evaluate the outcome of repeat stereotactic radiosurgery (SRS) for acoustic neuromas, we assessed tumor control, clinical outcomes, and the risk of adverse radiation effects in patients whose tumors progressed after initial management.

Methods and Materials
During a 21-year experience at our center, 1,352 patients underwent SRS as management for their acoustic neuromas. We retrospectively identified 6 patients who underwent SRS twice for the same tumor. The median patient age was 47 years (range, 35–71 years). All patients had imaging evidence of tumor progression despite initial SRS. One patient also had incomplete surgical resection after initial SRS. All patients were deaf at the time of the second SRS. The median radiosurgery target volume at the time of the initial SRS was 0.5 cc and was 2.1 cc at the time of the second SRS. The median margin dose at the time of the initial SRS was 13 Gy and was 11 Gy at the time of the second SRS. The median interval between initial SRS and repeat SRS was 63 months (range, 25–169 months).

Results
At a median follow-up of 29 months after the second SRS (range, 13–71 months), tumor control or regression was achieved in all 6 patients. No patient developed symptomatic adverse radiation effects or new neurological symptoms after the second SRS.

Conclusions
With this limited experience, we found that repeat SRS for a persistently enlarging acoustic neuroma can be performed safely and effectively.

Stereotactic radiosurgery as a therapeutic strategy for intracranial metastatic prostate carcinoma.

Intracranial metastatic prostate carcinoma is rare. We sought to determine the clinical outcomes after Gamma Knife® stereotactic radiosurgery (GKSRS) for patients with intracranial prostate carcinoma metastases. We studied data from 10 patients who underwent radiosurgery for 15 intracranial metastases (9 dural-based and 6 parenchymal). Six patients had radiosurgery for solitary tumors and four had multiple tumors. The primary pathology was adenocarcinoma (eight patients) and small cell carcinoma (two patients). All patients received multimodality management for their primary tumor (including resection, radiation therapy, androgen deprivation therapy) and eight patients had evidence of systemic disease at time of radiosurgery. The mean tumor volume was 7.7 cm3 (range 1.1-17.2 cm3) and a median margin dose of 16 Gy was administered. Two patients had progressive intracranial disease in spite of fractionated partial brain radiation therapy (PBRT) prior to SRS. A local tumor control rate of 85% was achieved (including patients receiving boost, upfront and salvage SRS). New remote brain metastases developed in three patients (33%) and one patient had repeat SRS for tumor recurrence. The median survival after radiosurgery was 13 months and the 1-year survival rate was 60%. SRS was a well tolerated and effective therapy either alone or as a boost to fractionated radiation therapy in the management of patients with intracranial prostate carcinoma metastases. © 2009 Springer Science+Business Media, LLC.

Foxp3+ T cells induce Perforin-Dependent Dendritic Cell Death in Tumor-Draining Lymph Nodes.

Regulatory T (Treg) cells limit the onset of effective antitumor immunity, through yet-ill-defined mechanisms. We showed the rejection of established ovalbumin (OVA)-expressing MCA101 tumors required both the adoptive transfer of OVA-specific CD8(+) T cell receptor transgenic T cells (OTI) and the neutralization of Foxp3(+) T cells. In tumor-draining lymph nodes, Foxp3(+) T cell neutralization induced a marked arrest in the migration of OTI T cells, increased numbers of dendritic cells (DCs), and enhanced OTI T cell priming. Using an in vitro cytotoxic assay and two-photon live microscopy after adoptive transfer of DCs, we demonstrated that Foxp3(+) T cells induced the death of DCs in tumor-draining lymph nodes, but not in the absence of tumor. DC death correlated with Foxp3(+) T cell-DC contacts, and it was tumor-antigen and perforin dependent. We conclude that Foxp3(+) T cell-dependent DC death in tumor-draining lymph nodes limits the onset of CD8(+) T cell responses.

