Cemaliye Boylu Akyerli

HerCanPred, a machine-learning-based pathogenicity classifier specifically optimized for 63 cancer-predisposition genes, was developed to improve the interpretation of missense variants in hereditary cancer syndromes. This model integrates sequence conservation with structural features derived from AlphaFold2 (AF2) structures. HerCanPred achieved a strong performance, outperforming 23 established predictors. SHAP analysis identified AF2-derived structural features, specifically local pLDDT confidence scores and relative solvent accessible area, as the strongest predictors of variant impact. Benchmarking the strengths and limitations of HerCanPred against existing methods showed that misclassification of pathogenic variants was concentrated in disordered and surface-exposed regions, whereas benign failures were more broadly distributed. HerCanPred and three established predictors were also applied to over 57,000 variants of uncertain significance (VUS) from the same gene set. Notably, 166 VUS were reassigned as pathogenic and 75 as benign, with an enrichment of the NF1, FH, and MLH1 genes. By combining gene-specific training with 3D structural information, HerCanPred provides a robust framework for reducing diagnostic uncertainty. Our findings demonstrate that targeted, structure-aware tools can contribute to resolving VUS, providing a rational basis for systematic variant reinterpretation and more informed medical management in hereditary cancer care.

Cholangiocarcinoma is a rare but highly lethal cancer of the biliary epithelium, marked by heterogeneous molecular subtypes, unclear etiology, and poor five-year survival, highlighting the need for new diagnostic and therapeutic strategies; therefore, this study integrates genomic, transcriptomic, single-cell, methylomic, and molecular-dynamics data to pinpoint pathogenic variants. We performed an integrative multi-omics analysis of publicly available datasets. Somatic variants from 23 tumor samples in The Cancer Genome Atlas were annotated with 11 pathogenicity tools (AUC ≥ 0.86 across EVE, REVEL, SIFT, AlphaMissense, DEOGEN2 were the most stringent). Differential gene expression was assessed in matched bulk RNA-seq (tumor vs. non-tumor) using DESeq2 with Benjamini–Hochberg FDR correction. A single-cell RNA-seq dataset comprising 23,782 cells from an intrahepatic cholangiocarcinoma was clustered with marker genes identified by Wilcoxon rank-sum tests. Illumina 450 K methylation arrays (52 tumors, 12 normal livers) were analyzed with limma and DMRcate to detect differentially methylated probes and regions. AlphaFold3 models of wild-type and MAP2K1R49C were subjected to 50 ns all-atom molecular-dynamics simulations in GROMACS; conformational shifts were quantified by RMSD/RMSF and stability tested with FoldX5. Twenty-four tumor-specific missense variants were detected. The four highest-confidence pathogenic substitutions (EVE, REVEL, SIFT, AlphaMissense, DEOGEN2) occurred in TUBB3, FLNC, ABCA1, and MAP2K1. Bulk RNA-seq confirmed significant dysregulation of these genes and enrichment of extracellular-matrix organization, cytoskeletal remodeling, MAPK signaling, and cholesterol-efflux pathways. Single-cell analysis resolved 23 transcriptionally distinct clusters; proliferative malignant cholangiocytes selectively over-expressed ABCA1 and MAP2K1, indicating tumor-cell specificity. Methylome profiling identified 148,928 DMPs and 7040 DMRs; promoter hypomethylation of TUBB3 and ABCA1 correlated with their transcriptional activation. Substituting Arg-49 with Cys in MAP2K1 dismantles the Arg-centred hydrogen-bond/salt-bridge cluster, reduces hydrophobic packing, and, corroborated by 50 ns MD (Welch’s t = −58.06, p = 3.17 × 10−230) and FoldX5 (ΔΔG = +2.3 kcal mol−1), significantly destabilises the protein, manifesting as higher backbone RMSD and increased local flexibility relative to wild type. This multi-omics, public data-driven synthesis delineates a coherent network of genomic, epigenomic, transcriptomic, and structural vulnerabilities, offering a rational framework for therapeutic targeting of cholangiocarcinoma. This study reveals novel bile duct-associated variations that expand our understanding of cholangiocarcinoma pathogenesis and provide potential targets for precision medicine approaches.

Background/Objectives: Triple-negative breast cancer (TNBC) is a highly aggressive subtype with limited therapeutic options, and identifying reliable biomarkers for diagnosis and prognosis is crucial for improving patient outcomes. Mitochondrial DNA (mtDNA) copy number has been linked to an increased risk of developing various types of cancer, including breast cancer. However, there is a lack of understanding regarding how mtDNA copy number variations may influence the development and progression of TNBC. Methods: This study investigated mtDNA copy number in TNBC tumors and corresponding normal breast tissues from 23 TNBC patients who received neoadjuvant chemotherapy. The relative mtDNA copy number was estimated using quantitative PCR for the NADH dehydrogenase subunit 1 (ND1) and subunit 5 (ND5) regions. Results: The results showed a significant decrease in mtDNA copy number in TNBC tumor tissues compared to corresponding normal breast tissue. However, no significant correlation was found between mtDNA content and clinical parameters such as age, tumor size, or chemotherapy response. Conclusions: These results suggest that while mtDNA content decreases in TNBC tumors, it may not directly influence these clinical characteristics. Despite some inconsistencies in the literature regarding mtDNA dynamics in cancer, this study supports the potential of mtDNA as a biomarker for TNBC. Larger cohort studies are needed to further validate these results and explore the role of mtDNA in guiding personalized treatment strategies for TNBC patients.

