Detection of PIK3CA hotspot mutations in canine mammary tumors using droplet digital PCR: tissue validation and liquid biopsy feasibility.

Domestic dogs (Canis lupus familiaris) serve as valuable translational models for human cancer research due to their biological similarities. Canine mammary tumors (CMTs), frequently diagnosed in female dogs, share various characteristics with human breast cancers. This study investigates the PIK3CA (H1047R) mutation in CMTs using droplet digital PCR (ddPCR) and explores the potential of liquid biopsy for non-invasive detection. We analyzed 80 formalin-fixed, paraffin-embedded (FFPE) CMT tissue samples and compared ddPCR results with next-generation sequencing (NGS) data, achieving high concordance. Plasma and serum samples were also assessed for mutation concordance with tissue results. Our findings indicate a higher frequency of the PIK3CA (H1047R) mutations in benign and grade I malignant CMTs compared to more aggressive malignancies. The ddPCR assay demonstrated high sensitivity and specificity, with plasma testing showing 78.6% sensitivity and 87.5% specificity, and serum testing showing 66.7% sensitivity and 90.0% specificity. These results highlight the viability of liquid biopsy as a minimally invasive method for monitoring PIK3CA mutations in canine patients. The study suggests that liquid biopsy techniques hold significant promise for improving the early detection and monitoring of canine cancers, warranting further research to refine these methods and explore their applications in canine cancer diagnostics and treatment.

Characterization of Molecular Subtypes of Rat Mammary Cancer and Their Association With Environmental Exposures.

BACKGROUND/AIM: Breast cancer is a heterogeneous disease with many subtypes, and the association between these subtypes and exposure to environmental factors such as radiation remains controversial. Although the rat is used widely for research into human breast cancer, the heterogeneity and subtype definitions are unclear. Here, we leveraged an archive of rat mammary cancer samples and gene expression microarray data to classify tumors and examine their association with exposures. MATERIALS AND METHODS: Eighty-four mammary cancer and 12 normal mammary tissue samples were obtained from previous experiments in which rats were exposed to different types of radiation, chemical carcinogens, and diets. Tumors were then subjected to immunohistochemical (IHC) analysis of conventional biomarkers, as well as gene expression profiling; they were then classified by three approaches based on IHC results, the PAM50 classifier algorithm, and unsupervised clustering of gene expression profiles. RESULTS: IHC identified four subtypes (luminal A-like, luminal B-like 1, luminal B-like 2, and triple-negative), while PAM50 identified six (luminal A, luminal B, basal-like, HER2-enriched, normal-like, and claudin-low). Unsupervised clustering divided the tumors into three large, statistically significant, groups (named "luminal A", "luminal B", and "non-luminal" clusters). The results of the three approaches were significantly associated with each other. Exposure to radiation and chemical carcinogens during post-pubertal development was significantly associated with an increased risk of developing luminal A tumors, whereas exposure to a high corn-oil diet was associated with a higher likelihood of luminal B tumors. CONCLUSION: Rat mammary cancer subtypes resemble those in humans and are related to environmental factors.

R-loop functions in Brca1-associated mammary tumorigenesis.

Deleterious accumulation of R-loops, a DNA-RNA hybrid structure, contributes to genome instability. They are associated with BRCA1 mutation-related breast cancer, an estrogen receptor α negative (ERα-) tumor type originating from luminal progenitor cells. However, a presumed causality of R-loops in tumorigenesis has not been established in vivo. Here, we overexpress mouse Rnaseh1 (Rh1-OE) in vivo to remove accumulated R-loops in Brca1-deficient mouse mammary epithelium (BKO). R-loop removal exacerbates DNA replication stress in proliferating BKO mammary epithelial cells, with little effect on homology-directed repair of double-strand breaks following ionizing radiation. Compared to their BKO counterparts, BKO-Rh1-OE mammary glands contain fewer luminal progenitor cells but more mature luminal cells. Despite a similar incidence of spontaneous mammary tumors in BKO and BKO-Rh1-OE mice, a significant percentage of BKO-Rh1-OE tumors express ERα and progesterone receptor. Our results suggest that rather than directly elevating the overall tumor incidence, R-loops influence the mammary tumor subtype by shaping the cell of origin for Brca1 tumors.

