Exploring p53 protein expression and its link to TP53 mutation in myelodysplasia-related malignancies-Interpretive challenges and potential field of applications.

AIMS: TP53 alterations have a significant prognostic effect in myeloid neoplasms. Our objective was to investigate the TP53 gene mutation status, p53 protein expression and their relationship in dysplasia-related myeloid neoplasms with varying levels of myeloblast counts. METHODS AND RESULTS: A total of 76 bone marrow biopsy samples with different blast counts were analysed. Total and strong (3+) p53 expression was determined. Dual immunohistochemical staining was performed to determine the cell population associated with p53 expression. NGS analysis was performed using the Accel-Amplicon Comprehensive TP53 panel. Both p53 expression and TP53 VAF showed a significant correlation with the myeloblast ratio (P < 0.0001); however, p53 expression was also present in other cell lineages. The VAF value exhibited a significant correlation with p53 expression. A high specificity (0.9800) was observed for TP53 mutation using the ≥ 10% strong (3+) p53 cut-off value, although the sensitivity (0.4231) was low. CONCLUSIONS: Strong (3+) p53 expression using a ≥ 10% cut-off value accurately predicts TP53 mutation but does not reveal the allelic state. The p53 expression is significantly influenced by myeloblast count, and histological interpretation should consider the presence of intermixed non-neoplastic marrow cells with varying physiological p53 expression.

Molecular Identification and In Silico Protein Analysis of a Novel BCOR-CLGN Gene Fusion in Intrathoracic BCOR-Rearranged Sarcoma.

BCOR (BCL6 corepressor)-rearranged sarcomas (BRSs) are a heterogeneous group of sarcomas previously classified as part of the group of "atypical Ewing" or "Ewing-like" sarcomas, without the prototypical ESWR1 gene translocation. Due to their similar morphology and histopathological features, diagnosis is challenging. The most common genetic aberrations are BCOR-CCNB3 fusion and BCOR internal tandem duplication (ITD). Recently, various new fusion partners of BCOR have been documented, such as MAML3, ZC3H7B, RGAG1, and KMT2D, further increasing the complexity of such tumor entities, although the molecular pathogenetic mechanism remains to be elucidated. Here, we present an index case of intrathoracic BRS that carried a novel BCOR-CLGN (calmegin) gene fusion, exhibited by a 52-year-old female diagnosed initially by immunohistochemistry due to the positivity of a BCOR stain; the fusion was identified by next-generation sequencing and was confirmed by Sanger sequencing. In silico protein analysis was performed to demonstrate the 3D structure of the chimera protein. The physicochemical properties of the fusion protein sequence were calculated using the ProtParam web-server tool. Our finding further broadens the fusion partner gene spectrum of BRS. Due to the heterogeneity, molecular ancillary tests serve as powerful tools to discover these unusual variants, and an in silico analysis of the fusion protein offers an appropriate approach toward understanding the exact pathogenesis of such a rare variant.

Correlation Analyses between Histological Staging and Molecular Alterations in Tumor-Derived and Cell-Free DNA of Early-Stage Primary Cutaneous Melanoma.

Here, we investigate the correlation and statistical analyses between histological staging and molecular alterations in tumor-derived (tdDNA) and cell-free DNA (cfDNA) obtained from early-stage primary cutaneous melanoma (PCM) patients using digital PCR (dPCR) for the detection of the BRAF p.V600E somatic pathogenic variant. In the prospective study, a total of 68 plasma and paired tdDNA samples, and in the retrospective cohort, a total of 100 tdDNA samples were analyzed using dPCR and reverse hybridization StripAssay. The Breslow depth (BD) and Clark level were applied to categorize the study population. Our results demonstrate that dPCR is a highly sensitive and specific method for the detection of BRAF p.V600E somatic variants in cfDNA samples from PCM patients. A strong correlation was detected between BD and cfDNA concentration in all mutant and negative cases, between the tdDNA concentration and the tumor-derived variant allele frequency (VAF) of BRAF p.V600E, between the tdVAF and the cfVAF in all cases, and between the cfDNA and cfVAF in mutant cases. The tdVAF and cfVAF of BRAF p.V600E and cfDNA concentration were the highest in Clark's V category. The cfDNA concentration was statistically significantly higher in Clark's III, IV, and V groups compared to cases with a better prognosis. It can also be explained by the fact that cases with a more advanced stage classification release more cfDNA into the peripheral circulation.

Deep Molecular and In Silico Protein Analysis of p53 Alteration in Myelodysplastic Neoplasia and Acute Myeloid Leukemia.

BACKGROUND: Mutation of the TP53 gene is one of the major drivers of myelodysplastic neoplasias (MDS) and acute myeloid leukemia with myelodysplasia-related changes (AML-MR). TP53 mutations present in these hematopoietic malignancies form a distinct molecular genetic cluster with a worse prognosis than without the alteration. However, besides well-characterized hot-spot variants, a significant proportion of TP53 alterations are of uncertain clinical significance. METHODS: To enlighten so far unknown aspects, bone-marrow samples from altogether 77 patients are analyzed retrospectively with the diagnosis of AML-MR (26 cases), MDS-IB (12 cases), and MDS-LB (39 cases) according to WHO 2022 guidelines. Next-generation sequencing results are correlated with histological, cytogenetic, and survival data. RESULTS: Twenty out of the 30 TP53 mutation types detected by NGS are not categorized in current public databases; thus, their clinical significance remained mysterious. Because of the interpretation difficulties and the absence of clinical correlations, pathogenicity is established based on in silico approaches. The 12 pathogenicity classification systems, as well as protein stability, protein-DNA, protein-protein interaction, and post-translational modification analyses are applied. We found statistically significant differences between AML/MDS groups considering p53 pathogenicity, protein structural changes, and overall survival. The largest number of abnormalities with the most severe consequences are found in AML-MR cases. CONCLUSIONS: These molecular and in silico protein data further support that MDS with increased-blast (MDS-IB) is an intermediate group between AML-MR and MDS with low-blast (MDS-LB) patients, which frequently progresses to AML and is therefore considered a pre-leukemic condition.