Mitochondria generate energy through cellular respiration and regulate various cellular processes such as heat production, generation and detoxification of reactive oxygen species, metabolism, apoptosis, and calcium homeostasis. In human cells, large numbers of mitochondria are present, each containing multiple copies of mitochondrial DNA. Variations in mitochondrial DNA have been associated with the onset and progression of various diseases, including neurological, cardiovascular, and metabolic disorders and also several cancers. These variants can be important drivers of cancer and may play a crucial role in tumor development. Additionally, mitochondrial copy number changes and structural variations, such as deletions can be associated with different types of cancer. Therefore, understanding the fundamental mechanisms is highly crucial. The molecular genetic correlations of mitochondrial DNA alterations and cancer, emphasize the importance of mitochondrial integrity in maintaining cellular homeostasis. Gaining knowledge of these associations can help us comprehend cancer processes as well as potential routes for targeted treatments and prevention, while further investigation is still required.

Identifying pathogenic missense variants in hereditary cancer is critical to the efforts of patient surveillance and risk-reduction strategies. For this purpose, many different gene panels consisting of different number and/or set of genes are available and we are particularly interested in a panel of 26 genes with a varying degree of hereditary cancer risk consisting of ABRAXAS1, ATM, BARD1, BLM, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, MEN1, MLH1, MRE11, MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53, and XRCC2. In this study, we have compiled a collection of the missense variations reported in any of these 26 genes. More than a thousand missense variants were collected from ClinVar and the targeted screen of a breast cancer cohort of 355 patients which contributed to this set with 160 novel missense variations. We analyzed the impact of the missense variations on protein stability by five different predictors including both sequence- (SAAF2EC and MUpro) and structure-based (Maestro, mCSM, CUPSAT) predictors. For the structure-based tools, we have utilized the AlphaFold (AF2) protein structures which comprise the first structural analysis of this hereditary cancer proteins. Our results agreed with the recent benchmarks that computed the power of stability predictors in discriminating the pathogenic variants. Overall, we reported a low-to-medium-level performance for the stability predictors in discriminating pathogenic variants, except MUpro which had an AUROC of 0.534 (95% CI [0.499–0.570]). The AUROC values ranged between 0.614–0.719 for the total set and 0.596–0.682 for the set with high AF2 confidence regions. Furthermore, our findings revealed that the confidence score for a given variant in the AF2 structure could alone predict pathogenicity more robustly than any of the tested stability predictors with an AUROC of 0.852. Altogether, this study represents the first structural analysis of the 26 hereditary cancer genes underscoring 1) the thermodynamic stability predicted from AF2 structures as a moderate and 2) the confidence score of AF2 as a strong descriptor for variant pathogenicity.

Amaç: Hücre için başta enerji metabolizması ve yapı taşlarının üretimi olmak üzere oldukça önemli bir role sahip olan mitokondrinin kanserdeki rolü henüz tam olarak bilinmemektedir. Mitokondriyal genomdaki varyasyonlar ve polimorfizm kombinasyonları kullanılarak belirlenen haplogrupların farklı kanserlerdeki etkisi araştırılmaya devam edilmektedir. Bu nedenle, mitokondriyal genomu çoğaltarak yeni nesil yöntemlerle dizileme protokolünü optimize ederek kanser çalışmalarında kullanmayı amaçladık.Materyal ve Metod: Gliom tümör örneklerinden elde edilmiş genomik DNAlar’dan mitokondriyal genom iki ayrı reaksiyon halinde Takara LA Taq DNA polimeraz ile çoğaltılmıştır. “Nextera XT DNA Library Prep Kit” kullanılarak kütüphane oluşturulmuş ve yeni nesil dizileme MiSeq FGx cihazında gerçekleştirilmiştir. Elde edilen veriler “mtDNA Server” veritabanı kullanılarak mitokondriyal varyasyonlar, heteroplazmi oranları ve haplogruplar açısından analiz edilmiştir.Bulgular: Mitokondriyal genom için yeni nesil dizileme protokolü optimize edilmiştir. Detaylı analiz edilen iki tümör örneğinde çeşitli varyasyonlar ve bunların heteroplazmi oranları tespit edilebilmiştir. Ayrıca, örneklere ait haplogruplar da saptanmıştır.Sonuç: Mitokondriyal varyasyonların kanser biyolojisi üzerindeki etkisi hala araştırılmaktadır. Yeni nesil dizileme teknolojileri özellikle heteroplazmi oranı düşük tümörlerin analizinde kullanılabilecek güvenilir bir yöntemdir.