Exploring the role of microRNAs as diagnostic and prognostic biomarkers in canine mammary tumors.

Canine mammary tumors (CMTs) represent a significant health concern in dogs, with a high incidence among intact female dogs. CMTs are a promising comparative model for human breast cancer, due to sharing several pathophysiological features. Additionally, CMTs have a strong genetic correlation with their human counterpart, including the expression of microRNAs (miRNAs). MiRNAs are a class of non-coding RNAs that play important roles in post-translational regulation of gene expression, being implicated in carcinogenesis, tumor progression, and metastasis. Moreover, miRNAs hold promise as diagnostic, prognostic, and metastatic biomarkers. Understanding the molecular mechanisms underlying CMTs is crucial for improving diagnosis, prognosis, and monitoring of treatments. Herein, we provide a comprehensive overview of the current knowledge on miRNAs in CMTs, highlighting their roles in carcinogenesis and their potential as biomarkers. Additionally, we highlight the current limitations and critically discuss the overarching challenges in this field, emphasizing the need for future research to translate miRNA findings into veterinary clinical practice.

p53R172H and p53R245W Hotspot Mutations Drive Distinct Transcriptomes in Mouse Mammary Tumors Through a Convergent Transcriptional Mediator.

Aggressive breast cancers harbor TP53 missense mutations. Tumor cells with TP53 missense mutations exhibit enhanced growth and survival through transcriptional rewiring. To delineate how TP53 mutations in breast cancer contribute to tumorigenesis and progression in vivo, we created a somatic mouse model driven by mammary epithelial cell-specific expression of Trp53 mutations. Mice developed primary mammary tumors reflecting the human molecular subtypes of luminal A, luminal B, HER2-enriched, and triple-negative breast cancer with metastases. Transcriptomic analyses comparing MaPR172H/- or MaPR245W/- mammary tumors to MaP-/- tumors revealed (1) differences in cancer-associated pathways activated in both p53 mutants and (2) Nr5a2 as a novel transcriptional mediator of distinct pathways in p53 mutants. Meta-analyses of human breast tumors corroborated these results. In vitro assays demonstrate mutant p53 upregulates specific target genes that are enriched for Nr5a2 response elements in their promoters. Co-immunoprecipitation studies revealed p53R172H and p53R245W interact with Nr5a2. These findings implicate NR5A2 as a novel mediator of mutant p53 transcriptional activity in breast cancer. SIGNIFICANCE: Our findings implicate NR5A2 as a novel mediator of mutant p53 transcriptional activity in breast cancer. NR5A2 may be an important therapeutic target in hard-to-treat breast cancers such as endocrine-resistant tumors and metastatic triple-negative breast cancers harboring TP53 missense mutations.

Molecular and Pathobiology of Canine Mammary Tumour: Defining a Translational Model for Human Breast Cancer.

Canine mammary tumours (CMT) have histological, clinicopathological and molecular resemblances to human breast cancer (HBC), positioning them as viable models for studying the human disease. CMT initiation and progression occur spontaneously in immune-competent animals, which challenge the suggested limitations of genetically modified mice, also enabling the evaluation of immunotherapies in canine patients. Dogs have shorter life expectancy compared to humans, and cancer advances more rapidly in this species. This makes it possible to perform studies about the clinical efficacy of new therapeutic modalities in a much shorter time than in human patients. The identification of biomarkers for tumour subtypes, progression and treatment response paves the way for the development of novel therapeutic and diagnostic approaches. This review addresses the similarities between CMT and HBC and the molecular signatures identified in CMT samples that have been explored to date. We proposed a detailed molecular exploration of the CMT stroma using state-of-the-art methods in transcriptomics and proteomics. Using CMT as an analog for HBC not only helps to understand the complexities of the disease, but also to advance comparative oncology to the next level to prove the claim of dogs as a valid translational model.

