The study, published in the journal Veterinary Oncology, was led by Ingrid Marie Moberg and colleagues at the Norwegian University of Life Sciences, Oslo University Hospital, and the University of Oslo. The team analyzed RNA sequencing data from 179 canine mammary tumors, drawn from a cohort of naturally occurring tumors removed from companion dogs during routine surgery. Their goal was twofold: to define molecular subgroups of canine mammary tumors without reference to histology, and then to ask whether tumors from the same dog share those molecular identities. The answer to the second question, in most cases, was no.

To classify the tumors, the researchers first reduced their high-dimensional gene expression data using principal component analysis, then applied unsupervised consensus clustering, an algorithm that groups samples based purely on expression similarity and stability across repeated resampling. This revealed five robust transcriptomic clusters. Crucially, the clusters did not map cleanly onto histological diagnosis. Each cluster contained a mixture of benign and malignant tumors, confirming that the microscopic appearance of these tumors tells only part of the story of their underlying biology.

Gene set enrichment analysis then revealed that the five clusters bear a remarkable resemblance to molecular subtypes long recognized in human breast cancer. Three of the clusters showed enrichment of hormone-related gene programs, including estrogen response, and were found to harbor the transcription factor GATA3, a classic marker of luminal breast cancer in humans. One cluster combined cell-cycle proliferation with immune and interferon signaling, evoking the aggressive basal-like or triple-negative phenotype, while a fifth cluster was defined by epithelial-mesenchymal transition, angiogenesis, and inflammatory modules, reminiscent of the claudin-low subtype. The parallel with human breast cancer taxonomy is not merely cosmetic; it strengthens the case for dogs as a comparative model in which the biology of mammary cancer can be studied in a spontaneously arising disease.

Beyond clustering, the team constructed a gene co-expression network using the hCoCena framework, identifying ten modules of genes that are expressed together and that encode distinct biological processes. One module captured hormone signaling, two captured immune and interferon responses, three reflected metabolism and proliferation through glycolysis, PI3K-AKT-mTOR signaling, oxidative phosphorylation, and MYC targets, and others traced epithelial differentiation through WNT signaling, epithelial-mesenchymal transition, cell-cycle regulation through E2F targets and the G2M checkpoint, and tumor microenvironment features such as angiogenesis. Transcription factor enrichment within the modules pointed to GATA3 as a regulator of the hormonal program and E2F1 as a driver of the proliferative module, providing candidate master switches behind the observed phenotypes.

The heart of the study, however, lies in its analysis of synchronous tumors. The researchers focused on 45 dogs that each carried exactly two tumors, yielding 90 paired samples classified as benign-benign, malignant-benign, or malignant-malignant. When they compared cluster assignments within each pair, concordance was low. Only about 45 percent of tumor pairs landed in the same transcriptomic cluster overall, with malignant-malignant pairs showing the highest agreement at 56 percent, benign-benign pairs at 44 percent, and mixed malignant-benign pairs at just 36 percent. In other words, the majority of dogs carried two tumors with fundamentally different molecular identities.

To quantify this divergence at the level of individual genes, the team calculated intraclass correlation coefficients for every gene across the paired samples. Genes were scored as low, moderate, or high in correlation between a dog’s two tumors. The results were unambiguous: between roughly 76 and 90 percent of genes showed low correlation across all diagnostic categories, and fewer than one percent of genes were highly correlated within any category. This pattern of widespread discordance held regardless of whether both tumors were benign, both malignant, or one of each, indicating that molecular independence between synchronous tumors is the norm rather than the exception.

A small set of exceptions proved informative. The analysis identified a handful of genes, including OMD and EN1, whose expression is strongly correlated within certain categories of synchronous tumor pairs and which have been reported as prognostic markers in human cancers. The authors suggest that these genes may point to shared disease processes or protective mechanisms against malignant transformation, and that they deserve further investigation, potentially at the DNA level, to uncover any genetic factors underlying their coordinated behavior. Meanwhile, examination of module-level variation showed that programs linked to cell-cycle activity, hormone signaling, and immune responses fluctuated most between paired tumors, while modules tied to epithelial differentiation and metabolism remained comparatively stable within individuals.

The clinical implications of this work reach in two directions at once. For veterinary medicine, the findings suggest that each tumor in a multi-tumor patient should be evaluated as a biologically independent lesion rather than assumed to be representative of its neighbors. Because molecular subtypes in human breast cancer drive dramatically different treatment decisions, from endocrine therapy for hormone receptor-positive disease to chemotherapy and PARP inhibitors for triple-negative tumors, a biology-driven approach could eventually refine the limited therapeutic options currently available for canine patients. The authors note, for instance, that identifying hormone-positive subtypes could inform whether ovariohysterectomy offers real benefit at the time of tumor removal, a decision veterinarians currently make without molecular guidance.

