S.G. Calazans; S.M. Rodigheri; S.C. Fernandes; S.S. Costa; T.D. Munhoz; R. Laufer-Amorim; J.L. Sequeira; J.A.D. Sena; C.R. Daleck
Via de Acesso Prof. Paulo Donato Castellane s/n, Jaboticabal, SP, Brazil
Lymphoma is the most common hematopoietic neoplasm in dogs. (Dhaliwal et al. 2003). Canine lymphoma has some similarities to human lymphoma and it has long been used as a model for investigations of cancer (Greenle et al. 1990). The tumor-suppressor gene TP53 prevents the accumulation of oncogenic mutations and genomic instability by arresting the cell cycle at G1/S phase, activating repair mechanisms or inducing apoptosis. Therefore, p53 inactivation could result in tumor development (Vogelstein et al. 2000). Canine TP53 is similar both in structure and function to human TP53 (Veldhoen & Milner 1998). The majority of TP53 mutations reported in human cancers are clustered in highly conserved domains, including exons 5-9 of this gene (Greenblatt et al. 1994). Although the TP53 status has been already investigated in several types of canine neoplasms, it is still poorly defined in lymphomas (Sokolowska et al. 2005). The relationship of introns to cancer has also been studied and several functions for introns have already been identified (Bergman 2001). However, the inotropic sequences of TP53 gene are as yet unknown in canine lymphoma. Thus, the aim of this study was to analyze the TP53 status in dogs with lymphoma and its relationship to anatomical classification, clinical stage, histological grade, immunophenotype and survival time.
Materials and Methods
Twelve dogs with lymphoma referred to Oncology Unit in the Veterinary Hospital of São Paulo State University (UNESP/Jaboticabal) were included in this study. Control group consisted of five healthy dogs. Clinical staging was accomplished, (stage I--involvement limited to a single lymph node, or lymphoid tissue in a single organ; stage II--involvement of many lymph nodes in regional area; stage III--generalized lymph node involvement; stage IV--liver and/or spleen involvement or stage V--manifestations in the blood and involvement of bone marrow and/or other organ systems), as well as anatomical classification (multicentric, alimentary, cutaneous, mediastinal or extranodal). Patients received chemotherapy protocol with L-asparaginase, doxorubicin, cyclophosphamide, vincristine and prednisone. Tumor samples were collected by means of incisional or excisional biopsy. Histopathological diagnosis was performed on the slides stained with hematoxylin and eosin, according to Kiel classification. Immunohistochemistry was performed to identify T or B cell origin, using anti-CD3 polyclonal antibody (Dako) and anti-CD79a monoclonal antibody (Dako), respectively. Genomic DNA was isolated from tumor sections using a DNeasy Blood and Tissue kit (Qiagen). PCR oligonucleotides for amplification of the region that encompasses exons 4-9 (including introns) were designed on basis of canine partial sequence, GenBank Accession No. U62133. One hundred ng of genomic DNA was used as a template for each PCR to amplify approx. 500bp fragment. The PCRs were performed in the presence of 10μM of each primer, 4mM of Magnesium Sulfate, 0.2mM of each dNTP (dATP, dCTP, dGTP, dTTP), 1U of Taq DNA Polymerase high fidelity (Invitrogen) and 1X PCR buffer. The thermocycle program included a denaturation step at 95°C (5min), 40 cycles of 95°C (60 s), 64 to 67°C (30s), 72°C (60s) and a final elongation step at 72°C (5 min). The product of amplification were then purified using a QIAquick PCR Purification kit (Qiagen) and sent to Center for Studies of the Human Genome, (University of São Paulo), where sequencing were performed twice, using the MegaBACE 1000 (GE Healthcare technology) and the DYEnamic ET Dye Terminator Kit (with Thermo Sequenaseô II DNA Polymerase). The homology of sequences was verified by Molecular Evolutionary Genetics Analysis (MEGA) software version 4.
The mean age of dogs was 5.83 (±2,12). Seven dogs (58.33%) were male and five dogs (41.67%) were female. Three dogs were mixed breeds and nine were purebred dogs (2 Poodles, 2 American Pit bulls, 2 Rottweilers, 1 Boxer, 1 Bull Terrier and 1 Basset Hound). Three dogs presented with stage II, two with stage III and seven with stage V. Fifty per cent of dogs presented systemic signs. The most common anatomical form was the multicentric lymphoma (six dogs). There were five dogs with cutaneous form and one had alimentary lymphoma. High-grade malignant lymphoma was observed in ten dogs and the other two dogs presented low-grade lymphoma according to histopathological findings. Eight lymphomas were of B-lymphocyte origin and four were of T cell origin. The mean survival time was 96,87 days (±137.13). Four fragments were amplified by PCR reactions (558, 578, 570 and 579bp), corresponding to region that encompasses exons 4-9. Gene mutations were not observed in any sequence.
Discussion and Conclusions
All dogs evaluated were adults. Lymphoma in adult dogs is more frequent that in younger dogs (Vail & Young 2007). Male dogs were prevalent although gender does not seem to be related to lymphoma in dogs (Jacobs et al., 2002). Previous studies reported that Boxer, Basset Hound and Rottweiler are at higher risk than others breeds (Vonderhaar & Morrison 2002, Vail & Young 2007). Despite these breeds have been included in the present study, mixed dogs were prevalent because of their high frequency in Veterinary Hospital. Several studies verified that lymphoma is commonly diagnosed at advanced clinical stage (stages IV and V) (Greenlee et al. 1990, Teske 1994, Vonderhaar & Morrison 2002, Vail & Young 2007). Advanced clinical stage was confirmed in almost all dogs (83,33%) and all dogs presented clinical signs. Multicentric lymphomas were the most common presentation, as reported by others (Teske 1994, Vonderhaar & Morrison 2002, Vail & Young 2007). Histological classification defined high-grade lymphomas in ten dogs, suggesting the prevalence of aggressive lymphomas in dogs. It was also concluded in previous studies (Greenlee 1990; Teske 1994, Fournel-Fleury et al. 1997, Ponce et al. 2004). Lymphomas of B-lymphocyte origin were more frequent and this finding is usually found (Teske 1994, Fournel-Fleury et al. 2002). Sokolowska et al 2005 have shown that the expression of p53 in canine lymphomas is rare. However, some studies have reported TP53 gene mutations in dogs with lymphoma. Veldhoen et al. (1998) reported the first example of germ line TP53 mutation in a Bull Mastiff which has been shown to exhibit a high incidence of familial lymphoma (Onions 1984). The same authors verified two base pair insertion within exon 7, resulting in a frameshift mutation at codon 234 and incorporation of a stop codon at position 247, which is sufficient to abolish expression of the p53 protein. Nasir & Argyle (1998) detected a somatic transition substitution mutation in exon 5 of TP53 gene, which resulted in a codon change from CGC to CAC and a predicted amino acid substitution of arginine to histidine. Many investigations have been focused on relationship of introns to cancer, According to previous studies, introns could produce transcripts that affect protooncogenes expression and induce cancer (Wang-Gohrke 1999, Reis et al. 2004). In the present study, TP53 gene mutations were not observed in any sequence, suggesting that they are rare in dogs with lymphoma. In summary, although mutations in dogs with lymphoma are rare, the pathways involving TP53 are not yet understood and must be investigated.
Authors gratefully acknowledge the financial support of the Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp)
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