AGR2-mediated unconventional secretion of 14-3-3ε and α-actinin-4, responsive to ER stress and autophagy, drives chemotaxis in canine mammary tumor cells.

BACKGROUND: Canine mammary tumors (CMTs) in intact female dogs provide a natural model for investigating metastatic human cancers. Our prior research identified elevated expression of Anterior Gradient 2 (AGR2), a protein disulfide isomerase (PDI) primarily found in the endoplasmic reticulum (ER), in CMT tissues, highly associated with CMT progression. We further demonstrated that increased AGR2 expression actively influences the extracellular microenvironment, promoting chemotaxis in CMT cells. Unraveling the underlying mechanisms is crucial for assessing the potential of therapeutically targeting AGR2 as a strategy to inhibit a pro-metastatic microenvironment and impede tumor metastasis. METHODS: To identify the AGR2-modulated secretome, we employed proteomics analysis of the conditioned media (CM) from two CMT cell lines ectopically expressing AGR2, compared with corresponding vector-expressing controls. AGR2-regulated release of 14-3-3ε (gene: YWHAE) and α-actinin 4 (gene: ACTN4) was validated through ectopic expression, knockdown, and knockout of the AGR2 gene in CMT cells. Extracellular vesicles derived from CMT cells were isolated using either differential ultracentrifugation or size exclusion chromatography. The roles of 14-3-3ε and α-actinin 4 in the chemotaxis driven by the AGR2-modulated CM were investigated through gene knockdown, antibody-mediated interference, and recombinant protein supplement. Furthermore, the clinical relevance of the release of 14-3-3ε and α-actinin 4 was assessed using CMT tissue-immersed saline and sera from CMT-afflicted dogs. RESULTS: Proteomics analysis of the AGR2-modulated secretome revealed increased abundance in 14-3-3ε and α-actinin 4. Ectopic expression of AGR2 significantly increased the release of 14-3-3ε and α-actinin 4 in the CM. Conversely, knockdown or knockout of AGR2 expression remarkably reduced their release. Silencing 14-3-3ε or α-actinin 4 expression diminished the chemotaxis driven by AGR2-modulated CM. Furthermore, AGR2 controls the release of 14-3-3ε and α-actinin 4 primarily via non-vesicular routes, responding to the endoplasmic reticulum (ER) stress and autophagy activation. Knockout of AGR2 resulted in increased α-actinin 4 accumulation and impaired 14-3-3ε translocation in autophagosomes. Depletion of extracellular 14-3-3ε or α-actinin 4 reduced the chemotaxis driven by AGR2-modulated CM, whereas supplement with recombinant 14-3-3ε in the CM enhanced the CM-driven chemotaxis. Notably, elevated levels of 14-3-3ε or α-actinin 4 were observed in CMT tissue-immersed saline compared with paired non-tumor samples and in the sera of CMT dogs compared with healthy dogs. CONCLUSION: This study elucidates AGR2's pivotal role in orchestrating unconventional secretion of 14-3-3ε and α-actinin 4 from CMT cells, thereby contributing to paracrine-mediated chemotaxis. The insight into the intricate interplay between AGR2-involved ER stress, autophagy, and unconventional secretion provides a foundation for refining strategies aimed at impeding metastasis in both canine mammary tumors and potentially human cancers.

GATA3 functions downstream of BRCA1 to promote DNA damage repair and suppress dedifferentiation in breast cancer.

