PhD in Veterinary Sciences for Animal Health and Food Safety

Contacts

Supervisor

Fulvio Riondato

Phd thesis

Background

In veterinary medicine, the diagnosis of neoplastic conditions in the absence of tissue biopsies can be challenging. Effusions, when present, represent a valid alternative-one already used in human medicine but often overlooked in veterinary practice.

This study aims to enhance the diagnostic potential of effusions in dogs, particularly in cases of carcinomas, mesotheliomas, and other non-hematopoietic (NH) neoplasms, using a combination of flow cytometry (FC), immunohistochemistry (IHC), next-generation sequencing (NGS), and droplet digital PCR (ddPCR).

Materials and Methods

The study is divided into three parts:

1. reactive and neoplastic effusions were analyzed using FC and IHC on cell blocks to test and identify differentially expressed markers in NH cells. Starting with the optimization of a panel including cytokeratin (CK), vimentin (VIM), and desmin (DES), additional markers such as CD44, calretinin, WT1, podoplanin, and Epcam were tested.
2. NGS was performed using a panel of 120 genes known to be involved in both canine and human cancers, on 9 tissue biopsies of canine mesothelioma, a tumor that had not been previously sequenced.
3. a ddPCR test was developed for the detection of a specific HER2 mutation, commonly found in canine pulmonary carcinomas, using effusions as a liquid biopsy sample.

Results

1. Comparison of FC and IHC for CK, VIM and DES panel, yielded an agreement of 76.6% (33/43 cases), with discrepancies mainly related to VIM expression (8/10 cases). By testing the new antibodies, CD44 and podoplanin were identified as potential markers to differentiate NH cells and distinguish reactive and neoplastic effusions.
2. NGS revealed 10 single nucleotide variants (SNV), 39 copy number variants (CNV) and 8 internal tandem duplications (ITD), with mutations in genes such as NF2, KMT2D and BRCA2 showing relevance to the pathogenesis of human mesothelioma.
3. Finally, ddPCR, tested on effusions with suspected or confirmed diagnosis of carcinoma or differential diagnosis between carcinoma and mesothelioma, successfully detected the HER2 mutation in 20.4% of cases.

Conclusion

Effusions represent a promising matrix for diagnostic investigations in veterinary oncology.

1. FC has proven to be a valid alternative to IHC on cell blocks. The currently available panel (CK, VIM, DES, CD44, PDPN) allows for the discrimination of non-hematopoietic (NH) cells into mesothelial, epithelial, reactive, and neoplastic categories; however, the addition of further markers would be necessary to improve specificity.
2. NGS provided preliminary data on the mutational profile of canine mesothelioma. A considerable number of mutations were identified, revealing similarities in pathways and genes involved with the corresponding human tumor. Although further data are needed, this represents a significant starting point.
3. The ddPCR test demonstrated its potential for identifying target and recurrent mutations, proving especially useful in the analysis of heterogeneous samples such as effusions, with the ability to detect low-frequency mutations below 1%. In the future, similar tests could be developed for other tumors, including mesothelioma, and assist in diagnosis when tissue biopsy is not available.

Overall, these findings contribute to the advancement of diagnostic capabilities in veterinary medicine, offering less invasive and more practical approaches for diagnosing neoplastic conditions in dogs, utilizing effusions as an alternative diagnostic matrix.

 

• Bibliography

1. Michael, C. W. Serous fluid cytopathology: Past, present, and future. Diagn. Cytopathol. 49, 577–581 (2021).
2. Milne, E. M. et al. Comparison of effusion cell block and biopsy immunohistochemistry in mesothelial hyperplasia, mesothelioma, and carcinoma in dogs. Vet. Clin. Pathol. 1–13 (2021) doi:10.1111/vcp.13002.
3. Marcos, R. et al. The cell tube block technique and an immunohistochemistry panel including Wilms tumor 1 to assist in diagnosing cavitary effusions in dogs and cats. Vet. Clin. Pathol. 48, 50–60 (2019).
4. Pillai, V. & Dorfman, D. M. Flow cytometry of nonhematopoietic neoplasms. Acta Cytol. 60, 336–343 (2016).
5. Siravegna, G., Marsoni, S., Siena, S. & Bardelli, A. Integrating liquid biopsies into the management of cancer. Nat. Rev. Clin. Oncol. 14, 531–548 (2017).
6. Churg, A. & Naso, J. R. The Separation of Benign and Malignant Mesothelial Proliferations. Am. J. Surg. Pathol.44, e100–e112 (2020).
7. Pinto, D. & Schmitt, F. Current applications of molecular testing on body cavity fluids. Diagn. Cytopathol. 48, 840–851 (2020).
8. Kruglyak, K. M. et al. Blood-Based Liquid Biopsy for Comprehensive Cancer Genomic Profiling Using Next-Generation Sequencing: An Emerging Paradigm for Non-invasive Cancer Detection and Management in Dogs. Front. Vet. Sci. 8, 1–8 (2021).
9. Chibuk, J. et al. Horizons in Veterinary Precision Oncology: Fundamentals of Cancer Genomics and Applications of Liquid Biopsy for the Detection, Characterization, and Management of Cancer in Dogs. Front. Vet. Sci. 8, (2021).

