CBIO-16. SUPPRESSION OF HISTONE H3.3 MITOTIC PHOSPHORYLATION DRIVES DEVELOPMENT OF H3K27M-MUTANT DIFFUSE MIDLINE GLIOMAS

Abstract Pediatric midline gliomas – including DIPG – are lethal brain tumors in children, with poor prognosis and limited treatment options that provide only short-term benefits. The majority have a lysine-to-methionine substitution at residue 27 (H3K27M) in genes expressing histone H3 – predominantly in the H3.3 variant. This causes a global reduction in H3 Lys27 tri-methylation (H3K27Me3), comprehensive epigenetic reprogramming, and is a key driver in gliomagenesis. We show that the H3.3K27M mutation also induces chromosome segregation defects, which in high-grade tumors, results in extensive copy number alterations (CNAs). Ser31 is one of five amino acid substitutions differentiating H3.3 from canonical H3.1. Mitotic phosphorylation of H3.3 Ser31 by Chk1 kinase is restricted to pericentromeric heterochromatin, where it plays a role in chromosome segregation. We show that the K27M mutation affects neighboring Ser31 phosphorylation and pericentromeric heterochromatin organization. We demonstrate that (i) H3.3 K27M protein is defective for Ser31 phosphorylation by Chk1 kinase in vitro; (ii) DIPG cell lines have significantly decreased mitotic Ser31 phosphorylation, and are chromosomally unstable; and (iii) CRISPR-reversion of H3.3K27M to Lys27 restores phospho-Ser31 (and Lys27 tri-methylation) and significantly decreases chromosome instability. Expression of H3.3K27M or non-phosphorylatable H3.3S31A mutants in WT cells results in chromosome missegregation; this is suppressed by co-expression of phospho-mimetic H3.3K27M/S31E. In normal cells, chromosome missegregation stimulates p53-dependent cell cycle arrest in G1 to prevent the proliferation of aneuploid daughters. However, cells expressing H3.3 K27M or S31A failed to arrest following missegregation - despite having WT p53. Finally, in a novel mouse model of glioma, mean survival of mice with tumors induced with H3.3K27M and H3.3S31A was 81 and 68 days: 100% of H3.3S31A mice developed high-grade tumors. H3.3 WT controls developed only low-grade tumors and all survived 100 days. H3.3S31A is WT for Lys27 tri-methylation and thus, loss of Ser31 phosphorylation alone is oncogenic.

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Assessment of TP53 mutation using purified tissue samples of ovarian serous carcinomas reveals a higher mutation rate than previously reported and does not correlate with drug resistance

International Journal of Gynecological Cancer ◽

10.1111/j.1525-1438.2007.01039.x ◽

2008 ◽

Vol 18 (3) ◽

pp. 487-491 ◽

Cited By ~ 88

Author(s):

R. SALANI ◽

R. J. KURMAN ◽

R. GIUNTOLI ◽

G. GARDNER ◽

R. BRISTOW ◽

...

Keyword(s):

Drug Resistance ◽

Mutation Frequency ◽

Clinical Stage ◽

Serous Carcinoma ◽

Low Grade ◽

High Grade ◽

Mutation Status ◽

Tumor Dna ◽

Serous Tumors

The TP53 mutation frequency in ovarian serous carcinomas has been reported to range between 50% and 80%, but a stringent analysis of TP53 using purified epithelial samples has not yet been performed to accurately assess the mutation frequency and to correlate it with the histologic grade. The purpose of this study was to assess the TP53 mutational profile in a relatively large series of high-grade (53 primary and 18 recurrent) and 13 low-grade ovarian serous tumors using DNA isolated from affinity-purified tumor cells and to correlate it with in vitro drug resistance. All samples were affinity purified, and the tumor DNA was analyzed for TP53 mutations in exons 4–9. In vitro drug resistance assays to carboplatin, cisplatin, paclitaxel, and taxotere were performed on the same tumor samples and correlated with the TP53 mutation status. TP53 mutations were detected in 57 (80.3%) of 71 high-grade carcinomas and in one (7.8%) of 13 low-grade serous tumors (an invasive low-grade serous carcinoma). The mutations were predominantly missense mutations (59.6%). TP53 mutations were associated with high-grade serous carcinomas and recurrent disease (P < 0.0001). There was no statistically significant correlation between TP53 mutation status and drug resistance assays or clinical stage (P > 0.25). The frequency of TP53 mutations using purified tumor DNA from ovarian serous carcinomas was 80.3%, which is much higher than previously reported. Furthermore, we found that TP53 is not directly involved in the development of drug resistance in high-grade ovarian serous carcinomas.

