The BCL2 gene is a member of a family of genes that regulate apoptosis. Overexpression of BCL2 protein has been associated with increased relapse rates, shorter TTP, and decreased survival times in human patients with non-Hodgkin lymphoma.1–3 Three-year survival rates of 30% and 70% have been reported for human patients with CLL and follicular forms of non-Hodgkin lymphoma that have high or low BCL2 expression in biopsy samples, respectively2–4 As an antiapoptotic protein, BCL2 can contribute to resistance to chemotherapeutic agents and radiation.5–7
Peptide receptor targeting for diagnosis and treatment of cancers has become a focus of nuclear medicine in humans. However, these technologies are rarely used in veterinary patients. Peptide-based agents can target cells in vivo by agonist-induced receptor internalization and intracellular retention of the radiolabeled agent in tumor cells.8,9 A peptide receptor commonly used for in vivo cancer targeting is the somatostatin receptor, which can be highly expressed in neuroendocrine tumors and have lower expression levels in lymphoproliferative disorders, including lymphoma.10–12
Lymphoma is a common neoplastic disease in dogs and was reported to affect 1.2% of all dogs in a large retrospective study13 that included records from veterinary teaching hospitals spanning a 38-year period. Current treatment recommendations consist of multidrug protocols that result in 60% to 90% complete remission rates and median survival times ranging from 6 to 12 months.14–16 Unfortunately, reported response rates to conventional chemotherapy in canine patients have not changed in > 20 years. Several prognostic indicators have been identified for lymphoma in dogs, including immunophenotype, World Health Organization substage, and mediastinal involvement.17–19 Results of 1 study20 in 23 dogs with multicentric lymphoma indicated that BCL2 expression was not correlated with resistance to chemotherapeutic agents; however, TTP and other outcome measures were not evaluated. Results of experimental research in rodents as well as human cancer research have indicated that BCL2 has a role in drug resistance, suggesting that further investigation is warranted.2–5
Noninvasive imaging of BCL2 expression can potentially be used to identify high-risk patients with lymphoma that may be resistant to conventional chemotherapeutic agents. Molecular imaging of BCL2 expression has been described in immunodeficient mice engrafted with Mec-1 (human B-cell CLL) cells by investigators who used an RaPP to target BCL2 gene expression.21 In that study,21 the radiolabeled PNA-somatostatin analogue conjugate, γ-emitting 111In-DOTA-anti–BCL2–PNA-Tyr3-octreotate, was confirmed to be internalized and to have specific receptor-mediated cell uptake and targeting of human BCL2 mRNA.
Favorable biodistribution and tumor-specific uptake of the somatostatin analogue, 111In-Tyr3-octreotate, has been demonstrated in a variety of tumor tissues in rats and mice. In a study22 performed to evaluate tumor-specific uptake of various 111In-labeled somatostatin analogues, Tyr3-octreotate had the highest uptake in somatostatin receptor–positive tumors and the greatest degree of binding and internalization by tumor cells in vivo, compared with other compounds tested.
Importantly, the radionuclide coupled to the conjugate is internalized by the targeted cell after binding to the somatostatin receptor. The internalization of somatostatin analogues occurs via G-protein coupling, and peptide analogues are targeted for degradation during receptor recycling.23 In contrast, PNA analogues are resistant to peptidases and remain highly stable in biological systems. Specificity of the RaPP binding to BCL2 mRNA within the cell is accomplished via the PNA, which is a DNA-like antisense molecule that directs the conjugate to the mRNA of interest.
The purpose of the study reported here was to determine whether whole-body nuclear scintigraphy could be used to detect neoplastic lymphocytes in dogs with histologically confirmed B-cell lymphoma following IV administration of the RaPP 111In-DOTA-anti–BCL2–PNA-Tyr3-octreotate and to assess associations among RaPP uptake, TTP, and BCL2 mRNA expression in an affected lymph node. We hypothesized that dogs with naturally occurring B-cell lymphoma would have specific uptake of the RaPP in neoplastic tissues and that increased specific uptake of the RaPP in predetermined ROIs would be directly correlated with BCL2 mRNA expression and negatively associated with TTP.
B-cell leukemia-lymphoma 2
Chronic lymphocytic leukemia
Peptide nucleic acid
Radiolabeled peptide nucleic acid–peptide conjugate
Region of interest
Time to tumor progression
Equistand II planar gamma camera, Diagnostic Imaging, Middlesex, NJ.
Mirage version 54x, Diagnostic Services, Sussex, NJ.
Open Biosystems, Huntsville, Ala.
Wizard plus SV Minipreps DNA Purification System, Promega Corp, Madison, Wis.
STAT-60 reagent, Tel-Test Inc, Friendswood, Tex.
SUPERase-In, Applied Biosystems, Carlsbad, Calif.
ND-1000 spectrophotometer, NanoDrop Technologies, Wilmington, Del.
RNeasy MinElute cleanup kit, Qiagen, Valencia, Calif.
Superscript III Reverse Transcriptase, Invitrogen, Carlsbad, Calif.
TaqMan Gene Expression Assay, assay No. Cf02622425_m1, Applied Biosystems, Carlsbad, Calif.
7500 Thermocycler, Applied Biosystems, Carlsbad, Calif.
FAM fluorescent dye, Invitrogen, Carlsbad, Calif.
ROX fluorescent dye, Invitrogen, Carlsbad, Calif.
Sequence Detection Systems, version 1.2.3, Applied Biosystems, Carlsbad, Calif.
R, version 2.12.2, R Foundation for Statistical Computing, Vienna, Austria. Available at: www.r-project.org/. Accessed Jan 6, 2011.
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