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DIPG AND WT1
- 1. By: Manuela PabónBernal Molecular Biology Third semester Universidad Pontificia Bolivariana 2019-02
- 2. Diffuse Intrinsic Pontine Glioma(DIPG) • A rare, fast-growing, highly aggressive tumor that forms in glial cells in the pons, that tend to spread to nearby tissue and other parts of the brain stem, are hard to treat, and have a poor prognosis. • They usually occur in children (diagnosed 5-9 years), equally in boys and girls. • Makes up 10-15% of all brain tumors in children, with about 100-300 new cases diagnosed each year in the United States. • Problems with eye movement; Facial weakness; Sudden appearance of hearing problems; Trouble chewing or swallowing; Limb weakness, difficulty standing or walking, abnormal gaits, clumsiness, unbalanced limb movements; Headache; and, Nausea and vomiting from brain edema.
- 3. Wilm’s Tumor Protein •Is encoded by the WT1 gene on chromosome 11p. • Transcription factor that plays an important role in cellular development and cell survival. • Regulates the expression of numerous target genes, including EPO. • Plays an essential role for development of the urogenital system. • It has a suppressor as well as an oncogenic role in tumor formation. • Known as a protein highly associated with various cancers including adult glioblastomas. • WT1 has been ranked by the NCI as the Number 1 target for cancer inmunotherapy.
- 4. General objective The differential expressionof Wilms’ Tumor Protein in Diffuse Intrinsic Pontine Glioma
- 5. Methods. Quantitative RT- PCR •The RNA amplification through its copy DNA synthesized a priori, using a reverse transcriptase, followed by several cycles of conventional PCR. This technique can determine the expression of genes in various tissues. • Performed qRT-PCR using cDNA from fresh frozen DIPG specimens, which confirmed the significantly higher expression of WT1 in tumor specimens compared with adjacent healthy brain samples. Data showed higher expression (6.4-fold) in the H3.3-mutant samples.
- 6. Methods. Immunohistochemistry • Uses antibodiesto check for certain antigens (markers) in a sample of tissue. The antibodies are usually linked to an enzyme or a fluorescent dye. After the antibodies bind to the antigen in the tissue sample, the enzyme or dye is activated, and the antigen can then be seen under a microscope. • WT1 expression is associated with the mutation status of oncohistone H3. • They observed that WT1 expression, at both the mRNA and protein level, was significantly higher in a number of DIPG specimens. This led them to expand their investigation to a cohort of 37 tumor specimens and 20 adjacent healthy specimens from 37 subjects diagnosed with midline glioma and probed for WT1 expression by IHC.
- 7. Methods. Western Blot Assays •Identify specific proteins from a complex mixture of proteins extracted from cells. The technique uses three elements to accomplish this task: (1) separation by size, (2) transfer to a solid support, and (3) marking target protein using a proper primary and secondary antibody to visualize. • Using protein extracts from DIPG subjects for whom frozen tumor and adjacent normal tissue was available. Western blot analysis showed significant overexpression of WT1 in DIPG specimens compared with adjacent normal samples. • Western blot analysis using human DIPG primary cells showed significantly higher level of WT1 in DIPG tumor cells compared with healthy brain tissue specimens from pediatric patients without CNS disease.
- 8. Methods. Immunofluorescence • Based onthe identification of the reaction between an specific antigen and antibody. The reaction is targeted with a fluorescent reagent which evidences the reaction itself. The intensity of the fluorescence indicates the reaction intensity as well. • Using antibodies against H3K27M, they probed for mutant histones and found that 94.6% (35/37) of subjects harbored the H3K27M mutation and corroborated with the conclusion which stated that WT1 expression is associated with the mutation status of oncohistone H3. • When they performed IF using human DIPG primary cells, WT1 was present in the cells but was localized to the cytosol in both human DIPG primary cells tested. Given the fact that WT1 is known to be a nuclear protein, this finding was surprising.
