PhD in Veterinary Sciences for Animal Health and Food Safety

Contacts

Supervisor

Fulvio Riondato

Phd thesis

• Rationale

Cytological evaluation of cavity effusions is a standard practice in the diagnostic process, both in veterinary and human medicine. Although it is a key point to differentiate neoplastic causes from others, for the cytologists the differentiation of specific cytomorphological characteristics of different cell types is often challenging making it difficult to distinguish between epithelial and mesothelial cells and between reactive and neoplastic elements ¹. Immunohistochemistry (IHC) on cell blocks can help in the diagnosis but it is time spending and specific markers have not been identified in veterinary medicine ^2 3. Flow cytometry (FC) represents a very good tool for effusion analysis but markers for non-hematopoietic (NH) lineage (e.g. epithelial and mesothelial) are lacking and there are not yet validated antibodies in dogs ⁴. Thus, surgical biopsies and specialized immunohistochemical or immunocytochemical stains are mandatory in many cases.  Unfortunately, obtaining a sample for histology is often impossible in the veterinary clinical setting and cytology still represents the last diagnostic step ⁵.

In this scenario, the improvement of FC panels by the identification of markers for NH cell lineages would provide a very useful tool in routine lab diagnostics and clinical management of patients ^6 7. Furthermore, most of the limitations can be definitively overcome by the application of more advanced and specific technique such as genomic analyses, using effusion as liquid biopsy ^8 9.

• Goals

The aim of the study is to improve the laboratory diagnostics for canine effusions with the final goal of providing lab tests to differentiate NH cell lineages and identify neoplastic conditions.  The present PhD project aims are to provide the basis to develop and validate such tests through the identification of new NH cell markers for FC analysis and the description and detection of recurrent mutations in canine carcinomas and mesotheliomas.

• Materials and methods

Samples and inclusion criteria - For the first part of the project all the suspected neoplastic effusions will be analyzed according to standard laboratory methods  and classified as reactive, neoplastic, and doubtful cases. Hemopoietic neoplasms will be ruled out by flow cytometry (FC). All the non-hematopoietic effusion will be stored.For the molecular part, we will be use only the effusions classified into carcinoma, mesothelioma and sarcoma.  Samples from primary masses with or without effusions will be also collected and processed for FC or stored for eventual genomic analysis.

Flow cytometry - As a first step, different commercially available clones of possible markers already used in humans (e.g., calretinin and Epcam) will be screened to find a product cross-reacting in dogs and/or working in FC. The selected clones will then use to label included effusions and primary masses.

Mutational profile - NGS will be performed on samples with final diagnosis reported above on the NH population sorted in the first instance or on the pellet for samples in which it was not possible to perform the sorting. Primary masses will be sequenced if the minimum effusion number established for this study cannot be collected.

Based on the data obtained from the bioinformatic analysis, a PCR test will be developed and optimized for the most representative mutations found. Available effusion samples (cell pellets and supernatants) will be used as pilot samples for PCR application.

• Expected results

The expected results are:

• identification of new cellular NH markers for FC analysis.
• definition of a FC panel for NH lineages characterization
• description of the mutational profile of effusion associated tumors, especially carcinoma and mesothelioma.
• identification of target mutations to be used as a target in carcinomas and mesotheliomas

optimization of a PCR or ddPCR test for identified driver mutations to be used as target in carcinomas and mesotheliomas diagnosis using effusions as liquid biopsy. These results will provide new diagnostic tools, setting the basis for perspective studies on a larger court of patients to validate the methods as routine diagnostic tests for canine effusions.

• 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

- Full articles in ISI journals:

Riondato F., Colitti B., Rosati S., Sini F., Martini V.  A method to test antibody cross-reactivity toward animal antigens for flow cytometry. Cytometry. September 2022. PMID: 36161760.  DOI: 10.1002/cyto.a.24691

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, 10, 374. https://doi.org/10.3390/vetsci10060374

- Congresses abstract:

Poster: Sini F, Miniscalco B, Valenti P, Melega M, Poggi A, Riondato F.

Flow cytometric cd44 and cd45 expression in neoplastic and reactive non-hemopoietic cells in canine effusions. Veterinary Pathology and Veterinary Clinical Pathology (ESVP/ECVP/ESVCP/ECVCP, 31 August – 2 September 2023, Lisbon) - Best Research Poster 

Co-author (oral presentation): Ubiali A, Moretti P, Riondato F, Sini F, De Maria R, Stefanello D, Comazzi S, Aresu L, Marconato L, Martini V.                             

Evaluation of PD-L1 expression in canine lymphomas using flow cytometry. Research Communication 33rd ECVIM-CA Congress 21-23 September 2023, Barcelona, Spain.