Here we are, even in dogs we can now start to evaluate the mutation status of tumors in diagnostic routine: here is what you can do and how TP53 gene mutation analysis in dogs with DLBCL can help to determine prognosis and therapeutic approach.

DLBCL – The Most Common Lymphoma in Dogs

Among the canine lympho-proliferative diseases, DLBCL is the most common and represents around 70% of all lymphomas in this species. DLBCL is characterized by an aggressive biological behavior and is mostly presented in multicentric nodal form, with the multiple involvement of secondary lymphatic organs. In the most advanced clinical stages, the infiltration of target organs such as the liver, spleen, bone marrow and peripheral blood can also be observed. According to the criteria of the classification of canine lymphomas, established by the World Health Organization (WHO), nodal DLBCL is characterized by the uncontrolled and widespread growth of a neoplastic cell population in the lymph node context, with a total impairment of the normal anatomy of the organ. The neoplastic cells are of medium/large size and often present multiple peripheral nucleoli or a single central nucleolus. This population is related to centroblasts and immunoblasts (FIG 1). The neoplastic lymphocytes are of B immunophenotype and are identified via immunohistochemestry with the positivity to the antibodies CD20, CD79 or PAX5 (FIG 1).

The first line therapy for canine Diffuse Large B-Cell Lymphoma (DLBCL) is a CHOP (Cyclophosphamide, Doxorubicin, Vincristine, Prednisone) chemotherapy regimen, with the potential addition of APAVAC vaccine in the chemo-immunotherapy protocol.

Classic microscopic/cytological picture of DLBCL in dogs. A predominantly medium/large lymphoid population with prominent nucleoli and moderate basophilic cytoplasm is noted
Fig. 1 – Classic microscopic/cytological picture of DLBCL in dogs. A predominantly medium/large lymphoid population with prominent nucleoli and moderate basophilic cytoplasm is noted
Histological and immunohistochemical examination of canine DLBCL. The lymph node appears diffusely infiltrated by a neoplastic population, which overtakes the normal architecture of the organ (image on the left). The neoplastic cells appear positive for CD20 (B-lymphocyte marker) on immunohistochemical examination (image on the right)
Fig. 2 – Histological and immunohistochemical examination of canine DLBCL. The lymph node appears diffusely infiltrated by a neoplastic population, which overtakes the normal architecture of the organ (image on the left). The neoplastic cells appear positive for CD20 (B-lymphocyte marker) on immunohistochemical examination (image on the right)

DLBCL – The Clinical-Pathological Classification

DLBCL has historically been classified as “high-grade” or “aggressive” lymphoma, but in recent years research in veterinary oncology has revealed new targets and therapeutic approaches, which have improved survival and disease-free time in dogs with this cancer. Unfortunately, the response to therapy is still very variable and unpredictable at diagnosis. Some dogs remain in clinical remission for only a few months after treatment, while others have much longer remission and survival times, in some cases even several years. This different biological behavior is partly justified by some measurable clinical variables, such as stage, clinical sub-stage and neoplastic infiltration of the bone marrow by flow cytometry. Although very useful, these parameters do not always discriminate the prognosis of the dog, in fact, other variables may affect the response to therapy, such as the role of the immune system in response to therapy and the genetic or molecular profile of the tumor itself.

DLBCL – Genetic Classification: Here We Go!

It is therefore clear that the only clinical-pathological classification can no longer be sufficient to clinically characterize canine DLBCL. For this reason, similarly to human medicine, studies on canine DLBCL in recent years have mainly focused on explaining the molecular and genetic mechanisms underlying the development and progression of the disease. Several aberrations have been identified in genes involved in activation, differentiation and cellular proliferation mechanisms. In some cases, mutations in the DNA have been discovered that potentially make lymphoid cells capable of proliferating more quickly, escaping the immune control and being resistant to programmed cell death (apoptosis). In some cases, such mutations have been correlated to clinical variables and response to therapy. A study in 2022 (Giannuzzi et al., 2022) has in fact demonstrated how TP53 gene mutations are an independent and negative prognostic factor in canine DLBCL.

Due to its role, TP53 has recently been referred to as “the guardian of the genome”, referring to its ability to repair damaged DNA by inducing cell growth arrest, apoptosis and cellular senescence. Therefore, it is easily understandable how, in a neoplastic or pre-neoplastic context, any mutations affecting this gene can suppress this activity, favoring carcinogenesis.

Analysis of TP53 in Dogs with DLBCL: Here’s How to Do It!

In dogs with a suspected DLBCL, based on cytological and cytofluorimetric examination of an enlarged lymph node, it will always be necessary to perform a histological and immunohistochemical (CD3, CD20) examination of the entire excised lymph node for a definitive confirmation. It will be important, when the lymphadenectomy is performed, to isolate a small fragment of neoplastic tissue (0.5 cm3) and to keep it in a vial with physiological solution in order to perform the TP53 mutation test (FIG 2). The vial must be sent to the laboratory within 24-48 hours to proceed with the test. At the same time, it is always necessary to send an aliquot of blood in an EDTA vial (FIG 3).

For the mutation test, it is necessary to send both the lymph node biopsy in physiological solution and the peripheral blood of the patient
Fig. 3 – For the mutation test, it is necessary to send both the lymph node biopsy in physiological solution and the peripheral blood of the patient

Therefore, based on the results obtained from the clinical staging, histopathological/immunohistochemical examination, and TP53 screening, vets can consult the prognostic algorithm developed based on Giannuzzi et all. (go to your private area of our Mylav website). Through this algorithm, it will also be possible to identify the best therapy based on the selected clinical variables.

Bibliography

Diana Giannuzzi , Laura Marconato, Antonella Fanelli , Luca Licenziato , Raffaella De Maria, Andrea Rinaldi, Luca Rotta , Nicole Rouquet, Giovanni Birolo , Piero Fariselli, Afua A. Mensah, Francesco Bertoni  and Luca Aresu. (2022) The genomic landscape of canine diffuse large B-cell lymphoma identifies distinct subtypes with clinical and therapeutic implications. Lab Animals 51: 191-202.

Luca Aresu, University of Turin, Head of the Pathology Team at MYLAV
Walter Bertazzolo, Med. Vet. EBVS European Specialist in Veterinary Clinical Pathology (Dipl. ECVCP); Scientific Director of MYLAV