Glioblastoma Recurrence Correlates With Increased APE1 and Polarization Toward an Immuno-Suppressive Microenvironment.

While treatment with surgery, radiotherapy and/or chemotherapy may prolong life for patients with glioblastoma, recurrence is inevitable. What is still being discovered is how much these treatments and recurrence of disease affect the molecular profiles of these tumors and how these tumors adapt to withstand these treatment pressures. Understanding such changes will uncover pathways used by the tumor to evade destruction and will elucidate new targets for treatment development. Nineteen matched pre-treatment and post-treatment glioblastoma tumors were subjected to gene expression profiling (Fluidigm, TaqMan assays), MGMT promoter methylation analysis (pyrosequencing) and protein expression analysis of the DNA repair pathways, known to be involved in temozolomide resistance (immunohistochemistry). Gene expression profiling to molecularly subtype tumors revealed that 26% of recurrent post-treatment specimens did not match their primary diagnostic specimen subtype. Post-treatment specimens had molecular changes which correlated with known resistance mechanisms including increased expression of APEX1 (p < 0.05) and altered MGMT methylation status. In addition, genes associated with immune suppression, invasion and aggression (GPNMB, CCL5, and KLRC1) and polarization toward an M2 phenotype (CD163 and MSR1) were up-regulated in post-treatment tumors, demonstrating an overall change in the tumor microenvironment favoring aggressive tumor growth and disease recurrence. This was confirmed by in vitro studies that determined that glioma cell migration was enhanced in the presence of M2 polarized macrophage conditioned media. Further, M2 macrophage-modulated migration was markedly enhanced in post-treatment (temozolomide resistant) glioma cells. These findings highlight the ability of glioblastomas to evade not only the toxic onslaught of therapy but also to evade the immune system suggesting that immune-altering therapies may be of value in treating this terrible disease.

Intratumoral heterogeneity identified at the epigenetic, genetic and transcriptional level in glioblastoma.

Heterogeneity is a hallmark of glioblastoma with intratumoral heterogeneity contributing to variability in responses and resistance to standard treatments. Promoter methylation status of the DNA repair enzyme O(6)-methylguanine DNA methyltransferase (MGMT) is the most important clinical biomarker in glioblastoma, predicting for therapeutic response. However, it does not always correlate with response. This may be due to intratumoral heterogeneity, with a single biopsy unlikely to represent the entire lesion. Aberrations in other DNA repair mechanisms may also contribute. This study investigated intratumoral heterogeneity in multiple glioblastoma tumors with a particular focus on the DNA repair pathways. Transcriptional intratumoral heterogeneity was identified in 40% of cases with variability in MGMT methylation status found in 14% of cases. As well as identifying intratumoral heterogeneity at the transcriptional and epigenetic levels, targeted next generation sequencing identified between 1 and 37 unique sequence variants per specimen. In-silico tools were then able to identify deleterious variants in both the base excision repair and the mismatch repair pathways that may contribute to therapeutic response. As these pathways have roles in temozolomide response, these findings may confound patient management and highlight the importance of assessing multiple tumor biopsies.

Factors that may influence the willingness of cancer patients to consent for biobanking.

BACKGROUND: Broad or general consent given by cancer patients for their tissue, blood, and clinical information to be stored in institutional biorepositories is fundamental to enable future ethical translational cancer research. The decision to consent for biobanking will contribute to the development of advanced diagnostic and prognostic tests, as well as new therapies to improve patient outcomes. While the rate of patient participation in biobanking programs is generally reported as high worldwide, few studies have investigated factors that may influence this decision. Biobanking at our medical research institute, an associated public (government-run) university hospital, and private hospital has been established for over 20 years, with collection of certain tumor types embedded in the research culture of these institutions. In this study, we investigated factors that may influence a cancer patient's decision to give broad consent for biobanking of their specimens. METHODS: Data on patient consent were collected over a 6-month period from both government and private hospitals associated with our medical research institute. Factors considered included gender, patient age at surgery, type of malignancy (breast, duodenal, cervical, endometrial, gastric, liver, esophageal, ovarian, pancreatic, pelvic, uterine, or vulval), type of institution where surgery was performed, and timing of consent. RESULTS: Of 171 cancer patients, 159 (93%) gave broad consent for biobanking of their tissue and blood specimens for future cancer research projects receiving ethical and scientific approval. None of the factors analyzed was shown to influence a patient's decision to contribute biological specimens and clinical data to a biorepository for future medical research. CONCLUSION: Biobanking for future ethically and scientifically approved research projects in an established institution is an initiative that receives strong support from patients undergoing cancer surgery, independent of factors including gender, age, type of tumor, type of institution where surgery was performed, or timing of consent.

