Revisiting TP53 Mutations and Immunohistochemistry--A Comparative Study in 157 Diffuse Gliomas.
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ABSTRACT:
SUBMITTER: Takami H
PROVIDER: S-EPMC8028857 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Diffuse intrinsic pontine gliomas (DIPGs) are high-grade tumors of the brainstem that often occur in children, with a median overall survival of less than one year. Given the fact that DIPGs are resistant to chemotherapy and are not amenable to surgical resection, it is imperative to develop new therapeutic strategies for this deadly disease. The p53 pathway is dysregulated by TP53 (~ 60%) or PPM1D gain-of-function mutations (~ 30%) in DIPG cases. PPM1D gain-of-function mutations suppress p53 activity and result in DIPG tumorigenesis. While MDM2 is a major negative regulator of p53, the efficacy of MDM2 inhibitor has not been tested in DIPG preclinical models. In this study, we performed a comprehensive validation of MDM2 inhibitor RG7388 in patient-derived DIPG cell lines established from both TP53 wild-type/PPM1D-mutant and TP53 mutant/PPM1D wild-type tumors, as well in TP53 knockout isogenic DIPG cell line models. RG7388 selectively inhibited the proliferation of the TP53 wild-type/PPM1D mutant DIPG cell lines in a dose- and time-dependent manner. The anti-proliferative effects were p53-dependent. RNA-Seq data showed that differential gene expression induced by RG7388 treatment was enriched in the p53 pathways. RG7388 reactivated the p53 pathway and induced apoptosis as well as G1 arrest. In vivo, RG7388 was able to reach the brainstem and exerted therapeutic efficacy in an orthotopic DIPG xenograft model. Hence, this study demonstrates the pre-clinical efficacy potential of RG7388 in the TP53 wild-type/PPM1D mutant DIPG subgroup and may provide critical insight on the design of future clinical trials applying this drug in DIPG patients.
| S-EPMC8565061 | biostudies-literature
Project description:Abstract Introduction: The updated 2016 edition of the WHO Classification of Tumor of the Central Nervous System has shifted from the traditional approach primarily based on microscopic features to that on genetic hallmarks. The classification of diffuse gliomas is now defined in accordance with their IDH and 1p/19q status. Although that shift has resulted in a more objective definition of diffuse gliomas, genetic testings, which are not always available worldwide, has become mandatory for WHO diagnosis. Immunohistochemistry (IHC) for mutant IDH1R132H and fluorescence in situ hybridization (FISH) for 1p/19q are the most popular methods, but IDH1R132H does not detect other loci and currently available probes for 1p36 and 19q13 are known to yield false positive results (about 15%). To overcome such disadvantages, we verified the utility of combined IHC for p53 and ATRX in addition to IDH1R132H, since about 90% of IDH mutated astrocytomas carry TP53 mutations followed by ATRX mutations in 80% of TP53 mutants and TP53 or ATRX mutations, which are detectable on IHC, are mutually exclusive to 1p/19q co-deletion. Materials and Methods: Archived 97 grade II-IV diffuse gliomas of adults with known 1p/19q status on FISH testing (Vysis/Abbotte DNA FISH probes, Chicago, IL) were served for study. Using Envision kit (Dako, Santa Clara, CA), IDHR132H (H09; Dianova, Hamburg, Germany), p53 (DO-7; DAKO) and ATRX (polyclonal HPA001906; Sigma-Aldrich, St. Louis, MO) were applied formalin-fixed, paraffin-embedded sections. Tumors with strongly positive nuclei > 10% of tumor cells on p53 IHC was regarded as positive (TP53 mutated) based on previous studies. Results: All ATRX-lost gliomas were 1p/19q non-deleted. Seven IDH1R132H-negative tumors were p53-positive and ATRX-lost. Six IDH1R132H-positive and ATRX-lost tumors were p53-negative. Of ATRX-intact 53 tumors, 19 tumors were p53-positive; 18 of which were 1p/19q non-deleted. Of 13 co-deleted tumors, two were IDH1R132H-negative. Conclusions: ATRX loss in gliomas was equivalent to 1p/19q non-deletion. IDH1R132H-negative, p53-positive and ATRX-lost gliomas were highly suspicious of carrying IDH mutations other than IDH1R132H. IDH1R132H-positive, ATRX-lost and p53-negative gliomas were suggested to have nonsense or frame-shift mutations of TP53. Although when IDH1R132H and p53 are negative and ATRX is intact it is difficult to predict 1p/19q status, the combined immunohistochemistry for IDH1R132H, p53 and ATRX improves the molecular classification of diffuse gliomas in daily practice.