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Cytotoxicity of Protein-Carbon Nanotubes on J774 Macrophages Is a Functionalization Grade-Dependent Effect

BioMed Research International ◽

10.1155/2015/796456 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 1

Author(s):

Silvia Lorena Montes-Fonseca ◽

Blanca Sánchez-Ramírez ◽

Antonia Luna-Velasco ◽

Carlos Arzate-Quintana ◽

Macrina Beatriz Silva-Cazares ◽

...

Keyword(s):

Carbon Nanotubes ◽

Cytotoxic Effect ◽

Surface Protein ◽

Low Grade ◽

High Grade ◽

Dependent Effect ◽

Chemical Substances ◽

Raman And Infrared Spectroscopy

Carbon nanotubes (CNTs) are used as carriers in medicine due to their ability to be functionalized with chemical substances. However, cytotoxicity analysis is required prior to use forin vivomodels. The aim of this study was to evaluate the cytotoxic effect of CNTs functionalized with a 46 kDa surface protein fromEntamoeba histolytica(P46-CNTs) on J774A macrophages. With this purpose, CNTs were synthesized by spray pyrolysis and purified (P-CNTs) using sonication for 48 h. A 46 kDa protein, with a 4.6–5.4 pI range, was isolated fromE. histolyticaHM1:IMSS strain trophozoites using an OFFGEL system. The P-CNTs were functionalized with the purified 46 kDa protein, classified according to their degree of functionalization, and characterized by Raman and Infrared spectroscopy.In vitrocytotoxicity was evaluated by MTT, apoptosis, and morphological assays. The results demonstrated that P46-CNTs exhibited cytotoxicity dependent upon the functionalized grade. Contrary to what was expected, P46-CNTs with a high grade of functionalization were more toxic to J774 macrophages than P46-CNTs with a low grade of functionalization, than P-CNTs, and had a similar level of toxicity as UP-CNT. This suggests that the nature of the functionalized protein plays a key role in the cytotoxicity of these nanoparticles.

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Novel and emerging approaches in the management of non-muscle invasive urothelial carcinoma

Therapeutic Advances in Medical Oncology ◽

10.1177/17588359211039052 ◽

2021 ◽

Vol 13 ◽

pp. 175883592110390

Author(s):

Omid Yassaie ◽

Cyrus Chehroudi ◽

Peter C. Black

Keyword(s):

Treatment Options ◽

Unmet Need ◽

Invasive Disease ◽

Intravesical Therapy ◽

Low Grade ◽

High Grade ◽

Bcg Therapy ◽

Muscle Invasive ◽

Considerable Morbidity ◽

Intravesical Bcg Therapy

Non-muscle invasive bladder cancer (NMIBC) has traditionally been managed with transurethral resection followed by intravesical chemotherapy and/or bacillus Calmette–Guerin (BCG) in a risk-adapted manner. These tumors commonly recur and can progress potentially to lethal muscle invasive disease. A major unmet need in the field of NMIBC is bladder preserving therapy for recurrent high-grade NMIBC after adequate intravesical BCG therapy. The current gold standard treatment for these BCG-unresponsive patients is radical cystectomy, which is associated with considerable morbidity and mortality, particularly in older and frailer patients. It is therefore critical to provide alternative treatment options with acceptable oncological outcomes. In this review we explore novel bladder-sparing treatment options including combination intravesical therapy, enhanced instillation methods, immunotherapy, gene therapy, targeted therapy, photodynamic therapy and BCG variants across the spectrum of NMIBC disease states, ranging from low grade BCG-naïve patients through to high-grade BCG-unresponsive NMIBC.

