Lymphomas are a heterogenous group of cancers, which collectively account for nearly 15% of all neoplasia in dogs.1 The most common of these is DLBCL, which represents 30% to 60% of the lymphomas in dogs.2,3,a Most lymphomas in dogs are intermediate to high grade in nature and have a multicentric anatomic distribution in which peripheral lymph nodes are primarily affected, with secondary involvement of the liver, spleen, bone marrow, and other organs. Standard treatment for these cancers involves a combination of chemotherapeutics (eg, CHOP).
Treatment with CHOP results in cancer remission rates of 70% to 90% and median survival times of 9 to 14 months; however, CHOP rarely results in a permanent cure, and nearly all dogs with these lymphomas will die of a relapse of cancer.4–7 In contrast, treatment with CHOP results in cures in approximately 30% to 40% of humans with DLBCL.8 This disparity in cure rates is likely attributable, at least in part, to the lower intensity for CHOP used in dogs, compared with that for CHOP used in humans; dogs generally are treated at lower drug doses with longer intervals between successive treatments. Unfortunately, increasing the dosing intensity is unlikely to be a feasible strategy for successful treatment of multicentric lymphomas in dogs because associated treatment-related toxicoses would be unacceptable to many dog owners.9
Thus, improving the cure rate for intermediate- to high-grade multicentric lymphomas in dogs will likely necessitate adding additional chemotherapeutics to CHOP or substituting more targeted drugs for some component of the treatment, which increases the dose intensity without substantially augmenting the toxic effects. Drugs that target FRs represent a promising method for meeting this therapeutic goal.10–13
Folate is a B vitamin that is essential for many cellular processes, including DNA and RNA synthesis. Folate is hydrophilic; thus, transport of folate across biological membranes must be facilitated by various mechanisms. The 3 principle mechanisms by which folate is transported into cells are the RFC system, which is the predominant transporter in most adult and fetal tissues; proton-coupled folate transporters, which function mainly in the intestinal absorption of dietary folates as well as in folate uptake into the CNS; and FRs, which play an important role in neural tube development during embryogenesis but which do not serve a clearly defined function in adult tissues.11
Two primary FR isoforms are found in healthy tissues obtained from adults (FRα and FRβ)10–12,14 The FRα isoform is expressed primarily on the apical surface of polarized epithelial cells, such as the epithelial cells of the renal proximal tubules. Importantly, the apical expression of FRs in these cells limits the exposure of the receptor to ligands in the circulating plasma. The FRβ isoform is expressed predominantly by activated macrophages, but lower amounts of FRβ can also be detected on proinflammatory monocytes and their precursors. Curiously, FRβ can exist in both a functional and nonfunctional form. The functional form (which is expressed by activated macrophages and some types of cancers) binds folate with high affinity, but the nonfunctional form of FRβ has no affinity for folate.10–12,14
Despite their limited expression in physiologically normal tissues, FRs are highly expressed in several cancers in humans.10–12,14 The FRα isoform is overexpressed primarily in carcinomas, especially those of the ovary and uterus. Also, FRα is expressed in non–small cell lung cancer, breast cancer, testicular tumors, and renal tumors. Increased FRβ expression is most often evident in hematopoietic tumors, particularly myeloid leukemias. The FRs may be detected in vitro by several methods, including immunohistochemical analysis, folate ligand binding assays, PCR assays, quantitative in situ hybridization, and flow cytometry.11 The FRs can be detected in vivo through the use of various diagnostic imaging modalities, such as nuclear scintigraphy or positron emission tomography, which detect tissue uptake of folate-conjugated radiopharmaceuticals.10,11,15
The overexpression of FRs in certain tumor types, combined with limited FR expression in physiologically normal tissues, makes FRs attractive diagnostic and therapeutic targets. Both FR-targeted diagnostic imaging and FR-targeted therapeutic agents have been used successfully in preclinical studies11–16 in laboratory animal species and in clinical trials17 involving human cancer patients. Recently, FR expression was also characterized in dogs with naturally occurring TCC of the urinary bladder and urethra.18 Investigators used immunohistochemical analysis to identify FR expression in 76% of primary TCCs as well as 58% of metastatic tumors in lymph nodes and 48% of metastatic tumors in the lungs. Nuclear scintigraphy also revealed uptake of a folate-conjugated radiopharmaceutical (99mTc-EC20) in 12 of 13 dogs with TCC.18 Subsequent FR-targeted chemotherapy of 9 dogs with FR-expressing tumors in that study18 resulted in partial remission in 5 dogs and stable disease in 4 dogs.
These results in TCCs of dogs indicate the potential use of FR-targeted drugs in the treatment of dogs with cancers. However, identifying the expression of FRs on the cancer cell of interest or detecting tumoral uptake of folate in vivo is a prerequisite for FR-targeted chemotherapy, and the extent of FR expression and folate uptake in lymphomas of dogs is unknown. Therefore, the purposes of the study reported here were to determine the expression of FRs in multicentric lymphomas in dogs (as determined by immunohistochemical analysis) and to determine the extent of folate uptake by these cancers in vivo (as determined with nuclear scintigraphy). Our hypothesis was that a proportion of lymphomas would express FRs or take up 99mTc-EC20 (or both). A secondary objective was to evaluate the therapeutic response and treatment-related toxic effects for a folate-conjugated chemotherapeutic agent after administration to dogs with FR-expressing lymphomas.
Technetium Tc 99m
Cyclophosphamide, doxorubicin, vincristine, and prednisone
Diffuse large B-cell lymphoma
Peripheral T-cell lymphoma, not otherwise specified
Reduced folate carrier
Transitional cell carcinoma
Raskin RE, Fox LE. Clinical relevance of the World Health Organization classification of lymphoid neoplasms in dogs (abstr). Vet Clin Pathol 2003;32:151.
Target retrieval solution, Dako Corp, Carpinteria, Calif.
Background Sniper, Biocare Medical Inc, Walnut Creek, Calif.
Purdue University, West Lafayette, Ind.
Universal negative control serum, Biocare Medical Inc, Walnut Creek, Calif.
MACH4 Universal HRP-polymer, Biocare Medical Inc, Walnut Creek, Calif.
Vector Laboratories Inc, Burlingame, Calif.
Richard-Allan Scientific, Kalamazoo, Mich.
Etarfolatide, Endocyte Inc, West Lafayette, Ind.
EC20, Endocyte Inc, West Lafayette, Ind.
MiE equine scanner H.R., Scintron VI, Elk Grove Village, Ill.
EC0905, Endocyte Inc, West Lafayette, Ind.
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