| S-EPMC5463599 | biostudies-literature
Project description:BackgroundRecently, molecular classifications of gastric cancer (GC) have been proposed that include TP53 mutations and their functional activity. We aimed to demonstrate the correlation between p53 immunohistochemistry (IHC) and TP53 mutations as well as their clinicopathological significance in GC.MethodsDeep targeted sequencing was performed using surgical or biopsy specimens from 120 patients with GC. IHC for p53 was performed and interpreted as strong, weak, or negative expression. In 18 cases (15.0%) with discrepant TP53 mutation and p53 IHC results, p53 IHC was repeated.ResultsStrong expression of p53 was associated with TP53 missense mutations, negative expression with other types of mutations, and weak expression with wild-type TP53 (p<.001). The sensitivity for each category was 90.9%, 79.0%, and 80.9%, and the specificity was 95.4%, 88.1%, and 92.3%, respectively. The TNM stage at initial diagnosis exhibited a significant correlation with both TP53 mutation type (p=.004) and p53 expression status (p=.029). The Kaplan-Meier survival analysis for 109 stage II and III GC cases showed that patients with TP53 missense mutations had worse overall survival than those in the wild-type and other mutation groups (p=.028). Strong expression of p53 was also associated with worse overall survival in comparison to negative and weak expression (p=.035).ConclusionsResults of IHC of the p53 protein may be used as a simple surrogate marker of TP53 mutations. However, negative expression of p53 and other types of mutations of TP53 should be carefully interpreted because of its lower sensitivity and different prognostic implications.
| S-EPMC7483024 | biostudies-literature
Project description:AimsThe aim of this study was to establish p53 immunohistochemistry (IHC) patterns to predict TP53 mutations in gastrointestinal neuroendocrine neoplasms (GI-NENs) and to determine whether p53 IHC patterns could be used for the differential diagnosis of neuroendocrine neoplasms.MethodsTP53 gene sequencing and p53 IHC were performed on formalin-fixed paraffin-embedded (FFPE) tissue samples from 92 patients diagnosed with GI-NENs from five medical centers.ResultsThe cohort included 35 well-differentiated neuroendocrine tumors and 57 poorly differentiated neuroendocrine carcinomas. Gene sequencing revealed 38 wild-type TP53 and 54 TP53 mutations. p53 expression was interpreted as follows: pattern A, p53 was absent from all tumor cells; pattern B, scattered and weak p53 expression in 1-20% of tumor cells; and pattern C was subclassified as pattern C1: variable p53 staining intensity in 21-60% of tumor cells and tumor cell nests with focal strong positive p53 staining and pattern C2: strong p53 staining in more than 60% of tumor cells. p53 IHC patterns were evaluated as a binary classifier where pattern B predicted wild-type TP53, and patterns A and C predicted TP53 mutations. The sensitivity, specificity, and overall accuracy of this binary classification to predict TP53 status were 0.963, 0.868, and 0.924, respectively. p53 IHC patterns were also correlated with TP53 mutation types. Most cases with pattern A harboured loss-of-function (LOF) mutations, whereas patterns B and C tended to indicate wild-type TP53 and gain-of-function (GOF) mutations, respectively. Furthermore, most of the well-differentiated NETs showed pattern B, whereas pattern C2 was more common in poorly differentiated NECs. Finally, staining interpretation between different observers also yielded high reproducibility.Conclusionsp53 IHC patterns may be used as predictors of TP53 gene mutations and therefore could be potential surrogate markers for TP53 mutations in GI-NENs and could distinguish between well-differentiated NETs and poorly differentiated NECs.