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Environmental stresses induce karyotypic instability in colorectal cancer cells

Molecular Biology of the Cell ◽

10.1091/mbc.e18-10-0626 ◽

2019 ◽

Vol 30 (1) ◽

pp. 42-55 ◽

Cited By ~ 2

Author(s):

Zhihao Tan ◽

Yong Jie Andrew Chan ◽

Ying Jie Karen Chua ◽

Samuel D. Rutledge ◽

Norman Pavelka ◽

...

Keyword(s):

Colorectal Cancer ◽

Drug Resistance ◽

Cancer Cells ◽

Chromosome Segregation ◽

Spindle Assembly Checkpoint ◽

Chromosome Instability ◽

Chemotherapeutic Agents ◽

Tumor Evolution ◽

Chromosomal Changes

Understanding how cells acquire genetic mutations is a fundamental biological question with implications for many different areas of biomedical research, ranging from tumor evolution to drug resistance. While karyotypic heterogeneity is a hallmark of cancer cells, few mutations causing chromosome instability have been identified in cancer genomes, suggesting a nongenetic origin of this phenomenon. We found that in vitro exposure of karyotypically stable human colorectal cancer cell lines to environmental stress conditions triggered a wide variety of chromosomal changes and karyotypic heterogeneity. At the molecular level, hyperthermia induced polyploidization by perturbing centrosome function, preventing chromosome segregation, and attenuating the spindle assembly checkpoint. The combination of these effects resulted in mitotic exit without chromosome segregation. Finally, heat-induced tetraploid cells were on the average more resistant to chemotherapeutic agents. Our studies suggest that environmental perturbations promote karyotypic heterogeneity and could contribute to the emergence of drug resistance.

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Role of MCM as an adjuvant to urine cytology in the diagnosis of bladder cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.e15011 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. e15011-e15011

Author(s):

Nadira Narine ◽

Bensita M.V Thottakam ◽

Alessia Donnini ◽

Sushant Dhanvijay ◽

Kasra Saeb-Parsy ◽

...

Keyword(s):

Bladder Cancer ◽

Positive Outcome ◽

Urine Cytology ◽

Low Grade ◽

High Grade ◽

Minichromosome Maintenance ◽

Molecular Tests ◽

In Vitro Diagnostic ◽

Mcm Proteins

e15011 Background: Urine cytology, a cheap non invasive first line test (PAP) has traditionally been used for the diagnosis of urothelial carcinoma (UC) as it has excellent sensitivity for the detection of high grade and in situ lesions. However, sensitivity for low grade carcinoma is rather varied as this is a problematic area with various mimics leading to both over and under diagnoses. To overcome this dilemma, a number of biomarkers and molecular tests have been employed with mixed results and varied financial implications. We report on a relatively cheap and easily adopted in vitro diagnostic test, Minichromosome maintenance (MCM) proteins, as an adjuvant to cytology in the diagnosis of both low and high grade bladder cancer (BC). MCM proteins play an important regulatory role in eukaryotic DNA replication and is expressed only as normal cells progress from G0 into G1/S phase of the cell cycle. However, over expression has been demonstrated in neoplasia in a range of sites including urothelium. Methods: 106 patients from gross haematuria (GH, 39) and cystoscopic surveillance (CS, 67) clinics were investigated for newly diagnosed and recurrent BC respectively by MCM and cytology with histological outcome being used as the gold standard. Results: Using biopsy positive outcome, MCM and cytology had a sensitivity of 91.7%, specificity of 86.6%, PPV of 66.7% and NPV of 97.3%. There was noticeably improved correlation of MCM and cytology with increasing grade of BC (Table). Conclusions: We showed the combination of MCM and cytology to be highly sensitive and specific for the determination of CIS, G2, and G3 BC with improved detection of G1 bladder cancer. The combination of MCM and cytology suggests that this offers promise as a novel diagnostic biomarker in GH and CS patients. [Table: see text]

