Supervised Machine Learning Approach for Prediction of Occult Lymph Node Metastasis in T1-T2 Papillary Thyroid Carcinoma

This study aimed to assess and compare four machine learning (ML) based classifiers in predicting occult cervical lymph node metastasis (LNM) in clinically node-negative (cN0), T1-T2 papillary thyroid carcinoma (PTC) patients. The study cohort included 288 PTC patients who underwent total thyroidectomy and prophylactic central neck dissection with sentinel lymph node biopsy performed for lateral LNM identification. The classifiers, namely k-Nearest Neighbor (k-NN), Support Vector Machines, Decision Tree, and Logistic Regression were developed using patients’ demographic and clinicopathological variables. Evaluation metrics such as area under the receiver operating characteristic curve (AUC), sensitivity, specificity, positive and negative predictive values (PPV and NPV), accuracy, and F1 and F2 scores were utilized for model comparison. The final ML classifier was selected based on achieving the highest specificity and the lowest degree of overfitting while maintaining a sensitivity of 95%. Among the evaluated models, the k-NN emerged as the best-performing, demonstrating an AUC of 0.72. The sensitivity, specificity, PPV, NPV, F1, and F2 scores were 98%, 27%, 56%, 93%, 72%, and 85%, respectively Furthermore, a web application was developed allowing users to predict the potential of cervical LNM and explore possibilities for further model development. The k-NN classifier incorporating patients’ clinicopathological information shows potential in predicting LNM. Improved prediction models are necessary to identify patients at higher risk of LNM, guiding appropriate postsurgical treatment for high-risk individuals while minimizing unnecessary interventions for low-risk patients.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

Comparison and imputation-aided integration of five commercial platforms for targeted DNA methylome analysis

Targeted bisulfite sequencing (TBS) has become the method of choice for the cost-effective, targeted analysis of the human methylome at base-pair resolution. In this study, we benchmarked five commercially available TBS platforms-three hybridization capture-based (Agilent, Roche and Illumina) and two reduced-representation-based (Diagenode and NuGen)-across 11 samples. Two samples were also compared with whole-genome DNA methylation sequencing with the Illumina and Oxford Nanopore platforms. We assessed workflow complexity, on/off-target performance, coverage, accuracy and reproducibility. Although all platforms produced robust and reproducible data, major differences in the number and identity of the CpG sites covered make it difficult to compare datasets generated on different platforms. To overcome this limitation, we applied imputation and show that it improves interoperability from an average of 10.35% (0.8 million) to 97% (7.6 million) common CpG sites. Our study provides guidance on which TBS platform to use for different methylome features and offers an imputation-based harmonization solution that allows comparative, integrative analysis

Evaluation of variant calling tools for detection of SNVs in BRCA1 and BRCA2 genes in patients from the Institute of Oncology and Radiology of Serbia

Serbia has one of the world’s highest incidences and mortality rates of ovarian cancer. Germline or somatic mutations in BRCA1 and BRCA2 genes, such as single nucleotide variants (SNVs), indels, insertions, deletions, commonly lead to development of breast and ovary cancer. Targeted therapy with PARP inhibitors is the current standard of care for serous epithelial BRCA-mutated ovarian cancer and depends on the accurate detection of mutations in these genes. In this study, a subset of patient specimens from Institute of Oncology and Radiology were sequenced on MiSeq Illumina sequencer, raw data were analysed bioinformatically, which included checking quality control of raw FASTQ sequences, trimming, mapping them on reference genome(hg19), target coverage quality control and variant calling. We tested various variant calling tools including Mutect2, GATK HaplotypeCaller, FreeBayes, VarDict and MuSe callers. We evaluated the relative performance- concordance rate, false positive and false negative rates between the callers for SNV/indel detection in BRCA1 and BRCA2 genes.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

Molecular signature of response to preoperative radiotherapy in locally advanced breast cancer