BACKGROUND: Inadequate DNA damage repair promotes aberrant differentiation of mammary epithelial cells. Mammary luminal cell fate is mainly determined by a few transcription factors including GATA3. We previously reported that GATA3 functions downstream of BRCA1 to suppress aberrant differentiation in breast cancer. How GATA3 impacts DNA damage repair preventing aberrant cell differentiation in breast cancer remains elusive. We previously demonstrated that loss of p18, a cell cycle inhibitor, in mice induces luminal-type mammary tumors, whereas depletion of either Brca1 or Gata3 in p18 null mice leads to basal-like breast cancers (BLBCs) with activation of epithelial-mesenchymal transition (EMT). We took advantage of these mutant mice to examine the role of Gata3 as well as the interaction of Gata3 and Brca1 in DNA damage repair in mammary tumorigenesis. RESULTS: Depletion of Gata3, like that of Brca1, promoted DNA damage accumulation in breast cancer cells in vitro and in basal-like breast cancers in vivo. Reconstitution of Gata3 improved DNA damage repair in Brca1-deficient mammary tumorigenesis. Overexpression of GATA3 promoted homologous recombination (HR)-mediated DNA damage repair and restored HR efficiency of BRCA1-deficient cells. Depletion of Gata3 sensitized tumor cells to PARP inhibitor (PARPi), and reconstitution of Gata3 enhanced resistance of Brca1-deficient tumor cells to PARP inhibitor. CONCLUSIONS: These results demonstrate that Gata3 functions downstream of BRCA1 to promote DNA damage repair and suppress dedifferentiation in mammary tumorigenesis and progression. Our findings suggest that PARP inhibitors are effective for the treatment of GATA3-deficient BLBCs.

eEF1A2 promotes PTEN-GSK3ß-SCF complex-dependent degradation of Aurora kinase A and is inactivated in breast cancer.

The translation elongation factor eEF1A promotes protein synthesis. Its methylation by METTL13 increases its activity, supporting tumor growth. However, in some cancers, a high abundance of eEF1A isoforms is associated with a good prognosis. Here, we found that eEF1A2 exhibited oncogenic or tumor-suppressor functions depending on its interaction with METTL13 or the phosphatase PTEN, respectively. METTL13 and PTEN competed for interaction with eEF1A2 in the same structural domain. PTEN-bound eEF1A2 promoted the ubiquitination and degradation of the mitosis-promoting Aurora kinase A in the S and G2 phases of the cell cycle. eEF1A2 bridged the interactions between the SKP1-CUL1-FBXW7 (SCF) ubiquitin ligase complex, the kinase GSK3ß, and Aurora-A, thereby facilitating the phosphorylation of Aurora-A in a degron site that was recognized by FBXW7. Genetic ablation of Eef1a2 or Pten in mice resulted in a greater abundance of Aurora-A and increased cell cycling in mammary tumors, which was corroborated in breast cancer tissues from patients. Reactivating this pathway using fimepinostat, which relieves inhibitory signaling directed at PTEN and increases FBXW7 expression, combined with inhibiting Aurora-A with alisertib, suppressed breast cancer cell proliferation in culture and tumor growth in vivo. The findings demonstrate a therapeutically exploitable, tumor-suppressive role for eEF1A2 in breast cancer.

Genetic analysis of PALB2 gene WD40 domain in canine mammary tumour patients.

BACKGROUND: DNA repair mechanisms are essential for tumorigenesis and disruption of HR mechanism is an important predisposing factor of human breast cancers (BC). PALB2 is an important part of the HR. There are similarities between canine mammary tumours (CMT) and BCs. As its human counterpart, PALB2 mutations could be a predisposing factor of CMT. OBJECTIVES: In this study, we aimed to investigate the impacts of PALB2 variants on tumorigenesis and canine mammary tumor (CMT) malignancy. METHODS: We performed Sanger sequencing to detect germline mutations in the WD40 domain of the canine PALB2 gene in CMT patients. We conducted in silico analysis to investigate the variants, and compared the germline PALB2 mutations in humans that cause breast cancer (BC) with the variants detected in dogs with CMT. RESULTS: We identified an intronic (c.3096+8C>G) variant, two exonic (p.A1050V and p.R1354R) variants, and a 3' UTR variant (c.4071T>C). Of these, p.R1354R and c.4071T>C novel variants were identified for the first time in this study. We found that the p.A1050V mutation had a significant effect. However, we could not determine sufficient similarity due to the differences in nucleotide/amino acid sequences between two species. Nonetheless, possible variants of human sequences in the exact location as their dog counterparts are associated with several cancer types, implying that the variants could be crucial for tumorigenesis in dogs. Our results did not show any effect of the variants on tumor malignancy. CONCLUSIONS: The current project is the first study investigating the relationship between the PALB2 gene WD40 domain and CMTs. Our findings will contribute to a better understanding of the pathogenic mechanism of the PALB2 gene in CMTs. In humans, variant positions in canines have been linked to cancer-related phenotypes such as familial BC, endometrial tumor, and hereditary cancer predisposition syndrome. The results of bioinformatics analyses should be investigated through functional tests or case-control studies.