Little is known about the mutational processes that shape the genetic landscape of gliomas. Numerous mutational processes leave marks on the genome in the form of mutations, copy number alterations, rearrangements or their combinations. To explore gliomagenesis, we hypothesized that gliomas with different underlying oncogenic mechanisms would have differences in the burden of various forms of these genomic alterations. This was an analysis on adult diffuse gliomas, but IDH-mutant gliomas as well as diffuse midline gliomas H3-K27M were excluded to search for the possible presence of new entities among the very heterogenous group of IDH-WT glioblastomas. The cohort was divided into two molecular subsets: (1) Molecularly-defined GBM (mGBM) as those that carried molecular features of glioblastomas (including TERT promoter mutations, 7/10 pattern, or EGFR-amplification), and (2) those who did not (others). Whole exome sequencing was performed for 37 primary tumors and matched blood samples as well as 8 recurrences. Single nucleotide variations (SNV), short insertion or deletions (indels) and copy number alterations (CNA) were quantified using 5 quantitative metrics (SNV burden, indel burden, copy number alteration frequency-wGII, chromosomal arm event ratio-CAER, copy number amplitude) as well as 4 parameters that explored underlying oncogenic mechanisms (chromothripsis, double minutes, microsatellite instability and mutational signatures). Findings were validated in the TCGA pan-glioma cohort. mGBM and “Others” differed significantly in their SNV (only in the TCGA cohort) and CNA metrics but not indel burden. SNV burden increased with increasing age at diagnosis and at recurrences and was driven by mismatch repair deficiency. On the contrary, indel and CNA metrics remained stable over increasing age at diagnosis and with recurrences. Copy number alteration frequency (wGII) correlated significantly with chromothripsis while CAER and CN amplitude correlated significantly with the presence of double minutes, suggesting separate underlying mechanisms for different forms of CNA.

Objective: Despite the recent advances in diagnosis and treatment of hepatocellular carcinoma (HCC), it is still a major health problem. Therefore, understanding the molecular mechanism is very important. Our aim is to investigate the molecular basis of aflatoxin B1 (AFB1) induced HCC other than the hotspot TP53 p.Arg249Ser (c.747G>T) (R249S) mutation. Methods: 525 genes previously reported to be involved in carcinogenesis with mutations in different cancer types were analyzed by next generation sequencing for 525 cancer-gene panel (Roche/NimbleGen) in one tumor sample (T29) and one cell line (MAHLAVU) carrying TP53 R249S mutation. Additionally, ARID2 and BCORL1 genes were analyzed by Sanger sequencing for MAHLAVU and Primary Liver Carcinoma/Poliomyelitis Research Foundation/5 (PLC/PRF/5) cell lines. Results: No other common gene mutations were found in the analyzed T29 and MAHLAVU samples and also no genetic variation possibly associated with AFB1 was detected in PLC/PRF/5 cell line and 68 COSMIC HCC samples. Likewise, no pathogenic mutation was detected in ARID2 and BCORL1 genes of MAHLAVU and PLC/PRF/5 cell lines. Conclusion: No fingerprint mutations were detected in the analyzed genes. To the best of our knowledge, other hotspot mutations appear to be absent if not at a very low frequency in HCC carrying TP53 R249S mutation.

In a retrospective cohort, we investigated the presence of the JAK2V617F mutation in chronic non-cirrhotic portal vein thrombosis (PVT) patients, irrespective of the presence or absence of myeloproliferative diseases (MPDs). We identified 25 patients in whom thrombophilia workup was completed. The diagnoses of MPDs were made according to the World Health Organization (WHO) criteria. JAK2V617F mutation analysis was performed by allele-specific polymerase chain reaction (PCR). There were 9 male and 16 female patients. Prior to JAK2V617F analysis, there were one or more thrombophilic risk factors in 19 patients (76%). The JAK2V617F mutation analysis revealed the presence of this mutation (all in the heterozygote state) in six patients (24%; two male, four female). Five of the six cases with prior clinical diagnosis of MPDs were found to have wild-type JAK2. We found that the addition of JAK2V617F analysis into the thrombophilia workup in patients with chronic PVT contributes to a 4% increase in the diagnosis of thrombophilic conditions.

SOCS‐1, an important protein in the JAK/STAT pathway, has a role in the down stream of BCR‐ABL protein kinase. We investigated 56 CML patients and 16 controls for the methylation status of SOCS‐1 gene promoter and Exon 2 regions. Exon 2 was found to be methylated in 58.9% of the patients and 93.8% of the controls [P = 0.020, OR = 0.121(0.015–0.957)%95CI]. The promoter region was found unmethylated in all patient samples and controls. Although previous studies revealed a relation between SOCS1 gene Exon‐2 hypermethylation and CML development or progression, the results of this study showed no such correlation. On the contrary, our results might be indicating hypomethylation in CML patients, this hypothesis need to be studied in larger study population. Am. J. Hematol., 2007.