For comparative oncology, the study reinforces the value of the canine model in a way that human cohorts cannot easily replicate. Synchronous bilateral breast cancer occurs in only around one percent of human patients, whereas synchronous mammary tumors affect the majority of affected dogs. Studying these paired tumors within the same genetic background eliminates many host-specific confounders, offering a natural experiment in tumor evolution and inter-individual heterogeneity. The authors acknowledge limitations inherent to bulk RNA sequencing, which averages expression across all cells in a tissue and may reflect tissue composition as much as tumor biology, and they caution that a single RNA sample may not represent an entire tumor. Even so, their conclusion stands firm: histopathology alone does not capture the molecular reality of canine mammary tumors, and expression-based profiling offers a more faithful map of the biological terrain that clinicians and researchers alike will need to navigate.

Subject of Research: Transcriptomic heterogeneity of synchronous canine mammary tumors and their molecular resemblance to human breast cancer subtypes

Article Title: High molecular heterogeneity in synchronous canine mammary tumors detected by transcriptomic analysis

Article References: Moberg, I. M., Murphy, S. L., Hansen, N., Borge, K. S., Gunnes, G., Sørlie, T., Lingaas, F., Bergholtz, H., & Solbakken, M. H. (2026). High molecular heterogeneity in synchronous canine mammary tumors detected by transcriptomic analysis. Veterinary Oncology, 3(1), Article 11. https://doi.org/10.1186/s44356-026-00066-3

Image Credits: AI Generated

DOI: 10.1186/s44356-026-00066-3

Keywords: canine mammary tumors, transcriptomics, RNA sequencing, tumor heterogeneity, synchronous tumors, breast cancer subtypes, gene co-expression network, consensus clustering, comparative oncology, veterinary pathology, High, molecular

L. martiniquensis has attracted growing concern in Southeast Asia as an emerging cause of visceral leishmaniasis, a disease that can be fatal particularly among patients with advanced HIV infection whose immune systems cannot contain the parasite. Amphotericin B remains a mainstay of therapy, yet clinicians in Thailand have documented relapse and treatment failure in patients infected with this species, raising the possibility that some parasite populations carry or acquire reduced susceptibility to the drug. Until now, the molecular machinery underlying such reduced susceptibility in L. martiniquensis has remained largely unexplored, making paired isolates drawn from the same patient at different clinical time points an unusually valuable window into how the parasite changes under drug pressure.

The research team, led by investigators at Chulalongkorn University in Bangkok in collaboration with Hatyai Hospital and King Chulalongkorn Memorial Hospital, obtained one isolate before treatment, designated CULE7.2, and a second, CULE8, after the patient relapsed. They first confirmed the biological difference between the two isolates using in vitro susceptibility assays: CULE8 displayed significantly higher half-maximal and 90 percent inhibitory concentrations of amphotericin B than CULE7.2, with the dose-response curve shifted markedly to the right. Transmission electron microscopy of CULE7.2 promastigotes exposed acutely to the drug revealed the expected cellular damage, including prominent cytoplasmic vacuolization, lipid-like inclusions, mitochondrial swelling, and focal disruption of the nuclear membrane, consistent with amphotericin B’s mechanism of binding membrane sterols and destabilizing cellular membranes.

To explore the transcriptional landscape underlying these phenotypic differences, the researchers profiled both isolates across two developmental stages of the parasite, promastigotes and axenic amastigotes, using RNA sequencing. Importantly, the two isolates were cultured under different conditions: CULE8 was maintained under continuous exposure to a sublethal 0.3 micromolar concentration of amphotericin B, while CULE7.2 was cultured in parallel without the drug. Differential expression analysis, hierarchical clustering, and functional enrichment were performed independently within each stage. This design choice means the observed patterns likely reflect a combination of intrinsic isolate-associated characteristics and the transcriptional response to ongoing drug exposure, an important caveat the authors emphasize in interpreting their results.

In the promastigote stage, the post-relapse isolate CULE8 showed predominant downregulation of genes involved in sterol and lipid metabolism, membrane transport, protein synthesis, motility, and broader metabolic and regulatory processes. Among the affected genes were sterol C24 reductase, an enzyme central to the parasite’s membrane sterol composition and a known determinant of amphotericin B activity, together with upstream enzymes of the mevalonate pathway. At the same time, CULE8 promastigotes displayed increased expression of genes associated with thiol-based redox homeostasis, suggesting a shift toward enhanced management of oxidative stress, a plausible countermeasure to drug-induced membrane and mitochondrial damage.