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Pathobiology and treatment of lymphomatoid granulomatosis, a rare EBV-driven disorder

Blood ◽

10.1182/blood.2019000933 ◽

2020 ◽

Vol 135 (16) ◽

pp. 1344-1352 ◽

Cited By ~ 4

Author(s):

Christopher Melani ◽

Elaine S. Jaffe ◽

Wyndham H. Wilson

Keyword(s):

B Cell ◽

Immune Modulation ◽

Treatment Options ◽

Bone Marrow Involvement ◽

Immune Surveillance ◽

Lymphoproliferative Disease ◽

Lymphomatoid Granulomatosis ◽

Low Grade ◽

High Grade ◽

Hematopoietic Stem

Abstract Lymphomatoid granulomatosis (LYG) is a rare Epstein-Barr virus (EBV)–driven B-cell lymphoproliferative disease (LPD). This disease is hypothesized to result from defective immune surveillance of EBV, with most patients showing evidence of immune dysfunction, despite no known primary immunodeficiency. Pathologically, LYG is graded by the number and density of EBV+ atypical B cells, and other characteristic findings include an angioinvasive/angiodestructive reactive T-cell infiltrate and various degrees of necrosis. Clinically, LYG universally involves the lungs with other common extranodal sites, including skin, central nervous system, liver, and kidneys. Nodal and/or bone marrow involvement is extremely rare and, if present, suggests an alternative diagnosis. Treatment selection is based on histologic grade and underlying pathobiology with low-grade disease hypothesized to be immune-dependent and typically polyclonal and high-grade disease to be immune-independent and typically oligoclonal or monoclonal. Methods of augmenting the immune response to EBV in low-grade LYG include treatment with interferon-α2b, whereas high-grade disease requires immunochemotherapy. Given the underlying defective immune surveillance of EBV, patients with high-grade disease may have a recurrence in the form of low-grade disease after immunochemotherapy, and those with low-grade disease may progress to high-grade disease after immune modulation, which can be effectively managed with crossover treatment. In patients with primary refractory disease or in those with multiple relapses, hematopoietic stem cell transplantation may be considered, but its efficacy is not well established. This review discusses the pathogenesis of LYG and highlights distinct histopathologic and clinical features that distinguish this disorder from other EBV+ B-cell LPDs and lymphomas. Treatment options, including immune modulation and combination immunochemotherapy, are discussed.

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Reliability and Accuracy of Peri-Interventional Stenosis Grading in Peripheral Artery Disease Using Color-Coded Quantitative Fluoroscopy: A Phantom Study Comparing a Clinical and Scientific Postprocessing Software

BioMed Research International ◽

10.1155/2018/6180138 ◽

2018 ◽

Vol 2018 ◽

pp. 1-11 ◽

Cited By ~ 2

Author(s):

Patrick Ghibes ◽

Sasan Partovi ◽

Gerd Grözinger ◽

Petros Martirosian ◽

Fritz Schick ◽

...

Keyword(s):