Erythropoietin-Induced Activation of the JAK2/STAT5, PI3K/Akt, and Ras/ERK Pathways Promotes Malignant Cell Behavior in a Modified Breast Cancer Cell Line

Abstract Erythropoietin (Epo), the major regulator of erythropoiesis, and its cognate receptor (EpoR) are also expressed in nonerythroid tissues, including tumors. Clinical studies have highlighted the potential adverse effects of erythropoiesis-stimulating agents when used to treat cancer-related anemia. We assessed the ability of EpoR to enhance tumor growth and invasiveness following Epo stimulation. A benign noninvasive rat mammary cell line, Rama 37, was used as a model system. Cell signaling and malignant cell behavior were compared between parental Rama 37 cells, which express few or no endogenous EpoRs, and a modified cell line stably transfected with human EpoR (Rama 37-28). The incubation of Rama 37-28 cells with pharmacologic levels of Epo led to the rapid and sustained increases in phosphorylation of signal transducers and activators of transcription 5, Akt, and extracellular signal-regulated kinase. The activation of these signaling pathways significantly increased invasion, migration, adhesion, and colony formation. The Epo-induced invasion capacity of Rama 37-28 cells was reduced by the small interfering RNA-mediated knockdown of EpoR mRNA levels and by inhibitors of the phosphoinositide 3-kinase/Akt and Ras/extracellular signal-regulated kinase signaling pathways with adhesion also reduced by Janus-activated kinase 2/signal transducers and activators of transcription 5 inhibition. These data show that Epo induces phenotypic changes in the behavior of breast cancer cell lines and establishes links between individual cell signaling pathways and the potential for cancer spread.

Raman microscopy in the diagnosis and prognosis of surgically resected nonsmall cell lung cancer

The main curative therapy for patients with nonsmall cell lung cancer is surgery. Despite this, the survival rate is only 50%, therefore it is important to more efficiently diagnose and predict prognosis for lung cancer patients. Raman spectroscopy is useful in the diagnosis of malignant and premalignant lesions. The aim of this study is to investigate the ability of Raman microscopy to diagnose lung cancer from surgically resected tissue sections, and predict the prognosis of these patients. Tumor tissue sections from curative resections are mapped by Raman microscopy and the spectra analzsed using multivariate techniques. Spectra from the tumor samples are also compared with their outcome data to define their prognostic significance. Using principal component analysis and random forest classification, Raman microscopy differentiates malignant from normal lung tissue. Principal component analysis of 34 tumor spectra predicts early postoperative cancer recurrence with a sensitivity of 73% and specificity of 74%. Spectral analysis reveals elevated porphyrin levels in the normal samples and more DNA in the tumor samples. Raman microscopy can be a useful technique for the diagnosis and prognosis of lung cancer patients receiving surgery, and for elucidating the biochemical properties of lung tumors. (C) 2010 Society of Photo-Optical Instrumentation Engineers. [DOI: 10.1117/1.3323088]

Chemotherapy-Induced Activation of ADAM-17: A Novel Mechanism of Drug Resistance in Colorectal Cancer

Purpose: We have shown previously that exposure to anticancer drugs can trigger the activation of human epidermal receptor survival pathways in colorectal cancer (CRC). In this study, we examined the role of ADAMs (a disintegrin and metalloproteinases) and soluble growth factors in this acute drug resistance mechanism.

Experimental Design: In vitro and in vivo models of CRC were assessed. ADAM-17 activity was measured using a fluorometric assay. Ligand shedding was assessed by ELISA or Western blotting. Apoptosis was assessed by flow cytometry and Western blotting.

Results: Chemotherapy (5-fluorouracil) treatment resulted in acute increases in transforming growth factor-a, amphiregulin, and heregulin ligand shedding in vitro and in vivo that correlated with significantly increased ADAM-17 activity. Small interfering RNA–mediated silencing and pharmacologic inhibition confirmed that ADAM-17 was the principal ADAM involved in this prosurvival response. Furthermore, overexpression of ADAM-17 significantly decreased the effect of chemotherapy on tumor growth and apoptosis. Mechanistically, we found that ADAM-17 not only regulated phosphorylation of human epidermal receptors but also increased the activity of a number of other growth factor receptors, such as insulin-like growth factor-I receptor and vascular endothelial growth factor receptor.

Conclusions: Chemotherapy acutely activates ADAM-17, which results in growth factor shedding, growth factor receptor activation, and drug resistance in CRC tumors. Thus, pharmacologic inhibition of ADAM-17 in conjunction with chemotherapy may have therapeutic potential for the treatment of CRC.