 

 

Research activities

National Congress attendance (as the first author)

1. VIII Congresso Nazionale ISCCA, 2024 - VERONA (Italy) - Poster Prentation: Flow cytometric characterization of non-hemopoietic cells in canine and feline effusions. Sini F., Melega M., Cannizzo F.T., Miniscalco B., Valenti P., Riondato F.
2. 77° Congresso SISVET, 2024 - PARMA (Italy) - Oral Presentation: Flow cytometric CD44 and CD45 expression to characterize non-hemopoietic neoplastic, reactive, epithelial and mesothelial cells in canine effusions. Sini F., Miniscalco B., Valenti P., Melega M., Poggi A., Goldoni M., Riondato F.

International Congress attendance (as the first author)

1. Veterinary Pathology and Veterinary Clinical Pathology (ESVP/ECVP/ESVCP/ECVCP), 2023 - LISBON (Portugal) - Poster Presentation: Flow cytometric cd44 and cd45 expression in neoplastic and reactive non-hemopoietic cells in canine effusions. Sini F., Miniscalco B., Valenti P., Melega M., Poggi A., Riondato F.

Awards

Best Poster Research - Veterinary Pathology and Veterinary Clinical Pathology (ESVP/ECVP/ESVCP/ECVCP), 2023 - LISBON (Portugal). Flow cytometric cd44 and cd45 expression in neoplastic and reactive non-hemopoietic cells in canine effusions. Sini F., Miniscalco B., Valenti P., Melega M., Poggi A., Riondato F. 

Period Abroad

1. University of Cambridge, MRC Toxicology Unit - Internetional PREDICT-meso Project (1 month).
2. University of Oxford, Laboratory of Respiratory Translational Research, Nuffield Department of Medicine, University of Oxford (1 month).
3. IMP Diagnostic and Vedis Laboratory, Porto (2 months).

Pubblications (during the PhD period)

1. Riondato F, Colitti B, Rosati S, Sini F, Martini V. A method to test antibody cross-reactivity toward animal antigens for flow cytometry. Cytometry A. 2023 May;103(5):455-457. doi: 10.1002/cyto.a.24691.
2. Riondato F, Poggi A, Miniscalco B, Sini F, Marconato L, Martini V. Flow Cytometric Features of B- and T-Lmphocytes in Reactive Lymph Nodes Compared to Their Neoplastic Counterparts in Dogs. Vet Sci. 2023 May 26;10(6):374. doi: 10.3390/vetsci10060374.
3. Martini V, Moretti P, Sini F, Ubiali A, Poggi A, Riondato F. Factors correlating with circulating T-zone-like cells of undetermined significance (TZUS) in dogs resident in Italy. Vet J. 2024 Jun;305:106143. doi: 10.1016/j.tvjl.2024.106143.
4. Ubiali A, Cesar Conti L, Dall'Ara P, De Maria R, Aresu L, Moretti P, Sini F, Riondato F, Stefanello D, Comazzi S, Martini V. Exploring the dynamics of Programmed Death-Ligand 1 in canine lymphoma: unraveling mRNA amount, surface membrane expression and plasmatic levels. Front Vet Sci. 2024 Jul 26;11:1412227. doi: 10.3389/fvets.2024.1412227.
5. Sini F, Melega M, Cannizzo FT, Miniscalco B, Valenti P, Riondato F. Flow cytometry of non-hematopoietic cells in canine effusions. Front Vet Sci. 2024 sep 24; 11: 14114271. doi:11. 10.3389/fvets.2024.1414271.