Correlation of MicroRNA 132 Up-regulation with an Unfavorable Clinical Outcome in Patients with Primary Glioblastoma Multiforme Treated with Radiotherapy Plus Concomitant and Adjuvant Temozolomide Chemotherapy.

BACKGROUND: MicroRNA 132 (miR-132) is dysregulated in a range of human malignancies; however, its role in glioma has not been reported. The aim of this study was to profile miR-132 expression in a cohort of patients with primary glioblastoma multiforme (GBM) treated with the Stupp regimen and to correlate microRNA levels with patient outcome. METHODS: miR-132 levels relative to RNU44 were assessed by quantitative reverse transcription-polymerase chain reaction in 43 GBMs and normal brain tissue. The cohort comprised patients less than 72 years of age with Eastern Cooperative Oncology Group (ECOG) scores between 0 and 2 who had undergone 6-week concomitant radiation and temozolomide followed by adjuvant temozolomide. Survival data were available for all cases. Tumors were characterized for O6-methylguanine-DNA methyltransferase (MGMT) methylation and isocitrate dehydrogenase (IDH) 1/2 mutation status. Associations between miR-132 expression and clinical indicators were analyzed. RESULTS: Tumor miR-132 levels ranged from 0.07- to 40.4-fold increase (mean = 5.5-fold increase) relative to normal brain. High-level miR-132 (above the mean) independently predicted for a significantly shorter overall survival (P = .008). miR-132 was a stronger prognostic indicator than ECOG score (P = .012) and age at diagnosis (P = .026) but did not correlate with MGMT methylation status or extent of tumor resection. Cox regression analysis confirmed high miR-132 as the strongest predictor of outcome (P = .010) with a hazard ratio of 2.8. CONCLUSIONS: This study identified high miR-132 expression as a biomarker of poor prognosis in patients with primary GBM treated with the Stupp regimen.

Pancreatic stellate cells: partners in crime with pancreatic cancer cells.

Pancreatic stellate cells (PSC) produce the stromal reaction in pancreatic cancer, but their role in cancer progression is not fully elucidated. We examined the influence of PSCs on pancreatic cancer growth using (a) an orthotopic model of pancreatic cancer and (b) cultured human PSCs (hPSC) and human pancreatic cancer cell lines MiaPaCa-2 and Panc-1. Athymic mice received an intrapancreatic injection of saline, hPSCs, MiaPaCa-2 cells, or hPSCs + MiaPaCa-2. After 7 weeks, tumor size, metastases, and tumor histology were assessed. In vitro studies assessed the effect of cancer cell secretions on PSC migration and the effect of hPSC secretions on cancer cell proliferation, apoptosis, and migration. Possible mediators of the effects of hPSC secretions on cancer cell proliferation were examined using neutralizing antibodies. Compared with mice receiving MiaPaCa-2 cells alone, mice injected with hPSCs + MiaPaCa-2 exhibited (a) increased tumor size and regional and distant metastasis, (b) fibrotic bands (desmoplasia) containing activated PSCs within tumors, and (c) increased tumor cell numbers. In vitro studies showed that, in the presence of pancreatic cancer cells, PSC migration was significantly increased. Furthermore, hPSC secretions induced the proliferation and migration, but inhibited the apoptosis, of MiaPaCa-2 and Panc-1 cells. The proliferative effect of hPSC secretions on pancreatic cancer cells was inhibited in the presence of neutralizing antibody to platelet-derived growth factor. Our studies indicate a significant interaction between pancreatic cancer cells and stromal cells (PSCs) and imply that pancreatic cancer cells recruit stromal cells to establish an environment that promotes cancer progression.

Reversible binding of kynurenine to lens proteins: potential protection by glutathione in young lenses.