Antibody Targeting of Camptothecin-Loaded PLGA Nanoparticles to Tumor Cells

Antibody targeting of drug substances can improve the efficacy of the active molecule, improving distribution and concentration of the drug at the site of injury/disease. Encapsulation of drug substances into polymeric nanoparticles can also improve the therapeutic effects of such compounds by protecting the molecule until its action is required. In this current study, we have brought together these two rationales to develop a novel immunonanoparticle with improved therapeutic effect against colorectal tumor cells. This nanoparticle comprised a layer of peripheral antibodies (Ab) directed toward the Fas receptor (CD95/Apo-1) covalently attached to poly(lactide-co-glycolide) nanoparticles (NP) loaded with camptothecin. Variations in surface carboxyl density permitted up to 48.5 mu g coupled Ab per mg of NP and analysis of nanoparticulate cores showed efficient camptothecin loading. Fluorescence visualization studies confirmed internalization of nanoconstructs into endocytic compartments of HCT 116 cells, an effect not evident in NP without superficial Ab. Cytotoxicity studies were then carried out against HCT116 cells. After 72 h, camptothecin solution resulted in an IC50 of 21.8 ng mL(-1). Ab-directed delivery of NP-encapsulated camptothecin was shown to be considerably more effective with an IC50 of 0.37 ng mL(-1). Calculation of synergistic ratios for these nanoconstructs demonstrated synergy of pharmacological relevance. Indeed, the results in this paper suggest that the attachment of anti-Fas antibodies to camptothecin-loaded nanoparticles may result in a therapeutic strategy that could have potential in the treatment of tumors expressing death receptors.

Oncogenic Kras Promotes Chemotherapy-Induced Growth Factor Shedding via ADAM17.

Oncogenic mutations in Kras occur in 40% to 45% of patients with advanced colorectal cancer (CRC). We have previously shown that chemotherapy acutely activates ADAM17, resulting in growth factor shedding, growth factor receptor activation, and drug resistance in CRC tumors. In this study, we examined the role of mutant Kras in regulating growth factor shedding and ADAM17 activity, using isogenic Kras mutant (MT) and wild-type (WT) HCT116 CRC cells. Significantly higher levels of TGF-a and VEGF were shed from KrasMT HCT116 cells, both basally and following chemotherapy treatment, and this correlated with increased pErk (phosphorylated extracellular signal regulated kinase)1/2 levels and ADAM17 activity. Inhibition of Kras, MEK (MAP/ERK kinase)1/2, or Erk1/2 inhibition abrogated chemotherapy-induced ADAM17 activity and TGF-a shedding. Moreover, we found that these effects were not drug or cell line specific. In addition, MEK1/2 inhibition in KrasMT xenografts resulted in significant decreases in ADAM17 activity and growth factor shedding in vivo, which correlated with dramatically attenuated tumor growth. Furthermore, we found that MEK1/2 inhibition significantly induced apoptosis both alone and when combined with chemotherapy in KrasMT cells. Importantly, we found that sensitivity to MEK1/2 inhibition was ADAM17 dependent in vitro and in vivo. Collectively, our findings indicate that oncogenic Kras regulates ADAM17 activity and thereby growth factor ligand shedding in a MEK1/2/Erk1/2-dependent manner and that KrasMT CRC tumors are vulnerable to MEK1/2 inhibitors, at least in part, due to their dependency on ADAM17 activity.

Tumor Risks and Genotype-Phenotype-Proteotype Analysis in 358 Patients With Germline Mutations in SDHB and SDHD

Succinate dehydrogenase B (SDHB) and D (SDHD) subunit gene mutations predispose to adrenal and extraadrenal pheochromocytomas, head and neck paragangliomas (HNPGL), and other tumor types. We report tumor risks in 358 patients with SDHB (n = 295) and SDHD (n = 63) mutations. Risks of HNPGL and pheochromocytoma in SDHB mutation carriers were 29% and 52%, respectively, at age 60 years and 71% and 29%, respectively, in SDHD mutation carriers. Risks of malignant pheochromocytoma and renal tumors (14% at age 70 years) were higher in SDHB mutation carriers; 55 different mutations (including a novel recurrent exon 1 deletion) were identified. No clear genotype-phenotype correlations were detected for SDHB mutations. However, SDHD mutations predicted to result in loss of expression or a truncated or unstable protein were associated with a significantly increased risk of pheochromocytoma compared to missense mutations that were not predicted to impair protein stability (most such cases had the common p.Pro81Leu mutation). Analysis of the largest cohort of SDHB/D mutation carriers has enhanced estimates of penetrance and tumor risk and supports in silicon protein structure prediction analysis for functional assessment of mutations. The differing effect of the SDHD p.Pro81Leu on HNPGL and pheochromocytoma, risks suggests differing mechanisms of tumorigenesis in SDH-associated HNPGL and pheochromocytoma. Hum Mutat 31:41-51, 2010. (C) 2009 Wiley-Liss, Inc.