Background: Radiation therapy is an indispensable part of various treatment modalities for breast cancer. Specifically, for non-inflammatory locally advanced breast cancer (LABC) patients, preoperative radiotherapy (pRT) is currently indicated as a second line therapy in the event of lack of response to neoadjuvant chemotherapy. Still approximately one third of patients fails to respond favourably to pRT. The aim of this study was to explore molecular mechanisms underlying differential response to radiotherapy (RT) to identify predictive biomarkers and potential targets for increasing radiosensitivity.Methods: The study was based on a cohort of 134 LABC patients, treated at the Institute of Oncology and Radiology of Serbia (IORS) with pRT, without previous or concomitant systemic therapy. Baseline transcriptional profiles were established using Agilent 60 K microarray platform in a subset of 23 formalin-fixed paraffin-embedded (FFPE) LABC tumour samples of which 11 radiotherapy naïve and 3 post-radiotherapy samples passed quality control and were used for downstream analysis. Biological networks and signalling pathways underlying differential response to RT were identified using Ingenuity Pathways Analysis software. Predictive value of candidate genes in the preoperative setting was further validated by qRT-PCR in an independent subset of 60 LABC samples of which 42 had sufficient quality for data analysis, and in postoperative setting using microarray data from 344 node-negative breast cancer patients (Erasmus cohort, GSE2034 and GSE5327) treated either with surgery only (20%) or surgery with RT (80%).Results: We identified 192 significantly differentially expressed genes (FDR < 0.10) between pRT-responsive and non-responsive tumours, related to regulation of cellular development, growth and proliferation, cell cycle control of chromosomal replication, glucose metabolism and NAD biosynthesis II route. APOA1, MAP3K4, and MMP14 genes were differentially expressed (FDR < 0.20) between pRT responders and non-responders in preoperative setting, while MAP3K4 was further validated as RT-specific predictive biomarker of distant metastasis free survival (HR = 2.54, [95%CI:1.42–4.55], p = 0.002) in the postoperative setting.Conclusions: This study pinpoints MAP3K4 as a putative biomarker of response to RT in both preoperative and postoperative settings and a potential target for radiosensitising combination therapy, warranting further pre-clinical studies and prospective clinical validation

Nischarin expression may have differing roles in male and female melanoma patients

Due to the development of resistance to previously effective therapies, there is a constant need for novel treatment modalities for metastatic melanoma. Nischarin (NISCH) is a druggable scaffolding protein reported as a tumor suppressor and a positive prognostic marker in breast and ovarian cancers through regulation of cancer cell survival, motility and invasion. The aim of this study was to examine the expression and potential role of nischarin in melanoma. We found that nischarin expression was decreased in melanoma tissues compared to the uninvolved skin, and this was attributed to the presence of microdeletions and hyper-methylation of the NISCH promoter in the tumor tissue. In addition to the previously reported cytoplasmic and membranous localization, we observed nischarin in the nuclei in melanoma patients’ tissues. NISCH expression in primary melanoma had favorable prognostic value for female patients, but, unexpectedly, high NISCH expression predicted worse prognosis for males. Gene set enrichment analysis suggested significant sex-related disparities in predicted association of NISCH with several signaling pathways, as well as with different tumor immune infiltrate composition in male and female patients. Taken together, our results imply that nischarin may have a role in melanoma progression, but that fine-tuning of the pathways it regulates is sex-dependent.This is the submitted version of the article: Ostojić, M.; Jevrić, M.; Mitrović-Ajtić, O.; Živić, K.; Tanić, M.; Čavić, M.; Srdić-Rajić, T.; Grahovac, J. Nischarin Expression May Have Differing Roles in Male and Female Melanoma Patients. J Mol Med 2023, 101 (8), 1001–1014. https://doi.org/10.1007/s00109-023-02339-y

The evolution of lung cancer and impact of subclonal selection in TRACERx

Lung cancer is the leading cause of cancer-associated mortality worldwide. Here we analysed 1,644 tumour regions sampled at surgery or during follow-up from the first 421 patients with non-small cell lung cancer prospectively enrolled into the TRACERx study. This project aims to decipher lung cancer evolution and address the primary study endpoint: determining the relationship between intratumour heterogeneity and clinical outcome. In lung adenocarcinoma, mutations in 22 out of 40 common cancer genes were under significant subclonal selection, including classical tumour initiators such as TP53 and KRAS. We defined evolutionary dependencies between drivers, mutational processes and whole genome doubling (WGD) events. Despite patients having a history of smoking, 8% of lung adenocarcinomas lacked evidence of tobacco-induced mutagenesis. These tumours also had similar detection rates for EGFR mutations and for RET, ROS1, ALK and MET oncogenic isoforms compared with tumours in never-smokers, which suggests that they have a similar aetiology and pathogenesis. Large subclonal expansions were associated with positive subclonal selection. Patients with tumours harbouring recent subclonal expansions, on the terminus of a phylogenetic branch, had significantly shorter disease-free survival. Subclonal WGD was detected in 19% of tumours, and 10% of tumours harboured multiple subclonal WGDs in parallel. Subclonal, but not truncal, WGD was associated with shorter disease-free survival. Copy number heterogeneity was associated with extrathoracic relapse within 1 year after surgery. These data demonstrate the importance of clonal expansion, WGD and copy number instability in determining the timing and patterns of relapse in non-small cell lung cancer and provide a comprehensive clinical cancer evolutionary data resource