Programmed Death-Ligand (PD-L1), Epidermal Growth Factor (EGF), Relaxin, and Matrix Metalloproteinase-3 (MMP3): Potential Biomarkers of Malignancy in Canine Mammary Neoplasia.

Gene expression has been suggested as a putative tool for prognosis and diagnosis in canine mammary neoplasia (CMNs). In the present study, 58 formalin-fixed paraffin-embedded (FFPE) paraffined canine mammary neoplasias from 27 different bitches were included. Thirty-seven tumours were classified as benign, whereas thirty-one were classified as different types of canine carcinoma. In addition, mammary samples from three healthy bitches were also included. The gene expression for vascular endothelial growth factor-α (VEGFα), CD20, progesterone receptor (PGR), hyaluronidase-1 (HYAL-1), programmed death-ligand 1 (PD-L1), epidermal growth factor (EGF), relaxin (RLN2), and matrix metalloproteinase-3 (MMP3) was assessed through RT-qPCR. All the assessed genes yielded a higher expression in neoplastic mammary tissue than in healthy tissue. All the evaluated genes were overexpressed in neoplastic mammary tissue, suggesting a role in the process of tumorigenesis. Moreover, PD-L1, EGF, relaxin, and MMP3 were significantly overexpressed in malignant CMNs compared to benign CMNs, suggesting they may be useful as malignancy biomarkers.

PD-L1 mRNA and protein expression in canine mammary carcinomas: Correlation with histopathological grade and molecular markers.

Programmed death ligand 1 (PD-L1) is an immune checkpoint molecule that plays a crucial role in regulating antitumor immune responses. Canine mammary carcinomas (CMCs) are common tumors of dogs. Despite extensive studies on the heterogeneity of CMCs, there is still a lack of effective precision therapies for the treatment of CMCs. In this study, we aimed to investigate the correlation between PD-L1 mRNA and protein expression in CMCs and explore its association with histopathological grade and molecular markers, including the estrogen receptor, epidermal growth factor receptor 2, and cytokeratin 5/6 (CK5/6). Formalin-fixed paraffin-embedded samples were evaluated for PD-L1 mRNA expression using RNA in situ hybridization and PD-L1 protein expression using immunohistochemistry. We observed no substantial correlation between PD-L1 mRNA and protein expression in CMCs; however, PD-L1 mRNA levels were significantly higher in grade 3 than in grade 1 tumors (P = .001). In addition, we observed a positive correlation between PD-L1 protein expression and CK5/6 expression in CMCs (P = .032). These findings suggest that PD-L1 expression in CMCs is heterogeneous and may be regulated post-transcriptionally. Further studies are needed to explore the prognostic and therapeutic implications of PD-L1 expression in different molecular subtypes of CMCs and their potential as predictive biomarkers for immunotherapy.

Time of day bias for biological sampling in studies of mammary cancer.