| S-EPMC8695011 | biostudies-literature
Project description:Diffuse low-grade gliomas (LGG) have been reclassified based on molecular mutations, which require invasive tumor tissue sampling. Tissue sampling by biopsy may be limited by sampling error, whereas non-invasive imaging can evaluate the entirety of a tumor. This study presents a non-invasive analysis of low-grade gliomas using imaging features based on the updated classification. We introduce molecular (MGMT methylation, IDH mutation, 1p/19q co-deletion, ATRX mutation, and TERT mutations) prediction methods of low-grade gliomas with imaging. Imaging features are extracted from magnetic resonance imaging data and include texture features, fractal and multi-resolution fractal texture features, and volumetric features. Training models include nested leave-one-out cross-validation to select features, train the model, and estimate model performance. The prediction models of MGMT methylation, IDH mutations, 1p/19q co-deletion, ATRX mutation, and TERT mutations achieve a test performance AUC of 0.83?±?0.04, 0.84?±?0.03, 0.80?±?0.04, 0.70?±?0.09, and 0.82?±?0.04, respectively. Furthermore, our analysis shows that the fractal features have a significant effect on the predictive performance of MGMT methylation IDH mutations, 1p/19q co-deletion, and ATRX mutations. The performance of our prediction methods indicates the potential of correlating computed imaging features with LGG molecular mutations types and identifies candidates that may be considered potential predictive biomarkers of LGG molecular classification.
| S-EPMC7048831 | biostudies-literature
Project description:BackgroundMutations in telomerase reverse transcriptase promoter (TERTp) and isocitrate dehydrogenase 1 and 2 (IDH) offer objective markers to assist in classifying diffuse gliomas into genetic subgroups. However, traditional mutation detection techniques lack sensitivity or have long turnaround times or high costs. We developed GliomaDx, an allele-specific, locked nucleic acid-based quantitative PCR assay to overcome these limitations and sensitively detect TERTp and IDH mutations.MethodsWe evaluated the performance of GliomaDx on cell line DNA and frozen tissue diffuse glioma samples with variable tumor percentage to mimic use in clinical settings and validated low percentage variants using sensitive techniques including droplet digital PCR (ddPCR) and next-generation sequencing. We also developed GliomaDx Nest, which incorporates a high-fidelity multiplex pre-amplification step prior to allele-specific PCR for low-input formalin-fixed paraffin embedded (FFPE) samples.ResultsGliomaDx detects the TERTp and IDH1 alterations at an analytical sensitivity of 0.1% mutant allele fraction, corresponding to 0.2% tumor cellularity. GliomaDx identified TERTp/IDH1 alterations in a cohort of frozen tissue samples with variable tumor percentage of all major diffuse glioma histologic types. GliomaDx Nest is able to detect these hotspot mutations with similar sensitivity from pre-amplified samples and was successfully tested on a cohort of clinical FFPE samples. Testing of a cohort of previously identified TERTpWT-IDHWT gliomas (by Sanger sequencing) revealed that 26.3% harbored low-percentage mutations. Analysis by ddPCR and whole exome sequencing of these tumors confirmed the low mutant fraction of these alterations and overall mutation-based tumor purity.ConclusionsOur results show that GliomaDx can rapidly detect TERTp/IDH mutations with high sensitivity, identifying cases that might be missed due to the lack of sensitivity of other techniques. This approach may facilitate more objective classification of diffuse glioma samples in clinical settings such as intraoperative diagnosis or in testing cases with low tumor purity.
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Project description:H3K27M diffuse intrinsic pontine gliomas (DIPGs) are fatal and lack treatments. They mainly harbor H3.3K27M mutations resulting in H3K27me3 reduction. Integrated analysis in H3.3K27M cells, tumors, and in vivo imaging in patients showed enhanced glycolysis, glutaminolysis, and tricarboxylic acid cycle metabolism with high alpha-ketoglutarate (α-KG) production. Glucose and/or glutamine-derived α-KG maintained low H3K27me3 in H3.3K27M cells, and inhibition of key enzymes in glycolysis or glutaminolysis increased H3K27me3, altered chromatin accessibility, and prolonged survival in animal models. Previous studies have shown that mutant isocitrate-dehydrogenase (mIDH)1/2 glioma cells convert α-KG to D-2-hydroxyglutarate (D-2HG) to increase H3K27me3. Here, we show that H3K27M and IDH1 mutations are mutually exclusive and experimentally synthetic lethal. Overall, we demonstrate that H3.3K27M and mIDH1 hijack a conserved and critical metabolic pathway in opposing ways to maintain their preferred epigenetic state. Consequently, interruption of this metabolic/epigenetic pathway showed potent efficacy in preclinical models, suggesting key therapeutic targets for much needed treatments.