Flow Behavior ◽

Image Acquisition ◽

Area Under The Curve ◽

Vessel Diameter ◽

Frame Rate ◽

Low Grade ◽

High Grade ◽

Flat Detector ◽

Time To Peak

Purpose. To assess quantitative stenosis grading by color-coded fluoroscopy using an in vitro pulsatile flow phantom. Methods. Three different stenotic tubes (80%, 60%, and 40% diameter restriction) and a nonstenotic reference tube were compared regarding their different flow behavior by using contrast-enhanced fluoroscopy with a flat-detector system for visualisation purposes. Time-density curves (TDC), area under the curve (AUC), time-to-peak (TTP), and different ROI sizes were analyzed in three independent measurements using two different postprocessing software solutions. In addition, exemplary TDCs of a patient with a high-grade stenosis before and after stent angioplasty were acquired. Results. Color-coded fluoroscopy enabled depiction of differences in AUC and TDC between high-grade (80%), middle (60%), low-grade (40%), and nonstenotic tubes. The best correlation between high-, middle-, and low-grade stenosis was appreciated in ROIs behind the stenosis. This effect was enhanced by using longer integration times (5s, 7s) and a maximum frame rate of image acquisition for analysis (correlation coefficient rho=0.9284 at 5s). TTP showed no significant differences between high- and low-grade stenosis. Conclusions. Various clinical studies in the literature already demonstrated reproducible and reliable stenosis grading by analyzing TDCs acquired with color-coded fluoroscopy. In contrast to TTP, AUC values derived in ROIs behind the stenosis proved to be reliable parameters for stenosis grading. However, our results also demonstrate that several factors are able to significantly impact the evaluation of AUC values. More precisely, accuracy of acquired AUC values can be improved by choosing longer integration times, a large ROI size adapted to the vessel diameter, and a higher frame rate of image acquisition.

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TMOD-36. THE DEVELOPMENT OF A NOVEL MOUSE MODEL TO STUDY THE ROLE OF HISTONE MUTATIONS AND MODIFICATIONS IN PEDIATRIC HIGH-GRADE GLIOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa215.986 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii235-ii235

Author(s):

Florina Grigore ◽

Charles Day ◽

Nicholas Hanson ◽

Alyssa Langfald ◽

Jann Sarkaria ◽

...

Keyword(s):

Mouse Model ◽

Clonal Selection ◽

Mutant Cell ◽

High Grade Glioma ◽

Early Event ◽

Low Grade ◽

High Grade ◽

Newborn Mice

Abstract Pediatric glioblastoma and diffuse intrinsic pontine glioma are high-grade gliomas of children (pHGG) with a median overall survival of under 15 months and among the most lethal cancers. Mutations in histone H3.3 and H3.1 occur as an early event in pHGG. H3.3G34R/V-mutations occur in pHGG of cerebral hemispheres, and H3.3K27M mutations occur in midline pHGGs. Post-translational histone modifications (PTMs) serve to regulate gene expression by relaxing or compacting chromatin and by recruiting proteins, with subsequent silencing or activating effects. H3.3 Serine 31 (S31) shows reduced phosphorylation during mitosis in H3.3G34R/V and H3.3K27M mutant cell. Phosphorylation at S31 is restored in wildtype H3.3K27 CRISPR revertants. Serine to alanine (A) mutant H3.3 S31A are nonphosphorylatable in vitro. To study the influence of histone mutations and the role of altered PTM and including the loss of methylation and phosphorylation on tumorigenesis, we have developed an innovative model based on the RCAS/N-TVA mouse model. In this system, the expression of an oncogenic driver is linked to mutant histone expression using a self-cleaving peptide, and tumors develop following viral delivery to neural stem cells in newborn mice. This approach is necessary, as otherwise, clonal selection could prevent tumors from forming with mutations detrimental to growth. To establish the model, N-TVA mice were injected with RCAS H3.3K27M-P2A-PDGFB, RCAS H3.3G34R-P2A-PDGFB, or H3.3WT-P2A-PDGFB. The mean survival of mice injected with H3.3K27M and H3.3S31A was 81 and 68 days, respectively, and 100% of S31A mice developed HGG. In contrast, H3.3WT caused only low-grade tumors in 46% of the mice, and all mice survived until 100 days. In ongoing experiments with H3.3G34R, 23% of mice succumb to tumors by 80 days. These results provide mechanistic insights into the early establishment of pHGGs and established a new mouse model to study the role of histone mutation and PTMs in tumor development.