High incidence of proviral integrations in the Hoxa locus in a new model of E2a-PBX1-induced B-cell leukemia

Relevant mouse models of E2a-PBX1-induced pre-B cell leukemia are still elusive. We now report the generation of a pre-B leukemia model using E2a-PBX1 transgenic mice, which lack mature and precursor T-cells as a result of engineered loss of CD3epsilon expression (CD3epsilon(-/-)). Using insertional mutagenesis and inverse-PCR, we show that B-cell leukemia development in the E2a-PBX1 x CD3epsilon(-/-) compound transgenic animals is significantly accelerated when compared to control littermates, and document several known and novel integrations in these tumors. Of all common integration sites, a small region of 19 kb in the Hoxa gene locus, mostly between Hoxa6 and Hoxa10, represented 18% of all integrations in the E2a-PBX1 B-cell leukemia and was targeted in 86% of these leukemias compared to 17% in control tumors. Q-PCR assessment of expression levels for most Hoxa cluster genes in these tumors revealed an unprecedented impact of the proviral integrations on Hoxa gene expression, with tumors having one to seven different Hoxa genes overexpressed at levels up to 6600-fold above control values. Together our studies set the stage for modeling E2a-PBX1-induced B-cell leukemia and shed new light on the complexity pertaining to Hox gene regulation. In addition, our results show that the Hoxa gene cluster is preferentially targeted in E2a-PBX1-induced tumors, thus suggesting functional collaboration between these oncogenes in pre-B-cell tumors.

CCN3-A key regulator of the hematopoietic compartment

CCN3, a founding member of the CCN family of growth regulators, was linked with hematology in 2003(1) when it was detected in human serum. CCN3 is expressed and secreted by hematopoietic progenitor cells in normal bone marrow. CCN3 acts through the core stem cell signalling pathways including Notch and Bone Morphogenic Protein, connecting CCN3 with the modulation of self-renewal and maturation of a number of cell lineages including hematopoietic, osteogenic and chondrogenic. CCN3 expression is disrupted in Chronic Myeloid Leukemia as a consequence of the BCR-ABL oncogene and allows the leukemic clone to evade growth regulation. In contrast, naive cord blood progenitors undergo enhanced clonal expansion in response to CCN3. Altered CCN3 expression is associated with numerous solid tumors including glioblastoma, melanoma. adrenocortical tumours, prostate cancer and bone malignancies including osteosarcoma. Mature CCN3 protein has five distinct modules and truncated protein variants with altered function are found in many cancers. Regulation by CCN3 is therefore cell type and isoform specific. CCN3 has emerged as a key player in stem cell regulation, hematopoiesis and a crucial component within the bone marrow microenvironment. (c) 2008 Elsevier Ltd. All rights reserved.

Low cytosine triphosphate synthase 2 expression renders resistance to 5-fluorouracil in colorectal cancer

Understanding the determinants of resistance of 5-fluorouracil (5FU) is of significant value to optimizing administration of the drug, and introducing novel agents and treatment strategies. Here, the expression of 92 genes involved in 5FU transport, metabolism, co-factor (folate) metabolism and downstream effects was measured by real-time PCR low density arrays in 14 patient-derived colorectal cancer xenografts characterized for 5FU resistance. Candidate gene function was tested by siRNA and uridine modulation, and immunoblotting, apoptosis and cell cycle analysis. Predictive significance was tested by immunohistochemistry of tumors from 125 stage III colorectal cancer patients treated with and without 5FU. Of 8 genes significantly differentially expressed between 5FU sensitive and resistant xenograft tumors, CTPS2 was the gene with the highest probability of differential expression (p = 0.008). Reduction of CTPS2 expression by siRNA increased the resistance of colorectal cancer cell lines DLD1 and LS174T to 5FU and its analog, FUDR. CTPS2 siRNA significantly reduced cell S-phase accumulation and apoptosis following 5FU treatment. Exposure of cells to uridine, a precursor to the CTPS2 substrate uridine triphosphate, also increased 5FU resistance. Patients with low CTPS2 did not gain a survival benefit from 5FU treatment (p = 0.072), while those with high expression did (p = 0.003). Low CTPS2 expression may be a rationally-based determinant of 5FU resistance.