| S-EPMC7494613 | biostudies-literature
Project description:Stimulating the innate immune system has been explored as a therapeutic option for the treatment of gliomas. Inactivating mutations in ATRX, defining molecular alterations in IDH-mutant astrocytomas, have been implicated in dysfunctional immune signaling. However, little is known about the interplay between ATRX loss and IDH mutation on innate immunity. To explore this, we generated ATRX-deficient glioma models in the presence and absence of the IDH1R132H mutation. ATRX-deficient glioma cells are sensitive to dsRNA-based innate immune agonism and exhibit impaired lethality and increased T-cell infiltration in vivo. However, the presence of IDH1R132H dampens baseline expression of key innate immune genes and cytokines in a manner restored by genetic and pharmacological IDH1R132H inhibition. IDH1R132H co-expression does not interfere with the ATRX deficiency-mediated sensitivity to dsRNA. Thus, ATRX loss primes cells for recognition of dsRNA, while IDH1R132H reversibly masks this priming. This work reveals innate immunity as a therapeutic vulnerability of astrocytomas.
| S-EPMC10810843 | biostudies-literature
Project description:PurposeDiffuse midline gliomas (DMG) with H3K27M mutations have been identified as a rare distinctive entity with unique genetic features, varied molecular alterations, and poor prognosis. The current study aimed to evaluate the clinical characteristics and profile of molecular markers on patients with a DMG harboring H3K27M mutations, and explore the impact of this genetic makeup on overall survival.MethodsWe retrospectively analyzed 43 consecutive patients diagnosed with a DMG harboring H3K27M mutations (age range 3 to 75 years) and treated in a tertiary institution within China between January 2017 to December 2019. Various clinical and molecular factors were evaluated to assess their prognostic value in this unique patient cohort.ResultsThe median overall survival (OS) was 12.83 months. Preoperative Karnofsky Performance Score (KPS) and adjuvant radiotherapy were found to be independent clinical parameters influencing the OS by multivariate analysis (p = 0.027 and p < 0.001 respectively). Whereas extent of tumor resection failed to demonstrate statistical significance. For molecular markers, P53 overexpression was identified as a negative prognostic factor for overall survival by multivariate analysis (p = 0.030).ConclusionLow preoperative KPS, absence of radiotherapy and P53 overexpression were identified as predictors of a dismal overall survival in patients with DMG and H3K27M mutations.
| S-EPMC7917281 | biostudies-literature
Project description:We evaluate the topographic distribution of diffuse midline gliomas and hemispheric high-grade gliomas in children with respect to their normal gene expression patterns and pathologic driver mutation patterns. We identified 19 pediatric patients with diffuse midline or high-grade glioma with preoperative MRI from tumor board review. 7 of these had 500 gene panel mutation testing, 11 patients had 50 gene panel mutation testing and one 343 gene panel testing from a separate institution were included as validation set. Tumor imaging features and gene expression patterns were analyzed using Allen Brain Atlas. Twelve patients had diffuse midline gliomas and seven had hemispheric high-grade gliomas. Three diffuse midline gliomas had the K27M mutation in the tail of histone H3 protein. All patients undergoing 500 gene panel testing had additional mutations, the most common being in ACVR1, PPM1D, and p53. Hemispheric high-grade gliomas had either TP53 or IDH1 mutation and diffuse midline gliomas had H3 K27M-mutation. Gene expression analysis in normal brains demonstrated that genes mutated in diffuse midline gliomas had higher expression along midline structures as compared to the cerebral hemispheres. Our study suggests that topographic location of pediatric diffuse midline gliomas and hemispheric high-grade gliomas correlates with driver mutations of tumor to the endogenous gene expression in that location. This correlation suggests that cellular state that is required for increased gene expression predisposes that location to mutations and defines the driver mutations within tumors that arise from that region.
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