Despite its demonstrated biological significance, time of day is a broadly overlooked biological variable in preclinical and clinical studies. How time of day affects the influence of peripheral tumors on central (brain) function remains unspecified. Thus, we tested the hypothesis that peripheral mammary cancer tumors alter the transcriptome of immune responses in the brain and that these responses vary based on time of day; we predicted that time of day sampling bias would alter the interpretation of the results. Brain tissues collected at mid dark and mid light from mammary tumor-bearing and vehicle injected mice were analyzed using the Nanostring nCounter immune panel. Peripheral mammary tumors significantly affected expression within the brain of over 100 unique genes of the 770 represented in the panel, and fewer than 25% of these genes were affected similarly across the day. Indeed, between 65 and 75% of GO biological processes represented by the differentially expressed genes were dependent upon time of day of sampling. The implications of time-of-day sampling bias in interpretation of research studies cannot be understated. We encourage considering time of day as a significant biological variable in studies and to appropriately control for it and clearly report time of day in findings.

Advancing canine mammary tumor diagnostics: Unraveling the diagnostic potential of Cytokeratin 19 through droplet digital PCR analysis.

Cytokeratin 19 (CK19) is a complex intracytoplasmic cytoskeletal protein primarily localized in the ducts of the mammary gland and skin epithelial cells. In humans, the expression of CK19 gene within circulating tumor cells (CTCs) extracted from blood samples of breast cancer patients reflects tumor cell activity, offering valuable insights for predicting early metastatic relapse or monitoring treatment effectiveness. However, knowledge of serum tumor markers is limited in veterinary oncology. Recently, droplet digital PCR (ddPCR), has been employed to explore rare target genes due to its heightened sensitivity and accuracy as a novel molecular diagnostic tool. The objectives of this study were to investigate the expression of the CK19 mRNA in CTCs, non-neoplastic mammary tissues, and both benign and malignant canine mammary tumors (CMTs) through ddPCR analysis. In Study I, we optimized the discard volume for blood samples to reduce CK19 contamination from skin epithelial cells post-venipuncture. The results revealed that discarding the initial 3 mL of blood was adequate and effective in eliminating CK19 mRNA contamination. In Study II, after the removal of the initial 3 mL of blood, we investigated CK19 mRNA-positive CTCs in the peripheral blood of normal healthy dogs, including those with benign and malignant CMTs. Intriguingly, CK19 mRNA was undetectable in all blood samples. The expression of CK19 mRNA in mammary tissues was investigated in Study III. The copy number (CN) ratios of the CK19 gene in non-neoplastic mammary tissues (14.77 ± 14.65) were significantly higher (P < 0.05) than those in benign (4.23 ± 3.35) and malignant groups (6.56 ± 5.64). Notably, no difference was observed between the benign and malignant groups. In conclusion, CK19 mRNA appeared unlikely to be a suitable candidate as a biomarker in the peripheral blood of CMTs, while the CN ratio in mammary tissues could serve as a potential discriminator between non-neoplastic and CMT groups, complementing the gold standard of histopathological examination.

YRNA and tRNA fragments can differentiate benign from malignant canine mammary gland tumors.

Mammary gland tumors (MGT) are the most common tumors in sexually intact female dogs. The functional regulation of miRNAs, a type of noncoding RNAs (ncRNAs), in canine MGT has been extensively investigated. However, the expression of other ncRNAs, such as YRNAs and transfer RNA-derived fragments (tRFs) in canine MGT is unknown. We investigated ncRNAs other than miRNAs from our small RNA project (PRJNA716131) in different canine MGT histologic subtypes. This study included benign tumors (benign mixed tumor, complex adenoma) and malignant tumors (carcinoma in benign tumor and carcinoma with metastasis) samples. Aberrantly expressed ncRNAs were examined by comparisons among MGT subtypes. The relative expression trends were validated in canine MGT tissues, plasma, extracellular vesicles, and MGT cell lines using quantitative reverse transcription PCR. Three aberrantly expressed ncRNAs were identified by comparisons among MGT subtypes. YRNA and tRNA-Gly-GCC distinguished benign mixed tumor from other MGT histologic subtypes, while tRNA-Val differentiated complex adenoma, carcinoma in benign tumors, and carcinoma with metastasis. The ROC curve of the three ncRNAs showed they might be potential biomarkers to discriminate malignant from benign MGT. YRNA and tRFs expression levels were decreased in metastatic compared with primary canine MGT cell lines. To the best of our knowledge, this is the first investigation of YRNA and tRFs in canine MGT. The three identified ncRNAs may be biomarkers for differentiating MGT histologic subtypes. Suggested Reviewers: Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporatio.

