Detecting the H3F3A mutant allele found in high-grade pediatric glioma by real-time PCR.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brain tumors with a median survival of 1 year after diagnosis. It has been reported recently that about 80% of cases DIPG and 70% of glioblastomas middle line contained a mutation in one allele of the gene H3F3A (encoding the H3 histone variant H3.3), replacing the lysine 27 to methionine (K27M). In order to facilitate the diagnosis of patients with DIPG, rapid and reliable method for identifying mutations K27M H3F3A required.

Here, we describe a real-time PCR-based procedure involving a specific mutant primer, oligonucleotide blockers, and the reverse primer can distinguish samples with H3F3A K27M mutation of those who do not. We first tested four different primer specific mutants for their ability to selectively Strengthening H3F3A K27M-mutant alleles and found that one mutant allele primer amplified more efficiently than others. We then determined the optimal concentration of oligo blocker that significantly enhances amplification H3F3A K27M-mutant allele.

The optimized using real-time PCR assay, we analyzed eleven samples, two of which contain H3F3A K27M mutation, and found that two different samples are amplified from nine others. In addition, we were able to distinguish H3F3A K27M mutation in pediatric glioblastoma brain stem sample of newly acquired that status previously unknown H3.3, and in three other DIPG samples and paraffin embedded samples. These results demonstrate that we have developed new reliable procedure for detecting mutations K27M H3F3A in glioblastoma samples of pediatric patients.

Detecting the H3F3A mutant allele found in high-grade pediatric glioma by real-time PCR.
Detecting the H3F3A mutant allele found in high-grade pediatric glioma by real-time PCR.

analysis of real-time PCR based on the BRAF V600E mutation in the low and medium grade lymphoma assert common in hairy cell leukemia.

Hairy cell leukemia (HCL) is a rare type of B-cell non-Hodgkin lymphoma (B-NHL), which is not known to be associated with recurrent abnormality karyotype characteristics. A recent study used massively parallel whole exome sequencing to identify mutations in the BRAF V600E enabled, which appears on the HCL. Here, we confirmed the specificity of BRAF V600E for HCL among low- and middle-grade B-NHL and describes a method of polymerase chain reaction real-time to detect this mutation in cases with a low tumor burden.

The V600E mutation does not appear associated with microsatellite instability, unlike the case of colorectal cancer. Thus, in conjunction with previous data, our results suggest the incorporation of BRAF V600E mutation analysis in the diagnostic workup of cases HCL. In addition, targeting the Ras-Raf-Mek-Erk-Map kinase should be investigated as a potential therapeutic strategy for patients with this disease.

OBJECTIVE
Prominin1 / CD133 has been the ideal marker for cancer stem cells (CSC) detection in human tumors. In this study we investigated the expression of this marker in some breast cancer specimens with CSC percentage associate with risk factors for this neoplasia.


METHOD
We examine specimens from 12 patients using CD133 and CD44 antibodies for the detection of CSC immunohistochemistry and for flow cytometry analysis. For each patient, we also perform immunohistochemical staining to evaluate the expression of estrogen receptor, progesterone receptor, marker c-erbB-2, Ki67, and E-cadherin. A TaqMan Probes for CD133 is used for mRNA quantification by real-time polymerase chain reaction.

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RESULTS
Prominin-1 expression is heterogenous different carcinomas but striking hyperexpressed in tubulolobular variant of breast cancer. Results were confirmed by all three methods.