Breast cancer is the most frequent cancer with a high fatality rate amongst women worldwide. Diagnosing at an early stage is challenging, and due to the limitations of the currently used techniques, including mammography and imaging diagnostics, it still remains unascertained. Serum biomarkers can be a solution for this as they can be isolated in a less painful, more cost-effective, and minimally invasive manner. In this study, we shed light on the relevant role of multiple microRNAs (miRNAs) as potential biomarkers in breast cancer diagnosis. We monitored the expressional changes of 15 pre-selected miRNAs in a large cohort, including 65 patients with breast cancer and 42 healthy individuals. We performed thorough statistical analyses on the cohort sample set and determined the diagnostic accuracy of individual and multiple miRNAs. Our study reveals a potential improvement in diagnostics by implicating the monitoring of miR-15a+miR-16+miR-221 expression in breast cancer management.

Reproductive abnormalities have been observed in fallow deer populations in Hungary. We supposed mycotoxin contamination to be one of the possible causes because multi-mycotoxin contamination is known to be dangerous even at low toxin levels, especially for young animals. We investigated the spatial pattern of mycotoxin occurrences and the relationship between maternal and fetal mycotoxin levels. A total of 72 fallow deer embryos and their mothers were sampled in seven forested regions in Hungary in the 2020/2021 hunting season. We analyzed Aflatoxin (AF), Zearalenone (ZEA), Fumonizin B1 (FB1), DON, and T2-toxin concentrations in maternal and fetal livers by ELISA. AF was present in 70% and 82%, ZEA in 41% and 96%, DON in 90% and 98%, T2-toxin in 96% and 85%, and FB1 in 84% and 3% of hind and fetus livers, respectively. All mycotoxins passed into the fetus, but only Fumonizin B1 rarely passed. The individual variability of mycotoxin levels was extremely high, but the spatial differences were moderate. We could not prove a relation between the maternal and fetal mycotoxin concentrations, but we found an accumulation of ZEA and DON in the fetuses. These results reflect the possible threats of mycotoxins to the population dynamics and reproduction of wild fallow deer.

Germline mutations in the BRCA1 and BRCA2 genes are responsible for hereditary breast and ovarian cancer syndrome. Germline and somatic BRCA1/2 mutations may define therapeutic targets and refine cancer treatment options. However, routine BRCA diagnostic approaches cannot reveal the exact time and origin of BRCA1/2 mutation formation, and thus, the fine details of their contribution to tumor progression remain less clear. Here, we establish a diagnostic pipeline using high-resolution microscopy and laser microcapture microscopy to test for BRCA1/2 mutations in the tumor at the single-cell level, followed by deep next-generation sequencing of various tissues from the patient. To demonstrate the power of our approach, here, we describe a detailed single-cell-level analysis of an ovarian cancer patient we found to exhibit constitutional somatic mosaicism of a pathogenic BRCA2 mutation. Employing next-generation sequencing, BRCA2 c.7795G>T, p.(Glu2599Ter) was detected in 78% of reads in DNA extracted from ovarian cancer tissue and 25% of reads in DNA derived from peripheral blood, which differs significantly from the expected 50% of a hereditary mutation. The BRCA2 mutation was subsequently observed at 17–20% levels in the normal ovarian and buccal tissue of the patient. Together, our findings suggest that this mutation occurred early in embryonic development. Characterization of the mosaic mutation at the single-cell level contributes to a better understanding of BRCA mutation formation and supports the concept that the combination of single-cell and next-generation sequencing methods is advantageous over traditional mutational analysis methods. This study is the first to characterize constitutional mosaicism down to the single-cell level, and it demonstrates that BRCA2 mosaicism occurring early during embryogenesis can drive tumorigenesis in ovarian cancer.