Conditional in vivo deletion of LYN kinase has little effect on a BRCA1 loss-of-function-associated mammary tumour model.

LYN kinase is expressed in BRCA1 loss-of-function-dependent mouse mammary tumours, in the cells of origin of such tumours, and in human breast cancer. Suppressing LYN kinase activity in BRCA1-defective cell lines as well as in in vitro cultures of Brca1-null mouse mammary tumours is deleterious to their growth. Here, we examined the interaction between LYN kinase and BRCA1 loss-of-function in an in vivo mouse mammary tumour model, using conditional knockout Brca1 and Lyn alleles. Comparison of Brca1 tumour cohorts showed little difference in mammary tumour formation between animals that were wild type, heterozygous or homozygous for the conditional Lyn allele, although this was confounded by factors including incomplete Lyn recombination in some tumours. RNA-sequencing analysis demonstrated that tumours with high levels of Lyn gene expression had a slower doubling time, but this was not correlated with levels of LYN staining in tumour cells themselves. Rather, high Lyn expression and slower tumour growth were likely a result of B-cell infiltration. The multifaceted role of LYN indicates that it is likely to present difficulties as a therapeutic target in breast cancer.

Research progress of good markers for canine mammary carcinoma.

PURPOSE: Mammary gland tumors are the most common neoplastic diseases in elderly female dogs, about 50% of which are considered to be malignant. Canine mammary tumors are similar to human breast cancers in many respects, so canine mammary tumors are frequently studied alongside human breast cancer. This article mentioned KI-67, HER-2, COX-2, BRCA1, BRCA2, P53, CA15-3, MicroRNA, Top2α and so on. All these markers are expected to have an important role in the clinic. METHODS: Existing markers of canine mammary carcinoma are reviewed, and the expression of each marker and its diagnostic role for this tumor are described in detail. RESULTS: This article introduced several effective markers of canine mammary tumors, among them, antigen KI-67 (KI-67), human epidermal growth factor receptor 2 (HER-2), cyclooxygenase 2 (COX-2) are promising and can be detected in both serum and tissue samples. Breast cancer caused by mutations in the breast cancer 1 gene (BRCA1) and breast cancer 2 gene (BRCA2) is also a hot topic of research. In addition to the above symbols, tumor protein p53 (p53), cancer antigen15-3 (CA15-3), MicroRNA (miRNA), topoisomerase πα (Top2α), proliferating cell nuclear antigen (PCNA), epidermal growth factor receptor (EGFR) and E-cadherin will also be involved in this paper. We will also mention Mammaglobin, which has been rarely reported so far.

Human basal-like breast cancer is represented by one of the two mammary tumor subtypes in dogs.