PURPOSE: Human ultraviolet light (UV) filters, such as kynurenine (Kyn), readily deaminate to reactive unsaturated ketones that covalently modify proteins in older human lenses. The aim of this study was to examine in vitro rates of formation and decomposition of the three major Kyn-amino acid adducts and possible consequences for the lens. METHODS: The t-Boc-protected Kyn-His, Kyn-Lys, and Kyn-Cys adducts and Kyn-Cys were synthesized from the corresponding amino acids and Kyn. Calf lens proteins were modified with Kyn by incubation at pH 7. Stability and competition studies of the adducts were conducted under physiological conditions. Kyn-amino acids and their decomposition products were quantified using HPLC. RESULTS: At physiological pH, Kyn-Cys adducts formed more rapidly than either Lys or His adducts, but they also decomposed readily. By contrast, His adducts were stable. Cysteine (Cys) residues in beta-crystallins were major sites of modification. The Kyn moiety, initially bound to Cys residues, was found to transfer to other amino acids. Glutathione promoted the breakdown of Kyn-Cys. CONCLUSIONS: These data may help explain why proteins in young lenses are not modified by UV filters in situ. The initial phase of the modification of proteins in the human lens by UV filters may be a dynamic process. In lenses, Cys residues of crystallins modify preferentially, but these adducts also decompose to release deaminated Kyn. This can then potentially react with other amino acids. Glutathione, which is present in high concentrations in the lenses of young people, may play a vital role in keeping proteins free from modification by intercepting reactive deaminated kynurenines formed by the spontaneous breakdown of free UV filters, promoting the decomposition of Kyn-Cys residues, and sequestering the unsaturated ketones once they are released from modified proteins.

Protein-bound kynurenine is a photosensitizer of oxidative damage.

Human lens proteins become progressively modified by tryptophan-derived UV filter compounds in an age-dependent manner. One of these compounds, kynurenine, undergoes deamination at physiological pH, and the product binds covalently to nucleophilic residues in proteins via a Michael addition. Here we demonstrate that after covalent attachment of kynurenine, lens proteins become susceptible to photo-oxidation by wavelengths of light that penetrate the cornea. H2O2 and protein-bound peroxides were found to accumulate in a time-dependent manner after exposure to UV light (lambda > 305-385 nm), with shorter-wavelength light giving more peroxides. Peroxide formation was accompanied by increases in the levels of the protein-bound tyrosine oxidation products dityrosine and 3,4-dihydroxyphenylalanine, species known to be elevated in human cataract lens proteins. Experiments using D2O, which enhances the lifetime of singlet oxygen, and azide, a potent scavenger of this species, are consistent with oxidation being mediated by singlet oxygen. These findings provide a mechanistic explanation for UV light-mediated protein oxidation in cataract lenses, and also rationalize the occurrence of age-related cataract in the nuclear region of the lens, as modification of lens proteins by UV filters occurs primarily in this region.

Protein-bound kynurenine decreases with the progression of age-related nuclear cataract.

PURPOSE: Posttranslational modification by UV filters is a key event in human lenses that appears to be largely responsible for normal age-dependent yellowing. It has been proposed that subsequent reactions of these covalently bound UV filters may also be involved in the genesis of age-related nuclear cataract. To examine this hypothesis, the levels of kynurenine-lysine and kynurenine-histidine were measured in both normal and cataractous human lenses. METHODS: Proteins isolated from the nuclei of normal lenses and lenses with and types I to IV nuclear cataract were hydrolyzed in 6 M HCl, and the levels of kynurenine-lysine and kynurenine-histidine were determined by HPLC. RESULTS: The content of kynurenine-lysine and kynurenine-histidine decreased substantially with the progression of age-related nuclear cataract. On average, levels of both kynurenine adducts were four times lower in advanced cataract (type IV) than in normal lenses. Simple autoxidation of the derivatives did not appear to be responsible for this decrease, because incubation in the presence of oxygen or H(2)O(2) did not affect adduct stability. CONCLUSIONS: Although protein-bound kynurenine accumulates over time in normal lenses, the levels attached to the proteins decrease significantly with the progression of age-related nuclear cataract. This finding suggests that in cataract there is a breakdown of the protein-bound adducts. Such further reactions of bound UV filters may contribute to the etiology of age-related nuclear cataract.