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Revisiting TP53 Mutations and Immunohistochemistry--A Comparative Study in 157 Diffuse Gliomas.

ABSTRACT: The association between p53 immunohistochemistry and TP53 mutation status has been controversial. The present study aims to re-evaluate the efficacy of p53 immunohistochemistry to predict the mutational status of TP53. A total of 157 diffuse gliomas (World Health Organization grades II-IV) were assessed by exon-by-exon DNA sequencing from exon 4 through 10 of TP53 using frozen tissue samples. Immunohistochemistry with a p53 antibody (DO-7) on paired formalin-fixed paraffin-embedded materials was assessed for the extent and intensity of reactivity in all cases. A total of 72 mutations were detected in 66 samples. They included 60 missense mutations, five nonsense mutations, four deletions and three alterations in the splicing sites. A receiver operating characteristic curve analysis revealed that strong p53 immunoreactivity in more than 10% of cells provided the most accurate prediction of mutation. Using this cutoff value, 52 of 55 immunopositive cases harbored a mutation, whereas only 14 of 102 immunonegative cases showed mutations, sensitivity and specificity being 78.8% and 96.7%. Tumors with frameshift mutations frequently showed negative immunostaining. Staining interpretation by an independent observer yielded comparable accuracy. We thus propose p53 immunohistochemistry as a moderately sensitive and highly specific marker to predict TP53 mutation.

SUBMITTER: Takami H

PROVIDER: S-EPMC8028857 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Revisiting TP53 Mutations and Immunohistochemistry--A Comparative Study in 157 Diffuse Gliomas.

Takami Hirokazu H Yoshida Akihiko A Fukushima Shintaro S Arita Hideyuki H Matsushita Yuko Y Nakamura Taishi T Ohno Makoto M Miyakita Yasuji Y Shibui Soichiro S Narita Yoshitaka Y Ichimura Koichi K

Brain pathology (Zurich, Switzerland) 20141029 3

The association between p53 immunohistochemistry and TP53 mutation status has been controversial. The present study aims to re-evaluate the efficacy of p53 immunohistochemistry to predict the mutational status of TP53. A total of 157 diffuse gliomas (World Health Organization grades II-IV) were assessed by exon-by-exon DNA sequencing from exon 4 through 10 of TP53 using frozen tissue samples. Immunohistochemistry with a p53 antibody (DO-7) on paired formalin-fixed paraffin-embedded materials was ...[more]

PMID: 25040820

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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.

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.

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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Revisiting TP53 Mutations and Immunohistochemistry--A Comparative Study in 157 Diffuse Gliomas.

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Revisiting TP53 Mutations and Immunohistochemistry--A Comparative Study in 157 Diffuse Gliomas.

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