BACKGROUND: About 20% of breast cancers in humans are basal-like, a subtype that is often triple-negative and difficult to treat. An effective translational model for basal-like breast cancer is currently lacking and urgently needed. To determine whether spontaneous mammary tumors in pet dogs could meet this need, we subtyped canine mammary tumors and evaluated the dog-human molecular homology at the subtype level. METHODS: We subtyped 236 canine mammary tumors from 3 studies by applying various subtyping strategies on their RNA-seq data. We then performed PAM50 classification with canine tumors alone, as well as with canine tumors combined with human breast tumors. We identified feature genes for human BLBC and luminal A subtypes via machine learning and used these genes to repeat canine-alone and cross-species tumor classifications. We investigated differential gene expression, signature gene set enrichment, expression association, mutational landscape, and other features for dog-human subtype comparison. RESULTS: Our independent genome-wide subtyping consistently identified two molecularly distinct subtypes among the canine tumors. One subtype is mostly basal-like and clusters with human BLBC in cross-species PAM50 and feature gene classifications, while the other subtype does not cluster with any human breast cancer subtype. Furthermore, the canine basal-like subtype recaptures key molecular features (e.g., cell cycle gene upregulation, TP53 mutation) and gene expression patterns that characterize human BLBC. It is enriched in histological subtypes that match human breast cancer, unlike the other canine subtype. However, about 33% of canine basal-like tumors are estrogen receptor negative (ER-) and progesterone receptor positive (PR+), which is rare in human breast cancer. Further analysis reveals that these ER-PR+ canine tumors harbor additional basal-like features, including upregulation of genes of interferon-γ response and of the Wnt-pluripotency pathway. Interestingly, we observed an association of PGR expression with gene silencing in all canine tumors and with the expression of T cell exhaustion markers (e.g., PDCD1) in ER-PR+ canine tumors. CONCLUSIONS: We identify a canine mammary tumor subtype that molecularly resembles human BLBC overall and thus could serve as a vital translational model of this devastating breast cancer subtype. Our study also sheds light on the dog-human difference in the mammary tumor histology and the hormonal cycle.

A genetic mosaic mouse model illuminates the pre-malignant progression of basal-like breast cancer.

Basal-like breast cancer (BLBC) is highly aggressive, and often characterized by BRCA1 and p53 deficiency. Although conventional mouse models enabled the investigation of BLBC at malignant stages, its initiation and pre-malignant progression remain understudied. Here, we leveraged a mouse genetic system known as mosaic analysis with double markers (MADM) to study BLBC initiation by generating rare GFP+Brca1, p53-deficient mammary cells alongside RFP+ wild-type sibling cells. After confirming the close resemblance of mammary tumors arising in this model to human BLBC at both transcriptomic and genomic levels, we focused our studies on the pre-malignant progression of BLBC. Initiated GFP+ mutant cells showed a stepwise pre-malignant progression trajectory from focal expansion to hyper-alveolarization and then to micro-invasion. Furthermore, despite morphological similarities to alveoli, hyper-alveolarized structures actually originate from ductal cells based on twin-spot analysis of GFP-RFP sibling cells. Finally, luminal-to-basal transition occurred exclusively in cells that have progressed to micro-invasive lesions. Our MADM model provides excellent spatiotemporal resolution to illuminate the pre-malignant progression of BLBC, and should enable future studies on early detection and prevention for this cancer.

Deciphering Stromal Changes between Metastatic and Non-metastatic Canine Mammary Carcinomas.

Cancer-associated stroma (CAS) is widely recognized to influence development and progression of epithelial tumours including breast cancer. Canine mammary tumours (CMTs) such as simple canine mammary carcinomas represent valuable models for human breast cancer also with respect to stromal reprogramming. However, it remains unclear whether and how CAS changes in metastatic tumours compared to non-metastatic ones. To characterize stromal changes between metastatic and non-metastatic CMTs and identify potential drivers of tumour progression, we analysed CAS and matched normal stroma from 16 non-metastatic and 15 metastatic CMTs by RNA-sequencing of microdissected FFPE tissue. We identified 1438 differentially regulated genes between CAS and normal stroma, supporting previous results demonstrating stromal reprogramming in CMTs to be comparable with CAS in human breast cancer and validating deregulation of pathways and genes associated with CAS. Using primary human fibroblasts activated by treatment with TGFß, we demonstrate some of the strongest expression changes to be conserved in fibroblasts across species. Furthermore, we identify 132 differentially expressed genes between CAS from metastatic and non-metastatic tumours, with strong changes in pathways including chemotaxis, regulation of apoptosis, immune response and TGFß signalling and validate deregulation of several targets using RT-qPCR. Finally, we identify specific upregulation of COL6A5, F5, GALNT3, CIT and MMP11 in metastatic CAS, suggesting high stromal expression of these targets to be linked to malignancy and metastasis of CMTs. In summary, our data present a resource supporting further research into stromal changes of the mammary gland in relation to metastasis with implications for both canine and human mammary cancer.