Hepatocellular adenoma (HCA) and hepatocellular carcinoma (HCC) can be difficult to differentiate but must be diagnosed correctly as treatment and prognosis for these tumors differ markedly. Relevant diagnostic biomarkers are thus needed, and those identified in dogs may have utility in human medicine because of the similarities between human and canine HCA and HCC. A tRNA-derived fragment (tRF), tRNA-Val, is a promising potential biomarker for canine mammary gland tumors but has not previously been investigated in hepatic tumors. Accordingly, we aimed to elucidate the potential utility of tRNA-Val as a biomarker for canine HCA and HCC using clinical samples (tumor tissue and plasma extracellular vesicles [EVs]) and tumor cell lines with qRT-PCR assays. We also investigated relevant functions and signaling pathways with bioinformatic analyses (Gene Ontology and Kyoto Encyclopedia of Genes and Genomes). tRNA-Val was markedly downregulated in HCC tumor tissue versus HCA tumor tissue and normal liver tissue, and a similar trend was shown in plasma EVs and HCC cell lines versus healthy controls. Based on areas under the receiver operating characteristic curves (AUCs), tRNA-Val significantly distinguished HCC (AUC = 1.00, p = 0.001) from healthy controls in plasma EVs and HCC from HCA (AUC = 0.950, p = 0.01). Bioinformatics analysis revealed that tRNA-Val may be primarily involved in DNA repair, mRNA processing, and splicing and may be linked to the N-glycan and ubiquitin-mediated proteasome pathways. This is the first report on the expression of tRNA-Val in canine HCC and HCA and its possible functions and signaling pathways. We suggest that tRNA-Val could be a promising novel biomarker to distinguish canine HCC from HCA. This study provides evidence for a greater understanding of the role played by tRNA-Val in the development of canine HCC.

Lymphadenomegaly is well described in dogs with neoplastic diseases but can also reflect inflammation. However, its role in assessments of inflammation is little reported in the veterinary literature. Lymph nodes are most easily assessed superficially through palpation, but computed tomography (CT) enables the evaluation of deep lymph nodes. Accordingly, we investigated associations between the maximal lymph node diameter and plasma concentrations of C-reactive protein (CRP) and white blood cell (WBC) count, as inflammatory markers, targeting three deep lymph nodes (sternal, cranial mediastinal, and internal iliac). We evaluated data from small dogs with non-neoplastic diseases that underwent synchronous CT and blood biochemistry at our institution. We found that the sternal lymph node diameter was significantly greater in dogs with clinically elevated CRP than those with clinically unremarkable CRP (p = 0.04) and significantly correlated with CRP (r = 0.32; p < 0.01). We found no significant association with CRP for the cranial mediastinal or internal iliac lymph node. Thus, we consider CT-measured sternal lymphadenomegaly a clinically valuable finding for the comprehensive evaluation of systemic inflammation. We anticipate further research may highlight the utility of CT lymph node measurements for such evaluations.

Hepatocellular adenoma (HCA) and hepatocellular carcinoma (HCC) can be difficult to differentiate but must be diagnosed correctly as treatment and prognosis for these tumors differ markedly. Relevant diagnostic biomarkers are thus needed, and those identified in dogs may have utility in human medicine because of the similarities between human and canine HCA and HCC. A tRNA-derived fragment (tRF), tRNA-Val, is a promising potential biomarker for canine mammary gland tumors but has not previously been investigated in hepatic tumors. Accordingly, we aimed to elucidate the potential utility of tRNA-Val as a biomarker for canine HCA and HCC using clinical samples (tumor tissue and plasma extracellular vesicles [EVs]) and tumor cell lines with qRT-PCR assays. We also investigated relevant functions and signaling pathways with bioinformatic analyses (Gene Ontology and Kyoto Encyclopedia of Genes and Genomes). tRNA-Val was markedly downregulated in HCC tumor tissue versus HCA tumor tissue and normal liver tissue, and a similar trend was shown in plasma EVs and HCC cell lines versus healthy controls. Based on areas under the receiver operating characteristic curves (AUCs), tRNA-Val significantly distinguished HCC (AUC=1.00, P=0.001) from healthy controls in plasma EVs and HCC from HCA (AUC=0.950, P=0.01). Bioinformatics analysis revealed that tRNA-Val may be primarily involved in DNA repair, mRNA processing, and splicing and may be linked to the N-glycan and ubiquitin-mediated proteasome pathways. This is the first report on the expression of tRNA-Val in canine HCC and HCA, and its possible functions and signaling pathways. We suggest that tRNA-Val could be a promising novel biomarker to distinguish canine HCC from HCA. This study provides evidence for a greater understanding of the role played by tRNA-Val in the development of canine HCC.