The evolution of non-small cell lung cancer metastases in TRACERx

Metastatic disease is responsible for the majority of cancer-related deaths. We report the longitudinal evolutionary analysis of 126 non-small cell lung cancer (NSCLC) tumours from 421 prospectively recruited patients in TRACERx who developed metastatic disease, compared with a control cohort of 144 non-metastatic tumours. In 25% of cases, metastases diverged early, before the last clonal sweep in the primary tumour, and early divergence was enriched for patients who were smokers at the time of initial diagnosis. Simulations suggested that early metastatic divergence more frequently occurred at smaller tumour diameters (less than 8 mm). Single-region primary tumour sampling resulted in 83% of late divergence cases being misclassified as early, highlighting the importance of extensive primary tumour sampling. Polyclonal dissemination, which was associated with extrathoracic disease recurrence, was found in 32% of cases. Primary lymph node disease contributed to metastatic relapse in less than 20% of cases, representing a hallmark of metastatic potential rather than a route to subsequent recurrences/disease progression. Metastasis-seeding subclones exhibited subclonal expansions within primary tumours, probably reflecting positive selection. Our findings highlight the importance of selection in metastatic clone evolution within untreated primary tumours, the distinction between monoclonal versus polyclonal seeding in dictating site of recurrence, the limitations of current radiological screening approaches for early diverging tumours and the need to develop strategies to target metastasis-seeding subclones before relapse

Genomic–transcriptomic evolution in lung cancer and metastasis

Intratumour heterogeneity (ITH) fuels lung cancer evolution, which leads to immune evasion and resistance to therapy. Here, using paired whole-exome and RNA sequencing data, we investigate intratumour transcriptomic diversity in 354 non-small cell lung cancer tumours from 347 out of the first 421 patients prospectively recruited into the TRACERx study. Analyses of 947 tumour regions, representing both primary and metastatic disease, alongside 96 tumour-adjacent normal tissue samples implicate the transcriptome as a major source of phenotypic variation. Gene expression levels and ITH relate to patterns of positive and negative selection during tumour evolution. We observe frequent copy number-independent allele-specific expression that is linked to epigenomic dysfunction. Allele-specific expression can also result in genomic–transcriptomic parallel evolution, which converges on cancer gene disruption. We extract signatures of RNA single-base substitutions and link their aetiology to the activity of the RNA-editing enzymes ADAR and APOBEC3A, thereby revealing otherwise undetected ongoing APOBEC activity in tumours. Characterizing the transcriptomes of primary–metastatic tumour pairs, we combine multiple machine-learning approaches that leverage genomic and transcriptomic variables to link metastasis-seeding potential to the evolutionary context of mutations and increased proliferation within primary tumour regions. These results highlight the interplay between the genome and transcriptome in influencing ITH, lung cancer evolution and metastasis

Antibodies against endogenous retroviruses promote lung cancer immunotherapy

B cells are frequently found in the margins of solid tumours as organized follicles in ectopic lymphoid organs called tertiary lymphoid structures (TLS). Although TLS have been found to correlate with improved patient survival and response to immune checkpoint blockade (ICB), the underlying mechanisms of this association remain elusive. Here we investigate lung-resident B cell responses in patients from the TRACERx 421 (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy) and other lung cancer cohorts, and in a recently established immunogenic mouse model for lung adenocarcinoma. We find that both human and mouse lung adenocarcinomas elicit local germinal centre responses and tumour-binding antibodies, and further identify endogenous retrovirus (ERV) envelope glycoproteins as a dominant anti-tumour antibody target. ERV-targeting B cell responses are amplified by ICB in both humans and mice, and by targeted inhibition of KRAS(G12C) in the mouse model. ERV-reactive antibodies exert anti-tumour activity that extends survival in the mouse model, and ERV expression predicts the outcome of ICB in human lung adenocarcinoma. Finally, we find that effective immunotherapy in the mouse model requires CXCL13-dependent TLS formation. Conversely, therapeutic CXCL13 treatment potentiates anti-tumour immunity and synergizes with ICB. Our findings provide a possible mechanistic basis for the association of TLS with immunotherapy response

Upotreba tečne biopsije za dijagnostiku malignih bolesti : Principi i trenutna praksa

&lt;p&gt;For years, the focus of scientists' research has been the mechanisms that lead to the onset of cancer, as a basis for the further development of diagnostic approaches in its early detection, as well as the development of personalized therapy and increasing the survival rate of patients suffering from cancer. The current gold standard of diagnosis is biopsy of tumor tissue, which has several limitations, and the development of other diagnostic approaches is expanding. In recent years, liquid biopsy has become the center of research in clinical oncology, both because of its non-invasive approach and because it opens the door to a much larger area of investigation than tissue biopsy. The term liquid biopsy refers to the sampling of biological material that does not include solid tumor tissue, such as blood, urine, saliva, cerebrospinal fluid, etc. There are three basic markers of liquid biopsy, circulating tumor cells, circulating DNA and exosomes, which together form a huge area of research that offers enormous progress in the field of diagnosing, monitoring diseases and predicting adequate targeted therapy.&lt;/p&gt