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Article Commentary: Management of Barrett's Esophagus

The American Surgeon ◽

10.1177/000313481207801123 ◽

2012 ◽

Vol 78 (11) ◽

pp. 1193-1200 ◽

Cited By ~ 2

Author(s):

Vic Velanovich

Keyword(s):

Barrett’S Esophagus ◽

Barrett's Esophagus ◽

Antireflux Surgery ◽

Prolonged Exposure ◽

Treatment Options ◽

Standard Of Care ◽

High Grade Dysplasia ◽

Low Grade ◽

High Grade ◽

Endoscopic Ablation

Barrett's esophagus is a pathologic change of the normal squamous epithelium of the esophagus to specialized columnar metaplasia. Barrett's esophagus is a result of prolonged exposure of the esophagus to gastroduodenal refluxate. Although Barrett's itself is not symptomatic, and, in fact, patients with Barrett's esophagus may be completely asymptomatic, it does identify patients at higher risk of developing esophageal adenocarcinoma. Traditionally, antireflux surgery was reserved for patients with symptoms, because it was believed that antireflux surgery did not eliminate Barrett's esophagus and reduce cancer risk. Rationale for the treatment of Barrett's esophagus beyond treating symptoms of gastroesophageal reflux disease stems from the hope to decrease, if not eliminate, the risk of adenocarcinoma. Treatment options ranged from medical acid suppression without surveillance to resection. Ablation, particularly endoscopic radio-frequency ablation, has become the standard of care for Barrett's esophagus with high-grade dysplasia. It role in nondysplastic or low-grade dysplastic Barrett's is less clear. Combined endoscopic mucosal resection with ablation is effective in nodular high-grade Barrett's esophagus. Resection should be reserved for patients with persistent high-grade dysplasia despite multiple attempts at endoscopic ablation or resection or for patients with evidence of carcinoma.

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MNGI-11. HIGH-GRADE AND LOW-GRADE MENINGIOMAS HARBOR DIFFERING METABOLOMIC PROFILES

Neuro-Oncology ◽

10.1093/neuonc/noz175.593 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi141-vi142

Author(s):

Garrett Fitzpatrick ◽

Maryam Rahman ◽

Timothy Garrett ◽

Jesse Kresak

Keyword(s):

Brain Tumor ◽

Treatment Options ◽

Disease Free Survival ◽

Metastatic Potential ◽

Primary Brain Tumor ◽

Sphingolipid Metabolism ◽

Low Grade ◽

Tumor Type ◽

High Grade ◽

Who Grade

Abstract BACKGROUND Meningiomas are the most common primary brain tumor in adults. While the majority of meningiomas are low-grade and effectively treated by resection alone, there is a subset of tumors that have a high incidence of recurrence, metastatic potential, and morbidity. Radiation has been employed with variable success for high-grade meningiomas. No chemotherapeutic approaches have proven effective against these tumors to date. There is a need for a better understanding of this tumor type in order to provide our patients with better treatment options. OBJECTIVE The purpose of this study is to investigate the metabolomic profile of meningiomas with a focus on comparing low- and high-grade tumors and identifying biologically significant metabolites which could correlate with overall and disease-free survival. METHODS Ten tumor samples of each meningioma grade (WHO grades I-III) were collected from the Florida Center for Brain Tumor Research. Global metabolomic profiling by liquid chromatography mass spectrometry was performed on the frozen tumor samples. Statistical analyses were performed using the Southeast Center for Integrated Metabolomics Galaxy interface. Select metabolites which significantly differed between low-grade (WHO Grade I) and high-grade (WHO grade II-III) were identified using the Human Metabolome Database. RESULTS Differing metabolomic profiles between low-grade and high-grade meningiomas were confirmed by multivariate analysis and demonstrated by unsupervised hierarchical clustering. Notably, lysophospholipid and sphingolipid metabolism was increased in the high-grade tumors, while FAPy-adenine, an oxidized nucleoside which may serve as a tumor marker, was decreased. Guanine was found to be consistently decreased in patients with negative outcomes. CONCLUSIONS High-grade and low-grade meningiomas harbor different metabolomic profiles. The significance of these specific differences requires further investigation.

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