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- 12. Author What itsaid Match with the article Nakatsuka S, Oji Y, Horiuchi T, et al. Oji Y, Suzuki T, Nakano Y, et al. “Our cytosolic detection of WT1 is not surprising as previous studies have also shown cytosolic localization of WT1 in glioblastoma cells and lung cancer cells”. NO Chiba Y, Hashimoto N, Tsuboi A, et al. ”WT1-directed immunotherapeutic trials in adults with glioblastoma have indicated better survival of patients with higher WT1 expression levels receiving WT1-specific immunotherapy”. YES O’Rourke D, Nasrallah M, Desai A, et al. “There has been a rapid increase in studies investigating immunotherapy in CNS tumors. For instance, vaccines YES Discussio n
- 13. • In myopinion this study was very well performed, used a wide variety of methods and had coherent results with what they were looking for and acknowledging now a days advances with genetic therapies, it seems like a promising pathway in order to develop health promoting tools, such as treatments or pharmaceutical aids for patients with DIPG. • Molecular biology’s importance stands up in every single conclusion they had, and enhances the value of knowing the origin of diseases from the simplest and smallest factors, which can fully help doctors to improve not just these, but every patients’ quality of life. Conclution s
- 15. Bibliograph y https://www.cancer.gov/publications/dictionaries/cancer- terms https://www.uniprot.org/uniprot/P19544 Biologia Molecular, Sánchez.Lina. 2016. Universidad Pontificia Bolivarian
Editor's Notes
- #3 The pons controls many of the body’s most vital functions such as breathing, blood pressure and heart rate Given the location of the tumor and the infiltration of tumor cells with normal tissue, DIPGs are not amenable to meaningful surgical resection. Chemo- therapy has thus far shown little evidence of benefit (2, 3), and radiation therapy provides only a temporary clinical stabiliza- tion.
- #4 a protein highly associated with various cancers including adult glioblasto- mas (9). The WT1 gene encodes the WT1 protein, which is a transcription factor containing 4 zinc-finger DNA binding domains, essential for embryonic development of the spleen, kidneys, gonads, and cardiac vasculature A report by the National Cancer Institute identi- fied WT1 as the protein with the highest potential for cancer immunotherapy (12), and a recent phase I clinical trial showed that WT1 peptide vaccine therapy in adult glioblas- tomas was safe, which induced cellular and humoral immune response
- #6 para generar una gran cantidad de copias de ADN, proceso llamado "amplificación". En la RT-PCR, se retrotranscribe una hebra de ARN en ADN complementario (ADNc) usando una enzima llamada transcriptasa inversa o transcriptasa reversa, y el resultado se amplifica mediante una PCR tradicional. El término PCR en transcripción reversa no debe confundirse con la PCR en tiempo real, también denominada PCR cuantitativa (Q-PCR) Una de las características más importantes es que en el proceso de RT-PCR, el ADNc generado ya no lleva los intrones que sí tendría el ADN original. De este modo, al expresar el ADNc producto de la RT-PCR, se generará un ARNm formado exclusivamente por exones CDNA. DNA complementario, copia del RNA mensajero sintetizada en el laboratorio por medio de la transcriptasa inversa.
- #8 We performed addi- tional validation assays by Western blot using protein extracts from DIPG subjects (n 1⁄4 9) for whom frozen tumor and adja- cent normal tissue was available (Table). Western blot analy- sis showed significant overexpression (7.4-fold, p < 0.0001) of WT1 in DIPG specimens compared with adjacent normal samples (Fig. 1C).
- #9 When we performed IF using human DIPG primary cells, WT1 was present in the cells but was localized to the cytosol in both human DIPG primary cells tested (Fig. 4D). Given the fact that WT1 is known to be a nuclear protein (21), this find- ing was surprising.
- #10 WT1 expression is associated with pediatric CNS cancers WT1 overexpression in tumor tissues was validated by Western blot assays using DIPG tumor specimens (n 1⁄4 9) and adjacent normal specimens (n 1⁄4 9). Quantification of the Western blot assays of DIPG tumor versus normal showed significantly higher expression of WT1 in the tumors (7.4-fold, p < 0.0001). They observed that WT1 expression, at both the mRNA and protein level, was significantly higher in a number of DIPG specimens (Fig. 1B, C).
- #11 Immunohistochemistry of a large cohort of DIPGs indicated high expression of WT1 in H3.3 subtype compared with H3.1 subtype DIPGs. (A) IHC of a large cohort of DIPGs and pediatric midline gliomas showed WT1 overexpression in tumor compared with adjacent healthy brain tissue was valid. Scale bar 1⁄4 50 mm.
- #12 Validation of WT1 differential expression in human DIPG primary cells. ( A) A Western blot assay of WT1 using human DIPG primary cells (n1⁄46) and healthy brain tissue (HBT) lysates mmunofluorescence staining of human DIPG primary cells (n 1⁄4 2) showed WT1 expression in the tumor cells and localization of WT1, mainly in the cytoplasm of the tumor cells. The specificity of the antibody was validated by staining K562 cells, which showed WT1 localization in the nucleus. Scale bar 1⁄4 30 mm.