Circulating tumor DNA (ctDNA) is increasingly employed in the screening, follow-up, and monitoring of the continuously evolving tumor; however, most ctDNA assays validated for clinical use cannot maintain the right balance between sensitivity, coverage, sample requirements, time, and cost. Here, we report our BC-monitor, a simple, well-balanced ctDNA diagnostic approach using a gene panel significant in breast cancer and an optimized multiplex PCR-based NGS protocol capable of identifying allele variant frequencies below 1% in cell-free plasma DNA. We monitored a cohort of 45 breast cancer patients prospectively enrolled into our study receiving neoadjuvant chemotherapy or endocrine therapy or palliative therapy for metastatic diseases. Their tumor mutation status was examined in the archived tumor samples and plasma samples collected before and continuously during therapy. Traceable mutations of the used 38-plex NGS assay were found in approximately two-thirds of the patients. Importantly, we detected new pathogenic variants in follow-up plasma samples that were not detected in the primary tumor and baseline plasma samples. We proved that the BC-monitor can pre-indicate disease progression four–six months earlier than conventional methods. Our study highlights the need for well-designed ctDNA monitoring during treatment and follow-up, integrated into a real-time treatment assessment, which could provide information on the active tumor DNA released into the blood.

Cervical intraepithelial neoplasia (CIN) represents a spectrum of preinvasive squamous lesions within the cervical epithelium, whose identification is a diagnostic challenge due to subtle histomorphological differences among its categories. This study explores ORF1p, a nucleic acid-binding protein derived from long interspersed nuclear element-1 (LINE-1), as a potential biomarker for enhancing CIN diagnosis. A comprehensive analysis of 143 cervical specimens, encompassing CIN I (n=20), CIN II (n=46), CIN III (n=14), invasive cancer (n=32), and nondysplastic cases (normal cervical epithelia (n=24) and atrophy (n=7) were conducted. ORF1p, Ki67, and p16 expressions were evaluated using immunohistochemistry. ORF1p immunopositivity was detected in the vast majority [110/112 (98.2%)] of dysplastic and neoplastic (CIN and invasive cancer) specimens, whereas 19/24 (79.2%) of normal cervical specimens lacked ORF1p expression. The observed pattern of ORF1p expression showed a progressively increasing extent and intensity with advancing CIN grades. CIN I exhibited mild ORF1p expression in the lower one or two-thirds of the cervical epithelium [14/16 (87.5%)], whereas CIN II demonstrated moderate to strong ORF1p expression spanning the lower two-thirds [29/46 (63.0%)]. Pronounced transepithelial ORF1p immunopositivity characterized CIN III cases [13/14 (92.8%)] and cervical cancer [30/32 (93.8%)]. These findings propose ORF1p as a valuable indicator even for detecting CIN I, effectively discerning them from normal cervical tissue (p < 0.0001). Our findings underscore the potential of ORF1p as an early diagnostic marker for cervical neoplasia.

Reproductive abnormalities have been observed in fallow deer populations in Hungary. We supposed mycotoxin contamination to be one of the possible causes because multi-mycotoxin contamination is known to be dangerous even at low toxin levels, especially for young animals. We investigated the spatial pattern of mycotoxin occurrences and the relation of maternal and foetal mycotoxin levels. 72 fallow deer embryos and their mothers were sampled in seven forested regions in Hungary in the 2020/2021 hunting season. We analyzed Aflatoxin (AF), Zearalenone (ZEA), Fumonizin B1 (FB1), DON, T2-toxin concentrations in maternal and foetal liver by ELISA. AF was present in 70% and 82%, ZEA in 41% and 96%, DON in 90% and 98%, T2 in 96% and 85%, FB1 in 84% and 3% of hind and foetus livers, respectively. All mycotoxins passed into the foetus, but Fumonizin B1 only rarely. The individual variability of mycotoxin levels was extremely high, but the spatial differences were moderate. We could not prove a relation between the maternal and foetal mycotoxin concentrations, but we found accumulation of ZEA and DON in the foetuses. These results reflect the possible threats of mycotoxins to the population dynamics and reproduction of wild fallow deer.