The axenic amastigote stage, the form that proliferates inside the mammalian host, told a different story. Here, CULE8 showed increased expression of genes linked to lipid metabolism, redox homeostasis, protein synthesis, and proteostasis, alongside broader downregulation of transport genes and genes involved in genome maintenance. Despite the predominantly stage-specific patterns, a small subset of genes showed concordant regulation across both life stages, including genes involved in lipid metabolism and transport. Genes tied to thiol-based redox homeostasis stood out for their elevated expression in both stages, marking this antioxidant capacity as a consistent candidate feature of the relapse-associated parasite and a potential target for functional validation studies.

The virological dimension of the study may prove equally consequential. De novo assembly of RNA-seq reads that failed to map to the parasite genome recovered complete L and S genomic segments of a genetically distinct leishbuvirus from CULE8. This represents, to the authors’ knowledge, the first molecular characterization of a leishbuvirus in a Southeast Asian L. martiniquensis isolate, expanding the known geographic and host range of these viruses. Comparative structural modeling using predicted aligned error matrices supported conservation of the RNA-dependent RNA polymerase core and its Motif C between the CULE8 virus and a reference variant, while negative-stain transmission electron microscopy revealed structures consistent with virus-like particles. Viral RNA abundance was higher in CULE8 than in CULE7.2 across both developmental stages.

The significance of leishbuviruses remains a subject of active debate in the field. Related viruses, including Leishmania RNA virus 1, have been associated in some studies with heightened virulence and treatment failure in other Leishmania species, particularly through modulation of the host inflammatory response, although such associations are not universal and their mechanisms are incompletely understood. The authors are careful to note that the higher leishbuvirus RNA abundance observed in the post-relapse isolate has no established causal relationship with reduced amphotericin B susceptibility or with the patient’s clinical relapse. The finding is best viewed as an observation warranting further investigation in independent isolates rather than as evidence of a viral driver of drug resistance.

Indeed, the authors frame the entire study as hypothesis-generating. Because the paired isolates were examined under different drug-exposure conditions, the transcriptional differences they report cannot be attributed cleanly to either intrinsic isolate biology or adaptive response to amphotericin B, and the findings will require validation in independent isolates under matched experimental conditions. Nevertheless, the breadth of the observed changes, encompassing sterol biosynthesis, lipid handling, membrane transport, redox metabolism, protein homeostasis, and genome maintenance, broadens the molecular landscape associated with reduced amphotericin B susceptibility in this species and identifies multiple candidate pathways for future functional studies, including gene knockout or overexpression experiments that could disentangle correlation from causation.

For a disease that disproportionately strikes immunocompromised patients in resource-limited settings, the stakes of understanding amphotericin B failure are high. Visceral leishmaniasis caused by L. martiniquensis in Thailand is emerging in HIV-coinfected populations, and the therapeutic arsenal is narrow. By pairing rigorous susceptibility testing and ultrastructural imaging with stage-resolved transcriptomics and sensitive viral discovery, this study demonstrates how a single patient’s clinical course can illuminate molecular processes that no laboratory strain could fully recapitulate. The identified candidate pathways, from mevalonate-dependent sterol metabolism to thiol-based antioxidant defenses, now provide concrete starting points for the functional experiments and epidemiological surveillance needed to determine how widespread reduced drug susceptibility might be, and whether hidden viral passengers within these parasites play any role in the clinical outcomes that matter most to patients.

Subject of Research: Transcriptomic analysis of paired Leishmania martiniquensis clinical isolates to identify molecular pathways associated with reduced amphotericin B susceptibility

Article Title: Transcriptomic profiling of paired clinical Leishmania (Mundinia) martiniquensis isolates reveals candidate pathways associated with reduced amphotericin B susceptibility and identifies a novel leishbuvirus variant

Article References: Phadungsaksawasdi, K., Pongpanich, M., Yuanlae, S., Lerona, P. G. E., Sricharoensuk, C., Songumpai, N., Siriyasatien, P., Asawanonda, P., Shotelersuk, V., & Preativatanyou, K. (2026). Transcriptomic profiling of paired clinical Leishmania (Mundinia) martiniquensis isolates reveals candidate pathways associated with reduced amphotericin B susceptibility and identifies a novel leishbuvirus variant. Parasites & Vectors. https://doi.org/10.1186/s13071-026-07628-2

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07628-2

Keywords: Leishmania martiniquensis, Mundinia, amphotericin B, drug susceptibility, transcriptomics, leishbuvirus, visceral leishmaniasis, Thailand, RNA sequencing, parasite virome, sterol metabolism, redox homeostasis