Biliary tract cancer (BTC) is a rare malignancy with a long disease course and an overall poor prognosis. Despite multiple chemotherapy agents, there is no defined second-line treatment opportunity for advanced BTCs. In the era of precision oncology, NGS plays an important role in identifying mutations that may predict the molecular pathomechanism and manage the BTC therapy. The peripheral blood liquid biopsy (LB) of cancer patients represents variable amounts of cell-free DNA (cfDNA) released from tumor foci of any anatomical location. Our study aimed to identify somatic mutations and tumor variant burden (TVB) in cell-free and matched tumor DNA. We found a positive correlation between the estimated tumor volume and cfDNA yield (r = 0.9326, p < 0.0001). Comparing tissue and LB results, similar TVB was observed. SNVs were proven in 84% of the cases, while in two cases, only the LB sample was informative for molecular analysis. The most important aberrations in BTCs, such as FGFR2, IDH1, IDH2, KRAS, and TP53, could be detected in matched LB samples. Our prospective study demonstrates a minimally invasive testing approach to identify molecular genetic alterations in cholangiocarcinoma and gallbladder cancers. Clinical applications of cfDNA reflect by capturing the outstanding spatial tumor heterogeneity and guarantee novel aspects for the precision oncology treatment.

Merkel cell carcinoma (MCC) is a rare, high-grade, aggressive cutaneous neuroendocrine malignancy most commonly associated with sun-exposed areas of older individuals. A relatively newly identified human virus, the Merkel cell polyomavirus (MCPyV) has been implicated in the pathogenesis of MCC. Our study aimed to examine nine MCC cases and randomly selected 60 melanoma cases to identify MCPyV status and to elucidate genetic differences between virus-positive and -negative cases. Altogether, seven MCPyV-positive MCC samples and four melanoma samples were analyzed. In MCPyV-positive MCC RB1, TP53, FBXW7, CTNNB1, and HNF1A pathogenic variants were identified, while in virus-negative cases only benign variants were found. In MCPyV-positive melanoma cases, besides BRAF mutations the following genes were also affected: PIK3CA, STK11, CDKN2A, SMAD4, and APC. In contrast to studies found in the literature, a higher tumor burden was detected in virus-associated MCC compared to MCPyV-negative cases. No association was identified between virus infection and tumor burden in melanoma samples. We concluded that analyzing the key morphologic and immunohistological features of MCC is critical to avoid confusion with other cutaneous malignancies. Molecular genetic investigations such as next-generation sequencing (NGS) enable molecular stratification, which may have future clinical impact.

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.

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.

BCOR (BCL-6 corepressor) rearranged small round cell sarcoma (BRS) represents an uncommon soft tissue malignancy, frequently characterized by the BCOR::CCNB3 fusion. Other noteworthy fusions include BCOR::MAML3, BCOR::CLGN, BCOR::MAML1, ZC3H7B::BCOR, KMT2D::BCOR, CIITA::BCOR, RTL9::BCOR, and AHR::BCOR. The BCOR gene plays a pivotal role in the Polycomb Repressive Complex 1 (PRC1), essential for histone modification and gene silencing. It interfaces with the Polycomb group RING finger homolog (PCGF1). This study employed comprehensive in silico methodologies to investigate the structural and functional effects of BCOR fusion events in BRS. The analysis revealed significant alterations in the domain architecture of BCOR, which resulted in the loss of BCL6-regulated transcriptional repression. Furthermore, IUPred3 prediction indicated a significant increase in disorder in the C-terminal regions of the BCOR in the fusion proteins. A detailed analysis of the physicochemical properties by ProtParam revealed a decrease in isoelectric point, stability, and hydrophobicity. The analysis of protein structures predicted by AlphaFold3 using the PRODIGY algorithm revealed statistically significant deviations in binding affinities for BCOR-PCGF1 dimers and a non-canonical PRC1 variant tetramer compared to the wild-type BCOR. The findings provide a comprehensive summary and elucidation of the fusion proteome associated with BRS, suggesting a substantial impact on the stability and functionality of the fusion proteins, thereby contributing to the oncogenic mechanisms underlying BRS. In this study, we provide the first compilation and comparative analysis of the known BCOR fusions of BRS and introduce a new in silico approach to enhance a better understanding of the molecular basis of BRS.

BCOR (BCL-6 corepressor)-rearranged small round cell sarcoma (BRS) is a rare soft tissue tumor, mostly featuring the BCOR :: CCNB3 fusion, with other fusions like BCOR :: MAML3 , BCOR :: CLGN , ZC3H7B :: BCOR , KMT2D :: BCOR , CIITA :: BCOR , and RTL9 - BCOR also reported. BCOR, a Polycomb Repressive Complex 1 (PRC1) component, influences histone modifications. It dimerizes with Polycomb group RING finger homolog (PCGF1) via its PCGF ubiquitin-like fold discriminator (PUFD) domain interacting with PCGF1’s RING finger and WD40-associated ubiquitin-like (RAWUL) domain. We used various in silico tools to explore the impact of fusion events on BCOR’s functionality and RAWUL-PUFD dimer binding affinity. Changes were found in the domain landscapes, physicochemical properties, GO terms and significant increases in the disordered regions within the PUFD domain of the fusion proteins. Structural predictions indicated modified intermolecular contacts (ICs) and a significant reduction in binding affinity in fusion protein RAWUL-PUFD dimers. These findings align with expression data showing PRC1-regulated gene upregulation in BRS, likely due to reduced RAWUL-PUFD binding affinity, impacting dimer formation and PRC1 assembly. Our findings enhance the understanding of BRS oncogenesis and identify potential therapeutic targets.