Impact of Sample Heterogeneity on Methylation Analysis

The recent emergence of high-throughput arrays for methylation analysis has made the influence of tumor content on the interpretation of methylation levels increasingly pertinent. However, to what degree does tumor content have an influence, and what degree of tumor content makes a specimen acceptable for accurate analysis remains unclear. Taking a systematic approach, we analyzed 98 unselected formalin-fixed and paraffin-embedded gastric tumors and matched normal tissue samples using the Illumina GoldenGate methylation assay. Unsupervised hierarchical clustering showed 2 separate clusters with a significant difference in average tumor content levels. The probes identified to be significantly differentially methylated between the tumors and normals also differed according to the tumor content of the samples included, with the sensitivity of identifying the

Claudin-1 Has Tumor Suppressive Activity and Is a Direct Target of RUNX3 in Gastric Epithelial Cells

BACKGROUND & AIMS: The transcription Factor RUNX3 is a gastric tumor suppressor. Tumorigenic Runx3(-/-) gastric epithelial cells attach weakly to each other, compared with nontumorigenic Runx3(+/+) cells. We alined to identify RUNX3 target genes that promote cell-cell contact to Improve our understanding of RUNX3's role in Suppressing gastric carcinogenesis. METHODS: We compared gene expression profiles of Runx3(+/+) and Runx3(-/-) cells and observed down-regulation of genes associated with cell-cell adhesion in Runx3(-/-) cells. Reporter, mobility shift, and chromatin immunoprecipitation assays were used to examine the regulation of these genes by RUNX3. Tumorigenesis assays and immunohistologic, analyses of human gastric tumors were performed to confirm the role of the candidate genes ill gastric tumor development. RESULTS: Mobility shift and chromatin immunoprecipitation assays revealed that the promoter activity of the gene that encodes the tight Junction protein claudin-1 was up-regulated via the binding of RUNX3 to the RUNX consensus sites. The tumorigenicity of gastric epithelial cells From Runx3(-/-) mice was significantly reduced by restoration of claudin-1 expression, whereas knockdown of claudin-1. increased the tumorigenicity of human gastric cancer cells. Concomitant expression of RUNX3 and claudin-1 was observed in human normal gastric epithelium and cancers. CONCLUSIONS: The tight junction protein claudin-1 has gastric tumor suppressive activity and is a direct transcriptional target of RUNX3. Claudin-1 is down-regulated during the epithelial-mesenchymal transition; RUNX3 might therefore act as a tumor suppressor to antagonize the epithelial-mesenchymal transition.

Runx2, p53, and pRB Status as Diagnostic Parameters for Deregulation of Osteoblast Growth and Differentiation in a New Pre-Chemotherapeutic Osteosarcoma Cell Line (OS1)

Osteosarcomas are the most prevalent primary bone tumors found in pediatric patients. To understand their molecular etiology, cell culture models are used to define disease mechanisms under controlled conditions. Many osteosarcoma cell lines (e.g., SAOS-2, U2OS, MG63) are derived from Caucasian patients. However, patients exhibit individual and ethnic differences in their responsiveness to irradiation and chemotherapy. This motivated the establishment of osteosarcoma cell lines (OS1, OS2, OS3) from three ethnically Chinese patients. OS1 cells, derived from a pre-chemotherapeutic tumor in the femur of a 6-year-old female, were examined for molecular markers characteristic for osteoblasts, stem cells, and cell cycle control by immunohistochemistry, reverse transcriptase-PCR, Western blotting and flow cytometry. OS I have aberrant G-banded karyotypes, possibly reflecting chromosomal abnormalities related to p53 deficiency. OS I had ossification profiles similar to human fetal osteoblasts rather than SAOS-2 which ossifies ab initio, (P