Background/Objectives: Hepatocellular carcinoma (HCC) is associated with altered microRNA (miRNA) expression, but small RNA sequencing (sRNA-seq) analyses usually aggregate reads by annotated features. This aggregation can mask changes in individual miRNA sequence variants (isomiRs) and in fragments derived from transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), and other non-coding RNAs. We aimed to characterize HCC-associated small RNA changes at exact-sequence resolution. Methods: We analyzed five liver sRNA-seq datasets from dogs, humans, and mice. The main canine, human (hsa-Tissue 1) and mouse (mmu-Tissue 1) comparisons included only three biological samples per group. Each unique read sequence was retained as the analytical unit and annotated afterwards against species-specific RNAcentral references. We compared sequence-level and feature-level differential expression, characterized fragment terminal positions, and examined sequence sharing and fold-change concordance between datasets. Results: In canine HCC, 3520 of 40,231 retained sequences were differentially abundant. Sequence-level analysis resolved HCC-associated isomiRs and tRNA-derived fragments that were not apparent in feature-level summaries, and additional changes involved rRNA-derived and unannotated sequences. Several RNA classes showed recurrent terminal patterns compatible with selective processing or stabilization. Exact sequences shared between datasets showed dataset-dependent concordance of HCC-associated changes, including limited agreement between the two human datasets. Conclusions: Exact-sequence analysis complements feature-level quantification by distinguishing HCC-associated changes among individual small RNA sequences and provides candidate fragments for functional studies. The small sample sizes and differences in disease context, control tissue, and library preparation limit the interpretation of individual candidates and cross-dataset comparisons.

Hypoxia-driven regulatory mechanisms play a critical role in tumor progression and therapeutic resistance in hepatocellular carcinoma (HCC), yet hypoxia-responsive microRNAs (HRMs) remain incompletely characterized. This study aimed to identify HRMs in canine HCC to evaluate their diagnostic potential and translational relevance to human disease. Next-generation sequencing of two canine HCC cell lines under normoxic and hypoxic conditions identified 332 and 321 differentially expressed miRNAs, respectively. Integrating these with tumor tissue data revealed 11 HRMs, featuring consistent upregulation of cfa-miR-210 and cfa-miR-34a, which was validated via RT-qPCR in hypoxic cells and clinical tissues. Both miRNAs were significantly elevated in plasma-derived extracellular vesicles (EVs), highlighting their value as promising circulating biomarkers (AUC 1.00 for miR-210; 0.98 for miR-34a). Target gene and pathway analyses identified shared regulatory nodes, including TGIF2 and SPRED1; and enrichment of MAPK, Ras, PI3K-Akt, and Rap1 signaling, broadly linked to hypoxia adaptation, cellular metabolism, and stress-response signaling. Cross-species comparison with human HCC datasets showed that, while miR-210 associates with poor prognosis in human HCC, miR-34a exhibits tumor-suppressive features. These findings define complementary HRM programs in canine HCC, reflecting conserved adaptive and stress-limiting regulatory mechanisms with potential diagnostic and translational relevance to human HCC.

Lymphadenomegaly is well described in dogs with neoplastic diseases, but it can also reflect in-flammation. However, its role in assessments of inflammation is little reported in the veterinary literature. Lymph nodes are most easily assessed superficially through palpation, but computed tomography (CT) enables evaluation of deep lymph nodes. Accordingly, we investigated associ-ations between maximal lymph node diameter and plasma concentrations of C-reactive protein (CRP), a sensitive inflammatory marker, targeting three deep lymph nodes (sternal, cranial me-diastinal, and internal iliac). We evaluated data from small dogs with non-neoplastic diseases that underwent synchronous CT and blood biochemistry at our institution. We found that cranial mediastinal lymph node diameter was significantly greater in dogs with clinically elevated CRP than those with clinically unremarkable CRP (CRP 0.7-7.0< mg/dL vs.